JPH0685A - Gene or DNA fragment derived from hepatitis C virus, polypeptide encoded thereby and method for producing the same - Google Patents

Abstract

(57) Abstract: A novel gene or DNA fragment derived from hepatitis C virus (HCV), a polypeptide encoded by the DNA fragment, and a host cell transformed with a vector expressing the polypeptide are provided. A method for producing an HCV antigenic peptide by culturing. [Effect] The above HCV antigenic peptides are useful for the development of a diagnostic method or a novel vaccine capable of detecting HCV antibodies at a high rate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel hepatitis C virus (hereinafter referred to as "HCV")-derived gene or DNA.
The present invention relates to a fragment and a novel polypeptide corresponding thereto, and further relates to a DNA fragment encoding the polypeptide and a method of using the polypeptide.

[0002]

2. Description of the Related Art The human hepatitis virus has been
Type B, type D, and type D have been discovered, and serological diagnostic methods have been established for each, but hepatitis of unknown cause still existed and remained a problem (Digestive Diseases and Sciences, 31 , 122S. −
132S, 1986, Seminars In River Diseases, 6 , 56-66, 1986). On the other hand, in the middle of 1970, a specific diagnostic technique for detecting hepatitis A virus (HAV) and hepatitis B virus (HBV) was developed,
It came to be put to practical use. As a result, it was gradually revealed that most hepatitis associated with blood transfusion was caused by a pathogen, unlike HAV, HBV, and other viruses that grow in hepatocytes, and these hepatitis were named as non-A non-B hepatitis. Was given. In the United States, post-transfusion hepatitis occurs at a frequency of 1 to 10% of blood transfusion cases at present, and non-A non-B hepatitis accounts for 90% or more thereof (Experimental Medicine, 8, (3), 1).
5-18, 1990). Also in Japan, 10 to 10 transfusion cases
Post-transfusion hepatitis develops in 20% (about 200,000 a year), of which 95% is non-A non-B hepatitis. In addition, 4 out of about 300,000 sporadic hepatitis cases occur annually
0 to 50% is non-A non-B hepatitis, and many of these, including non-A non-B hepatitis that are prevalent only in one region, have no clear infectious route of blood transfusion. It is considered to have been infected by another route of infection (Experimental Medicine, 8, (3), 13-14, 1990).

The causative virus of non-A non-B hepatitis, which is the main cause of post-transfusion hepatitis, was reported by Chiron in May 1988.
It succeeded in obtaining the gene fragment by a method completely different from the conventional traditional virus search method, and was named hepatitis C virus. After that, in addition to Chiron, Shimo-Tono et al. (Proc Natl. Acad. Sci. US, Proceedings of National Academy of Science)
A), 87, 9524-9528, 1990), Takamizawa et al. Of Micro Lab, Osaka University (Journal of Birology (J
individual of Virology, 65, 3, 1
105-1113, 1991)), the sequences of all genes of the structural and nonstructural proteins of hepatitis C virus have been published. Many patent applications have been filed for the gene (European Patent Publication 318).
Nos. 216, 388232, 398748, International Patent Application Publication WO 91/01376, and European Patent Publication 4
19182, British Patent 2239245, European Patent Publication 450931, International Patent Application Publication WO 91/155.
16, European Patent Publication Nos. 464287 and 463848.
No., 468657).

Chiron Co., Ltd. is a fusion protein having a C-terminal fusion polypeptide of the region (363 residues) from NS3 to NS4 which is a part of the non-structural protein of hepatitis C virus (N-terminal side is human superhuman). Oxide dismutase) is expressed in yeast (Japanese Patent Publication No. 2-500880, European Patent Publication No. 318216), and using this recombinant antigen, an ELISA (enzyme-linked immunosorbent assay [enzyme- li
naked immunosorbent assa
y]) was developed. Japan's Ministry of Health is the first in the world to authorize the use of this antibody detection kit to screen blood for transfusion and aid in the diagnosis of hepatitis C, and the import of this test kit was approved (December 26, 1989). From the next day, the Japanese Red Cross Society will nationwide
Screening for CV antibody started. 1 in Japan
About 1.7 million patients receive blood transfusions annually. The hepatitis C incidence rate before the introduction of this test drug was 12.3%, and after the introduction,
It is estimated to be about 3%, and the number of hepatitis C patients due to blood transfusion, which was 173,000 per year, has been reduced to about one-fourth (Asahi Shinma Morning Edition, May 2, 1991).

By the way, the C100-3 clone, which is a recombinant antigen used by Chiron, has a homology in the nucleotide sequence and amino acid sequence which may differ from that cloned in Japan by almost 20%, which is the cause. It cannot be denied that the HCV antibody cannot be detected in. Furthermore, it has been reported variously that in other regions (for example, a part of each region of NS1, NS2, NS3, NS5 according to Chiron, or most of them), there is a homology of 70% or less. It is easily presumed that there are some specimens that cannot be detected with the kit. In other words, it should be noted here that HCV has a high frequency of gene mutations,
It is speculated that this mutation also mutates the epitope site of the HCV-derived protein and affects the antibody against the HCV-related antigen (particularly, the HCV-derived protein) presented in serum. In addition, it is during the transition to chronic hepatitis among hepatitis that the above-mentioned test kit developed by Ortho Co.
45, 12, 2331-2336, 1990), and the positive period for this antibody was as late as 24.7 weeks after transfusion (medical history 151, 892-896). The development of a diagnostic method capable of detecting HCV antibody in hepatitis, etc.) is awaited. Furthermore, even if chronic hepatitis progresses to liver cirrhosis and liver cancer, HCV is often detected in liver cells.
HCV infection was observed in nearly 60%, and studies on the relationship between the onset of liver cancer and HCV have begun to be actively conducted, but it has not been clarified yet. In particular, a test kit that can always detect a high proportion of HCV carriers who have not developed hepatitis, acute hepatitis patients, chronic hepatitis patients, and patients with advanced disease states, or However, no test kit has yet been developed that can indicate when the patient's serum of hepatitis is present.

[0006] In addition, the number of patients already infected with HCV has spread all over the world. For example, in Japan,
HCV was detected in the serum of about 12 out of 000 people, and about 0.8% in the United States, Italy, Spain, etc.
Infection rates of 1.5% and 1.2% have been reported, causing social problems (protein, nucleic acid, enzyme 36, 10,
1679-1691, 1991). None of these antiviral agents for removing or eliminating HCV from HCV patients have been developed.

[0007]

Problems to be Solved As described in the prior art, it is considered that the gene of HCV (hepatitis C virus) is very susceptible to mutation. Due to this mutation, the subtypes of HCV are largely divided, and various sites are mutated including not only the surface antigen site of the protein constituting the virus but also the important site responsible for characterizing the protein. These viruses are thought to cause so-called hepatitis C when infecting humans, but the symptoms may be somewhat different depending on the type. In other words, it can be said that different viruses have low homology. Moreover, we have isolated a plurality of viruses having different amino acid sequences and nucleotide sequences from one hepatitis C patient as shown in the sequence listing. The susceptibility to these mutations is 1 in the HCV gene.
It seems that this is due to the fact that it is a double-stranded RNA.

[0008]

[Means for Solving the Problems] The present inventors have acquired a novel virus different from the one found so far for the diagnosis of hepatitis C patients and the development of a vaccine, and have made a diagnosis developed so far. H with a character different from that of the system, especially in the range that could not be detected until now, or at a higher rate than before
In order to develop a diagnostic method capable of detecting CV antibody and a new vaccine, the inventors have conducted intensive studies to obtain a novel HCV gene in the following manner, and have obtained such a gene and a DNA fragment thereof. Furthermore, the present inventors have
The fragments could also be used to produce by recombinant DNA technology a protein derived from the virus useful for such purposes. In addition, by synthesizing + -strand RNA from a double-strand cDNA fragment in the protease gene region derived from hepatitis C virus and its peripheral region, HC was synthesized in vitro.
Synthesizing a polypeptide containing a V-derived protease,
By examining the specificity, activity and various functions of the protease, it has become possible to prepare a screening system for a substance that specifically suppresses the processing of the HCV-derived precursor polypeptide by the protease. By expressing an HCV-derived precursor polypeptide in Escherichia coli or animal cells using the cDNA fragment, a processing system for the polypeptide in the cell and a screening system for a substance capable of specifically suppressing the processing can be prepared. did.

Further, a HCV-derived polypeptide is expressed in Escherichia coli or animal cells using a double-stranded cDNA fragment of a gene region predicted to be an RNA-dependent RNA polymerase gene region derived from hepatitis C virus, It was also possible to create a screening system for substances that suppress the enzyme activity of polypeptides. That is, the gist of the present invention is to provide SEQ ID NOs: 1-43, 64-75 and 101-10 of the sequence listing.
It exists in the gene or DNA fragment encoded by the amino acid sequence shown in 4 or represented by the nucleotide sequence, and the polypeptide encoded thereby. BEST MODE FOR CARRYING OUT THE INVENTION To explain the present invention below, the novel HCV-derived gene or DNA fragment of the present invention and the polypeptide encoded by the same are represented by SEQ ID NOs: 1 to 1 in Sequence Listing.
43, 64-75 and 101-104, wherein the polypeptide specifically immunochemically reacts with hepatitis C patient serum. Of course, in the present invention, a modification obtained by removing, inserting, modifying or adding a part of amino acids or nucleic acids within a range not impairing the reactivity with the serum of the hepatitis C patient is also a novel feature of the present invention. Included in polypeptides and DNA fragments.

Further, said polypeptide which immunochemically reacts with serum of a hepatitis C patient with different specificity at a part of the amino acid sequences represented by SEQ ID NOs: 1 to 104 in the sequence listing (for example, claims) Claims 119, 121, 123 of the scope,
125, 127, 129, 131-136, 138, 1
40 to 154, 157 to 179, 185 to 199) are also modified by removing, inserting, modifying or adding a part of amino acids within a range that does not impair the reactivity with such hepatitis C patient serum. Those are also included in the novel polypeptide of the present invention. [1] (1) Method for obtaining cDNA clone derived from serum of hepatitis C patient shown in SEQ ID NOS: 1 to 12 of sequence listing and method for determining nucleotide sequence of the clone (core to envelope region) Shown in sequence listing of the present invention The gene or DNA fragment encoding the novel polypeptide represented by the nucleotide sequence can be obtained, for example, by the following method.

[0011] This HCV is present in the serum in a very small amount, and the gene is expected to be RNA.
Since it was expected that it would be difficult to clone by a method based on the Okayama-Berg method or the Gubler-Hoffman method, which is a NA cloning method, in order to reliably clone the gene having many mutations from a small amount of serum, According to the method. That is, nucleic acids are extracted from the serum of hepatitis C patients, as described in Example 1 below. As the serum, one having an OD of 3.5 or more as measured by a test kit of Ortho Co. is usually used, but it is not limited to this value. It is preferable that serum is mixed with transfer RNA (tRNA) as a carrier of viral RNA. The carrier is not necessarily limited to tRNA, and any polyribonucleoside can be used instead. However, the use of tRNA has the advantage that it is possible to rapidly confirm by electrophoresis whether or not the required amount of tRNA having an intact length is present, and by confirming this, at least transfer RNA can be used as a carrier for viral RNA. From the mixing step, it can be confirmed whether or not there is degradation of viral RNA.

As a means for cloning cDNA from such nucleic acid, a polymerase chain reaction method developed by Saiki et al. {(PCR method) Nature, 324 , 126, (1986)}
Improve and use. First, reverse transcriptase is reacted using viral RNA as a template. At this time, a commercially available random primer may be used as the primer, or a synthetic DNA having a base sequence as shown below may be used. 5'3 'S1; CTCCACCATAGATACACTCC (SEQ ID NO: 105) S2; AGGTCTAGTAGAGCCGTGC (SEQ ID NO: 106) S3; AGGAAGACTTCCGAGCGG (SEQ ID NO: 107) S4; CGTGAACTATGCAACAGGG (SEQ ID NO: 108ACT AC; GACAGTG (SEQ ID NO: 108ACT AC; GCTACT: GACATCT: GACATCT: GACATCT: GACATCT: GACATCT: GCTAC: GCTAC: GACATCT: GCTAC: GCTAC: GAGCTCT: GAGCTCT: GACATCT: GACAGCT (CT: GACATCT: GACAGCT: GACAGCT (G)). GGGCAAGTTCCCTGTTGC (SEQ ID NO: 110) AS3; GCTGGATTCTCTGAGACG (SEQ ID NO: 111)

The PCR method is specifically carried out under the conditions as described in Example 2. The first strand complementary strand DNA (1st cDNA) thus obtained is used as a template to carry out the PCR method as in Example 2 to obtain the desired DNA fragment. At this time, the PCR conditions may vary depending on the type, combination, length of amplification, etc. of the primers, and therefore the conditions may be changed depending on the situation.
Specific examples of the primer combination include the following. S1-AS1 S1-AS2 S1-AS3 S2-AS1 S2-AS2 S2-AS3 S3-AS2 S3-AS3 S4-AS3

The minimum amount of serum required for cloning the DNA fragment according to the method of Example 2 [2] varies depending on the content of the virus in the serum used. When it is 3 or more, it is about 5 to 7 μl. When a random primer was used as a primer when synthesizing the first-strand complementary strand DNA (Example 2 [2]), a uniquely designed synthetic DN was used.
It was confirmed, for example, by the method as in Example 8 that the final nucleotide sequences were the same when A was used (Example 2 [3]). That is, the region described in SEQ ID NO: 1 in the sequence listing (clone N1-1) was obtained by the above two methods. Among the clones obtained from the same patient serum, three clones independently obtained had the nucleotide sequences shown in SEQ ID NO: 1 in the sequence listing when random primers were used. When the synthetic DNAs shown in S1 and AS1 described above were used, two of the three clones independently obtained had the same nucleotide sequence as shown in SEQ ID NO: 1 in the sequence listing, and the remaining one was In the nucleotide sequence of SEQ ID NO: 1 of the sequence listing, the 345th A was C, the 332nd A was T, and the 95th A was C at 3 positions. This mutation indicates that there is more than one type of virus that infects one patient.

The DNA fragments thus obtained independently determine the nucleotide sequences of three or more clones for both strands. Fluorescence Sequencer Genesis 2000 (GENESIS 2000) manufactured by DuPont is used to determine the nucleotide sequence.
The system can be easily determined by following the protocol for the system from DuPont. Where the nucleotide sequence is difficult to determine, or the DNA fragment to be determined is about 18
When there are 0 or more base pairs, subcloning may be performed according to a conventional method. The nucleotide sequences of the DNA fragments obtained in the examples described below, which were determined as described above, are as shown in SEQ ID NOS: 1 to 12 of the sequence listing. In the present invention, a part of the base sequence of these DNA fragments may be mutated. Specifically, for example, SEQ ID NO: 1 in the sequence listing
345th A is C and 332nd A is
May be mutated to T, and the 95th A may be mutated to C, respectively. (2) Expression of Polypeptide Encoded by Clone Next, a DNA fragment represented by the nucleotide sequences of SEQ ID NOs: 1 to 12 in the sequence listing is expressed to obtain an HCV-derived recombinant hepatitis C-specific antigen protein.

The expression vector of the present invention has a promoter at a position where the DNA encoding the HCV-derived recombinant hepatitis C-specific antigen protein obtained as described above can be transcribed. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector is a promoter,
It is preferably composed of a ribosome binding (SD) sequence, a recombinant hepatitis C-specific antigen protein gene derived from HCV, a transcription termination factor, and a gene that controls a promoter.
The promoter is derived from Escherichia coli, phage and the like, for example, tryptophan synthase (trp), lactose operon (lac), lambda phage P _L ,
P _{R, T 5} early gene _{P 25, P 26} promoter. Also, these may be uniquely designed sequences. The ribosome binding sequence may be derived from Escherichia coli, phage or the like, but a consensus sequence of 4 or more bases in which a sequence complementary to the 3'terminal region of 16S ribosomal RNA prepared by synthesis of a uniquely designed sequence is continuous. It may be a thing with. The transcription terminator is not always necessary, but it is desirable to have a ρ-independent one such as a lipoprotein terminator or a trp operon terminator.

Further, the sequence of the factors required for the expression on the expression plasmid is as follows: from the 5'upstream, the promoter, the SD sequence, the HCV-derived recombinant hepatitis C-specific antigen protein gene, and the transcription termination factor. It is desirable to line up. As an expression vector, commercially available pKK233-2
(Made by Pharmacia) can be used. To express the fusion protein, the expression vector pGEX series (Pharmacia) is used. The host can be transformed by the protocol of TOYOBO as shown in Example 3 or by a conventional method. Culture of transformants was carried out by molecular cloning (Mol
The method described in "Electrical Cloning, 1982)" may be used as a reference. The culture temperature is about 28 ° C to 42 ° C. The basic units constituting the expression vector in eukaryotes such as insect cells and animal (mammalian) cells are the same as those of the aforementioned microorganisms, but the following units are preferable.

The insect cells include, for example, MAXBAC ^™ , BACULOVIRUS, which is a baculovirus expression manual of Invitrogen.
EXPRESSION SYSTEM MANUAL
Use this kit according to VERSION 1.4). At this time, it is desirable to change the distance from the promoter of polyhedrin to the initiation codon in order to increase the expression level. As the promoter in animal cells, SV40 early promoter, SV40 late promoter, apolipoprotein E gene promoter and the like are used. Basically, the expression vector PKCR (Proceedings
Of National Academy of Sciences USA, 78 , 1528, (1981)) or p that has been improved to the extent that its basic function is not impaired
BPV MT1 (Proceedings of National Academy of Sciences USA, 80 , 398, (1983)) and the like can be used. Animal cells transformed with the expression vector include CHO
Cells, COS cells, and the like. To identify the recombinant polypeptide expressed in microorganisms such as Escherichia coli, insect cells, and animal cells as an HCV-derived antigen,
Utilizes immunological reactivity with patient serum. That is, it is identified by the Western blotting method.

As a result of measurement by this method, the polypeptides having the amino acid sequences set forth in SEQ ID NOS: 1 to 12 of the sequence listing of the present invention are hepatitis C-related antigens, It was found to be the expressed polypeptide. Among these polypeptides, the amino acid sequences of SEQ ID NOs: 5, 6 and 8 in the sequence listing are 1
The polypeptide consisting of 191 amino acids from position 191 to position 191 is presumed to be the polypeptide obtained after being cleaved by being subjected to processing in expression in insect cells. In other words, from the 174th position to the 188th position in the amino acid sequence of SEQ ID NOs: 5, 6 and 8 (region A)
Amino acids having a hydrophobic side chain with a large molecular weight are mainly followed by alanine having a side chain with a small molecular weight at 189th and 191st. This sequence pattern retains the property of the signal region recognized by the signal peptidase in animal (including insect) cells. In addition, the sequences before and after these sequences have larger substitutions of base sequences or mutations of the corresponding amino acid sequences than the base sequences of other known hepatitis C virus genes that have been cloned so far. However, regions A and B have very few mutations. Therefore, it is presumed that the C-terminal side of the 191st alanine is cleaved by the signal peptidase.

Further, SEQ ID NOs: 5, 6 and 8 in this sequence listing
The 1st to 191st polypeptides in the amino acid sequence described in 1. are homologous to the amino acid sequences of the polypeptides of the core and matrix regions of Japanese encephalitis virus and yellow fever virus reported so far, And seems to be a matrix protein. In the present invention, such a polypeptide consisting of 191 amino acids is referred to as "core protein" or "core region". A polypeptide consisting of 18 amino acids from the 1st to 18th in the amino acid sequences of SEQ ID NOs: 1, 9, 10, 11 and 12 and 34 from the 40th to 73rd in the amino acid sequence of SEQ ID NO: 3 And a polypeptide consisting of 35 amino acids from the 81st position to the 115th position are known other hepatitis C virus genes that have been cloned up to now (the aforementioned Chiron Co., Shimotono). Et al. And Takamizawa et al. Have a high homology and a relatively high hydrophilicity compared to the amino acid sequence predicted from the cloned HCV gene), and thus are useful as an antigenic peptide for diagnosis or as a vaccine.

Further, in the amino acid sequence of SEQ ID NO: 4 in the sequence listing, a polypeptide consisting of 18 amino acids 40 to 57 and a polypeptide consisting of 12 amino acids 240 to 251 are Since it has extremely low homology and relatively high hydrophilicity as compared with the amino acid sequences predicted from other known hepatitis C virus genes that have been cloned, it is useful as an antigen peptide for diagnosis. In addition, these polypeptides may be produced by gene recombination technology or may be produced by a synthetic method. Furthermore, in the amino acid sequence of SEQ ID NO: 3 in the sequence listing, a polypeptide consisting of 115 amino acids from the 1st to 115th, which is the epitope region of the core protein, and the entire core protein from the 1st to 191st A polypeptide consisting of 191 amino acids representing a region can be mass-produced by gene recombination technology and is useful for diagnosis or as a vaccine. On the other hand, among these polypeptides, the polypeptide consisting of 192 amino acids from the 31st position to the 222nd position in the amino acid sequence of SEQ ID NO: 4 in the sequence listing is processed by cleavage in expression in insect cells, and after cleavage, It is presumed to be the obtained polypeptide. In other words, in the amino acid sequence shown in SEQ ID NO: 4 of the Sequence Listing, amino acids having hydrophobic and large molecular weight side chains mainly continue from the 13th to the 29th (region A), and the 30th side chain having the smallest molecular weight. Is followed by alanine with. Also, from the 210th to 221
Up to the (region B), the amino acid having a hydrophobic side chain having a large molecular weight is also mainly followed by the glycine having a side chain having a small molecular weight at 222nd. This sequence pattern retains the property of a signal region recognized by a signal peptidase in animal (including insect) cells.

Further, the sequences before and after these sequences have a large substitution of the base sequence or a corresponding mutation of the amino acid sequence as compared with the base sequences of other known hepatitis C virus genes cloned so far. However, area A
And B have very few mutations. Therefore, it is presumed that the C-terminal side of the 30th alanine and the C-terminal side of the 222nd alanine are cleaved by the signal peptidase. At least this polypeptide has a low homology with the base sequence corresponding to this region of the base sequences of other known hepatitis C virus genes that have been cloned so far.
It may be the envelope protein of CV itself, or a part thereof. In the present invention, such 1
A polypeptide consisting of 92 amino acids was designated as "M-gp3.
5 "," M-gp35 protein "or" M-gp35 region ". 1 in the amino acid sequence of SEQ ID NO: 4 in the sequence listing
Polypeptide consisting of 19 amino acids from 34th to 152nd, 1 from 223rd to 239th
The polypeptide consisting of 7 amino acids and the polypeptide consisting of 18 amino acids from the 92nd position to the 109th position have the amino acid sequences predicted from other known hepatitis C virus genes cloned so far. By comparison, because of its high homology and relatively high hydrophilicity,
It is useful as a diagnostic antigenic peptide or as a vaccine.

Further, in the amino acid sequence of SEQ ID NO: 4 in the sequence listing, a polypeptide consisting of 18 amino acids from 40th to 57th and a polypeptide consisting of 12 amino acids from 240th to 251st are Since it has extremely low homology and relatively high hydrophilicity as compared with the amino acid sequences predicted from other known hepatitis C virus genes that have been cloned so far, it is useful as a diagnostic antigen peptide. These polypeptides are
It may be produced by a synthetic method without using the technique of recombinant DNA. Furthermore, in the amino acid sequence of SEQ ID NO: 4 in the sequence listing, a polypeptide consisting of 76 amino acids from 31st to 106th and a polypeptide consisting of 36 amino acids from 134th to 169th are M- g
Recombinant D, which is the epitope region of p35 protein
It can be mass-produced by NA technology and is useful for diagnosis or as a vaccine.

[2] (1) C shown in Sequence Listing 13 to 27
Method for obtaining cDNA clone derived from serum of hepatitis C patient and method for determining nucleotide sequence of the clone (NS1 (gp70)
Region) The gene or DNA fragment encoding the novel polypeptide represented by the nucleotide sequences shown in Sequence Listing 13 to 27 of the present invention can be obtained, for example, by the following method. This HCV is present in the serum in a very small amount, and the gene is R
It is expected to be NA, and the conventional cDNA cloning method of Okayama-Berg method or Gubler-
Since it was expected to be difficult to clone by the method based on the Hoffman method, the following method was used in order to reliably clone the gene having many mutations from a small amount of serum. That is, nucleic acids are extracted from the serum of hepatitis C patients, as described in Example 1 below. The serum is usually
An OD of 3.5 or higher as measured by an Ortho test kit is used, but the OD is not limited to this value. Transfer R as a carrier of viral RNA in serum
It is preferable to mix NA (tRNA). The carrier is not necessarily limited to tRNA, and any polyribonucleoside can be used instead. However, if tRNA is used, there is an advantage that it is possible to rapidly confirm by electrophoresis whether or not a necessary amount of tRNA having an intact length is present.
From the step of mixing transfer RNA as the carrier of A, it can be confirmed whether or not viral RNA is degraded.

As a means for cloning cDNA from such nucleic acid, the polymerase chain reaction method {(PCR method) Nature, 324, 126, (1986)} developed by Saiki et al. Is used. First, reverse transcriptase is reacted using viral RNA as a template. At this time, a commercially available random primer may be used as the primer, or MS1 among the primers shown below may be used.
23, MS157, MS148, synthetic DNA having a nucleotide sequence may be used. 5 '3'MS122; GAGGCCGTGAACTGCGATGA (SEQ ID NO: 112) MS148; TTCTCTAAGGTGGCNTCNGCNTG (SEQ ID NO: 113) MS157; CCGGACGCGTTGAANCTNTGNGT (SEQ ID NO: 114) MS123; CATCCAGGTACAACCGAACCA (SEQ ID NO: 11 5) MS146; AACACACGGCCGCCNCANGGNAA (SEQ ID NO: 116) MS156; CCGGATCCCACAAGCCGTNGTNGA (SEQ ID NO: 117)

The "N" in this sequence represents inosine. In these sequences, the base on the 5'side may be changed from several bases to another sequence, but preferably within a few bases by mutation within 10 bases from the 5'side, more preferably on the 5'side. Mutations within 5 bases are preferable. Also, in these sequences, the 5'side sequence may lack 4 to 5 bases, but preferably lacks several bases from the 5'side. Further, an arbitrary sequence may be added to the 5'side as long as it has about 8 to 12 bases, preferably 5 to 6 bases, more preferably several bases. Specifically, the PCR method is carried out under the conditions described in Example 9.
The first strand complementary strand DNA (1st
The desired DNA fragment can be obtained by performing the PCR method as in Example 9 using (cDNA) as a template. At this time,
The conditions for PCR may vary depending on the type, combination, length of amplification, etc. of the primers, so the conditions may be appropriately changed depending on the situation. As the combination of primers,
Specific examples include the following.

MS122-MS123 MS157-MS156 MS148-MS146 The DNA fragment thus obtained is incorporated into one of the cloning sites of the cloning vector (eg pUC19) (eg Sma I site) by a conventional method. Using the plasmid having this DNA fragment, the nucleotide sequences of three or more clones are independently determined for both strands. Fluorescence Sequencer Genesis 2000 (GENESIS 2000) manufactured by DuPont is used to determine the nucleotide sequence.
The system can be easily determined by following the protocol for the system from DuPont. Where the nucleotide sequence is difficult to determine, or the DNA fragment to be determined is about 18
When there are 0 or more base pairs, subcloning may be performed according to a conventional method. The DN determined in this way
The base sequence of the A fragment is as shown in SEQ ID NOs: 13 to 27 of the sequence listing.

Clone N19-1,2,3 and clone N27-1,2,3 were obtained from the serum of one patient N, and H19-2,4,10 and Y19-4,6,7 were also obtained. It is a clone obtained from the serum of each independent patient H and Y. MX24-4, 5, 13 were obtained from multiple hepatitis C patient serum pools. Further primer MS15
The clones shown in SEQ ID NOs: 13 to 15 of the sequence listing obtained from the combination of 7 and primer MS156 correspond to the same region on the HCV gene (hereinafter, this region is referred to as N27).
Area called). Also, for the same reason, primer MS122
The clones of SEQ ID NOs: 16 to 24 in the sequence listing obtained by using the primer and MS123 are also in the same region on the HCV gene (hereinafter, this region is referred to as N19 region), and are shown in SEQ ID NOs: 25 to 27 in the sequence listing. Three clones were also obtained from primer MS148 and primer MS146,
It corresponds to the same region on the V gene (hereinafter, this region is referred to as MX24 region). In addition, National Cancer Center Kato et al. Announced (Proceedings of National Academy of Sciences USA (Pr
oc Natl. Acad. Sci. USA), 87 ,
9524-9528, 1990), Takamizawa et al. Of the Osaka University Micro Lab (Journa of Birology).
l of Virology, 65 , 3, 1105-1
113, 1991)) based on the homology with the sequence of the hepatitis C virus gene, the regions of each of the obtained clones are the N27 region, the N19 region and the MX24 region in order from the 5'on the designated HCV gene. They are lined up, and there are overlapping parts in each area,
Utilizing this overlapping region, each DNA fragment was ligated as follows.

(2) Ligation of clones shown in SEQ ID NOS: 13 to 27 of the Sequence Listing The cDNA clones derived from sera of hepatitis C patients of SEQ ID NOs: 13 to 27 are ligated as described below. The clones of SEQ ID NOS: 13 to 15 in the sequence listing and the concatenations of SEQ ID NOS: 16 to 24 in the sequence listing Cleavage at the Mlu I site located at the 330th to 335th positions from the 5'side of the nucleotide sequence of SEQ ID NOS: 13 to 15 in the sequence listing Then
D of the nucleotide sequences of SEQ ID NOs: 16 to 24 in the sequence listing, respectively
The Mlu I site at the 71st position from the 5'side of NA was ligated by ligation reaction, and N27-1, 2,
3 derived DNA fragment, clone N19-1 on the 3'side,
27 clones having DNA fragments derived from clones H19-2, 4, 10 and clones Y19-4, 6, 7 were obtained. For example, the clone obtained by ligating N27-3 and N19-1 is SEQ ID NO: 28 in the sequence listing.
Clone N27N19-1 shown in FIG. Concatenation of clones of SEQ ID Nos. 16 to 24 in the sequence listing with clones of SEQ ID Nos. 25 to 27 in the sequence listing

The clones of SEQ ID NOs: 16 to 24 in the sequence listing and the clones of sequence listings 13 to 15 were ligated together using the PCR method. 54 clones were obtained by this method. That is, 27 patterns are 1 from N-terminal (amino terminal) to C-terminal of SEQ ID NOs: 16 to 24 in the sequence listing.
It has an amino acid sequence consisting of 31 residues at the N-terminal and 132
DNA encoding a polypeptide having an amino acid sequence from the 16th to the C-terminal of the amino acid sequences of SEQ ID NOs: 25 to 27 in the sequence listing at the 1st to the C-terminal (carboxyl terminal)
Fragment, for example, when N19-1 and MX24-4 are used, clone N19M shown in SEQ ID NO: 29 in the sequence listing
X24A-1 was obtained. The amino acid sequences of N-terminal (amino terminal) to 116th of SEQ ID NOS: 16 to 24 in the sequence listing are at the N-terminal, and 117th to C-terminal (carboxyl terminal) have the amino acid sequences of SEQ ID NOs: 25 to 27 of the sequence listing. A DNA fragment encoding a polypeptide having an amino acid sequence consisting of 209 residues from the N-terminus to the C-terminus of
For example, when N19-1 and MX24-4 were used, clone N19MX24B-1 shown in SEQ ID NO: 30 in the sequence listing
Got

Concatenation of clones of SEQ ID Nos. 13 to 27 in the Sequence Listing Cleavage at the Mlu I site at the 330th to 335th positions from the 5'side in the SEQ ID Nos. 13 to 15 of the SEQ ID Nos. 71st to 76th in the sequence
By ligation with the Mlu I site by ligation reaction, clone N27MX24 series was obtained. For example, N2
The clone obtained by ligating 7-3 and N19MX24A-1 is clone N shown in SEQ ID NO: 31 of the sequence listing.
27MX24A-1. Also, N27-3 and N19
The clone obtained by ligating MX24B-1 is clone N27MX24B shown in SEQ ID NO: 32 of the sequence listing.
-1. Kato et al. (Proceedings of National Academy of Sciences US
A (Proc. Natl. Acad. Sci. US
A), 88 , 5547-5551, 1991) and Hijikata et al. (1990 Japanese Society for Molecular Bioscience, November 29).
Clone N27N19MX24A by homology with the entire amino acid sequence derived from HCV gene reported by
-1 contains the entire gene of the polypeptide corresponding to gp70 reported by Kato et al. That is, the 46th to 395th amino acid numbers of SEQ ID NOs: 31 and 32 in the sequence listing correspond to the gp70 polypeptide reported by Kato et al.

The amino acid numbers 1 to 45 of SEQ ID NOs: 13 to 15 in the sequence listing are Hijikata et al. (1990).
The polypeptide gp35 reported by the Japanese Molecular Biology Society (November 29) corresponds to the C-terminal side, and the 46th to the end correspond to the N-terminal side of gp70 reported by Hijikata et al. Further, in SEQ ID NOS: 13 to 15 of the sequence listing, the 46th to C-terminal amino acid numbers correspond to the N-terminal to 67th amino acid of gp70 reported by Kato et al. SEQ ID NOs: 16 to 24 in the sequence listing are amino acid numbers, and N-terminal to C-terminal are 42 to 172 of gp70.
It corresponds to the th amino acid and is a partial polypeptide of gp70 reported by Kato et al. SEQ ID Nos. 25 to 27 in Sequence Listing
The first amino acid number in the sequence corresponds to the 158th amino acid counted from the N-terminus of gp70 reported by Hijikata et al. The amino acid number is 350th, and the amino acid numbers 194 to C in the sequence numbers 25 to 27 of the sequence list correspond to the N-terminal region of the hepatitis C virus nonstructural protein NS2.

The various ligated clones obtained in (2) also encode part or all of the polypeptide corresponding to gp70 reported by Hijikata et al. For example, from the 46th position in the amino acid number of SEQ ID NO: 31 or 32 in the sequence listing
The 395th polypeptide is gp7 reported by Hijikata et al.
Equivalent to 0. In addition, SEQ ID NO: 31 or 32 in the sequence listing
The protein expressed from the HCV gene in the region corresponding to the polypeptide from the 46th amino acid number to the 395th amino acid number is referred to as gp70. The gp70 obtained by the present invention is hereinafter particularly referred to as M-gp70, and Hijikata et al. Reported at the Japan Society for Molecular Biology 1990 November 29
Distinguish from p70. (3) Expression of the polypeptide encoded by the clone obtained in (1) and (2) Next, the polypeptide encoded by the clone obtained in (1) and (2) was expressed. A recombinant hepatitis C-specific antigen protein derived from HCV is obtained. The expression vector of the present invention contains a promoter at a position where the DNA encoding the recombinant hepatitis C-specific antigen protein derived from HCV obtained as described above can be transcribed. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector includes a promoter, a ribosome binding (SD) sequence, H
A recombinant hepatitis C-specific antigen protein gene derived from CV,
It preferably comprises a transcription termination factor and a gene that controls a promoter.

The promoter is derived from Escherichia coli, phage or the like, for example, tryptophan synthase (tr).
p), lactose operon (lac), lambda phage PL, PR, T5 early gene P25, P26 promoter and the like. Also, these may be uniquely designed sequences. The ribosome binding sequence may be derived from Escherichia coli, phage or the like, but a consensus sequence of 4 or more bases in which a sequence complementary to the 3'terminal region of 16S ribosomal RNA prepared by synthesis of a uniquely designed sequence is continuous. It may be a thing with. The transcription terminator is not always necessary, but it is desirable to have a ρ-independent one such as a lipoprotein terminator or a trp operon terminator. Furthermore, the sequence order of the factors required for expression on the expression plasmid is:
It is desirable that the promoter, the SD sequence, the recombinant hepatitis C-specific antigen protein gene derived from HCV, and the transcription termination factor are arranged in this order from 5 ′ upstream. As an expression vector,
Commercially available pKK233-2 (manufactured by Pharmacia) can be used. To express the fusion protein, the expression vector pGEX series (Pharmacia) is used.

The host can be transformed by the protocol of Toyobo Co., Ltd. as shown in Example 10 or by a conventional method. Culture of the transformant was carried out by molecular cloning (Molecular Cloni).
ng, 1982). The culture temperature is about 28 ° C to 42 ° C. The basic unit constituting the expression vector in a eukaryote such as an insect cell is the same as that of the above-mentioned microorganism, but the following units are preferable. Insect cells include, for example, MAXBACTM, BA, which is a baculovirus expression manual of Invitrogen.
CULOVIRS EXPRESSION SYSTEM
This kit is used according to EM MANUAL VERSION 1.4). At this time, it is desirable to change the distance from the promoter of polyhedrin to the initiation codon in order to increase the expression level.

When the clone is introduced into an expression vector in Escherichia coli or a eukaryote, it may be aligned in the frame of the start codon derived from the vector, and even if the start codon derived from the vector is not used, The initiation codon may be added to the 5'side so as to match the translation frame and then introduced into the expression vector. In this case, the translation frame of the clone is, for example, a base sequence divided into three corresponding to one amino acid in SEQ ID NOS: 13 to 32 of the sequence listing shown as a representative clone. It is a framework. Microorganisms such as E. coli,
To identify the recombinant polypeptide expressed in insect cells as an HCV-derived antigen, immunological reactivity with HCV patient serum is used. That is, it is identified by the Western blotting method. Also, in E. coli, the above (1),
When the clone obtained in (2) is expressed, gp3
5, a single polypeptide is expressed without cleavage between the respective regions called gp70, NS2. When expressed in insect cells, it is cleaved between the respective regions called the respective polypeptides gp35, gp70 and NS2. Clone N27MX24A-
1. When the clone N27MX24B-1 was expressed in insect cells and animal cells, the clone M27g derived from the clones N27MX24A-1 and clone N27MX24B-1
p70 was processed and expressed. Also the M
-Gp70 was a glycoprotein.

A polypeptide consisting of 13 amino acids from 143rd to 155th in the amino acid sequence of SEQ ID NO: 31 or 32 in the sequence listing, 171st to 191st (at least 182nd to 187th) Polypeptide consisting of 21 amino acids, 202
2nd to 215th (at least, 202nd to 2nd
(Including up to the 09th), a polypeptide consisting of 14 amino acids, a polypeptide consisting of 13 amino acids from the 244th to the 256th, and 299th to 3rd.
The polypeptide consisting of 21 amino acids up to the 19th has relatively high homology and hydrophilicity compared to the amino acid sequences predicted from other known hepatitis C virus genes cloned so far. High Also,
M-gp70 is a glycoprotein adjacent to the C-terminal side of the envelope protein (M-gp35) on the HCV gene. It also has a membrane passage estimation region. from these things,
If all or part of gp70 whose function is still unknown is part of the viral envelope protein, the above-mentioned 5 peptide fragments, and the amino acid sequences of SEQ ID Nos. 21
A polypeptide consisting of 106 amino acids up to the 4th position and a polypeptide consisting of 92 amino acids up to the 233rd to 324th positions are useful as vaccines.

Further, in the amino acid sequences of SEQ ID NOs: 31 and 32 of the sequence listing, which are predicted to be the epitope site of M-gp70, the 252nd to 261st amino acids (at least 25
Polypeptide consisting of 10 amino acids, including 2nd to 256th), 250th to 283rd (including at least 273rd to 279th)
Polypeptide consisting of 34 or less amino acids, Polypeptide consisting of 20 amino acids 77 to 96th, Polypeptide consisting of 18 amino acids 306 to 323, and 122th to 137
The polypeptide of 16 amino acids up to the
It is useful as a vaccine. In addition, SEQ ID NO: 3 in the sequence listing
A polypeptide consisting of 12 amino acids from the 136th to the 147th amino acids in the amino acid sequence of 1 or 32 (including at least the 136th to 142nd amino acids), 45
The 71st to 71st (at least the 53rd to 69th
The polypeptide consisting of 27 amino acids and the polypeptide consisting of 9 amino acids 193 to 201 are predicted from other known hepatitis C virus genes cloned so far. It has extremely low homology and a relatively high degree of hydrophilicity as compared with the amino acid sequence described above. These polypeptides are recombinant D
It may be produced by a synthetic method without depending on the technique of NA.

A polypeptide consisting of 106 amino acids 109 to 214 in the amino acid sequence of SEQ ID NO: 31 or 32 in the above sequence listing, and 233 to
A 92-amino acid polypeptide up to the 324th position can also be mass-produced by recombinant DNA technology.

[3] (1) SEQ ID NOs: 33 to 44 in the sequence listing
Method for obtaining cDNA clone derived from serum of hepatitis C patient shown in 1) and method for determining nucleotide sequence of the clone (NS2-4
Region) The gene or DNA fragment encoding the novel polypeptide represented by the nucleotide sequence shown in the sequence listing of the present invention can be obtained, for example, by the following method. This HCV is
Owing to the conventional cDNA cloning method, the gene is expected to be RNA in addition to the trace amount present in serum.
kayama-Berg method, Gubler-Hoff
Since it was expected to be difficult to clone by the method based on the man method, the following method was used to surely clone the gene having many mutations from a small amount of serum.
That is, nucleic acids are extracted from the serum of hepatitis C patients, as described in Example 1 below. As the serum, one having an OD of 3.5 or more as measured by a test kit of Ortho Co. is usually used, but it is not limited to this value. Transfer RNA as a carrier of viral RNA in serum
It is preferable to mix (tRNA). The carrier is not necessarily limited to tRNA, and any polyribonucleoside can be used instead. However, if tRNA is used, there is an advantage that it is possible to rapidly confirm by electrophoresis whether or not a required amount of tRNA having an intact length is present. By performing this confirmation, at least viral RNA
From the step in which transfer RNA is mixed as a carrier, it is possible to confirm whether or not viral RNA is degraded.

As a means for cloning cDNA from such a nucleic acid, the polymerase chain reaction method {(PCR method) Nature, 324, 126, (1986)} developed by Saiki et al. Can be used. First, reverse transcriptase is reacted using viral RNA as a template. At this time, as the primer, a commercially available random primer may be used, or among the primers shown below, MS49, MS152, MS158, MS132,
You may use the synthetic DNA of a base sequence like MS100 and MS88.

5 ′ 3 ′ MS49; GACATGCATGTCATCATGATGTA (SEQ ID NO: 118) MS88; GGCTGCAGGCGGTTCATCCACTGCAC (SEQ ID NO: 119) MS100; No .: 122) MS158; ATGGGCCCGGGNGANAGNAGNCCTCCCCC TNCTNTC (SEQ ID NO: 123) MS48; GGCTATACCGGCGACTTCGA (SEQ ID NO: 124) MS86; No .: 125) MS97; GCGGATCCTCCACCCTCCATCGTG (SEQ ID NO: 126) MS135; CTGCTGTCGCCCNGNCCCAT (SEQ ID NO: 127) MS151;

"N" in this sequence represents inosine. In these sequences, some bases of the 5'side sequence may be changed to other bases, but in this case, preferably the mutation is within 10 bases from the 5'side and within several bases, and more preferably, Mutations within 5 bases from the 5'side are preferable. In addition, although 4 to 5 bases may be deleted from the 5'side sequence in these sequences, it is preferable that a few bases are deleted from the 5'side. Further, an arbitrary sequence may be added to the 5'side as long as it has about 8 to 12 bases, preferably 5 to 6 bases, more preferably several bases. In addition, MS86, MS97, MS88 and MS100
8 bases containing a restriction enzyme recognition sequence at the 5'end (in MS88, 5'GGCTGCAG3 ', MS86 and MS
97 and MS100, 5'GCGGATCC
3 ') is added, this sequence is not always necessary to obtain the desired DNA fragment. The PCR method is
Specifically, it is carried out under the conditions as described in the fifteenth embodiment. The first strand complementary strand DNA (1
st cDNA) as a template and PC as in Example 15.
The desired DNA fragment can be obtained by performing R method. At this time, the PCR conditions are:
Since it may vary depending on the length of amplification and the like, the conditions may be appropriately changed depending on the situation. Specific examples of the primer combination include the following.

MS48-MS49 MS86-MS100 MS97-MS88 MS135-MS132 MS155-MS152 MS151-MS158 The DNA fragment thus obtained is used as one of the clonin sites of a cloning vector (for example, pUC19) by a conventional method ( ( Sma I site, for example). Using the plasmid having this DNA fragment, the nucleotide sequences of three or more clones are independently determined for both strands. The nucleotide sequence can be easily determined by using a fluorescent sequencer Genesis 2000 system manufactured by DuPont, according to the protocol for the system manufactured by DuPont. Where the nucleotide sequence is difficult to determine, or the DNA fragment to be determined is approximately 180
When there are more base pairs, subcloning may be performed according to a conventional method. The DN determined in this way
The nucleotide sequences of the A fragments are SEQ ID NOs: 33 to 39 in the sequence listing,
44 to 55, 103 and 104.

In addition, clone N13-1 and clone N1
5-1 and clone N16 and clone N23 were obtained from the serum of one patient N, and O26 and U16-4 were also clones obtained from the sera of independent patients O and U, respectively.
Clone MX25 was obtained from multiple hepatitis C patient serum pools. Furthermore, primer MS48 and primer MS
The clone shown in SEQ ID NO: 37 of the sequence listing obtained from the combination of 49 and the clones shown in SEQ ID NO: 53 to 55 of the sequence listing correspond to the same region on the HCV gene (hereinafter, this region is referred to as N16 region). . SEQ ID NO: 4 in the sequence listing obtained using primer MS155 and primer MS152
The region of the nucleotide sequence derived from the HCV gene of clones 4 to 46 is designated as MX25 region, and SEQ ID NOS: 47 to 4 in the sequence listing
The clones O26 and N23 shown in 9, 50 to 52 are also combinations of the primer MS151 and the primer MS158, the primer MS135 and the primer MS132, respectively.
The regions obtained from the combination of the above and corresponding to each other are the O26 region and the N23 region for the same reason. In addition, clones N13-1, N15-1, O15-1, and O15-2 shown in SEQ ID NOs: 38, 39, 103, and 104 of the sequence listing.
Also primer MS86 and primer MS10, respectively
0 combination, primer MS97 and primer MS8
The regions obtained from the combination of 8 and corresponding to each other for the same reason were designated as N13 region and N15 region.

Kato et al., National Cancer Center, also announced (Proceedings of National Academy of Sciences USA).
atl Acad. Sci. USA), 87,9524
-9528, 1990), Takamizawa et al. Of Micro Lab., Osaka University (Journal of Virology (Journal of V
irology, 65,3,1105-1113,19
91)) reported homology with the sequences of the hepatitis C virus gene, the regions of each of the obtained clones were MX25 region, 026 region, N23 region, N1 in order from the 5 ′ side.
It is lined up with 6 areas, N13 area, and N15 area.
Each region has an overlapping portion, and each DNA fragment was ligated as follows using this overlapping region. In order to obtain the clone N16 of SEQ ID NO: 36 in the sequence listing, a plasmid having DNA fragments of three N16 regions was independently isolated from the serum of one patient N, and the entire nucleotide sequence of the HCV-derived DNA fragment was determined. did. Each of these DNA fragments had a high homology, but was a DNA fragment encoding a different base sequence and a different amino acid sequence. This indicates that multiple hepatitis C viruses are present in one patient. In addition, this is SEQ ID NO: 3 in the sequence listing.
The same applies to the clones shown in 3, 34, and 35. 5'of the core protein region and core protein gene
The region located on the side is considered to be a region with few mutations in the hepatitis C virus gene, but when cloned by the same method as described above, there was almost no mutation between clones.

From this, it was also found that the MX25 region, the O26 region, the N23 region, and the N16 region were much more likely to be mutated than the core protein region and the 5'side region thereof. In particular, it was found that the MX25 region is easily mutated in both the nucleotide sequence and the amino acid sequence among the clones. (2) Ligation of clones shown in SEQ ID NOs: 33 to 39 of the Sequence Listing The cDNA clones derived from the sera of hepatitis C patients of SEQ ID NOs: 33 to 37 and 39 are ligated as described below. Clone N16 of SEQ ID NO: 36 in the sequence listing, linked clone N15-1 of SEQ ID NO: 39 in the sequence listing example, the nucleotide sequence of SEQ ID NO: 36 in the sequence listing, Bst EII site in the 576 th ～582 position from the 5 'side Cleavage with, 1 from the 5'side of the nucleotide sequence of SEQ ID NO: 39 in the Sequence Listing
The 14th to 120th Bst EII sites were ligated with each other by a ligation reaction, and a DNA fragment derived from clone N16 on the 5'side and a DNA derived from clone N16 on the 3'side.
A clone with the fragment is obtained. The nucleotide sequences of such clones are collectively shown in SEQ ID NO: 41 of the sequence listing.

Sequence Listing SEQ ID NO: 33 clone MX2
5 and ligation of clone O26 of SEQ ID NO: 34 in the sequence listing Each nucleotide sequence shown in clone MX25 of SEQ ID NO: 33 of the sequence listing and each clone O26 of SEQ ID NO: 34 in the sequence listing are ligated by PCR method. . For example,
2 from N-terminal (amino terminal) to C-terminal of SEQ ID NO: 33
285 with an amino acid sequence consisting of 84 residues at the N-terminus
A DNA fragment encoding a polypeptide having an amino acid sequence from the 32nd to the C-terminal of the amino acid sequence of SEQ ID NO: 34 at the 1st to C-terminal (carboxyl terminal), and the N-terminal (amino terminal) to the 252nd of SEQ ID NO: 33 Up to the N-terminal of the amino acid sequence of SEQ ID NO: 34 from the 253rd position to the C-terminal (carboxyl terminal).
A DNA fragment encoding a polypeptide having an amino acid sequence consisting of 174 residues up to the C-terminus can be mentioned. These base sequences are collectively shown in SEQ ID NO: 40 in the sequence listing.
Also, the clones of SEQ ID NOs: 36 and 39 of the sequence listing or SEQ ID NOs: 37 and 39 of the sequence listing can be similarly ligated by the PCR method, and the clones thus obtained are also shown in the sequence listing together with the nucleotide sequences shown in. SEQ ID NO: 41 of Further, the clone MX25 of SEQ ID NO: 33 in the sequence listing, which consists of DNA fragments having different base sequences, and the clone O of SEQ ID NO: 34 in the sequence listing, which comprises DNA fragments of different base sequences,
26 is similarly linked and has a plurality of different base sequences D
A clone MX25O26 consisting of the NA fragment is obtained.
This base sequence is collectively shown in SEQ ID NO: 40 in the sequence listing.

Clone N23 and clone N16N15 are ligated by the same method as the method shown in the ligation of the clones of SEQ ID NO: 35 of the sequence listing and SEQ ID NO: 41 of the sequence listing. The nucleotide sequence of the obtained DNA fragment is shown in SEQ ID NO: 42 in the sequence listing, and the clone names are collectively designated as N23N15. For example, an amino acid sequence consisting of 307 residues from the N-terminal (amino terminal) to the C-terminal of SEQ ID NO: 35 in the sequence listing summarizing the nucleotide sequences of DNA fragments has at the N-terminal, and the 308th to C-terminal (carboxyl terminal) And a DNA fragment encoding a polypeptide having an amino acid sequence from the 17th amino acid sequence to the C-terminal of the amino acid sequence of SEQ ID NO: 41 in the sequence listing, and the N-terminal (amino terminal) to the 291st amino acid sequence of SEQ ID NO: 33 in the sequence listing Has an amino acid sequence at the N-terminal, and the 292nd to C-terminal (carboxyl terminal)
At the N-terminal to C of the amino acid sequence of SEQ ID NO: 41 in the sequence listing.
A plurality of kinds of DNA fragments encoding a polypeptide having an amino acid sequence consisting of 477 residues up to the end are obtained.

SEQ ID NO: 40 and SEQ ID NO: 41 in the sequence listing
The clone MX25O26 was cloned in the same manner as described for the ligation.
And clone N23N15 are ligated. The nucleotide sequences of the DNA fragments obtained here are shown together in SEQ ID NO: 43 in the sequence listing. The N-terminal domain of the nonstructural protein NS3 of flavivirus, which is related to HCV, has protease activity (Proc. Natl. Acad. Sci. USA),
87, 8898-8902, 1990), etc.
It is expected that the N-terminal domain of the region presumed to be the NS3 region of V also has protease activity. Also,
The entire amino acid sequence derived from HCV gene announced by Kato et al. (Procedures of National Academy of Science USA (Proc.
tl. Acad. Sci. USA), 87, 9524-
9528, 1990) and Hijikata et al. (1991 Japan Cancer Society), the clone MX25N15 contains the entire region having the HCV-derived protease activity.

The region presumed to be the NS3 region of HCV is not certain at both the N-terminal and C-terminal, but the primary structure of the amino acid sequence of the flavivirus cleavage region and the pattern of hydrophilic / hydrophobic regions. Prediction and Hught
on et al. (Hepatology
y), 14, 2, 381-388, 1991), it is deduced that the 276th to 884th amino acid numbers of clone MX25N15 shown in SEQ ID NO: 43 of the sequence listing correspond to the NS3 region. This area will be referred to as MK / N
Called the S3 area. Similarly, the region presumed to be NS2 is clone MX25N shown in SEQ ID NO: 43 of the sequence listing.
15 and the clone MX25O shown in the sequence listing under SEQ ID NO: 40.
It is considered to be a region of the polypeptide consisting of the 3rd to 275th amino acids in the 26 amino acid number. Below, this area is MK /
It is called the NS2 area. (3) Expression of the polypeptide encoded by the clone obtained in (1) and (2) Next, the polypeptide encoded by the clone obtained in (1) and (2) was expressed. A recombinant hepatitis C-specific antigen protein derived from HCV is obtained.

The expression vector of the present invention contains a promoter at a position where the DNA encoding the HCV-derived recombinant hepatitis C-specific antigen protein obtained as described above can be transcribed. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector is a promoter,
It is preferably composed of a ribosome binding (SD) sequence, a recombinant hepatitis C-specific antigen protein gene derived from HCV, a transcription termination factor, and a gene that controls a promoter.
The promoter is derived from Escherichia coli, phage or the like, for example, tryptophan synthase (trp), lactose operon (lac), lambda phage PL, P.
R, T5 early gene P25, P26 promoter and the like. Also, these may be uniquely designed sequences. The ribosome binding sequence may be derived from Escherichia coli, phage or the like, but a consensus sequence of 4 or more bases in which a sequence complementary to the 3'terminal region of 16S ribosomal RNA prepared by synthesis of a uniquely designed sequence is continuous. It may be a thing with. The transcription terminator is not always necessary, but it is desirable to have a ρ-independent one such as a lipoprotein terminator or a trp operon terminator.

Furthermore, the sequence of the factors required for the expression on the expression plasmid is as follows: from the 5'upstream, the promoter, the SD sequence, the HCV-derived recombinant hepatitis C-specific antigen protein gene, and the transcription termination factor. It is desirable to line up. As an expression vector, commercially available pKK233-2
(Made by Pharmacia) can be used. To express the fusion protein, the expression vector pGEX series (Pharmacia) is used. The host can be transformed by the protocol of Toyobo Co., Ltd. as shown in Example 16 or by a conventional method. Culture of transformants was performed by molecular cloning (Mole
The method described in "Clar Cloning, 1982)" may be used as a reference. The culture temperature is about 28 ° C to 42 ° C. The basic unit constituting an expression vector in eukaryotes such as insect cells and animal (mammalian) cells is the same as that of the above-mentioned microorganisms, but the following units are preferable.

Examples of insect cells include MAXBACTM, BACULOVIRUS, which is a baculovirus expression manual manufactured by Invitrogen.
EXPRESSION SYSTEM MANUAL
Use this kit according to VERSION 1.4). At this time, it is desirable to change the distance from the promoter of polyhedrin to the initiation codon in order to increase the expression level. As a promoter in animal cells, an active promoter based on the adenovirus EIA gene (Secondary Chemistry Experimental Course 1 Gene Research Method II, 189-190, 19)
86), SV40 early promoter, SV40 late promoter, apolipoprotein E gene promoter,
SRα promoter (Molecular and Cellular
Biology, 8, 1, 466-472, 1988) and the like are used. Basically, the expression vector PKCR (Proceedings of National Academy of Sciences USA, 78, 1528,
(1981)) or pBPV MT1 (Proceedings of National Academy of Science USA, 80, 398, (1983)) which is improved to the extent that its basic function is not impaired.

Animal cells transformed with the expression vector include CHO cells, COS cells, CV-1 cells, mouse RIII-derived cell C127 strain and the like. When the clone is introduced into an expression vector in Escherichia coli or a eukaryote, it suffices to match the frame of the start codon derived from the vector, and even when the start codon derived from the vector is not used, the translation frame of the clone The start codon may be added to the 5'side so as to match and then introduced into the expression vector. In this case, the translation frame of the clone is, for example, a base sequence divided into three corresponding to one amino acid in SEQ ID NOS: 33 to 43 of the sequence listing shown as a representative clone. It is a framework. To identify the recombinant polypeptide expressed in microorganisms such as Escherichia coli, insect cells, and animal cells as an HCV-derived antigen, immunological reactivity with HCV patient serum is used. That is, it is identified by the Western blotting method.

2 in the amino acid sequence of SEQ ID NO: 43 in the sequence listing
A polypeptide consisting of 19 amino acids from 47 to 265; at least 300 to 30 in the same sequence
A polypeptide comprising a polypeptide represented by 8 amino acids up to the 7th position, which is derived from the amino acid sequence of SEQ ID NO: 43 and has a length of 8 to 25 amino acid residues; at least the 410th position to the 428th position in the same sequence A polypeptide consisting of 13 to 25 amino acid residues derived from the amino acid sequence of SEQ ID NO: 43;
A polypeptide of 10 amino acids up to the second;
Polypeptides consisting of 14 amino acids from 477th to 490th; 1 from 498th to 512th
A polypeptide consisting of 4 amino acids;
A polypeptide consisting of 12 amino acids up to the 550th position; a polypeptide represented by 21 amino acids from the 747th position to the 767th position in the amino acid sequence of SEQ ID NO: 43; at least 841th to 852 positions in the amino acid sequence of SEQ ID NO: 43 A polypeptide represented by 12 amino acids up to the 12th amino acid; a polypeptide represented by 12 amino acids from at least 867th to 878th in the amino acid sequence of SEQ ID NO: 43; at least 665th in the amino acid sequence of SEQ ID NO: 43 To the 672nd amino acid sequence represented by the 8 amino acid sequence, the polypeptide consisting of 8 to 25 amino acid residues in length derived from the amino acid sequence set forth in SEQ ID NO: 41; and the 315th to 327th positions Polypeptide consisting of up to 15 amino acids Is hydrophilicity relatively high, than helix or β-sheet structure also to α in the prediction of the conformation of the polypeptide, established to take a so-called turn structure is higher than the other.

Since the above-described amino acid sequence-derived polypeptide of the present invention serves as an antigenic site as an HCV antigen in a more complete range than in other regions, its original higher-order structure in serum is unknown, and therefore not characteristic. Therefore, not all of them have good reactivity, but it seems that the possibility of specifically reacting with antisera against HCV-related antigens of HCV patients is sufficiently high. These polypeptides may be produced synthetically, rather than by recombinant DNA technology. 2 from 461st to 726th in the amino acid sequence of SEQ ID NO: 43.
A polypeptide consisting of 66 amino acids or a polypeptide containing all or part thereof;
A polypeptide consisting of 74 amino acids up to the 50th position; a polypeptide consisting of 42 amino acids up to the 963rd to 1004th positions; and 283rd to 32nd positions
A polypeptide consisting of 45 amino acids up to the 7th amino acid can also be mass-produced by recombinant DNA technology.

[4] (1) SEQ ID NOs: 64-75 in the sequence listing
Method for obtaining cDNA clone derived from serum of hepatitis C patient shown in 1) and method for determining nucleotide sequence of the clone (NS4 to N
S5 region) The gene or DNA fragment encoding the novel polypeptide represented by the nucleotide sequence shown in the sequence listing of the present invention can be obtained, for example, by the following method. This HCV is
Owing to the conventional cDNA cloning method, the gene is expected to be RNA in addition to the trace amount present in serum.
kayama-Berg method, Gubler-Hoff
Since it was expected to be difficult to clone by the method based on the man method, the following method was used in order to reliably clone the gene having many mutations from a small amount of serum.
That is, nucleic acids are extracted from the serum of hepatitis C patients, as described in Example 1 below. As the serum, one having an OD of 3.5 or more as measured by a test kit of Ortho Co. is usually used, but it is not limited to this value. Transfer RNA as a carrier of viral RNA in serum
It is preferable to mix (tRNA). The carrier is not necessarily limited to tRNA, and any polyribonucleoside can be used instead. However, if tRNA is used, there is an advantage that it is possible to rapidly confirm by electrophoresis whether or not a required amount of tRNA having an intact length is present. By performing this confirmation, at least viral RNA
From the step in which transfer RNA is mixed as a carrier, it is possible to confirm whether or not viral RNA is degraded.

As a means for cloning cDNA from such nucleic acid, the polymerase chain reaction method {(PCR method) Nature, 324, 126, (1986)} developed by Saiki et al. Can be used. First, reverse transcriptase is reacted using viral RNA as a template. At this time, a commercially available random primer may be used as the primer, or MS126, MS119, MS161, MS16 among the primers shown below.
2, synthesis of base sequences such as MS163, MS121 D
NA may be used.

Synthetic DNA used in the present invention 5'3 'MS126; GGTGAGCATGGAGGGTGACCAC (SEQ ID NO: 130) MS119; TCATCCTCCTCCGCCTCGAAGC (SEQ ID NO: 131) GTGGACGCCTCTNCACNTCTG: GCTGACTCTGTG: GCTGACCTTNGTG: GCTGACTCTGTG: GCTGACTCTGTG: GCTGACTCTGTG: GGTGAGCATGACCTTNG: GCTGACTCTGTG: GCTGACGTCTGTG: GCTGACCTCTG: GCTGACCTCTGCTGAG: 13 3) MS121; GGCGGAATTCCTGGTCATAGCCCTCCGTGA A (SEQ ID NO: 134) MS163; GGGGNATGGCCTATTTGGCTG (SEQ ID NO: 135) MS127; 137) MS159; GTGGTANTCCTGGACTCNTTNGA (SEQ ID NO: 138) MS160; ACTACCGNGACGTGCTNAANGA (SEQ ID NO: 1 39) MS120; TGGGGATCCCGTATGATACCCGCTGCTTT G (SEQ ID NO: 140) MS174; ATTGTCAGATCTACGGGGCCACTT (SEQ ID NO: 141) MS175; GCAAGCTTAAAAAAAAAAAAGGGGGATGG CCTATTGGCCTGGA (SEQ ID NO: 142)

"N" in this sequence represents inosine. In these sequences, some bases of the 5'side sequence may be changed to other bases, but in this case, preferably the mutation is within 10 bases from the 5'side and within several bases, and more preferably, Mutations within 5 bases from the 5'side are preferable. In addition, although 4 to 5 bases may be deleted from the 5'side sequence in these sequences, it is preferable that a few bases are deleted from the 5'side. Further, an arbitrary sequence may be added to the 5'side as long as it has about 8 to 12 bases, preferably 5 to 6 bases, more preferably several bases. PCR
The method is specifically carried out under the conditions as described in Example 21. The first strand complementary strand DN thus obtained
The desired DNA fragment can be obtained by performing the PCR method as in Example 21 using A (1st cDNA) as a template. At this time, the PCR conditions may vary depending on the type, combination, length of amplification, etc. of the primers, and therefore the conditions may be changed depending on the situation. Specific examples of the primer combination include the following.

MS127-MS126 MS118-MS119 MS159-MS161 MS160-MS162 MS120-MS163 MS120-MS121 The DNA fragment thus obtained is one of the cloning sites of a cloning vector (for example, pUC19) by a conventional method ( For example, SmaI site). Using the plasmid having this DNA fragment, the nucleotide sequences of three or more clones are independently determined for both strands. Fluorescence Sequencer Genesis 2000 (GENESIS 2000) manufactured by DuPont is used to determine the nucleotide sequence.
The system can be easily determined by following the protocol for the system from DuPont. Where the nucleotide sequence is difficult to determine, or the DNA fragment to be determined is about 18
When there are 0 or more base pairs, subcloning may be performed according to a conventional method. The D determined in this way
The nucleotide sequences of the NA fragments are SEQ ID NOs: 64 to 69 in the sequence listing.
And 76 to 100.

Clone N22-1, 3 and clones N17-1, 2, 3 and clones N29-1, 2, 3 and clones N18-2, 3, 4 were obtained from the serum of one patient N, Clone H22-3, 8, 9 and clone H17
-1,3 and clones H18-1,2,3 were obtained from the sera of one patient H, and clones O28-1,
2, 4 and clones O30-2, 3, 4 are one patient O
Obtained from the serum of. On the other hand, in the case of the core protein region and the region located further 5 ′ to the gene of the core protein, it is said that the region having few mutations in the hepatitis C virus gene is the same as the above, but the core protein region or core Even when the region located further 5'to the gene of the protein was cloned, there were almost no mutations among the clones. However, in the present invention, a plurality of clones having the same region on the HCV gene were obtained from the same patient, and each of them has high homology,
DN encoding different base sequences and different amino acid sequences
It was the A fragment. This indicates that multiple hepatitis C viruses are present in one patient. From this, the N22 region, N17 region, O28 region, N18 region, N29 region, and O30 region are much more likely to be mutated as a result of examining the nucleotide sequence from the HCV core protein region or the 5'upstream region thereof. all right.

The HCV gene region corresponding to each clone was named as follows. Clone N22-1, 3
And clones H22-3,8,9 are primers MS127
And the MS126 combination. The region on this HCV gene is designated as N22 region. Similarly, clone N
17-1,2,3 and clones H17-1,3 were obtained from the combination of primers MS118 and MS119, clones O28-1,2 and 4 were from the combination of primers MS159 and MS161 and clone N29-.
1, 2, 3 are combinations of primers MS160 and MS162, clones N18-2, 3, 4 and clone H18-
1, 2, and 3 are combinations of primers MS120 and MS121, clones O30-2, 3, and 4 are primers MS1.
20 and MS163, respectively.
Each region on these HCV genes is designated as N17, O28,
N29, N18, and O30.

Furthermore, the National Cancer Center Kato et al. Announced (Proceedings of National Academy of Sciences USA).
atl. Acad. Sci. USA), 87,9524
-9528, 1990), Takamizawa et al. Of Micro Lab., Osaka University (Journal of Virology (Journal of V
irology, 65,3,1105-1113,19
91)) reported the homology with the sequence of hepatitis C virus gene, the obtained regions of each clone are N22 region, N17 region, O28 region, N29 in order from the 5 ′ side.
The region and the O30 region are lined up (the N18 region is a part of the O30 region), and each adjacent region has an overlapping portion. The overlapping region is used as follows. Each DNA fragment was ligated to.

(2) Concatenation of clones shown in SEQ ID NOS: 64 to 69 of the region of clone N15 (see [3] above), N22 region, N17 region, O28 region, N29 region, N18 region, O30 region CDNA clones derived from hepatitis C patient sera are ligated as described below using PCR or restriction enzyme sites. 1) Ligation of N17 region and O28 region For ligation of N17 region and O28 region, for example, the nucleotide sequence shown in clone N17-3 of SEQ ID NO: 81 in the sequence listing and the clone O28-1 of SEQ ID NO: 86 in the sequence listing are used. . The PCR method is used for ligation. Clone N17-3
And the DNA fragment of clone O28-1 as a template were added to the same reaction solution in about equimolar amounts to prepare primers MS118 and MS.
The PCR reaction is performed by 161. This makes clone N
Clone 1 in which 17-3 and clone O28-1 are ligated
728 is obtained. 2) N29 region and N18 region ligation In the same manner as in 1), N29 region and N18 region are ligated to each other, for example, clone N29-1 of SEQ ID NO: 89 in the sequence listing and DNA N18-4 of SEQ ID NO: 92 in the sequence listing. Clone 2 ligated using the fragment, primers MS160 and MS121
918 is obtained.

3) Ligation from N17 region to N18 region The DNA fragments of clone 1728 and clone 2918 were used, the primers were MS118 and MS121, and 5 '
The clone 1718 connected to N17, O28, N29, and N18 from the side is ligated by the PCR method. Clone 17
18 was cloned into the Sma I site of the plasmid pUC19 and constructed so that the Eco RI site of pUC19 would be located at the 5'end side (although the N17 region side is the 5'end side of the HCV gene). This plasmid is called pUC
1718. 4) Ligation of N22 region and N17 region In the same manner as in 1), for example, clone N22-1 of SEQ ID NO: 76 in the sequence listing and clone N17- of SEQ ID NO: 81 in the sequence listing.
Using the DNA fragment of 3 and the primers MS127 and MS119, a DNA fragment composed of the N22 region and the N17 region is obtained from the 5'side by PCR reaction. This clone was designated as clone 2217. As in 4), pU is added to the 5'end side.
Plasmid pU so that the Eco RI site of C19 comes
Built into C19. The plasmid is designated as pUC2217.

5) Clone 2217 and Clone 1718
Concatenation of. Clone 1718 is cut at only one site at the Xba I site in the N17 region. Therefore, pUC1718
Cut with X ba I with Xba I site from C19, the cut-out clones derived N17 region of pUC2217 a DNA fragment from 1718 Xba I site and p
The UC19-derived Xba I site was inserted so that the nucleotide sequence of the N17 region was connected. This plasmid is called pUC2
218. 6) Forward ligation of N15 region and O30 region corresponding to HCV 3'end DNA fragment of O30 region is, for example, clone O30-3.
Is shown as SEQ ID NO: 98 in the sequence listing. The DNA fragment of clone O30-3 having this sequence is the S of pUC19.
Eco site on the 5'side of clone O30-3 at the ma I site.
The RI site is inserted in one direction (pUCO3
0). In addition, like the clone O30-3, the HCV gene of clone N15 (the above [3]) is inserted in the Sma I site of pUC 19 in the direction having the Eco RI site on the 5 ′ side (pUCN15). pUCO30
Cleavage with Sac I followed by blunt end and Hin dI
The HCV gene from DNA fragments cut out in II, X
After being cut with ba I, blunt ended, Hin dII
DNA derived from pUCN15 vector excised with I
The fragments were ligated. The obtained plasmid was designated as pUCI5-
Call 30. The plasmid is Bgl II and Hin dII
Utilizing not be only cut at each one place in I, PCR was performed as follows in the primers MS174 containing Bgl II site at the sequence from clone O30-3, poly to 3 'end of clone O30-3 U addition was performed.

A PCR reaction was carried out using pUC15-30 as a template and primers MS174 and MS175, and
After cutting the R fragment Bgl II, with Hin dIII, Bgl
II, was cut with Hin dIII pUCO15-30
Was ligated with the DNA fragment containing the vector part of the above to obtain a plasmid pUC15-30U containing a DNA fragment having poly U on the 3′-end side of clone O30-3. 7) Linkage from N15 region to O30 region Apa I site exists in the common region of N15 region and N22 region. Also, in the common area of the N18 area and the O30 area, A
There is a pa I site. PUC2 so that both fit together
The DNA fragment from clone 2218 was cut from 218 Apa I to yield the inserted plasmid pUC15-30U the Apa I site of PUC1530U. The N15 to O30 regions thus linked encode an amino acid sequence highly homologous to NS5, a part of the nonstructural protein NS4 of flavivirus, which is related to HCV. In addition to the above-mentioned Chiron, Shimotono et al. Of the National Cancer Center, Takamizawa et al.
From the sequence of all genes of hepatitis C virus, the region is NS4
Since it has homology with the gene sequence encoding the entire NS5 from the middle of the above, it is expected that the clones described in the sequence listing of the present invention are the gene sequences of the NS4 region and the NS5 region of hepatitis C virus. Therefore, it was attempted to incorporate the clone into an expression plasmid to produce a polypeptide encoded by the clone and immunoreact with the serum of a hepatitis C patient. (3) Expression of the polypeptide encoded by the clone obtained in (2) Next, the polypeptide encoded by the clone obtained in (2) is expressed to express HCV-derived recombinant hepatitis C. Obtain a specific antigen protein.

The expression vector of the present invention contains a promoter at a position where the DNA encoding the HCV-derived recombinant hepatitis C-specific antigen protein obtained as described above can be transcribed. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector is a promoter,
It is preferably composed of a liposome binding (SD) sequence, a recombinant hepatitis C-specific antigen protein gene derived from HCV, a transcription termination factor, and a gene that controls a promoter.
The promoter is derived from Escherichia coli, phage or the like, for example, tryptophan synthase (trp), lactose operon (lac), lambda phage PL, P.
R, T5 early gene P25, P26 promoter and the like. Also, these may be uniquely designed sequences. The ribosome binding sequence may be derived from Escherichia coli, phage or the like, but a consensus sequence of 4 or more bases in which a sequence complementary to the 3'terminal region of 16S ribosomal RNA prepared by synthesis of a uniquely designed sequence is continuous. It may be a thing with.

Although a transcription termination factor is not always necessary,
It is desirable to have a ρ-independent one such as a lipoprotein terminator or a trp operon terminator. Furthermore, the sequence of the factors required for expression on the expression plasmid is that the promoter, the SD sequence, the HCV-derived recombinant hepatitis C-specific antigen protein gene, and the transcription termination factor are arranged in this order from 5 ′ upstream. desirable. As an expression vector, commercially available pKK233-2
(Made by Pharmacia) can be used. To express the fusion protein, the expression vector pGEX series (Pharmacia) is used. The host can be transformed by the protocol of Toyobo Co., Ltd. as shown in Example 22, or by a conventional method. Culture of transformants was performed by molecular cloning (Mole
The method described in "Clar Cloning, 1982)" may be used as a reference. The culture temperature is about 28 ° C to 42 ° C.

The basic units constituting the expression vector in eukaryotes such as insect cells and animal (mammalian) cells are the same as those of the above-mentioned microorganisms, but the following ones are preferable. Examples of insect cells include Maxbac ( ^TM , BACULO), which is a baculovirus expression manual of Invitrogen.
VIRUS EXPRESSION SYSTEM M
Use this kit according to ANUAL VERSION 1.4). At this time, it is desirable to change the distance from the promoter of polyhedrin to the initiation codon in order to increase the expression level. As a promoter in animal cells,
Activated promoter based on adenovirus EIA gene (Secondary Chemistry Laboratory 1 Gene Research Method II, 189-1
90, 1986), SV40 early promoter, SV4
0 late promoter, apolipoprotein E gene promoter, SRα promoter (Molecular and Cellular Biology, 8, 1, 466-472, 1,
988) and the like are used. Basically, the expression vector p
KCR (Proceedings of National Academy of Sciences USA, 78, 1
528, (1981)) or pBPV MT1 (Proceedings of National Academy of Sciences USA, 80, 398, (198), which has been improved to the extent that its basic functions are not impaired to the minimum.
3)) etc. can be used. Animal cells transformed with the expression vector include CHO cells, COS cells, CV-1.
Cells, mouse-derived cell C127 strain and the like.

When the clone is introduced into an expression vector in Escherichia coli or a eukaryote, it may be aligned with the frame of the start codon derived from the vector, and even if the start codon derived from the vector is not used, The initiation codon may be added to the 5'side so as to match the translation frame and then introduced into the expression vector. In this case, the translation frame of the clone is, for example, a base sequence divided into three corresponding to one amino acid in SEQ ID NOS: 64 to 75 of the sequence listing shown as a representative clone. It is a framework. Microorganisms such as E. coli,
To identify the recombinant polypeptide expressed in insect cells and animal cells as an HCV-derived antigen, immunological reactivity with HCV patient serum is used. That is, it is identified by the Western blotting method.

A polypeptide consisting of 20 amino acids from at least 324 to 343 in the amino acid sequence of SEQ ID NO: 75 in the sequence listing; a polypeptide consisting of 14 amino acid residues from at least 356 to 369 in the same sequence Peptide; at least 584 to 60 in the same sequence
A polypeptide represented by 18 amino acids up to the first position; a polypeptide consisting of 10 amino acids from the 588th position to the 597th position; a polypeptide consisting of 10 amino acids from the 620th position to the 629th position; 901 The polypeptide consisting of 18 amino acids from the 9th position to the 918th position, and the polypeptide consisting of a maximum of 25 amino acids derived from the amino acid sequence of SEQ ID NO: 75 in the sequence listing including at least the polypeptides described above are: It has a relatively high degree of hydrophilicity, and it is expected that the probability of taking a so-called turn structure is higher than that of α-helix or β-sheet structure in predicting the higher-order structure of a polypeptide. Since the conditions for the above-described amino acid sequence-derived polypeptide of the present invention to serve as an antigenic site as an HCV antigen are more complete than those in other regions, the original higher-order structure in serum is unknown, so it is not a character. Does not always have good reactivity, but H
It seems highly likely that CV patients will react specifically with antisera to HCV-related antigens.

These polypeptides may be produced synthetically, rather than by recombinant DNA technology. In the amino acid sequence of SEQ ID NO: 75, a polypeptide consisting of 74 amino acids from 413th to 486th or a polypeptide containing all or a part thereof; a polypeptide consisting of 997th amino acids from 415th to 1411th Peptide; Polypeptide consisting of 74 amino acids 655th to 728th; 858th to 955th
A polypeptide consisting of 98 amino acids up to the th position; a polypeptide consisting of 92 amino acids from the 1009th to 1100th positions; a polypeptide consisting of 66 amino acids from the 1160th to 1225th positions;
A polypeptide consisting of 54 amino acids 763rd to 816th can also be mass-produced by recombinant DNA technology.

[5] Method for obtaining cDNA clone T7N1-30U derived from serum of hepatitis C patient represented by SEQ ID NO: 101 in the sequence listing The novel polypeptide represented by the nucleotide sequence represented by SEQ ID NO: 101 Gene encoding or DN
The A fragment can be obtained, for example, by the following method.
Clone N19MX24A-1 and clone MX25-1
Overlaps by ligation using PCR method
A DNA fragment in which either the 3'portion of the X24 region or the 5'portion of the MX25 region is preferentially obtained is obtained (this obtained clone is referred to as clone 1925). In addition, restrictions on the 5 'side of the clone N1-1 enzyme site Hin dIII,
Spe I and T7 promoter sequences are introduced in this order from the 5 ′ side, so that synthetic DNA is prepared and a clone T7N1-1 is obtained by cassette ligation. Also clone N
1N3N10 was obtained in the same way as clone N
Clone T7N1N3N10 is obtained by using clone T7N1-1 instead of 1-1. This clone and clone N27N19-1 are ligated with a restriction enzyme Bam HI to obtain clone T7N119. This clone T7
N119 and clone 1925 have an N19 region, and by utilizing the restriction enzyme site Pvu I in this N19 region, these two clones were ligated to each other to obtain clone T7N1-2.
Get 5.

Next, the clone 153 obtained in 5) of [4]
Hin d at the 3 'end of pUC1530U with 0U
A clone 1530UN having a Not I site at the 3'end of clone 1530U by introducing an Eco RI- Not I- Bam HI adapter (manufactured by Toyobo Co., Ltd.) into the III site.
get ot. Clone T7N1-25 and clone 153
Clone MX25N15-1 obtained with 0UNot and [3]
Clones T7N1-25 and MX25 to ligate
Three DNA fragments are ligated into one at the Pst I site in the MX25 region where N15-1 overlaps and the Eco T22I site in the N15 region where MX25N15-1 and clone 1530UNot overlap. Also, clone T7N1
-25 has a Spe I site at the 5'end, and clone 1
530UNot has a Not I site at the 3'end.

The gene sequence of HCV is clone T7N1.
-25, clone MX25N15-1, clone 153
It can be created by connecting in the order of 0UNot without overlapping. Therefore, clone T7N1-25 was cloned into Spe
I and PstI, the clone MX25N15-1 Pst I
Eco and Eco T22I, the clone 1530UNot
A DNA fragment having an HCV gene sequence between the Spe I site and Not I site of λZapII by cutting T22I and Not I, and λZapII (manufactured by Stratagene) with Spe I and Not I, respectively, and then ligating them. Only one was inserted (from the 5'end, clone T7N1-25, clone MX25N15-1, clone 1530UNot
To obtain phage (ligated HCV)
The derived clone is designated as T7N1-30U). λZap
II (Stratagene), phage DNA isolation, Bluescript II (pBlue
subcloning into escript II)
It can be performed according to the attached protocol. Also,
Gigapack II is a package for making phage particles
Gold (Gigapack II Packagi
ng Extracts / manufactured by Stratagene) according to the attached protocol.
This clone T7N1-30U is a cDNA derived from HCV.
Is a DNA fragment having a T7 phage promoter sequence artificially introduced at the 5'end and a poly T sequence at the 3'end.

[6] Fusion expression in which polypeptide encoded by cDNA derived from hepatitis C patient serum is linked [1], [2], [3], and poly encoded by each clone obtained in [4] Peptides and HC obtained in [5]
A recombinant hepatitis C-specific antigen protein derived from HCV can be obtained by expressing a polypeptide in a specific region from a DNA sequence encoding all V-derived proteins.
The expression vector of the present invention is the H vector obtained as described above.
A promoter is contained at a position where the DNA encoding the CV-derived recombinant hepatitis C-specific antigen protein can be transcribed. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector controls a promoter, a ribosome binding (SD) sequence, a HCV-derived recombinant hepatitis C-specific antigen protein gene, a transcription termination factor, and a promoter. It is desirable that the gene consists of Examples of the promoter include those derived from Escherichia coli, phage and the like, for example, tryptophan synthase (trp), lactose operon (lac), lambda phage PL, PR, T5 early gene P25, P26 promoter and the like. Also,
These may be uniquely designed arrays.

The ribosome binding sequence may be derived from Escherichia coli, phage or the like, but a sequence complementary to the 3'terminal region of 16S ribosomal RNA prepared by synthesis of a uniquely designed sequence is 4 or more consecutive bases. It may have a consensus sequence of. The transcription terminator is not always necessary, but it is desirable to have a ρ-independent one such as a lipoprotein terminator or a trp operon terminator. Furthermore, the sequence order of the factors required for expression on the expression plasmid is:
It is desirable that the promoter, the SD sequence, the recombinant hepatitis C-specific antigen protein gene derived from HCV, and the transcription termination factor are arranged in this order from 5 ′ upstream. As an expression vector,
Commercially available pKK233-2 (manufactured by Pharmacia) can be used. To express the fusion protein, the expression vector pGEX series (Pharmacia) is used. The host can be transformed by the protocol of Toyobo Co., Ltd. as shown in Example 30 or by a conventional method.

Culture of transformants was carried out by molecular cloning (Molecular Cloning, 19).
The method described in 82) may be used as a reference. Culture temperature is 2
It is about 8 to 42 ° C. Also, eukaryotes, for example,
The basic units constituting the expression vector in insect cells, animal (mammalian) cells and the like are the same as those of the above-mentioned microorganisms, but the following ones are preferable. Examples of insect cells include MAXBAC (MAXBA, which is a baculovirus expression manual manufactured by Invitrogen).
C ^TM , BACULOVIUS EXPRESSIO
N SYSTEM MANUAL VERSION
Use this kit according to 1.4). At this time, it is desirable to change the distance from the promoter of polyhedrin to the initiation codon in order to increase the expression level. As the promoter in animal cells, an active promoter based on the adenovirus EIA gene (sequel biochemistry experimental course 1 gene research method II, 189-190, 1986), SV40 early promoter, SV40 late promoter, apolipoprotein E gene promoter, SRα Promoter (Molecular and Cellular Biology, 8
(1), 466-472, 1988) and the like are used.
Basically, the expression vector pKCR (Proceedings of National Academy of Sciences USA, 78, 1528, 1981) or p which is improved to the extent that the basic function thereof is not impaired
BPV MT1 (Proceedings of National Academy of Sciences USA)
80, 398, 1983) and the like can be used.

Animal cells transformed with the expression vector include CHO cells, COS cells, CV-1 cells, mouse RIII-derived cell C127 strain and the like. When the clone is introduced into an expression vector in Escherichia coli or a eukaryote, it suffices to match the frame of the start codon derived from the vector, and even when the start codon derived from the vector is not used, the translation frame of the clone The start codon may be added to the 5'side so as to match and then introduced into the expression vector. In this case, the translation frame of the clone refers to, for example, a base sequence divided into three corresponding to one amino acid in SEQ ID NOS: 1 to 104 of the sequence listing shown as a representative clone. It is a framework. To identify the recombinant polypeptide expressed in microorganisms such as Escherichia coli, insect cells, and animal cells as an HCV-derived antigen, immunological reactivity with HCV patient serum is used. That is, it is identified by the Western blotting method.

Regions that are predicted to have high immunological reactivity with HCV patient serum with the polypeptide encoded by the cDNA derived from hepatitis C patient serum were ligated and expressed in large amounts in various cells. For example, the expression vector pCZCORE was used to express a polypeptide consisting of the 1st to 115th amino acid sequences of the amino acid numbers of SEQ ID NO: 3 in the sequence listing shown in [1]. The polypeptide encoded by clone N23 is ligated to the C-terminal side of the residue and expressed as a fusion protein. Further, a region that is expected to have high immunological reactivity with HCV patient serum, such as ligation to the C-terminal side thereof, of a polypeptide containing the amino acid sequence of the 963rd to 1005th amino acid numbers of SEQ ID NO: 43 is expected. cDN
It is ligated on A and incorporated into an expression vector to express the polypeptide. Specifically, as shown in Example 30 described below, an epitope of the HCV-derived core protein and a polypeptide which is a part of the nonstructural protein region NS3 and is encoded by clone N23 are immunoreactive with HCV patient serum. The polypeptide CN23, which is linked to a region containing an epitope expected to be highly expressed, was directly expressed in Escherichia coli in a large amount.
That is, Sac in the core gene of pCZCORE
Clone N23 so that the translation frame matches the II site
From the 107th G. An expression vector capable of expressing as a fusion protein by connecting a part of clone N23 to the 3'end side of the N-terminal gene of the core protein and linking the epitope sites of the polypeptide encoded by the clone N23 with the core protein. pCZCN23 was created. Furthermore, by tandemly connecting a DNA fragment having an SD sequence on the 5 ′ side and encoding an HCV-derived protein, the target polypeptide could be expressed in a larger amount. The fusion protein in which the core protein and the epitope site of the N23 region were linked with each other had an extremely high probability of immunological reaction with HCV patient serum.

[0084]

INDUSTRIAL APPLICABILITY The HCV-derived gene or DN of the present invention
The A fragment is novel, and the HCV antigenic peptides obtained by using these DNAs or the polypeptides encoded by these DNAs can be used for the development of a diagnostic method that can detect HCV anti-rest at a high rate and can be detected specifically. It is useful.

[0085]

The present invention will be described in more detail with reference to the following examples, but the invention is not limited to the following examples as long as the gist thereof is not exceeded. In addition, Embodiment 1 to Embodiment 3
In the experimental operation of, basically, attention should be paid to the contamination of the sample.For example, the tips and pipettes used in all the operations such as sample preparation for each reaction solution and reagent preparation should be
It was replaced after every single use. In addition, the basic genetic manipulations described below, which were performed without describing the method in particular, were performed according to the protocol attached to the experiment manuals and reagent sales companies shown in parentheses after each manipulation. Electrophoresis of nucleic acids (Molecular Cl
oning) 1982 Cold Spring Harbor, Cleavage of DNA fragments by restriction enzymes (Molecular Cloning 1982)
Cold Spring Harbor (Cold Spr
ing Harbor), or Toyobo Co., Ltd. general catalog for reagents engineering research 1990, 199
1), ligation reaction between DNA fragments (TAKARA Biotechnology C, manufactured by Takara Shuzo)
atalog 1991 vol. 1) Extraction of DNA fragment from acrylamide gel or agarose gel (Molecular Cloning
1982 Cold Spring Harbor, culture of E. coli transformed with the introduced plasmid on agarose plates and isolation of colonies from it, E. coli transformed with the plasmid (Molecular・
Cloning (Molecular Cloning)
1982 Cold Spring Harbor (Cold
Spring Harbor))

Example 1 Nucleic Acid Extraction from Serum of Hepatitis C Patient Serum of Hepatitis C Patient (This serum is HCV E manufactured by Ortho Co.)
OD = 3.5 or more in IA kit) 10m
Tris buffer (50 mM Tris-HCl (pH
8.0), 1 mM EDTA, 100 mM NaCl)
Add 25 ml, mix and mix for 20,000 g, 20 ° C for 20
Centrifuge for 10 minutes and add 100,000g of supernatant to 20 ℃
It was centrifuged for 5 hours. The proteinase K solution (1
% Sodium dodecyl sulfate, 10 mM EDTA, 10
mM Tris-HCl (pH 7.5), Proten
asek (Pharmacia) 2 mg / ml, yeast
After adding 1.5 ml of tRNA mixture (6.6 μg) and dissolving, incubate at 45 ° C. for 90 minutes, add an equal amount of phenol / chloroform, mix vigorously and collect the aqueous phase containing nucleic acid by centrifugation. This operation (hereinafter abbreviated as “phenol / chloroform treatment”) was performed 4 times or more. Further, the chloroform treatment was performed twice or more. To the aqueous phase thus obtained, 1/10 of 3M sodium acetate or an equal amount of 4M ammonium acetate and 2.5 volumes of ethanol of the aqueous phase were added and mixed, and the mixture was mixed at -20 ° C overnight or- After standing at 80 ℃ for 15 minutes or more, SW41
Nucleic acid was recovered as a precipitate by centrifugation at 35,000 rpm for 4 hours using a Ti rotor (manufactured by Beckman).

Example 2 Synthesis of cDNA [1] Preparation of RNA Sample After drying the nucleic acid obtained in Example 1, 30 μl of water and ribonuclease inhibitor (100 units / μ)
l, manufactured by Takara Shuzo) was added and dissolved. The cDNA synthesis shown below was performed using this nucleic acid aqueous solution. [2] Synthesis of cDNA using Langmum primer Random primer (0.170D, Amersham Co.) 2.7 μl was added to 2 μl of the aqueous nucleic acid solution prepared in [1].
xPCR (Mg) buffer (100 mM Tris-HC
1 (pH 8.3), 60 mM MgCl ₂ , 500 mM
KCl) 2 μl, 1.25 mM 4dNTP 8 μ
l, 2 μl of water was added, and the mixture was kept at 65 ° C. for 5 minutes and then at 25 ° C. for 5 minutes, then, reverse transcriptase (manufactured by Life Science Co., Ltd.) 25 units 1 μl, ribonuclease inhibitor (100 units / μl, Takara Shuzo) 1 μl were added 37 ℃ 20 minutes,
Next, after moisturizing at 42 ° C for 30 minutes and finally at 95 ° C for 5 minutes,
It was cooled to 0 ° C. (complementary strand DNA synthesis). This DNA {using 2 μl of sample, the method of Saiki et al. [Nature (Na
Pure), 324 , 126, (1986)], and a DNA having a specific sequence was amplified by the so-called PCR method. That is, 2 μl of this DNA sample, 10x PCR
Buffer solution (100 mM Tris-HCl (pH 8.
3), 500 mM KCl, 150 mM MgCl ₂ ,
1% gelatin) 10 μl, 2.5 mM 4dNTP 8
μl, two kinds of synthetic DNA primers (S1-AS1, S
A combination consisting of 2-AS1, S2-AS2 or S4-AS3 was used), 50 pmole each, and 100 μl of water was added so that the total amount became 100 μl. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ^™ Takara Shuzo) was added and mixed, and then mineral oil was overlaid. This sample was subjected to DNA Thermal CyCler manufactured by Perkin Elmer Cetus at 95 ° C. 1
Min, 40 to 55 ° C. for 1 minute, and 72 ° C. for 1 to 5 minutes.

This reaction aqueous solution was treated with phenol / chloroform and subjected to ethanol precipitation (ethanol precipitation means 1/10 volume of 3M sodium acetate or an equal volume of 4M in the aqueous phase).
Ammonium acetate and 2.5 times the volume of ethanol of the aqueous phase were added and mixed, using a rotor with a radius of about 5 cm for 15,000.
A process of cooling and centrifuging at 0 rpm and 4 ° C. for 15 minutes to dry the precipitate was performed) to obtain amplified DNA fragments derived from various combinations of primers.

[3] c using antisense primer
Synthesis of DNA In 2 μl of the aqueous solution of nucleic acid cleaved in [1], an antisense primer (synthetic DNA primer AS1, AS2 or A
S3, 15 pmols / μl) 1 μl, 10 × RT buffer (100 mM Tris-HCl (pH 8.3), 5
00 mM KCl) 2 μl, 25 mM MgCl ₂ 4
μl, 2.5 mM 4dNTP 8 μl, water 1 μl were added, and the mixture was kept at 65 ° C. for 5 minutes and then at room temperature for 5 minutes, 1 unit of reverse transcriptase (manufactured by Life Science) 25 units, ribonuclease inhibitor (100 units / μl, manufactured by Takara Shuzo) ) Add 1 μl at 37 ° C for 20 minutes, then 42 ° C for 30 minutes,
Finally, after keeping the temperature at 95 ° C for 2 minutes, it was immediately cooled to 0 ° C (complementary strand DNA synthesis). Using 10 μl of this DNA sample, Sa
iki et al. [Nature, 32]
4 , 126, (1986)], so-called PCR
DNA having a specific sequence was amplified by the method. That is, 10 μl of this DNA sample, 10x PCR buffer (100 m
M Tris-HCl (pH 8.3), 500 mM K
Cl, 15 mM MgCl ₂ , 1% gelatin) 10μ
1, 2.5 mM 4dNTP 8 μl, synthetic DNA primer used for complementary strand DNA synthesis (150 pmol
s / μl) 2 μl, synthetic DN corresponding to this primer
A primer (15 pmols / μl, paired with the synthetic DNA primer used during complementary strand DNA synthesis)
Water was added to 3 μl so that the total amount became 100 μl. First, the mixture was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, Taq DNA polymerase (7 units / μl,
AmpliTaq ^™ Takara Shuzo Co., Ltd. (0.5 μl) was added and mixed, and then overlaid with mineral oil. This sample was used for DNA Therma manufactured by Perkin Elmer Cetus.
l Cycler 95 ° C 1 minute, 40-55 ° C 1 minute, 7
It was treated 25 times at 2 ° C. for 1 to 5 minutes. Finally, after incubating at 72 ° C. for 7 minutes, the reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol.
One-third the amount of 3M sodium acetate or an equal amount of 4M ammonium acetate and 2.5 times volume of ethanol of the aqueous phase were added and mixed, and a rotor having a radius of about 5 cm was used to obtain 15,000.
A process of cooling and centrifugation for 15 minutes at 4 ° C. at rpm and drying the precipitate was performed to obtain amplified DNA fragments derived from various combinations of primers.

Example 3 Cloning of Amplified DNA Fragment and Determination of Nucleotide Sequence At least 1 pmolc of the DNA fragment obtained by the method of [2] and [3] of Example 2 was prepared, and this DNA was transformed into T4.
Blunted with DNA polymerase (manufactured by TOYOBO) (Molecular cloning (Molecular)
Cloning) 1982 Cold Spring
Harbor (Cold Spring Harbo
r)), after introducing a phosphate group at the 5'end with a polynucleotide kinase (manufactured by TOYOBO) (Molecular Cloning) 1
982 Cold Spring Harbor (Cold
(Spring Harbor)) and a ligation kit (manufactured by Takara Shuzo Co., Ltd.) were used to integrate the SUC I site in the multi-cloning site of the pUC19 cloning vector. (The vector DNA used for ligation at this time was 5 ng to 1
0 ng was used. That is, the pUC19 cloning vector was cleaved with the restriction enzyme Sma I (manufactured by TOYOBO),
After phenol / chloroform treatment and ethanol precipitation, the 5'end was further dephosphorylated with alkaline phosphatase (Boehringer Mannheim) (Molecular Cloning).
ng) 1982 Cold Spring Harbor (C
old Spring Harbor)), phenol / chloroform treatment, and ethanol precipitation. ) Escherichia coli JM109 or DH5 was transformed with the thus-prepared DNA (at this time, competent cells made by TOYOBO were used).

The method of transformation is as follows: COMPETENF HIGH from TOYOBO.
Followed the protocol. In this way, from the combination of the respective primers, [2] and [3] of Example 2 were obtained.
The transformant transformed with the pUC19 cloning vector having the DNA fragment obtained by the method of at least 20 can obtain at least 20 each, and a plasmid DNA is prepared from the obtained transformant by a conventional method, Sequences were determined using a DuPont fluorescent sequencer GENESIS 2000 system. The following two synthetic primers 5'd (GTAA
AACGACGGCCAGT) 3 '(SEQ ID NO: 14)
3). 5'd (CAGGAAACAGCTATGAC)
3 '(SEQ ID NO: 144) was used to
The base sequence of the chain and the single chain was determined. The DNA fragment had a base sequence as shown in SEQ ID NOs: 1 to 4 and 9 to 12 in the sequence listing. Sequence ID Nos. 1, 2, 3, 4, of the sequence listing,
The nucleotide sequences shown in 9, 10, 11 and 12 are clones N1-1 and N2 among the transformants obtained above.
-1, N3-1, N10-1, N1-2, S1-1 S
The nucleotide sequences of the + chains of 1-2 and S1-3 are shown. These clones are double-stranded DNAs obtained according to the methods of Example 2 and Example 3 based on the primers of the four combinations shown in Example 2 [2]. The plasmids used to determine the nucleotide sequences of these clones were designated as pUCN1-1, pUCN2-1 and pUCN2-1, respectively.
UCN3-1, pUCN10-1, pUCN1-2, p
UCS1-1, pUCS1-2, pUCS1-3. Each of these plasmids had one molecule of the corresponding DNA fragment.

These sequences represent the nucleotide sequences of clones cloned from cDNA prepared from RNA derived from sera of hepatitis C patients. Not get or
The cycle of the PCR method shown in Example 2 [2] and [3] was tripled or quadrupled (the cycle was probably 60-100).
Even if there is a DNA fragment obtained by amplification, the sequence of the DNA fragment has no homology with the base sequences shown in SEQ ID NOS: 1 to 4 of the sequence listing corresponding to the above-mentioned four combinations. I only got it. That is,
Clone N1-1, N2-1, N3-obtained here
1, N10-1, N1-2, S1-1, S1-2, S1
It was found that the nucleotide sequence of -3 is specific to that derived from the serum of hepatitis C patients. Then, the obtained DNA
In order to investigate whether the polypeptide formed by the open reading frame of the fragment can be an antigen that reacts with an antibody derived from the serum of a hepatitis C patient, the DNA for expressing the polypeptide is exemplified as described in Example 4 below. Was modified and its expression was performed.

Example 4 Method for Obtaining Clone N1N3N10 or N3N10 [1] Method for Obtaining Clone N3N10 1 μl each of DNA fragments N3-1 and N10-1 from the clones N3-1 and N10-1 (about 200
~ 300 ng) in 10x PCR buffer (100 mM T
ris-HCl (pH 8.3), 500 mM KCl,
15 mM MgCl ₂ , 1% gelatin) 10 μl, 2.
8 μl of 5 mM 4dNTP, synthetic primers S2 and A
5 μl each of S3 (both 20 pmol / μl),
76.5 μl of water was added to the reaction solution. After mixing well, the temperature was first kept at 95 ° C for 5 minutes and then rapidly cooled to 0 ° C. 1 minute later, Ta
qDNA polymerase (7 units / μl, Ampli
0.5 μl of Taq ^™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was used as DNA Thermal Cycle manufactured by Perkin Elmer Cetus.
er for 25 minutes at 95 ° C. for 1 minute, 40 ° C. for 1 minute, and 72 ° C. for 2 minutes. Then, after keeping the temperature at 97 ° C. for 2 minutes, it was rapidly cooled to 0 ° C. and kept for 2 minutes. Further, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTAq ^™ Takara Shuzo) was added to the reaction solution, and the same procedure was performed at 95 ° C. for 1 minute and 50 ° C. for 1 minute.
Min, 72 ° C., 2 minutes, 25 times.

Finally, after keeping the temperature at 72 ° C. for 7 minutes, this reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. The amplified DNA sample was separated by agarose gel electrophoresis (Molecular Cloning 1982).
Cold Spring Harbor (Cold Spri
ng Harbor), a DNA fragment of the desired length was excised from the gel (Molecular Cloning 1982 Cold Spring Harbor.
g Harbor)). The obtained DNA fragment was obtained in Example 3
Was treated in the same manner as above and incorporated into the Sma I site in the multiple cloning site of the pUC19 cloning vector. This was cloned and screened (method is the same as in Example 3), and plasmid pUCN3N.
10 obtained, and thus obtained cD derived from serum of hepatitis C patient
NA was designated as clone N3N10. The base sequence is shown in SEQ ID NO: 5 in the sequence listing.

[2] Method for Obtaining Clone N1N3N10 Overlapping Clone N1-1 and Clone N3N
In order to ligate 10 without overlapping, it was utilized that the restriction enzyme cleavage sites common to both and only one were at overlapping positions. That is, the restriction enzyme
Clone N1-1 is 455th G by BssH II
The clone N3N10 is cut at the 3'side of the 159th G. Also, the plasmid pUCN
In consideration of the fact that the DNA fragments are in the same orientation in both 1 and pUCN3N10, pUCN1 was replaced with Hind II.
I, BssH II, and a DNA fragment having a length of 492 bp (this DNA fragment is located on the 5'side of the 455 bp 5'side of the clone N1-1 of the cDNA derived from hepatitis C patient serum, from the Hind III site of the plasmid pUC19 to the Sma I site. (Which is the one to which the DNA fragment up to
159 bp Hind III of pUCN3N10,
By replacing with the DNA fragment excised with BssH II, cloning and screening, the plasmid p
UCN1N3N10 could be obtained. This plasmid is clone N1N361 in which clones N1-1, N3-1 and N10-1 are ligated without overlapping.
I had 0. The nucleotide sequence of clone N1N3N10 is shown in SEQ ID NO: 6 in the sequence listing.

Example 5 Clone N3-1, N3N10
Modification of DNA for expressing HCV-derived polypeptide encoded by Escherichia coli [1] Modification of DNA for expressing HCV-derived polypeptide encoded by clone N3-1 in Escherichia coli Clone N3-1 It is considered that the 22nd A to HCV structural proteins are encoded, and it is expressed using the initiation codon ATG at the 22nd to 24th positions. DNA
The PCR method was used for modification. At this time, the following synthetic DNA was used as the primer. The base sequence for the 5'primer is 5'GCAAGCTTTATGAGCAACAATCCA
AAAACCCCAAAAGA3 '(SEQ ID NO: 145). The base sequence for the 3'primer is 5'GCGAATTCAGATCTTTCACCTAG
CCGGGGGTCCGTGGGG3 '(SEQ ID NO: 14
6) 20 pmol / μl of these two synthetic DNAs
Was prepared.

This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCN was used as template DNA.
100 ng of 3 was used, and 2 μl of the solution prepared as described above was used as a primer for both 5'and 3'primers. First, the prepared sample was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ^™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler (Perkin Elmer Cetus) at 95 ° C. 1
Min, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. Phenol / chloroform treatment of this sample by a conventional method,
Ethanol-precipitated and sampled this DNA with Hind II
After digestion with I and EcoR I, a DNA fragment having a desired length was separated by acrylamide gel and extracted (Molecular Cloning).
g) 1982 Cold Spring Harbor (Co
ld Spring Harbor)). Furthermore, Hind III and EcoR of cloning vector pUC19
I site was ligated by a conventional method, cloned, and screened to obtain plasmid pUCHN3,
The base sequence was determined. The nucleotide sequence of the clone HN3 obtained by this method is as shown in SEQ ID NO: 7 in the sequence listing and has a Hind III site at the 5'end, followed by an initiation codon ATG. Also,
There is an EcoRI site at the 3'end and Bgl inside it .
It has the II site and the stop codon TGA.

[2] Modification of DNA for expressing HCV-derived polypeptide encoded by clone N3N10 in Escherichia coli Clone N3N10 is also considered to encode HCV structural protein from the 22nd position. It was expressed using the initiation codon ATG at the 24th position. DNA
The PCR method was used for modification. At this time, the brimer used was the following synthetic DNA. The base sequence for the 5'primer is 5'GCAAGCTTTATGAGCAACAATCCA
AAAACCCCAAAAGA3 '(SEQ ID NO: 145). The base sequence for the 3'primer is 5'GCGAATTCAGACTCTCAGATTCT.
CTGAGACGCGCCCTCGT3 '(SEQ ID NO: 1
47). 20 pmol / μl of these two synthetic DNAs
Was prepared.

[1] Preparation of PCR reaction solution and post-treatment [1]
As well as done. However, the template DNA is pU
CN3N10, the above two primers were used,
PCR cycle is 95 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C 5
After 10 minutes, 95 ℃ 1 minute, 65 ℃ 1 minute, 72 ℃
It was performed 20 times in 5 minutes. This DNA sample was used for Hind II
After digestion with I and EcoR I, a DNA fragment having a desired length was separated by acrylamide gel and extracted (Molecular Cloning).
g) 1982 Cold, Spring Harbor (Co
ld Spring Harbor)). Furthermore, Hind III and EcoR of cloning vector pUC19
The I site was ligated by a conventional method, cloned and screened to obtain a plasmid pUCHN3N10, and its nucleotide sequence was determined.

Clone HN3 obtained by this method
N10 has a Hind III site at the 5'end , followed by the start codon ATG. Also, at the end of 3 '
There is an EcoR I site, which has a Bgl II site and a stop codon TGA inside. Clone HN3
To eliminate the BamH I site is in the N10, 5'GGAT the nucleotide sequence 5'GGATCC3 'of the site
Modified to AC3 '. PCR was used to modify the DNA. At this time, the synthetic D shown below was used as the primer.
NA was used. The base sequence for the 5'primer is 5'GCTACTCCGGATACCAC3 '(SEQ ID NO: 148). The base sequence for the 3'primer is 5'GTAAAACGACGGCCAGT3 '(SEQ ID NO: 143). 20 pmol / μl of these two synthetic DNAs
Was prepared.

The 5'-most G of this 5'primer
Is the position corresponding to the 1016th G in the base sequence of clone N3N10. The 3'primer is a sequence derived from the plasmid pUC19 and is the same as one of the sequence primers used in Example 3. PC
The cycle of the R method was 95 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and was repeated 25 times. As the template DNA, 100 ng of the plasmid pUCHN3N10 was used, and 3 μl of each primer was used. Phenol / chloroform treating the sample by a conventional method, ethanol precipitated, and the DNA sample Mro I, Bgl II, BamH
After digestion with I, a 226 bp DNA fragment of the desired length was separated on an acrylamide gel and extracted (Molecular Cloning).
g) 1982 Cold Spring Harbor (Co
ld Spring Harbor)). Furthermore, Mro I, ligated and cloned by a conventional method to Bgl II site of plasmid PUCHN3N10, and screening to give a plasmid PUCHN3N10derutaB, and their nucleotide sequences were determined. The nucleotide sequence of the clone HN3N10ΔB obtained by this method is as shown in SEQ ID NO: 8 in the sequence listing.

[3] Modification of DNA for expressing HCV-derived polypeptide encoded by clone N3N10 in insect cells Clone N3N1 encoding polypeptide to be expressed
0 contains all HCV-derived protein genes (core, envelope (M-gp35)), and it seems that the 22nd A to HCV structural proteins are encoded,
It was expressed in insect cells using the initiation codon ATG at the 22nd to 24th positions. In insect cells, the core and the envelope (M-gp35) are first connected and expressed in the cell as a precursor, and then processed in the cell to be divided into the core and the envelope (M-gp35), and at least the envelope (M-gp35). M-gp35) was modified incompletely with a sugar chain, expressed as a mature protein, and accumulated in cells. Clone N to construct expression vector
The PCR method was used for modification of 3N10 DNA. At this time,
The synthetic DNA shown below was used as the primer.

The base sequence for the 5'primer is MS106: 5'GCGTCGGACGCTAGCATG.
AGCACAAATCCAAAAACCC3 '(SEQ ID NO: 149) MS107: 5'GCGTCGGACGCTAGCAGG
TCTCTAGAGACCGTGCCATC3 '(SEQ ID NO: 150). The base sequence for the 3'primer is MS108: 5'GCGAATTCGCTAGCTCA.
GGATTCTCTGAGACGGCCCTCGA3 '
(SEQ ID NO: 151). 20 pmol / μl of these three synthetic DNAs
Was prepared.

[1] Preparation of PCR reaction solution and post-treatment
As well as done. However, the template DNA is pU
CN3N10 uses MS106 or MS107 as the 5'primer and M3 as the 3'primer.
S108 was used. PCR cycle is 95 ℃ 1
Minutes, 50 ° C. for 1 minute, 72 ° C. for 5 minutes, which was repeated 10 times, and then 95 ° C. for 1 minute, 65 ° C. for 1 minute, 72 ° C. for 5 minutes were repeated 20 times. When MS106 is used as the 5'primer,
The desired DNA fragments 106-108 having a length of 1265 bp were obtained. When MS107 was used as the 5'primer, a 1286 bp DNA fragment 107-1 was similarly obtained.
I got 08. After cutting these DNA samples with Nhe I,
DNA fragments of the desired length were separated on acrylamide gel and extracted (Molecular Cloning (Mol
Equal Cloning) 1982 Cold ・
Cold Spring Ha
rbor)). Furthermore, the transfer vector pBl
The plasmid pBlueN3N10-1 and the DNA fragment 107-1 derived from the DNA fragments 106-108 were ligated to the Nhe I site of ueBac (Invitrogen) by a conventional method, cloned and screened.
The 08-derived plasmid pBlueN3N10-2 was obtained.

[0105] Plasmid pBlueN3N10-1 plasmid PBlueN3N10-2, have <br/> with Nhe I is then completely cut with BamH I, the DNA fragment is inserted by one into Nhe I site of the transfer vector pBlueBac It was confirmed. Regarding the expression unit of these plasmids, the cloning site Nhe I downstream of the polyhedrin promoter can be referred to by referring to those shown in the protocol of Invitrogen.
The cleavage site had a structure in which HCV-derived structural genes (core, envelope) were linked in the forward direction. Example 6 Expression of HCV-derived polypeptide encoded by clones HN3 and HN3N10ΔB [1] Expression in Escherichia coli of polypeptide encoded by clone HN3 The polypeptide encoded by clone HN3 is C
It is a part of a polypeptide encoded by cDNA derived from hepatitis C patient sera, which was directly expressed in E. coli. As shown below, this is pC as an expression vector.
Z44 (JP-A-1-124387) was used, and it was carried out by subcloning the cDNA of clone HN3 into pCZ44.

First, clone HN3 was cloned into Hind III,
Are sufficiently cut by Bgl II, treated with phenol / chloroform, ethanol precipitated, both ends respectively Hin
The cD that became the protruding end of the d III or Bgl II site
A DNA fragment having NA was separated by acrylamide gel and extracted (Molecular Cloning (Mol
Equal Cloning) 1982 Cold ・
Spring Harbor (Cold Spring Ha)
rbor)). Similarly, the expression vector pCZ44 is also Hi.
Hind II prepared from clone HN3 was prepared by preparing a longer DNA fragment sufficiently digested with nd III and Bgl II and having an expression vector ability in the same manner as described above.
I and BglII fragments were ligated so as to have one fragment, and further cloned according to a conventional method. The resulting plasmid was named pCZCORE. Further, as another attempt, an expression vector was constructed using an expression vector pGEX-2T (manufactured by Pharmacia) that can express a fusion protein instead of pCZ44. In this case, the operation was basically performed according to the instructions of Pharmacia. First, the expression vector pGEX-2T was cleaved with BamHI and then HindIII.
Insert the linker to fit the reading frame of the amino acid and codon of clone N3-1, 3 'side of the Ec oR I site and the 5' (made with linker) side of the Hind III site in HN3 of Hind III, EcoR The I fragment was inserted. Thus, the expression vector pGEXCORE was obtained.

Escherichia coli derived from strain K12 (for example, strain JM109 or strain KS476) or strain B carrying plasmid pCZCORE was transformed into L-Broth (Molecular Cloning).
3) at Cold Spring Harbor in 1982.
It was cultured overnight at 7 ° C. This was inoculated into new L-Broth to be diluted 50-fold, and after shaking culture at 30 ° C. for 2 hours, IPTG (isopropyl-β-D-galactopyranoside) was adjusted to 1 or 2 mM. The mixture was added to the medium and further cultured for 3 hours or more. Thus, by culturing the bacterium in the presence of IPTG, a recombinant derived from a single clone (in this case, Escherichia coli transformed only with the plasmid pCZCORE) was cloned using the plasmid pCZCORE derived from the clone HN3. HCV-derived CO
Was induced to express the RE-N3 polypeptide.
The nucleotide sequence of the cDNA of clone HN3 and the deduced amino acid sequence of the polypeptide encoded by the sequence are shown in SEQ ID NO: 7 in the sequence listing.

Similarly, the plasmid pGEXCORE was similarly analyzed for β-glutathione-S-transferase (G
The expression product can be obtained as a fusion protein with ST). In this case, the GST has a site that is specifically cleaved by thrombin at the C-terminal as described in Pharmacia, and a polypeptide derived from clone HN3 similar to pCZCORE is connected to the C-terminal side. In order to obtain this fusion protein GST-CORE, Escherichia coli transformed with the plasmid pGEXCORE was cultured in the presence of IPTG in the same manner as the above-mentioned protocol for expressing HCV-derived CORE-N3 polypeptide from pCZCORE, and HCV-derived Was induced to express the fusion protein GST-CORE having the C protein at the C-terminus.

[2] Expression of Polypeptide Encoded by Clone HN3N10ΔB The polypeptide (CME-N3N10ΔB) was directly expressed in Escherichia coli from the cDNA derived from hepatitis C patient serum contained in clone HN3N10ΔB. The expression method was similar to that described in [1].
However, as a material for HCV cDNA, clone HN3 is used.
It is a cDNA derived from the serum of a hepatitis C patient contained in N10ΔB, which is used in Example 3, Example 4 [1], and Example 5.
It was isolated and the nucleotide sequence was determined as described in [2]. Therefore, the expression plasmid used was the plasmid pUCHN3N10 obtained in Example 5 [2].
From ΔB, a DNA fragment containing the cDNA having both Hind III and Bgl II site protruding ends at p
In the same way that CZCORE was obtained, Hi of pCZ44 was obtained.
It was obtained as a plasmid pCZCMEΔB by subcloning the cDNA into the nd III and Bgl II sites so that only one cDNA was in the correct orientation. Whether or not the expression unit of this plasmid pCZCMEΔB was reconstructed correctly was confirmed by nucleotide sequencing and creation of a restriction enzyme cleavage map.

By culturing the fungus in the presence of IPTG, a single clone derived (eg, plasmid pCZC
A recombinant of Escherichia coli JM109 strain transformed only with MEΔB was modified from clone N3N10 and derived from clone HN3N10ΔB.
Using CMEΔB, HCV-derived CME-N3N10Δ
Was induced to express the B polypeptide. The nucleotide sequence of cDNA derived from hepatitis C patient serum of clone HN3N10ΔB and the deduced amino acid sequence of the polypeptide encoded by the sequence are shown in SEQ ID NO: 8 in the sequence listing. (Clone HN
The deduced amino acid sequence of the polypeptide encoded by the nucleotide sequence of 3N10ΔB and the definite amino acid sequence from clone N3N10 before modification are the same. ) As in the case of [1], construct the plasmid pGEXCMEΔB, and according to the protocol of Pharmacia, the above plasmid pCZ.
Similarly to CMEΔB, the fusion protein GST-CME-N3N10ΔB was expressed by inducing with IPTG.

[3] Expression of HCV-derived polypeptide encoded by clone N3N10 in insect cells Plasmid pBlueN3N constructed in Example 5 [3]
10-1 and the HCV-derived structural protein (core, envelope (M-gp35)) encoded by the plasmid pBlueN3N10-2 are expressed in insect cells by the Maxbac (MAXBAC) baculovirus expression manual of Invitrogen. ^TM BA
CULOVIRS EXPRESSION SYSTEM
EM MANUAL VERSION 1.4, hereinafter referred to as Maxback. ) Was basically referred to. H was inserted at the restriction enzyme Nhe I cleavage site of the transfer vector pBlueBac (Maxback page 37) prepared in Example 5 [3].
Plasmid pBlueN3N10-1 and plasmid pBlueN in which a DNA fragment containing the CV structural gene was inserted
3N10-2 was recovered and purified from recombinant Escherichia coli according to the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 86-96 (1982)) to obtain HCV structural gene transfer plasmid DNA. Got a lot. MaxBack according to the method on page 27, kit AcNPV
1 μg of virtual DNA and plasmid pBlueN
3N10-1 or plasmid pBlueN3N10
2 was mixed with 2 μg and co-infected with Sf9 cells to give Sf9
The cells were transformed.

That is, first, 2 × 10 5 Sf9 cells were placed in a TMN-FH medium (Invitrogen) containing 10% FCS (fetal bovine serum) in a petri dish having a diameter of 6 cm.
^It culture | cultivated until it became ⁶ / sheet. Next, from the petri dish, TMN-
Grace (Grace) medium (GIBC (GIBC) containing 10% of FCS of 0.75 ml except FH medium)
O)) was added. Add 0.75 ml of the transfection buffer included in the kit to the above-mentioned mixed DNA, mix well by vortexing, and then Grace.
The DNA solution was slowly added dropwise over the medium. 27 ° C 4
Allow to stand for a period of time, then remove Grace medium and
Add 3 ml of TMN-FH medium containing 2% to a Petri dish and 2
It was cultured at 7 ° C for 6 days. On day 3, polyhedra were observed in some cells, and on day 6, most cells formed polyhedra. Transfer this culture supernatant to a centrifuge tube and
The supernatant obtained by centrifugation at 00 rpm for 10 minutes was used as a co-infection virus solution.

This co-infectious virus solution contained 10 ⁸ viruses per ml, and about 0.5% of them was recombinant. The plaque isolation method was used to isolate the recombinant virus. The method is as follows. The co-infected virus solution was diluted to 10 ⁻⁴ , 10 ⁻⁵ . 1.5 x 10 ⁶ per piece in 6 cm Petri dish in advance
After culturing individual cells and adsorbing them, the medium was completely removed, and 100 μl of each of 10 ⁻⁴ solution and 10 ⁻⁵ solution was added to each petri dish. In order to prevent the cells from drying, the petri dish was tilted every 15 minutes so that the virus solution was spread over the entire surface. Thus, 2.5% baculovirus agarose (manufactured by Invitrogen) was used for 10 hours while being infected at room temperature for 1 hour.
The mixture was mateclaved at 5 ° C for 10 minutes, mixed with TMN-FH medium containing 10% FCS warmed at 46 ° C at a ratio of 1: 3, and kept warm at 46 ° C. The final concentration of 150 μg / μl was added to this warmed medium.
Bromo-4-chloro-3-indolyl-β-D-galactoside was added (according to Maxbac, pages 16-17) and this medium was called agarose-containing x-gal medium.

After the infection, the virus solution was completely sucked up, and the agarose-containing x-gal medium which had been kept warm was gently added in an amount of 4 ml per petri dish so that the cells would not come off. The petri dish was allowed to stand with the lid slightly displaced until the agarose was solidified and dried, and then, the lid was put on and turned over, and incubation was carried out at 27 ° C. After culturing for 6 days, blue plaques in which polyhedra were not formed were found by a phase contrast microscope. The recombinant plaque is sucked up with agarose with a bath tool pipette and 1 ml of TMN-
The virus was suspended by pipetting many times in FH medium. This series of operations (an operation of infecting and then culturing for 6 days to isolate a virus having the transfer plasmid DNA) is called a plaque method. The same plaque method was performed using 100 μl of the virus suspension. These series of operations are repeated three times, and HCV free from contamination by wild strains.
A recombinant virus having a derived structural protein gene was obtained. The virus plaque was absorbed with a Pasteur pipette and mixed well with 1 ml of TMN-FH medium. Since this primary recombinant virus solution has a low virus concentration, 100 μl of it was taken, and after adsorbing to Sf9 cells cultured in a 6 cm dish at half confluence, 4 ml of TMN-FH medium containing 10% FCS was added, and after 3 days, the culture supernatant was added. Was collected and used as a recombinant virus solution for infection.

In order to produce the HCV-derived structural protein, 5 × 10 ⁶ Sf9 cells were suspended in 10 ml of TMN-FH medium containing 10% FCS in advance, and the suspension was spread on a 9 cm Petri dish and adsorbed for 1 hour. The medium was removed from the petri dish, 250 μl of the recombinant virus solution for infection was added, the whole was spread, 10 ml of TMN-FH medium containing 10% FCS was added, and the mixture was allowed to stand at 27 ° C. for 4 days. The cells were thoroughly suspended in 1,000 ml of phosphate buffered saline. Thus, the HCV-derived structural protein was expressed in Sf9 cells infected with the virus. In addition, the plasmid pBlueN3N10
-1 infected cells and plasmid pBlueN3N
The same HCV-derived polypeptide was expressed in cells infected with 10-2. Example 7 Identification of the expressed product as an HCV-derived antigen

Human sera infected with hepatitis C virus were treated with the polypeptide expressed in Example 6 (in this case
The CORE-N3 polypeptide and CME-N3N10ΔB polypeptide shown in [1] and [2], and the HCV-derived polypeptide encoded by the clone N3N10 expressed in insect cells shown in [3]. The polypeptide was identified as a hepatitis C-related antigen because it immunologically reacts with. This identification was performed by the Western blotting method shown below. First, Example 6
Expression plasmids pCZCORE and pCZCMEΔB encoding the CORE-N3 polypeptide and CME-N3N10ΔB polypeptide described in [1] and [2] were transformed into E. coli in the presence of IPTG for 3 hours or as in Example 6. It was induced to express the polypeptide by culturing overnight. This culture solution 1,000μ
1, 6,500 r. p. m. The recombinant strains were harvested by centrifugation for 10 minutes. SD of this precipitate
Sample processing solution for S-polyacrylamide gel electrophoresis (2
50% SDS, 5% mercaptoethanol, 10% glycerin and 0.005% bromophenol blue 50
mM Tris-HCl buffer, pH 6.8) with a final volume of 0.2
It was dissolved and prepared so as to become ml.

Further, the infected cells (having the virus which has undergone the Braak method three times or more) obtained in Example 6 [3] were thoroughly suspended in 1,000 ml of physiological saline phosphate buffer, and 100 μl of the suspension was suspended. 6,50
0r. p. m. A precipitate of the cells was obtained by centrifugation at 10 minutes. This precipitate was dissolved and prepared in a sample treatment solution for SDS-polyacrylamide gel electrophoresis so that the final amount was 0.2 ml. Next, these samples were boiled at 100 ° C. for 10 minutes. 10 μl of the sample thus obtained was added to 0.1% SDS-15.0% polyacrylamide gel (70 × 85 × 1 mm). At that time, "LMW KitE" (low molecular weight marker protein) manufactured by Pharmacia was used as a marker protein. Tris buffer (25 mM as electrode solution
Tris pH 8.3, 192 mM glycine, 0.1
% SDS), and after running for 45 minutes at a constant current of 30 mA, the gel was overlaid with a nitrocellulose filter (BA-83) manufactured by S & S Co. according to a conventional method, and the gel side was used as a cathode and the nitrocellulose filter side was used as an anode. , 120mA
The constant current was applied for about 20 minutes to transfer. The transferred nitrocellulose filter was cut so as to separate the marker protein portion and the sample portion, the marker protein portion filter was stained with 0.1% (W / V) Amido Black 10B, and the sample portion filter was also removed. ,
Immersion was performed in 0.01 M phosphate saline (PBS) pH 7.4 containing 5% (W / V) bovine serum albumin. next,
10 ml of hepatitis C patient serum diluted 50 times with 0.01 M phosphate saline (PBS) pH 7.4 containing 5% (W / V) bovine serum albumin is placed on the filter of the sample portion at room temperature. After standing for 2 hours at 0.1% (V / V) S
It was washed 3 times for 20 minutes each with PBS containing Tween 20 manufactured by igma.

Next, PBS is placed on the filter of the sample portion.
Place 10 ml of peroxidase-labeled anti-human IgG (manufactured by Gappel) diluted 1,000 times with the solution at 37 ° C.
The reaction was carried out for 1 hour and the plate was washed with PBS containing 0.1% (V / V) Tween 20 three times for 20 minutes each. Next, the sample part filter 100 ml of peroxygose color developing solution (6
It was immersed in 0 mg 4-chloro-1-naphthol, 20 ml methanol, 80 ml PBS, 20 μl hydrogen peroxide).
The colored filter was washed with distilled water and then compared with the marker protein portion. The proteins developed by this are CORE-N3 polypeptide and CME-N3N10.
There is only one in ΔB polypeptide, and it is almost the same in plasmid pCZCORE and pCZCMEΔ from the molecular weight.
The protein expressed from the cDNA derived from the serum of the hepatitis C patient encoded by B had a reasonable size and could be identified as a hepatitis C-related antigen. Further, the HCV-derived polypeptide encoded by the clone N3N10 expressed in insect cells shown in Example 6 [3] was obtained by the plasmid p
The same pattern of HCV-derived polypeptides was expressed in cells infected with BlueN3N10-1 and in cells infected with the plasmid pBlueN3N10-2. That is, the molecular weight when expressed as a polypeptide from the core gene of clone N3N10 was calculated to be about 22 kD (when not modified), but a protein of about 22 kD corresponding to this was expressed. H
The polypeptide was predicted to be derived from the core protein of CV and could be identified as a hepatitis C-related antigen.

Example 8 Examination of differences between clones obtained by the methods of Example 2 [2] and [3] Example 2 was performed using serum collected from a hepatitis C patient.
According to [2], 3 clones were independently cloned in the region corresponding to SEQ ID NO: 1 in the sequence listing, and the nucleotide sequences were determined for each. Also, using serum collected from the same patient,
This time, the nucleotide sequences of 3 clones were similarly determined by the method of Example 2 [3]. Among the clones obtained from the same patient serum, three clones independently obtained had the nucleotide sequences shown in SEQ ID NO: 1 in the sequence listing when random primers were used. In addition, S of the aforementioned synthetic DNA primer
When 1 and AS1 were used, two of the three clones independently obtained were the nucleotide sequences shown in SEQ ID NO: 1 of the sequence listing, and the remaining one clone was the nucleotide sequence of SEQ ID NO: 1 of the sequence listing. 345th A is C, 332nd A is T,
The 95th A was different from C at 3 points. This mutation indicates that there are more than one of the virus that infects one patient. From this result, it was found that there is essentially no difference between the obtained clones due to the difference between the methods of Example 2 [2] and [3].

Example 9 Synthesis of cDNA [1] Preparation of RNA Sample After drying the nucleic acid obtained in Example 1, 30 μl of water and ribonuclease inhibitor (100 units / μ)
l, manufactured by Takara Shuzo) was added and dissolved. The cDNA synthesis shown below was performed using this nucleic acid aqueous solution. [2] Synthesis of cDNA Using Antisense Primer Antisense primer (synthetic DNA primer MS122, MS15) was added to 2 μl of the aqueous nucleic acid solution prepared in [1].
7, MS148, etc. are antisense primers.
15 pmols / μl) 1 μl, 10 × RT buffer (100 mM Tris-HCl (pH 8.3), 50
0 mM KCl) 2 μl, 25 mM MgCl 2 4 μ
1, 2.5 mM 4dNTP 8 μl, water 1 μl were added, and the mixture was kept at 65 ° C. for 5 minutes and then at room temperature for 5 minutes. ) 1 μl was added and the mixture was kept at 37 ° C. for 20 minutes, then at 42 ° C. for 30 minutes, and finally at 95 ° C. for 2 minutes, and immediately cooled to 0 ° C. (complementary strand DNA synthesis). Using 10 μl of this DNA sample, Sai
ki et al. [Nature, 324,
126, (1986)], a DNA having a specific sequence was amplified by the so-called PCR method. That is, this D
NA sample 10 μl, 10x PCR buffer (100 mM
Tris-HCl (pH 8.3), 500 mM KC
1, 15 mM MgC12, 1% gelatin) 10 μl,
8 μl of 2.5 mM 4dNTP, synthetic DNA primer used in complementary strand DNA synthesis (150 pmols /
μl) 2 μl, a synthetic DNA primer corresponding to this primer (15 pmols / μl, which composes a pair with the synthetic DNA primer used in the synthesis of complementary strand DNA, and has the above-mentioned primers MS122-MS123 and MS1.
The primer combination of 57-MS156, MS148-MS146 was used. ) Water was added to 3 μl so that the total amount became 100 μl. First, the mixture was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler (Perkin Elmer Cetus) at 95 ° C. 1
Min, 40 to 55 ° C. for 1 minute, and 72 ° C. for 1 to 5 minutes.

Finally, after incubating at 72 ° C. for 7 minutes, this reaction aqueous solution was treated with phenol / chloroform and subjected to ethanol precipitation (ethanol precipitation means 1/10 volume of 3M sodium acetate or equivalent volume of 4M ammonium acetate). And 2.5 times the volume of the aqueous phase of ethanol are added and mixed, and the radius is 5 cm.
Rotor 15,000 rpm at 4 ℃ for 15
Amplified DN derived from various combinations of primers was subjected to cooling centrifugation for 5 minutes and drying the precipitate).
A fragment was obtained.

Example 10 Cloning of Amplified DNA Fragment and Determination of Nucleotide Sequence At least 1 pmole of the DNA fragment obtained by the method of Example 9 [2] was prepared, and this DNA was treated with T4 DNA polymerase (manufactured by Toyobo Co., Ltd.). Blunt-ended (Molecular Cloning
g) 1982 Cold Spring Harbor (Col
d Spring Harbor)) and a polynucleotide kinase (manufactured by Toyobo Co., Ltd.) after introducing a phosphate group at the 5 ′ end (Molecular Cloning).
Cloning) 1982 Cold Spring Harbor),
Using the ligation kit (Takara Shuzo), pUC
It was incorporated into the Sma I site within the multiple cloning site of the 19 cloning vector. (At this time, 5 ng to 10 ng of vector DNA used for ligation was prepared as follows. That is, pUC19.
The cloning vector was cleaved with a restriction enzyme Sma I (Toyobo Co., Ltd.), treated with phenol / chloroform and precipitated with ethanol, and then further dephosphorylated at the 5'end with alkaline phosphatase (Boehringer Mannheim) (Molecular).・ Cloning (Molecul
ar Cloning) 1982 Cold Spring Harbor
r)), phenol / chloroform treatment and ethanol precipitation. ) Escherichia coli JM109 or DH5 was transformed with the thus-prepared DNA (at this time, a competent cell manufactured by Toyobo Co., Ltd. was used).

The method of transformation was according to the protocol of COMPETENTHlGH from Toyobo Co., Ltd. As described above, at least 20 transformants were obtained by transforming the pUC19 cloning vector having the DNA fragment obtained by the method of [2] of Example 2 from each primer combination.
I got more than one. Plasmid DNA was prepared from the obtained transformant by a conventional method, and the sequence was determined using a fluorescent sequencer GENESIS 2000 system manufactured by DuPont. The following two synthetic primers 5'd (GTAAAACGAC) are used as sequence primers.
GGCCAGT) 3 '(SEQ ID NO: 143), 5'd
(CAGGAAACAGCTATGAC) 3 '(SEQ ID NO: 144) was used to determine the nucleotide sequences of the + strand and single strand of the DNA fragment. The DNA fragment is SEQ ID NO: 1 in the sequence listing.
It had a nucleotide sequence as shown in 3 to 27. The nucleotide sequences shown in SEQ ID NOs: 13 to 27 in the Sequence Listing are the H sequences incorporated in the transformant plasmids obtained above.
The nucleotide sequence of the + chain of the CV-derived gene is shown. Further, the clone is a double-stranded DNA, and for example, clone N19
The plasmids used for determining the nucleotide sequences of -1, N19-2, and N19-3 were pUCN19- and
1, pUCN19-2 and pUCN19-3. Each of these plasmids had one molecule of the corresponding DNA fragment. Hereinafter, the plasmid having one of the clones and used for determining the nucleotide sequence is represented by the clone name after pUC.

These sequences represent the nucleotide sequences of clones cloned from cDNA prepared from RNA derived from sera of hepatitis C patients, and the sera of normal humans can be used in any manner by this method. Not get or
The cycle of the PCR method shown in Example 9 [2] and [3] was tripled or quadrupled (the cycle was probably 60 to 100).
Even if there is a DNA fragment obtained by amplification, the sequence of the DNA fragment has no homology with the nucleotide sequences shown in SEQ ID NOs: 13 to 27 of the sequence listing corresponding to the above-mentioned four combinations. I only got it. That is, it was found that the nucleotide sequences of the clones shown in SEQ ID NOS: 13 to 27 of the sequence listing obtained here are specific to those derived from the serum of hepatitis C patients. Therefore, the nucleotide sequence of the obtained DNA fragment was compared with the previously reported gene sequence of hepatitis C virus. In addition, Example 9
In [2], for example, MS122 and MS123 were used as primers, three clones were obtained from the serum of the same patient, and the clones N19-1, N19-2, and N19-3 whose nucleotide sequences were determined were one patient serum. It shows that the patient has the hepatitis C patient serum-derived nucleotide sequence obtained from the above, that is, one patient has a plurality of viruses.

Example 11 Clone N27MX24A-
1 and method for obtaining clone N27MX24B-1 [1] Clone N19MX24A-1 and clone N19
Method for Obtaining MX24B-1 The DNA fragments from the clone N19-1 and the clone MX24-4 were each added to 1 μl (about 0.5 to 1).
μg / μl) in 10 × PCR buffer (100 mM
Tris-HCl (pH 8.3), 500 mM KC
1, 15 mM MgCl2, 1% gelatin) 10 μl,
2.5 mM 4dNTP 8 μl, synthetic primer S2
And AS3 (both 20 pmol / μl) each 5μ
1 and 76.5 μl of water were added to the reaction solution. After mixing well,
First, the temperature was kept at 95 ° C for 5 minutes and then rapidly cooled to 0 ° C. One minute later,
Taq DNA polymerase (7 units / μl, Am
0.5 μl of pliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample is Perkin
Elmer Cetus DNA ThermalCy
Cler for 1 minute at 95 ° C, 1 minute at 40 ° C, 2 minutes at 72 ° C for 25 minutes
Processed twice. Then, after keeping the temperature at 97 ° C. for 2 minutes, it was rapidly cooled to 0 ° C. and kept for 2 minutes. In addition, Taq DNA polymerase (7 units / μl, AmpliTaqTM) was added to the reaction solution.
Takara Shuzo) Add 0.5 μl and in the same manner at 95 ° C for 1 minute, 50
The treatment was carried out 25 times at 1 ° C. for 1 minute and 72 ° C. for 2 minutes. Finally, after keeping the temperature at 72 ° C. for 7 minutes, this reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. This amplified D
NA samples were separated by agarose electrophoresis (Molecular Cloning).
Cloning) 1982 Cold Spring Harbor,
DNA fragments with the desired length were excised from the gel (Molecular Cloning).
Cloning) 1982 Cold Spring Harbor).

The obtained DNA fragment was treated in the same manner as in Example 10 and incorporated into the Sma I site in the multicloning site of the pUC19 cloning vector.
This is cloned and screened (this method
Same as Example 10. ) Plasmid pUCN19M
X24A-1 and pUCN19MX24B-1 were obtained, and the cDNA derived from the serum of the hepatitis C patient thus obtained was clone N19MX24A-1 and clone N19MX24B-1.
I named it. The nucleotide sequences are shown in SEQ ID NOs: 29 and 3 of the sequence listing.
It shows in 0. [2] Method for obtaining clone N27N19-1 Overlapping clone N27-3 and clone N1
In order to ligate 9-1 without overlapping, it was utilized that the restriction enzyme cleavage sites that are common to both of them and have only one were at the overlapping sites. That is, the clone N27-3 is cut at the 3'side of the 330th A by the restriction enzyme Mlu I, and the clone N19-1 is cut at the 3'side of the 51st A. Also, the plasmid pUCN
In consideration of the fact that the DNA fragments are in the same orientation for both 27-3 and pUCN19-1, pUCN27-3 was
Hin dIII, with Mlu I, 'DNA fragment (DNA fragment containing the side, 5 clones N27-3 patients with hepatitis C serum-derived cDNA' 5 clones N27-3 Sm from Hind III site of plasmid pUC19 to side
cut out a I is obtained by adding the DNA fragments up site), Hin of pUCN19-1 dIII, Mlu
D containing the 3'side of clone N19-1 cut out by I
The plasmid pUCN27N19-1 could be obtained by exchanging the NA fragment, cloning and screening. This plasmid is clone N27-3 and N
19-1 carried clone N27N19-1 which was ligated without overlap.

The nucleotide sequence of clone N27N19-1 is shown in SEQ ID NO: 28 in the sequence listing. [3] Clone N27MX24A-1 and clone N27
Method for obtaining MX24B-1 Overlapping clone N27-3 and clone N1
In order to ligate 9MX24A-1 and clone N19MX24B-1 without overlapping, it was utilized that both have common and only one restriction enzyme cleavage site. That is, the restriction enzyme Mlu
Clone N27-3 is cleaved by I at the 3'side of A at the 330th position, and clone N19MX24A-1 and clone N19MX24B-1 are cleaved at the 3'side of A at the 71st position. In addition, plasmids pUCN27-3 and pUC
N19MX24A-1, pUCN19MX24B-1
Are both in consideration of the fact that the DNA fragment is in the same direction, Hin a pUCN27-3 dIII, Mlu
I, a DNA fragment of 363 bp in length (this DNA fragment is a clone N27-of cDNA derived from hepatitis C patient serum).
Is obtained by adding a DNA fragment from Hin dIII site of plasmid pUC19 to Sma I site is) cut out in 3 of 5 'side, pUCN19MX24A-1,
Hin of pUCN19MX24B-1 with a length of 363 bP
Clone N19MX excised with dIII and Mlu I
The plasmid pUCN27NMX24A-1 was obtained by exchanging 24A-1 and the DNA fragment containing clone MX24B-1, respectively, and cloning and screening.
And plasmid pUCN27NMX24B-1 could be obtained. This plasmid is clone N27-3, clone N19MX24A-1, clone N19MX24B
Clone N27MX24A-1 and clone N27MX24B-1 in which -1 was ligated without overlapping
Had. The nucleotide sequences of clone N27MX24A-1 and clone N27MX24B-1 are shown in SEQ ID NOs: 31 and 32 of the sequence listing.

Example 12 Clone N27MX24A-
1. Modification of DNA for expressing HCV-derived polypeptide encoded by clone N27MX24B-1 [1] Clone N27MX24A-1 and clone N27
Modification of DNA for expressing HCV-derived polypeptide encoded by MX24B-1 in E. coli Clone N27MX24A-1 and clone N27MX2
It is considered that 4B-1 encodes an open reading frame (ORF) derived from the HCV gene from the second C, and the start codon ATG was introduced at the 5 ′ upstream side of the gene in accordance with this frame and expressed. Let When the start codon ATG is introduced at the 5 ′ upstream side of the gene, a polypeptide other than the polypeptide encoded by the HCV gene is added to the N-terminus (amino terminal side) of the amino acid sequences set forth in SEQ ID NOs: 31 and 32 of the sequence listing. Peptides may be added. Further, when the clone is introduced into a vector having a start codon derived from an expression vector in Escherichia coli, the frame of the clone may be aligned so as to match the frame of the start codon derived from the vector. The PCR method was used to modify the vector-derived DNA. At this time, the following synthetic DNA was used as the primer. Also, the following is clone N2
An example is shown using 7MX24A-1, but it can also be carried out using clone N27MX24B-1 in exactly the same manner.

The base sequence for the 5'primer is MS2724-1; 5'GCAAGCTTATGCGGG.
ATCCCACAAGCCGTGGTGGGAT3 '(SEQ ID NO: 152). The base sequence for the 5'primer when the DNA fragment is introduced into the vector having the initiation codon derived from the expression vector is MS2724-2; 5'CGGATCCCCACAAGC.
CGTGGTGGGAT3 '((SEQ ID NO: 153). The base sequence for the 3'primer is MS2724-3; 5'GCGAATTCAGACTCT.
TCATCACTCTAAGGTGGCGTCGGCG
TGGG3 '(SEQ ID NO: 154). 20 pmol / μl of these two synthetic DNAs
Was prepared. This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCN was used as template DNA.
100 ng of 27MX24A-1 was used, and 2 μl of the solution prepared as described above was used as a primer for both 5 ′ and 3 ′ primers. First, prepare the prepared sample
After keeping the temperature at 0 ° C for 5 minutes, it was rapidly cooled to 0 ° C. 1 minute later, Taq
DNA polymerase (7 units / μl, AmpliT
0.5 μl of aqTM Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample is a DNA Thermal Cycle manufactured by Perkin Elmer Cetus.
It was treated with r at 25 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 5 minutes.

[0130] Phenol / chloroform treatment by a conventional method the samples were ethanol precipitated, this DNA sample Hin dIII, in the case of using MS2724-2 as cutting (5 'primer Eco RI, blunted with T4DNA polymerase Then, after digestion with Eco RI), a DNA fragment of a desired length was separated by acrylamide gel and extracted (Molecular Cloning 1982 Cold Spring Harbor (Cold Spring).
Harbor)). Further cloning vector pU
Using Hin dIII (MS2724-2 of C19
In this case, Sma I site) and Eco RI site were ligated by a conventional method, cloned, and screened to obtain plasmid pUCHN27MX24A-1 (M
When S2724-2 was used, plasmid pUCH2
N27MX24A-1 was obtained) and its nucleotide sequence was determined. Clone HN obtained by this method
In the nucleotide sequence of 27MX24A-1, the following DNA fragments are added to the 5'side of the DNA fragment obtained by removing the C at the 5'end of SEQ ID NOs: 31 and 32,

5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: 15
5) 5 'end has a Hin dIII site, owns followed by a start codon ATG. At the 3'end, the DNA fragment shown below is the A at the 3'end of SEQ ID NO: 31 in the sequence listing.
Is added to the 3'side of the DNA fragment from which the two have been removed.
(SEQ ID NO: 156) There is an Eco RI site from the 3'side , and B is located inside it.
It carries the gl I site followed by two stop codons TGA. Primers MS2724-2 and MS2724-
Clone H2N27MX24 created by the combination of 3
Nothing was added to the base sequence of A-1 at the 5'end, and the same base sequence as that of clone HN27MX24A-1 was added to the 3'end.

[2] An HCV-derived polypeptide consisting of 106 amino acid sequences from 109th to 214th of the amino acid sequences set forth in SEQ ID NOs: 31 and 32 of the Sequence Listing for expressing in Escherichia coli Modification of the DNA fragment encoding the: The HCV gene-derived open reading frame (ORF) in the polypeptide represented by 106 amino acids 109 to 214 of the amino acid sequences shown in SEQ ID NOs: 31 and 32 of the sequence listing. ) Is coded, the start codon ATG is introduced at the 5 ′ upstream side of the gene in accordance with this frame and expressed. When the start codon ATG is introduced at the 5 ′ upstream side of the gene, HCV is added to the N-terminus (amino terminal side) of the amino acid sequence of the polypeptide.
A polypeptide other than the gene-encoded polypeptide may be added. Further, when the clone is introduced into a vector having a start codon derived from an expression vector in Escherichia coli, the frame of the clone may be aligned so as to match the frame of the start codon derived from the vector. PCR was used to modify the DNA. At this time, the following synthetic DNA was used as the primer.

The base sequence for the 5'primer is MSHNS1-1; 5'GCAAGCTTATGTTC.
AACGCGTCCGGATGTCCGGA3 '(SEQ ID NO: 157). The base sequence for the 5'primer when the DNA fragment is introduced into the vector having the initiation codon derived from the expression vector is MSHNS1-2; 5'TTCAACGCGTCCGG
ATGTCCGGA 3 '(SEQ ID NO: 158) The base sequence for the 3'primer is MSHNS1-3; 5'GCGAATTCAGATCT
TCATCAACAACCGAACCAGTTTGCCC
TGCG3 '(SEQ ID NO: 159). 20 pmol / μl of these two synthetic DNAs
Was prepared. This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCN was used as template DNA.
27MX24A-1 (or plasmid pUCN2
7MX24B-1) was used in an amount of 100 ng, and the solution prepared as described above was used as a primer in an amount of 2 μl each for the 5 ′ and 3 ′ primers. First, prepare the prepared sample
After keeping the temperature at 0 ° C for 5 minutes, it was rapidly cooled to 0 ° C. 1 minute later, Taq
DNA polymerase (7 units / μl, AmpliT
0.5 μl of aqTM Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample is a DNA Thermal CyCle manufactured by Perkin Elmer Cetus.
It was treated with r at 25 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 5 minutes.

[0134] conventional manner phenol / chloroform treatment the samples were ethanol precipitated, cutting the DNA sample Hin dIII, with Eco RI (MSHNS1-
When 2 was used, after blunting with T4 DNA polymerase and cutting with Eco RI), a DNA fragment having a desired length was separated by acrylamide gel and extracted (Molecular cloning (Molecular cloning)).
Cloning) 1982 Cold Spring
Harbor (Cold Spring Harbo
r)). Furthermore, Hi of cloning vector pUC19
n dIII (when using MSHNS1-2, Sma
I site) and Eco RI site were ligated by a conventional method, cloned, and screened to obtain plasmid pUCH48 (MSHNS1-2 and MSHNS1).
-3, the plasmid pUCH4
8-2), the base sequence was determined. The base sequence of the clone H48 obtained by this method is DN shown below.
A fragment is added to the 5'side of the 326th T in the nucleotide sequence of the DNA fragment of SEQ ID NO: 31 or 32 in the sequence listing,

5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: 15
5) 5 'end has a Hin dIII site, owns followed by a start codon ATG. (The base sequence of clone H48-2 obtained using MSHNS1-2 is D
No NA fragment was added, but clone H48 on the 3'side.
Was the same as ) Also, the DN shown below is attached to the 3'end.
The A fragment is added to the 3'side of the 643th T end in the nucleotide sequence of SEQ ID NO: 31 or 32 in the sequence listing, and is most 3 '
It has an EcoRI site from the side, and has a BglI site inside it, followed by two stop codons TGA. 5'TGATGAAGATCTGAATTCGC 3'3'ACTACTTCTAGACTTAAGCG 5 '
(SEQ ID NO: 156)

[3] An HCV-derived polypeptide consisting of 92 amino acid sequences from the 233rd position to the 324th position among the amino acid sequences shown in SEQ ID NOs: 31 and 32 of the Sequence Listing for expressing in Escherichia coli Modification of the DNA fragment encoding the: The open reading frame (ORF) derived from the HCV gene is added to the polypeptide represented by 96 amino acids from the 233rd position to the 324th position among the amino acid sequences shown in SEQ ID NOs: 31 and 32 of the sequence listing. ) Is coded, and if the primers used have the sequences shown below, the 109th to 214th positions of the amino acid sequences shown in SEQ ID NOs: 31 and 32 of the sequence listing used in [2] are used. Up to the modification of the DNA fragment for expressing the polypeptide represented by 106 amino acids in E. coli. It was done in the operation. The clone obtained by this method was clone H49 (primers MSNS1-4 and MSNS1-
6) and clone H49-2 (primer MSNS1-5)
And MSNS1-6).

The base sequence for the 5'primer is MSNS1-4; 5'GCAAGCTTATGATCG
GGGGGGGTCGGCAACAATAC3 '(SEQ ID NO: 160). The base sequence for the 5'primer when the DNA fragment is introduced into the vector having the initiation codon derived from the expression vector is MSNS1-5; 5'ATCGGGGGGGTCCGGC.
AACAATAC 3 '(SEQ ID NO: 161) The base sequence for the 3'primer is MSNS1-6; 5'GCGAATTCAGATCTT
CATCAAAGCTCTGATCTATCCCTGT
CCT 3 '(SEQ ID NO: 162). 20 pmol / μl of these two synthetic DNAs
Was prepared.

[4] Modification of DNA for expressing HCV-derived polypeptide encoded by clone N27MX24A-1, clone N27MX24B-1, clone H48-2 and clone H49-2 in insect cells Clone N27MX24A-1 And clone N27MX2
It is considered that 4B-1 encodes the open reading frame (ORF) derived from the second C to HCV gene. Also, clone H48-2 and clone H49
In -2, the ORF starts from the first 5'end. The initiation codon ATG is introduced upstream of the 5'side of the gene in accordance with this frame and expressed. When the start codon ATG was introduced 5'to the upstream side of the gene, clone N27M
X24A-1, clone N27MX24B-1, clone H48-2 and clone H49-2 with a polypeptide other than the polypeptide encoded by the HCV gene added to the N-terminus (amino terminal side) of the amino acid sequence encoded by the clone H49-2 Is also good. Moreover, when the clone is introduced into a vector having a start codon derived from an expression vector in insect cells, the frame of the clone may be aligned with the frame of the start codon derived from the vector. Also in this case, clone N27MX24A-1, clone N27MX24
A polypeptide other than the polypeptide encoded by the HCV gene may be added to the N-terminus (amino terminal side) of the amino acid sequences encoded by B-1, clone H48-2 and clone H49-2. In addition, vector-derived DNA
The PCR method was used for modification. The following is clone N27MX
Examples are shown using 24A-1, but the same procedure can be performed using clone N27MX24B-1.

Clone N27MX24A in insect cells
-1, and the polypeptide encoded by the clone N27MX24B-1 are linked and expressed intracellularly as a precursor, and then processed in the cell to be a mature protein, which is at least partially modified with a sugar chain, and which is a mature protein. It was expressed as a 70 K dalton glycoprotein and accumulated intracellularly. Clone N2 for constructing expression vector
7MX24A-1, clone N27MX24B-1, H
The PCR method was used to modify the DNAs of 48-2 and H49-2. At this time, the synthetic D shown below was used as the primer.
NA was used. The base sequence for the 5'primer is MS2724-4; 5'GCGTCGACGCCATAGC
ATGCGGATCCCCACAAGCCGTGGTGGG
AT 3 '(SEQ ID NO: 163) MSNS1-7; 5'GCGTCGGACGCTAGCA
TGTTCAACGCGTCCGGATGTCCGGA
3 '(SEQ ID NO: 164) MSNS1-8; 5'GCGTCGGACGCTAGCA
TGATCGGGGGGGGTCGGCAACAAATAC
3 '(SEQ ID NO: 165).

The base sequence for the 3'primer is MS2724-5; 5'GCGAATTCGCTAGC.
TCACTCTAAGGTGGCGTCCGGCGTGG
G 3 '(SEQ ID NO: 166) MSNS1-9; 5'GCGAATTCGCTAGCT
CAACAACCGAACCAGTTTGCCCTGCG
3 '(SEQ ID NO: 167) MSNS1-10; 5'GCGAATTCGCTAGC
TCAAAGCTCTGATCTATCCCCTGTCC
T3 '(SEQ ID NO: 168). 20 pmol / μl of these three synthetic DNAs
Was prepared. Preparation of PCR reaction solution and post-treatment [1]
As well as done. However, the plasmid pUCHN27MX24A-1 (primer MS
2724-4 and MS2724-5 combination),
Plasmid pUCH48 (using the combination of primers MSNS1-7 and MSNS1-9) or plasmid pUCH49 (primers MSNS1-8 and MSNS)
1-10 combinations were used). The cycle of PCR is 95 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 5 minutes,
After repeating this 10 times, 95 ° C for 1 minute, 65 ° C for 1 minute, 7
2 minutes 5 minutes was repeated 20 times. The plasmid pUCHN27MX24A-1 was used as the template DNA, and the target length 1 was set using the primers MS2724-4 and MS2724-5.
A 268 bp DNA fragment was obtained. Also, template D
Using the plasmid pUCH48 for NA, primer MS
A target DNA fragment having a length of 352 bp was obtained with NS1-7 and MSNS1-9. Plasmid p for template DNA
Using UCH49, primers MSNS1-8 and MSN
A target DNA fragment of 322 bp in length was obtained in S1-10.

After cutting these DNA samples with Nhe I,
DNA fragments of the desired length were separated on acrylamide gel and extracted (Molecular Cloning (Mol
Equal Cloning) 1982 Cold ・
Spring Harbor (Cold Spring Ha)
rbor)). Furthermore, the transfer vector pBl
The DNA was ligated and cloned at a NheI site of ueBac (Invitrogen) by a conventional method.
A clone in which only one fragment was inserted at the NheI site was screened and the primer MS2724-
No. 4 and MS2724-5, the D of the desired length of 1268 bp
Plasmid pBlueN27MX24A derived from NA fragment
-1, a plasmid pBlueH48 derived from the DNA fragment having a target length of 352 bp with primers MSNS1-7 and MSNS1-9, and primers MSNS1-8 and MS.
A plasmid pBlueH49 derived from the DNA fragment having a desired length of 322 bp was obtained by NS1-10. The expression unit of these plasmids has a structure in which a DNA fragment derived from the HCV gene is ligated in the forward direction at the cloning site Nhe I cleavage site downstream of the polyhedrin promoter, referring to the one shown in the protocol of Invitrogen. Was there.

Example 13 Clone HN27MX24A
-1, clone HN27MX24B-1, clone H2
N27MX24A-1, clone H2N27MX24B
-1, clone H48, clone H48-2, clone H49, expression of HCV-derived polypeptide encoded by clone H49-2 [1] clone HN27MX24A-1, clone HN
27MX24B-1, clone H48, clone H49
Expression of the polypeptide encoded by E. coli in Escherichia coli The polypeptide encoded by the clone is a part of the polypeptide encoded by cDNA derived from hepatitis C patient serum, which was directly expressed in E. coli. This is expressed as pCZ44 as an expression vector as shown below.
(JP-A-1-124387) was used, and the cDNA of the clone was subcloned into pCZ44. First of all, the clone is Hin dIII,
Are sufficiently cut by Bgl II, treated with phenol / chloroform, ethanol precipitated, both ends respectively Hin
dIII, the became cohesive ends of Bgl II site cD
A DNA fragment having NA was separated by acrylamide gel and extracted (Molecular Cloning (Mol
Equal Cloning) 1982 Cold ・
Spring Harbor (Cold Spring Ha)
rbor)). Similarly, the expression vector pCZ44 is also Hi.
n dIII, are sufficiently cut by Bgl II, a longer DNA fragment with an expression vector ability similarly prepared above, this prepared from the clones were Hin dIII,
It was ligated so as to have one BglII fragment, and further cloned according to a conventional method. The obtained plasmids were clone HN27MX24A-1, clone HN, respectively.
27MX24B-1, clone H48, clone H49
Derived plasmids pCZ2724A-1, pCZ272
It was named 4B-1, pCZ48, pCZ49.

As another attempt, an expression vector pGEX-2T capable of expressing a fusion protein instead of pCZ44
(Manufactured by Pharmacia) was used to construct an expression vector. In this case, the operation was basically performed according to the instructions of Pharmacia. First, the expression vector pGEX-2T
After cutting with Bam HI, a Hin dIII linker was inserted so that the amino acid and codon reading frames of the clone would match, and the clone was inserted into the EcoRI site on the 3'side and the Hin dIII site on the 5'side (made by the linker). Hin dIII which is prepared from the DNA fragment was inserted Eco RI fragment. Thus, the expression vector pGEX2724A-
1, pGEX2724B-1, pGEX48, pGEX
49 was obtained.

Plasmids pCZ2724A-1, pCZ
The E. coli JM109 strain carrying 2724B-1, pCZ48 and pCZ49 was cloned into L-Broth (Molecular Cloning).
g) 1982 Cold Spring Harbor (Co
ld Spring Harbor)), the cells were cultured overnight at 37 ° C. Use a new L-
Inoculate Broth, shake culture at 30 ° C. for 2 hours,
IPTG (isopropyl-β-D-galactopyranoside) was added to the medium to 2 mM, and the mixture was further cultured for 3 hours or more. By culturing the bacterium in the presence of IPTG in this manner, a recombinant derived from a single clone (in this case, Escherichia coli JM109 strain transformed with only the plasmid) is used, using the plasmid derived from the clone. And was induced to express an HCV-derived polypeptide. Clone HN27MX24A-1, clone H
The nucleotide sequence of N27MX24B-1 cDNA and the deduced amino acid sequence of the polypeptide encoded by the sequence are shown in SEQ ID NO: 31 of the sequence listing. The nucleotide sequences of the cDNAs of clones H48 and H49 and the deduced amino acid sequences of the polypeptides encoded by the sequences are the amino acid sequences 109 to 214 of the amino acid sequence set forth in SEQ ID NO: 31 of the Sequence Listing, respectively. And 233 to 32
It is the amino acid sequence up to the fourth position.

Similarly, clone HN27MX24 was prepared.
When clone HN27MX24B-1 is used instead of A-1, the polypeptide encoded by the clone is obtained. The deduced amino acid sequence of the polypeptide is shown in SEQ ID NO: 32 in the sequence listing. Also, the plasmid pGEX2724
A-1, pGEX2724B-1, pGEX48, pG
Similarly, EX49 can be obtained as an expression product as a fusion protein with β-glutathione-S-transferase (GST). In this case, the GST C-terminal has a site that is specifically cleaved with thrombin as described in the manual of Pharmacia, and clone HN27MX24A-1 and clone HN27MX24 are further present at the C-terminal side.
Polypeptides derived from B-1, clone H48 and clone H49 are linked. To obtain the fusion protein, pCZ2724A-1, pCZ2724B-1,
IPTG as in the above protocol performed when the HCV-derived polypeptide is expressed from pCZ48, pCZ49
In the presence of pGEX2724A-1, pGE
Escherichia coli transformed with X2724B-1, pGEX48 and pGEX49 was cultured and induced to express the fusion protein having a HCV-derived protein at the C terminus.

[2] Clone H2N27MX24A-
1, clone H2N27MX24B-1, clone H4
8-2, Expression of polypeptide encoded by clone H49-2 Clone H2N27MX24A-1, clone H2N2
7MX24B-1, clone H48-2, clone H4
From the cDNA derived from hepatitis C patient serum contained in 9-2,
The polypeptide was directly expressed in E. coli. The expression method was similar to that described in [1]. However, as a material for HCV cDNA, clone H is used.
2N27MX24A-1, clone H2N27MX24
It is a cDNA derived from the serum of a hepatitis C patient contained in B-1, clone H48-2, and clone H49-2, and this clone was isolated and the nucleotide sequence was determined. The expression plasmid used was pOFA (JP-A-2-841).
No. 95 public information) was used. First, the DNA fragment of each of the above clones was blunt-ended with T4 DNA polymerase. After the expression plasmid pOFA was treated with Kpn I, and that both ends were blunted with T4DNA polymerase, blunt-ended the clones, ligated and cloned by a conventional method, the DNA fragment is inserted only one The clones were screened. That is, the plasmid pOFA2724A was obtained by subcloning each cDNA derived from HCV so that the + chain expressing the HCV-derived protein is correctly inserted in the direction of translation.
-1, pOFA2724B-1, pOFA48, pOF
Obtained as A49. Whether or not the HCV-derived cDNA in the plasmid was reconstructed correctly was confirmed by nucleotide sequencing and creation of a restriction enzyme cleavage map.

By culturing the bacterium in the presence of IPTG, a recombinant derived from a single clone (for example, Escherichia coli JM109 strain transformed with the plasmid) expresses the HCV gene-derived polypeptide encoded by the clone. Was invited to do. Clone H2N27MX24
The nucleotide sequences of A-1, clone H2N27MX24B-1, clone H48-2, and clone H49-2 derived from the sera of hepatitis C patient sera and the deduced amino acid sequences of the polypeptides encoded by the sequences are SEQ ID NOs in the sequence listing, respectively. The amino acid sequence of 31 or 32 and the amino acid sequence of 109th to 214th and the amino acid sequence of 233rd to 324th of the amino acid sequence of SEQ ID NO: 31. [3] Clone N27MX24A-1, Clone N27
MX24B-1, clone H48-2 and clone H49
Expression of HCV-Derived Polypeptide Encoded by C-2 in Insect Cells Plasmid pBlueN2 constructed in Example 12 [4]
In order to express the HCV-derived glycoprotein encoded by 7MX24A-1, plasmid pBlueH48 and plasmid pBlueH49 in insect cells, Maxbac (MAXBACTM BACULOVI) which is a baculovirus expression manual of Invitrogen.
RUS EXPRESSION SYSTEM MAN
UALVERSION 1.4, hereafter referred to as Maxback. ) Was basically referred to.

The plasmid prepared in Example 12 [4] and having the DNA fragment containing the HCV gene inserted into the restriction enzyme Nhe I cleavage site of the transfer vector pBlueBac (Maxback page 37) was inserted into Mania.
The method of tis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 86-
HCV gene transfer plasmid DN, which was recovered and purified from recombinant Escherichia coli according to page 96 (1982).
Obtained a large amount of A. 1 μg of AcNPV viral DNA in the kit according to the method on page 27
And 2 μg of the plasmid containing the DNA fragment derived from the HCV gene were mixed and co-infected with Sf9 cells to transform Sf9 cells. That is, first, TMN-F containing 10% FCS (fetal bovine serum) in a petri dish having a diameter of 6 cm.
2x Sf9 cells in H medium (Invitrogen)
The cells were cultured to 10 ⁶ / sheet. Next from the petri dish TM
The N-FH medium was removed and 0.75 ml of FCS was added thereto.
% Grace medium (GIBCO (GIB
CO) product) was added. 0.75 ml of the transfection buffer included in the kit is added to the mixed DNA described above.
Add well by vortexing, and then mix with Grace.
e) The DNA solution was slowly added dropwise over the medium. 27
The mixture was left to stand still at 4 ° C. for 4 hours, then the Grace medium was removed and 3 ml of TMN-FH medium containing 10% FCS was placed in a petri dish and cultured at 27 ° C. for 6 days. On day 3, polyhedra were observed in some cells, and on day 6, most cells formed polyhedra. Transfer this culture supernatant to a centrifuge tube,
The supernatant obtained by centrifugation at 1,000 rpm for 10 minutes was used as a co-infection virus solution.

This co-infected virus solution contained 10 ⁸ viruses per ml, and about 0.5% of them was recombinant. The plaque isolation method was used to isolate the recombinant virus. The method is as follows. The co-infected virus solution was diluted to 10 ⁻⁴ , 10 ⁻⁵ . 1.5 x 10 ⁶ per piece in 6 cm Petri dish in advance
After culturing individual cells and adsorbing them, the medium was completely removed, and 100 μl of each of 10 ⁻⁴ solution and 10 ⁻⁵ solution was added to each petri dish. In order to prevent the cells from drying, the petri dish was tilted every 15 minutes so that the virus solution was spread over the entire surface. As described above, 105% of baculovirus agarose (manufactured by Invitrogen) was used while infecting at room temperature for 1 hour.
Autoclave at 10 ° C for 10 minutes, mix with TMN-FH medium containing 10% FCS warmed at 46 ° C at a ratio of 1 to 3, and keep warm at 46 ° C. To this incubated medium, 5-bromo-4-chloro-3-indolyl-β-D-galactoside was added to a final concentration of 150 μg / μl (according to Maxback's pages 16-17). The medium was called agarose-containing x-gal medium.

After the infection was completed, the virus solution was completely sucked up, and the agarose-containing x-gal medium which had been kept warm was gently added in an amount of 4 ml per dish so that the cells would not come off. The petri dish was allowed to stand with the lid slightly displaced until the agarose was solidified and dried, and then, the lid was put on and turned over, and incubation was carried out at 27 ° C. After culturing for 6 days, blue plaques in which polyhedra were not formed were found by a phase contrast microscope. This recombinant plaque is sucked up with agarose using a Pasteur pipette and 1 ml of TMN-
The virus was suspended by pipetting many times in FH medium. This series of operations (an operation of infecting and then culturing for 6 days to isolate a virus having the transfer plasmid DNA) is called a plaque method. The same plaque method was performed using 100 μl of the virus suspension. These series of operations are repeated three times, and HCV free from contamination by wild strains.
A recombinant virus carrying the derived glycoprotein gene was obtained.
Absorb the virus plaque with a Pasteur pipette,
It was mixed well with 1 ml of TMN-FH medium. This primary recombinant virus solution has a low virus concentration, so
After taking 00 μl and adsorbing to Sf9 cells cultured in a 6 cm dish at half confluence, T containing 10% FCS
4 ml of MN-FH medium was added, and after 3 days, the culture supernatant was collected and used as a recombinant virus solution for infection. In order to produce HCV-derived glycoprotein, 5 × 10 ⁶ S was previously prepared.
10 m of TMN-FH medium containing f9 cells and 10% FCS
It was suspended in 1 and sprinkled on a 9 cm Petri dish and adsorbed for 1 hour. Remove the medium from the dish and add 250 μl of the recombinant virus solution for infection to spread it over the whole area, then 10%
10 ml of TMN-FH medium containing FCS was added, and the mixture was allowed to stand at 27 ° C for 4 days. The cells were thoroughly suspended in 1,000 ml of phosphate buffered saline. Thus, the HCV-derived glycoprotein was expressed in Sf9 cells infected with the virus.

Example 14 Identification of Expressed Product as HCV Derived Antigen Human serum infected with hepatitis C virus
The polypeptide was identified as a hepatitis C-related antigen because it immunologically reacts with the polypeptide expressed in E. coli. This identification was performed by the Western blotting method shown below. First, Example 13 [1], [2],
Clone HN27MX24A-1, clone HN27MX24B-1, clone H2N27M described in [3]
X24A-1, clone H2N27MX24B-1, clone H48, clone H48-2, clone H49,
Plasmids pCZ2724A-1 and pCZ2724 for expressing each polypeptide encoded by clone H49-2
B-1, pCZ48, pCZ49, plasmid pOFA
2724A-1, pOFA2724B-1, pOFA4
8, pOFA49, pGEX2724A-1, pGEX
2724B-1, pGEX48, pGEX49, pBl
ueN27MX24A-1, plasmid pBlueH4
Escherichia coli transformed with 8 and the plasmid pBlueH49 was induced to express the polypeptide by culturing in the presence of IPTG as in Example 13 for 3 hours or overnight.

1,000 μl of this culture solution was added to 6,500
r. p. m. The recombinant strains were harvested by centrifugation for 10 minutes. This precipitate was treated with a sample treatment solution for SDS-polyacrylamide gel electrophoresis (2% SDS, 5%
Mercaptoethanol, 10% glycerin and 0.00
It was prepared by dissolving it in 50 mM Tris-HCl buffer containing 5% bromophenol blue, pH 6.8) so that the final volume was 0.2 ml. In addition, the infected cells (having a virus that has undergone the plaque method three times or more) obtained in Example 13 [3] are scratched, and physiological saline phosphate buffer 1,
Suspend well in 000ml, of which 100μ
1, 6,500 r. p. m. A precipitate of the cells was obtained by centrifugation at 10 minutes. This precipitate was dissolved and prepared in a sample treatment solution for SDS-polyacrylamide gel electrophoresis so that the final amount was 0.2 ml. Next, these samples were boiled at 100 ° C. for 10 minutes. 10 μl of the sample thus obtained was added to 0.1% SDS-15.
Loaded on 0% polyacrylamide gel (70 × 85 × 1 mm). At that time, as a marker protein, Phar
"LMW KitE" (low molecular weight marker protein) manufactured by macia was used. A Tris buffer solution (25 mM Tris pH 8.3, 192 mM glycine, 0.1% SDS) was used as an electrode solution, and a constant current of 30 mA was applied to generate 45
After electrophoresis for minutes, the gel was overlaid with a S & S nitrocellulose filter (BA-83) according to a conventional method, and the gel side was used as a cathode and the nitrocellulose filter side was used as an anode.
A constant current of 20 mA was applied for about 20 minutes for transfer.

The transferred nitrocellulose filter was cut out so as to separate the marker protein portion and the sample portion, and the marker protein portion filter was stained with 0.1% (W / V) Amido Black 10B. Of the filter of 0.01M phosphate saline (PB containing 5% (W / V) bovine serum albumin)
S) Soaked in pH 74. Next, 0.01M phosphate saline (PB containing 5% (W / V) bovine serum albumin)
S) 10 ml of hepatitis C patient serum diluted 50 times with pH 7.4 is placed on the filter of the sample portion, and it is kept at room temperature for 2 hours.
After standing for 0.1 hours (V / V) Tween manufactured by Sigma
The cells were washed with PBS containing n20 three times for 20 minutes each. Next, a peroxidase-labeled anti-human IgG (Ga) diluted 1,000 times with PBS was placed on the sample part filter.
10 ml of the product (manufactured by Ppel Co.) was put on the plate, reacted at 37 ° C. for 1 hour, and washed with PBS containing 0.1% (V / V) Tween 20 three times for 20 minutes each. Next, 100 ml of peroxidase color developing solution (60 mg 4-chloro-1-naphthol, 20 ml methanol, PBS8) of the sample part filter.
It was immersed in 0 ml of hydrogen peroxide (20 μl). The colored filter was washed with distilled water and then compared with the marker protein portion.

The protein developed in this manner was obtained by the plasmid p
CZ2724A-1, pCZ2724B-1, pCZ4
8, any of which was expressed by pCZ49, had a reasonable molecular weight as the introduced HCV-derived gene product and could be identified as a hepatitis C-related antigen. Plasmid p
OFA2724A-1, pOFA2724B-1, pO
Expression products derived from plasmid pOFA, such as FA48 and pOFA49, are obtained by adding an E. coli OmpF signal peptide to the N-terminus of an HCV-derived polypeptide. Plasmids pGEX2724A-1, pGEX27
Expression products derived from the plasmid pGEX, such as 24B-1, pGEX48, and pGEX49, are those in which a cleavage site for GST and thrombin is added to the N-terminus of the HCV-derived polypeptide. Both of them had a reasonable molecular weight as a gene product of the clone and could be identified as hepatitis C-related antigens. In addition, clone N27MX24A-1, clone N2 expressed in insect cells shown in Example 13 [3]
7MX24B-1, clone H48-2 and clone H4
The HCV-derived polypeptide encoded by clone 9-2 was obtained as a glycoprotein of about 70 kDa in cells infected with the plasmids pBlueN27MX24A-1 and pBlueN27MX24B-1. The amino acid sequence of M-gp70 is from the 46th position to the 395th position set forth in SEQ ID NO: 31 or 32 of the Sequence Listing, respectively.
Corresponds to the first array.

[0156] In addition, in cells infected with plasmids pBlueH48 and pBlueH49, HCV
The derived glycoprotein was expressed. This glycoprotein is a polypeptide represented by 106 amino acids from the 109th position to the 214th position of the amino acid sequences shown in SEQ ID NOs: 31 and 32 of the sequence listing encoded by clone H48-2 and clone H49-2. 233rd to 3rd
It is a polypeptide represented by 96 amino acids up to the 24th position and could be identified as a hepatitis C-related antigen.

Example 15 Synthesis of cDNA [1] Preparation of RNA Sample After drying the nucleic acid obtained in Example 1, 30 μl of water and ribonuclease inhibitor (100 units / μ)
l, manufactured by Takara Shuzo) was added and dissolved. The cDNA synthesis shown below was performed using this nucleic acid aqueous solution. [2] Synthesis of cDNA Using Antisense Primer Antisense primer (synthetic DNA primer MS158, MS15) was added to 2 μl of the aqueous nucleic acid solution prepared in [1].
2, MS132, MS49, MS88, MS100, etc. are antisense primers. 15 pmols /
μl) 1 μl, 10 × RT buffer (100 mM Tri
s-HCl (pH 8.3), 500 mM KCl) 2μ
1, 25 mM MgCl2 4 μl, 2.5 mM 4d
After adding 8 μl of NTP and 1 μl of water and incubating at 65 ° C. for 5 minutes and then at room temperature for 5 minutes, reverse transcriptase (Life Science) 25
1 μl unit, ribonuclease inhibitor (1
00 unit / μl, Takara Shuzo Co., Ltd.) 1 μl added, 37 ° C
Incubate for 20 minutes, then 42 ° C for 30 minutes, and finally 95 ° C for 2 minutes,
It was immediately cooled to 0 ° C. (complementary strand DNA synthesis). This DN
Method of Saiki et al. Using 10 μl of A sample [Nature, 324, 126, (1986)]
D having a specific sequence by the so-called PCR method according to
NA was amplified.

That is, 10 μl of this DNA sample, 10 × P
CR buffer (100 mM Tris-HCl (pH 8.
3), 500 mM KCl, 15 mM MgCl2, 1
% Gelatin) 10 μl, 2.5 mM 4dNTP 8 μ
l, 2 μl of synthetic DNA primer (150 pmols / μl) used for complementary strand DNA synthesis, synthetic DNA primer corresponding to this primer (15 Pmols / μl)
l, which composes a pair with the synthetic DNA primer used in the synthesis of complementary strand DNA,
5, MS152, MS151-MS158, MS135
-MS132, MS48-MS49, MS97-MS8
8, a combination of MS86-MS100 primers was used. ) Water was added to 3 μl so that the total amount became 100 μl. First, the mixture was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was used as DNA Th manufactured by Perkin Elmer Cetus.
95 ° C for 1 minute, 40-55 ° C with the Ermal Cycler
It was treated 25 times at 72 ° C. for 1 to 5 minutes for 1 minute. Finally 72 ℃
After incubating at room temperature for 7 minutes, the reaction aqueous solution is treated with phenol / chloroform and precipitated with ethanol.
To the aqueous phase, 1/10 volume of 3M sodium acetate or an equal volume of 4M ammonium acetate and 2.5 volumes of ethanol of the aqueous phase were added and mixed, and the mixture was mixed with a rotor having a radius of about 5 cm.
The solution was subjected to cooling centrifugation at 5,000 rpm at 4 ° C. for 15 minutes to dry the precipitate), and amplified DNA fragments derived from various combinations of primers were obtained.

Example 16 Cloning of Amplified DNA Fragment and Determination of Nucleotide Sequence At least 1 pmole of the DNA fragment obtained by the method of Example 15 [2] was prepared, and this DNA was transformed into T4DNA.
Blunt-end with polymerase (Toyobo Co., Ltd.) (Molecular Clon
ing) 1982 Cold Spring Harbor and a polynucleotide kinase (manufactured by Toyobo Co., Ltd.) after introducing a phosphate group at the 5'end (Molecular Cloning (Molecular Cloning)).
(Clar Cloning) 1982 Cold Spring Harbor (Ccld Spring Har)
bor)), using a ligation kit (Takara Shuzo Co., Ltd.), incorporated into the Sma I site within the multiple cloning site of pUC19 cloning vector.
(At this time, 5 ng to 10 ng of the vector DNA used for ligation was prepared as follows. That is, the pUC19 cloning vector was used as a restriction enzyme Sma.
After cutting with I (manufactured by Toyobo Co., Ltd.), phenol / chloroform treatment and ethanol precipitation, the 5'end was further dephosphorylated with alkaline phosphatase (manufactured by Boehringer Mannheim) (Molecular Cloning (M
1982. Cold Spring Harbor (Cold Spring)
Harbor)), phenol / chloroform treatment, ethanol precipitation). DNA prepared in this way
Was used to transform Escherichia coli JM109 or DH5 (at this time, a competent cell manufactured by Toyobo Co., Ltd. was used). The method of transformation is the COMPETENT HIG of Toyobo Co., Ltd.
The protocol of H) was followed.

Thus, at least 20 or more transformants were transformed with the pUC19 cloning vector having the DNA fragment obtained by the method of [2] of Example 15 from each combination of primers. I was able to get it. A plasmid DNA was prepared from the obtained transformant by a conventional method, and using a fluorescence sequencer GENESIS 2000 system manufactured by DuPont,
The sequence was determined. The following 2 as a sequence primer
Seed synthetic primer 5'd (GTAAAACGACGG
CCAGT) 3 '(SEQ ID NO: 143), 5'd (CA
GGAAACAGCTATGAC) 3 '(SEQ ID NO: 1)
44) was used to determine the nucleotide sequences of the + strand and single strand of the DNA fragment. The respective nucleotide sequences are shown in SEQ ID NOs: 37 to 39, 44 to 55, 103 and 104 in the sequence listing.
The obtained clones organized by region are the nucleotide sequences shown in SEQ ID NOs: 33 to 36 in the sequence listing, and the obtained plural types of clones are shown together. For example, the sequence shown in SEQ ID NO: 33 in the sequence listing is clone MX25-1,
It is a group of three clones, clone MX25-2 and clone MX25-3. Hereinafter, similarly, the sequences of the respective clones represented by SEQ ID NOS: 34 to 36 in the sequence listing are the sequences represented by SEQ ID NOS: 47 to 55 in the sequence listing,
It is shown collectively for each clone obtained.

The nucleotide sequences shown in SEQ ID NOs: 33 to 39, 44 and 55 of the Sequence Listing are + of the HCV-derived gene incorporated in the plasmid of the transformant obtained above.
The base sequence of the chain is shown. The clone is a double-stranded DNA
And, for example, clone MX25-1, clone MX
25-2, the plasmid used to determine the nucleotide sequence of clone MX25-3 was pUCMX25, respectively.
-1, pUCMX25-2, pUCMX25-3. Each of these plasmids had one molecule of the corresponding DNA fragment. Below, plasmid D, which has one of the clones and was used to determine the nucleotide sequence
NA is indicated by pUC followed by the clone name. These sequences represent the nucleotide sequences of clones cloned from cDNA prepared from RNA derived from sera of hepatitis C patients, and nothing can be obtained from sera of healthy individuals by this method. Alternatively, Example 15
3 times or 4 times the cycle of PCR method shown in [2] and [3]
If the DNA fragment obtained by doubling (probably 60 to 100 times) amplification is obtained, its DNA fragment
Only the sequence of the A fragment having no homology with the base sequences shown in SEQ ID NOs: 33 to 43 of the sequence listing corresponding to the above-mentioned four combinations was obtained. That is, it was found that the nucleotide sequences of the clones shown in SEQ ID NOs: 33 to 43 of the sequence listing obtained here are specific to those derived from the serum of hepatitis C patients.

Therefore, the nucleotide sequence of the obtained DNA fragment was compared with the previously reported gene sequence of hepatitis C virus. In Example 15 [2], for example, MS155 and MS152 were used as primers, three clones were obtained from the serum of the same patient, and their nucleotide sequences were determined.
-3 indicates that the patient has a hepatitis C patient serum-derived nucleotide sequence obtained from one patient serum, that is, one patient has a plurality of viruses. Example 17 Clone MX25O26A-1 and Clone MX25O26B-1, Clone N16N15-1, Clone N16N15A-1 and Clone N16N15B-
1. Clone U16N15A-1 and clone U16N1
5B-1, clone N23N15A-1 and clone N2
Method for obtaining 3N15B-1, clone MX25N15A-1 and clone MX25N15B-1

[1] Method for Obtaining Clone MX25O26A-1 and Clone MX25O26B-1 The DNA fragments from clone MX25-1 and clone O26-1 obtained in Example 16 were each added in an amount of 1 μl (about 0.5 to 1 μg / μl) each in 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 50
10 μl of 0 mM KCl, 15 mM MgCl ₂ , 1% gelatin), 8 μl of 2.5 mM 4dNTP, synthetic primers MS155 and MS158 (both 20 pmo)
1 / μl) was added to each reaction solution of 5 μl and 76.5 μl of water. After mixing well, first incubate at 95 ℃ for 5 minutes,
Quenched to 0 ° C. 1 minute later, Taq DNA polymerase (7 units / μl, AmpliTaqTM Takara Shuzo)
0.5 μl was added and mixed, and then overlaid with mineral oil.
This sample was subjected to DNA Thermal Cycler manufactured by Perkin Elmer Cetus at 95 ° C. for 1 minute,
It was treated 25 times at 37 ° C. for 1 minute and 72 ° C. for 2 minutes. Next 97 ° C
After being kept warm for 2 minutes, it was rapidly cooled to 0 ° C. and kept warm for 2 minutes. Further, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaqTM Takara Shuzo) was added to the reaction aqueous solution, and similarly, 95 ° C. for 1 minute, 50 to 55 ° C. for 1 minute, 72 ° C. 2
Treated 25 times in minutes. Finally, after incubating at 72 ℃ for 7 minutes,
This reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. This amplified DNA sample
Separated by agarose electrophoresis (Molecular
Cloning (Molecular Cloning)
1982 Cold Spring Harbor (Cold
(Spring Harbour)), a DNA fragment having the desired length was excised from the gel (Molecular Cloning 1).
982 Cold Spring Harbor (Cold
Spring Harbor)).

The obtained DNA fragment was treated in the same manner as in Example 16 and incorporated into the Sma I site in the multicloning site of the pUC19 cloning vector.
This is cloned and screened (this method
The same as Example 16. ) Plasmid pUCMX25
O26A-1 and pUCMX25O26B-1 were obtained, and the cDNAs derived from the sera of hepatitis C patients thus obtained were clone MX25O26A-1 and clone MX25O26B-1.
I named it. The nucleotide sequences are shown together in SEQ ID NO: 40 in the sequence listing. The nucleotide sequences and amino acid sequences of clone MX25O26A-1 and clone MX25O26B-1 are shown in SEQ ID NOs: 56 and 57 of the Sequence Listing, respectively. That is, clone MX25O26A-1 is clone MX2
It shows that the overlapping region of 5-1 and clone O26-1 is a sequence derived from clone MX25-1,
In the clone MX25O26B-1, the region is clone O.
It is shown that the sequence is derived from 26-1. Similarly, clone N16N15A-1 and clone N16N15
B-1, clone U16N15A-1 and clone U16
N15B-1 was obtained. In this case, the clone used was N16 of SEQ ID NO: 36 or U1 of SEQ ID NO: 37 in the sequence listing.
6-4 and clone N15-1 of SEQ ID NO: 39 in the sequence listing, the nucleotide sequences of the ligated DNA fragments are collectively shown in SEQ ID NO: 41 in the sequence listing. Clone N16N15A
The nucleotide sequences and amino acid sequences of No. 1 and clone N16N15B-1 are shown in SEQ ID NOs: 58 and 59 of the sequence listing, respectively.
Shown in.

[2] Method for obtaining clone N16N15-1 Overlapping clone N16-1 and clone N1
In order to ligate with 5-1 without overlapping, it was utilized that the restriction enzyme cleavage sites that are common to both of them and have only one are at the overlapping positions. That is, the clone N16-1 was cleaved by the restriction enzyme BSt EII at the 3'side of G at the 576th position of SEQ ID NO: 36 in the sequence listing,
The clone N15-1 is cleaved at the 3'of G at the 114th position in SEQ ID NO: 39 in the sequence listing. Also, the plasmid pUCN
16-1 and pUCN15-1 both take pUCN16- in consideration of the fact that the DNA fragments are in the same orientation.
1 with Hin dIII, BSt EII, length 609 bp
DNA fragment (the DNA fragment is obtained by adding a DNA fragment from Hin dIII site of plasmid pUC19 to Sma I sites 5 'to the cloning N16-1 patients with hepatitis C serum-derived cDNA) cut out of,
The length of the pUCN15-1 Hin of 147bp dIII,
The DNA fragment containing clone N15-1 cut out with Bst EII was replaced with each other, cloned and screened to obtain plasmid pUCN16N15.
-1 was obtained. This plasmid is clone N
16-1 and clone N15-1 harbored clone N16N15-1 which was ligated without overlapping. The nucleotide sequences obtained in the same manner as for clone N16N15-1 are collectively shown in SEQ ID NO: 41 in the sequence listing. The nucleotide sequence and amino acid sequence of clone N16N15-1 are
It is shown in SEQ ID NO: 60 in the sequence listing.

[3] Method for Obtaining Clone N23N15A-1 and Clone N23N15B-1 Clone N23-1 obtained in Example 16 and Example 17
Clone N16N15A-1, N obtained in [1] and [2]
16N15B-1, and the DN represented by N16N15-1
1 μl of each A fragment (about 0.5 to 1 μg / μ
l) each in 10x PCR buffer (100 mM Tris
-HCl (pH 8.3), 500 mM KCl, 15 m
M MgCl2, 1% gelatin) 10 μl, 2.5 mM
8 μl of 4dNTP, synthetic primers MS135 and M
S88 (both 20 pmol / μl) 5μ each
1 and 76.5 μl of water were added to the reaction solution. After mixing well,
First, the temperature was kept at 95 ° C for 5 minutes and then rapidly cooled to 0 ° C. One minute later,
Taq DNA polymerase (7 units / μl, Am
0.5 μl of pliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample is Perkin
Elmer Cetus DNA Thermal C
Cycler 95 ° C for 1 minute, 37 ° C for 1 minute, 72 ° C
Treated 25 times in 3.5-4 minutes. Then, after keeping the temperature at 97 ° C. for 2 minutes, it was rapidly cooled to 0 ° C. and kept for 2 minutes. Furthermore, Taq DNA polymerase (7 units / μl, A
(mpliTaqTM Takara Shuzo) 0.5 μl was added in the same manner, 95 ° C. for 1 minute, 50 to 55 ° C. for 1 minute, 72 ° C. 3.5.
Processed 25 times in ~ 4 minutes.

Finally, after keeping the temperature at 72 ° C. for 7 minutes, this reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. The amplified DNA sample was separated by agarose gel electrophoresis (Molecular Cloning 1982).
Cold Spring Harbor (Cold Spri
ng Harbor), a DNA fragment with the desired length was excised from the gel (Molecular Cloning 1982 Cold Spring Harbor.
g Harbor)). The obtained DNA fragment was obtained in Example 1.
It was treated in the same manner as 6 and incorporated into the Sma I site in the multi-cloning site of the pUC19 cloning vector. This was cloned and screened (this method is similar to that in Example 16), and plasmid pU was used.
CN23N15A-1 and pUCN23N15B-1 were obtained, and the cDNAs derived from the sera of hepatitis C patients thus obtained were clone N23N15A-1 and clone N23N15B-.
I named it 1. The nucleotide sequences are shown together in SEQ ID NO: 42 in the sequence listing. The nucleotide sequences and amino acid sequences of clone N23N15A-1 and clone N23N15B-1 are shown in SEQ ID NOs: 61 and 62 of the sequence listing, respectively.

That is, clone N23N15A-1 was clone N23-1 and clones N16N15A-1 and N1.
It is shown that the overlapping regions of 6N15B-1 and N16N15-1 are sequences derived from clone N23-1, and clone N23N15B-1 shows that the regions are clones N16N15A-1, N16N15B-1 and N16.
It shows that the sequence is derived from N15-1. [4] Method for Obtaining Clone MX25N15-1 In order to ligate the overlapping clones MX25O26A-1 and Clone N23N15A-1 without overlapping, a restriction enzyme cleavage site common to both of them was used. That is, the restriction enzyme Apa
Clone MX25O26A-1 was 1277 by I.
Cleavage at the 3'side of the th C, clone N23N15A
-1 is truncated at the 17th 3 '. In addition, plasmids pUCMX25O26A-1 and pUCN23N15
In A-1, both of the DNA fragments have the plasmid pUC19.
Considering that the Sma I site in the multi-cloning site of pUCX25 has the same orientation, pUCMX25
The O26A-1 Hin dIII, with Apa I, length 13
DNA fragment of 10 bp (this DNA fragment is obtained by adding a DNA fragment from Hin dIII site of plasmid pUC19 to Sma I sites 5 'to the cloning MX25O26A-1 patients with hepatitis C serum-derived cDNA) cut out , PUCN23N15A-1 length 5
Hin of bp dIII, replaced respectively a DNA fragment containing the clone N15-1 cut out by Apa I, it was possible to obtain a plasmid pUCMX25N15-1 by cloning and screening. This plasmid harbored clone MX25N15-1 in which clone MX25O26A-1 and clone N23N15A-1 were ligated without overlapping.
The nucleotide sequence and amino acid of clone MX25N15-1 are shown in SEQ ID NO: 63 of the sequence listing.

Also, ligation was performed using the PCR method as in [3]. The nucleotide sequences of the DNA fragments obtained by all of these methods are shown together in SEQ ID NO: 43 of the sequence listing. Example 18 DN for expressing HCV-derived polypeptide encoded by clone MX25N15-1
Modification of A [1] Modification of DNA for expressing HCV-derived polypeptide encoded by clone MX25N15-1 in Escherichia coli In clone MX25N15, the seventh nucleotide in the nucleotide sequence shown in SEQ ID NO: 43 of the sequence listing is used. Open reading frame N from HCV gene from T to 825th G N
S2 (hereinafter referred to as MK / NS2) is considered to be coded, and the 826th G to 265
It is considered that the HCV gene-derived open reading frame NS3 (hereinafter referred to as MK / NS3) is encoded up to the second G. The initiation codon ATG is introduced upstream of the 5'side of the gene in accordance with this frame and expressed. In addition, MK / NS2 or MK /
When a part of the amino acid sequence of NS3 was to be expressed, the start codon ATG was introduced upstream of the gene on the 5'side in accordance with the frame of the amino acid sequence, and the stop codon was also introduced in the same frame. When the start codon ATG is introduced upstream of the gene on the 5 ′ side, SEQ ID NO: 4 in the sequence listing
A polypeptide other than the polypeptide encoded by the HCV gene may be added to the N-terminus (amino terminal side) of the amino acid sequence described in 3. Further, when the clone is introduced into a vector having a start codon derived from an expression vector in Escherichia coli, the frame of the clone may be aligned so as to match the frame of the start codon derived from the vector.

Further, modification of vector-derived DNA is carried out by PCR.
The method was used. First, the following synthetic DNA was used as the primer used to obtain the DNA fragment used to express the polypeptide MK / NS2. The base sequence for the 5'primer is MSNS2-1; 5'GCAAGCTTATGTGGT
TGTGGATGATGCTGCTG 3 '(SEQ ID NO: 169). The base sequence for the 5'primer when the DNA fragment is introduced into a vector having a start codon derived from the expression vector is MSNS2-2; 5'TGGTTGTGGATGAGTG.
CTG CTG 3 '(SEQ ID NO: 170). The base sequence for the 3'primer is MSNS2-3; 5'GCGAATTCAGACTTTT.
CATCATCCTCCGGGCGGAGACNGGNA
GNCC 3 '(SEQ ID NO: 171; N represents inosine). 20 pmol / μl of these two synthetic DNAs
Was prepared.

This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCM was used as template DNA.
X25N15-1 (or plasmid pUCMX2
5O26A-1 can be used because it also contains the same region), and the primers were 2 μl each of the solution prepared as described above for both 5'and 3'primers. First, the prepared sample was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler manufactured by Perkin Elmer Cetus at 95 ° C. 1
Min, 60 ° C. for 1 minute, and 72 ° C. for 3 minutes, 25 times. The sample conventional manner phenol / chloroform treatment and ethanol precipitated, and the DNA sample Hin dIII,
Cleaved with Eco RI (if MSNS2-2 is used, T
After blunting with 4 DNA polymerase, Eco RI
DNA fragment having the desired length was separated by acrylamide gel and extracted (Molecular Cloning) 1.
982 Cold Spring Harbor (Cold
Spring Harbor)). In addition Hin of the cloning vector pUC19 dIII (MSNS2-2
In the case of using the plasmid pUCHNS2-1 (MSN), Sma I site) and Eco RI site were ligated by a conventional method, cloned and screened.
When S2-2 was used, plasmid pUCH2NS2
-1 was obtained) and the nucleotide sequence was determined. In the nucleotide sequence of the clone HNS2-1 obtained by this method, the DNA fragment shown below is added to the 5'side of the DNA fragment shown from 7th to 825th of SEQ ID NO: 43 in the sequence listing.

5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: 15
5) 5 'end has a Hin dIII site, owns followed by a start codon ATG. At the 3'end, the DNA fragments shown below are from 7th to 8th of SEQ ID NO: 43 in the sequence listing.
5'TGATGAAGATCTGAATTCGC 3'3'ACTTACTCTAGACTTAAGCG 5'added to the 3'side of the 25th DNA fragment
(SEQ ID NO: 156) There is an Eco RI site from the 3rd and 3rd side, and B is inside it.
It carries the gl II site followed by two stop codons TGA. Primers MSNS2-2 and MSNS2-3
The nucleotide sequence of clone H2NS2-1 produced by the combination of No. 1 and No 2 was not added to the 5'end, and the same nucleotide sequence was added to the 3'end of clone HNS2-1.

The DNA fragment used to express MK / NS3 was obtained using the following synthetic DNA as a primer. The method is based on the plasmid PUCHNS2-
1. The same procedure as for obtaining the plasmid pUCH2NS2-1 was performed, and MSNS3-1, 2 and 3 correspond to the primers MSNS2-1, 2 and 3, respectively. MSNS3-1; 5'GCAAGCTTATGGGCA
ACGAGNTNCTNCTNGG 3 '(SEQ ID NO: 172) MSNS3-2; 5'GGCAACGAGNNTNCTN
CTNGG 3 '(SEQ ID NO: 173) MSNS3-3; 5'GCGAATTCAGACTTTT
CATCACTTCAGCCGTATGAGACACT
T 3 '(SEQ ID NO: 174) The resulting plasmid was plasmid pUCHNS2-
1. Corresponding to plasmid pUCH2NS2-1, plasmid pUCHNS3-1 and plasmid pUCH2NS
It is 3-1.

[2] 305 from the 422nd position to the 726th position in the amino acid sequence of SEQ ID NO: 43 in the sequence listing
HCV-derived polypeptide (M
K1) is expressed in Escherichia coli in a modified DNA fragment encoding the polypeptide. 42 out of the amino acid sequences set forth in SEQ ID NO: 43 in Sequence Listing
The initiation codon ATG is introduced upstream of the 5 ′ side of the gene in synchronization with the frame of the open reading frame (ORF) of the polypeptide MK1 represented by 305 amino acids from the 2nd position to the 726th position and expressed. When the start codon ATG is introduced upstream of the gene on the 5 ′ side, HC is added to the N-terminus (amino terminal side) of the amino acid sequence of the polypeptide.
A polypeptide other than the polypeptide encoded by the V gene may be added. Further, when the clone is introduced into a vector having a start codon derived from an expression vector in Escherichia coli, the frame of the clone may be aligned so as to match the frame of the start codon derived from the vector. DNA
The PCR method was used for modification. At this time, the following synthetic DNA was used as the primer.

The base sequence for the 5'primer is MSMK1-1; 5'GCAAGCTTATGCTGT
CGCCCGGGCCCCATTC 3 '(SEQ ID NO:
175). The base sequence for the 5'primer when the DNA fragment is introduced into the vector having the initiation codon derived from the expression vector is MSMK1-2; 5'CTGTCGCGCCCGGGCCC.
ATCTC 3 '(SEQ ID NO: 176) The base sequence for the 3'primer is MSMK1-3; 5'GCGAATTCAGATCTT
CATCAACATGTGTTGCAGTCGATCA
C3 '(SEQ ID NO: 177). 20 pmol / μl of these two synthetic DNAs
Was prepared.

PCR reaction, cloning subcloning, etc. are carried out according to the method carried out in [1], and the DNA fragment obtained by using the combination of MSMK1-1 and MSMK1-3 in the PCR reaction is cloned. In this case, the plasmid pUCMK1 was obtained (MSMK1-2 and MSMK1-3
Plasmid pUCMK1-
2), the base sequence was determined. In the base sequence of the clone MK1 obtained by this method, the DNA fragment shown below is 1 in the base sequence of the DNA fragment of SEQ ID NO: 43 in the sequence listing.
It is added to the 5'side of the 264th G, 5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: 15
5) 5 'end has a Hin dIII site, owns followed by a start codon ATG. (The base sequence of clone MK1-2 obtained by using MSMK1-2 is DN
There was no addition of the A fragment, but the 3'side was the same as clone MK1. ) In addition, the 3'end has the DNA shown below.
The fragment is added to the 3'side of the 2178th end of G in the nucleotide sequence of SEQ ID NO: 43 in the sequence listing, and Ec from the most 3'side
There is an o RI site, which has a Bgl II site inside, followed by two stop codons TGA. 5'TGATGAAGATCTGAATTCGC 3'3'ACTACTTCTAGACTTAAGCG 5 '
(SEQ ID NO: 156)

[3] 32 from the 712th position to the 1033rd position of the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing
Modification of a DNA fragment encoding an HCV-derived polypeptide (referred to as MK2) consisting of two amino acid sequences in E. coli 71 of the amino acid sequences set forth in SEQ ID NO: 43 of the Sequence Listing
The polypeptide MK2 represented by 322 amino acids from the 2nd position to the 1033th position is considered to be an HCV-derived antigen protein that immunologically reacts with the serum of HCV patients at a high rate. In order to express the region, the primer to be used has the sequence described below, and the region is expressed by the same operation as the modification of the DNA fragment for expressing the polypeptide MK1 in Escherichia coli in [2]. Was obtained. The clone obtained by this method is clone MK
2 (primers MSMK2-1 and MSMK2-3) and clone MK2-2 (primers MSMK2-2 and MSM)
K2-3). The base sequence for the 5'primer is MSMK2-1; 5'GCAAGCTTATGGGGCT
ATACCGGNGACTTNGAC3 '(SEQ ID NO:
178). The base sequence for the 5'primer when the DNA fragment is introduced into the vector having the initiation codon derived from the expression vector is MSMK2-2; 5'GGCTATACCGGNGAC
TTNGAC 3 '(SEQ ID NO: 179) The base sequence for the 3'primer is MSMK2-3; 5'GCGAATTCAGATCTT
CAGTGCTTCGCCCAGAAGGT 3 '(SEQ ID NO: 180). 2 of these two synthetic DNAs
It was adjusted to 0 pmol / μl.

[4] Modification of DNA for expressing a part of HCV-derived polypeptide encoded by clone MX25N15-1 in insect cells In clone MX25N15, an open reading derived from HCV gene from the first T The frame (ORF) seems to be coded. The initiation codon ATG is introduced upstream of the 5'side of the gene in accordance with this frame and expressed. When the start codon ATG is introduced upstream of the gene on the 5th side, other than the polypeptide encoded by the HCV gene at the N-terminus (amino terminal side) of the amino acid sequence encoded by clone MX25N15 (or part thereof). May be added. Moreover, when the clone is introduced into a vector having a start codon derived from an expression vector in insect cells, the frame of the clone may be aligned with the frame of the start codon derived from the vector. Also in this case, a polypeptide other than the polypeptide encoded by the HCV gene may be added to the N-terminus (amino terminal side) of the amino acid sequence (or part thereof) encoded by the clone MX25N15. In insect cells, the polypeptide encoded by clone MX25N15 is an HCV-derived polypeptide containing at least the amino acid sequence from the 167th position to the 502nd position at the amino acid number, and is linked and expressed intracellularly as a precursor. It was processed in cells, modified with sugar chains, and then expressed and accumulated in cells. To construct the expression vector, the DNA of clone MX25N15 was modified by P
The CR method was used. At this time, the following synthetic DNA was used as the primer used.

The base sequence for the 5'primer is MS2515-1; 5'GCCGCTAGCATGTGG.
TTGTGGATGATGGCTG 3 '(SEQ ID NO: 1
81). The base sequence for the 3'primer is MS2515-2; 5'GCGAATTCGCTAGC.
TCACACGCCGGTTCATCCACTGCAC
3 '(SEQ ID NO: 182). 20 pmol / μl of these three synthetic DNAs
Was prepared. Preparation of PCR reaction solution and post-treatment [1]
As well as done. However, plasmid pUCMX25N15-1 was used as template DNA, and MS was used as primer.
A combination of 2515-1 and MS2515-2 was used. PCR cycle is 95 ° C for 1 minute, 50 ° C for 1 minute, 72
℃ 5 minutes, after repeating this 10 times, 95 ℃ 1 minute,
Repeated 1 minute at 65 ° C and 5 minutes at 72 ° C 20 times to obtain the desired length of 3
A 586 bp DNA fragment was obtained. Nhe the DNA sample
After cutting with I, the DNA fragment of the desired length was separated by acrylamide gel and extracted (Molecular Cloning 1982).
Cold Spring Harbor (ColdSpri
ng Harbor)). In addition, the transfer vector pBlueBac (Invitrogen) Nhe
I site is ligated and cloned by a conventional method,
A clone in which only one of the DNA fragments is inserted at the NheI site is screened, and the primer MS2
The plasmid pBlueMX25N15-1 derived from the DNA fragment obtained from 515-1 and MS2515-2 was obtained.

The expression units of these plasmids are referred to those shown in the protocol of Invitrogen, and the HCV gene-derived DN is located at the cloning site Nhe I cleavage site downstream of the polyhedrin promoter.
It had a structure in which the A fragment was linked in the forward direction. Example 19 H encoded by clone MX25N15
CV-derived polypeptides and polypeptides MK / NS2, M
Expression of K / NS3, MK1, MK2 [1] Clone HNS2-1, Clone HNS3-1,
Expression of Polypeptides Encoded by Clone MK1 and Clone MK2 in Escherichia coli The polypeptide encoded by the clone is a part of the polypeptide encoded by cDNA derived from hepatitis C patient serum. Was expressed in. As shown below, pCZ44 (Japanese Unexamined Patent Publication No. 1-124387) was used as an expression vector, and it was performed by subcloning the cDNA of the clone into pCZ44. First of all, the clone is Hin dIII, Bgl I
Are sufficiently cut by I, phenol / chloroform treatment, and ethanol precipitation, Hin dII both ends each
A DNA fragment having the cDNA as the protruding ends of the I and Bgl II sites was separated by acrylamide gel and extracted (Molecular cloning (Molecular cloning)).
ar Cloning) 1982 Cold Spring Harbor
r)). Similarly, the expression vector pCZ44 also contains Hin dI.
II, are sufficiently cut by Bgl II, a longer DNA fragment with an expression vector ability similarly prepared above, this prepared from the clones were Hin dIII, Bgl
It was ligated so as to have one II fragment, and further cloned according to a conventional method. The obtained plasmids are the plasmids pCZHN derived from the clones HNS2-1, clone HNS3-1, clone MK1 and clone MK2, respectively.
S2-1, pCZHNS3-1, pCZMK1, pCZ
It was named MK2.

As another attempt, an expression vector pGEX-2T capable of expressing a fusion protein instead of pCZ44
(Manufactured by Pharmacia) was used to construct an expression vector. In this case, the operation was basically performed according to the instructions of Pharmacia. First, the expression vector pGEX-2T
After cutting with Bam HI, a Hin dIII linker was inserted so that the reading frames of the amino acid and codon of the clone would match so that the Eco RI site on the 3'side and the Hin dIII site on the 5'side (made by the linker) were Hin was prepared from the DNA fragment of clone dIII, was inserted Eco RI fragment. Thus the expression vector pGEXHNS2-
1, pGEXHNS3-1, pGEXMK1, pGEX
MK2 was obtained. Plasmid pCZHNS2-1, p
E. coli JM109 strain harboring CZHNS3-1, pCZMK1, and pCZMK2 was transformed into L-Broth (Molecular Cloning).
ing) 1982 Cold Spring Harbor, 3
It was cultured overnight at 7 ° C. This was inoculated into new L-Broth so as to be diluted 50-fold, shake-cultured at 30 ° C. for 2 hours, and IPTG (isopropyl-β-D-galactopyranoside) was added to the medium to 2 mM. In addition, the cells were further cultured for 3 hours or more. Thus, by culturing the bacterium in the presence of IPTG, a single clone was derived (in this case, Escherichia coli JM109 strain transformed only with the plasmid).
Recombinant was induced to express an HCV-derived polypeptide using the plasmid derived from the clone. Clone HNS2-1, clone HNS3
-1, cDNA derived from clone MK1 and clone MK2
The deduced amino acid sequence of the polypeptide encoded by: is the amino acid sequence from the 422nd position to the 726th position among the amino acid sequences set forth in SEQ ID NO: 43 of the sequence listing, and 7 respectively.
It is the amino acid sequence from the 12th to the 1033rd.

Further, the plasmid pGEXHNS2-1,
pGEXHNS3-1, pGEXMK1, pGEXMK
Similarly, with respect to 2, an expression product can be obtained as a fusion protein with β-glutathione-S-transferase (GST). In this case, as described in the manual of Pharmacia, GST has a site that is specifically cleaved by thrombin at the C-terminus, and clone HN is present at the C-terminus.
The polypeptides derived from S2-1, clone HNS3-1, clone MK1, and clone MK2 are linked. To obtain the fusion protein, the plasmid pCZHN
S2-1, pCZHNS3-1, pCZMK1, pCZ
Similar to the above-mentioned protocol for expressing the HCV-derived polypeptide from MK2, the plasmids pGEXHNS2-1, pGEXHNS3-1, p in the presence of IPTG were used.
Escherichia coli transformed with GEXMK1 and pGEXMK2 was cultured and induced to express the fusion protein having an HCV-derived protein at the C-terminal of GST. [2] Clone H2NS2-1, clone H2NS3-
1. Expression of polypeptide encoded by clone MK1-2, clone MK2-2 5 clones of hepatitis C patient serum-derived cDNA contained in clone H2NS2-1, clone H2NS3-1, clone MK1-2, clone MK2-2 The polypeptide was expressed in E. coli in a form in which genes for other proteins were linked to the 'side. For example, it is the outer membrane protein of Escherichia coli as shown below.
A signal peptide such as Omp F is used for this. The expression method was similar to that described in [1]. However, the material for the HCV cDNA is cDNA derived from hepatitis C patient serum contained in clone H2NS2-1, clone H2NS3-1, clone MK1-2, and clone MK2-2. This clone was isolated and its nucleotide sequence was determined. It is a thing.

The expression plasmid used was pOFA.
(Japanese Patent Laid-Open No. 2-84195) was used. First, the DNA fragment of each of the above clones was blunt-ended with T4 DNA polymerase. Expression plasmid pOFA
Was treated with Kpn I and blunt-ended at both ends with T4 DNA polymerase, and the blunt-ended clone was ligated by a conventional method to clone the DNA.
Clones with only one fragment inserted were screened. That is, each HCV-derived cDNA is HC
By subcloning so that the + chain expressing the V-derived protein can be correctly inserted in the direction of translation, plasmids pOFANS2-1, pOFANS3-1, and pOFAM can be obtained.
Obtained as K1, pOFAMK2. Whether or not the HCV-derived cDNA in the plasmid was correctly reconstructed was confirmed by nucleotide sequencing and creation of a restriction enzyme cleavage map.

By culturing the bacterium in the presence of IPTG, a recombinant derived from a single clone (for example, Escherichia coli JM109 strain transformed with the plasmid) expresses the HCV gene-derived polypeptide encoded by the clone. Was invited to do. Clone H2NS2-1, clone H2NS3-1, clone MK1-2, and clone MK2-2 have the nucleotide sequences of cDNAs derived from the sera of hepatitis C patients and the deduced amino acid sequences of the polypeptides encoded by the sequences, respectively. Of the amino acid sequence described in No. 43, they are the 422nd to 726th amino acid sequences and the 712nd to 1033th amino acid sequences. That is, the polypeptide derived from this clone expressed by this method was constructed so that the polypeptide consisting of the amino acid sequence encoded by each clone would be translated as a fusion protein with the signal peptide of OmpF.

[3] Expression of HCV-derived polypeptide encoded by clone MX25N15 in insect cells Plasmid pBlueMX constructed in Example 18 [4]
In order to express the HCV-derived protein encoded by 25N15 in insect cells, the baculovirus expression manual of Invitrogen Co., Ltd., Maxvac (M
AXBACTMBACULOVEIRUS EXPRESS
SION SYSTEM MANUAL VERSION
1.4, hereinafter referred to as Maxback. ) Was basically referred to. Example 1
8 [4], transfer vector p
Restriction enzyme N of BlueBac (Maxback page 37)
The plasmid in which a DNA fragment containing the HCV gene was inserted at the he I cleavage site was prepared by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pages 86-96 (1982)).
According to the procedure described above, the recombinant Escherichia coli was recovered and purified to obtain a large amount of HCV gene transfer plasmid DNA. MaxBack according to the method on page 27, kit AcNPV
1 μg of viral DNA and D derived from HCV gene
2 μg of the plasmid containing NA fragment was mixed with Sf
Sf9 cells were transformed by co-infection with 9 cells.

That is, first, 2 × 10 5 of Sf9 cells were placed in a TMN-FH medium (Invitrogen) containing 10% FCS (fetal bovine serum) in a petri dish having a diameter of 6 cm.
^It culture | cultivated until it became ⁶ / sheet. Next, from the petri dish, TMN-
Grace (Grace) medium (GIBC (GIBC) containing 10% of FCS of 0.75 ml except FH medium)
O)) was added. Add 0.75 ml of the transfection buffer included in the kit to the above-mentioned mixed DNA, mix well by vortexing, and then Grace.
The DNA solution was slowly added dropwise over the medium. 27 ° C 4
Allow to stand for a period of time, then remove Grace medium and
Add 3 ml of TMN-FH medium containing 2% to a Petri dish and 2
It was cultured at 7 ° C for 6 days. On day 3, polyhedra were observed in some cells, and on day 6, most cells formed polyhedra. Transfer this culture supernatant to a centrifuge tube and
The supernatant obtained by centrifugation at 00 rpm for 10 minutes was used as a co-infection virus solution.

[0187] This co-infectious virus solution contained 10 ⁸ viruses per ml, and about 0.5% of them was recombinant. The plaque isolation method was used to isolate the recombinant virus. The method is as follows. The co-infected virus solution was diluted to 10-4 and 10-5. 1.5 x 10 ⁶ per piece in 6 cm Petri dish in advance
After culturing individual cells and adsorbing them, the medium was completely removed, and 100 μl of each of 10 ⁻⁴ solution and 10 ⁻⁵ solution was added to each petri dish. In order to prevent the cells from drying, the petri dish was tilted every 15 minutes so that the virus solution was spread over the entire surface. As described above, 105% of baculovirus agarose (manufactured by Invitrogen) was used while infecting at room temperature for 1 hour.
Autoclave at 10 ° C for 10 minutes, mix with TMN-FH medium containing 10% FCS warmed at 46 ° C at a ratio of 1 to 3, and keep warm at 46 ° C. To this incubated medium, 5-bromo-4-chloro-3-indolyl-β-D-galactoside was added to a final concentration of 150 μg / μl (according to Maxback's pages 16-17). The medium was called agarose-containing x-gal medium.

After the infection, the virus solution was completely sucked up, and the agarose-containing x-gal medium which had been kept warm was gently added in an amount of 4 ml per petri dish so that the cells would not come off. The petri dish was allowed to stand with the lid slightly displaced until the agarose was solidified and dried, and then, the lid was put on and turned over, and incubation was carried out at 27 ° C. After culturing for 6 days, blue plaques in which polyhedra were not formed were found by a phase contrast microscope. This recombinant plaque is sucked up with agarose using a Pasteur pipette and 1 ml of TMN-
The virus was suspended by pipetting many times in FH medium. This series of operations (an operation of infecting and then culturing for 6 days to isolate a virus having the transfer plasmid DNA) is called a plaque method. The same plaque method was performed using 100 μl of the virus suspension. These series of operations are repeated three times, and HCV free from contamination by wild strains.
A recombinant virus carrying the derived glycoprotein gene was obtained.
Absorb the virus plaque with a Pasteur pipette,
It was mixed well with 1 ml of TMN-FH medium. This primary recombinant virus solution has a low virus concentration, so
After taking 00 μl and adsorbing to Sf9 cells cultured in a 6 cm dish at half confluence, T containing 10% FCS
4 ml of MN-FH medium was added, and after 3 days, the culture supernatant was collected and used as a recombinant virus solution for infection.

In order to produce HCV-derived glycoprotein, 5 × 10 6 Sf9 cells were previously suspended in 10 ml of TMN-FH medium containing 10% FCS, and the suspension was spread on a 9 cm Petri dish and adsorbed for 1 hour. The medium was removed from the petri dish, 250 μl of the recombinant virus solution for infection was added, the whole was spread, 10 ml of TMN-FH medium containing 10% FCS was added, and the mixture was allowed to stand at 27 ° C. for 4 days. The cells were thoroughly suspended in 1,000 ml of phosphate buffered saline. Thus, the HCV-derived glycoprotein was expressed in Sf9 cells infected with the virus. Example 20 Identification of Expressed Product as HCV-Derived Antigen Since human serum infected with hepatitis C virus reacts immunologically with the polypeptide expressed in Example 6,
The polypeptide was identified as a hepatitis C-related antigen.
This identification was performed by the Western blotting method shown below. First, for example, Example 19 [1], [2]
Clone HNS2-1 and clone HNS3- described in 1.
1, clone MK1, clone MK2, clone H2N
S2-1, clone H2NS3-1, clone MK1-
2. Plasmids pCZHNS2-1 and pCZHNS for expressing each polypeptide encoded by clone MK2-2
3-1, pCZMK1, pCZMK2, pGEXHNS
2-1, pGEXHNS3-1, pGEXMK1, pG
EXMK2, pOFANS2-1, pOFANS3-
E. coli transformed with 1, pOFAMK1 and pOFAMK2 were induced to express the polypeptide by culturing in the presence of IPTG for 3 hours or overnight as in Example 19.

1,000 μl of this culture solution was added to 6,500
r. p. m. The recombinant strains were harvested by centrifugation for 10 minutes. This precipitate was treated with a sample treatment solution for SDS-polyacrylamide gel electrophoresis (2% SDS, 5%
Mercaptoethanol, 10% glycerin and 0.00
It was prepared by dissolving it in 50 mM Tris-HCl buffer containing 5% bromophenol blue, pH 6.8) so that the final volume was 0.2 ml. Then, these samples are
Boiled for 10 minutes at ℃. 10 μl of the sample thus obtained was added to 0.1% SDS-15.0% polyacrylamide gel (70 × 85 × 1 mm). At that time, as a marker protein, "LMW KitE" manufactured by Pharmacia (low molecular weight marker protein)
It was used. Tris buffer (25 mM Tris pH 8.3, 192 mM glycine, 0.1% S as an electrode solution)
DS), and after running for 45 minutes at a constant current of 30 mA, the gel was overlaid with a Nitrocellulose filter (BA-83) manufactured by S & S Co. according to a conventional method, and the gel side was used as a cathode and the nitrocellulose filter side was used as an anode. A constant current of 120 mA was applied for about 20 minutes for transfer. The transferred nitrocellulose filter was cut so as to separate the marker protein portion and the sample portion, and the marker protein portion filter was filtered with 0.1% (W / V) amide black 1
Stain with 0B and filter the sample part by 5%
(W / V) Bovine serum albumin in 0.01 M phosphate saline (PBS) pH 7.4 was immersed. Then 5%
(W / V) 10 ml of hepatitis C patient serum diluted 50 times with 0.01 M phosphate saline (PBS) pH 7.4 containing (W / V) bovine serum albumin is placed on the filter of the sample portion, and 2 After standing for 0.1 hours (V / V) Sig
20 minutes each with PBS including Ma Tween 20 3
Washed twice.

Next, PBS is placed on the filter of the sample portion.
Place 10 ml of peroxidase-labeled anti-human IgG (manufactured by Gappel) diluted 1,000 times with the solution at 37 ° C.
The reaction was carried out for 1 hour and the plate was washed with PBS containing 0.1% (V / V) Tween 20 three times for 20 minutes each. Next, 100 ml of the peroxidase coloring solution (6
It was immersed in 0 mg 4-chloro-1-naphthol, 20 ml methanol, 80 ml PBS, 20 μl hydrogen peroxide).
The colored filter was washed with distilled water and then compared with the marker protein portion. The proteins developed in this manner are plasmids pCZHNS2-1 and pCZHNS3-.
Any of those expressed in 1, pCZMK1 and pCZMK2 had an appropriate molecular weight as a product derived from the introduced HCV gene and could be identified as a hepatitis C-related antigen. Plasmids pOFANS2-1, pOFANS3-1, pO
Plasmid pOFA such as FAMK1 and pOFAMK2
The derived expression product is an HCV-derived polypeptide to which the OmpF signal peptide of Escherichia coli has been added to the N-terminus. Plasmid pGEXHNS2-1, pGEXHN
Expression products derived from the plasmid pGEX, such as S3-1, pGEXMK1 and pGEXMK2, are those in which a cleavage site for GST and thrombin is added to the N-terminus of the HCV-derived polypeptide. Both of them had a reasonable molecular weight as a gene product of the clone and could be identified as hepatitis C-related antigens.

Example 21 Synthesis of cDNA All water used in Example 21 was obtained by autoclaving ultrapure water twice. [1] Preparation of RNA sample After drying the nucleic acid obtained in Example 1, it was dissolved in 50 μl of a 0.3 M sodium acetate (pH 7.0) aqueous solution and subjected to phenol / chloroform treatment once, and then 2. 5
A double volume of ethanol was added and mixed, and the mixture was centrifuged at room temperature with a rotor having a radius of about 5 cm for 15,000 rpm for 20 minutes to obtain a nucleic acid precipitate. This precipitate is dried and 30 μl of water
And ribonuclease inhibitor (100 units / μ
l, manufactured by Takara Shuzo) was added and dissolved. The cDNA synthesis shown below was performed using this nucleic acid aqueous solution. [2] Synthesis of cDNA Using Antisense Primer One kind of synthetic DNA primer MS126, MS119, MS161, MS162, M shown in one kind of antisense primer (Table 1) per 2 μl of the nucleic acid aqueous solution prepared in [1]
S121 and MS163 are antisense primers. The concentration of each primer was adjusted to 15 pmols / μl. ) 1 μl, 10 × RT buffer (100 mM Tr
is-HCl (pH 8.3), 500 mM KCl) 2
μl, 25 mM MgCl 2 4 μl, 2.5 mM 4
dNTP 8 μl and water 1 μl were added, and the temperature was 65 to 70 ° C. for 5 minutes.
Next, after incubating at room temperature for 5 minutes, 1 unit of 25 units of reverse transcriptase (manufactured by Life Science) and 1 μl of ribonuclease inhibitor (100 units / μl, manufactured by Takara Shuzo) were added, 20 minutes at 37 ° C, then 30 minutes at 42 ° C, and finally 95 ℃ 2
After keeping the temperature for minutes, it was immediately cooled to 0 ° C. (complementary strand DNA synthesis). Using 10 μl of this DNA sample, the method of Saiki et al. [Nature, 324, 126,
(1986)], a DNA having a specific sequence was amplified by the so-called PCR method.

That is, 10 μl of this DNA sample, 10 × P
CR buffer (100 mM Tris-HCl (pH 8.
3), 500 mM KCl, 15 mM MgCl2, 1
% Gelatin) 10 μl, 2.5 mM 4dNTP 8 μ
l, 2 μl of synthetic DNA primer (15 pmols / μl) used for complementary strand DNA synthesis, synthetic DNA primer corresponding to this primer (15 pmols / μl)
l, which is paired with the synthetic DNA primer used in the synthesis of complementary strand DNA, and is a combination of the above-mentioned primers MS127-MS126, MS118-MS119, M
S159-MS161, MS160-MS162, MS
120-MS163 and MS120-MS121 were considered. ) Water was added to 3 μl so that the total amount became 100 μl. First, the mixture was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C.
After 1 minute, Taq DNA polymerase (7 units / μ
1, AmpliTaq ™ Takara Shuzo) (0.5 μl) was added and mixed, and then overlaid with mineral oil. This sample is a DNA Therm manufactured by Perkin Elmer Cetus
al Cycler at 95 ° C for 1 minute, 40-55 ° C for 1 minute,
It was treated 25 times at 72 ° C. for 1 to 5 minutes. Finally, after incubating at 72 ° C. for 7 minutes, the reaction aqueous solution was treated with phenol / chloroform and then subjected to ethanol precipitation.
Ammonium acetate and 2.5 times the volume of ethanol of the aqueous phase were added and mixed, using a rotor with a radius of about 5 cm for 15,000.
A process of cooling and centrifuging at 0 rpm and 4 ° C. for 15 minutes to dry the precipitate was performed) to obtain amplified DNA fragments derived from various combinations of primers.

Example 22 Cloning of Amplified DNA Fragment and Determination of Nucleotide Sequence At least 1 pmole of the DNA fragment obtained by the method of Example 21 [2] was prepared, and this DNA was transformed into T4DNA.
Blunt ends with a polymerase (manufactured by Toyobo Co., Ltd.) (Molecular Clon
ing) 1982 Cold Spring Harbor and a polynucleotide kinase (manufactured by Toyobo Co., Ltd.) after introducing a phosphate group at the 5'end (Molecular Cloning (Molecular Cloning)).
1982. Cold Cloning Harbour. Cold Spring Harbour.
bor)), using a ligation kit (Takara Shuzo Co., Ltd.), incorporated into the Sma I site within the multiple cloning site of pUC19 cloning vector.
(At this time, 5 ng to 10 ng of the vector DNA used for ligation was prepared as follows. That is, the pUC19 cloning vector was used as a restriction enzyme Sma.
After cutting with I (Toyobo Co., Ltd.), phenol / chloroform treatment, and ethanol precipitation, the 5'end was dephosphorylated with alkaline phosphatase (Boehringer Mannheim) (Molecular Cloning (M
1982. Cold Spring Harbor (Cold Spring)
Harbor)), phenol / chloroform treatment, and ethanol precipitation. ) DNA prepared in this way
Was used to transform Escherichia coli JM109 or DH5 (at this time, the competent cells used were those manufactured by Toyobo Co., Ltd.). The method of transformation was according to the protocol of COMPETENT HIGH of Toyobo Co., Ltd.

In this manner, at least 20 or more transformants were transformed with the pUC19 cloning vector having the DNA fragment obtained by the method of [21] of Example 21 from each combination of primers. I was able to get it. A plasmid DNA was prepared from the obtained transformant by a conventional method, and using a fluorescence sequencer GENESIS 2000 system manufactured by DuPont,
The sequence was determined. The following 2 as a sequence primer
Seed synthetic primer 5'd (GTAAAACGACGG
CCAGT) 3 '(SEQ ID NO: 143), 5'd (CA
GGAAACAGCTATGAC) 3 '(SEQ ID NO: 1)
44) was used to determine the nucleotide sequences of the + strand and single strand of the DNA fragment. When the DNA fragment was as long as 200 base pairs or more, it was subcloned into a clone of the deletion mutant according to a conventional method to perform reliable sequencing. In order to organize each DNA fragment obtained from the combination of each primer of Example 2, each DNA fragment was named as follows in the present invention.

[0196]

[Table 1] Combination of primers Clone name whose nucleotide sequence was determined MS127-MS126 N22-1, N22-3, N22-4, H22-3, H22-8, H22-9 MS118-MS119 N17-1, N17- 2, N17-3, H17-1, H17-3 MS159-MS161 O28-1, O28-2, O28-4 MS160-MS162 N29-1, N29-2, N29-3 MS120-MS163 O30-2, O30- 3, O30-4 MS120-MS121 N18-2, N18-3, N18-4, H18-1, H18-2, H18-3 Moreover, the alphabet of each clone name is the hepatitis C patient serum used in Example 1. Distinguished by. Based on the determined nucleotide sequence, all of these clones have already been reported as HCV.
It has been found that it has a homology with the gene sequence of
The region name of the A fragment on the HCV gene was set as follows.

[0197]

[Table 2] Combination of primers Region name MS127-MS126 N22 MS118-MS119 N17 MS159-MS161 O28 MS160-MS162 N29 MS120-MS163 O30 MS120-MS121 N18 Clones N22-1, N17- among the obtained clones.
3, O28-1, N29-1, N18-4, O30-3
The nucleotide sequence of and the amino acid sequence encoded by it are shown in SEQ ID NOs: 76, 81, 86, 89, 92 and 98 in the sequence listing. Furthermore, the base sequences of other clones obtained in the same manner are shown below, arranged under the clone shown in the sequence number in the sequence listing. Each clone shown in the sequence number in the sequence listing is shown at the top, and the same base sequence is shown by a dot. The number in the table is the base number on the left of the line. The left is the 5'side. In each clone, the 5'end side was designated as base number 1.

[0198]

[Table 3]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

The obtained clones are arranged in each region and are represented by SEQ ID NOs: 64-69 in the sequence listing. The nucleotide sequences shown in SEQ ID NOs: 64-69 and 76-100 in the sequence listing are
The respective nucleotide sequences of the + chains of the HCV gene-derived DNA fragments incorporated in the transformant plasmids obtained above are shown below. The clone is a double-stranded DNA, and for example, the plasmid used to determine the nucleotide sequence of clone N22-1 was pUCN22-1. Each of these plasmids had one molecule of the corresponding DNA fragment. The plasmid DNA used to determine the nucleotide sequence of one of the clones is shown below.
The clone name is given after C.

These sequences represent the nucleotide sequences of clones cloned from cDNA prepared from RNA derived from sera of hepatitis C patients, and from the sera of healthy persons, this method can be used for any Not get or
The PCR method shown in Example 21 [2] was amplified by triple or quadruplicate the cycle (probably 60 to 100 times), and if any DNA fragment was obtained, its D
The sequences of the NA fragments are SEQ ID NOS: 64-69, 76-
Only those having no homology with the nucleotide sequence shown in 100 were obtained. That is, the nucleotide sequences of the clones shown in SEQ ID NOS: 64-69 and 76-100 of the sequence listing obtained here are specific to those derived from the serum of hepatitis C patients. Further, from the above, it can be seen that one patient has multiple viruses. Example 23 Method for obtaining clone 1530U [1] Method for obtaining clone 1728, clone 2217, clone 2918 Clone N17-3 and clone O obtained in Example 22
The overlapping region of 28-1 is used to ligate by PCR reaction. 1 μl (about 0.5 to 1 μg / μl) of each of the DNA fragments (311 base pairs and 740 base pairs, respectively) having the nucleotide sequences of clone N17-3 and clone O28-1 was added to each of 10 × PCR buffer (100 mM Tris-). HCl (pH 8.3), 50
10 μl of 0 mM KCl, 15 mM MgCl2, 1% gelatin), 8 μl of 2.5 mM 4dNTP, synthetic primers MS118 and MS161 (both 20 pmo)
1 / μl) was added to each reaction solution of 5 μl and 76.5 μl of water. After mixing well, first incubate at 95 ℃ for 5 minutes,
Quenched to 0 ° C. 1 minute later, Taq DNA polymerase (7 units / μl, AmpliTaqTM Takara Shuzo)
0.5 μl was added and mixed, and then overlaid with mineral oil.

This sample was treated with a DNA Thermal Cycler manufactured by Perkin Elmer Cetus Co., Ltd. 25 times at 95 ° C. for 1 minute, 37 ° C. for 1 minute, and 72 ° C. for 2 minutes. Then, after keeping the temperature at 97 ° C. for 2 minutes, it was rapidly cooled to 0 ° C. and kept for 2 minutes. Furthermore, Taq DNA polymerase (7 units / μl, AmpliTaqTM Takara Shuzo) was added to the reaction solution.
Add 0.5 μl and do the same at 95 ° C for 1 minute, 55 ° C for 1 minute,
It was treated 25 times at 72 ° C. for 2 minutes. Finally, after keeping the temperature at 72 ° C for 7 minutes, the reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. The two types of DNA fragments are P
It was also ligated and amplified by CR reaction. Concatenated D
NA samples were separated by agarose electrophoresis (Molecular Cloning).
Cloning 1982 Cold Spring Harbor, a DNA fragment with a length of approximately 1000 base pairs was excised from the gel (Molecu Cloning).
lar Cloning) 1982 Cold Spring Harbor (Cold Spring Harbo)
r)). The obtained DNA fragment was treated in the same manner as in Example 22 and incorporated into the Sma I site in the multicloning site of the pUC19 cloning vector. This was cloned and screened (the method is the same as in Example 22) to obtain the plasmid pUC1728. The hepatitis C patient serum-derived clone thus obtained was designated as clone 1728. The nucleotide sequences are shown together in SEQ ID NO: 8 in the sequence listing.

Similarly, the clones N22-1 and N17-3 were ligated using the PCR reaction to obtain the plasmid pUC2217. This plasmid is Sma I
It has a DNA fragment derived from hepatitis C patient serum at the site. The orientation of the DNA fragment was such that there was an Eco RI site of pUC19 on the 5'side of the nucleotide sequence derived from clone N22-1 and Hin dI on the 3'side of the nucleotide sequence derived from clone N17-3.
There is a II site. The nucleotide sequence and amino acid sequence of the clone 2217 are shown in SEQ ID NO: 70 of the sequence listing, respectively. Similarly, clone N29-1 and clone N18-
Clone 2918 was obtained from 4. The nucleotide sequence and amino acid sequence of the linked clones are shown in SEQ ID NO: 72 in the sequence listing. [2] Method for obtaining clone 1718 Clone 1728 and clone 2918 obtained in [1]
Have 43 base pairs of overlap. DNA fragments each having a length of 1004 base pairs and 857 base pairs were ligated by PCR reaction. The reaction conditions and procedures were based on the method described in [1], but Taq poly in the PCR reaction was used.
The reaction time of the merase extension reaction step at 72 ° C. was changed to 5 minutes. This is 1818 base pairs long,
Sma DNA fragment having a nucleotide sequence derived from HCV gene
A plasmid pUC1718 having an I site was obtained. At this time, the N17 region side is 5'side on the HCV gene than the N18 region, but pUC19 is located on the 5'side of clone N17-3.
PUC171 that comes with the Eco RI site of
It was set to 8. The clone was designated as clone 1718, and its nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 73 of the sequence listing.
It was shown to.

[3] Method for Obtaining Clone 2218 Overlapping Clone 2217 and Clone 171
In order to ligate 8 and 8 without overlapping, it was utilized that both have common and only one restriction enzyme cleavage site at the overlapping position. That is, the restriction enzyme
By Xba I pUC2217 was cloned N17-
A large DNA fragment pUC22 having the most sequence derived from the vector and clone 2217, which was cleaved at two positions of the sequence derived from 3 and the sequence derived from pUC19, and a short DNA fragment of about 40 base pairs or less was separated by agarose electrophoresis.
17 / Xba I was extracted by a conventional method. pUC1718
DNA fragment 1718/1545 bp containing the majority of the sequence derived from clone 1718, which is cleaved at two positions, the sequence derived from clone N17-3 and the sequence derived from pUC19.
Xba I was separated and extracted by agarose electrophoresis by a conventional method. Plasmids pUC2217 and pUC17
In consideration of the fact that both DNA fragments are in the same direction, pUC2217 / Xba I 10 ng and 1718 / Xba I 50 ng were ligated with T4 DNA ligase, and this reaction solution was used for E. coli JM10.
9 competent cells, cloned from E. coli transformed as in Example 22,
Sequences derived from clone N17-3 are linked back to p
A transformant having the plasmid pUC2218 ligated at the Xba I sites derived from UC19 was obtained.

The plasmid was cloned into the Sma I site of pUC19 from the Eco RI site side to clones N22-1 and N1.
7-3, O28-1, N29-1, and N18-4, in which overlapping regions are ligated without overlapping regions
Have fragments. This clone is designated as clone 2218. The nucleotide sequence and amino acid sequence of clone 2218 are
It is shown in SEQ ID NO: 74 of the sequence listing. [4] Forward ligation of N15 region and O30 region corresponding to HCV 3 ′ end Clone O30-3 is shown in Sequence Listing 98. The DNA fragment of clone O30-3 having this sequence is pUC19.
To the Sma I site, E to the 5 'side of the clone O30-3
The co RI site is inserted in one direction (pUCO
30). Also, HCV of clone N15 (Example 16)
Like the clone O30-3, the gene was inserted in the Sma I site of pUC19 in the direction of the Eco RI site on the 5 ′ side in the forward direction of the gene (pUCN15). pUCO30
After There was cut with Sac I site from cloning site of pUC19, blunt ended by the conventional methods T4DNA polymerase, and further digested with Hin dIII from pUC19 cloning sites, HCV gene-derived DNA fragment O30 (SacT4 / Hind ) Got. Further, after cutting the plasmid pUCN15 with Xba I, blunt, and cut with Hin dIII pU
A DNA fragment pUCN15 (XbaT4 / Hin containing most of the C19 region and sequences derived from clone N15-1
d) was obtained. DNA fragment O30 (SacT4 / Hin
d) About 80 ng and DNA fragment pUCN15 (XbaT
Approximately 20 ng of 4 / Hind) was ligated with T4 DNA ligase in a reaction solution of 20 μl according to a conventional method. This reaction solution was incubated with Competent High (manufactured by Toyobo Co., Ltd.) JM109 and transformed according to the attached protocol, followed by the desired plasmid pU.
Transformants with C15-30 were isolated. Using the said plasmid is not only cut at each one place in Bgl II and Hin dIII, a primer comprising a Bgl II site at the sequence from clone O30-3 MS17
PCR was performed in 4 as follows, and poly U was added to the 3'end of clone O30-3.

PCR was carried out using 10 ng of pUC1530 as a template and primers MS174 and MS175.
After cutting the PCR fragment Bgl II, with Hin dIII, Bgl II, was cut with Hin dIII pUCO
A plasmid pUC15-30U containing a DNA fragment containing poly U at the 3'end of clone 30-3 was obtained by ligating with the DNA fragment containing 30 vector parts. [5] Linkage from N15 region to O30 region Apa I site exists in the common region of N15 region and N22 region. Also, in the common area of the N18 area and the O30 area, A
There is a pa I site. PUC2 so that both fit together
The DNA fragment from clone 2218 was cut from 218 Apa I and inserted into Apa I site PUC15-30U, resulting in plasmid PUC1530U. As an experimental condition at this time, a DNA step piece derived from clone 2218 cut out from pUC2218 with Apa I was electrophoresed on an agarose gel and then extracted by a conventional method to obtain pUC221.
30 μg of 8 / Apa I was obtained. 20 μg and pUC of a DNA fragment derived from the plasmid pUC15-30U which had been completely cleaved with Apa I and dephosphorylated at the 5'end.
30 μg of 2218 / ApaI was subjected to a ligation reaction at a final concentration of 10 μl, and the whole reaction solution was transformed with Competent High (Toyobo Co., Ltd.) DH5. From the obtained transformant, a DNA fragment clone 1530U ligated without overlapping sequences overlapping from N15 region to O30 region was used as plasmid pUC19.
The plasmid pUC1530U having the Sma I site in the multi-cloning site of was prepared. The nucleotide sequence of clone 1530U was determined by the same method as shown in Example 22, and its sequence is shown in SEQ ID NO: 75 of the sequence listing.
At the same time, the amino acid sequence of the HCV-derived polypeptide encoded by the clone is also shown.

From the N15 region thus linked, O3
The 0 region encodes an amino acid sequence highly homologous to NS5, a part of flavivirus nonstructural protein NS4, which is related to HCV. In addition to the above-mentioned Chiron, from the sequence of all genes of hepatitis C virus reported by Shimo-Tono et al. Of National Cancer Center and Takamizawa et al. Of Osaka University / Micro Lab. Since it has a homology with the gene sequence encoding, the clones described in the sequence listing of the present invention are expected to be the gene sequences of NS4 region (part) and NS5 region of hepatitis C virus. Therefore, it was attempted to incorporate the clone into an expression plasmid to produce a polypeptide encoded by the clone and immunoreact with the serum of a hepatitis C patient. Example 24 H encoded by clone 1530U
Modification of DNA to express CV-derived polypeptide The HCV encoded by clone 1530U is shown below.
The means used to select a part of the derived polypeptide, or a partial region, or to express the whole, is shown. The following method is shown as an example, and any of these can be used to express in any region of clone 1530U.

[1] Modification of DNA for Expression in Escherichia coli of a Part of HCV-Derived Polypeptide Encoded by Clone 1530U This method was performed without adding an extra amino acid sequence in E. coli. Used when expressing only the polypeptide. An open reading frame (hereinafter referred to as NS5) derived from the HCV gene from the 1246th C to the 1692th C in the nucleotide sequence number of SEQ ID NO: 75 in the sequence listing.
I'll call it N).
The initiation codon ATG is introduced upstream of the 5'side of the gene in accordance with this frame and expressed. In addition, 1 of NS5N
When the partial amino acid sequence was expressed, the start codon ATG was introduced upstream of the gene in the frame of the amino acid sequence, and the stop codon was also introduced in the same frame. When the start codon ATG is introduced at the 5 ′ upstream side of the gene, if a sequence derived from pUC19 is inserted between the base sequence derived from ATG and HCV, N of the amino acid sequence shown in SEQ ID NO: 7 of the sequence listing will be used. A polypeptide other than the polypeptide encoded by the HCV gene may be added to the end (amino terminal side).
Further, PCR was used to modify the DNA. First, the primer used to obtain the DNA fragment used to express the polypeptide NS5N was synthesized D
NA was used.

The base sequence for the 5'primer is MSNS5-1; 5'GCAAGCTTATGCAGC.
GTGGGGTACAAGGGGGTT 3 '(SEQ ID NO: 183). The base sequence for the 3'primer is MSNS5-2; 5'GCGAATTCAGACTTTT.
CATCAGAGCTGTGACCCAACCGTAT
ATTGGGTT 3 '(SEQ ID NO: 184). 20 pmol / μl of these two synthetic DNAs
Was prepared. This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUC2 was used as template DNA.
217 (or the plasmid pUCN22-1 can be used because it also contains the same region).
The primer was prepared by using the solution prepared as described above in 5 ', 3'
2 μl was used for each of the primers. First, the prepared sample was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. 1
Minutes later, Taq DNA polymerase (7 units / μ
1, AmpliTaq ™ Takara Shuzo) (0.5 μl) was added and mixed, and then overlaid with mineral oil. This sample was used as DNATherma manufactured by Perkin Elmer Cetus.
l Cycler 1 min at 95 ° C, 1 min at 55 ° C, 1 ° C at 72 ° C
Treated 25 times in minutes.

This sample was treated with phenol /
Chloroform treatment, ethanol precipitated, was separated and extracted with acrylamide gel after cutting the DNA fragment Hin dIII, with Eco RI. Further Hin cloning vector pUC19 dIII, by a conventional method and ligated to the Eco RI site, cloned, and screened to obtain plasmid PUCNS5N, and their nucleotide sequences were determined. Clone N obtained by this method
In the nucleotide sequence of S5N, the DNA fragment shown below is added to the 5'side of the DNA fragment shown as 1246th to 1692nd in SEQ ID NO: 75 of the sequence listing. 5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 ' Number: 15
5) 5 'end has a Hin dIII site, owns followed by a start codon ATG. At the 3'end, the following DNA fragment is added to the 3'side of the DNA fragment shown from 1246th to 1692nd in SEQ ID NO: 75, 5'TGATGAAGATCTGAATTTCGC 3'3'ACTACTTCTAGACTTAAGCG 5 '.
(SEQ ID NO: 156) There is an Eco RI site from the 3'-most side, and B is inside it.
It carries the gl II site followed by two stop codons TGA.

[2] Modification of DNA for expressing MKCNS5 region in insect cells The MKCNS5 region is 4 in SEQ ID NO: 75 of the sequence listing.
HCV having the 15th to the 1411th amino acid sequence
Gene-derived open reading frame (ORF)
Is. The initiation codon ATG is introduced upstream of the 5'side of the gene in accordance with this frame and expressed. When the start codon ATG is introduced at the 5 ′ upstream side of the gene, the amino acid sequence encoded by the gene (or part thereof)
A polypeptide other than the polypeptide encoded by the HCV gene may be added to the N-terminus (amino terminal side) of.
Moreover, when the clone is introduced into a vector having a start codon derived from an expression vector in insect cells, the frame of the clone may be aligned with the frame of the start codon derived from the vector. Also in this case, a polypeptide other than the polypeptide encoded by the HCV gene may be added to the N-terminus (amino terminal side) of the amino acid sequence (or part thereof) encoded by the gene. In an insect cell, if the polypeptide is an HCV-derived polypeptide containing at least the amino acid sequence of amino acid numbers 415 to 1411 of SEQ ID NO: 75 in the sequence listing, it is expressed in the cell as a single polypeptide. , It was then processed by being modified with sugar chains and accumulated in cells. MK for constructing expression vector
PCR was used to modify the DNA corresponding to the CNS5 region. At this time, the synthetic D shown below was used as the primer.
NA was used.

The base sequence for the 5'primer is MKCNS5-1; 5'GCCGCTAGCATGGGG.
TACAAGGGGGTTTTGGCGGG 3 '(SEQ ID NO: 185). The base sequence for the 3'primer is MKCNS5-2; 5'GCCGCTAGCTCATCG.
GTTTGGGGAGGCAGGTAGAT 3 '(SEQ ID NO: 186). These primers have Nhe at both ends of the gene.
The I-site is designed to be introduced. Of course, the transfer vector pBlue shown below
This is to introduce the gene into the Nhe I site of Bac (Invitrogen). When it is introduced into another transfer vector for expressing insect cells, it can be similarly carried out by introducing a restriction enzyme site suitable for the vector into the gene fragment. 20 pmol of these two synthetic DNAs
/ Μl. Preparation of PCR reaction solution and post-treatment were performed in the same manner as in [1]. However, 20 ng of the plasmid pUC1530U was used as the template DNA, and the combination of MKCNS5-1 and MKCNS5-2 was used as the primers. PCR cycle is 95 ° C for 1 minute, 50 ° C for 1 minute
Min, 72 ° C. for 5 minutes, and after repeating this 10 times, 95
1 minute at 65 ° C., 1 minute at 65 ° C. and 5 minutes at 72 ° C. were repeated 20 times to obtain the desired DNA fragment with a length of 3013 bp.

After cutting the DNA sample with Nhe I, a DNA fragment having a desired length was separated by acrylamide gel,
Extracted. Furthermore, the transfer vector pBlue
The Nhe I site of Bac (Invitrogen) was ligated and cloned by a conventional method, and a clone having only one DNA fragment inserted in the Nhe I site was screened to obtain primers MKCNS5-1 and MKCNS5-1.
A plasmid pBlueMKCNS5 derived from the DNA fragment obtained from KCNS5-2 was obtained. The expression unit of these plasmids has a structure in which a HCV gene-derived DNA fragment is ligated in the forward direction at the cloning site Nhe I cleavage site downstream of the polyhedrin promoter, referring to the one shown in the protocol of Invitrogen. Was there. Example 25 Clone NS5N, Clone MKCNS4
Expression of HCV-Derived Polypeptide Encoded by bNS5 in Escherichia coli The polypeptide encoded by this clone is a part of the polypeptide encoded by cDNA derived from hepatitis C patient serum, which is expressed in E. coli as it is. Let As shown below, pCZ44 (Japanese Unexamined Patent Publication No. 1-124387) was used as an expression vector, and it was performed by subcloning the cDNA of the clone into pCZ44.

First, the clone NS obtained in Example 24
DNA fragments Hid having the sequence 5N III, Bgl I
Are sufficiently cut by I, phenol / chloroform treatment, and ethanol precipitation, Hin dII both ends each
A DNA fragment having the cDNA as the protruding ends of the I and Bgl II sites was separated by acrylamide gel and extracted. Similarly, the expression vector pCZ44 also contains Hin dI.
II, are sufficiently cut by Bgl II, expression vector the longer DNA fragment having the ability similarly prepared above, this prepared from the clones were Hin dIII, Bgl I
It was ligated so as to have one I fragment, and further cloned according to a conventional method. The resulting plasmid was named plasmid pCZNS5N derived from each clone NS5N. As another attempt, an expression vector pCEX-2T that can express a fusion protein instead of pCZ44
(Manufactured by Pharmacia) was used to construct an expression vector. In this case, the operation was basically performed according to the instructions of Pharmacia. First, the expression vector pGEX-2T
After cutting with Bam HI and Eco RI, a Hin dIII linker was inserted so that the amino acid and codon reading frames of the clone would match, and the Eco RI site on the 3'side and Hin dIII on the 5'side (made by the linker). A DNA fragment prepared from the DNA fragment of the clone was inserted into the site.

For example, an HCV-derived polypeptide (referred to as MKCNS4bNS5, which is composed of 863 amino acid sequences from the 306th position to the 1168th position of the amino acid sequence shown in SEQ ID NO: 75, which will be described below, further referred to as MKCNS4bNS5. Fragment Clone MKCNS4b
NS5. ) Is expressed in E. coli. The area is H
It is considered to be an HCV-derived antigen protein that immunologically reacts with the serum of CV patients at a high rate. P to express the region
An expression vector may be constructed in the same manner as in the method using CZ44, but here, GST (β-glutathione-S is used.
-Expressed as a fusion protein linked to transferase. Plasmid pUC2218 2μg the Hin d
After completely cleaving with III, Pvu II, and Ssp I, the DNA fragment containing the region derived from clone 2218 was separated and extracted by agarose gel to obtain about 200 ng. This is blunt-ended and the DNA fragment 2218 (Hin / Pv
from u / T4), it was inserted into the Hin dIII site of pCEXH10. In other words, pGEXH10 is pGEX-
2T the Bam HI site and the Eco RI nucleotide sequence of over the site 5 'GGATCC CCCC AAGCTT GGGG GAATTC 3 ' Bam HI Hin dIII Eco RI ( SEQ ID NO: 187) is adapted to the. 1 μg of pCEXH10 was Hin
After being completely cleaved with dIII, blunt-ended to obtain pCEX
20 ng of H10-derived DNA fragment and DNA fragment 2218
Ligation reaction with 50 µg of (Hin / Pvu / T4), transformation and cloning by a conventional method, and N2 of DNA fragment 2218 (Hin / Pvu / T4) on the GST side.
It was possible to obtain the plasmid pGEX2218 constructed so as to come to the site corresponding to the 2 region.

E. coli JM109 strain harboring the plasmid pCZNS5N was transformed into L-Broth (Molecular
Cloning (Molecular Cloning)
1982 Cold Spring Harbor (Cold
(Spring Harbor)), and cultured overnight at 37 ° C. This is diluted with new L-Br so that it is diluted 50 times.
After inoculation into oth and shaking culture at 30 ° C. for 2 to 4 hours, IPTG (isopropyl-β-D-galactopyranoside) was added to the medium to 2 mM, and the mixture was further cultured for 3 hours or more. By culturing the bacterium in the presence of IPTG in this manner, a recombinant derived from a single clone (in this case, Escherichia coli JM109 strain transformed with only the plasmid) is used, using the plasmid derived from the clone. And was induced to express the HCV-derived polypeptide NS5N. The deduced amino acid sequence of the polypeptide encoded by the cDNA derived from clone NS5N is 124 of the amino acid sequences set forth in SEQ ID NO: 75 in the sequence listing.
It is the amino acid sequence from the 6th position to the 1692nd position. Similarly, with the plasmid pGEX2218, an expression product can be obtained as a fusion protein of β-glutathione-S-transferase (GST) and the polypeptide MKCNS4bNS5. In this case, GST has a site that is specifically cleaved by thrombin at the C-terminal as described in the manual of Pharmacia, and further contains the sequence of clone 2218 at the C-terminal side (in this case, the sequence derived from pUC19 is also a little The polypeptide is connected (although I brought it in).

In order to obtain the fusion protein, Escherichia coli transformed with the plasmid pGEX2218 was cultured in the presence of IPTG in the same manner as the above-described protocol for expressing the HCV-derived polypeptide from the plasmid pCZNS5N, and the HCV-derived Protein is GST C
It was induced to express the fusion protein with the termini. Example 26 Expression of MKCNS5 region in insect cells Plasmid pBlueMK constructed in Example 24 [2]
In order to express the HCV-derived protein encoded by CNS5 in insect cells, Maxbac (MA), a baculovirus expression manual of Invitrogen, was used.
XBACTMBACULOVEIRUS EXPRESS
ION SYSTEM MANUAL VERSION
1.4, hereinafter referred to as Maxback. ) Was basically referred to. Example 24
Transfer vector pB produced by [2]
Restriction enzyme Nh of lueBac (Maxback page 37)
The plasmid in which a DNA fragment containing an HCV gene was inserted at the eI cleavage site was assembled according to the method of Maniatis et al. (“Molecular cloning”, Cold Spring Harbor Laboratory, pages 86 to 96 (1982)). The recombinant Escherichia coli was recovered and purified to obtain a large amount of HCV gene transfer plasmid DNA. MaxBack according to the method on page 27, kit AcNPV
1 μg of viral DNA and DN derived from HCV gene
2 μg of the plasmid containing the A fragment was mixed with Sf9
The cells were co-infected to transform Sf9 cells.

That is, first, 2 × 10 5 of Sf9 cells were placed in a TMN-FH medium (Invitrogen) containing 10% FCS (fetal bovine serum) in a petri dish having a diameter of 6 cm.
^It culture | cultivated until it became ⁶ / sheet. Next, from the petri dish, TMN-
Grace (Grace) medium (GIBC (GIBC) containing 10% of FCS of 0.75 ml except FH medium)
O)) was added. Add 0.75 ml of the transfection buffer included in the kit to the above-mentioned mixed DNA, mix well by vortexing, and then Grace.
The DNA solution was slowly added dropwise over the medium. 27 ° C 4
Allow to stand for a period of time, then remove Grace medium and
Add 3 ml of TMN-FH medium containing 2% to a Petri dish and 2
It was cultured at 7 ° C for 6 days. On day 3, polyhedra were observed in some cells, and on day 6, most cells formed polyhedra. Transfer this culture supernatant to a centrifuge tube and
The supernatant obtained by centrifugation at 00 rpm for 10 minutes was used as a co-infection virus solution.

[0224] This co-infectious virus solution contained 10 ⁸ viruses per ml, and about 0.5% of them was recombinant. The plaque isolation method was used to isolate the recombinant virus. The method is as follows. The co-infected virus solution was diluted to 14 ⁻⁴ , 10 ⁻⁵ . 1.5 x 10 ⁶ per piece in 6 cm Petri dish in advance
After culturing individual cells and adsorbing them, the medium was completely removed, and 100 μl of each of 10 ⁻⁴ solution and 10 ⁻⁵ solution was added to each petri dish. In order to prevent the cells from drying, the petri dish was tilted every 15 minutes so that the virus solution was spread over the entire surface. As described above, 105% of baculovirus agarose (manufactured by Invitrogen) was used while infecting at room temperature for 1 hour.
The mixture was autoclaved at 10 ° C for 10 minutes, mixed with TMN-FH medium containing 10% FCS warmed at 46 ° C at a ratio of 1: 3, and kept at 46 ° C. To this incubated medium, 5-bromo-4-chloro-3-indolyl-β-D-galactoside was added to a final concentration of 150 μg / μl (according to Maxback's pages 16-17). The medium was called agarose-containing x-gal medium.

After the infection was completed, the virus solution was completely sucked up, and the agarose-containing x-gal medium, which was kept warm, was gently added in an amount of 4 ml per dish so that the cells would not come off. The petri dish was allowed to stand with the lid slightly displaced until the agarose was solidified and dried, and then, the lid was put on and turned over, and incubation was carried out at 27 ° C. After culturing for 6 days, blue plaques in which polyhedra were not formed were found by a phase contrast microscope. This recombinant plaque is sucked up with agarose using a Pasteur pipette and 1 ml of TMN-
The virus was suspended by pipetting many times in FH medium. This series of operations (an operation of infecting and then culturing for 6 days to isolate a virus having the transfer plasmid DNA) is called a plaque method. The same plaque method was performed using 100 μl of the virus suspension. These series of operations are repeated three times, and HCV free from contamination by wild strains.
A recombinant virus carrying the derived glycoprotein gene was obtained.
Absorb the virus plaque with a Pasteur pipette,
It was mixed well with 1 ml of TMN-FH medium. This primary recombinant virus solution has a low virus concentration, so
After taking 00 μl and adsorbing to Sf9 cells cultured in a 6 cm dish at half confluence, T containing 10% FCS
4 ml of MN-FH medium was added, and after 3 days, the culture supernatant was collected and used as a recombinant virus solution for infection. In order to produce HCV-derived glycoprotein, 5 × 10 ⁶ S was previously prepared.
10 m of TMN-FH medium containing f9 cells and 10% FCS
It was suspended in 1 and sprinkled on a 9 cm Petri dish and adsorbed for 1 hour. Remove the medium from the dish and add 250 μl of the recombinant virus solution for infection to spread it over the whole area, then 10%
10 ml of TMN-FH medium containing FCS was added, and the mixture was allowed to stand at 27 ° C for 4 days. The cells were thoroughly suspended in 1,000 ml of phosphate buffered saline.

Thus, the HCV-derived glycoprotein was expressed in Sf9 cells infected with the virus. Example 27 Identification of Expressed Product as HCV Derived Antigen Hepatitis C virus-infected human serum
The polypeptide was identified as a hepatitis C-related antigen because it immunologically reacts with the polypeptide expressed at 5,26. This identification was performed by the Western blotting method shown below. First, for example, Escherichia coli transformed with pCZNS5N and pCEX2218 described in Example 25 was cultured for 3 hours or overnight in the presence of IPTG to induce expression of the polypeptide. 1,000 μl of this culture solution was added to 6,500 r. p. m. The recombinant strains were harvested by centrifugation for 10 minutes. This precipitate is SDS-
Sample processing solution for polyacrylamide gel electrophoresis (2% S
50 mM containing DS, 5% mercaptoethanol, 10% glycerin and 0.005% bromophenol blue
0.2 ml final volume in Tris-HCl buffer, pH 6.8)
Was prepared by dissolving. Next, these samples were boiled at 100 ° C. for 10 minutes. 10 μl of the sample thus obtained was added to 0.1% SDS-15.0% polyacrylamide gel (70 × 85 × 1 mm). At that time, as a marker protein, Pharmac
"LMW KitE" (low molecular weight marker protein) manufactured by ia was used. Tris buffer (25
mM Tris pH 8.3, 192 mM glycine,
Using 0.1% SDS), the gel was electrophoresed for 45 minutes at a constant current of 30 mA, and then the gel was overlaid with a S & S nitrocellulose filter (BA-83) according to a conventional method, with the gel side serving as the cathode and the nitrocellulose filter side. 12 as the anode
Transfer was performed by applying a constant current of 0 mA for about 20 minutes.

The transferred nitrocellulose filter was cut so as to separate the marker protein portion and the sample portion, and the marker protein portion filter was stained with 0.1% (W / V) Amido Black 10B. Of the filter of 0.01M phosphate saline (PB containing 5% (W / V) bovine serum albumin)
S) Soaked in pH 7.4. Next, 0.01M phosphate saline (PB containing 5% (W / V) bovine serum albumin)
S) 10 ml of hepatitis C patient serum diluted 50 times with pH 7.4 is placed on the filter of the sample portion, and it is kept at room temperature for 2 hours.
After standing for 0.1 hours (V / V) Tween manufactured by Sigma
The cells were washed with PBS containing n20 three times for 20 minutes each. Next, a peroxidase-labeled anti-human IgG (Ga) diluted 1,000 times with PBS was placed on the sample part filter.
10 ml of the product (manufactured by Ppel Co.) was put on the plate, reacted at 37 ° C. for 1 hour, and washed with PBS containing 0.1% (V / V) Tween 20 three times for 20 minutes each. Next, 100 ml of peroxidase color developing solution (60 mg 4-chloro-1-naphthol, 20 ml methanol, PBS8) of the sample part filter.
It was immersed in 0 ml of hydrogen peroxide (20 μl). The colored filter was washed with distilled water and then compared with the marker protein portion. The protein colored in this manner had a reasonable molecular weight as a product derived from the HCV gene introduced with any of the proteins expressed from the plasmids pCZNS5N and pGEX2218, and could be identified as a hepatitis C-related antigen. The plasmid pCEX2218 was derived from pGEX, and the expression product was GS at the N-terminus of the HCV-derived polypeptide.
It is provided by the cleavage site of T and thrombin.

Example 28 Method for Obtaining Clone T7N1-25 [1] Method for Obtaining Clone 1925 Clone N19MX24A-1 obtained in Example 11 [1]
And the overlapping region of clone MX25-1 is used to ligate by PCR reaction. Clone clone N19MX24A-1 and clone MX25-1
The DNA fragment having the nucleotide sequence of (977 base pairs and 849
1 μl (about 0.5 to 1 μg / μ)
l) each, 10x PCR buffer (100 mM Tris
-HCl (pH 8.3), 500 mM KCl, 15 m
M MgCl2, 1% gelatin) 10 μl, 2.5 mM
8 μl of 4dNTP, synthetic primers MS122 and M
S152 (both 20 pmol / μl) 5μ each
1 and 76.5 μl of water were added to the reaction solution. After mixing well,
First, the temperature was kept at 95 ° C for 5 minutes and then rapidly cooled to 0 ° C. One minute later,
Taq DNA polymerase (7 units / μl, Am
0.5 μl of pliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample is Perkin
Elmer Cetus DNA Thermal C
1 cycle at 95 ° C for 1 minute, 37 ° C for 1 minute, 72 ° C for 5 minutes
Treated 0 times. Then, after keeping the temperature at 97 ° C. for 2 minutes, it was rapidly cooled to 0 ° C. and kept for 2 minutes. Furthermore, Taq DNA was added to the reaction solution.
Polymerase (7 units / μl, AmpliTaqT
M Takara Shuzo) Add 0.5 μl in the same manner at 95 ° C for 1 minute, 5
It was treated 15 times at 5 ° C. for 1 minute and 72 ° C. for 5 minutes. Finally 72 ℃
After keeping the temperature for 7 minutes, the reaction aqueous solution was treated with phenol / chloroform and precipitated with ethanol. 2 types of DN
The A fragment was ligated and amplified by PCR reaction. The ligated DNA sample was separated by agarose electrophoresis (Molecular Cloning).
ural Cloning 1982 Cold Spring Harb
or)), a DNA fragment having a length of about 1000 base pairs was excised from the gel (Molecular Cloning 1982 Cold Spring Harbor (ColdSpring).
Harbor)). The obtained DNA fragment was treated in the same manner as in Example 3 and incorporated into the SmaI site within the multicloning site of the pUC19 cloning vector. This was cloned and screened (the method is the same as in Example 3), and the plasmid pUC19 is used.
I got 25. The hepatitis C patient serum-derived clone thus obtained was named clone 1925.

[2] Method for Obtaining Clone T7N119 pUCN1-1 has cDNA clone N1-1 derived from hepatitis C patient serum in the SmaI site of plasmid pUC19, and its insertion direction is 5'side in the translation direction of HCV gene. Has the HindIII site of the plasmid pUC19. This plasmid pUCN1-1 was Hin
It was completely digested with dIII and NcoI, and the larger DNA fragment pUCN1HN having a vector function was excised.
10 ng of the DNA fragment was ligated with the synthetic DNA shown below. MS168 AGCTTACTAGTTTAATACGA
CTCACTATAGG (31 bases, SEQ ID NO: 18
8) MS169 CTGGCACCCTAGATAGTGAGT
CGTATTAACTAGTA (33 bases, SEQ ID NO:
189) MS170 TGCCAGCCCCCTGATGGGGG
GCGACACTCCACCATAGATCACTCC
(44 bases, SEQ ID NO: 190) MS171 TCACAGGGGAGTGATCTAT
GGTGGAGTGTCGCCCCCATCATGGGG
G (45 bases, SEQ ID NO: 191) MS172 CCTGTGAGGAACTACTGTC
TTCACGCAGAAAGCGTCTAGC (40 bases, SEQ ID NO: 192) MS173 CATGGCTAGACGCTTTCTG
CGTGAAGACAGTAGTTCC (37 bases, SEQ ID NO: 193) (These sequences are written in order from the 5 ′ side to the 3 ′ side.)

That is, except for MS 168 and MS 173, phosphoric acid is kaitinated to the 5 ′ side, and kinated MS 169, MS 170, MS 171, MS 172.
100 pmol of each and MS168, MS173 of 20 pmol each are added, ligated by a conventional method with T4 DNA ligase, phenol treatment and ethanol precipitation. 1/4 of the obtained DNA sample was added to pUCN1.
Ligation reaction with 10 ng of HN, 5'of clone N1-1
Side, HindIII site, SpeI site, T7R
A plasmid pUCT7N1 was constructed in the order of the NA polymerase promoter sequence, the 5'untranslated sequence of the HCV gene, and the DNA fragment encoding the gene of the HCV core protein N-terminal region. This plasmid pUCT7N1
Has a clone T7N1 between the HindIII site and the SmaI site. By using this plasmid pUCT7N1 instead of pUCN1-1 having clone N1-1, clone T7N1N3N10 was obtained by the same construction method as in Example 4 [2]. This clone T7N1
N3N10 and clone N2 obtained in Example 11 [2]
In order to ligate the overlapping regions of 7N19-1 without overlapping, it was utilized that both have common and only one restriction enzyme cleavage site.

That is, the clone N1N3N10 was cut at the 3'side of the 1332nd G by the restriction enzyme BamHI, and the clone N27N19-1 was cut at the 3'th 3'of the G. In addition, the plasmid pUCN1N3N10,
Considering that both pUCN27N19-1 have the same DNA fragment in the translation direction of the HCV gene (HindI of pUC19 at the 5'side on the HCV gene).
II), pUCN27N19-1 with EcoRI,
In BamHI, a DNA fragment containing the 5'side of clone N27N19-1 (this DNA fragment is the DNA fragment from the EcoRI site to the SmaI site of plasmid pUC19 at the 3'side of clone N27N19-1 of cDNA derived from hepatitis C patient serum). The plasmid pUCN119 could be obtained by ligating to the DNA fragment containing the vector function, which was excised with EcoRI and BamHI of pUCN1N3N10, cloned and screened. This plasmid is composed of HindIII site, SpeI site, T7 RNA polymerase promoter sequence, H
Part of the 5'untranslated sequence of the CV gene, clone N1-
Clone T7N in which 1, N3-1, N10-1, N27-3 and N19-1 were ligated without overlapping.
I had 119.

[3] Method for Obtaining Clone T7N1-25 Since clone 1925 obtained in [1] and [2] clone T7N119 which are overlapping with each other are ligated without overlapping, common to both, and only one The fact that there are no restriction enzyme cleavage sites that overlapped was used. That is, the clone T7 was cloned by the restriction enzyme PvuI.
N119 is the 3'of the 288th T (SEQ ID NO: 16 in the sequence listing) in the base sequence of clone N19-1 in the N19 region.
It is cut only at one side, and clone 1925 is also cut only at the 3'1 position of the 288th T. In addition, the plasmids pUCT7N119 and pUC1925 were
Considering that the NA fragments are in the same direction, pU
C1925 is a DNA fragment encoding an HCV-derived gene with PvuI and EcoRI (this DNA fragment is C
Plasmid pU on the 3'side of cDNA derived from hepatitis patient serum
D from C19 EcoRI site to SmaI site
NA fragment was added) and pUCT
7N119 was replaced with a region containing the 3'portion of the N19 region between the PvuI site and the EcoRI site, cloned and screened to obtain plasmid pUC
T7N1-25 could be obtained. This plasmid is clone T7 without overlapping N19 region.
N119 and Clone 1925 have restriction enzyme site Pvu
Had clone T7N1-25 linked by I.

Example 29 Method for Obtaining Clone T7N1-30U [1] Method for Obtaining Clone 1530UNot Hind of Clone 1530U obtained in Example 23 [5]
The III site is immediately 3'to the HCV-derived cDNA sequence. To introduce a NotI site into this HindIII site, pUC1530U was completely cleaved with HindIII, and then blunt-ended with T4 DNA polymerase by a conventional method, 10 ng fragment and EcoRI
-NotI-BamHI adapter (Takara Shuzo) 1
The reaction mixture was ligated with T4 DNA ligase by a conventional method after adding a 100-fold molar amount. After phenol treatment and ethanol precipitation, the product was further cleaved with NotI and ligated. Plasmid pU by cloning and screening
C1530UNot could be obtained. [2] Method for obtaining clone T7N1-30U Overlapping clone T7N obtained in Example 28
1-25 and clone MX25N obtained in Example 17 [4]
15-1 and the clone 1530U obtained in [1] of this example.
In order to ligate the Nots without overlapping, it was utilized that both restriction sites, which are common to both, and where there was only one restriction enzyme site were overlapping.

That is, pUCT7N1-25 was cleaved with restriction enzymes SpeI and PstI to give clone T7N1-2.
DNA fragment T7N1-25SP containing most of 5 was cut out from the gel by a conventional method to obtain about 1 μg. Then pU
Restricting CMX25N15-1 to the restriction enzymes PstI and EcoT2
After cutting with 2I, a DNA fragment MX25N15-1PE containing most of clone MX25N15-1 was cut out from the gel by a conventional method to obtain about 1 μg. Finally, pUC15
DNA fragment 1 containing most of clone 1530U obtained by cutting 30UNot with restriction enzymes EcoT22I and NotI
Approximately 1 μg of 530 UEN was cut out from the gel by a conventional method
Got The above three DNA fragments (T7N1-25SP,
MX25N15-1PE, 1530UEN) was digested with about 200 ng of each of restriction enzymes SpeI and NotI, and λZapII (Stratagene) was used.
1 μg (manufactured by ne) was ligated according to the attached protocol. Furthermore, GIGAPACKII PACKI
Packaging was performed using NG EXTRACTS and GOLD (manufactured by Stratagene). From ligation to titer check, lambda DNA amplification, isolation,
All operations, such as packaging, followed the attached protocol. For the screening of recombinant λ phage, 20 white plaque phages were isolated, and the plasmid pBLUESCRIPT SK (−) was isolated using a helper phage.
The DNA fragment inserted after subcloning was examined. Plasmid pBLUESCRIPT SK
Two clones out of 20 clones subcloned in (-) are plasmid pBLUESCRIPT SK.
Between the (-) SpeI site and NotI site, HC
Three DNA fragments derived from V are T7N from the SpeI site side.
1-25SP, MX25N15-1PE, 1530UE
They were connected one by one in the order of N. P
It was named T7N1-30U. Plasmid pT7N1-
30U is ligated without overlapping three DNA fragments derived from HCV, and has clone T7N1-30U between the SpeI site and the NotI site. Sequence listing 101 shows the nucleotide sequence of clone T7N1-30U and the amino acid sequence of the HCV-derived polypeptide encoded thereby.

Example 30 Large expression of polypeptide CORE and polypeptide C + N23 [1] Method for obtaining clone CN23 pUCN2 having clone N23-1 obtained in Example 16
Clone N23-1 by PCR using 3-1 as a template
A region to be expressed was created. First, the following synthetic DNA was used as the primer used. The base sequence for the 5'primer is MS165; 5'GCAAGCTTATGCTGCTG.
TCGCCCGGGGCCCATCT3 '(SEQ ID NO:
194). The base sequence for the 3'primer is MS166; 5'GCGAATTCAGATCTTCA.
TCATGTGTTGCAGTCGATCAC 3 '
(SEQ ID NO: 195). 20 pmol / μl of these two synthetic DNAs
Was prepared. This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCN was used as template DNA.
23 ng of 100 ng was used, and 2 μl of the solution prepared as described above was used for the 5'and 3'primers.
Used one by one. First, the prepared sample was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler manufactured by Perkin Elmer Cetus at 95 ° C. 1
Min, 55 ° C. 1 minute, 72 ° C. 1 minute. further,
The treatment was performed 17 times at 95 ° C. for 1 minute, 65 ° C. for 1 minute, and 72 ° C. for 1 minute. This sample was treated with phenol / chloroform and ethanol-precipitated by a conventional method, and the DNA fragment was Hin-treated.
After cleaving with dIII and EcoRI, it was separated and extracted with acrylamide gel. Further cloning vector pUC
The HindIII and EcoRI sites of 19 were ligated by a conventional method, cloned and screened to obtain plasmid pUCN23A, and its nucleotide sequence was determined. Clone N23A obtained by this method
The nucleotide sequence of 5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: : 15
5) It has a HindIII site at the 5'end, followed by a start codon ATG. At the 3'end, the following DNA fragments are added to the 3'sides of the DNA fragments shown from 1st to 915th of SEQ ID NO: 50 in the sequence listing,

5'TGATGAAGATCTGAATTCGC 3'3'ACTACTTCTAGACTTAAGCG 5 '
(SEQ ID NO: 156) There is an EcoRI site from the 3'most side, and B is inside it.
It carries the glII site followed by two stop codons TGA. In addition, pCZCO obtained in Example 6 [1]
Cleavage RE with the restriction enzyme SacII and perform T4D
Blunt-ended with NA polymerase. Further BglI
It was cleaved with I, and a DNA fragment pCZCORE / SB having the vector part of pCZ and the N-terminal part of the core protein of HCV was separated and extracted on an acrylamide gel to obtain the fragment. Similarly, the plasmid pUCN23A was completely digested with restriction enzymes SmaI and BglII, and the DNA fragment having the sequence of clone N23-1 was separated and extracted with acrylamide gel to obtain DNA fragment N23A / SB. The 5'end of this DNA fragment corresponds to the 107th G of SEQ ID NO: 50 in the sequence listing and has a sequence from there to the 915th A of SEQ ID NO: 50, and the 3'side thereof has two stop codons and a BglII site. have. DNA fragment pCZCORE / SB 10 ng
And DNA fragment N23A / SB (100 ng) were ligated with T4 DNA ligase by a conventional method to obtain plasmid pCZC.
N23 was obtained. The plasmid has SEQ ID NO: 10 in the sequence listing between the HindIII site and the BglII site.
It has a clone CN23 having the base sequence shown in 2.

[2] Improvement of Expression Vector In order to improve expression efficiency by using a plurality of expression units in the expression vector, plasmid pCZCORE,
pCZCN23 was completely cleaved with the restriction enzymes BamHI and BglII, and the obtained DNA fragments CORE / BB and CN23 / BB encoding the polypeptide derived from HCV were recovered. Only 100 ng of the DNA fragment CORE / BB was used for 3 hours at 12 ° C. using T4 DNA ligase by a conventional method.
The ligation reaction was performed for 0 minutes. Phenol treatment, ethanol precipitation, digestion with restriction enzymes BamHI and BglII,
Plasmid pCZCORE completely digested with BglII and dephosphorylated by alkaline phosphatase by a conventional method.
Ligation reaction with 10 ng of plasmid pCZCORE
The expression unit of the polypeptide CORE sandwiched between the BamHI site and the BglII site of
Plasmid pCZCORE tandems 2, 3, 4, 8, 16 in which 4, 6, 12 were linked in tandem were obtained.
Similarly, DNA fragment CN23 / BB and plasmid pCZC
The combination of N23 yielded the plasmid pCZCN23 tandem 2, 3, 4, 6.

[3] Direct large-scale expression of polypeptide CORE and polypeptide CN23 [0238] The polypeptide CORE was prepared by using each of the expression vectors obtained in this Example [2] in the same manner as in Example 6 [1].
And the polypeptide CN23 was expressed in E. coli. In this case, first, the induction time in the system transformed with pCZCORE, the type of host, the number of tandems,
The culture temperature was examined. For example, the host is JM10
This was carried out using strains derived from K12 such as 9, DH5 and KS476, and strains derived from B strain. The expression level varied considerably depending on the host, and the strain from B strain and KS476 were the best, and DH5
And the expression level per medium was about 8 to 10 times. In addition, there was a considerable difference in the expression level depending on the time of induction. That is, 1 day of the recombinant (OD600 = about 1.5) cultured for 1 day was placed in an L-shaped tube for 10 ml.
0 ml of bactopeptone medium (manufactured by Difco; bactopeptone 20 g / 1, glycerin 0.2% v /
v, 0.1 M MgSO4, NaCl 10 g / 1,
Add 0.5 ml of 0.1% thiamine hydrochloride (160 μl / 1, ampicillin 100 mg / l) and incubate at 30 ° C. The turbidity of the culture solution is OD600 = about 0.5, about 0.8,
Induction was performed by adding IPTG at 5 points of about 1.2, about 2.0, and about 3.0. Among them, about OD600 = 0.
The number 1 expression was high in those obtained by applying induction to No. 5. Furthermore, the tandem number does not necessarily mean that the expression amount is large as much as the tandem number.
Even if we had up to 3 units of NA fragment CORE / BB in tandem, the expression was low and it started to increase suddenly from 4 and 8 and 16 were almost saturated and did not change much. Therefore, in summary, in order to express a large amount of the polypeptide CORE, a strain derived from strain B or KS476 was used as a host in a system transformed with pCZCORE tandem 8, and OD600 = about 0.5 was used for induction.
Culturing was carried out overnight at 0 ° C. In addition, of the plasmid pCZCN23 tandems 2, 3, 4, and 6 prepared for expressing the polypeptide CN23, the tandem 6 was used,
After that, it was expressed under the same conditions as pCZCORE tandem 8.

[0239]

[Sequence list]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

SEQ ID NO: 105 Sequence Length: 19 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CTCCACCATAGATCACTCC 19

SEQ ID NO: 106 Sequence Length: 18 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence AGGTCTAGTAGACACCGTGC 18

SEQ ID NO: 107 Sequence Length: 18 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence AGGAAGACTTCCGAGCGG 18

SEQ ID NO: 108 Sequence Length: 19 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CGTGAACTATGCAACAGGG19

SEQ ID NO: 109 Sequence Length: 18 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence ACCGCTCCGGAAGTCTTCC 18

SEQ ID NO: 110 Sequence Length: 18 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGGCAAGTTCCCTGTTGC 18

SEQ ID NO: 111 Sequence Length: 18 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCTGGATTCTCTGAGACG 18

SEQ ID NO: 112 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GAGGCCGTGAACTGCCGATGA 23

SEQ ID NO: 113 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TTCTCTAAGGTGGCNTCNGCNTG 23 N: Inosine

SEQ ID NO: 114 Sequence length: 21 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence CCGGACGCGTGTGAANCTNGNGT 21 N: Inosine

SEQ ID NO: 115 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CATCCAGGTACACACCGAACCA 23

SEQ ID NO: 116 Sequence Length: 24 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virns Sequence AACACACGGCCGCCCANCANGNAA 24 N: Inosine

SEQ ID NO: 117 Sequence length: 19 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence CCGGATCCCACAAGCCGTNGTNGA 19 N: Inosine

SEQ ID NO: 118 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence GACATGCATGTCATGATGGTA 20

SEQ ID NO: 119 Sequence Length: 26 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGCTGCAGCCGGTTTCATCCACTGCAC 26

SEQ ID NO: 120 Sequence Length: 26 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGGATCCTGCTTCGCCCCAGAAGGTC 26

SEQ ID NO: 121 Sequence length: 22 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence GACACATGTGTTGCAGTCGATC 22

SEQ ID NO: 122 Sequence length: 24 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence CGGTCCNAGNAGATATCTCNTTNCC 24 N: Inosine

SEQ ID NO: 123 Sequence Length: 35 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence ATGGGGCCCGGGGNANAGNAGNCTCCCCCTNCTNTC 35 N: Inosine

SEQ ID NO: 124 sequence length: 20 sequence type: nucleic acid topology: linear sequence type: cDNA origin: Hepatitis C virus sequence GGCTATACCGGGCACTTCGA 20

SEQ ID NO: 125 Sequence Length: 27 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGGATCCGGGCCTCACCCACATATAGG 27

SEQ ID NO: 126 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGGATCTCCCACCCTCCATCGTG 23

SEQ ID NO: 127 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence CTGCTCTCGCCCNGNCCCCAT 20 N: Inosine

SEQ ID NO: 128 Sequence length: 23 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence ATCACGGTGGGGNGCAGANACNGC 23 N: Inosine

SEQ ID NO: 129 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGTGCCTGNTTTNTGATGAGTG 21 N: Inosine

SEQ ID NO: 130 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGTGAGCATGGAGGGTGACCAC 21

SEQ ID NO: 131 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TCATCCCTCCCTCCGCTCGAAGC 21

SEQ ID NO: 132 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GTGGACGCCCTTNGCCTTTCATNTC 23 N: Inosine

SEQ ID NO: 133 sequence length: 21 sequence type: nucleic acid topology: linear sequence type: cDNA origin: Hepatitis C virus sequence ACGGATGTCNTTTCTCNGTNAC 21 N: inosine

SEQ ID NO: 134 Sequence Length: 30 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGCGGAATTCCTGGTCATAGCCTCCGTGAA 30

SEQ ID NO: 135 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGGGNATGGCCTATTGGCCTG 21 N: Inosine

SEQ ID NO: 136 sequence length: 21 sequence type: nucleic acid topology: linear sequence type: cDNA origin: Hepatitis C virus sequence GGCATGTGGGGCCCAGGGGAGGG 21

SEQ ID NO: 137 Sequence Length: 20 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGTGAGCCCGAACCGGATGT 20

SEQ ID NO: 138 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GTGGGTANTCCTGGACTCNTTNGA 23 N: Inosine

SEQ ID NO: 139 Sequence Length: 22 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence ACTACCGNGACGTGCTNAANGA 22 N: Inosine

SEQ ID NO: 140 Sequence Length: 30 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGGGGGATCCCGTATGATACCCGCTGCTCTTG 30

SEQ ID NO: 141 Sequence Length: 24 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence ATTGTCAGATATCACGGGGCCACTT 24

SEQ ID NO: 142 Sequence Length: 43 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTAAAAAAAAAAAAGGGGGATGGCCTTTTGGCCTGGA 43

SEQ ID NO: 143 Sequence Length: 17 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GTAAAACGACGGCCAGT 17

SEQ ID NO: 144 Sequence Length: 17 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CAGGAAACAGCTATGAC 17

SEQ ID NO: 145 Sequence Length: 35 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGAGCACAAATCCAAAACCCCCAAAGA 35

SEQ ID NO: 146 Sequence Length: 38 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGATCTTTCACCTACGCCCGGGGTCCCGT GGG 38

SEQ ID NO: 147 Sequence Length: 39 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTCTCAGATTCTCTGAGACGGCCC TCGT 39

SEQ ID NO: 148 Sequence Length: 17 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCTACTCCGGATACCAC 17

SEQ ID NO: 149 Sequence Length: 35 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGTCGGACGCTAGCATGAGCACAAATCCAAAACCCC 35

SEQ ID NO: 150 Sequence Length: 35 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGTCGGACGCTAGCAGGTCTCGTAGACCGGTGCATC 35

SEQ ID NO: 151 Sequence length: 40 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence GCGAATTTCGCTAGCTCAGGATTCTCTGAGACGGGCC CTCGA 40

SEQ ID NO: 152 Sequence Length: 35 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGCGGATCCACAAGCCGTGGTGGGAT 35

SEQ ID NO: 153 Sequence Length: 24 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CGGATCCCACAAGCCGTGGTGGGAT 24

SEQ ID NO: 154 Sequence Length: 43 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTTCTCATCACTCTAAGGTGGCGTGCGGTGGGG 43

SEQ ID NO: 155 Sequence Length: 11 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATG 11

SEQ ID NO: 156 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGATGAAGATCTGAATTCGC 20

SEQ ID NO: 157 Sequence Length: 34 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGTTCAACGCGTCCGGATGTCCGGA 34

SEQ ID NO: 158 Sequence Length: 23 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TTCAACGCGTCCGGATGTCCGGA23

SEQ ID NO: 159 Sequence Length: 43 Column Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGATCTTCATCATCAACAACCGAACCAGTT GCCCTGCG 43

SEQ ID NO: 160 Sequence Length: 34 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGATCGGGGGGTGCGGCAACAATAC 34

SEQ ID NO: 161 Sequence length: 23 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence ATCGGGGGGGGTCGGCAACAATAC 23

SEQ ID NO: 162 Sequence Length: 43 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGATCTTCATCATAAAGCTCTGATCATTC CCTGTCCT 43

SEQ ID NO: 163 Sequence length: 41 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence GCGTCGACGCTAGAGCATCGGATCCCCACAAGCCGTG GTGGAT 41

SEQ ID NO: 164 Sequence Length: 40 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGTCGACGCCATAGCATGTCAACGCGTCCGGATGT CCGGA 40

SEQ ID NO: 165 Sequence length: 40 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence GCGTCGGACGCTAGCATGATCCGGGGGGTGCGGCAAC AATAC 40

SEQ ID NO: 166 Sequence Length: 40 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCGCTAGCTCACTCTAAGGTGCGTCGGGCGTGGG 40

SEQ ID NO: 167 Sequence length: 40 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCGGCTAGCTCAACCCGAACCAGTTGCC CTGCG 40

SEQ ID NO: 168 Sequence Length: 40 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCGCTAGCTCAAAGCTCTGACTTATCCCT GTCCT 40

SEQ ID NO: 169 Sequence Length: 32 Sequence Type: Nucleic Acid Topology: Linear Sequence Insertion: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGTGGTGTGGATGATGCTGCTG 32

SEQ ID NO :: 170 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGGTTGTGGATGATGCTGCTG 21

SEQ ID NO: 171 Sequence length: 44 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence GCGAATTCAGACTCTCATCATCCTCCGGGCGAGAC NGGNAGNCC 44 N: Inosine

SEQ ID NO: 172 Sequence Length: 31 Sequence Type: Nucleic Acid Topology: Linear Sequence Citrus: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGGGCAACGAGNTNCTNCTNGG 31 N: Inosine

SEQ ID NO: 173 Sequence Length: 20 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGCAACGAGNTNCTNCTNGG 20 N: Inosine

SEQ ID NO: 174 Sequence Length: 41 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTTCTCATCACTCAGCCGTATGAG ACACTT 41

SEQ ID NO: 175 Sequence Length: 31 Sequence Type: Nucleic Acid Topology: Linear Sequence Persimmon: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGCTGTCGCCCGGGCCCATCTC 31

SEQ ID NO: 176 Sequence Length: 20 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: HePatitis C virus Sequence CTGTCGGCCCGGGGCCCATCTC 20

SEQ ID NO: 177 Sequence Length: 41 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTTCTCATCAACATGTGTTGCAGTC GATCAC 41

SEQ ID NO: 178 Sequence Length: 32 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGGGCTATACCGGACTTNGAC 32 N: Inosine

SEQ ID NO: 179 Sequence Length: 21 Sequence Type: Nucleic Acid Topology: Linear Sequence Insertion: cDNA Origin: Hepatitis C virus Sequence GGCTATACCGGNGACTTNGAC 21 N: Inosine

SEQ ID NO: 180 Sequence length: 35 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGATCTTCAGTGCTTCGCCCAGAAGGT 35

SEQ ID NO: 181 Sequence Length: 29 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGCCTAGCATGTGGGTGTGGATGATGGCTG 29

SEQ ID NO: 182 Sequence Length: 38 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCGCTAGCTCACAGCCGGTTCATCCACTG CAC 38

SEQ ID NO: 183 Sequence Length: 32 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGCAGCGTGGGTACAAGGGGGTT 32

SEQ ID NO: 184 Sequence Length: 47 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGATCTTCATCATAGAGCTGACCCAACCG TATTATTGGTT 47

SEQ ID NO: 185 Sequence Length: 33 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence: GGCCTAGCATGGGGTACAAGGGGTTTTGGCGG G 33

SEQ ID NO: 186 Sequence length: 32 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence GGCCTAGCTCATCGGTTGGGGAGCAGGTAGAT, 32

SEQ ID NO: 187 Sequence Length: 26 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GGATCCCCCCAAGCTTGGGGGAATTC 26

SEQ ID NO: 188 Sequence Length: 31 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence AGCTTACTAGTATAACACGACTCACTATAGAGG 31

SEQ ID NO: 189 Sequence Length: 33 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CTGGCACCCTATAGTGAGTCGTATTTAACTAGTA 3 3

SEQ ID NO: 190 Sequence Length: 44 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence TGCCAGCCCCCTGATGGGGGCGACACTCCACCATACATACCACTCC 44

SEQ ID NO: 191 Sequence Length: 45 Sequence Type: Nucleic Acid Topology: Linear Sequence Insertion: cDNA Origin: Hepatitis C virus Sequence TCACAGGGGGAGTGATCTATGGTGGAGTGTCGCCCC CATCAGGGGGG 45

SEQ ID NO: 192 Sequence Length: 40 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CCTGTGAGGAACTACTGTCTTCACCGCAGAAAGCGT CTAGC 40

SEQ ID NO: 193 Sequence Length: 37 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence CATGGCTAGACGCCTTTCTGCTFGAAGACAGTAGTT CC 37

SEQ ID NO: 194 Sequence Length: 33 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCAAGCTTATGCTGCTGTCGCCCCGGGCCCATCT 3 3

SEQ ID NO: 195 Sequence Length: 38 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTCTCATCATCATGTGTGCAGTCGATC AC 38

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0313

[Correction method] Change

[Correction content]

SEQ ID NO: 75 Sequence length: 4296 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: 1530U Sequence GCGGATCCT CCA CCT CCA TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG 51 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg 1 5 10 CTG AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA 99 Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly 15 20 25 30 GCC GTT CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC 147 Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile 35 40 45 ATG GCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG 195 Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val 50 55 60 CTG GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG 243 Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr 65 70 75 GGC AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC 291 Gly S er Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala 80 85 90 GTT ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA 339 Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu 95 100 105 110 GAG TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC 387 Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala 115 120 125 GAG CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG 435 Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys 130 135 140 CAA GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT 483 Gln Ala Glu Ala Ala Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu 145 150 155 GAG ACC TTC TGG GCG AAG CAC ATG TGG AAT TTC ATC AGC GGG ATA CAG 531 Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 160 165 170 TAC TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA 579 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser 175 180 185 190 CTG ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC ACC CAA T AT 627 Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr 195 200 205 ACC CTC CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC 675 Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala 210 215 220 CCC CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG 723 Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala 225 230 235 GCT GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG 771 Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala 240 245 250 GGT TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG 819 Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met 255 260 265 270 AGC GGT GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC 867 Ser Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala 275 280 285 ATC CTC TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA 915 Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile 290 295 300 CTG CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG C AG TGG ATG AAC 963 Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn 305 310 315 CGG CTG ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC 1011 Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His 320 325 330 TAT GTG CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC 1059 Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser 335 340 345 350 AAC CTT ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT 1107 Asn Leu Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn 355 360 365 GAG GAC TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG 1155 Glu Asp Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp 370 375 380 GAC TGG ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC 1203 Asp Trp Ile Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser 385 390 395 AAG CTC CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT 1251 Lys Leu Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg 400 405 410 GGG TAC AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC 1299 Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys 415 420 425 430 CCA TGT GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG 1347 Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg 435 440 445 ATC GTT GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC 1395 Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro 450 455 460 ATC AAC GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC 1443 Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn 465 470 475 TAT TCC AGG GCG TTG TGG CGG GTG GCC ATT GAG GAG TAT GTG GAG GTC 1491 Tyr Ser Arg Ala Leu Trp Arg Val Ala Ile Glu Glu Tyr Val Glu Val 480 485 490 ACG CGG GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC 1539 Thr Arg Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn 495 500 505 510 GTG AAA TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG 1587 Val Lys Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Thr Glu Leu 515 520 525 GAT GGG GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG 1635 Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu 530 535 540 CGG GAT GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG 1683 Arg Asp Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly 545 550 555 TCA CAG CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC 1731 Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser 560 565 570 ATG CTC ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG 1779 Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg 575 580 585 590 CTG GCC AGA GGG TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG 1827 Leu Ala Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln 595 600 605 TTG TCT GCG CTT TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GGC GCC 1875 Leu Ser Ala Leu Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala 610 615 620 CCA GAC ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG 1923 Pro Asp Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met 6 25 630 635 GGC GGA AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA 1971 Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu 640 645 650 GAC TCT TTT GAA CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC 2019 Asp Ser Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser 655 660 665 670 GTT GCG GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG 2067 Val Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met 675 680 685 CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG 2115 Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp 690 695 700 AAG AAC CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG 2163 Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro 705 710 715 CCT ACC AAG GCC CCT CCA ATA CCA CCT CCA CGA AGA AAG AGA ACG GTT 2211 Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val 720 725 730 GTC CTG ACA GAA TCC TCC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA 2259 Val Leu Thr Glu Ser Ser Val Ser Ser Ala Leu Al a Glu Leu Ala Thr 735 740 745 750 AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG 2307 Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala 755 760 765 ACC GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC 2355 Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp 770 775 780 GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC 2403 Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp 785 790 795 CCC GAT CTC AGC GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC 2451 Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser 800 805 810 GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA 2499 Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu 815 820 825 830 ATT ACA CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG 2547 Ile Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu 835 840 845 AGC AAC CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC 2595 Ser Asn Pro Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser 850 855 860 CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA 2643 Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln 865 870 875 GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG 2691 Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys 880 885 890 GCG TCC ACA GTT AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG 2739 Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys 895 900 905 910 CTG ACG CCC CCA CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG 2787 Leu Thr Pro Pro His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys 915 920 925 GAC GTC CAG AGC CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG 2835 Asp Val Gln Ser Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val 930 935 940 TGG AAG GAC TTG CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC 2883 Trp Lys Asp Leu Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile 945 950 955 ATG GCA AAA AAT GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC 2931 Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg 960 965 970 AAG CCA GCT CGC CTT ATC GTA TTC CCA GAC TTG GGG GTT CGT GTG TGC 2979 Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys 975 980 985 990 GAG AAA ATG GCC CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG 3027 Glu Lys Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val 995 1000 1005 ATG GGC TCC TCA TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG 3075 Met Gly Ser Ser Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu 1010 1015 1020 TTC CTG GTG AAT GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA 3123 Phe Leu Val Asn Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala 1025 1030 1035 TAT GAC ACC CGC TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT 3171 Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg 1040 1045 1050 ACT GAG GAG TCA ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA 3219 Thr Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg 1055 1060 1065 1070 CAG GCC ATA AGG TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC CTG 3267 Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu 1075 1080 1085 ACT AAT TCA AAG GGG CAG AAC TGC GGT TAT CGC CGG TGC CGC GTC AGC 3315 Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Val Ser 1090 1095 1100 GGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG 3363 Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys 1105 1110 1115 GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC TGC ACG ATG CTT 3411 Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu 1120 1125 1130 GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAT AGC GCG GGA CAG 3459 Val Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser Ala Gly Thr Gln 1135 1140 1145 1150 GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT ATG ACT AGG TAC 3507 Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr 1155 1160 1165 TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC GAC TTG GAG CTG 3555 Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu 1170 1175 1180 ATA ACA TCA TGT TCC TCC AAT GTG TCG GTC GCG CAC GAC GCA TCA GGC 3603 Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Ala Ser Gly 1185 1190 1195 AAA CGG GTG TAC TAT CTC ACC CGT GAC CCC ACC ACC CCC CTA GCG CGG 3651 Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg 1200 1205 1210 GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC TCC TGG CTA GGC 3699 Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly 1215 1220 1225 1230 AAC ATC ATC ATG TAC GCG CCC ACC TTA TGG GCA AGG ATG ATT CTG ATG 3747 Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met 1235 1240 1245 ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA CTT GAA AAA GCC 3795 Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala 1250 1255 1260 CTA GAT TGT CAG ATC TAC GGG GCC ACT TAC TCC ATT GAG CCA CTT GAC 3843 Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile Glu Pro Leu Asp 1265 1270 1275 CTA CCT CAG ATC ATT CAA CGA CTC CAC GGT CTT AGC GCA TTT TCA CTC 3891 Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu 1280 1285 1290 CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT TCA TGC CTC AGG 3939 His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg 1295 1300 1305 1310 AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT CGG GCC AGA AGC 3987 Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser 1315 1320 1325 GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC GCC ACC TGT GGC 4035 Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly 1330 1335 1340 AAA TAC CTC TTC AAC TGG GCA GTA AAG ACC AAG CTC AAA CTC ACT CCA 4083 Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro 1345 1350 1355 ATC CCA GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG TTC GTT GCT GGT 4131 Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly 1360 1365 1370 TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT GCC CGA CCC CGC 4179 Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg 1375 1380 1385 1390 TGG TTC ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG GTA GGC ATC TAC 4227 Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr 1395 1400 1405 CTG CTC CCC AAC CGA TGA GCGGG GAGCTAAACA CTCCAGGCCA ATAGGCCATC 4280 Leu Leu Prou Asn Arg Stop 1410 CCCCTTTTTTTTTTTTTTT
4296

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0319

[Correction method] Change

[Correction content]

SEQ ID NO: 81 Sequence length: 311 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N17-3 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly 20 25 30 TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG TCT GCG CTT 144 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Leu 35 40 45 TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GGC GCC CCA GAC ACT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp Thr Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT G A 311 Pro Leu Arg Ala Glu Glu Asp 100

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0320

[Correction method] Change

[Correction content]

SEQ ID NO: 82 Sequence length: 311 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N17-1 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly 20 25 30 TCT CCT CCT TCT TTG GCC AGC TCT TCA GCT AGC CAG TTG TCT GCG CCA 144 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro 35 40 45 TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GAC TCC CCA GAC GCT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGG AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTG ATT CTA GAC TCT TCT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Ser Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT G A 311 Pro Leu Arg Ala Glu Glu Asp 100

[Submission date] September 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0321

[Correction method] Change

[Correction content]

SEQ ID NO: 83 Sequence length: 311 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N17-2 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATC ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Thr Arg Gly 20 25 30 TCT CCT CCT TCT TTG GCC AGC TCT TCA GCT AGT CAG TTG TCT GCG CCA 144 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro 35 40 45 TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GAC TCC CCA GAC GCT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGG AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT GA 311 Pro Leu Arg Ala Glu Glu Asp 100 ───────────── ──────────────────────────────────────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 125

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0235

[Correction method] Change

[Correction content]

Example 30 Large expression of polypeptide CORE and polypeptide C + N23 [1] Method for obtaining clone CN23 pUCN2 having clone N23-1 obtained in Example 16
Clone N23-1 by PCR using 3-1 as a template
A region to be expressed was created. First, the following synthetic DNA was used as the primer used. The base sequence for the 5'primer is MS165; 5'GCAAGCTTATGCTGCTG.
TCGCCCGGGGCCCATCT3 '(SEQ ID NO:
194). The base sequence for the 3'primer is MS166; 5'GCGAATTCAGATCTTTA.
TCATGTGTTGCAGTCGATCAC 3 '
(SEQ ID NO: 195). 20 pmol / μl of these two synthetic DNAs
Was prepared. This PCR reaction was performed according to the method of Saiki et al. This reaction was performed so that the final volume was 100 μl, and the plasmid pUCN was used as template DNA.
23-1 was used at 100 ng, and the solution prepared as described above was used as a primer for 2'of 5'and 3'primers.
Used one by one. First, the prepared sample was kept at 95 ° C. for 5 minutes and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ Takara Shuzo) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler (Perkin Elmer Cetus) at 95 ° C. 1
Min, 55 ° C. 1 minute, 72 ° C. 1 minute. further,
The treatment was performed 17 times at 95 ° C. for 1 minute, 65 ° C. for 1 minute, and 72 ° C. for 1 minute. This sample was treated with phenol / chloroform and ethanol-precipitated by a conventional method, and the DNA fragment was Hin-treated.
After cleaving with dIII and EcoRI, it was separated and extracted with acrylamide gel. Further cloning vector pUC
The HindIII and EcoRI sites of 19 were ligated by a conventional method, cloned and screened to obtain plasmid pUCN23A, and its nucleotide sequence was determined. Clone N23A obtained by this method
The nucleotide sequence of 5'GCAAGCTTATG 3'3'CGTTCGAATAC 5 '(SEQ ID NO: : 15
5) It has a HindIII site at the 5'end, followed by a start codon ATG. At the 3'end, the following DNA fragments are added to the 3'sides of the DNA fragments shown from 1st to 915th of SEQ ID NO: 50 in the sequence listing,

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0236

[Correction method] Change

[Correction content]

5'TGATGAAGATCTGAATTCGC 3'3'ACTACTTCTAGACTTAAGCG 5 '
(SEQ ID NO: 156) 5'TGATAAAGATCTGAATTCGC 3'3'ACTATTTTCTAGACTTAAGCG 5 '
(SEQ ID NO: 196) There is an EcoRI site from the 3'most side and B is inside
It carries a glII site followed by two stop codons. Further, pCZCORE obtained in Example 6 [1] was cleaved with a restriction enzyme SacII and blunt-ended with T4 DNA polymerase by a conventional method. It was further digested with BglII, and a DNA fragment pCZCORE / SB having the vector portion of pCZ and the N-terminal portion of the core protein of HCV was separated and extracted with an acrylamide gel to obtain the fragment. Similarly, plasmid pU
CN23A was completely cleaved with restriction enzymes SmaI and BglII, and the DNA fragment having the sequence of clone N23-1 was separated and extracted with acrylamide gel to obtain DNA fragment N23.
A / SB was obtained. The 5'end of this DNA fragment corresponds to the 107th G of SEQ ID NO: 50 in the sequence listing, and has a sequence from there to the 915th A of SEQ ID NO: 50, 3 '
The side has two stop codons and a BglII site. DNA fragment pCZCORE / SB 10ng and DN
100 ng of A fragment N23A / SB was subjected to T4D by a conventional method.
Ligated with NA ligase and plasmid pCZCN23
Was obtained. The plasmid has a clone CN23 having the nucleotide sequence shown in SEQ ID NO: 102 in the sequence listing between the HindIII site and the BglII site.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0239

[Correction method] Change

[Correction content]

[0239]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 483 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Sequence type: cDNA to genomic RNA Origin: Hepatitis C virus direct Origin Clone name: N1-1 sequence CTCCACCATA GATCACTCCC CTGT
GAGGAA CTACTGTCTT CACGCAGAAA GCGTCTAGCC 60ATGGCG
TTAG TATGAGTGTC GTGCAGCCTC CAGGACCCCC CCTCCCGGGA G
AGCCATAGT 120GGTCTGCGGA ACCGGTGAGT ACACCGGAAT TGC
CAGGACG ACCGGGTCCT TTCTTGGATC 180AACCCGCT
CA ATGCCTGGAG ATTTGGGGCGT
GCCCCCCGCGA GACTGCTAGC CGA
GTAGGTGT240TGGGTCGCGA AA
GGCCTTGT GGTACTGCCT GATAG
GGTGC TTGCGAGTGC CCCGGGAG
GT 300CTCGTAGACC GTGCATC
ATG AGC ACA AAT CCA AAA
CCC CAA AGA AAA ATC 3
50Met Ser Thr Asn Pro Lys
Pro Gln Arg Lys Ile1
5
10 AAA CGT AAC ACC AA
C CGC CGC CCA CAG GAC GTT
AAG TTC CCG GGC GGT 3
98 Lys Arg Asn Thr Asn Arg
Arg Pro Gln Asp Val Lys
Phe Pro Gly Gly15
20
25GGT CAG ATC GTT GG
T GGA GTT TAC CTG TTG CCG
CGC AGG GGC CCC AGG 4
46Gly Gln Ile Val Gly Gly
Val Tyr Leu Leu Pro Arg
Arg Gly Pro Arg30
35
40TTG GGT GTG CGC GC
G ACT AGG AAG ACT TCC GAG
CGG T 4
83Leu Gly Val Arg Ala Thr
Arg Lys Thr Ser Glu Arg

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0241

[Correction method] Change

[Correction content]

SEQ ID NO: 3 Sequence length: 531 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N3-1 sequence AGGTCTCGTA GACCGTGCAT C ATG AGC ACA
AAT CCA AAA CCC CAA AGA AAA ACC 54 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr 1 5 10 AAA CGT AAC ACC AAC CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT 102 Lys Arg Asn Thr Asn Arg Arg Pro Gln As Val Lys Phe Pro Gly Gly 15 20 25 GGT CAG ATC GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG 150 Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 30 35 40 TTG GGT GTG CGC GCG ACT AGG AAG ACT TCC GAG CGG CCG CAA CCT CGT 198 Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Pro Gln Pro Arg 45 50 55 GGA AGG CGA CAA CCT ATC CCC AAG GCT CGC CAA CCC GAG GGT AGG GCC 246 Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala 60 65 70 75 TGG GCT CAG CCC GGG TAC CCT TGG CCC CTC TAT GGC AAT GAG GGC TTG 294 Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu 80 85 90 GGG TGG GCA GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG 342 Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp 95 100 105 GGC CCC ACG GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC 390 Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile 110 115 120 GAT ACC CTC ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC 438 Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu 125 130 135 GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG GCT CTA GCG CAT GGC GTC 486 Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val 140 145 150 155 CGG GTT CTG GAG GAC GGC GTG AAC TAC GCA ACA GGG AAC TTG CCC 531 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro 160
165 170

[Submission date] September 11, 1992

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0243

[Correction method] Change

[Correction content]

SEQ ID NO: 5 Sequence length: 1258 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N3N10 sequence AGGTCTCGTA GACCGTGCAT CATG AGC ACA
AAT CCA AAA CCC CAA AGA AAA ATC 54 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Ile 1 5 10 AAA CGT AAC ACC AAC CGC CGC CCA CAG GAC GTT AAG TTC CCG GGC GGT 102 Lys Arg Asn Thr Asn Arg Arg Pro G As Val Lys Phe Pro Gly Gly 15 20 25 GGT CAG ATC GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG 150 Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 30 35 40 TTG GGT GTG CGC GCG ACT AGG AAG ACT TCC GAG CGG CCG CAA CCT CGT 198 Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Pro Glu Pro Arg 45 50 55 GGA AGG CGA CAA CCT ATC CCC AAG GCT CGC CAA CCC GAG GGT AGG GCC 246 Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala 60 65 70 75 TGG GCT CAG CCC GGG TAC CCT TGG CCC CTC TAT GGC AAT GAG GGC TTG 294 Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu 80 85 90 GGG TGG GCA GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG 342 Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp 95 100 105 GGC CCC ACG GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC 390 Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile 110 115 120 GAT ACC CTC ACA TGC GGC TTC GCC GAT CTC ATG GGG TAC ATT CCG CTC 438 Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu 125 130 135 GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG GCT CTA GCG CAT GGC GTC 486 Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val 140 145 150 155 CGG GTT CTG GAG GAC GGC GTG AAC TAT GCA ACA GGG AAC CTG CCT GGT 534 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly 160 165 170 TGC TCC TTT TCT ATC TTC CTT TTG GCT TTG CTG TCC TGT TTG ACC ATC 582 Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile 175 180 185 CCA GCT TCC GCC TAC CAA GTG CGC AAC GCG TCC GGG GTG TAC CAT GTC 630 Pro Ala Ser Ala Tyr Gln Val Arg Asn Ala Ser Gly Val Tyr His Val 190 195 200 ACG AAC GAC TGC TCC AAC TCA AGT ATT GTG TAT GAG GCG GCG GAC GTG 678 Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val 205 210 215 ATT ATG CAC ACC CCC GGG TGC GTG CCC TGC GTC CGG GAG AAC AAT TCC 726 Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser 220 225 230 235 TCC CGC TGC TGG GTA GCG CTC ACT CCC ACG CTT GCG GCC AGG AAC AGC 774 Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser 240 245 250 AGC ATC CCC ACT ACG ACA ATA CGG CGT CAT GTC GAC TTG CTC GTT GGG 822 Ser Ile Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly 255 260 265 GCA GCT GCT CTC TGT TCC GCT ATG TAT GTG GGG GAT TTT TGC GGA TCT 870 Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Phe Cys Gly Ser 270 275 280 GTT TTC CTC GTC TCC CAG CTG TTC ACT TTC TCA CCT CGC CGG TAT GAG 918 Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu 285 290 295 ACG GTG CAA GAC TGC AAT TGC TCA ATC TAT CCC GGC CAT GTA TCA GGC 966 Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly 300 305 310 315 CAT CAT CGC ATG GCT TGG GAT ATG ATA ATG AAT TGG TCA CCT ACA ACA GCC 1014 His Arg Met Ala Trp Asp Met Ile Met Asn Trp Ser Pro Thr Th r Ala 320 325 330 CTA GTG GTA TCG CAG CTA CTC CGG ATC CCA CAA GCC GTC GTG GAT ATG 1062 Leu Val Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met 335 340 345 GTG GCG GGG GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC 1110 Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser 350 355 360 ATG GTG GGG AAC TGG GCT AAG GTC TTG GTT GTG ATG CTG CTC TTC GCC 1158 Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 365 370 375 GGT GTT GAC GGG GGG ACC CAC GTG ACA GGG GGA AAG GTA GCC TAC ACC 1206 Gly Val Asp Gly Gly Thr His Val Thr Gly Gly Lys Val Ala Tyr Thr 380 385 390 395 ACC CAG AGC TTT ACA TCC TTC TTT TCA CGA GGG CCG TCT CAG AGA ATC 1254 Thr Gln Ser Phe Thr Ser Phe Phe Ser Arg Gly Pro Ser Gln Arg Ile 400 405 410 CAGC 1258 Gln

[Submission date] September 11, 1992

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0244

[Correction method] Change

[Correction content]

SEQ ID NO: 6 Sequence length: 1554 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N1N3N10 Sequence CTCCACCATA GATCACTCCC CTGTGAGGAA AAA CTA
CTGTCTT CACGCAGAAA GCGTCTAGCC 60 ATGGCGTTAG TATGAGTGTC GTGCAGCCTC CAGGACCCCC CCTCCCGGGA GAGCCATAGT 120 GGTCTGCGGA ACCGGTGAGT ACACCGGAAT TGCCAGGACG ACCGGGTCCT TTCTTGGATC 180 AACCCGCTCA ATGCCTGGAG ATTTGGGCGT GCCCCCGCGA GACTGCTAGC CGAGTAGTGT 240 TGGGTCGCGA AAGGCCTTGT GGTACTGCCT GATAGGGTGC TTGCGAGTGC CCCGGGAGGT 300 CTCGTAGACC GTGCATC ATG AGC ACA AAT CCA AAA CCC CAA AGA AAA ATC 350 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Ile 1 5 10 AAA CGT AAC ACC AAC CGC CGC CCA CAG GAC GTT AAG TTC CCG GGC GGT 398 Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly 15 20 25 GGT CAG ATC GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG 446 Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 30 35 40 TTG GTT GTG CGC GCG ACT AGG AAG ACT TCC GAG CGG CCG CAA CCT CGT 494 Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Pro Gln Pro Arg 45 50 55 GGA AGG CGA CAA CCT ATC CCC AAG GCT CGC CAA CCC GAG GGT AGG GCC 542 Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala 60 65 70 75 TGG GCT CAG CCC GGG TAC CCT TGG CCC CTC TAT GGC AAT GAG GGC TTG 590 Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu 80 85 90 GGG TGG GCA GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG 638 Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp 95 100 105 GGC CCC ACG GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC 686 Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile 110 115 120 GAT ACC CTC ACA TGC GGC TTC GCC GAT CTC ATG GGG TAC ATT CCG CTC 734 Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu 125 130 135 GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG GCT CTA GCG CAT GGC GTC 782 Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val 140 145 150 155 CGG GTT CTG GAG GAC GGC GTG AAC TAT GCA ACA GGG AAT CTG CCT GGT 830 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly 160 165 170 TGC TCC TTT TCT ATC TTC CTT TTG GCT TTG CTG TCC TGT TTG ACC ATC 878 Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile 175 180 185 CCA GCT TCC GCC TAC CAA GTG CGC AAC GCG TCC GGG GTG TAC CAT GTC 926 Pro Ala Ser Ala Tyr Gln Val Arg Asn Ala Ser Gly Val Tyr His Val 190 195 200 ACG AAC GAC TGC TCC AAC TCA AGT ATT GTG TAT GAG GCG GCG GAC GTG 974 Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val 205 210 215 ATT ATG CAC ACC CCC GGG TGC GTG CCC TGC GTC CGG GAG AAC AAT TCC 1022 Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser 220 225 230 235 TCC CGC TGC TGG GTA GCG CTC ACT CCC ACG CTT GCG GCC AGG AAC AGC 1070 Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser 240 245 250 AGC ATC CCC ACT ACG ACA ATA CGG CGT CAT GTC GAC TTG CTC GTT GGG 1118 Ser Ile Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly 255 260 265 GCA GCT GCT CTC TGT TCC GCT ATG TAT GTG GGG GAT TTT TGC GGA TCT 1166 Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Phe Cys Gly Ser 270 275 280 GTT TTC CTC GTC TCC CAG CTG TTC ACT TTC TCA CCT CGC CGG TAT GAG 1214 Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu 285 290 295 ACG GTG CAA GAC TGC AAT TGC TCA ATC TAT CCC GGC CAT GTA TCA GGC 1262 Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly 300 305 310 315 CAT CGC ATG GCT TGG GAT ATG ATA ATG AAT TGG TCA CCT ACA ACA GCC 1310 His Arg Met Ala Trp Asp Met Ile Met Asn Trp Ser Pro Thr Thr Ala 320 325 330 CTA GTG GTA TCG CAG CTA CTC CGG ATC CCA CAA GCC GTC GTG GAT ATG 1358 Leu Val Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met 335 340 345 GTG GCG GGG GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC 1406 Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser 350 355 360 ATG GTG GGG AAC TGG GCT AAG GTC TTG GTT GTG ATG CTG CTC TTC GCC 1454 Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 365 370 375 GGT GTT GAC GGG GGG ACC CAC GTG ACA GGG GGA AAG GTA GCC TAC ACC 1502 Gly Val Asp Gly Gly Thr His Val Thr Gly Gly Lys Val Ala Tyr Thr 380 385 390 395 ACC CAG AGC TTT ACA TCC TTC TTT TCA CGA G GG CCG TCT CAG AGA ATC 1550 Thr Gln Ser Phe Thr Ser Phe Phe Ser Arg Gly Pro Ser Gln Arg Ile 400 405 410 CAG C 1554 Gln

[Submission date] September 11, 1992

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0246

[Correction method] Change

[Correction content]

SEQ ID NO: 8 Sequence length: 1264 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Antisense: No Origin: Hepatitis C virus Direct origin Clone name: HN3N10ΔB sequence GCAAGCTT ATG AGC ACA AAT CCA AAA CCC
CAA AGA AAA ATC AAA CGT 47 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Ile Lys Arg 1 5 10 AAC ACC AAC CGC CGC CCA CAG GAC GTT AAG TTC CCG GGC GGT GGT CAG 95 Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln 15 20 25 ATC GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT 143 Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly 30 35 40 45 GTG CGC GCG ACT AGG AAG ACT TCC GAG CGG CCG CAA CCT CGT GGA AGG 191 Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Pro Gln Pro Arg Gly Arg 50 55 60 CGA CAA CCT ATC CCC AAG GCT CGC CAA CCC GAG GGT AGG GCC TGG GCT 239 Arg Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala 65 70 75 CAG CCC GGG TAC CCT TGG CCC CTC TAT GGC AAT GAG GGC TTG GGG TGG 287 Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp 80 85 90 GCA GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC 335 Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro 95 100 105 ACG GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC 383 Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr 110 115 120 125 CTC ACA TGC GGC TTC GCC GAT CTC ATG GGG TAC ATT CCG CTC GTC GGC 431 Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly 130 135 140 GCC CCC CTA GGG GGC GCT GCC AGG GCT CTA GCG CAT GGC GTC CGG GTT 479 Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val 145 150 155 CTG GAG GAC GGC GTG AAC TAT GCA ACA GGG AAC CTG GGT GGT TGC TCC 527 Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser 160 165 170 TTT TCT ATC TTC CTT TTG GCT TTG CTG TCC TGT TTG ACC ATC CCA GCT 575 Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala 175 180 185 TCC GCC TAC CAA GTG CGC AAC GCG TCC GGG GTG TAC CAT GTC ACG AAC 623 Ser Ala Tyr Gln Val Arg Asn Ala Ser Gly Val Tyr His Val Thr Asn 190 195 200 205 GAC TGC TCC AAC TCA AGT ATT GTG TAT GAG GCG GCG GAC GTG ATT ATG 671 Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met 210 215 220 CAC ACC CCC GGG TGC GTG CCC TGC GTC CGG GAG AAC AAT TCC TCC CGC 719 His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg 225 230 235 TGC TGG GTA GCG CTC ACT CCC ACG CTT GCG GCC AGG AAC AGC AGC ATC 767 Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile 240 245 250 CCC ACT ACG ACA ATA CGG CGT CAT GTC GAC TTG CTC GTT GGG GCA GCT 815 Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala 255 260 265 GCT CTC TGT TCC GCT ATG TAT GTG GGG GAT TTT TGC GGA TCT GTT TTC 963 Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Phe Cys Gly Ser Val Phe 270 275 280 285 CTC GTC TCC CAG CTG TTC ACT TTC TCA CCT CGC CGG TAT GAG ACG GTG 911 Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val 290 295 300 CAA GAC TGC AAT TGC TCA ATC TAT CCC GGC CAT GTA TCA GGC CAT CGC 959 Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg 305 310 315 ATG GCT TGG GAT ATG ATA ATG AAT TGG TCA CCT ACA ACA GCC CTA GTG 1007 Met Ala Trp Asp Met Ile Met Asn Trp Ser Pro Thr Thr Ala Leu Val 320 325 330 GTA TCG CAG CTA CTC CGG ATA CCA CAA GCC GTC GTG GAT ATG GTG GCG 1015 Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala 335 340 345 GGG GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG 1103 Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val 350 355 360 365 GGG AAC TGG GCT AAG GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT 1151 Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val 370 375 380 GAC GGG GGG ACC CAC GTG ACA GGG GGA AAG GTA GCC TAC ACC ACC CAG 1199 Asp Gly Gly Thr His Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln 385 390 395 AGC TTT ACA TCC TTC TTT TCA CGA GGG CCG TCT CAG AGA ATC 1247 Ser Phe Thr Ser Phe Phe Ser Arg Gly Pro Ser Gln Arg Ile 400 405 410 TGAAGATCTG AATTCGC
1264 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0253

[Correction method] Change

[Correction content]

SEQ ID NO: 15 Sequence length: 339 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N27-3 Sequence C CGG ATC CCA CAA GCC GTG GTG GAT ATG GTG GCA GGG GCC CAC TGG GGA 49 Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 5 10 15 GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG 97 Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 20 25 30 GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT GAC GGG GGG ACC CAC 145 Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly Thr His 35 40 45 GTG ACA GGG GGG AAG GTA GCC TAC ACC ACC CAG GGC TTT ACA TCC TTC 193 Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln Gly Phe Thr Ser Phe 50 55 60 TTT TCA CGA GGG CCG TCT CAG AAA ATC CAA CTT GTA AAC ACT AAC GGC 241 Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr Asn Gly 65 70 75 80 AGC TGG CAC ATC AAT AGG ACT GCC CTC AAT TGC AAT GAC TCC CTT AAC 2 89 Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn 85 90 95 ACC GGG TTC CTT GCC GCG CTG TTC TAC ACC CAC AGC TTC AAC GCG TCC GG 339 Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Ser Phe Asn Ala Ser 100 105
110

[Submission date] September 11, 1992

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0263

[Correction method] Change

[Correction content]

SEQ ID NO: 25 Sequence length: 629 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX24-4 Sequence AAC ACA CGG CCG CCG CAG GGG AAC TGG TTT GGC TGT ACA TGG ATG AAT 48 Asn Thr Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn 1 5 10 15 GGC ACT GGG TTC ACA AAG ACG TGC GGG GGC CCC CCG TGC AAC ATC GGG 96 Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly 20 25 30 GGG GTC GGC AAC AAT ACC TTG ACT TGC CCC ACG GAC TGC TTC CGG AAG 144 Gly Val Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys 35 40 45 CAC CCC GAG GCC ACT TAC ACA AAA TGT GGT TCG GGG CCT TGG TTG ACA 192 His Pro Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr 50 55 60 CCT AGG TGC CTA GTT CAT TAC CCA TAC AGG CTC TGG CAC TAT CCC TGC 240 Pro Arg Cys Leu Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys 65 70 75 80 ACT GTC AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT GTG GGG GGC GTG 288 Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val 85 90 95 GAA CAC AGG CTT GAA GCT GCA TGC AAT TGG ACC CGA GGA GAG CGT TGT 336 Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys 100 105 110 GAC TTG GAG GAC AGG GAT AGA TCA GAG CTT AGC CCG CTA TTG CTG TCC 384 Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser 115 120 125 ACA ACA GAG TGG CAG GTA CTG CCC TGT TCC TTC ACC ACC CTG CCG GCT 432 Thr Thr Glu Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala 130 135 140 CTG TCC ACT GGT TTG ATT CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA 480 Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln 145 150 155 160 TAT CTG TAC GGC ATA GGG TCG GCG GTT GTC TCC TTC GCA ATC AAA TGG 528 Tyr Leu Tyr Gly Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp 165 170 175 GAA TAT ATT CTG TTG CTT TTC CTC CCC CTG GCG GAC GCG CGC GTC TGT 576 Glu Tyr Ile Leu Leu Leu Phe Leu Pro Leu Ala Asp Ala Arg Val Cys 180 185 190 GCC TGC TTG TGG ATG ATG CTG CTG ATA GCC CAC GCC GAC GCC ACC TTA 624 Ala Cys Leu Trp Met Met Leu Leu Ile Ala His Ala Asp Ala Thr Leu 195 200 205 GAG AA
629 Glu ───────────────────────────────────────────────── ─────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0266

[Correction method] Change

[Correction content]

SEQ ID NO: 28 Sequence length: 652 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N27N19-1 Sequence C CGG ATC CCA CAA GCC GTG GTG GAT ATG GTG GCA GGG GCC CAC TGG GGA 49 Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 15 GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG 97 Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 20 25 30 GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT GAC GGG GGG ACC CAC 145 Val Leu Val Val Met Leu Leu P (e Ala GLy VAl Asp Gly Gly THr His 35 40 45 GTG ACA GGG GGG AAG GTA GCC TAC ACC ACC CAG GGC TTT ACA TCC TTC 193 Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln Gly Phe Thr Ser Phe 50 55 60 TTT TCA CGA GGG CCG TCT CAG AAA ATC CAA CTT GTA AAC ACT AAC GGC 241 Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr Asn Gly 65 70 75 80 AGC TGG CAC ATC AAT AGG ACT GCC CTC AAT TGC AAT GAC TCC CTT AAC 289 Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn 85 90 95 ACC GGG TTC CTT GCC GCG CTG TTC TAC ACC CAC AGC TTC AAC GCG TCC 337 Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Ser Phe Asn Ala Ser 100 105 110 GGA TGT CCG GAG CGT ATG GCC GGT TGC CGC CCC ATT GAC GAG TTC GCT 385 Gly Cys Pro Glu Arg Met Ala Gly Cys Arg Pro Ile Asp Glu Phe Ala 115 120 125 CAG GGG TGG GGT CCC ATC ACT CAT GTT GTG CCT AAC ATC TCG GAC CAG 433 Gln Gly Trp Gly Pro Ile Thr His Val Val Pro Asn Ile Ser Asp Gln 130 135 140 AGG CCC TAT TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT ATC GTA CCC 481 Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro 145 150 155 160 GCG TCG CAG GTG TGT GGT CCG GTG TAT TGC TTC ACC CCA AGC CCT GTT 529 Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 165 170 175 GTG GTG GGG ACG ACC GAT CGT TTC GGC GCC CCC ACG TAC AAC TGG GGA 577 Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Asn Trp Gly 180 185 190 AAC AAT GAG ACG GAT GTG CTA CTC CTC AAC AAC ACA CG G CCG CCG CAG 625 Asn Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro Pro Gln 195 200 205 GGC AAC TGG TTC GGT TGT ACC TGG ATG 652 Gly Asn Trp Phe Gly Cys Thr Trp Met 210 215

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0267

[Correction method] Change

[Correction content]

SEQ ID NO: 29 Sequence length: 977 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N19MX24A-1 Sequence GAG GCC GTG AAC TGC GAT GAC TCC CTT AAC ACC GGG TTC CTT GCC GCG 48 Glu Ala Val Asn Cys Asp Asp Ser Leu Asn Thr Gly Phe Leu Ala Ala 1 5 10 15 CTG TTC TAC ACG CAC AGG TTC AAC GCG TCC GGA TGT CCG GAG CGT ATG 96 Leu Phe Tyr Thr His Arg Phe Asn Ala Ser Gly Cys Pro Glu Arg Met 20 25 30 GCC GGT TGC CGC CCC ATT GAC GAG TTC GCT CAG GGG TGG GGT CCC ATC 144 Ala Gly Cys Arg Pro Ile Asp Glu Phe Ala Gln Gly Trp Gly Pro Ile 35 40 45 ACT CAT GTT GTG CCT AAC ATC TCG GAC CAG AGG CCC TAT TGC TGG CAC 192 Thr His Val Val Pro Asn Ile Ser Asp Gln Arg Pro Tyr Cys Trp His 50 55 60 TAC GCG CCT CGA CCG TGT GGT ATC GTA CCC GCG TCG CAG GTG TGT GGT 240 Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly 65 70 75 80 CCG GTG TAT TGC TTC ACC CCA AGC CCT GTT GTG GTG GGG ACG ACC GAT 288 Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp 85 90 95 CGT TTC GGC GCC CCC ACG TAC AAC TGG GGA AAC AAT GAG ACG GAT GTG 336 Arg Phe Gly Ala Pro Thr Tyr Asn Trp Gly Asn Asn Glu Thr Asp Val 100 105 110 CTA CTC CTC AAC AAC ACA CGG CCG CCG CAG GGC AAC TGG TTC GGT TGT 384 Leu Leu Leu Asn Asn Thr Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys 115 120 125 ACC TGG ATG AAT GGC ACT GGG TTC ACA AAG ACG TGC GGG GGC CCC CCG 432 Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro 130 135 140 TGC AAC ATC GGG GGG GTC GGC AAC AAT ACC TTG ACT TGC CCC ACG GAC 480 Cys Asn Ile Gly Gly Val Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp 145 150 155 160 TGC TTC CGG AAG CAC CCC GAG GCC ACT TAC ACA AAA TGT GGT TCG GGG 528 Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly 165 170 175 CCT TGG TTG ACA CCT AGG TGC CTA GTT CAT TAC CCA TAC AGG CTC TGG 576 Pro Trp Leu Thr Pro Arg Cys Leu Val His Tyr Pro Tyr Arg Leu Trp 180 185 190 CAC TAT CCC TGC ACT GTC AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT 624 His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr 195 200 205 GTG GGG GGC GTG GAA CAC AGG CTT GAA GCT GCA TGC AAT TGG ACC CGA 672 Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg 210 215 220 GGA GAG CGT TGT GAC TTG GAG GAC AGG GAT AGA TCA GAG CTT AGC CCG 720 Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro 225 230 235 240 CTA TTG CTG TCC ACA ACA GAG TGG CAG GTA CTG CCC TGT TCC TTC ACC 768 Leu Leu Leu Ser Thr Thr Glu Trp Gln Val Leu Pro Cys Ser Phe Thr 245 250 255 ACC CTG CCG GCT CTG TCC ACT GGT TTG ATT CAT CTC CAT CAG AAC ATC 816 Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile 260 265 270 GTG GAC GTG CAA TAT CTG TAC GGC ATA GGG TCG GCG GTT GTC TCC TTC 864 Val Asp Val Gln Tyr Leu Tyr Gly Ile Gly Ser Ala Val Val Ser Phe 275 280 285 GCA ATC AAA TGG GAA TAT ATT CTG TTG CTT TTC CTC CCC CTG GCG GAC 912 Ala Ile Lys Trp Glu Tyr Ile Leu Leu Leu Phe Leu Pro Leu Ala Asp 290 295 300 GCG CGC GTC TGT GCC TGC TTG TGG ATG ATG CTG CTG ATA GCC CAC GCC 960 Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala His Ala 305 310 315 320 GAC GCC ACC TTA GAG AA 977 Asp Ala Thr Leu Glu 325

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0268

[Correction method] Change

[Correction content]

SEQ ID NO: 30 Sequence length: 977 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N19MX24B-1 Sequence GAG GCC GTG AAC TGC GAT GAC TCC CTT AAC ACC GGG TTC CTT GCC GCG 48 Glu Ala Val Asn Cys Asp Asp Ser Leu Asn Thr Gly Phe Leu Ala Ala 1 5 10 15 CTG TTC TAC ACG CAC AGG TTC AAC GCG TCC GGA TGT CCG GAG CGT ATG 96 Leu Phe Tyr Thr His Arg Phe Asn Ala Ser Gly Cys Pro Glu Arg Met 20 25 30 GCC GGT TGC CGC CCC ATT GAC GAG TTC GCT CAG GGG TGG GGT CCC ATC 144 Ala Gly Cys Arg Pro Ile Asp Glu Phe Ala Gln Gly Trp Gly Pro Ile 35 40 45 ACT CAT GTT GTG CCT AAC ATC TCG GAC CAG AGG CCC TAT TGC TGG CAC 192 Thr His Val Val Pro Asn Ile Ser Asp Gln Arg Pro Tyr Cys Trp His 50 55 60 TAC GCG CCT CGA CCG TGT GGT ATC GTA CCC GCG TCG CAG GTG TGT GGT 240 Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly 65 70 75 80 CCG GTG TAT TGC TTC ACC CCA AGC CCT GTT GTG GTG GGG ACG ACC GAT 288 Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp 85 90 95 CGT TTC GGC GCC CCC ACG TAC AAC TGG GGA AAC AAT GAG ACG GAT GTG 336 Arg Phe Gly Ala Pro Thr Tyr Asn Trp Gly Asn Asn Glu Thr Asp Val 100 105 110 CTA CTC CTC AAC AAC ACA CGG CCG CCG CAG GGG AAC TGG TTT GGC TGT 384 Leu Leu Leu Asn Asn Thr Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys 115 120 125 ACA TGG ATG AAT GGC ACT GGG TTC ACA AAG ACG TGC GGG GGC CCC CCG 432 Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro 130 135 140 TGC AAC ATC GGG GGG GTC GGC AAC AAT ACC TTG ACT TGC CCC ACG GAC 480 Cys Asn Ile Gly Gly Val Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp 145 150 155 160 TGC TTC CGG AAG CAC CCC GAG GCC ACT TAC ACA AAA TGT GGT TCG GGG 528 Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly 165 170 175 CCT TGG TTG ACA CCT AGG TGC CTA GTT CAT TAC CCA TAC AGG CTC TGG 576 Pro Trp Leu Thr Pro Arg Cys Leu Val His Tyr Pro Tyr Arg Leu Trp 180 185 190 CAC TAT CCC TGC ACT GTC AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT 624 His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr 195 200 205 GTG GGG GGC GTG GAA CAC AGG CTT GAA GCT GCA TGC AAT TGG ACC CGA 672 Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg 210 215 220 GGA GAG CGT TGT GAC TTG GAG GAC AGG GAT AGA TCA GAG CTT AGC CCG 720 Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro 225 230 235 240 CTA TTG CTG TCC ACA ACA GAG TGG CAG GTA CTG CCC TGT TCC TTC ACC 768 Leu Leu Leu Ser Thr Thr Glu Trp Gln Val Leu Pro Cys Ser Phe Thr 245 250 255 ACC CTG CCG GCT CTG TCC ACT GGT TTG ATT CAT CTC CAT CAG AAC ATC 816 Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile 260 265 270 GTG GAC GTG CAA TAT CTG TAC GGC ATA GGG TCG GCG GTT GTC TCC TTC 864 Val Asp Val Gln Tyr Leu Tyr Gly Ile Gly Ser Ala Val Val Ser Phe 275 280 285 GCA ATC AAA TGG GAA TAT ATT CTG TTG CTT TTC CTC CCC CTG GCG GAC 912 Ala Ile Lys Trp Glu Tyr Ile Leu Leu Leu Phe Leu Pro Leu Ala Asp 290 295 300 GCG CGC GTC TGT GCC TGC TTG TGG AT G ATG CTG CTG ATA GCC CAC GCC 960 Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala His Ala 305 310 315 320 GAC GCC ACC TTA GAG AA 977 Asp Ala Thr Leu Glu 325

[Submission date] September 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0269

[Correction method] Change

[Correction content]

SEQ ID NO: 31 Sequence length: 1236 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N27MX24A-1 Sequence C CGG ATC CCA CAA GCC GTG GTG GAT ATG GTG GCA GGG GCC CAC TGG GGA 49 Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 15 GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG 97 Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 20 25 30 GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT GAC GGG GGG ACC CAC 145 Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly Thr His 35 40 45 GTG ACA GGG GGG AAG GTA GCC TAC ACC ACC CAG GGC TTT ACA TCC TTC 193 Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln Gly Phe Thr Ser Phe 50 55 60 TTT TCA CGA GGG CCG TCT CAG AAA ATC CAA CTT GTA AAC ACT AAC GGC 241 Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr Asn Gly 65 70 75 80 AGC TGG CAC ATC AAT AGG ACT GCC CTC AAT TGC AAT GAC T CC CTT AAC 289 Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn 85 90 95 ACC GGG TTC CTT GCC GCG CTG TTC TAC ACC CAC AGC TTC AAC GCG TCC 337 Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Ser Phe Asn Ala Ser 100 105 110 GGA TGT CCG GAG CGT ATG GCC GGT TGC CGC CCC ATT GAC GAG TTC GCT 385 Gly Cys Pro Glu Arg Met Ala Gly Cys Arg Pro Ile Asp Glu Phe Ala 115 120 125 CAG GGG TGG GGT CCC ATC ACT CAT GTT GTG CCT AAC ATC TCG GAC CAG 433 Gln Gly Trp Gly Pro Ile Thr His Val Val Pro Asn Ile Ser Asp Gln 130 135 140 AGG CCC TAT TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT ATC GTA CCC 481 Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro 145 150 155 160 GCG TCG CAG GTG TGT GGT CCG GTG TAT TGC TTC ACC CCA AGC CCT GTT 529 Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 165 170 175 GTG GTG GGG ACG ACC GAT CGT TTC GGC GCC CCC ACG TAC AAC TGG GGA 577 Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Asn Trp Gly 180 185 190 AAC AAT GAG ACG GAT GTG CTA CTC CTC AAC AAC ACA CGG CCG CCG CAG 625 Asn Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro Pro Gln 195 200 205 GGC AAC TGG TTC GGT TGT ACC TGG ATG AAT GGC ACT GGG TTC ACA AAG 673 Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys 210 215 220 ACG TGC GGG GGC CCC CCG TGC AAC ATC GGG GGG GTC GGC AAC AAT ACC 721 Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly Asn Asn Thr 225 230 235 240 TTG ACT TGC CCC ACG GAC TGC TTC CGG AAG CAC CCC GAG GCC ACT TAC 769 Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr 245 250 255 ACA AAA TGT GGT TCG GGG CCT TGG TTG ACA CCT AGG TGC CTA GTT CAT 817 Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu Val His 260 265 270 TAC CCA TAC AGG CTC TGG CAC TAT CCC TGC ACT GTC AAC TTT ACC ATC 865 Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile 275 280 285 TTC AAG GTT AGG ATG TAT GTG GGG GGC GTG GAA CAC AGG CTT GAA GCT 913 Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala 290 295 300 GCA TGC AAT TGG ACC CGA GGA GAG CGT TGT GAC TTG GAG GAC AGG GAT 961 Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp 305 310 315 320 AGA TCA GAG CTT AGC CCG CTA TTG CTG TCC ACA ACA GAG TGG CAG GTA 1009 Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Val 325 330 335 CTG CCC TGT TCC TTC ACC ACC CTG CCG GCT CTG TCC ACT GGT TTG ATT 1057 Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile 340 345 350 CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA TAT CTG TAC GGC ATA GGG 1105 His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Ile Gly 355 360 365 TCG GCG GTT GTC TCC TTC GCA ATC AAA TGG GAA TAT ATT CTG TTG CTT 1153 Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile Leu Leu Leu 370 375 380 TTC CTC CCC CTG GCG GAC GCG CGC GTC TGT GCC TGC TTG TGG ATG ATG 1201 Phe Leu Pro Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp Met Met 385 390 395 400 CTG CTG ATA GCC CAC GCC GAC GCC ACC TTA GAG AA 1236 Leu Leu Ile Ala His Ala Asp Ala Thr Leu Glu 405
410

[Submission date] September 11, 1992

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0270

[Correction method] Change

[Correction content]

SEQ ID NO: 32 Sequence length: 1236 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N27MX24B-1 Sequence C CGG ATC CCA CAA GCC GTG GTG GAT ATG GTG GCA GGG GCC CAC TGG GGA 49 Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 15 GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG 97 Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 20 25 30 GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT GAC GGG GGG ACC CAC 145 Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly Thr His 35 40 45 GTG ACA GGG GGG AAG GTA GCC TAC ACC ACC CAG GGC TTT ACA TCC TTC 193 Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln Gly Phe Thr Ser Phe 50 55 60 TTT TCA CGA GGG CCG TCT CAG AAA ATC CAA CTT GTA AAC ACT AAC GGC 241 Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr Asn Gly 65 70 75 80 AGC TGG CAC ATC AAT AGG ACT GCC CTC AAT TGC AAT GAC T CC CTT AAC 289 Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn 85 90 95 ACC GGG TTC CTT GCC GCG CTG TTC TAC ACC CAC AGC TTC AAC GCG TCC 337 Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Ser Phe Asn Ala Ser 100 105 110 GGA TGT CCG GAG CGT ATG GCC GGT TGC CGC CCC ATT GAC GAG TTC GCT 385 Gly Cys Pro Glu Arg Met Ala Gly Cys Arg Pro Ile Asp Glu Phe Ala 115 120 125 CAG GGG TGG GGT CCC ATC ACT CAT GTT GTG CCT AAC ATC TCG GAC CAG 433 Gln Gly Trp Gly Pro Ile Thr His Val Val Pro Asn Ile Ser Asp Gln 130 135 140 AGG CCC TAT TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT ATC GTA CCC 481 Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro 145 150 155 160 GCG TCG CAG GTG TGT GGT CCG GTG TAT TGC TTC ACC CCA AGC CCT GTT 529 Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 165 170 175 GTG GTG GGG ACG ACC GAT CGT TTC GGC GCC CCC ACG TAC AAC TGG GGA 577 Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Asn Trp Gly 180 185 190 AAC AAT GAG ACG GAT GTG CTA CTC CTC AAC AAC ACA CGG CCG CCG CAG 625 Asn Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro Pro Gln 195 200 205 GGG AAC TGG TTT GGC TGT ACA TGG ATG AAT GGC ACT GGG TTC ACA AAG 673 Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys 210 215 220 ACG TGC GGG GGC CCC CCG TGC AAC ATC GGG GGG GTC GGC AAC AAT ACC 721 Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly Asn Asn Thr 225 230 235 240 TTG ACT TGC CCC ACG GAC TGC TTC CGG AAG CAC CCC GAG GCC ACT TAC 769 Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr 245 250 255 ACA AAA TGT GGT TCG GGG CCT TGG TTG ACA CCT AGG TGC CTA GTT CAT 817 Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu Val His 260 265 270 TAC CCA TAC AGG CTC TGG CAC TAT CCC TGC ACT GTC AAC TTT ACC ATC 865 Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile 275 280 285 TTC AAG GTT AGG ATG TAT GTG GGG GGC GTG GAA CAC AGG CTT GAA GCT 913 Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala 290 295 300 GCA TGC AAT TGG ACC CGA GGA GAG CGT TGT GAC TTG GAG GAC AGG GAT 961 Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp 305 310 315 320 AGA TCA GAG CTT AGC CCG CTA TTG CTG TCC ACA ACA GAG TGG CAG GTA 1009 Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Val 325 330 335 CTG CCC TGT TCC TTC ACC ACC CTG CCG GCT CTG TCC ACT GGT TTG ATT 1057 Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile 340 345 350 CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA TAT CTG TAC GGC ATA GGG 1105 His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Ile Gly 355 360 365 TCG GCG GTT GTC TCC TTC GCA ATC AAA TGG GAA TAT ATT CTG TTG CTT 1153 Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile Leu Leu Leu 370 375 380 TTC CTC CCC CTG GCG GAC GCG CGC GTC TGT GCC TGC TTG TGG ATG ATG 1201 Phe Leu Pro Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp Met Met 385 390 395 400 CTG CTG ATA GCC CAC GCC GAC GCC ACC TTA GAG AA 1236 Leu Leu Ile Ala His Ala Asp Ala Thr Leu Glu 405
410

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0277

[Correction method] Change

[Correction content]

SEQ ID NO: 39 Sequence Length: 969 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Antisense: No Origin: Hepatitis C virus Direct Origin Clone Name: N15-1 Sequence GCGGATCCT CCA CCT CCA TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG 51 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg 1 5 10 CTG AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA 99 Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly 15 20 25 30 GCC GTT CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC 147 Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile 35 40 45 ATG GCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG 195 Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val 50 55 60 CTG GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG 243 Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr 65 70 75 GGC AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC 291 Gly Se r Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala 80 85 90 GTT ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA 339 Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu 95 100 105 110 GAG TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC 387 Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala 115 120 125 GAG CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG 435 Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys 130 135 140 CAA GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT 483 Gln Ala Glu Ala Ala Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu 145 150 155 GAG ACC TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG 531 Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 160 165 170 TAC TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA 579 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser 175 180 185 190 CTG ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC ACC CAA TA T 627 Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr 195 200 205 ACC CTC CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC 675 Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala 210 215 220 CCC CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG 723 Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala 225 230 235 GCT GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG 771 Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala 240 245 250 GGT TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG 819 Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met 255 260 265 270 AGC GGT GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC 867 Ser Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala 275 280 285 ATC CTC TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA 915 Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile 290 295 300 CTG CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CA G TGG ATG AAC 963 Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn 305 310 315 CGG CTG C AGCC 974 Arg Leu 320

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0279

[Correction method] Change

[Correction content]

SEQ ID NO: 41 Sequence length: 1431 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N16N15 Sequence GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGC AAC ACA TGT GTC 48 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1 5 10 15 ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC ATY GAG ACG 96 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 20 25 30 ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG 144 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg 35 40 45 ACT GGT AGG GGC AGR GGG GGC ATA TAC AGG TTT GTA ACT CCA GGG GAA 192 Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu 50 55 60 CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA TGT TAT GAC 240 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp 65 70 75 80 GCG GGC TGT GCT TGG TAC GAG CTC ACG YCC GCC GAG ACC TCG GTT AGG 288 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Xaa Ala Glu Thr Ser Val Arg 85 90 95 TTG CGG GCT TAC CTA AAY ACA CCT GGG CTG CCC GTC TGC CAG GAC CAT 336 Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His 100 105 110 CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC ATA GAT GCC 384 Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala 115 120 125 CAC TTC TTG TCC CAG ACY AAA CAG GCA GGA GAC AAC TTC CCC TAC CTG 432 His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu 130 135 140 GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT CCA CCT CCA 480 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro 145 150 155 160 TCG TGG GAT CAR ATG TGG AAG TGT CTC ATA CGG CTG AAG CCT ACG CTA 528 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu 165 170 175 CAC GGG CCA ACG CCC CTG TTG YAT AGG TTA GGA GCC GTT CAG AAC RAG 576 His Gly Pro Thr Pro Leu Leu Xab Arg Leu Gly Ala Val Gln Asn Xac 180 185 190 GTT RCC CTY ACA CAC CCY ATA ACC AAG TWC ATC ATG RC A TGC ATG TCG 624 Val Xad Leu Thr His Pro Ile Thr Lys Xae Ile Met Xaf Cys Met Ser 195 200 205 GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA GGC GGG GTC 672 Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val 210 215 220 CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC GTG GTC ATT 720 Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile 225 230 235 240 GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT CCC GAC AGG 768 Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg 245 250 255 GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC GCC TCG CAC 816 Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His 260 265 270 CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA TTC AAG CAG 864 Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln 275 280 285 AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG GAG GCT GCT 912 Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala 290 295 300 GCT CCC GTG GTG GAG TCC AAG TGG CGA GC C CTT GAG ACC TTC TGG GCG 960 Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala 305 310 315 320 AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA GCA GGC TTG 1008 Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu 325 330 335 TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG GCA TTC ACA 1056 Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr 340 345 350 GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC CTG TTT AAC 1104 Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu Leu Phe Asn 355 360 365 ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC AGT GCC GCT 1152 Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala 370 375 380 TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT GGC AGC ATA 1200 Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile 385 390 395 400 GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT GGA GCA GGG 1248 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 405 410 415 GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG AGC GGT GAC ATG CCC 1296 Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Asp Met Pro 420 425 430 TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC TCT CCT GGT 1344 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 435 440 445 GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT CGG CAT GTG 1392 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 450 455 460 GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 1431 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 465 470 475 Y: C or TR: A or GM: A or CK: G or TS: G or CW: A or TH: A or C or TB: G or T or C Xaa: Pro or Ser Xab: Tyr or His Xac: Glu or Lys Xad: Thr or Ala Xae: T
yr or Phe Xaf: Thr or Ala

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0280

[Correction method] Change

[Correction content]

SEQ ID NO: 42 Sequence length: 2304 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N23N15 Sequence CTG CTG TCG CCC GGG CCC ATC TCY TAC YTG AAG GGY TCC TCG GGT GGT 48 Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly 1 5 10 15 CCG CTG CYT TGC CCC TCG GGC CRT GTT GTG GGC ATC TTC CGG GCT GCY 96 Pro Leu Xaa Cys Pro Ser Gly Xab Val Val Gly Ile Phe Arg Ala Ala 20 25 30 GTG TGC ACC CGG GGG GTT GCG AAG GCG GTR GAC TTT GTG CCC GTT GAG 144 Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu 35 40 45 TCT ATG GAA ACC ACY ATG CGG TCT CCG GTC TTC RCG GAT AAC TCA ACC 192 Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Xac Asp Asn Ser Thr 50 55 60 CCC CCG GCC GTA CCG CAG WCA TTC CAA GTG GCC CAC CTA CAC GCT CCC 240 Pro Pro Ala Val Pro Gln Xad Phe Gln Val Ala His Leu His Ala Pro 65 70 75 80 ACT GGC AGC GGC AAA AGC ACC ARG GTG CCG GCT GCG TAT GCG GCC CAA 288 Thr Gly Ser Gly Lys Ser Thr Xae Val Pro Ala Ala Tyr Ala Ala Gln 85 90 95 GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT GCC ACT TTG GGC 336 Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 100 105 110 TTT GGG GCG TAY ATG TCC AAG GCA CAT GGT GTT GAC CCT AAC ATC AGA 384 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg 115 120 125 ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC RTC ACG TAC TCC ACC 432 Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Xaf Thr Tyr Ser Thr 130 135 140 TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG GGT GCC TAT GAC 480 Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp 145 150 155 160 ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG ACT TCC ATC TTG 528 Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu 165 170 175 GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGC CTT 576 Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu 180 185 190 GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC AC C GTG CCG CAT 624 Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His 195 200 205 CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA GAG ATC CCC TTC 672 Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe 210 215 220 TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG GGG AGG CAT CTC 720 Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu 225 230 235 240 AYT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC GCT GCG AAG CTG 768 Xag Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu 245 250 255 TCG GCC CTC GGA GTC AAY GCT GTA GCA TAY TAC CGG GGT CTT GAT GTG 816 Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val 260 265 270 TCC RTC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG GCA ACW GAC GCT 864 Ser Xah Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala 275 280 285 CTA ATG ACA GGC TAT ACC GGY GAC TTY GAC TCR GTG ATC GAC TGY AAC 912 Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn 290 295 300 ACA TGT GTC ACC CAA ACA GTC GAT TTC AG C TTG GAC CCT ACT TTC ACC 960 Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr 305 310 315 320 ATY GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG 1008 Ile Glu Thr Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 325 330 335 CGA GGC AGG ACT GGT AGG GGC AGR GGG GGC ATA TAC AGG TTT GTA ACT 1056 Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr 340 345 345 350 CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA 1104 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu 355 360 365 TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG YCC GCC GAG ACC 1152 Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Xai Ala Glu Thr 370 375 380 TCG GTT AGG TTG CGG GCT TAC CTA AAY ACA CCT GGG CTG CCC GTC TGC 1200 Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys 385 390 395 400 CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC 1248 Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His 405 410 415 ATA GAT GCC CAC TTC TTG TCC CAG ACY AAA CAG GCA GGA GAC AAC TTC 1296 Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe 420 425 430 CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT 1344 Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala 435 440 445 CCA CCT CCA TCG TGG GAT CAR ATG TGG AAG TGT CTC ATA CGG CTG AAG 1392 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys 450 455 460 CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG YAT AGG TTA GGA GCC GTT 1440 Pro Thr Leu His Gly Pro Thr Pro Leu Leu Xaj Arg Leu Gly Ala Val 465 470 475 480 CAG AAC RAG GTT RCC CTY ACA CAC CCY ATA ACC AAG TWC ATC ATG RCA 1488 Gln Asn Xak Val Xal Leu Thr His Pro Ile Thr Lys Xam Ile Met Xan 485 490 495 TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA 1536 Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val 500 505 510 GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC 1584 Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser 515 520 5 25 GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT 1632 Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile 530 535 540 CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC 1680 Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys 545 550 555 560 GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA 1728 Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln 565 570 575 TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG 1776 Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala 580 585 590 GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC 1824 Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr 595 600 605 TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA 1872 Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu 610 615 620 GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG 1920 Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Se r Leu Met 625 630 635 640 GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC 1968 Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu 645 650 655 CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC 2016 Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro 660 665 670 AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT 2064 Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val 675 680 685 GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT 2112 Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr 690 695 700 GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG AGC GGT 2160 Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly 705 710 715 720 GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC 2208 Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu 725 730 735 TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT 2256 Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg 740 745 750 CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 2304 Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 755 760 765 Y: C or TR: A or GM: A or CK: G or TS: G or CW: A or TH: A or C or TB: G or T or C Xaa: Leu or Pro Xab: His or Arg Xac: Thr or Ala Xad : Ser or Thr Xae: Lys or Arg Xaf: Ile or Val Xag: Thr or Ile Xah: Val or Ile Xai: Pro or Ser Xaj: Tyr or His Xak: Gln or Lys Xal: Thr or Ala Xam: Tyr or Phe Xan : T
hr Ala ──────────────────────────────────────────────── ──────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0281

[Correction method] Change

[Correction content]

SEQ ID NO: 43 Sequence length: 3564 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX25N15 Sequence TGT GCC TGG TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC 48 Cys Ala Trp Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala 1 5 10 15 TTG GAG AAC CTG GTG GTC CTC AAT GCA GCA TCC ATG GCS GGA GCG CAT 96 Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His 20 25 30 GGC ATC CTC TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA 144 Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 35 40 45 GGC AGG CTG GTC CCY GGG GCG RCA TAY GCT YTC TAT GGC GTA TGG CCG 192 Gly Arg Leu Val Pro Gly Ala Xaa Tyr Ala Xab Tyr Gly Val Trp Pro 50 55 60 CTG CTC CTG CTC TTG MTG GCG CTA CCS SCA CGG GCG TAC GCC ATG GAC 240 Leu Leu Leu Leu Leu Xac Ala Leu Pro Xad Arg Ala Tyr Ala Met Asp 65 70 75 80 CGG GAS ATG GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GT A 288 Arg Xae Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val 85 90 95 CTC YTG ACC TTG TCA CCA TAC TAC AAA GTG TTC CTC GCT ARG CTC ATA 336 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Xaf Leu Ile 100 105 110 TGG TGG TTR CAA TAT CTC ATC ACC AGR GCC GAG GCG CAC YTG CAA GTG 384 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val 115 120 125 TGG ATY CCC CCY CTY AAC GTY CGG GGR GGC CGC GAY GCC ATC ATC CTY 432 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 130 135 140 CTC ACR TGT GCG GTC CAY CCR GAG CTR ATY TTT GAC ATC ACC AAR CTY 480 Leu Thr Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu 145 150 155 160 YTG CTC GCC ATA CTC GGT CCG CTC ATG GTR CTC CAG GCT GSC MTA ACY 528 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Xag Xah Thr 165 170 175 MRA RTG CCG TAC TTY GTR CGY GCT CAA GGG CTC ATY CGT RYG TGC ATG 576 Xai Xaj Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Xak Cys Met 180 185 190 TTR GTG CGG AAA GYC GCY GGR GGT CAT TAT GTY CAR ATG G CY YTY RTG 624 Leu Val Arg Lys Xal Ala Gly Gly His Tyr Val Gln Met Ala Xam Xan 195 200 205 AAG CTG GCY GCR CTG ACA GGT ACG TAC RTT TAT GWC CAT CTT RCY CCA 672 Lys Leu Ala Ala Leu Thr Gly Thr Tyr Xao Tyr Xap His Leu Xaq Pro 210 215 220 CTG CAG SAY TGG GCC CAY GCG GGC CTA CGR GAC CTT GCG GTR GCR GTW 720 Leu Gln Xar Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val 225 230 235 240 GAG CCC GTT GYC TTC TCT GAY ATG GAG ACY AAG ATC ATC ACS TGG GGG 768 Glu Pro Val Xas Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly 245 250 255 GCA GAS ACB GCG GCG TGT GGG GAC ATC ATY TYG GGY CTA CCH GTY TCC 816 Ala Xat Thr Ala Ala Cys Gly Asp Ile Ile Xau Gly Leu Pro Val Ser 260 265 270 GCC CGR AGG GGS ARS GAG MTR CTY YTS GGR CCG GCC GAT AGT TTT GAC 864 Ala Arg Arg Gly Xav Glu Xaw Leu Xax Gly Pro Ala Asp Ser Phe Asp 275 280 285 GGG CAG GGG TGG CGA CTC CTT GCG CCT ATC ACG GCC TAC TCC CAG CAR 912 Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln 290 295 300 ACG CGG GGC CTG CTT GGT TGC ATC ATC ACY A GC CTT ACG GGC CGG GAT 960 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 305 310 315 320 AAR AAC CAG GTC GAG GGG GAG GTT CAA GTG GTC TCT ACC GCA ACA CAA 1008 Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln 325 330 335 TCT TTC CTG GCG ACC TGY RTC AAC GGC GTK TGC TGG ACT GTT TTC CAC 1056 Ser Phe Leu Ala Thr Cys Xay Asn Gly Val Cys Trp Thr Val Phe His 340 345 350 GGY GCC GGC TCG AAG ACC TTA GCC GGC CCA AAA GGC CCA ATC ACC CAA 1104 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln 355 360 365 ATG TAC ACC AAT GTR GAT CAG GAC CTC GTC GGY TGG TCG GCG CCC CCC 1152 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Ser Ala Pro Pro 370 375 380 SGG GCG CGT TCC TTG ACA CCW TGC ACC TGC GGC AGC TCG GAC CTT TAT 1200 Xaz Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 385 390 395 400 TTG GTC ACG AGR CAT GCT GAT GTC ATT CCG GTG CAC CGG CGG GGC GAC 1248 Leu Val Thr Arg His Ala Asp Val Ile Pro Val His Arg Arg Gly Asp 405 410 415 AGC AGG GGG AGC CT S CTS TCS CCC GGG CCC ATC TCY TAC YTG AAG GGY 1296 Ser Arg Gly Ser Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly 420 425 430 TCC TCG GGT GGT CCG CTG CYT TGC CCC TCG GGC CRT GTT GTG GGC ATC 1344 Ser Ser Gly Gly Pro Leu Xba Cys Pro Ser Gly Xbb Val Val Gly Ile 435 440 445 TTC CGG GCT GCY GTG TGC ACC CGG GGG GTT GCG AAG GCG GTR GAC TTT 1392 Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe 450 455 460 GTG CCC GTT GAG TCT ATG GAA ACC ACY ATG CGG TCT CCG GTC TTC RCG 1440 Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Xbc 465 470 475 480 GAT AAC TCA ACC CCC CCG GCC GTA CCG CAG WCA TTC CAA GTG GCC CAC 1488 Asp Asn Ser Thr Pro Pro Ala Val Pro Gln Xbd Phe Gln Val Ala His 485 490 495 CTA CAC GCT CCC ACT GGC AGC GGC AAA AGC ACC ARG GTG CCG GCT GCG 1536 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Xbe Val Pro Ala 500 505 510 TAT GCG GCC CAA GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT 1584 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 515 520 525 GCC ACT TTG GGC TTT GGG GCG TAY ATG TCC AAG GCA CAT GGT GTT GAC 1632 Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp 530 535 540 CCT AAC ATC AGA ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC RTC 1680 Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Xbf 545 550 555 560 ACG TAC TCC ACC TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG 1728 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 565 570 575 GGT GCC TAT GAC ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG 1776 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 580 585 590 ACT TCC ATC TTG GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT 1824 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 595 600 605 GGA GCG CGC CTT GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC 1872 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 610 615 620 ACC GTG CCG CAT CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA 1920 Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly 625 630 635 640 GAG ATC CCC TTC TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG 1968 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly 645 650 655 GGG AGG CAT CTC AYT TTC TGC TCC AAG AAG AAA TGT GAC GAG CTC 2016 Gly Arg His Leu Xbg Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 660 665 670 GCT GCG AAG CTG TCG GCC CTC GGA GTC AAY GCT GTA GCA TAY TAC CGG 2064 Ala Ala Lys Leu Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg 675 680 685 GGT CTT GAT GTG TCC RTC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG 2112 Gly Leu Asp Val Ser Xbh Ile Pro Thr Ser Gly Asp Val Val Val Val 690 695 700 GCA ACW GAC GCT CTA ATG ACA GGC TAT ACC GGY GAC TTY GAC TCA GTG 2160 Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val 705 710 715 720 ATC GAC TGC AAC ACA TGT GTC ACC CAA ACA GTC GAT TTC AGC TTG GAC 2208 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp 725 730 735 CCT ACT TTC ACC ATY GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG 2256 Pro Thr Phe Thr Ile Glu Thr Thr T hr Val Pro Gln Asp Ala Val Ser 740 745 750 CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG GGC AGR GGG GGC ATA TAC 2304 Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr 755 760 765 AGG TTT GTA ACT CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG 2352 Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser 770 775 780 GTC CTG TGT GAA TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG 2400 Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 785 790 795 800 YCC GCC GAG ACC TCG GTT AGG TTG CGG GCT TAC CTA AAY ACA CCT GGG 2448 Xbi Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly 805 810 815 CTG CCC GTC TGC CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC 2496 Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr 820 825 830 GGC CTC ACC CAC ATA GAT GCC CAC TTC TTG TCC CAG ACY AAA CAG GCA 2544 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala 835 840 845 GGA GAC AAC TTC CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC 2592 Gly Asp Asn Ph e Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala 850 855 860 AGG GCC AAG GCT CCA CCT CCA TCG TGG GAT CAR ATG TGG AAG TGT CTC 2640 Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu 865 870 875 880 ATA CGG CTG AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG YAT AGG 2688 Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Xbj Arg 885 890 895 TTA GGA GCC GTT CAG AAC RAG GTT RCC CTY ACA CAC CCY ATA ACC AAG 2736 Leu Gly Ala Val Gln Asn Xbk Val Xbl Leu Thr His Pro Ile Thr Lys 900 905 910 TWC ATC ATG RCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT 2784 Xbm Ile Met Xbn Cys Met Ser Ala Asp Leu Glu Val Thr Ser Thr 915 920 925 TGG GTG CTG GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG 2832 Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu 930 935 940 ACA ACG GGC AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG 2880 Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg 945 950 955 960 CCG GCC GTT ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA 2928 Pro Ala Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu 965 970 975 ATG GAA GAG TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG 2976 Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln 980 985 990 CTC GCC GAG CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC 3024 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala 995 1000 1005 ACC AAG CAA GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA 3072 Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg 1010 1015 1020 GCC CTT GAG ACC TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG 3120 Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly 1025 1030 1035 1040 ATA CAG TAC TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA 3168 Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile 1045 1050 1055 GCA TCA CTG ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC 3216 Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr 1060 1065 1070 CAA TAT ACC CTC CTG TTT A AC ATC TTG GGG GGA TGG GTG GCC GCC CAA 3264 Gln Tyr Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln 1075 1080 1085 CTC GCC CCC CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT 3312 Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala 1090 1095 1100 GGC GCG GCT GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT 3360 Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile 1105 1110 1115 1120 CTG GCG GGT TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG 3408 Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys 1125 1130 1135 GTC ATG AGC GGT GAC ATG CCC TCC ACC GAG GAG CTG GTC AAC TTA CTC 3456 Val Met Ser Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu 1140 1145 1150 CCC GCC ATC CTC TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA 3504 Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala 1155 1160 1165 GCA ATA CTG CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG 3552 Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp 1170 1175 1180 ATG AAC CGG CTG 3564 Met Asn Arg Leu 1185 Y: C or TR: A or GM: A or CK: G or TS: G or CW: A or TH: A or C or TB: G or T or C Xaa: Ala or Thr Xab: Phe or Leu Xac: Met or Leu Xad: Ala or Pro Xae: Glu or Asp Xaf: Lys or Arg Xag: Gly or Ala Xah: Leu or Ile Xai: Gln or Arg Xaj: Met or Val Xak: Met or Ala Xal: Ala or Val Xam: Leu or Phe Xan: Met or Val Xao: Val or Ile Xap: Asp or Val Xaq: Thr or Ala Xar: Asp or His Xas: Ala or Val Xat: Asp or Glu Xau: Leu or Ser Xav: Asn or Arg or Lys Xaw: Ile or Leu Xax: Leu or Phe Xay: Ile or Val Xaz: Gly or Arg Xba: Leu or Pro Xbb: His or Arg Xbc: Thr or Ala Xbd: Ser or Thr Xbe: Lys or Arg Xbf: Ile or Val Xbg: Thr or Ile Xbh: Val or Ile Xbi: Pro or Ser Xbj: Tyr or His Xbk: Glu or Lys Xbl: Thr or Ala Xbm: Tyr or Phe Xbn: Thr or Ala

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0282

[Correction method] Change

[Correction content]

SEQ ID NO: 44 Sequence length: 849 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX25-1 Sequence TGT GCC TGG TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC 48 Cys Ala Trp Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala 1 5 10 15 TTG GAG AAC CTG GTG GTC CTC AAT GCA GCA TCC ATG GCG GGA GCG CAT 96 Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His 20 25 30 GGC ATC CTC TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA 144 Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 35 40 45 GGC AGG CTG GTC CCT GGG GCG GCA TAC GCT TTC TAT GGC GTA TGG CCG 192 Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro 50 55 60 CTG CTC CTG CTC TTG ATG GCG CTA CCC GCA CGG GCG TAC GCC ATG GAC 240 Leu Leu Leu Leu Leu Met Ala Leu Pro Ala Arg Ala Tyr Ala Met Asp 65 70 75 80 CGG GAG ATG GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GTA 288 Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val 85 90 95 CTC TTG ACC TTG TCA CCA TAC TAC AAA GTG TTC CTC GCT AAG CTC ATA 336 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Lys Leu Ile 100 105 110 TGG TGG TTG CAA TAT CTC ATC ACC AGG GCC GAG GCG CAC TTG CAA GTG 384 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val 115 120 125 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTT 432 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 130 135 140 CTC ACA TGT GCG GTC CAC CCG GAG CTG ATC TTT GAC ATC ACC AAG CTC 480 Leu Thr Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu 145 150 155 160 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTA CTC CAG GCT GGC CTA ACC 528 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr 165 170 175 CAA ATG CCG TAC TTT GTG CGT GCT CAA GGG CTC ATT CGT ATG TGC ATG 576 Gln Met Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Met Cys Met 180 185 190 TTG GTG CGG AAA GCC GCT GGG GGT CAT TAT GTC CAG ATG GCT CTC ATG 624 Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met 195 200 205 AAG CTG GCT GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTT ACT CCA 672 Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro 210 215 220 CTG CAG GAC TGG GCC CAC GCG GGC CTA CGA GAC CTT GCG GTA GCA GTT 720 Leu Gln Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val 225 230 235 240 GAG CCC GTT GTC TTC TCT GAT ATG GAG ACT AAG ATC ATC ACC TGG GGG 768 Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly 245 250 255 GCA GAC ACT GCG GCG TGT GGG GAC ATC ATT TTG GGC CTA CCT GTC TCC 816 Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser 260 265 270 GCC CGG AGG GGC AAC GAG ATA CTC CTC GGA CCG 849 Ala Arg Arg Gly Asn Glu Ile Leu Leu Gly Pro 275 280

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0290

[Correction method] Change

[Correction content]

SEQ ID NO: 52 Sequence length: 921 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N23-3 Sequence CTG CTG TCG CCC GGG CCC ATC TCT TAC TTG AAG GGC TCC TCG GGT GGT 48 Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly 1 5 10 15 CCG CTG CTT TGC CCC TCG GGC CAT GTT GTG GGC ATC TTC CGG GCT GCC 96 Pro Leu Leu Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala 20 25 30 GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTG CCC GTT GAG 144 Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu 35 40 45 TCT ATG GAA ACC ACC ATG CGG TCT CCG GTC TTC GCG GAT AAC TCA ACC 192 Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Ala Asp Asn Ser Thr 50 55 60 CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAC CTA CAC GCT CCC 240 Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro 65 70 75 80 ACT GGC AGC GGC AAA AGC ACC AGG GTG CCG GCT GCG TAT GCG GCC C AA 288 Thr Gly Ser Gly Lys Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala Gln 85 90 95 GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT GCC ACT TTG GGC 336 Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 100 105 110 TTT GGG GCG TAC ATG TCC AAG GCA CAT GGT GTT GAC CCT AAC ATC AGA 384 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg 115 120 125 ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC GTC ACG TAC TCC ACC 432 Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr 130 135 140 TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG GGT GCC TAT GAC 480 Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp 145 150 155 160 ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG ACT TCC ATC TTG 528 Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu 165 170 175 GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGC CTT 576 Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu 180 185 190 GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCG CAT 624 Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His 195 200 205 CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA GAG ATC CCC TTC 672 Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe 210 215 220 TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG GGG AGG CAT CTC 720 Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu 225 230 235 240 ATT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC GCT GCG AAG CTG 768 Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu 245 250 255 TCG GCC CTC GGA GTC AAT GCT GTA GCA TAT TAC CGG GGT CTT GAT GTG 816 Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val 260 265 270 TCC ATC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG GCA ACA GAC GCT 864 Ser Ile Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala 275 280 285 CTA ATG ACA GGC TAT ACC GGT GAC TTT GAC TCG GTG ATC GAC TGT AAC 912 Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn 290 295 300 ACA TGT GTC 921 Thr Cys Val 305

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0294

[Correction method] Change

[Correction content]

SEQ ID NO: 56 Sequence length: 1280 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX25O26A-1 Sequence TGT GCC TGG TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC 48 Cys Ala Trp Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala 1 5 10 15 TTG GAG AAC CTG GTG GTC CTC AAT GCA GCA TCC ATG GCG GGA GCG CAT 96 Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His 20 25 30 GGC ATC CTC TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA 144 Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 35 40 45 GGC AGG CTG GTC CCT GGG GCG GCA TAC GCT TTC TAT GGC GTA TGG CCG 192 Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro 50 55 60 CTG CTC CTG CTC TTG ATG GCG CTA CCC GCA CGG GCG TAC GCC ATG GAC 240 Leu Leu Leu Leu Leu Met Ala Leu Pro Ala Arg Ala Tyr Ala Met Asp 65 70 75 80 CGG GAG ATG GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GTA 288 Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val 85 90 95 CTC TTG ACC TTG TCA CCA TAC TAC TAC AAA GTG TTC CTC GCT AAG CTC ATA 336 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Lys Leu Ile 100 105 110 TGG TGG TTG CAA TAT CTC ATC ACC AGG GCC GAG GCG CAC TTG CAA GTG 384 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val 115 120 125 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTT 432 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 130 135 140 CTC ACA TGT GCG GTC CAC CCG GAG CTG ATC TTT GAC ATC ACC AAG CTC 480 Leu Thr Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu 145 150 155 160 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTA CTC CAG GCT GGC CTA ACC 528 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr 165 170 175 CAA ATG CCG TAC TTT GTG CGT GCT CAA GGG CTC ATT CGT ATG TGC ATG 576 Gln Met Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Met Cys Met 180 185 190 TTG GTG CGG AAA GCC GCT GGG GGT CAT TAT GTC CA G ATG GCT CTC ATG 624 Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met 195 200 205 AAG CTG GCT GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTT ACT CCA 672 Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro 210 215 220 CTG CAG GAC TGG GCC CAC GCG GGC CTA CGA GAC CTT GCG GTA GCA GTT 720 Leu Gln Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val 225 230 235 240 GAG CCC GTT GTC TTC TCT GAT ATG GAG ACT AAG ATC ATC ACC TGG GGG 768 Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly 245 250 255 GCA GAC ACT GCG GCG TGT GGG GAC ATC ATT TTG GGC CTA CCT GTC TCC 816 Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser 260 265 270 GCC CGG AGG GGC AAC GAG ATA CTC CTC GGA CCG GCC GAT AGT TTT GAC 864 Ala Arg Arg Gly Asn Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Asp 275 280 285 GGG CAG GGG TGG CGA CTC CTT GCG CCT ATC ACG GCC TAC TCC CAG CAG 912 Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln 290 295 300 ACG CGG GGC CTG CTT GGT TGC ATC AT C ACT AGC CTT ACG GGC CGG GAT 960 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 305 310 315 320 AAG AAC CAG GTC GAG GGG GAG GTT CAA GTG GTC TCT ACC GCA ACA CAA 1008 Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln 325 330 335 TCT TTC CTG GCG ACC TGT GTC AAC GGC GTG TGC TGG ACT GTT TTC CAC 1056 Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Phe His 340 345 350 GGC GCC GGC TCG AAG ACC TTA GCC GGC CCA AAA GGC CCA ATC ACC CAA 1104 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln 355 360 365 ATG TAC ACC AAT GTA GAT CAG GAC CTC GTC GGC TGG TCG GCG CCC CCC 1152 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Ser Ala Pro Pro 370 375 380 CGG GCG CGT TCC TTG ACA CCT TGC ACC TGC GGC AGC TCG GAC CTT TAT 1200 Arg Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 385 390 395 400 TTG GTC ACG AGG CAT GCT GAT GTC ATT CCG GTG CAC CGG CGG GGC GAC 1248 Leu Val Thr Arg His Ala Asp Val Ile Pro Val His Arg Arg Gly Asp 405 410 415 AGC AGG GGG AGC CTC CTC TCC CCC GGG CCC AT 1280 Ser Arg Gly Ser Leu Leu Ser Pro Gly Pro 420 4
25

[Submission date] September 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0295

[Correction method] Change

[Correction content]

SEQ ID NO: 57 Sequence length: 1280 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX25O26B-1 Sequence TGT GCC TGG TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC 48 Cys Ala Trp Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala 1 5 10 15 TTG GAG AAC CTG GTG GTC CTC AAT GCA GCA TCC ATG GCG GGA GCG CAT 96 Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His 20 25 30 GGC ATC CTC TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA 144 Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 35 40 45 GGC AGG CTG GTC CCT GGG GCG GCA TAC GCT TTC TAT GGC GTA TGG CCG 192 Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro 50 55 60 CTG CTC CTG CTC TTG ATG GCG CTA CCC GCA CGG GCG TAC GCC ATG GAC 240 Leu Leu Leu Leu Leu Met Ala Leu Pro Ala Arg Ala Tyr Ala Met Asp 65 70 75 80 CGG GAG ATG GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GTA 288 Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val 85 90 95 CTC TTG ACC TTG TCA CCA TAC TAC TAC AAA GTG TTC CTC GCT AAG CTC ATA 336 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Lys Leu Ile 100 105 110 TGG TGG TTG CAA TAT CTC ATC ACC AGG GCC GAG GCG CAC TTG CAA GTG 384 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val 115 120 125 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTT 432 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 130 135 140 CTC ACA TGT GCG GTC CAC CCG GAG CTG ATC TTT GAC ATC ACC AAG CTC 480 Leu Thr Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu 145 150 155 160 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTA CTC CAG GCT GGC CTA ACC 528 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr 165 170 175 CAA ATG CCG TAC TTT GTG CGT GCT CAA GGG CTC ATT CGT ATG TGC ATG 576 Gln Met Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Met Cys Met 180 185 190 TTG GTG CGG AAA GCC GCT GGG GGT CAT TAT GTC CA G ATG GCT CTC ATG 624 Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met 195 200 205 AAG CTG GCT GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTT ACT CCA 672 Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro 210 215 220 CTG CAG GAC TGG GCC CAC GCG GGC CTA CGA GAC CTT GCG GTA GCA GTT 720 Leu Gln Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val 225 230 235 240 GAG CCC GTT GTC TTC TCT GAT ATG GAG ACT AAG ATC ATC ACG TGG GGG 768 Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly 245 250 255 GCA GAG ACG GCG GCG TGT GGG GAC ATC ATC TCG GGT CTA CCC GTT TCC 816 Ala Glu Thr Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser 260 265 270 GCC CGA AGG GGG AGG GAG CTG CTT TTG GGG CCG GCC GAT AGT TTT GAC 864 Ala Arg Arg Gly Arg Glu Leu Leu Leu Gly Pro Ala Asp Ser Phe Asp 275 280 285 GGG CAG GGG TGG CGA CTC CTT GCG CCT ATC ACG GCC TAC TCC CAG CAG 912 Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln 290 295 300 ACG CGG GGC CTG CTT GGT TGC ATC AT C ACT AGC CTT ACG GGC CGG GAT 960 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 305 310 315 320 AAG AAC CAG GTC GAG GGG GAG GTT CAA GTG GTC TCT ACC GCA ACA CAA 1008 Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln 325 330 335 TCT TTC CTG GCG ACC TGT GTC AAC GGC GTG TGC TGG ACT GTT TTC CAC 1056 Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Phe His 340 345 350 GGC GCC GGC TCG AAG ACC TTA GCC GGC CCA AAA GGC CCA ATC ACC CAA 1104 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln 355 360 365 ATG TAC ACC AAT GTA GAT CAG GAC CTC GTC GGC TGG TCG GCG CCC CCC 1152 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Ser Ala Pro Pro 370 375 380 CGG GCG CGT TCC TTG ACA CCT TGC ACC TGC GGC AGC TCG GAC CTT TAT 1200 Arg Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 385 390 395 400 TTG GTC ACG AGG CAT GCT GAT GTC ATT CCG GTG CAC CGG CGG GGC GAC 1248 Leu Val Thr Arg His Ala Asp Val Ile Pro Val His Arg Arg Gly Asp 405 410 415 AGC AGG GGG AGC CTC CTC TCC CCC GGG CCC AT 1280 Ser Arg Gly Ser Leu Leu Ser Pro Gly Pro 420 4
25 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0296

[Correction method] Change

[Correction content]

SEQ ID NO: 58 Sequence length: 1431 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N16N15A-1 Sequence GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGC AAC ACA TGT GTC 48 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1 5 10 15 ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC ATC GAG ACG 96 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 20 25 30 ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG 144 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg 35 40 45 ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA ACT CCA GGG GAA 192 Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu 50 55 60 CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA TGT TAT GAC 240 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp 65 70 75 80 GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG ACC TCG GTT AGG 288 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg 85 90 95 TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC TGC CAG GAC CAT 336 Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His 100 105 110 CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC ATA GAT GCC 384 Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala 115 120 125 CAC TTC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC TTC CCC TAC CTG 432 His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu 130 135 140 GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT CCA CCT CCA 480 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro 145 150 155 160 TCG TGG GAT CAG ATG TGG AAG TGT CTC ATA CGG CTG AAG CCT ACG CTA 528 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu 165 170 175 CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC GTT CAG AAC GAG 576 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu 180 185 190 GTT ACC CTT ACA CAC CCC ATA ACC AAG TAC ATC ATG ACA TGC ATG TCG 624 Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys Met Ser 195 200 205 GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA GGC GGG GTC 672 Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val 210 215 220 CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC GTG GTC ATT 720 Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile 225 230 235 240 GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT CCC GAC AGG 768 Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg 245 250 255 GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC GCC TCG CAC 816 Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His 260 265 270 CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA TTC AAG CAG 864 Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln 275 280 285 AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG GAG GCT GCT 912 Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala 290 295 300 GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC TTC TGG GCG 960 Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala 305 310 315 320 AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA GCA GGC TTG 1008 Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu 325 330 335 TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG GCA TTC ACA 1056 Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr 340 345 350 GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC CTG TTT AAC 1104 Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu Leu Phe Asn 355 360 365 ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC AGT GCC GCT 1152 Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala 370 375 380 TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT GGC AGC ATA 1200 Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile 385 390 395 400 GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT GGA GCA GGG 1248 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 405 410 415 GTG GCA GGC GCG C TC GTG GCC TTT AAG GTC ATG AGC GGT GAC ATG CCC 1296 Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Asp Met Pro 420 425 430 TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC TCT CCT GGT 1344 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 435 440 445 GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT CGG CAT GTG 1392 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 450 455 460 GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 1431 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 465 470
475

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0297

[Correction method] Change

[Correction content]

SEQ ID NO: 59 Sequence length: 1431 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N16N15B-1 Sequence GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGC AAC ACA TGT GTC 48 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1 5 10 15 ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC ATC GAG ACG 96 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 20 25 30 ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG 144 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg 35 40 45 ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA ACT CCA GGG GAA 192 Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu 50 55 60 CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA TGT TAT GAC 240 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp 65 70 75 80 GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG ACC TCG GTT AGG 288 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg 85 90 95 TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC TGC CAG GAC CAT 336 Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His 100 105 110 CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC ATA GAT GCC 384 Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala 115 120 125 CAC TTC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC TTC CCC TAC CTG 432 His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu 130 135 140 GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT CCA CCT CCA 480 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro 145 150 155 160 TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG CTG AAG CCT ACG CTA 528 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu 165 170 175 CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC GTT CAG AAC GAG 576 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu 180 185 190 GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC ATG GCA TGC ATG TCG 624 Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile Met Ala Cys Met Ser 195 200 205 GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA GGC GGG GTC 672 Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val 210 215 220 CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC GTG GTC ATT 720 Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile 225 230 235 240 GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT CCC GAC AGG 768 Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg 245 250 255 GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC GCC TCG CAC 816 Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His 260 265 270 CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA TTC AAG CAG 864 Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln 275 280 285 AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG GAG GCT GCT 912 Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala 290 295 300 GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC TTC TGG GCG 960 Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala 305 310 315 320 AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA GCA GGC TTG 1008 Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu 325 330 335 TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG GCA TTC ACA 1056 Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr 340 345 350 GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC CTG TTT AAC 1104 Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu Leu Phe Asn 355 360 365 ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC AGT GCC GCT 1152 Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala 370 375 380 TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT GGC AGC ATA 1200 Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile 385 390 395 400 GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT GGA GCA GGG 1248 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 405 410 415 GTG GCA GGC G CG CTC GTG GCC TTT AAG GTC ATG AGC GGT GAC ATG CCC 1296 Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Asp Met Pro 420 425 430 TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC TCT CCT GGT 1344 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 435 440 445 GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT CGG CAT GTG 1392 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 450 455 460 GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 1431 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 465 470
475 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0298

[Correction method] Change

[Correction content]

SEQ ID NO: 60 Sequence length: 1431 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N16N15-1 Sequence GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGC AAC ACA TGT GTC 48 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1 5 10 15 ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC ATC GAG ACG 96 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 20 25 30 ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG 144 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg 35 40 45 ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA ACT CCA GGG GAA 192 Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu 50 55 60 CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA TGT TAT GAC 240 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp 65 70 75 80 GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG ACC TCG GTT A GG 288 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg 85 90 95 TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC TGC CAG GAC CAT 336 Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His 100 105 110 CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC ATA GAT GCC 384 Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala 115 120 125 CAC TTC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC TTC CCC TAC CTG 432 His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu 130 135 140 GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT CCA CCT CCA 480 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro 145 150 155 160 TCG TGG GAT CAG ATG TGG AAG TGT CTC ATA CGG CTG AAG CCT ACG CTA 528 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu 165 170 175 CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC GTT CAG AAC GAG 576 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu 180 185 190 GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC ATG GCA TGC ATG TCG 624 Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile Met Ala Cys Met Ser 195 200 205 GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA GGC GGG GTC 672 Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val 210 215 220 CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC GTG GTC ATT 720 Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile 225 230 235 240 GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT CCC GAC AGG 768 Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg 245 250 255 GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC GCC TCG CAC 816 Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His 260 265 270 CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA TTC AAG CAG 864 Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln 275 280 285 AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG GAG GCT GCT 912 Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala 290 295 300 GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC TTC TGG GCG 960 Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala 305 310 315 320 AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA GCA GGC TTG 1008 Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu 325 330 335 TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG GCA TTC ACA 1056 Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr 340 345 350 GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC CTG TTT AAC 1104 Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu Leu Phe Asn 355 360 365 ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC AGT GCC GCT 1152 Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala 370 375 380 TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT GGC AGC ATA 1200 Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile 385 390 395 400 GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT GGA GCA GGG 1248 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 405 410 415 GTG GCA GGC GCG CT C GTG GCC TTT AAG GTC ATG AGC GGT GAC ATG CCC 1296 Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Asp Met Pro 420 425 430 TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC TCT CCT GGT 1344 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 435 440 445 GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT CGG CAT GTG 1392 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 450 455 460 GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 1431 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 465 470
475

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0299

[Correction method] Change

[Correction content]

SEQ ID NO: 61 Sequence length: 2304 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N23N15A-1 Sequence CTG CTG TCG CCC GGG CCC ATC TCT TAC TTG AAG GGT TCC TCG GGT GGT 48 Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly 1 5 10 15 CCG CTG CCT TGC CCC TCG GGC CGT GTT GTG GGC ATC TTC CGG GCT GCC 96 Pro Leu Pro Cys Pro Ser Gly Arg Val Val Gly Ile Phe Arg Ala Ala 20 25 30 GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTG CCC GTT GAG 144 Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu 35 40 45 TCT ATG GAA ACC ACC ATG CGG TCT CCG GTC TTC ACG GAT AAC TCA ACC 192 Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr 50 55 60 CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAC CTA CAC GCT CCC 240 Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro 65 70 75 80 ACT GGC AGC GGC AAA AGC ACC AGG GTG CCG GCT GCG TAT GCG GCC CAA 288 Thr Gly Ser Gly Lys Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala Gln 85 90 95 GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT GCC ACT TTG GGC 336 Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 100 105 110 TTT GGG GCG TAC ATG TCC AAG GCA CAT GGT GTT GAC CCT AAC ATC AGA 384 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg 115 120 125 ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC ATC ACG TAC TCC ACC 432 Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr 130 135 140 TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG GGT GCC TAT GAC 480 Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp 145 150 155 160 ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG ACT TCC ATC TTG 528 Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu 165 170 175 GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGC CTT 576 Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu 180 185 190 GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCG CAT 624 Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His 195 200 205 CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA GAG ATC CCC TTC 672 Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe 210 215 220 TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG GGG AGG CAT CTC 720 Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu 225 230 235 240 ATT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC GCT GCG AAG CTG 768 Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu 245 250 255 TCG GCC CTC GGA GTC AAC GCT GTA GCA TAT TAC CGG GGT CTT GAT GTG 816 Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val 260 265 270 TCC ATC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG GCA ACA GAC GCT 864 Ser Ile Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala 275 280 285 CTA ATG ACA GGC TAT ACC GGT GAC TTT GAC TCG GTG ATC GAC TGC AAC 912 Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn 290 295 300 ACA TGT GTC ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC 960 Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr 305 310 315 320 ATC GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG 1008 Ile Glu Thr Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 325 330 335 CGA GGC AGG ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA ACT 1056 Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr 340 345 350 CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA 1104 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu 355 360 365 TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG ACC 1152 Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr 370 375 380 TCG GTT AGG TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC TGC 1200 Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys 385 390 395 400 CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC 1248 Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His 405 410 415 ATA GAT GCC C AC TTC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC TTC 1296 Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe 420 425 430 CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT 1344 Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala 435 440 445 CCA CCT CCA TCG TGG GAT CAG ATG TGG AAG TGT CTC ATA CGG CTG AAG 1392 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys 450 455 460 CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC GTT 1440 Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val 465 470 475 480 CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC ATG GCA 1488 Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile Met Ala 485 490 495 TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA 1536 Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val 500 505 510 GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC 1584 Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser 515 52 0 525 GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT 1632 Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile 530 535 540 CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC 1680 Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys 545 550 555 560 GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA 1728 Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln 565 570 575 TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG 1776 Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala 580 585 590 GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC 1824 Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr 595 600 605 TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA 1872 Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu 610 615 620 GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG 1920 Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met 625 630 635 640 GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC 1968 Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu 645 655 655 CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC 2016 Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro 660 665 670 AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT 2064 Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val 675 680 685 GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT 2112 Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr 690 695 695 700 GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG AGC GGT 2160 Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly 705 710 715 720 GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC 2208 Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu 725 730 735 TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT 2256 Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg 740 745 750 CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 2304 Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 755 760
765 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0300

[Correction method] Change

[Correction content]

SEQ ID NO: 62 Sequence length: 2304 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N23N15B-1 Sequence CTG CTG TCG CCC GGG CCC ATC TCT TAC TTG AAG GGT TCC TCG GGT GGT 48 Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly 1 5 10 15 CCG CTG CCT TGC CCC TCG GGC CGT GTT GTG GGC ATC TTC CGG GCT GCC 96 Pro Leu Pro Cys Pro Ser Gly Arg Val Val Gly Ile Phe Arg Ala Ala 20 25 30 GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTG CCC GTT GAG 144 Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu 35 40 45 TCT ATG GAA ACC ACC ATG CGG TCT CCG GTC TTC ACG GAT AAC TCA ACC 192 Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr 50 55 60 CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAC CTA CAC GCT CCC 240 Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro 65 70 75 80 ACT GGC AGC GGC AAA AGC ACC AGG GTG CCG GCT GCG TAT GCG GCC CAA 288 Thr Gly Ser Gly Lys Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala Gln 85 90 95 GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT GCC ACT TTG GGC 336 Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 100 105 110 TTT GGG GCG TAC ATG TCC AAG GCA CAT GGT GTT GAC CCT AAC ATC AGA 384 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg 115 120 125 ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC ATC ACG TAC TCC ACC 432 Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr 130 135 140 TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG GGT GCC TAT GAC 480 Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp 145 150 155 160 ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG ACT TCC ATC TTG 528 Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu 165 170 175 GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGC CTT 576 Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu 180 185 190 GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCG CAT 624 Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His 195 200 205 CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA GAG ATC CCC TTC 672 Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe 210 215 220 TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG GGG AGG CAT CTC 720 Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu 225 230 235 240 ATT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC GCT GCG AAG CTG 768 Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu 245 250 255 TCG GCC CTC GGA GTC AAC GCT GTA GCA TAT TAC CGG GGT CTT GAT GTG 816 Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val 260 265 270 TCC ATC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG GCA ACA GAC GCT 864 Ser Ile Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala 275 280 285 CTA ATG ACG GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGC AAC 912 Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn 290 295 300 ACA TGT GTC ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC ACC 960 Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr 305 310 315 320 ATC GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG CGG 1008 Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 325 330 335 CGA GGC AGG ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA ACT 1056 Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr 340 345 350 CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT GAA 1104 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu 355 360 365 TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG ACC 1152 Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr 370 375 380 TCG GTT AGG TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC TGC 1200 Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys 385 390 395 400 CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC CAC 1248 Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His 405 410 415 ATA GAT GCC CAC T TC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC TTC 1296 Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe 420 425 430 CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG GCT 1344 Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala 435 440 445 CCA CCT CCA TCG TGG GAT CAG ATG TGG AAG TGT CTC ATA CGG CTG AAG 1392 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys 450 455 460 CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC GTT 1440 Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val 465 470 475 480 CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC ATG GCA 1488 Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile Met Ala 485 490 495 TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG GTA 1536 Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val 500 505 510 GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC AGC 1584 Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser 515 520 52 5 GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT ATT 1632 Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile 530 535 540 CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG TGC 1680 Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys 545 550 555 560 GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC GAG CAA 1728 Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln 565 570 575 TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA GCG 1776 Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala 580 585 590 GAG GCT GCT GCT GCT GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG ACC 1824 Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr 595 600 605 TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC TTA 1872 Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu 610 615 620 GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG ATG 1920 Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met 625 630 635 640 GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC CTC 1968 Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr Leu 645 650 655 CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC CCC 2016 Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro 660 665 670 AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT GTT 2064 Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val 675 680 685 GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT TAT 2112 Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr 690 695 700 GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG AGC GGT 2160 Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly 705 710 715 720 GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC CTC 2208 Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu 725 730 735 TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG CGT 2256 Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg 740 745 750 CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 2304 Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 755 760
765

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0301

[Correction method] Change

[Correction content]

SEQ ID NO: 63 Sequence length: 3564 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: MX25N15-1 Sequence TGT GCC TGG TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC 48 Cys Ala Trp Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala 1 5 10 15 TTG GAG AAC CTG GTG GTC CTC AAT GCA GCA TCC ATG GCG GGA GCG CAT 96 Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His 20 25 30 GGC ATC CTC TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA 144 Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 35 40 45 GGC AGG CTG GTC CCT GGG GCG GCA TAC GCT TTC TAT GGC GTA TGG CCG 192 Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro 50 55 60 CTG CTC CTG CTC TTG ATG GCG CTA CCC GCA CGG GCG TAC GCC ATG GAC 240 Leu Leu Leu Leu Leu Met Ala Leu Pro Ala Arg Ala Tyr Ala Met Asp 65 70 75 80 CGG GAG ATG GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GTA 288 Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val 85 90 95 CTC TTG ACC TTG TCA CCA TAC TAC TAC AAA GTG TTC CTC GCT AAG CTC ATA 336 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Lys Leu Ile 100 105 110 TGG TGG TTG CAA TAT CTC ATC ACC AGG GCC GAG GCG CAC TTG CAA GTG 384 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val 115 120 125 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTT 432 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 130 135 140 CTC ACA TGT GCG GTC CAC CCG GAG CTG ATC TTT GAC ATC ACC AAG CTC 480 Leu Thr Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu 145 150 155 160 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTA CTC CAG GCT GGC CTA ACC 528 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr 165 170 175 CAA ATG CCG TAC TTT GTG CGT GCT CAA GGG CTC ATT CGT ATG TGC ATG 576 Gln Met Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Met Cys Met 180 185 190 TTG GTG CGG AAA GCC GCT GGG GGT CAT TAT GTC CAG ATG GCT CTC ATG 624 Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met 195 200 205 AAG CTG GCT GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTT ACT CCA 672 Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro 210 215 220 CTG CAG GAC TGG GCC CAC GCG GGC CTA CGA GAC CTT GCG GTA GCA GTT 720 Leu Gln Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val 225 230 235 240 GAG CCC GTT GCC TTC TCT GAT ATG GAG ACT AAG ATC ATC ACC TGG GGG 768 Glu Pro Val Ala Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly 245 250 255 GCA GAC ACT GCG GCG TGT GGG GAC ATC ATT TTG GGC CTA CCT GTC TCC 816 Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser 260 265 270 GCC CGG AGG GGC AAC GAG ATA CTC CTC GGA CCG GCC GAT AGT TTT GAC 864 Ala Arg Arg Gly Asn Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Asp 275 280 285 GGG CAG GGG TGG CGA CTC CTT GCG CCT ATC ACG GCC TAC TCC CAG CAG 912 Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln 290 295 300 ACG CGG GGC CTG CTT GGT TGC ATC ATC ACT AGC CTT ACG GGC CGG GAT 960 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 305 310 315 320 AAG AAC CAG GTC GAG GGG GAG GTT CAA GTG GTC TCT ACC GCA ACA CAA 1008 Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln 325 330 335 TCT TTC CTG GCG ACC TGT GTC AAC GGC GTG TGC TGG ACT GTT TTC CAC 1056 Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Phe His 340 345 350 GGC GCC GGC TCG AAG ACC TTA GCC GGC CCA AAA GGC CCA ATC ACC CAA 1104 Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln 355 360 365 ATG TAC ACC AAT GTA GAT CAG GAC CTC GTC GGC TGG TCG GCG CCC CCC 1152 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Ser Ala Pro Pro 370 375 380 CGG GCG CGT TCC TTG ACA CCT TGC ACC TGC GGC AGC TCG GAC CTT TAT 1200 Arg Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 385 390 395 400 TTG GTC ACG AGG CAT GCT GAT GTC ATT CCG GTG CAC CGG CGG GGC GAC 1248 Leu Val Thr Arg His Ala Asp Val Ile Pro Val His Arg Arg Gly Asp 405 410 415 AGC AGG GGG A GC CTC CTC TCC CCC GGG CCC ATC TCT TAC TTG AAG GGT 1296 Ser Arg Gly Ser Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly 420 425 430 TCC TCG GGT GGT CCG CTG CCT TGC CCC TCG GGC CGT GTT GTG GGC ATC 1344 Ser Ser Gly Gly Pro Leu Pro Cys Pro Ser Gly Arg Val Val Gly Ile 435 440 445 TTC CGG GCT GCC GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT 1392 Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe 450 455 460 GTG CCC GTT GAG TCT ATG GAA ACC ACC ATG CGG TCT CCG GTC TTC ACG 1440 Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr 465 470 475 480 GAT AAC TCA ACC CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAC 1488 Asp Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His 485 490 495 CTA CAC GCT CCC ACT GGC AGC GGC AAA AGC ACC AGG GTG CCG GCT GCG 1536 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Arg Val Pro Ala Ala 500 505 510 TAT GCG GCC CAA GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT 1584 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 515 52 0 525 GCC ACT TTG GGC TTT GGG GCG TAC ATG TCC AAG GCA CAT GGT GTT GAC 1632 Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp 530 535 540 CCT AAC ATC AGA ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC ATC 1680 Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile 545 550 555 560 ACG TAC TCC ACC TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG 1728 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 565 570 575 GGT GCC TAT GAC ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG 1776 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 580 585 590 ACT TCC ATC TTG GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT 1824 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 595 600 605 GGA GCG CGC CTT GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC 1872 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 610 615 620 ACC GTG CCG CAT CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA 1920 Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly 625 630 635 640 GAG ATC CCC TTC TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG 1968 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly 645 650 655 GGG AGG CAT CTC ATT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC 2016 Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 660 665 670 GCT GCG AAG CTG TCG GCC CTC GGA GTC AAC GCT GTA GCA TAT TAC CGG 2064 Ala Ala Lys Leu Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg 675 680 685 GGT CTT GAT GTG TCC ATC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG 2112 Gly Leu Asp Val Ser Ile Ile Pro Thr Ser Gly Asp Val Val Val Val 690 695 700 GCA ACA GAC GCT CTA ATG ACA GGC TAT ACC GGT GAC TTT GAC TCG GTG 2160 Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val 705 710 715 720 ATC GAC TGC AAC ACA TGT GTC ACC CAA ACA GTC GAT TTC AGC TTG GAC 2208 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp 725 730 735 CCT ACT TTC ACC ATC GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG 2256 Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser 740 745 750 CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG GGC AGG GGG GGC ATA TAC 2304 Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr 755 760 765 AGG TTT GTA ACT CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG 2352 Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser 770 775 780 GTC CTG TGT GAA TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG 2400 Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 785 790 795 800 CCC GCC GAG ACC TCG GTT AGG TTG CGG GCT TAC CTA AAT ACA CCT GGG 2448 Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly 805 810 815 CTG CCC GTC TGC CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC 2496 Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr 820 825 830 GGC CTC ACC CAC ATA GAT GCC CAC TTC TTG TCC CAG ACC AAA CAG GCA 2544 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala 835 840 845 GGA GAC AAC TTC CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC 2592 Gly Asp A sn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala 850 855 860 AGG GCC AAG GCT CCA CCT CCA TCG TGG GAT CAG ATG TGG AAG TGT CTC 2640 Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu 865 870 875 880 ATA CGG CTG AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG 2688 Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg 885 890 895 TTA GGA GCC GTT CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG 2736 Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys 900 905 910 TTC ATC ATG GCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT 2784 Phe Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr 915 920 925 TGG GTG CTG GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG 2832 Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu 930 935 940 ACA ACG GGC AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG 2880 Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg 945 950 955 960 CCG GCC GTT ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA 2928 Pro Ala Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu 965 970 975 ATG GAA GAG TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG 2976 Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln 980 985 990 CTC GCC GAG CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC 3024 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala 995 1000 1005 ACC AAG CAA GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA 3072 Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg 1010 1015 1020 GCC CTT GAG ACC TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG 3120 Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly 1025 1030 1035 1040 ATA CAG TAC TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA 3168 Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile 1045 1050 1055 GCA TCA CTG ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC 3216 Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr 1060 1065 1070 CAA TAT ACC CTC CTG T TT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA 3264 Gln Tyr Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln 1075 1080 1085 CTC GCC CCC CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT 3312 Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala 1090 1095 1100 GGC GCG GCT GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT 3360 Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile 1105 1110 1115 1120 CTG GCG GGT TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG 3408 Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys 1125 1130 1135 GTC ATG AGC GGT GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC 3456 Val Met Ser Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu 1140 1145 1150 CCC GCC ATC CTC TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA 3504 Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala 1155 1160 1165 GCA ATA CTG CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG 3552 Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp 1170 1175 1180 ATG AAC CGG CTG 3564 Met Asn Arg Leu 1185 ───────────────────────────────────── ─────────────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0302

[Correction method] Change

[Correction content]

SEQ ID NO: 64 Sequence length: 818 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N22-1, N22 -3, H22-8, H22-9 Sequence GG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 47 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 1 5 10 15 ATA GCG TTY GCY TCG CGG GGY AAC CAY GTC TCC CCC ACG CAY TAT GTG 95 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 20 25 30 CCT GAR AGC GAC GCC GCR GCG CGY GTC ACC CAG ATC CTC TCC ARC CTY 143 Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Xaa Leu 35 40 45 ACC ATC ACT CAG YTG YTG AAG AGG CTY CAC CAG TGG ATT RAT GAK GAC 191 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asx Xac Asp 50 55 60 TGC TCC ACG CCA TGY TCY GGY TCG TGG CTC AGG GAT GTT TGG GAC TGG 239 Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 65 70 75 ATA TGC ACG GTR TTG RST GAY TTC AAG ACC TGG CTC CAG TCC AAG CTC 287 Ile Cys Thr Val Leu Xad Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu 80 85 90 95 CTG CCG CGG YTA CCG GGR GTC CCT TTY YTY TCA TGC CAR CGT GGG TAC 335 Leu Pro Arg Leu Pro Gly Val Pro Phe Xae Ser Cys Gln Arg Gly Tyr 100 105 110 AAG GGR GTY TGG CGG GGA GAY GGC ATC ATG YAD ACC ACC TGC CCA TGY 383 Lys Gly Val Trp Arg Gly Asp Gly Ile Met Xaf Thr Thr Cys Pro Cys 115 120 125 GGA GCA CAA ATC RCC GGA CAT GTC AAA AAY GGT TCY ATG AGG ATC RYT 431 Gly Ala Gln Ile Xag Gly His Val Lys Asn Gly Ser Met Arg Ile Xah 130 135 140 GGS CYY AGA ACC TGT AGC AAC ACG TGG CRC GGA ACR TTY CCC ATC AAC 479 Gly Xai Arg Thr Cys Ser Asn Thr Trp Xaj Gly Thr Phe Pro Ile Asn 145 150 155 GCG TAC ACC ACC AGC GGC CCC TGC ACA CCC TCY CCR GCG CCR AAC TAY TCY 527 Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser 160 165 170 175 ARG GCG TTR TGG CGG GTR GCY GCT GAG GAG TAT GTG GAG GTC ACG CGG 575 Xak Ala Leu Trp Arg Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg 180 185 190 GTG GGG GAY TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC KTR AAA 623 Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Xal Lys 195 200 205 TGC CCA TGC CAG GTY CCG GCC CCC GAA TTY TTC ACR GAR TTG GAT GGG 671 Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Thr Glu Leu Asp Gly 210 215 220 GTR CGG CTR CRC AGR TAC GCT CCG GCG TGC AAA CCT CTC CTR CGG GAT 719 Val Arg Leu Xaj Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp 225 230 235 GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TWY MCG GTT GGG TCR CAG 767 Glu Val Thr Phe Gln Val Gly Leu Asn Gln Xam Xan Val Gly Ser Gln 240 245 250 255 CTM CCA TGY GAG CCC GAA CCG GAT GTA RYR GTG GTC ACC TCC ATG CTC 815 Leu Pro Cys Glu Pro Glu Pro Asp Val Xao Val Val Thr Ser Met Leu 260 265 270 ACC 818 Thr Y: C or TR: A or GM: A or CK: G or TS: G or CW: A or TD: G or T or A Xaa: Asn or Ser Asx: Asn or Asp Xac: Glu or Asp Xad: Ala or Ser Xae: Phe or Leu Xaf: Tyr or Gln or His Xag: Thr or Ala Xah: Val or Thr Xai: Pro or Leu Xaj: His or Arg Xak: Arg or Lys Xal: Val or Leu Xam: Tyr or Phe X an: Thr or Pro Xao: T
hr or Met or Ala

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0304

[Correction method] Change

[Correction content]

SEQ ID NO: 66 Sequence length: 740 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O28-1, O28 -2, O28-4 Sequence GTG GTA GTC CTG GAC TCG TTG GAS CCG CTT CRA GCG RAG GAA GRT GAG 48 Val Val Val Leu Asp Ser Leu Xaa Pro Leu Xab Ala Xac Glu Xad Glu 1 5 10 15 AGG GAA GTG TCC GTT GCG GCG GAG ATC CTG CGR AAG ACC ARG AAA TTC 96 Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Thr Xae Lys Phe 20 25 30 CCC GCA GCG ATG CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA 144 Pro Ala Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu 35 40 45 CTA GAG TCT TGG AAG AAC CCG GAC TAC GTC CCT CCR GTG GTA CAC GGG 192 Leu Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly 50 55 60 TGC CCA TTG CCG CCT AYC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA 240 Cys Pro Leu Pro Pro Xaf Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg 65 70 75 80 AAG AGR ACG GTT GYC CTG ACA GAA TCC ACC GTG TCC T CT GCC TTG GCG 288 Lys Arg Thr Val Xag Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Ala 85 90 95 GAG CTT GCT ACA AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC 336 Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp 100 105 110 AGC GGC ACG GCG ACY GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT 384 Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp 115 120 125 GCA GGA TCC GAC GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA 432 Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly 130 135 140 GAG CCG GGG GAC CCY GAT CTC AAC GAC GGG TCT TGG TCT ACY GTA AGC 480 Glu Pro Gly Asp Pro Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser 145 150 155 160 GAG GAG GCC RGC GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG 528 Glu Glu Ala Xah Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp 165 170 175 ACA GGC GCC TTA ATT ACA CCA TGC RCC GCG GAG GAG AGC AAG CTG CCC 576 Thr Gly Ala Leu Ile Thr Pro Cys Xai Ala Glu Glu Ser Lys Leu Pro 180 185 190 ATT AAT GCG CTG AGC AAC YCT TTG CTG CGY CAC CAC AAC ATG GTC TAT 624 Ile Asn Ala Leu Ser Asn Xaj Leu Leu Arg His His Asn Met Val Tyr 195 200 205 GCC ACA ACA TCC CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT 672 Ala Thr Thr Ser Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe 210 215 220 GAC AGA CTG CAA GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC 720 Asp Arg Leu Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp 225 230 235 240 ATG AAG GCC AAG GCG TCC AC 740 Met Lys Ala Lys Ala Ser 245 Y: C or TR: A or GS: G or C Xaa: Glu or Asp Xab: Gln or Arg Xac: Lys or Glu Xad: Gly or Asp Xae: A
rg or Lys Xaf: Thr or Ile Xag: Val or Ala
Xah: Ser or Gly Xai: Ala or Thr Xaj: P
ro or Ser

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0307

[Correction method] Change

[Correction content]

SEQ ID NO: 69 Sequence length: 1171 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O30-2, O30 -3, O30-4 Sequence TG GGG ATC CCG TAT GAT ACC CGC TGC TTT GAC TCA ACR GTC ACT GAG 47 Gly Ile Pro Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu 1 5 10 15 AAT GAC ATC CGT GTY GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC 95 Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala 20 25 30 CCC GAG GCC AGA CAG GCC ATA AGG TCR CTC ACA GAG CGG CTT TAC ATC 143 Pro Glu Ala Arg Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile 35 40 45 GGG GGC CCC CTG ACT AAT TCA AAR GGG CAG AAC TGC GGY TAT CGC CGG 191 Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg 50 55 60 TGC CGC GYC AGC GGC GTG CTG ACG ACT AGC TGC GGY AAT ACC CTC ACA 239 Cys Arg Xaa Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr 65 70 75 TGT TAC TTG AAG GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC 287 Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp 80 85 90 95 TGC ACG ATG CTT GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAW AGC 335 Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Xab Ser 100 105 110 GCG GGA ACT CAG GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT 383 Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala 115 120 125 ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC 431 Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr 130 135 140 GAC TTG GAG CTG ATA ACA TCA TGC TCC TCC AAY GTG TCG GTC GCG CAC 479 Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 145 150 155 GAC GCA TCA GGC AAA CGG GTG TAC TAY CTC ACC CGT GAC CCC MCC ACC 527 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Xac Thr 160 165 170 175 CCC CTW GCG CGG GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC 575 Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn 180 185 190 TCC TGG CTA GGC AAC ATC ATC ATG TAY GCG CCC ACC TTA TGG GCA AGG 623 Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg 195 200 205 ATG ATT CTG ATG ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA 671 Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln 210 215 220 CTT GAA AAA GCC CTA GAT TGT CAG ATC TAY GGG GCC ACT TAC TCC ATT 719 Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile 225 230 235 GAG CCA CTT GAC CTA CCT CAG ATC ATT CAA CGA CTC CAY GGT CTT AGC 767 Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser 240 245 250 255 GCA TTT TCA CTC CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT 815 Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala 260 265 270 TCA TGC CTC AGG AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT 863 Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His 275 280 285 CGG GCC AGA AGC GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC 911 Arg Ala Arg Ser Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala 290 295 300 GCC ACC TGT GGC AAA TAC CTC TTC AAC TGG GCA GTA AAG ACC AAG CTC 959 Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 305 310 315 AAA CYC ACT CCA ATC CCR GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG 1007 Lys Xad Thr Pro Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp 320 325 330 335 TTC GTT GCT GGT TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT 1055 Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg 340 345 350 GCC CGA CCC CGC TGG TTY ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG 1103 Ala Arg Pro Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly 355 360 365 GTA GGC ATC TAC CTG CTC CCC AAC CGA TGA GCG GGG AGC TAA ACA CTC 1151 Val Gly Ile Tyr Leu Leu Pro Asn Arg Stop 370 375 CAG GCC AAT AGG CCA TCC CC 1171 Y: C or TR: A or GM: A or CW: A or T Xaa: Val or Ala Xab: Asp or Glu Xac: Thr or Pro Xad: Leu or Pro ──────────────────────────────────────── ──────────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0309

[Correction method] Change

[Correction content]

SEQ ID NO: 71 Sequence length: 1004 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: 1728 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG ACC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Thr Arg Gly 20 25 30 TCT CCC CCT TCC TCG ACC AGT TCT TCA GCT AGT CAG TTG TCT GCG CTT 144 Ser Pro Pro Ser Ser Thr Ser Ser Ser Ala Ser Gln Leu Ser Ala Leu 35 40 45 TCT TCG CAG GCA ACA TGC ACT ACC CAT CAG GGC GCC CCA GAC ACT GAC 192 Ser Ser Gln Ala Thr Cys Thr Thr His Gln Gly Ala Pro Asp Thr Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT GAA 2 88 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG GCG GAG 336 Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu 100 105 110 ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA TGG GCA 384 Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val Trp Ala 115 120 125 CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC CCG GAC 432 Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn Pro Asp 130 135 140 TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC AAG GCC 480 Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala 145 150 155 160 CCT CCA ATA CCA CCT CCA CGG AGA AAG AGA ACG GTT GTC CTG ACA GAA 528 Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu 165 170 175 TCC ACC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC TTT GGC 576 Ser Thr Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly 180 185 190 AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC CCT CCT 624 Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro 195 200 205 GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG TCG TAC 672 Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr 210 215 220 TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AAC 720 Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Asn 225 230 235 240 GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC GTC GTC 768 Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val 245 250 255 TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA CCA TGC 816 Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 260 265 270 GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC CCT TTG 864 Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro Leu 275 280 285 CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC GCA AGC 912 Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser 290 295 300 CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG GAT GAC 960 Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp 305 310 315 320 CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG GCG TCC AC 1004 His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys Ala Ser 325
330

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0311

[Correction method] Change

[Correction content]

SEQ ID NO: 73 sequence length: 1818 sequence type: nucleic acid number of strands: double-stranded topology: linear antisense: No origin: Hepatitis C virus direct origin clone name: 1718 sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG ACC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Thr Arg Gly 20 25 30 TCT CCC CCT TCC TCG ACC AGT TCT TCA GCT AGT CAG TTG TCT GCG CTT 144 Ser Pro Pro Ser Ser Thr Ser Ser Ser Ala Ser Gln Leu Ser Ala Leu 35 40 45 TCT TCG CAG GCA ACA TGC ACT ACC CAT CAG GGC GCC CCA GAC ACT GAC 192 Ser Ser Gln Ala Thr Cys Thr Thr His Gln Gly Ala Pro Asp Thr Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG GCG GAG 336 Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu 100 105 110 ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA TGG GCA 384 Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val Trp Ala 115 120 125 CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC CCG GAC 432 Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn Pro Asp 130 135 140 TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC AAG GCC 480 Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala 145 150 155 160 CCT CCA ATA CCA CCT CCA CGG AGA AAG AGA ACG GTT GTC CTG ACA GAA 528 Pro Pro Ile Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu 165 170 175 TCC ACC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC TTT GGC 576 Ser Thr Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly 180 185 190 AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC CCT CCT 624 Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro 195 200 205 GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG TCG TAC 672 Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr 210 215 220 TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AAC 720 Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Asn 225 230 235 240 GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC GTC GTC 768 Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val 245 250 255 TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA CCA TGC 816 Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 260 265 270 GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC CCT TTG 864 Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro Leu 275 280 285 CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC GCA AGC 912 Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser 290 295 300 CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG GAT GAC 960 Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp 305 310 315 320 CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG GCG TCC ACA GTT 1008 His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys Ala Ser Thr Val 325 330 335 AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG CTG ACG CCC CCA 1056 Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro 340 345 350 CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG GAC GTC CAG AGC 1104 His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys Asp Val Gln Ser 355 360 365 CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG TGG AAG GAC TTG 1152 Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu 370 375 380 CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC ATG GCA AAA AAT 1200 Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn 385 390 395 400 GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC AAG CCA GCT CGC 1248 Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg 405 410 415 CTT ATC GTA TTC CC A GAC TTG GGG GTT CGT GTG TGC GAG AAA ATG GCC 1296 Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala 420 425 430 CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG ATG GGC TCC TCA 1344 Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser 435 440 445 TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG TTC CTG GTG AAT 1392 Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn 450 455 460 GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA TAT GAC ACC CGC 1440 Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala Tyr Asp Thr Arg 465 470 475 480 TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT ACT GAG GAG TCA 1488 Cys Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Thr Glu Glu Ser 485 490 495 ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA CAG GCC ATA AGG 1536 Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg Gln Ala Ile Arg 500 505 510 TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC TTG ACC AAT TCA AAA 1584 Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys 515 520 525 GGG CAA AAC TGC GGC TAT CGC CGG TGC CGC GCC AGC GGC GTG CTG ACG 1632 Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr 530 535 540 ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG GCC TCT GCA GCC 1680 Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala 545 550 555 560 TGT CGA GCT GCG AAG CTC CAG GAC TGC ACG ATG CTC GTG TGC GGA GAC 1728 Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp 565 570 575 GAC CTT GTC GTT ATC TGT GAA AGC GCG GGA ACC CAG GAG GAC GCG GCA 1776 Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Ala Ala 580 585 590 AAC CTA CGA GTC TTC ACG GAG GCT ATG ACC AGG AAT TCC GCC 1818 Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg Asn Ser Ala 595 600
605

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0312

[Correction method] Change

[Correction content]

SEQ ID NO: 74 sequence length: 2591 sequence type: nucleic acid number of strands: double-stranded topology: linear antisense: No origin: Hepatitis C virus direct origin clone name: 2218 sequence GG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 47 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 1 5 10 15 ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC TAT GTG 95 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 20 25 30 CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC AAC CTT 143 Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu 35 40 45 ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT GAG GAC 191 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp 50 55 60 TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG GAC TGG 239 Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 65 70 75 ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC AAG CTC 287 I le Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu 80 85 90 95 CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT GGG TAC 335 Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr 100 105 110 AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC CCA TGT 383 Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys Pro Cys 115 120 125 GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG ATC GTT 431 Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 130 135 140 GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC ATC AAC 479 Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn 145 150 155 GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC TAT TCC 527 Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser 160 165 170 175 AGG GCG TTG TGG CGG GTG GCC ATT GAG GAG TAT GTG GAG GTC ACG CGG 575 Arg Ala Leu Trp Arg Val Ala Ile Glu Glu Tyr Val Glu Val Thr Arg 180 185 190 GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC G TG AAA 623 Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys 195 200 205 TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG GAT GGG 671 Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Phe Phe Thr Glu Leu Asp Gly 210 215 220 GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG CGG GAT 719 Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp 225 230 235 GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG TCA CAG 767 Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln 240 245 250 255 CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC 815 Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu 260 265 270 ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG ACC 863 Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Thr 275 280 285 AGA GGG TCT CCC CCT TCC TCG ACC AGT TCT TCA GCT AGT CAG TTG TCT 911 Arg Gly Ser Pro Pro Ser Ser Thr Ser Ser Ser Ala Ser Gln Leu Ser 290 295 300 GCG CTT TCT TCG CAG GCA ACA TGC ACT ACC CAT C AG GGC GCC CCA GAC 959 Ala Leu Ser Ser Gln Ala Thr Cys Thr Thr His Gln Gly Ala Pro Asp 305 310 315 ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA 1007 Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly 320 325 330 335 AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT 1055 Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser 340 345 350 TTT GAA CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG 1103 Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala 355 360 365 GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA 1151 Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val 370 375 380 TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC 1199 Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn 385 390 395 CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC 1247 Pro Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr 400 405 410 415 AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA AAG AGA ACG GTT GTC CTG 1295 Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu 420 425 430 ACA GAA TCC ACC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC 1343 Thr Glu Ser Thr Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr 435 440 445 TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC 1391 Phe Gly Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly 450 455 460 CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG 1439 Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu 465 470 475 TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT 1487 Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp 480 485 490 495 CTC AAC GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC 1535 Leu Asn Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp 500 505 510 GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA 1583 Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr 515 520 525 CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC 1631 Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn 530 535 540 CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC 1679 Pro Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser 545 550 555 GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG 1727 Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 560 565 570 575 GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG GCG TCC 1775 Asp Asp His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys Ala Ser 580 585 590 ACA GTT AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG CTG ACG 1823 Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr 595 600 605 CCC CCA CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG GAC GTC 1871 Pro Pro His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys Asp Val 610 615 620 CAG AGC CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG TGG AAG 1919 Gln Ser Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp L ys 625 630 635 GAC TTG CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC ATG GCA 1967 Asp Leu Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala 640 645 650 655 AAA AAT GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC AAG CCA 2015 Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro 660 665 670 GCT CGC CTT ATC GTA TTC CCA GAC TTG GGG GTT CGT GTG TGC GAG AAA 2063 Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys 675 680 685 ATG GCC CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG ATG GGC 2111 Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly 690 695 700 TCC TCA TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG TTC CTG 2159 Ser Ser Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu Phe Leu 705 710 715 GTG AAT GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA TAT GAC 2207 Val Asn Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala Tyr Asp 720 725 730 735 ACC CGC TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT ACT GAG 2255 Thr Arg Cys Phe Asp Ser Thr Val Thr Gl u Asn Asp Ile Arg Thr Glu 740 745 750 GAG TCA ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA CAG GCC 2303 Glu Ser Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg Gln Ala 755 760 765 ATA AGG TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC TTG ACC AAT 2351 Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn 770 775 780 TCA AAA GGG CAA AAC TGC GGC TAT CGC CGG TGC CGC GCC AGC GGC GTG 2399 Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 785 790 795 CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG GCC TCT 2447 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser 800 805 810 815 GCA GCC TGT CGA GCT GCG AAG CTC CAG GAC TGC ACG ATG CTC GTG TGC 2495 Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Cys 820 825 830 GGA GAC GAC CTT GTC GTT ATC TGT GAA AGC GCG GGA ACC CAG GAG GAC 2543 Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp 835 840 845 GCG GCA AAC CTA CGA GTC TTC ACG GAG GCT ATG ACC AGG AAT TCC GCC 2591 Ala Ala Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg Asn Ser Ala 850 855
860 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0313

[Correction method] Change

[Correction content]

SEQ ID NO: 75 Sequence length: 4296 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: 1530U Sequence GCGGATCCT CCA CCT CCA TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG 51 Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg 1 5 10 CTG AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA 99 Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly 15 20 25 30 GCC GTT CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC 147 Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile 35 40 45 ATG GCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG 195 Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val 50 55 60 CTG GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG 243 Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr 65 70 75 GGC AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC 291 Gly S er Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala 80 85 90 GTT ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA 339 Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu 95 100 105 110 GAG TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CTC GCC 387 Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala 115 120 125 GAG CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG 435 Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys 130 135 140 CAA GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT 483 Gln Ala Glu Ala Ala Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu 145 150 155 GAG ACC TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG 531 Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 160 165 170 TAC TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA 579 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser 175 180 185 190 CTG ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC ACC CAA T AT 627 Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr 195 200 205 ACC CTC CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC 675 Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala 210 215 220 CCC CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG 723 Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala 225 230 235 GCT GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG 771 Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala 240 245 250 GGT TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG 819 Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met 255 260 265 270 AGC GGT GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC 867 Ser Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala 275 280 285 ATC CTC TCT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA 915 Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile 290 295 300 CTG CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG C AG TGG ATG AAC 963 Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn 305 310 315 CGG CTG ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC 1011 Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His 320 325 330 TAT GTG CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC 1059 Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser 335 340 345 350 AAC CTT ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT 1107 Asn Leu Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn 355 360 365 GAG GAC TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG 1155 Glu Asp Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp 370 375 380 GAC TGG ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC 1203 Asp Trp Ile Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser 385 390 395 AAG CTC CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT 1251 Lys Leu Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg 400 405 410 GGG TAC AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC 1299 Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys 415 420 425 430 CCA TGT GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG 1347 Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg 435 440 445 ATC GTT GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC 1395 Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro 450 455 460 ATC AAC GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC 1443 Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn 465 470 475 TAT TCC AGG GCG TTG TGG CGG GTG GCC ATT GAG GAG TAT GTG GAG GTC 1491 Tyr Ser Arg Ala Leu Trp Arg Val Ala Ile Glu Glu Tyr Val Glu Val 480 485 490 ACG CGG GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC 1539 Thr Arg Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn 495 500 505 510 GTG AAA TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG 1587 Val Lys Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Thr Glu Leu 515 520 525 GAT GGG GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG 1635 Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu 530 535 540 CGG GAT GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG 1683 Arg Asp Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly 545 550 555 TCA CAG CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC 1731 Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser 560 565 570 570 ATG CTC ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG 1779 Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg 575 580 585 590 CTG ACC AGA GGG TCT CCC CCT TCC TCG ACC AGT TCT TCA GCT AGT CAG 1827 Leu Thr Arg Gly Ser Pro Pro Ser Ser Thr Ser Ser Ser Ala Ser Gln 595 600 605 TTG TCT GCG CTT TCT TCG CAG GCA ACA TGC ACT ACC CAT CAG GGC GCC 1875 Leu Ser Ala Leu Ser Ser Gln Ala Thr Cys Thr Thr His Gln Gly Ala 610 615 620 CCA GAC ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG 1923 Pro Asp Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met 6 25 630 635 GGC GGA AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA 1971 Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu 640 645 650 GAC TCT TTT GAA CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC 2019 Asp Ser Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser 655 660 665 670 GTT GCG GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG 2067 Val Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met 675 680 685 CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG 2115 Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp 690 695 700 AAG AAC CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG 2163 Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro 705 710 715 CCT ACC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA AAG AGA ACG GTT 2211 Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val 720 725 730 GTC CTG ACA GAA TCC ACC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA 2259 Val Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Al a Glu Leu Ala Thr 735 740 745 750 AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG 2307 Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala 755 760 765 ACC GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC 2355 Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp 770 775 780 GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC 2403 Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp 785 790 795 CCC GAT CTC AAC GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC 2451 Pro Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser 800 805 810 GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA 2499 Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu 815 820 825 830 ATT ACA CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG 2547 Ile Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu 835 840 845 AGC AAC CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC 2595 Ser Asn Pro Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser 850 855 860 CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA 2643 Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln 865 870 875 GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG 2691 Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys 880 885 890 GCG TCC ACA GTT AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG 2739 Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys 895 900 905 910 CTG ACG CCC CCA CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG 2787 Leu Thr Pro Pro His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys 915 920 925 GAC GTC CAG AGC CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG 2835 Asp Val Gln Ser Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val 930 935 940 TGG AAG GAC TTG CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC 2883 Trp Lys Asp Leu Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile 945 950 955 ATG GCA AAA AAT GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC 2931 Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg 960 965 970 AAG CCA GCT CGC CTT ATC GTA TTC CCA GAC TTG GGG GTT CGT GTG TGC 2979 Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys 975 980 985 990 GAG AAA ATG GCC CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG 3027 Glu Lys Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val 995 1000 1005 ATG GGC TCC TCA TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG 3075 Met Gly Ser Ser Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu 1010 1015 1020 TTC CTG GTG AAT GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA 3123 Phe Leu Val Asn Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala 1025 1030 1035 TAT GAC ACC CGC TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT 3171 Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg 1040 1045 1050 ACT GAG GAG TCA ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA 3219 Thr Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg 1055 1060 1065 1070 CAG GCC ATA AGG TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC CTG 3267 Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu 1075 1080 1085 ACT AAT TCA AAG GGG CAG AAC TGC GGT TAT CGC CGG TGC CGC GTC AGC 3315 Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Val Ser 1090 1095 1100 GGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG 3363 Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys 1105 1110 1115 GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC TGC ACG ATG CTT 3411 Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu 1120 1125 1130 GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAT AGC GCG GGA CAG 3459 Val Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser Ala Gly Thr Gln 1135 1140 1145 1150 GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT ATG ACT AGG TAC 3507 Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr 1155 1160 1165 TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC GAC TTG GAG CTG 3555 Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu 1170 1175 1180 ATA ACA TCA TGC TCC TCC AAT GTG TCG GTC GCG CAC GAC GCA TCA GGC 3603 Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Ala Ser Gly 1185 1190 1195 AAA CGG GTG TAC TAT CTC ACC CGT GAC CCC ACC ACC CCC CTA GCG CGG 3651 Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg 1200 1205 1210 GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC TCC TGG CTA GGC 3699 Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly 1215 1220 1225 1230 AAC ATC ATC ATG TAC GCG CCC ACC TTA TGG GCA AGG ATG ATT CTG ATG 3747 Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met 1235 1240 1245 ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA CTT GAA AAA GCC 3795 Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala 1250 1255 1260 CTA GAT TGT CAG ATC TAC GGG GCC ACT TAC TCC ATT GAG CCA CTT GAC 3843 Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile Glu Pro Leu Asp 1265 1270 1275 CTA CCT CAG ATC ATT CAA CGA CTC CAC GGT CTT AGC GCA TTT TCA CTC 3891 Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu 1280 1285 1290 CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT TCA TGC CTC AGG 3939 His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg 1295 1300 1305 1310 AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT CGG GCC AGA AGC 3987 Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser 1315 1320 1325 GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC GCC ACC TGT GGC 4035 Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly 1330 1335 1340 AAA TAC CTC TTC AAC TGG GCA GTA AAG ACC AAG CTC AAA CTC ACT CCA 4083 Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro 1345 1350 1355 ATC CCA GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG TTC GTT GCT GGT 4131 Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly 1360 1365 1370 TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT GCC CGA CCC CGC 4179 Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg 1375 1380 1385 1390 TGG TTC ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG GTA GGC ATC TAC 4227 Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr 1395 1400 1405 CTG CTC CCC AAC CGA TGA GCGGG GAGCTAAACA CTCCAGGCCA ATAGGCCATC 4280 Leu Leu Prou Asn Arg Stop 1410 CCCCTTTTTTTTTTTTTTT
4296

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0314

[Correction method] Change

[Correction content]

SEQ ID NO: 76 Sequence length: 818 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N22-1 Sequence GG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 47 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 1 5 10 15 ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC TAT GTG 95 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 20 25 30 CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC AAC CTT 143 Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu 35 40 45 ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT GAG GAC 191 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp 50 55 60 TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG GAC TGG 239 Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 65 70 75 ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC AAG CTC 287 I le Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu 80 85 90 95 CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT GGG TAC 335 Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr 100 105 110 AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC CCA TGT 383 Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys Pro Cys 115 120 125 GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG ATC GTT 431 Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 130 135 140 GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC ATC AAC 479 Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn 145 150 155 GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC TAT TCC 527 Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser 160 165 170 175 AGG GCG TTG TGG CGG GTG GCC GCT GAG GAG TAT GTG GAG GTC ACG CGG 575 Arg Ala Leu Trp Arg Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg 180 185 190 GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC G TG AAA 623 Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys 195 200 205 TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG GAT GGG 671 Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Phe Phe Thr Glu Leu Asp Gly 210 215 220 GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG CGG GAT 719 Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp 225 230 235 GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG TCA CAG 767 Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln 240 245 250 255 CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC 815 Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu 260 265 270 ACC 818 Thr ──────────────────────────────── ───────────────────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0324

[Correction method] Change

[Correction content]

SEQ ID NO: 86 Sequence length: 740 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O28-1 Sequence GTG GTA GTC CTG GAC TCG TTG GAG CCG CTT CAA GCG AAG GAA GGT GAG 48 Val Val Val Leu Asp Ser Leu Glu Pro Leu Gln Ala Lys Glu Gly Glu 1 5 10 15 AGG GAA GTG TCC GTT GCG GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC 96 Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe 20 25 30 CCC GCA GCG ATG CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA 144 Pro Ala Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu 35 40 45 CTA GAG TCT TGG AAG AAC CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG 192 Leu Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly 50 55 60 TGC CCA TTG CCG CCT ACC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA 240 Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg 65 70 75 80 AAG AGA ACG GTT GTC CTG ACA GAA TCC ACC GTG TCC TCT GCC TTG GCG 2 88 Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Ala 85 90 95 GAG CTT GCT ACA AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC 336 Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp 100 105 110 AGC GGC ACG GCG ACC GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT 384 Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp 115 120 125 GCA GGA TCC GAC GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA 432 Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly 130 135 140 GAG CCG GGG GAC CCC GAT CTC AAC GAC GGG TCT TGG TCT ACC GTA AGC 480 Glu Pro Gly Asp Pro Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser 145 150 155 160 GAG GAG GCC AGC GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG 528 Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp 165 170 175 ACA GGC GCC TTA ATT ACA CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC 576 Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro 180 185 190 ATT AAT GCG CTG AGC AAC CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT 624 Ile Asn Ala Leu Ser Asn Pro Leu Leu Arg His His Asn Met Val Tyr 195 200 205 GCC ACA ACA TCC CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT 672 Ala Thr Thr Ser Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe 210 215 220 GAC AGA CTG CAA GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC 720 Asp Arg Leu Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp 225 230 235 240 ATG AAG GCC AAG GCG TCC AC 740 Met Lys Ala Lys Ala Ser 245

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0325

[Correction method] Change

[Correction content]

SEQ ID NO: 87 Sequence length: 740 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O28-2 Sequence GTG GTA GTC CTG GAC TCG TTG GAC CCG CTT CGA GCG GAG GAA GAT GAG 48 Val Val Val Leu Asp Ser Leu Asp Pro Leu Arg Ala Glu Glu Asp Glu 1 5 10 15 AGG GAA GTG TCC GTT GCG GCG GAG ATC CTG CGA AAG ACC AAG AAA TTC 96 Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Thr Lys Lys Phe 20 25 30 CCC GCA GCG ATG CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA 144 Pro Ala Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu 35 40 45 CTA GAG TCT TGG AAG AAC CCG GAC TAC GTC CCT CCG GTG GTA CAC GGG 192 Leu Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly 50 55 60 TGC CCA TTG CCG CCT ACC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA 240 Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg 65 70 75 80 AAG AGG ACG GTT GCC CTG ACA GAA TCC ACC GTG TCC TCT GCC TTG GCG 288 Lys Arg Thr Val Ala Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Ala 85 90 95 GAG CTT GCT ACA AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC 336 Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp 100 105 110 AGC GGC ACG GCG ACT GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT 384 Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp 115 120 125 GCA GGA TCC GAC GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA 432 Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly 130 135 140 GAG CCG GGG GAC CCT GAT CTC AAC GAC GGG TCT TGG TCT ACT GTA AGC 480 Glu Pro Gly Asp Pro Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser 145 150 155 160 GAG GAG GCC GGC GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG 528 Glu Glu Ala Gly Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp 165 170 175 ACA GGC GCC TTA ATT ACA CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC 576 Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro 180 185 190 ATT AAT GCG CTG AGC AAC TCT TTG CTG CGC CAC CAC AAC ATG GTC TAT 624 Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Met Val Tyr 195 200 205 GCC ACA ACA TCC CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT 672 Ala Thr Thr Ser Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe 210 215 220 GAC AGA CTG CAA GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC 720 Asp Arg Leu Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp 225 230 235 240 ATG AAG GCC AAG GCG TCC AC 740 Met Lys Ala Lys Ala Ser 245

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0326

[Correction method] Change

[Correction content]

SEQ ID NO: 88 Sequence length: 740 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O28-4 Sequence GTG GTA GTC CTG GAC TCG TTG GAC CCG CTT CGA GCG GAG GAA GAT GAG 48 Val Val Val Leu Asp Ser Leu Asp Pro Leu Arg Ala Glu Glu Asp Glu 1 5 10 15 AGG GAA GTG TCC GTT GCG GCG GAG ATC CTG CGA AAG ACC AAG AAA TTC 96 Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Thr Lys Lys Phe 20 25 30 CCC GCA GCG ATG CCC GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA 144 Pro Ala Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu 35 40 45 CTA GAG TCT TGG AAG AAC CCG GAC TAC GTC CCT CCG GTG GTA CAC GGG 192 Leu Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly 50 55 60 TGC CCA TTG CCG CCT ATC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA 240 Cys Pro Leu Pro Pro Ile Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg 65 70 75 80 AAG AGG ACG GTT GTC CTG ACA GAA TCC ACC GTG TCC TCT GCC TTG G CG 288 Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Ala 85 90 95 GAG CTT GCT ACA AAG ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC 336 Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp 100 105 110 AGC GGC ACG GCG ACC GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT 384 Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp 115 120 125 GCA GGA TCC GAC GCT GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA 432 Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly 130 135 140 GAG CCG GGG GAC CCT GAT CTC AAC GAC GGG TCT TGG TCT ACT GTA AGC 480 Glu Pro Gly Asp Pro Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser 145 150 155 160 GAG GAG GCC GGC GAG GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG 528 Glu Glu Ala Gly Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp 165 170 175 ACA GGC GCC TTA ATT ACA CCA TGC ACC GCG GAG GAG AGC AAG CTG CCC 576 Thr Gly Ala Leu Ile Thr Pro Cys Thr Ala Glu Glu Ser Lys Leu Pro 180 185 190 ATT AAT GCG CTG AGC AAC TCT TTG CTG CGT CAC CAC AAC ATG GTC TAT 624 Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Met Val Tyr 195 200 205 GCC ACA ACA TCC CGC AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT 672 Ala Thr Thr Ser Arg Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe 210 215 220 GAC AGA CTG CAA GTC CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC 720 Asp Arg Leu Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp 225 230 235 240 ATG AAG GCC AAG GCG TCC AC 740 Met Lys Ala Lys Ala Ser 245

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0336

[Correction method] Change

[Correction content]

SEQ ID NO: 98 Sequence length: 1171 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O30-3 Sequence TG GGG ATC CCG TAT GAT ACC CGC TGC TTT GAC TCA ACG GTC ACT GAG 47 Gly Ile Pro Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu 1 5 10 15 AAT GAC ATC CGT GTC GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC 95 Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala 20 25 30 CCC GAG GCC AGA CAG GCC ATA AGG TCA CTC ACA GAG CGG CTT TAC ATC 143 Pro Glu Ala Arg Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile 35 40 45 GGG GGC CCC CTG ACT AAT TCA AAG GGG CAG AAC TGC GGT TAT CGC CGG 191 Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg 50 55 60 TGC CGC GTC AGC GGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA 239 Cys Arg Val Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr 65 70 75 TGT TAC TTG AAG GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC 287 Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp 80 85 90 95 TGC ACG ATG CTT GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAT AGC 335 Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser 100 105 110 GCG GGA ACT CAG GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT 383 Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala 115 120 125 ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC 431 Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr 130 135 140 GAC TTG GAG CTG ATA ACA TCA TGC TCC TCC AAT GTG TCG GTC GCG CAC 479 Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 145 150 155 GAC GCA TCA GGC AAA CGG GTG TAC TAT CTC ACC CGT GAC CCC ACC ACC 527 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr 160 165 170 175 CCC CTA GCG CGG GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC 575 Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn 180 185 190 TCC TGG CTA GGC AAC ATC ATC ATG TAC GCG CCC ACC TTA TGG GCA AGG 623 Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg 195 200 205 ATG ATT CTG ATG ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA 671 Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln 210 215 220 CTT GAA AAA GCC CTA GAT TGT CAG ATC TAC GGG GCC ACT TAC TCC ATT 719 Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile 225 230 235 GAG CCA CTT GAC CTA CCT CAG ATC ATT CAA CGA CTC CAC GGT CTT AGC 767 Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser 240 245 250 255 GCA TTT TCA CTC CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT 815 Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala 260 265 270 TCA TGC CTC AGG AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT 863 Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His 275 280 285 CGG GCC AGA AGC GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC 911 Arg Ala Arg Ser Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala 290 295 300 GCC ACC TGT GGC AAA TAC CTC TTC AAC TGG GCA GTA AAG ACC AAG CTC 959 Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 305 310 315 AAA CTC ACT CCA ATC CCA GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG 1007 Lys Leu Thr Pro Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp 320 325 330 335 TTC GTT GCT GGT TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT 1055 Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg 340 345 350 GCC CGA CCC CGC TGG TTC ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG 1103 Ala Arg Pro Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly 355 360 365 GTA GGC ATC TAC CTG CTC CCC AAC CGA TGA GCG GGG AGC TAA ACA CTC 1151 Val Gly Ile Tyr Leu Leu Pro Asn Arg Stop 370 375 CAG GCC AAT AGG CCA TCC CC
1171

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0337

[Correction method] Change

[Correction content]

SEQ ID NO: 99 Sequence length: 1170 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O30-2 Sequence G GGG ATC CCG TAT GAT ACC CGC TGC TTT GAC TCA ACG GTC ACT GAG 46 Gly Ile Pro Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu 1 5 10 15 AAT GAC ATC CGT GTT GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC 94 Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala 20 25 30 CCC GAG GCC AGA CAG GCC ATA AGG TCG CTC ACA GAG CGG CTT TAC ATC 142 Pro Glu Ala Arg Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile 35 40 45 GGG GGC CCC CTG ACT AAT TCA AAA GGG CAG AAC TGC GGC TAT CGC CGG 190 Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg 50 55 60 TGC CGC GTC AGC GGC GTG CTG ACG ACT AGC TGC GGC AAT ACC CTC ACA 238 Cys Arg Val Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr 65 70 75 TGT TAC TTG AAG GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC 286 C ys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp 80 85 90 95 TGC ACG ATG CTT GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAA AGC 334 Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser 100 105 110 GCG GGA ACT CAG GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT 382 Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala 115 120 125 ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC 430 Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr 130 135 140 GAC TTG GAG CTG ATA ACA TCA TGC TCC TCC AAC GTG TCG GTC GCG CAC 478 Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 145 150 155 GAC GCA TCA GGC AAA CGG GTG TAC TAC CTC ACC CGT GAC CCC ACC ACC 526 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr 160 165 170 175 CCC CTT GCG CGG GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC 574 Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn 180 185 190 TCC TGG CTA GGC AAC ATC ATC ATG TAT GCG CCC ACC TTA TGG G CA AGG 622 Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg 195 200 205 ATG ATT CTG ATG ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA 670 Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln 210 215 220 CTT GAA AAA GCC CTA GAT TGT CAG ATC TAT GGG GCC ACT TAC TCC ATT 718 Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile 225 230 235 GAG CCA CTT GAC CTA CCT CAG ATC ATT CAA CGA CTC CAT GGT CTT AGC 766 Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser 240 245 250 255 GCA TTT TCA CTC CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT 814 Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala 260 265 270 TCA TGC CTC AGG AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT 862 Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His 275 280 285 CGG GCC AGA AGC GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC 910 Arg Ala Arg Ser Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala 290 295 300 GCC ACC TGT GGC AAA TAC CTC TTC AAC TGG GCA G TA AAG ACC AAG CTC 958 Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 305 310 315 AAA CCC ACT CCA ATC CCG GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG 1006 Lys Pro Thr Pro Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp 320 325 330 335 TTC GTT GCT GGT TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT 1054 Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg 340 345 350 GCC CGA CCC CGC TGG TTT ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG 1102 Ala Arg Pro Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly 355 360 365 GTA GGC ATC TAC CTG CTC CCC AAC CGA TGA GCG GGG AGC TAA ACA CTC 1150 Val Gly Ile Tyr Leu Leu Pro Asn Arg Stop 370 375 CAG GCC AAT AGG CCA TCC CC
1170

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0338

[Correction method] Change

[Correction content]

SEQ ID NO: 100 Sequence length: 1171 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: O30-4 Sequence TG GGG ATC CCG TAT GAT ACC CGC TGC TTT GAC TCA ACA GTC ACT GAG 47 Gly Ile Pro Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu 1 5 10 15 AAT GAC ATC CGT GTT GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC 95 Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala 20 25 30 CCC GAG GCC AGA CAG GCC ATA AGG TCG CTC ACA GAG CGG CTT TAC ATC 143 Pro Glu Ala Arg Gln Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile 35 40 45 GGG GGC CCC CTG ACT AAT TCA AAG GGG CAG AAC TGC GGT TAT CGC CGG 191 Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg 50 55 60 TGC CGC GCC AGC GGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA 239 Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr 65 70 75 TGT TAC TTG AAG GCC TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC 2 87 Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp 80 85 90 95 TGC ACG ATG CTT GTG TGC GGA GAC GAC CTT GTC GTT ATC TGT GAA AGC 335 Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser 100 105 110 GCG GGA ACT CAG GAG GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT 383 Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala 115 120 125 ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC 431 Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr 130 135 140 GAC TTG GAG CTG ATA ACA TCA TGC TCC TCC AAT GTG TCG GTC GCG CAC 479 Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His 145 150 155 GAC GCA TCA GGC AAA CGG GTG TAC TAT CTC ACC CGT GAC CCC CCC ACC 527 Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Pro Thr 160 165 170 175 CCC CTT GCG CGG GCT GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC 575 Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn 180 185 190 TCC TGG CTA GGC AAC ATC ATC ATG TAC GCG CCC ACC TTA T GG GCA AGG 623 Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg 195 200 205 ATG ATT CTG ATG ACC CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA 671 Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln 210 215 220 CTT GAA AAA GCC CTA GAT TGT CAG ATC TAC GGG GCC ACT TAC TCC ATT 719 Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile 225 230 235 GAG CCA CTT GAC CTA CCT CAG ATC ATT CAA CGA CTC CAT GGT CTT AGC 767 Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser 240 245 250 255 GCA TTT TCA CTC CAT AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT 815 Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala 260 265 270 TCA TGC CTC AGG AAA CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT 863 Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His 275 280 285 CGG GCC AGA AGC GTC CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC 911 Arg Ala Arg Ser Val Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala 290 295 300 GCC ACC TGT GGC AAA TAC CTC TTC AAC TGG G CA GTA AAG ACC AAG CTC 959 Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 305 310 315 AAA CTC ACT CCA ATC CCG GAA GCG TCC CAG CTG GAC TTG TCC GGC TGG 1007 Lys Leu Thr Pro Ile Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp 320 325 330 335 TTC GTT GCT GGT TAC AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT 1055 Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg 340 345 350 GCC CGA CCC CGC TGG TTC ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG 1103 Ala Arg Pro Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly 355 360 365 GTA GGC ATC TAC CTG CTC CCC AAC CGA TGA GCG GGG AGC TAA ACA CTC 1151 Val Gly Ile Tyr Leu Leu Pro Asn Arg Stop 370 375 CAG GCC AAT AGG CCA TCC CC
1171

[Submission date] September 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0433

[Correction method] Change

[Correction content]

SEQ ID NO: 195 Sequence Length: 38 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: cDNA Origin: Hepatitis C virus Sequence GCGAATTCAGACTCTTTATCATGTGT
TGCAGTCGATCAC 38 SEQ ID NO: 196 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA Origin: Hepatitis C virus sequence TGATAAAGACTCTGAATTCGC 20 ─────── ─────────────────────────────────────────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0198

[Correction method] Change

[Correction content]

[0198]

[Table 3] N22 region nucleotide sequence of each clone N22-1. NUC 1: GGCATGTGGGGCCCA
GGGGAGGGGGGCTGTGCAGGTGGATGAACCGGCTGATA (SEQ ID NO: 76 in the Sequence Listing) N22-3.NUC 1:・ ・ ・ ・ (Sequence ID No. of Sequence Listing: 77) H22-3.NUC 1:・ ・ ・ ・ ・ ・ ・ ・ ・ (Sequence ID No. 78) H22-8.NUC 1:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ( Sequence number of the sequence listing: 79) H22-9.NUC 1:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ (Sequence ID No .: 80) N22-1.NUC 51: GCGTTTGCTTCGCGGG GCAACCATGTCTCCCCCACGCACTATGTGCCTGA N22-3.NUC 51: .....................・ ・ ・ ・ ・ ・ ・ ・ H22-3.NUC 51: ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-8.NUC 51: ・ ・ ・ ・ ・ C ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ C ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 51: ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1.NUC 101: AAGCGACGCCGCAGCGCGCGTCACCCAGATCCTCTCCAACCTTACCATCA N22-3.NUC 101: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-3.NUC 101 : G ... T ...・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-8.NUC 101: G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ T ・・ ・ ・ ・ G ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 101: G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1.NUC 151: CTCAGCTGTTGAAGAGGCTTCACCAGTGGATTAATGAGGACTGCTCCACG N22-3.NUC 151: ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ H22-3.NUC 151: ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ H22-8.NUC 151: ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ H22-9.NUC 151: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1.NUC 201: CCATGCTCCGGCTCGTGGCTCAGGGATGTTTGGGACTGGATATGCACGGT N22-3.NUC 201: ..................... H22-3.NUC 201: ・ ・ T ・ ・ T ・ ・ T・ ・ ・ ・ ・ ・ ・ H22-8.NUC 201: ・ ・ ・ ・ ・ ・ T ・ ・ T ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 201: ・ ・ ・ ・ ・ T ・ ・ T ・ ・ T ・ ・ ・ ・ ・ ・N22-1.NUC 251: ATTGGCTGATTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGGTTAC N22-3.NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・.....・ ・ ・ ・ H22-3.NUC 251: G ・ ・ ・ AG ・ ・ ・ C ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ C ・ ・ ・ H22-8.NUC 251: G ・ ・ ・ AG ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ H22-9.NUC 251: G ・ ・ ・ AG ・ ・ ・ C ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ N22-1.NUC 301: CGGGGGTCCCTTTTTTCTCATGCCAGCGTGGGTACAAGGGGGTTTGGCGG N22-3.NUC 301: ... C ... H22-3.NUC 301: ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ CC ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ C・ ・ ・ ・ ・ ・ H22-8.NUC 301: ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ CC ・ T ・ ・ ・ ・ ・ ・ ・ ・ A ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ C ・ ・ ・ ・ ・ ・ H22-9.NUC 301: ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ CC ・ ・ ・ ・ ・・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ C ・ ・ ・ ・ ・ ・ N22-1.NUC 351: GGAGATGGCATCATGTATACCACCTGCCCATGTGGAGCACAAATCACCGG N22-3.NUC 351: ・ ・ ・ ・ ・ C ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-3.NUC 351:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・H22-8.NUC 351: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ G ・ ・ ・ ・ H22-9.NUC 351: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ C ・ ・ ・・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ N22-1.NUC 401: ACATGTCAAAAACGGTTCTATGAGGATCGTTGGGCCTAGAACCTGTAGCA N2 2-3.NUC 401: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ H22-3.NUC 401: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ AC ・ ・ ・ C ・ ・ C・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-8.NUC 401: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ AC ・・ ・ C ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 401: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・・ ・ ・ ・ AC ・ ・ ・ C ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1.NUC 451: ACACGTGGCACGGAACATTTCCCATCAACGCGTACACCACAGGCCCCTGC N22-3.NUC 451: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-3.NUC 451: ・ ・ ・... G ... C ...・ ・ ・ ・ ・ ・ ・ ・ H22-8.NUC 451: ・ ・ ・ G ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 451: ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ G ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1.NUC 501: ACACCCTCCCCGGCGCCAAACTATTCCAGGGCGTTGTGGCGGGTGGCCGC N22-3.NUC 501: ・ ・ ・ ・ T ・ ・ A ・・ ・ ・ ・ G ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ H22-3.NUC 501: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ T ・ ・ H22-8.NUC 501 : ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ A ・ ・ T ・・ H22-9.NUC 501: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ T ・ A ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ A ・ ・ T ・ ・ N22-1.NUC 551: TGAGGAGTATGTGGAGGTCACGCGGGTGGGGGATTTCCACTACGTGAC ・ ・ N22-3.NUC 551: ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-3.NUC 551: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ H22-8.NUC 551: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 551 : ...・ N22-1.NUC 601: GCATGACCACTGACAACGTGAAATGCCCATGCCAGGTTCCGGCCCCCGAA N22-3.NUC 601: ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・.....................・ ・ H22-3.NUC 601: ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・・ ・ ・ ・ ・ ・ ・ H22-8.NUC 601: ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9.NUC 601: ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N22-1. NUC 651: TTCTTCACAGAATTG
GATGGGGGTCGGCTGCACAGGTTACCGCTCCGGCGTG N22-3. NUC 651: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... G ... H22-3. NUC 651: ・ ・ ・ ・ ・ ・ G ・ ・ G ・ ・
・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ H22-8. NUC 651: ・ ・ ・ ・ ・ ・ G ・ ・ G ・ ・
・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ H22-9. NUC 651: ・ ・ T ・ ・ ・ ・ ・ G ・ ・ G ・ ・
・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ N22-1. NUC 701: CAAACCTCTCCTGC
GGGATGAGGTCACATTCCAGGTCGGGCTCAACCAAT N22-3. NUC 701: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... H22-3. NUC 701 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・
..................... H22-8. NUC 701 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・
..................... H22-9. NUC 701 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・
..................... N22-1. NUC 751: ATACGGTTGGGTCA
CAGCTCCCCATGTGAGCCCGAACCGGATGTAACAGTG N22-3. NUC 751: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... G ... H22-3. NUC 751: TCC ... G
··········· T ··· H22-8. NUC 751: TCC ... G
················ H22-9. NUC 751: TCC ... G
... A ... C ... G ... N22-1. NUC 801: GTCACCTCCCATGCT
CACC N22-3. NUC 801: .....................
..... H22-3. NUC 801: .....................
..... H22-8. NUC 801: .....................
..... H22-9. NUC 801: .....................
...

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0200

[Correction method] Change

[Correction content]

Nucleotide sequence of each clone of O28 region O28-1. NUC 1: GTGGTAGTCCTGGA
CTCGTTGGAGCCGCTTCAAGCGAAGGAAGGTGAGAG (SEQ ID NO: 86 in the sequence listing) O28-2.NUC 1: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ G ・・ ・ ・ G ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ (Sequence ID No.:87) O28-4.NUC 1: ・ ・ ・ ・ ・ ・······················· G ······················································································ 28-2. NUC 51: GGAAGTGTCCCGTTG
CGGCGGAGATCCTGCCGAAGACCAGGAAATTCCCCG O28-2. NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
A ... A ... O28-4. NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
A ... A ... O28-1. NUC 101: CACGGATGCCCGTA
TGGGCACGCCCGGACTACAACCCCACCATTACTAGAG O28-2. NUC 101: .....................
..................... O28-4. NUC 101: .....................
........................... O28-1. NUC 151: TCTTGGAAGAACCC
GGACTACGTCCCTCCAGTGGGTACACGGGTGCCCATT O28-2. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ O28-4. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ O28-1. NUC 201: GCCGCCTACCAAGGG
CCCCTCCAATACCACCCTCCACGGAGAAAGAGAACGG O28-2. NUC 201: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... G ... O28-4. NUC 201: ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・
..................... G ... O28-1. NUC 251: TTGTCCCTGACAGAA
TCCACCGTGTCCTCTGCCTTGGGCGAGGCTTGCTACA O28-2. NUC 251 ： ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
........................... O28-1. NUC 301: AAGACCTTTGGCAG
TTCCGGATCGTCCGGCGTTCGACAGCGGCACGGCGAC O28-2. NUC 301: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 301: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
........................... O28-1. NUC 351: CGGCCCTCCTGACC
AGGCCTCCGCCGAAGGAGATGCAGGATCCGACGCTG O28-2. NUC 351: T ...
..................... O28-4. NUC 351: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
........................... O28-1. NUC 401: AGTCGTACTCCTCC
ATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGAT O28-2. NUC 401:
... T ... O28-4. NUC 401:
... T ... O28-1. NUC 451: CTCAACGACGGGTC
TTGGTCTACCGTAAAGCGAGGAGGCCAGCGAGGACGT O28-2. NUC 451: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O28-4. NUC 451: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ O28-1. NUC 501: CGTCTGCTGCTCGA
TGTCCTACACATGGACAGGCGCCTTAATTACACCAT O28-2. NUC 501 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 501 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
........................... O28-1. NUC 551: GCGCCGCGGAGGAG
AGCAAGCTGCCCCATTAATGCCGCTGAGCAACCCTTTG O28-2. NUC 551: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ O28-4. NUC 551: ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ O28-1. NUC 601: CTGCCGCCACCACAA
CATGGTCTATGCCACAACCATCCCGCAGCCGCAAGCCA O28-2. NUC 601: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 601: ... T ...
........................... O28-1. NUC 651: GCGGCAGAAAAAAG
TCACATTTGACAGAACTGCAAGTCCCTGGATGACCACT O28-2. NUC 651: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 651: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
........................... O28-1. NUC 701: ACCGGGACGTGTCTC
AAGGACATGAAGGCCAAGGCGTCCAC O28-2. NUC 701: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... O28-4. NUC 701: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
.....................

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0203

[Correction method] Change

[Correction content]

Nucleotide sequence of each clone in the O30 region O30-3. NUC 1: TGGGGATCCCCGTAT
GATACCCGCTGCTTTGAACTCAACGGTCACTGAGAAT (SEQ ID NO: 98 in the Sequence Listing) O30-2.NUC 1: (Sequence ID No .: 99) O30-4.NUC 1:・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ (Sequence ID No .: 100) O30-3.NUC 51: GACATCCGTGTCGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGA O30 -2.NUC 50: ・ ・ ・ ・ ・ ・ T・ ・ ・ ・ O30-4.NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 101: GGCCAGACAGGCCATAAGGTCACTCACAGAGCG GCTTTACATCGGGGGCC O30-2.NUC 100: ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 101: ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 151: CCCTGACTAATTCAAAGGGGCAGAACTGCGGTTATCGCCGGTGCCGCGTC O30-2.NUC 150: ・ ・ ・ ・ ・ A ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 151: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ C ・ O30-3.NUC 201: AGCGGCGTGCTGACGACTAGCTGCGGTAATACCCTCACATGTTACTTGAA O30-2.NUC 200: ... C ... ..O30-4.NUC 201: ... ..................................................................... O30-3 .NUC 251: GGCCTCTGCAGCCTGTCGAGCTGCAAAGCTCCAGGACTGCACGATGCTTG O30-2.NUC 250: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ O30-3.NUC 301: TGTGCGGAGACGACCTTGTCGTTATCTGTGATAGCGCGGGAACTCAGGAG O30-2.NUC 300: ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ O30-4.NUC 301: ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 351: GACGCGGCGAGCCTACGAGTCTTCACGGAGGCTATGACTAGGTA CTCTGC O30-2.NUC 350: ................................................ ... O30-4.NUC 351: ... O30-3.NUC 401: CCCCCCCGGGGACCCGCCCCAACCAGAATACGACTTGGAGCTGATAACAT O30-2.NUC 400: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 401: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 451: CATGCTCCTCCAATGTGTCGGTCGCGCACGACGCATCAGGCAAACGGGTG O30-2.NUC 450: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・..O30-4.NUC 451: ...・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3. NUC 501: TACTATCTCACCCGTGACCCCACCACCCCCCTAGCGCGGGCTGCGTGGGA O30-2.NUC 500: ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 501: ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・T ・ ・ ・ ・ ・ ・ O30-3.NUC 551: GACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAACATCATCATGT O30-2.NUC 550: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 551: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 601: ACGCGCCCACCTTATGGGCAAGGATGATTCTGATGACCCACTTCTTCTC C O30-2.NUC 600: ・ T ...・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 601: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・O30-3.NUC 651: ATCCTTCTAGCCCAGGAGCAACTTGAAAAAGCCCTAGATTGTCAGATCTA O30-2.NUC 650:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 651: ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 701: CGGGGCCACTTACTCCATTGAGCCACTTGACCTACCTCAGATCATTCAAC O30-2.NUC 700: T ... .... O30-4.NUC 701:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 751 : GACTCCACGGTCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAG O30-2.NUC 750: ・ ・ ・ ・ ・ ・ T・ ・ ・ ・ ・ ・ ・ O30-4.NUC 751: ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・O30-3.NUC 801: ATCAATAGGGTGGCTTCATGCCTCAGGAAACTTGGGGTACCGCCCTTGCG O30-2.NUC 800: ...・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 801: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 851: AGTCTGGAGACATCGGGCCAGAAGCGTCCGCGCTAAGCTACTGTCCCAGG O 30-2.NUC 850:・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 851: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 901: GGGGGAGGGCCGCCACCTGTGGCAAATACCTCTTCAACTGGGCAGTAAAG O30-2.NUC 900: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 901: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 951: ACCAAGCTCAAACTCACTCCAATCCCAGAAGCGTCCCAGCTGGACTTGTC O30-2.NUC 950 : ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ O30-4.NUC 951: ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 1001: CGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGT O30-2.NUC 1000: .....................・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 1001: O30-3.NUC 1051: CTCGTGCCCGACCCCGCTGGTTCATGTGGTGCCTACTCCTACTTTCCGTA O30-2.NUC 1050:・ ・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 1051: ・ ・ ・ ・ ・ ・O30-3.NUC 1101: GGGGTAGGCATCTACCTGCTCCCCAACCGATGAGCGGGGAGCTAAACA CT O30-2.NUC 1100: ...・ ・ ・ ・ ・ ・ ・ ・ O30-4.NUC 1101: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ O30-3.NUC 1151: CCAGGCCAATAGGCCATCCCC O30-2.NUC 1150: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・-O30-4. NUC 1151: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ──────────────────────────────────────────── ──────────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0199

[Correction method] Change

[Correction content]

Nucleotide sequence of each clone in N17 region N17-3.NUC 1: TGTGAGCCCGAACCGGATGTAACAGTGGTCACCTCCATGCTCACCGACCC (SEQ ID NO: 81 in Sequence Listing) N17-1.NUC 1: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ (Sequence ID No .: 82) N17-2.NUC 1 : ...・ (Sequence ID No.:83) H17-1.NUC 1: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ T・ ・ ・ ・ (Sequence ID No .: 84) H17-3.NUC 1:・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ (Sequence ID No .: 85) N17- . NUC 51: CTCCCACATTACAG
CAGAGGCGGCTAGGGCGTAGGCTGCGCCAGAGGGTCTC N17-1. NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... N17-2. NUC 51: ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・
..................... H17-1. NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... A ... A ... H17-3. NUC 51: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... G ... N17-3. NUC 101: CCCCTTCCTTGGCC
AGTTCTTCAGCTAGTCAGTTGTTCTGCGCTTTCTTTG N17-1. NUC 101: ・ T ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・
························ N17-2. NUC 101: ・ T ・ ・ ・ ・ ・ T ・ ・ ・ ・ ・ ・
···················· H17-1. NUC 101: ... C ...
..C ..................... CC ... CC ... H17-3. NUC 101: .....................
・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ CC ・ ・ ・ C ・ ・ N17-3. NUC 151: AAGGCGACATGCAC
TACCCATCATGGCGCCCCAGACACTGACCTCATCGA N17-1. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ T ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N17-2. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ T ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H17-1. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ T ・ ・ ・ ・ G ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H17-3. NUC 151 ： ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ A ・ T ・ ・ ・ ・ G ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N17-3. NUC 201: GGCCAACCTCCTGT
GGCGGCAGGAGATGGGCGGAAACATCACCCCGCTGGG N17-1. NUC 201: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... G ... N17-2. NUC 201: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
... G ... H17-1. NUC 201: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ A ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ ・ H17-3. NUC 201: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ A ・
A ... G ... N17-3. NUC 251: AGTCAGAGAACAAG
ATAGTAATTCTAGACTCTTTTTGAACCGCTTCCGAGCG N17-1. NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ N17-2. NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
..................... H17-1. NUC 251: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・
G ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ H17-3. NUC 251 ： ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・
G ・ ・ ・ ・ ・ ・ ・ ・ G ・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ N17-3. NUC 301: GAGGAGGATGA
N17-1. NUC 301:
N17-2. NUC 301:
H17-1. NUC 301:
H17-3. NUC 301:

[Submission date] September 11, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0303

[Correction method] Change

[Correction content]

SEQ ID NO: 65 Sequence length: 311 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: N17-1, N17 -2, N17-3, H17-1, H17-3 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG STC ACY TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Xaa Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATY ACA GCA GAG RCG GCT RRG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Xab Ala Xac Arg Arg Leu Ala Arg Gly 20 25 30 TCT CCY CCT YCY TTG GCC AGY TCT TCA GCT AGY CAG TTG TCT GCG CYH 144 Ser Pro Pro Xad Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Xae 35 40 45 TCY YTG AAG GCG ACA TGC ACT ACC CAT CAT GRC KCC CCR GAC RCT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Xaf Xag Pro Asp Xab Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTR TGG CGG CAG GAG ATG GGM GGR AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGY GTG GAG TCA GAG ARC AAG RTA G TR ATT CTR GAC TCT TYY GAM 288 Thr Arg Val Glu Ser Glu Xah Lys Xai Val Ile Leu Asp Ser Xaj Xak 85 90 95 CCG CTT CGA GCG GAG GAG GAT GA 311 Pro Leu Arg Ala Glu Glu Asp 100 Y: C or TR: A or GM: A or CK: G or TS: G or CH: A or T or CD: G or T or A Xaa: Val or Leu Xab: Ala or Thr Xac: Arg or Lys or Gly Xad: Pro or Ser Xae : Pro or Leu Xaf: Gly or Asp Xag: Ala or Ser Xah: Asn or Ser Xai: Ile or Val Xaj: P
he or Ser Xak: Glu or Asp

[Submission date] September 11, 1992

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0308

[Correction method] Change

[Correction content]

SEQ ID NO: 70 Sequence length: 1084 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: 2217 Sequence GG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 47 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 1 5 10 15 ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC TAT GTG 95 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 20 25 30 CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC AAC CTT 143 Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu 35 40 45 ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT GAG GAC 191 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp 50 55 60 TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG GAC TGG 239 Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 65 70 75 ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC AAG CTC 287 I le Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu 80 85 90 95 CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT GGG TAC 335 Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr 100 105 110 AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC CCA TGT 383 Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys Pro Cys 115 120 125 GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG ATC GTT 431 Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 130 135 140 GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC ATC AAC 479 Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn 145 150 155 GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC TAT TCC 527 Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser 160 165 170 175 AGG GCG TTG TGG CGG GTG GCC ATT GAG GAG TAT GTG GAG GTC ACG CGG 575 Arg Ala Leu Trp Arg Val Ala Ile Glu Glu Tyr Val Glu Val Thr Arg 180 185 190 GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC G TG AAA 623 Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys 195 200 205 TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG GAT GGG 671 Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Phe Phe Thr Glu Leu Asp Gly 210 215 220 GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG CGG GAT 719 Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp 225 230 235 GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG TCA CAG 767 Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln 240 245 250 255 CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC 815 Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu 260 265 270 ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC 863 Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala 275 280 285 AGA GGG TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG TCT 911 Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser 290 295 300 GCG CTT TCT TTG AAG GCG ACA TGC ACT ACC CAT C AT GGC GCC CCA GAC 959 Ala Leu Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp 305 310 315 ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA 1007 Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly 320 325 330 335 AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT 1055 Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser 340 345 350 TTT GAA CCG CTT CGA GCG GAG GAG GAT GA 1084 Phe Glu Pro Leu Arg Ala Glu Glu Asp 355 3
60

[Submission date] September 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0309

[Correction method] Change

[Correction content]

SEQ ID NO: 71 Sequence length: 1004 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: 1728 Sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly 20 25 30 TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG TCT GCG CTT 144 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Leu 35 40 45 TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GGC GCC CCA GAC ACT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp Thr Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG GCG GAG 336 Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu 100 105 110 ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA TGG GCA 384 Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val Trp Ala 115 120 125 CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC CCG GAC 432 Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn Pro Asp 130 135 140 TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC AAG GCC 480 Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala 145 150 155 160 CCT CCA ATA CCA CCT CCA CGA AGA AAG AGA ACG GTT GTC CTG ACA GAA 528 Pro Pro Ile Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu 165 170 175 TCC TCC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC TTT GGC 576 Ser Ser Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly 180 185 190 AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC CCT CCT 624 Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro 195 200 205 GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG TCG TAC 672 Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr 210 215 220 TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AGC 720 Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser 225 230 235 240 GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC GTC GTC 768 Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val 245 250 255 TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA CCA TGC 816 Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 260 265 270 GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC CCT TTG 864 Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro Leu 275 280 285 CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC GCA AGC 912 Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser 290 295 300 CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG GAT GAC 960 Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp 305 310 315 320 CAC TAC CGG GAC GTG CTC AAG GAC ATG AAG GCC AAG GCG TCC AC 1004 His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys Ala Ser 325
330

[Submission date] September 11, 1992

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0311

[Correction method] Change

[Correction content]

SEQ ID NO: 73 sequence length: 1818 sequence type: nucleic acid number of strands: double-stranded topology: linear antisense: No origin: Hepatitis C virus direct origin clone name: 1718 sequence TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC ACC GAC 48 Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp 1 5 10 15 CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC AGA GGG 96 Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly 20 25 30 TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG TCT GCG CTT 144 Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Leu 35 40 45 TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GGC GCC CCA GAC ACT GAC 192 Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp Thr Asp 50 55 60 CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA AAC ATC 240 Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile 65 70 75 80 ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT TTT G AA 288 Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser Phe Glu 85 90 95 CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG GCG GAG 336 Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu 100 105 110 ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA TGG GCA 384 Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val Trp Ala 115 120 125 CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC CCG GAC 432 Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn Pro Asp 130 135 140 TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC AAG GCC 480 Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala 145 150 155 160 CCT CCA ATA CCA CCT CCA CGA AGA AAG AGA ACG GTT GTC CTG ACA GAA 528 Pro Pro Ile Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu 165 170 175 TCC TCC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC TTT GGC 576 Ser Ser Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly 180 185 190 AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC CCT CCT 624 Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro 195 200 205 GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG TCG TAC 672 Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr 210 215 220 TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AGC 720 Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser 225 230 235 240 GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC GTC GTC 768 Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val 245 250 255 TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA CCA TGC 816 Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys 260 265 270 GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC CCT TTG 864 Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro Leu 275 280 285 CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC GCA AGC 912 Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser 290 295 300 CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG GAT GAC 960 Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp 305 310 315 320 CAC TAC CGG GAC GTG CTS AAG GAC ATG AAG GCC AAG GCG TCC ACA GTT 1008 His Tyr Arg Asp Val Xaa Lys Asp Met Lys Ala Lys Ala Ser Thr Val 325 330 335 AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG CTG ACG CCC CCA 1056 Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro 340 345 350 CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG GAC GTC CAG AGC 1104 His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys Asp Val Gln Ser 355 360 365 CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG TGG AAG GAC TTG 1152 Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu 370 375 380 CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC ATG GCA AAA AAT 1200 Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn 385 390 395 400 GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC AAG CCA GCT CGC 1248 Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg 405 410 415 CTT ATC GTA TTC CC A GAC TTG GGG GTT CGT GTG TGC GAG AAA ATG GCC 1296 Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala 420 425 430 CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG ATG GGC TCC TCA 1344 Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser 435 440 445 TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG TTC CTG GTG AAT 1392 Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn 450 455 460 GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA TAT GAC ACC CGC 1440 Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala Tyr Asp Thr Arg 465 470 475 480 TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT ACT GAG GAG TCA 1488 Cys Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Thr Glu Glu Ser 485 490 495 ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA CAG GCC ATA AGG 1536 Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg Gln Ala Ile Arg 500 505 510 TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC TTG ACC AAT TCA AAA 1584 Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys 515 520 525 GGG CAA AAC TGC GGC TAT CGC CGG TGC CGC GCC AGC GGC GTG CTG ACG 1632 Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr 530 535 540 ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG GCC TCT GCA GCC 1680 Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala 545 550 555 560 TGT CGA GCT GCG AAG CTC CAG GAC TGC ACG ATG CTC GTG TGC GGA GAC 1728 Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp 565 570 575 GAC CTT GTC GTT ATC TGT GAA AGC GCG GGA ACC CAG GAG GAC GCG GCA 1776 Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Ala Ala 580 585 590 AAC CTA CGA GTC TTC ACG GAG GCT ATG ACC AGG AAT TCC GCC 1818 Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg Asn Ser Ala 595 600
605

[Submission date] September 11, 1992

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0312

[Correction method] Change

[Correction content]

SEQ ID NO: 74 sequence length: 2591 sequence type: nucleic acid number of strands: double-stranded topology: linear antisense: No origin: Hepatitis C virus direct origin clone name: 2218 sequence GG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG CTG 47 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 1 5 10 15 ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC TAT GTG 95 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 20 25 30 CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC AAC CTT 143 Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu 35 40 45 ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT GAG GAC 191 Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp 50 55 60 TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG GAC TGG 239 Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp 65 70 75 ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC AAG CTC 287 I le Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu 80 85 90 95 CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT GGG TAC 335 Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr 100 105 110 AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC CCA TGT 383 Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys Pro Cys 115 120 125 GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG ATC GTT 431 Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 130 135 140 GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC ATC AAC 479 Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn 145 150 155 GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC TAT TCC 527 Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser 160 165 170 175 AGG GCG TTG TGG CGG GTG GCC ATT GAG GAG TAT GTG GAG GTC ACG CGG 575 Arg Ala Leu Trp Arg Val Ala Ile Glu Glu Tyr Val Glu Val Thr Arg 180 185 190 GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC G TG AAA 623 Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys 195 200 205 TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG GAT GGG 671 Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Phe Phe Thr Glu Leu Asp Gly 210 215 220 GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG CGG GAT 719 Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp 225 230 235 GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG TCA CAG 767 Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln 240 245 250 255 CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG CTC 815 Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu 260 265 270 ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG GCC 863 Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala 275 280 285 AGA GGG TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG TCT 911 Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser 290 295 300 GCG CTT TCT TTG AAG GCG ACA TGC ACT ACC CAT C AT GGC GCC CCA GAC 959 Ala Leu Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp 305 310 315 ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGA 1007 Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly 320 325 330 335 AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC TCT 1055 Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp Ser 340 345 350 TTT GAA CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT GCG 1103 Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala 355 360 365 GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC GTA 1151 Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro Val 370 375 380 TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG AAC 1199 Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asn 385 390 395 CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT ACC 1247 Pro Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr 400 405 410 415 AAG GCC CCT CCA ATA CCA CCT CCA CGA AGA AAG AGA ACG GTT GTC CTG 1295 Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu 420 425 430 ACA GAA TCC TCC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG ACC 1343 Thr Glu Ser Ser Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr 435 440 445 TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC GGC 1391 Phe Gly Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Gly 450 455 460 CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT GAG 1439 Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu 465 470 475 TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC GAT 1487 Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp 480 485 490 495 CTC AGC GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG GAC 1535 Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp 500 505 510 GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT ACA 1583 Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr 515 520 525 CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC AAC 1631 Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn 530 535 540 CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC AGC 1679 Pro Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser 545 550 555 GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC CTG 1727 Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 560 565 570 575 GAT GAC CAC TAC CGG GAC GTG CTS AAG GAC ATG AAG GCC AAG GCG TCC 1775 Asp Asp His Tyr Arg Asp Val Xaa Lys Asp Met Lys Ala Lys Ala Ser 580 585 590 ACA GTT AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG CTG ACG 1823 Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr 595 600 605 CCC CCA CAC TCG GCC AGA TCT AAA TTT GAC TAC GGG GCA AAG GAC GTC 1871 Pro Pro His Ser Ala Arg Ser Lys Phe Asp Tyr Gly Ala Lys Asp Val 610 615 620 CAG AGC CTG TCC AGC AAG GCC GTT AAC CAC ATC CAC TCC GTG TGG AAG 1919 Gln Ser Leu Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp L ys 625 630 635 GAC TTG CCG GAA GAC ACT GAG ACA CCA ATC GAC ACC ACC ATC ATG GCA 1967 Asp Leu Pro Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala 640 645 650 655 AAA AAT GAG GTT TTT TGT GTT CAA CCA GAG AAA GGA GGC CGC AAG CCA 2015 Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro 660 665 670 GCT CGC CTT ATC GTA TTC CCA GAC TTG GGG GTT CGT GTG TGC GAG AAA 2063 Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys 675 680 685 ATG GCC CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG ATG GGC 2111 Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly 690 695 700 TCC TCA TAC AGA TTT CAG TGC TCC CCT GGA CAG CGG GTC GAG TTC CTG 2159 Ser Ser Tyr Arg Phe Gln Cys Ser Pro Gly Gln Arg Val Glu Phe Leu 705 710 715 GTG AAT GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA TAT GAC 2207 Val Asn Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala Tyr Asp 720 725 730 735 ACC CGC TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT ACT GAG 2255 Thr Arg Cys Phe Asp Ser Thr Val Thr Gl u Asn Asp Ile Arg Thr Glu 740 745 750 GAG TCA ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA CAG GCC 2303 Glu Ser Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg Gln Ala 755 760 765 ATA AGG TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC TTG ACC AAT 2351 Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn 770 775 780 TCA AAA GGG CAA AAC TGC GGC TAT CGC CGG TGC CGC GCC AGC GGC GTG 2399 Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 785 790 795 CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG GCC TCT 2447 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser 800 805 810 815 GCA GCC TGT CGA GCT GCG AAG CTC CAG GAC TGC ACG ATG CTC GTG TGC 2495 Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Cys 820 825 830 GGA GAC GAC CTT GTC GTT ATC TGT GAA AGC GCG GGA ACC CAG GAG GAC 2543 Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp 835 840 845 GCG GCA AAC CTA CGA GTC TTC ACG GAG GCT ATG ACC AGG AAT TCC GCC 2591 Ala Ala Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg Asn Ser Ala 850 855
860 ────────────────────────────────────────────────── ────

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0339

[Correction method] Change

[Correction content]

SEQ ID NO: 101 Sequence length: 9471 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Antisense: No Origin: Hepatitis C virus Direct origin Clone name: T7N1-30 Sequence ACTAGTTAAT ACGACTCACT ATAGGGTGCC AGCCCCCTGA TGGGGGCGAC ACTCCACCAT 60 AGATCACTCC CCTGTGAGGA ACTACTGTCT TCACGCAGAA AGCGTCTAGC CATGGCGTTA 120 GTATGAGTGT CGTGCAGCCT CCAGGACCCC CCCTCCCGGG AGAGCCATAG TGGTCTGCGG 180 AACCGGTGAG TACACCGGAA TTGCCAGGAC GACCGGGTCC TTTCTTGGAT CAACCCGCTC 240 AATGCCTGGA GATTTGGGCG TGCCCCCGCG AGACTGCTAG CCGAGTAGTG TTGGGTCGCG 300 AAAGGCCTTG TGGTACTGCC TGATAGGGTG CTTGCGAGTG CCCCGGGAGG TCTCGTAGAC 360 CGTGCATC ATG AGC ACA AAT CCA AAA CCC CAA AGA AAA ATC AAA CGT AAC 410 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Ile Lys Arg Asn 1 5 10 ACC AAC CGC CGC CCA CAG GAC GTT AAG TTC CCG GGC GGT GGT CAG ATC 458 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 15 20 25 30 GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 506 Val G ly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 35 40 45 CGC GCG ACT AGG AAG ACT TCC GAG CGG CCG CAA CCT CGT GGA AGG CGA 554 Arg Ala Thr Arg Lys Thr Ser Glu Arg Pro Gln Pro Arg Gly Arg Arg 50 55 60 CAA CCT ATC CCC AAG GCT CGC CAA CCC GAG GGT AGG GCC TGG GCT CAG 602 Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln 65 70 75 CCC GGG TAC CCT TGG CCC CTC TAT GGC AAT GAG GGC TTG GGG TGG GCA 650 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala 80 85 90 GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC ACG 698 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 95 100 105 110 GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTC 746 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu 115 120 125 ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC 794 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 130 135 140 CCC CTA GGG GGC GCT GCC AGG GCT CTA GCG CAT GGC GTC CGG GTT CTG 842 Pro Le u Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu 145 150 155 GAG GAC GGC GTG AAC TAT GCA ACA GGG AAT CTG CCT GGT TGC TCC TTT 890 Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe 160 165 170 TCT ATC TTC CTT TTG GCT TTG CTG TCC TGT TTG ACC ATC CCA GCT TCC 938 Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 175 180 185 190 GCC TAC CAA GTG CGC AAC GCG TCC GGG GTG TAC CAT GTC ACG AAC GAC 986 Ala Tyr Gln Val Arg Asn Ala Ser Gly Val Tyr His Val Thr Asn Asp 195 200 205 TGC TCC AAC TCA AGT ATT GTG TAT GAG GCG GCG GAC GTG ATT ATG CAC 1034 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His 210 215 220 ACC CCC GGG TGC GTG CCC TGC GTC CGG GAG AAC AAT TCC TCC CGC TGC 1082 Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys 225 230 235 TGG GTA GCG CTC ACT CCC ACG CTT GCG GCC AGG AAC AGC AGC ATC CCC 1130 Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro 240 245 250 ACT ACG ACA ATA CGG CGT CAT GTC GAC TTG CTC GTT GGG GCA GCT GCT 1178 Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Valu Gly Ala Ala Ala 255 260 265 270 CTC TGT TCC GCT ATG TAT GTG GGG GAT TTT TGC GGA TCT GTT TTC CTC 1226 Leu Cys Ser Ala Met Tyr Val Gly Asp Phe Cys Gly Ser Val Phe Leu 275 280 285 GTC TCC CAG CTG TTC ACT TTC TCA CCT CGC CGG TAT GAG ACG GTG CAA 1274 Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln 290 295 300 GAC TGC AAT TGC TCA ATC TAT CCC GGC CAT GTA TCA GGC CAT CGC ATG 1322 Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met 305 310 315 GCT TGG GAT ATG ATA ATG AAT TGG TCA CCT ACA ACA GCC CTA GTG GTA 1370 Ala Trp Asp Met Ile Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val 320 325 330 TCG CAG CTA CTC CGG ATC CCA CAA GCC GTG GTG GAT ATG GTG GCA GGG 1418 Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly 335 340 345 350 GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG 1466 Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly 355 360 365 AAC TGG GCT AAG GTC TTG GTT GTG ATG CTG CTC TTC GCC GGT GTT GAC 1514 Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp 370 375 380 GG GGG ACC CAC GTG ACA GGG GGG AAG GTA GCC TAC ACC ACC CAG GGC 1562 Gly Gly Thr His Val Thr Gly Gly Lys Val Ala Tyr Thr Thr Gln Gly 385 390 395 TTT ACA TCC TTC TTT TCA CGA GGG CCG TCT CAG AAA ATC CAA CTT GTA 1610 Phe Thr Ser Phe Phe Ser Arg Gly Pro Ser Gln Lys Ile Gln Leu Val 400 405 410 AAC ACT AAC GGC AGC TGG CAC ATC AAT AGG ACT GCC CTC AAT TGC AAT 1658 Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn 415 420 425 430 GAC TCC CTT AAC ACC GGG TTC CTT GCC GCG CTG TTC TAC ACC CAC AGC 1706 Asp Ser Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Ser 435 440 445 TTC AAC GCG TCC GGA TGT CCG GAG CGT ATG GCC GGT TGC CGC CCC ATT 1754 Phe Asn Ala Ser Gly Cys Pro Glu Arg Met Ala Gly Cys Arg Pro Ile 450 455 460 GAC GAG TTC GCT CAG GGG TGG GGT CCC ATC ACT CAT GTT GTG CCT AAC 1802 Asp Glu Phe Ala Gln Gly Trp Gly Pro Ile Thr His Val Val Pro Asn 465 470 475 ATC TCG GAC CAG AGG CCC TAT TGC TGG CAC TAC GCG CCT CGA CCG TGT 1850 Ile Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys 480 485 490 GGT ATC GTA CCC GCG TCG CAG GTG TGT GGT CCG GTG TAT TGC TTC ACC 1898 Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr 495 500 505 510 CCA AGC CCT GTT GTG GTG GGG ACG ACC GAT CGT TTC GGC GCC CCC ACG 1946 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr 515 520 525 TAC AAC TGG GGA AAC AAT GAG ACG GAT GTG CTA CTC CTC AAC AAC ACA 1994 Tyr Asn Trp Gly Asn Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr 530 535 540 CGG CCG CCG CAG GGC AAC TGG TTC GGT TGT ACC TGG ATG AAT GGC ACT 2042 Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr 545 550 555 GGG TTC ACA AAG ACG TGC GGG GGC CCC CCG TGC AAC ATC GGG GGG GTC 2090 Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val 560 565 570 GGC AAC AAT ACC TTG ACT TGC CCC ACG GAC TGC TTC CGG AAG CAC CCC 2138 Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 575 580 5 85 590 GAG GCC ACT TAC ACA AAA TGT GGT TCG GGG CCT TGG TTG ACA CCT AGG 2186 Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 TGC CTA GTT CAT TAC CCA TAC AGG CTC TGG CAC TAT CCC TGC ACT GTC 2234 Cys Leu Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val 610 615 620 AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT GTG GGG GGC GTG GAA CAC 2282 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 AGG CTT GAA GCT GCA TGC AAT TGG ACC CGA GGA GAG CGT TGT GAC TTG 2330 Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu 640 645 650 GAG GAC AGG GAT AGA TCA GAG CTT AGC CCG CTA TTG CTG TCC ACA ACA 2378 Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 655 660 665 670 GAG TGG CAG GTA CTG CCC TGT TCC TTC ACC ACC CTG CCG GCT CTG TCC 2426 Glu Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 ACT GGT TTG ATT CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA TAT CTG 2474 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Va l Gln Tyr Leu 690 695 700 TAC GGC ATA GGG TCG GCG GTT GTC TCC TTC GCA ATC AAA TGG GAA TAT 2522 Tyr Gly Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr 705 710 715 ATT CTG TTG CTT TTC CTC CCC CTG GCG GAC GCG CGC GTC TGT GCC TGG 2570 Ile Leu Leu Leu Phe Leu Pro Leu Ala Asp Ala Arg Val Cys Ala Trp 720 725 730 TTG TGG ATG ATG CTG CTG ATA GCC CAA GCT GAG GCC GCC TTG GAG AAC 2618 Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn 735 740 745 750 CTG GTG GTC CTC AAT GCA GCA TCC ATG GCG GGA GCG CAT GGC ATC CTC 2666 Leu Val Val Leu Asn Ala Ala Ser Met Ala Gly Ala His Gly Ile Leu 755 760 765 TCT TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAA GGC AGG CTG 2714 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu 770 775 780 GTC CCT GGG GCG GCA TAC GCT TTC TAT GGC GTA TGG CCG CTG CTC CTG 2762 Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu 785 790 795 CTC TTG ATG GCG CTA CCC GCA CGG GCG TAC GCC ATG GAC CGG GAG ATG 2810 Leu Leu Met Ala Leu Pro Ala Arg Ala Tyr Ala Met Asp Arg Glu Met 800 805 810 GCT GCA TCG TGC GGA GGC GCG GTT TTT GTA GGT CTG GTA CTC TTG ACC 2858 Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr 815 820 825 830 TTG TCA CCA TAC TAC AAA GTG TTC CTC GCT AAG CTC ATA TGG TGG TTG 2906 Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Lys Leu Ile Trp Trp Leu 835 840 845 CAA TAT CTC ATC ACC AGG GCC GAG GCG CAC TTG CAA GTG TGG ATC CCC 2954 Gln Tyr Leu Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro 850 855 860 CCC CTC AAC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTT CTC ACA TGT 3002 Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys 865 870 875 GCG GTC CAC CCG GAG CTG ATC TTT GAC ATC ACC AAG CTC TTG CTC GCC 3050 Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Leu Ala 880 885 890 ATA CTC GGT CCG CTC ATG GTA CTC CAG GCT GGC CTA ACC CAA ATG CCG 3098 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr Gln Met Pro 895 900 905 910 TAC TTT GTG CGT GCT CAA GGG CTC ATT CGT ATG TGC ATG TTG GTG CGG 3146 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Met Cys Met Leu Val Arg 915 920 925 AAA GCC GCT GGG GGT CAT TAT GTC CAG ATG GCT CTC ATG AAG CTG GCT 3194 Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu Ala 930 935 940 GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTT ACT CCA CTG CAG GAC 3242 Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp 945 950 955 TGG GCC CAC GCG GGC CTA CGA GAC CTT GCG GTA GCA GTT GAG CCC GTT 3290 Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val 960 965 970 GTC TTC TCT GAT ATG GAG ACT AAG ATC ATC ACG TGG GGG GCA GAG ACG 3338 Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Glu Thr 975 980 985 990 GCG GCG TGT GGG GAC ATC ATC TCG AGT CTA CCC GTT TCC GCC CGA AGG 3386 Ala Ala Cys Gly Asp Ile Ile Ser Ser Leu Pro Val Ser Ala Arg Arg 995 1000 1005 GGG AGG GAG CTG CTT TTG GGA CCG GCC GAT AGT TTT GAC GGG CAG GGG 3434 Gly Arg Glu Leu Leu Leu Gly Pro Ala Asp Ser Phe Asp Gly Gln Gly 1010 1015 1020 TGG CGA CTC CTT GCG CCT ATC ACG GCC TAC TCC CAG CAG AG CG CGG GGC 3482 Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly 1025 1030 1035 CTG CTT GGT TGC ATC ATC ACC AGC CTT ACG GGC CGG GAT AAG AAC CAG 3530 Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln 1040 1045 1050 GTC GAG GGG GAG GTT CAA GTG GTC TCT ACC GCA ACA CAA TCT TTC CTG 3578 Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu 1055 1060 1065 1070 GCG ACC TGC ATC AAC GGC GTT TGC TGG ACT GTT TTC CAC GGC GCC GGC 3626 Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Phe His Gly Ala Gly 1075 1080 1085 TCG AAG ACC TTA GCC GGC CCA AAA GGC CCA ATC ACC CAA ATG TAC ACC 3674 Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr 1090 1095 1100 AAT GTA GAT CAG GAC CTC GTC GGC TGG TCG GCG CCC CCC GGG GCG CGT 3722 Asn Val Asp Gln Asp Leu Val Gly Trp Ser Ala Pro Pro Gly Ala Arg 1105 1110 1115 TCC TTG ACA CCT TGC ACC TGC GGC AGC TCG GAC CTT TAT TTG GTC ACG 3770 Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr 1120 1125 1130 AGG CAT GCT GAT GTC ATT CCG GTG CAC CGG CGG GGC GAC AGC AGG GGG 3818 Arg His Ala Asp Val Ile Pro Val His Arg Arg Gly Asp Ser Arg Gly 1135 1140 1145 1150 AGC CTC CTC TCC CCC GGG CCC ATC TCT TAC TTG AAG GGT TCC TCG GGT 3866 Ser Leu Leu Ser Pro Gly Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly 1155 1160 1165 GGT CCG CTG CCT TGC CCC TCG GGC CGT GTT GTG GGC ATC TTC CGG GCT 3914 Gly Pro Leu Pro Cys Pro Ser Gly Arg Val Val Gly Ile Phe Arg Ala 1170 1175 1180 GCC GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTG CCC GTT 3962 Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val 1185 1190 1195 GAG TCT ATG GAA ACC ACC ATG CGG TCT CCG GTC TTC ACG GAT AAC TCA 4010 Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser 1200 1205 1210 ACC CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAC CTA CAC GCT 4058 Thr Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala 1215 1220 1225 1230 CCC ACT GGC AGC GGC AAA AGC ACC AGG GTG CCG GCT GCG TAT GCG GCC 4106 Pro Thr Gly Ser Gly Lys Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala 1235 1240 1245 CAA GGG TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCT GCC ACT TTG 4154 Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 1250 1255 1260 GGC TTT GGG GCG TAC ATG TCC AAG GCA CAT GGT GTT GAC CCT AAC ATC 4202 Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile 1265 1270 1275 AGA ACT GGG GTG AGG ACC ATC ACC ACG GGC GCT CCC ATC ACG TAC TCC 4250 Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser 1280 1285 1290 ACC TAC GGT AAG TTC CTC GCC GAC GGT GGC TGT TCT GGG GGT GCC TAT 4298 Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr 1295 1300 1305 1310 GAC ATC ATA ATA TGT GAT GAG TGT CAT TCA ACT GAC TCG ACT TCC ATC 4346 Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile 1315 1320 1325 TTG GGC ATT GGT ACA GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGC 4394 Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg 1330 1335 1340 CTT GTC GTG CTC GCC ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCG 4442 Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro 1345 1350 1355 CAT CCT AAT ATT GAG GAG GTG GCC TTG TCC AAC ACT GGA GAG ATC CCC 4490 His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro 1360 1365 1370 TTC TAT GGC AAG GCC ATC CCC CTC GAG GCC ATC AAG GGG GGG AGG CAT 4538 Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His 1375 1380 1385 1390 CTC ATT TTC TGC CAT TCC AAG AAG AAA TGT GAC GAG CTC GCT GCG AAG 4586 Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys 1395 1400 1405 CTG TCG GCC CTC GGA GTC AAC GCT GTA GCA TAT TAC CGG GGT CTT GAT 4634 Leu Ser Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp 1410 1415 1420 GTG TCC ATC ATA CCG ACA AGC GGG GAC GTC GTT GTC GTG GCA ACA GAC 4682 Val Ser Ile Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp 1425 1430 1435 GCT CTA ATG ACA GGC TAT ACC GGT GAC TTT GAC TCG GTG ATC GAC TGC 4730 Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys 1440 1445 1450 AAC ACA TGT GTC ACC CAA ACA GTC GAT TTC AGC TTG GAC CCT ACT TTC 4778 Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe 1455 1460 1465 1470 ACC ATC GAG ACG ACG ACC GTA CCC CAA GAT GCG GTG TCG CGC TCG CAG 4826 Thr Ile Glu Thr Thr Thr Pro Gln Asp Ala Val Ser Arg Ser Gln 1475 1480 1485 CGG CGA GGC AGG ACT GGT AGG GGC AGG GGG GGC ATA TAC AGG TTT GTA 4874 Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val 1490 1495 1500 ACT CCA GGG GAA CGG CCC TCA GGC ATG TTC GAT TCT TCG GTC CTG TGT 4922 Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 1505 1510 1515 GAA TGT TAT GAC GCG GGC TGT GCT TGG TAC GAG CTC ACG CCC GCC GAG 4970 Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu 1520 1525 1530 ACC TCG GTT AGG TTG CGG GCT TAC CTA AAT ACA CCT GGG CTG CCC GTC 5018 Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val 1535 1540 1545 1550 TGC CAG GAC CAT CTG GAG TTC TGG GAG AGC GTC TTC ACC GGC CTC ACC 5066 Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr 1555 1560 1565 CAC ATA GAT GCC CAC TTC TTG TCC CAG ACC AAA CAG GCA GGA GAC AAC 5114 His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn 1570 1575 1580 TTC CCC TAC CTG GTA GCA TAC CAG GCT ACA GTG TGC GCC AGG GCC AAG 5162 Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys 1585 1590 1595 GCT CCA CCT CCA TCG TGG GAT CAG ATG TGG AAG TGT CTC ATA CGG CTG 5210 Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu 1600 1605 1610 AAG CCT ACG CTA CAC GGG CCA ACG CCC CTG TTG TAT AGG TTA GGA GCC 5258 Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala 1615 1620 1625 1630 GTT CAG AAC GAG GTT ACC CTC ACA CAC CCC ATA ACC AAG TTC ATC ATG 5306 Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Phe Ile Met 1635 1640 1645 GCA TGC ATG TCG GCT GAC CTA GAG GTC GTC ACT AGC ACT TGG GTG CTG 5354 Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu 1650 1655 1660 GTA GGC GGG GTC CTC GCG GCT CTG GCC GCG TAC TGC CTG ACA ACG GGC 5402 Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly 1665 1670 1675 AGC GTG GTC ATT GTG GGC AGG ATC ATC TTG TCC GGG AGG CCG GCC GTT 5450 Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val 1680 1685 1690 ATT CCC GAC AGG GAA GTT CTC TAC CAA GAG TTC GAT GAA ATG GAA GAG 5498 Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu 1695 1700 1705 1710 TGC GCC TCG CAC CTC CCT TAC ATC GAA CAA GGA ATG CAG CGA GCC GAG 5546 Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu 1715 1720 1725 CAA TTC AAG CAG AAG GCG CTC GGT TTG CTG CAA ACA GCC ACC AAG CAA 5594 Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln 1730 1735 1740 GCG GAG GCT GCT GCT CCC GTG GTG GAG TCC AAG TGG CGA GCC CTT GAG 5642 Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu 1745 1750 1755 ACC TTC TGG GCG AAG CAC ATG TGG AAT TTT ATC AGC GGG ATA CAG TAC 5690 Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr 1760 1765 1770 TTA GCA GGC TTG TCC ACT CTG CCT GGA AAC CCC GCA ATA GCA TCA CTG 5738 Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu 1775 1780 1785 1790 ATG GCA TTC ACA GCC TCT ATC ACC AGC CCG CTC ACC ACC CAA TAT ACC 5786 Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Tyr Thr 1795 1800 1805 CTC CTG TTT AAC ATC TTG GGG GGA TGG GTG GCC GCC CAA CTC GCC CCC 5834 Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro 1810 1815 1820 CCC AGT GCC GCT TCA GCC TTC GTG GGC GCC GGT ATA GCT GGC GCG GCT 5882 Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala 1825 1830 1835 GTT GGC AGC ATA GGC CTC GGG AAG GTG CTT GTG GAC ATT CTG GCG GGT 5930 Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly 1840 1845 1850 TAT GGA GCA GGG GTG GCA GGC GCG CTC GTG GCC TTT AAG GTC ATG AGC 5978 Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser 1855 1860 1865 1870 GGT GAC ATG CCC TCC ACC GAG GAC CTG GTC AAC TTA CTC CCC GCC ATC 6026 Gly Asp Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile 1875 1880 1885 CTC T CT CCT GGT GCC CTG GTC GTC GGG GTC GTG TGC GCA GCA ATA CTG 6074 Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu 1890 1895 1900 CGT CGG CAT GTG GGC CCA GGG GAG GGG GCT GTG CAG TGG ATG AAC CGG 6122 Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg 1905 1910 1915 CTG ATA GCG TTT GCT TCG CGG GGC AAC CAT GTC TCC CCC ACG CAC TAT 6170 Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr 1920 1925 1930 GTG CCT GAA AGC GAC GCC GCA GCG CGC GTC ACC CAG ATC CTC TCC AAC 6218 Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn 1935 1940 1945 1950 CTT ACC ATC ACT CAG CTG TTG AAG AGG CTT CAC CAG TGG ATT AAT GAG 6266 Leu Thr Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu 1955 1960 1965 GAC TGC TCC ACG CCA TGC TCC GGC TCG TGG CTC AGG GAT GTT TGG GAC 6314 Asp Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp 1970 1975 1980 TGG ATA TGC ACG GTA TTG GCT GAT TTC AAG ACC TGG CTC CAG TCC AAG 6362 Trp Ile Cys Thr Val Leu Ala Asp Phe Lys Th r Trp Leu Gln Ser Lys 1985 1990 1995 CTC CTG CCG CGG TTA CCG GGG GTC CCT TTT TTC TCA TGC CAG CGT GGG 6410 Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly 2000 2005 2010 TAC AAG GGG GTT TGG CGG GGA GAT GGC ATC ATG TAT ACC ACC TGC CCA 6458 Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr Cys Pro 2015 2020 2025 2030 TGT GGA GCA CAA ATC ACC GGA CAT GTC AAA AAC GGT TCT ATG AGG ATC 6506 Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile 2035 2040 2045 GTT GGG CCT AGA ACC TGT AGC AAC ACG TGG CAC GGA ACA TTT CCC ATC 6554 Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile 2050 2055 2060 AAC GCG TAC ACC ACA GGC CCC TGC ACA CCC TCC CCG GCG CCA AAC TAT 6602 Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr 2065 2070 2075 TCC AGG GCG TTG TGG CGG GTG GCC GCT GAG GAG TAT GTG GAG GTC ACG 6650 Ser Arg Ala Leu Trp Arg Val Ala Ala Glu Glu Tyr Val Glu Val Thr 2080 2085 2090 CGG GTG GGG GAT TTC CAC TAC GTG ACG GGC ATG ACC ACT GAC AAC GTG 6698 A rg Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Val 2095 2100 2105 2110 AAA TGC CCA TGC CAG GTT CCG GCC CCC GAA TTC TTC ACA GAA TTG GAT 6746 Lys Cys Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Thr Glu Leu Asp 2115 2120 2125 GGG GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA CCT CTC CTG CGG 6794 Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg 2130 2135 2140 GAT GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAT ACG GTT GGG TCA 6842 Asp Glu Val Thr Phe Gln Val Gly Leu Asn Gln Tyr Thr Val Gly Ser 2145 2150 2155 CAG CTC CCA TGT GAG CCC GAA CCG GAT GTA ACA GTG GTC ACC TCC ATG 6890 Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Thr Val Val Thr Ser Met 2160 2165 2170 CTC ACC GAC CCC TCC CAC ATT ACA GCA GAG GCG GCT AGG CGT AGG CTG 6938 Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu 2175 2180 2185 2190 GCC AGA GGG TCT CCC CCT TCC TTG GCC AGT TCT TCA GCT AGT CAG TTG 6986 Ala Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu 2195 2200 2205 TCT GCG CTT TCT TTG AAG GCG ACA TGC ACT ACC CAT CAT GGC GCC CCA 7034 Ser Ala Leu Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro 2210 2215 2220 GAC ACT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC 7082 Asp Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly 2225 2230 2235 GGA AAC ATC ACC CGC GTG GAG TCA GAG AAC AAG ATA GTA ATT CTA GAC 7130 Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu Asp 2240 2245 2250 TCT TTT GAA CCG CTT CGA GCG GAG GAG GAT GAG AGG GAA GTG TCC GTT 7178 Ser Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val 2255 2260 2265 2270 GCG GCG GAG ATC CTG CGG AAG ACC AGG AAA TTC CCC GCA GCG ATG CCC 7226 Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala Ala Met Pro 2275 2280 2285 GTA TGG GCA CGC CCG GAC TAC AAC CCA CCA TTA CTA GAG TCT TGG AAG 7274 Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys 2290 2295 2300 AAC CCG GAC TAC GTC CCT CCA GTG GTA CAC GGG TGC CCA TTG CCG CCT 7322 Asn Pro Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pr o 2305 2310 2315 ACC AAG GCC CCT CCA ATA CCA CCT CCA CGG AGA AAG AGA ACG GTT GTC 7370 Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val 2320 2325 2330 CTG ACA GAA TCC ACC GTG TCC TCT GCC TTG GCG GAG CTT GCT ACA AAG 7418 Leu Thr Glu Ser Thr Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys 2335 2340 2345 2350 ACC TTT GGC AGT TCC GGA TCG TCG GCC GTC GAC AGC GGC ACG GCG ACC 7466 Thr Phe Gly Ser Ser Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr 2355 2360 2365 GGC CCT CCT GAC CAG GCC TCC GCC GAA GGA GAT GCA GGA TCC GAC GCT 7514 Gly Pro Pro Asp Gln Ala Ser Ala Glu Gly Asp Ala Gly Ser Asp Ala 2370 2375 2380 GAG TCG TAC TCC TCC ATG CCC CCC CTT GAG GGA GAG CCG GGG GAC CCC 7562 Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro 2385 2390 2395 GAT CTC AAC GAC GGG TCT TGG TCT ACC GTA AGC GAG GAG GCC AGC GAG 7610 Asp Leu Asn Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu 2400 2405 2410 GAC GTC GTC TGC TGC TCG ATG TCC TAC ACA TGG ACA GGC GCC TTA ATT 7658 Asp Val Val Cys Cys S er Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile 2415 2420 2425 2430 ACA CCA TGC GCC GCG GAG GAG AGC AAG CTG CCC ATT AAT GCG CTG AGC 7706 Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser 2435 2440 2445 AAC CCT TTG CTG CGC CAC CAC AAC ATG GTC TAT GCC ACA ACA TCC CGC 7754 Asn Pro Leu Leu Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg 2450 2455 2460 AGC GCA AGC CAG CGG CAG AAA AAG GTC ACA TTT GAC AGA CTG CAA GTC 7802 Ser Ala Ser Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val 2465 2470 2475 CTG GAT GAC CAC TAC CGG GAC GTG CTG AAG GAC ATG AAG GCC AAG GCG 7850 Leu Asp Asp His Tyr Arg Asp Val Leu Lys Asp Met Lys Ala Lys Ala 2480 2485 2490 TCC ACA GTT AAG GCT AAA CTT CTA TCT GTA GAG GAA GCC TGC AAG CTG 7898 Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu 2495 2500 2505 2510 ACG CCC CCA CAC TCG GCC AGA TCT AAA TTT GGC TAC GGG GCA AAG GAC 7946 Thr Pro Pro His Ser Ala Arg Ser Lys Phe Gly Tyr Gly Ala Lys Asp 2515 2520 2525 GTC CGG AGC CTG TCC AGC AAG GCC GTT AAC CAC ATC CGC TCC GTG TGG 7994 Val Arg Ser Leu Ser Ser Lys Ala Val Asn His Ile Arg Ser Val Trp 2530 2535 2540 AAG GAC TTG CTG GAA GAC ACT GAG ACA CCA ATT GAC ACC ACC ATC ATG 8042 Lys Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met 2545 2550 2555 GCA AAA AAT GAG GTT TTC TGT GTT CAA CCA GAG AAA GGA GGC CGC AAG 8090 Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys 2560 2565 2570 CCA GCT CGC CTT ATC GTA TTC CCA GAC TTG GGG GTT CGT GTG TGC GAG 8138 Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu 2575 2580 2585 2590 AAA ATG GCC CTC TAC GAC GTG GTC TCC ACT CTT CCT CAG GCC GTG ATG 8186 Lys Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met 2595 2600 2605 GGC TCC TCA TAC GGA TTC CAG TAC TCC CCT GGA CAG CGG GTC GAG TTC 8234 Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe 2610 2615 2620 CTG GTG AAT GCC TGG AAG TCA AAG AAG AGC CCT ATG GGC TTT GCA TAT 8282 Leu Val Asn Ala Trp Lys Ser Lys Lys Ser Pro Met Gly Phe Ala Tyr 2625 2630 2635 GAC ACC CGC TGT TTT GAC TCA ACG GTC ACC GAG AAC GAC ATC CGT ACT 8330 Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Thr 2640 2645 2650 GAG GAG TCA ATT TAT CAA TGT TGT GAC TTG GAC CCC GAG GCC AGA CAG 8378 Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Asp Pro Glu Ala Arg Gln 2655 2660 2665 2670 GCC ATA AGG TCG CTC ACA GAG CGG CTT TAT ATC GGG GGC CCC CTG ACT 8426 Ala Ile Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr 2675 2680 2685 AAT TCA AAG GGG CAG AAC TGC GGT TAT CGC CGG TGC CGC GTC AGC GGC 8474 Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Val Ser Gly 2690 2695 2700 GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC TTG AAG GCC 8522 Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala 2705 2710 2715 TCT GCA GCC TGT CGA GCT GCA AAG CTC CAG GAC TGC ACG ATG CTT GTG 8570 Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val 2720 2725 2730 TGC GGA GAC GAC CTT GTC GTT ATC TGT GAT AGC GCG GGA ACT CAG GAG 8618 Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser Ala Gly Thr Gln Glu 2735 2740 2745 2750 GAC GCG GCG AGC CTA CGA GTC TTC ACG GAG GCT ATG ACT AGG TAC TCT 8666 Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser 2755 2760 2765 GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC GAC TTG GAG CTG ATA 8714 Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile 2770 2775 2780 ACA TCA TGC TCC TCC AAT GTG TCG GTC GCG CAC GAC GCA TCA GGC AAA 8762 Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Ala Ser Gly Lys 2785 2790 2795 CGG GTG TAC TAT CTC ACC CGT GAC CCC ACC ACC CCC CTA GCG CGG GCT 8810 Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala 2800 2805 2810 GCG TGG GAG ACA GCT AGA CAC ACT CCA GTC AAC TCC TGG CTA GGC AAC 8858 Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn 2815 2820 2825 2830 ATC ATC ATG TAC GCG CCC ACC TTA TGG GCA AGG ATG ATT CTG ATG ACC 8906 Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr 2835 2840 2845 CAC TTC TTC TCC ATC CTT CTA GCC CAG GAG CAA CTT GAA AAA GCC CTA 8954 His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu 2850 2855 2860 GAT TGT CAG ATC TAC GGG GCC ACT TAC TCC ATT GAG CCA CTT GAC CTA 9002 Asp Cys Gln Ile Tyr Gly Ala Thr Tyr Ser Ile Glu Pro Leu Asp Leu 2865 2870 2875 CCT CAG ATC ATT CAA CGA CTC CAC GGT CTT AGC GCA TTT TCA CTC CAT 9050 Pro Gln Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His 2880 2885 2890 AGT TAC TCT CCA GGT GAG ATC AAT AGG GTG GCT TCA TGC CTC AGG AAA 9098 Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys 2895 2900 2905 2910 CTT GGG GTA CCG CCC TTG CGA GTC TGG AGA CAT CGG GCC AGA AGC GTC 9146 Leu Gly Val Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val 2915 2920 2925 CGC GCT AAG CTA CTG TCC CAG GGG GGG AGG GCC GCC ACC TGT GGC AAA 9194 Arg Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys 2930 2935 2940 TAC CTC TTC AAC TGG GCA GTA AAG ACC AAG CTC AAA CTC ACT CCA ATC 9242 Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Ile 2945 2950 2955 CCA GAA GCG TCC CAG CT G GAC TTG TCC GGC TGG TTC GTT GCT GGT TAC 9290 Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr 2960 2965 2970 AGC GGG GGA GAC ATA TAT CAC AGC CTG TCT CGT GCC CGA CCC CGC TGG 9338 Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp 2975 2980 2985 2990 TTC ATG TGG TGC CTA CTC CTA CTT TCC GTA GGG GTA GGC ATC TAC CTG 9386 Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu 2995 3000 3005 CTC CCC AAC CGA TGA GCGGGGAGC TAAACACTCC AGGCCAATAG GCCATCCCCC 9440 Leu Pro Asn Arg Stop 3010 TTTTTTTTTT TTAAGCTGGA TCCGCGGCCG C 9471

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07K 7/10 8318-4H 13/00 8619-4H 15/12 8517-4H C12N 1/21 7236- 4B 5/10 15/11 15/70 15/85 C12P 21/02 C 8214-4B // A61K 39/00 H 9284-4C 39/29 9284-4C (C12N 1/21 C12R 1:19) (C12N 5 / 10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) C07K 99:00 (31) Priority claim number Japanese Patent Application No. 3-332329 (32) 3 (1991) December 16 (33) Priority claiming country Japan (JP) (31) Priority claim number Japanese Patent Application No. 4-99957 (32) Priority Day 4 (1992) April 20 (33) Priority Claiming country Japan (JP) (72) Inventor Tomoko Murakami 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa Sanryo Kasei Co., Ltd. Corporate Research Institute (72) Yutaka Teranishi, 1000 Kamoshida-cho, Midori-ku, Yokohama

Claims (223)

[Claims] 1. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. 2. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing. 3. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing. 4. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing. 5. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 5 in Sequence Listing. 6. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing. 7. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing. 8. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing. 9. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 9 in the sequence listing. 10. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing. 11. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing. 12. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 12 in the sequence listing. 13. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 13 in the sequence listing. 14. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing. 15. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 15 in the sequence listing. 16. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 16 in the sequence listing. 17. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 17 in the sequence listing. 18. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing. 19. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 19 in the sequence listing. 20. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 20 in the sequence listing. 21. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 21 in the sequence listing. 22. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 22 in the sequence listing. 23. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing. 24. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 24 in the sequence listing. 25. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing. 26. A gene encoding a polypeptide derived from hepatitis C virus, which is represented by the amino acid sequence set forth in SEQ ID NO: 26 in the sequence listing. 27. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing. 28. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 28 in the sequence listing. 29. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 29 in the sequence listing. 30. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 30 in the sequence listing. 31. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 31 of the Sequence Listing. 32. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 32 in the sequence listing. 33. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 33 of the Sequence Listing. 34. A gene encoding a polypeptide derived from hepatitis C virus, which is represented by the amino acid sequence set forth in SEQ ID NO: 34 of the Sequence Listing. 35. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 35 of the Sequence Listing. 36. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 36 in the sequence listing. 37. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 37 of the Sequence Listing. 38. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 38 of the Sequence Listing. 39. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 39 of the Sequence Listing. 40. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 40 in the sequence listing. 41. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 41 of the Sequence Listing. 42. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 42 in the sequence listing. 43. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing. 44. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 64 of the Sequence Listing. 45. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 65 of the Sequence Listing. 46. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 66 of the Sequence Listing. 47. A gene encoding a polypeptide derived from hepatitis C virus, which is represented by the amino acid sequence set forth in SEQ ID NO: 67 of the Sequence Listing. 48. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 68 of the Sequence Listing. 49. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 69 in the sequence listing. 50. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 70 of the Sequence Listing. 51. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 71 of the Sequence Listing. 52. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 72 of the Sequence Listing. 53. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 73 of the Sequence Listing. 54. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 74 of the Sequence Listing. 55. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 75 of the Sequence Listing. 56. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 101 of Sequence Listing. 57. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 102 of Sequence Listing. 58. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 103 of Sequence Listing. 59. A gene encoding a polypeptide derived from hepatitis C virus represented by the amino acid sequence set forth in SEQ ID NO: 104 of the Sequence Listing. 60. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 61. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 2 in Sequence Listing. 62. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 3 in the Sequence Listing. 63. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 64. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 5 in the Sequence Listing. 65. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 6 in Sequence Listing. 66. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 7 in Sequence Listing. 67. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 8 in Sequence Listing. 68. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 9 in Sequence Listing. 69. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 10 in Sequence Listing. 70. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 11 of the Sequence Listing. 71. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 12 in the Sequence Listing. 72. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 13 of the Sequence Listing. 73. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 14 of Sequence Listing. 74. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 15 of the Sequence Listing. 75. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 16 in Sequence Listing. 76. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 17 in Sequence Listing. 77. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 18 of the Sequence Listing. 78. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 19 of the Sequence Listing. 79. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 20 in the Sequence Listing. 80. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 21 of the Sequence Listing. 81. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 22 in the Sequence Listing. 82. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 23 of the Sequence Listing. 83. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 24 in the Sequence Listing. 84. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 25 of the Sequence Listing. 85. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 26 of Sequence Listing. 86. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 27 of the Sequence Listing. 87. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 28 of the Sequence Listing. 88. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 29 of the Sequence Listing. 89. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 30 in the sequence listing. 90. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 31 of the Sequence Listing. 91. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 32 of the Sequence Listing. 92. A DNA fragment derived from hepatitis C virus, which is represented by the nucleotide sequence set forth in SEQ ID NO: 33 of the Sequence Listing. 93. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 34 of the Sequence Listing. 94. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 35 of the Sequence Listing. 95. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 36 of the Sequence Listing. 96. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 37 of the Sequence Listing. 97. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 38 of the Sequence Listing. 98. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 39 of the Sequence Listing. 99. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 40 of the Sequence Listing. 100. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 41 of the Sequence Listing. 101. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 42 of the Sequence Listing. 102. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 43 of the Sequence Listing. 103. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 64 of the Sequence Listing. 104. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 65 of the Sequence Listing. 105. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 66 of the Sequence Listing. 106. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 67 of the Sequence Listing. 107. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 68 in the Sequence Listing. 108. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 69 in the sequence listing. 109. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 70 of the Sequence Listing. 110. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 71 of the Sequence Listing. 111. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 72 of the Sequence Listing. 112. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 73 of Sequence Listing. 113. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 74 of the Sequence Listing. 114. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 75 of the Sequence Listing. 115. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 101 of the Sequence Listing. 116. A hepatitis C virus-derived DNA fragment represented by the nucleotide sequence set forth in SEQ ID NO: 102 of the Sequence Listing. 117. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 103 of Sequence Listing. 118. A DNA fragment derived from hepatitis C virus represented by the nucleotide sequence set forth in SEQ ID NO: 104 of Sequence Listing. 119. SEQ ID NOs: 1, 9, 10, 1 in the sequence listing
1 to 1 of the amino acid sequences of 1 or 12
A polypeptide represented by 18 amino acids up to the eighth position. 120. A DNA fragment encoding the polypeptide according to claim 119. 121. A polypeptide represented by 34 amino acids from the 40th position to the 73rd position in the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. 122. A DNA fragment encoding the polypeptide of claim 121. 123. A polypeptide represented by 35 amino acids from the 81st position to the 115th position in the amino acid sequence of SEQ ID NO: 3 in the sequence listing. 124. A DNA fragment encoding the polypeptide according to claim 123. 125. A polypeptide represented by 191 amino acids from 1st to 171st of the amino acid sequence set forth in SEQ ID NO: 3 in Sequence Listing. 126. A DNA fragment encoding the polypeptide according to claim 125. 127. 115 to 115 of the amino acid sequences set forth in SEQ ID NOs: 3 and 7 of the Sequence Listing
A polypeptide represented by individual amino acids. 128. A DNA fragment encoding the polypeptide of claim 127. 129. A polypeptide represented by 192 amino acids from the 31st position to the 222nd position in the amino acid sequence of SEQ ID NO: 4 in the sequence listing. 130. A DNA fragment encoding the polypeptide according to claim 129. 131. A polypeptide represented by 19 amino acids from the 134th position to the 152nd position in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing. 132. A polypeptide represented by 17 amino acids from the 223rd position to the 239th position in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing. 133. A polypeptide represented by 18 amino acids 92 to 109 of the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 134. A polypeptide represented by 18 amino acids from the 40th position to the 57th position in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 135. A polypeptide represented by 12 amino acids 240 to 251 in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 136. A polypeptide represented by 76 amino acids from 31st position to 106th position in the amino acid sequence set forth in SEQ ID NO: 4 in Sequence Listing. 137. A DNA fragment encoding the polypeptide according to claim 136. 138. A polypeptide represented by 36 amino acids from the 134th position to the 169th position in the amino acid sequence of SEQ ID NO: 4 in the sequence listing. 139. A DNA fragment encoding the polypeptide of claim 138. 140. A polypeptide represented by 13 amino acids from position 143 to position 155 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 141. A polypeptide represented by 21 amino acids from the 171st position to the 191st position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 142. Amino acid sequence set forth in SEQ ID NO: 31 or 32, which comprises a polypeptide represented by 6 amino acids at least from 182nd position to 187th position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 A polypeptide derived from an amino acid sequence and consisting of 10 to 25 amino acid residues in length. 143. A polypeptide represented by 14 amino acids from 202 to 215 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 144. Amino acid sequence set forth in SEQ ID NO: 31 or 32, which comprises a polypeptide represented by at least eight amino acids 202 to 209 in the amino acid sequence set forth in SEQ ID NO: 31 or 32 A polypeptide derived from an amino acid sequence and consisting of 10 to 25 amino acid residues in length. 145. A polypeptide represented by 13 amino acids from the 244th position to the 256th position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 146. A polypeptide represented by 21 amino acids from the 299th position to the 319th position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 147. A polypeptide represented by 106 amino acids 109 to 214 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 148. A polypeptide represented by 92 amino acids from the 233rd position to the 324th position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 149. A polypeptide represented by 10 amino acids from 252nd to 261st of the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 150. Amino acid sequence set forth in SEQ ID NO: 31 or 32, which comprises a polypeptide represented by at least five amino acids 252 to 256 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 A polypeptide derived from an amino acid sequence and consisting of 10 to 25 amino acid residues in length. 151. A polypeptide represented by 34 amino acids from the 250th position to the 283rd position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 152. Amino acid sequence set forth in SEQ ID NO: 31 or 32, which comprises a polypeptide represented by at least 7 amino acids 273 to 279 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 A polypeptide derived from an amino acid sequence and consisting of 10 to 25 amino acid residues in length. 153. A polypeptide represented by 20 amino acids 77 to 96 in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 154. A polypeptide represented by 16 amino acids 122 to 137 of the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 155. A DNA fragment encoding the polypeptide according to claim 147. 156. A DNA fragment encoding the polypeptide of claim 148. 157. A polypeptide represented by 12 amino acids from the 136th position to the 147th position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 158. Among the amino acid sequences set forth in SEQ ID NO: 31 or 32 of the Sequence Listing, including a polypeptide represented by 7 amino acids at least from the 136th position to the 142nd position, the SEQ ID NO: 31 or 32 A polypeptide derived from an amino acid sequence and consisting of 10 to 25 amino acid residues in length. 159. A polypeptide represented by 27 amino acids from 45th position to 71st position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 160. A polypeptide represented by 18 amino acids from the 306th position to the 323rd position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 161. At least the sixth to the sixth amino acid sequences of the amino acid sequences of SEQ ID NO: 31 or 32 of the sequence listing.
A polypeptide comprising a polypeptide represented by 17 amino acids up to the 9th position, and having a length of 17 to 25 amino acid residues derived from the amino acid sequence of SEQ ID NO: 31 or 32. 162. A polypeptide represented by 9 amino acids from 193rd position to 201st position in the amino acid sequence set forth in SEQ ID NO: 31 or 32 of the Sequence Listing. 163. The 26th position from the 461st position to the 726th position in the amino acid sequence of SEQ ID NO: 43 in the sequence listing
A polypeptide represented by 6 amino acids or a polypeptide containing all or a part thereof. 164. The amino acid sequence of SEQ ID NO: 43 in the sequence listing, which is 4 from 963rd to 1004th.
A polypeptide represented by two amino acids or a polypeptide containing all or a part thereof. 165. The 247th to 265th 19th amino acids in the amino acid sequence of SEQ ID NO: 43 in the sequence listing
A polypeptide represented by individual amino acids. [166] The amino acid sequence of SEQ ID NO: 43 in the sequence listing, which is 45 from the 283rd position to the 327th position
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 167. Among the amino acid sequences set forth in SEQ ID NO: 43 of the Sequence Listing, 10 from 283rd position to 292nd position
A polypeptide represented by individual amino acids. 168. In the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing, comprising a polypeptide represented by at least eight amino acids from the 300th position to the 307th position,
A polypeptide derived from the amino acid sequence of SEQ ID NO: 43 and having a length of 8 to 25 amino acid residues. 169. The amino acid sequence of SEQ ID NO: 43 of the Sequence Listing, which is 15 from the 315th position to the 327th position.
A polypeptide represented by individual amino acids. 170. A polypeptide comprising a polypeptide represented by at least the 410th amino acid to the 428th amino acid in the amino acid sequence of SEQ ID NO: 43 of the Sequence Listing, which is derived from the amino acid sequence of SEQ ID NO: 43. Length 1
A polypeptide consisting of 3 to 25 amino acid residues. 171. A polypeptide represented by at least 74 amino acids from the 477th position to the 550th position in the amino acid sequence set forth in SEQ ID NO: 43 of the sequence listing, or a polypeptide comprising all or a part thereof. 172. Of the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing, 14 from the 477th position to the 490th position
A polypeptide represented by individual amino acids. 173. Among the amino acid sequences set forth in SEQ ID NO: 43 of the Sequence Listing, 14 from the 498th position to the 512th position
A polypeptide represented by individual amino acids. 174. The 12th amino acid sequence from the 538th position to the 550th position in the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing.
A polypeptide represented by individual amino acids. 175 includes a polypeptide represented by at least 8 amino acids from the 665th position to the 672nd position in the amino acid sequence of SEQ ID NO: 43 in the sequence listing,
A polypeptide derived from the amino acid sequence of SEQ ID NO: 43 and having a length of 8 to 25 amino acid residues. 176. The 21st amino acid sequence from the 747th position to the 767th position of the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing.
A polypeptide represented by individual amino acids. 177. The 12 from 841 to 852 of the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing.
A polypeptide represented by individual amino acids. 178. A polypeptide represented by at least 12 amino acids 867 to 878 in the amino acid sequence set forth in SEQ ID NO: 43 of the Sequence Listing. 179. The amino acid sequence of SEQ ID NO: 43 in the sequence listing, which is 61 from the 215th position to the 275th position.
A polypeptide represented by individual amino acids. 180. A DNA fragment encoding the polypeptide according to claim 163. 181. A DNA fragment encoding the polypeptide according to claim 164. 182. A DNA fragment encoding the polypeptide according to claim 166. 183. A DNA fragment encoding the polypeptide according to claim 171. 184. A DNA fragment encoding the polypeptide according to claim 179. 185. 74th from 413th to 486th of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 186. 9th from 415th to 1411th of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by 97 amino acids, or a polypeptide containing all or a part thereof. 187 (19) from the 247th position to the 265th position in the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 188. The 74th from 655th to 728th of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 189. The 54th amino acid sequence from the 763rd position to the 816th position of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 190. A polypeptide represented by 20 amino acids from at least 324 to 343 in the amino acid sequence of SEQ ID NO: 75 in the sequence listing, wherein the polypeptide has a length of at least 8 amino acid residues. peptide. 191. The 98 from the 858th position to the 955th position of the amino acid sequence of SEQ ID NO: 75 in the Sequence Listing.
A polypeptide represented by individual amino acids or a polypeptide containing all or a part thereof. 192 includes a polypeptide represented by 14 amino acids from at least the 356th position to the 369th position in the amino acid sequence of SEQ ID NO: 75 in the sequence listing, and is derived from the amino acid sequence of SEQ ID NO: 75. Length 1
A polypeptide consisting of 4 to 25 amino acid residues. 193. At least 1009 to 1100 of the amino acid sequences of SEQ ID NO: 75 in the sequence listing.
A polypeptide represented by the 92 amino acids up to the th position, or a polypeptide containing all or a part thereof. 194. A polypeptide represented by the 66 amino acids from 1160th to 1225th in the amino acid sequence of SEQ ID NO: 75 in the sequence listing, or a polypeptide containing all or a part thereof. 195, 18 from the 584th position to the 601st position, of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide consisting of 18 to 25 amino acid residues, which is derived from the amino acid sequence of SEQ ID NO: 75 and contains a polypeptide represented by 40 amino acids. 196. The 42 from the 615th position to the 656th position of the amino acid sequence of SEQ ID NO: 75 in the sequence listing
A polypeptide represented by individual amino acids. 197 includes a polypeptide represented by at least 11 amino acids from 326th position to 337th position in the amino acid sequence of SEQ ID NO: 75 in the sequence listing, and is derived from the amino acid sequence of SEQ ID NO: 75 Length 1
A polypeptide consisting of 1 to 25 amino acid residues. 198. The 10th position from the 588th position to the 597th position of the amino acid sequence set forth in SEQ ID NO: 75 of the Sequence Listing.
A polypeptide represented by individual amino acids. 199. 10 of the amino acid sequences set forth in SEQ ID NO: 75 of the Sequence Listing, from the 620th position to the 629th position.
A polypeptide represented by individual amino acids. 200. A DNA fragment encoding the polypeptide according to claim 185. 201. A DNA fragment encoding the polypeptide according to claim 186. 202. A DNA fragment encoding the polypeptide according to claim 188. 203. A DNA fragment encoding the polypeptide according to claim 191. 204. A DNA fragment encoding the polypeptide according to claim 193. 205. A DNA fragment encoding the polypeptide according to claim 194. 206. The method according to claim 60, which is downstream of the promoter.
61, 62, 63, 64, 65, 66, 67, 68, 6
DNA fragment derived from hepatitis C virus according to 9, 70, 71, 120, 122, 124, 126 or 128,
An expression vector in which a polypeptide encoded by the DNA fragment has been integrated so as to be expressed. 207. A method of culturing a host cell transformed with the expression vector of claim 206 to produce the polypeptide. 208. A host cell transformed with the expression vector of claim 206. 209. The method according to claim 12, which is downstream of the promoter.
An expression vector in which the DNA fragment derived from hepatitis C virus according to 9, 137 or 139 is incorporated so as to express the polypeptide encoded by the DNA fragment. 210. A method for producing the polypeptide by culturing a host cell transformed with the expression vector according to claim 209. 211. A host cell transformed with the expression vector of claim 209. 212. The method according to claim 72, which is downstream of the promoter.
73, 74, 75, 76, 77, 78, 79, 80, 8
1, 82, 83, 84, 85, 86, 87, 88, 8
The hepatitis C virus-derived DNA fragment described in 9, 90, 91, 155 or 156 is incorporated so that the polypeptide encoded by the DNA fragment is expressed, or the polypeptide containing the polypeptide is contained by the DNA containing the DNA. An expression vector in which the polypeptide has been incorporated so as to be expressed. 213. A method for producing the polypeptide by culturing a host cell transformed with the expression vector according to claim 212. 214. A host cell transformed with the expression vector of claim 212. 215. The method of claim 92, which is downstream of the promoter.
93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 117, 118, 171 or 184
DNA fragment derived from hepatitis C virus according to
An expression vector in which the polypeptide encoded by the fragment is incorporated so as to be expressed, or a DNA containing the DNA is incorporated so that the polypeptide containing the polypeptide is expressed. 216. A method for producing a polypeptide by culturing a host cell transformed with the expression vector according to claim 215. 217. A host cell transformed with the expression vector of claim 215. 218. The method according to claim 18, which is downstream of the promoter.
5, 186, 188, 189, 191, 193, 194
Alternatively, the hepatitis C virus-derived DNA fragment described in 196 is incorporated so that the polypeptide encoded by the DNA fragment is expressed, or D containing the DNA is incorporated.
An expression vector incorporated so that a polypeptide containing the polypeptide is expressed by NA. 219. A method for culturing a host cell transformed with the expression vector according to claim 218 to produce the polypeptide. 220. A host cell transformed with the expression vector of claim 218. 221. SEQ ID NOS: 1, 9, 10, 1 in the sequence listing
A single-stranded DNA fragment consisting of at least 15 bases selected from the 317 nucleotides from the 1st to the 317th in the nucleotide sequence of 1 or 12, or an antisense DNA fragment thereof. 222. The DNA fragment according to claim 221, which has a length of 16 to 30 bases. 223. The DNA fragment according to claim 221, which has a length of 17 to 23 bases.