JPH06500925A - Non-A, non-B hepatitis virus antigens, diagnostic methods and vaccines - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Non-A, non-B hepatitis virus antigen, diagnostic methods, and vaccine technology field The present invention provides deoxyribonucleic acid (DNA) encoding a non-A, non-B hepatitis structural protein. and recombinant DNA (rDAN) containing the DNA fragment. Main departure cells transformed with Ming rDNA and producing NANBV structural proteins. We will also plan ways to This invention provides NANBV structures useful for diagnostic methods and vaccines. Compositions containing the proteinogenic proteins are also described.

Background of the invention Non-A, non-B hepatitis (NANBH) is caused by hepatitis C virus (HCV) and non-A, non-B hepatitis. It is caused by a contagious virus both referred to as hepatitis virus (NANBV). It is believed that it will occur. Although this contagious disease was discovered many years ago, First, a complete characterization of the causative factors is still being developed.

Isolates of NANBV have already been obtained, and parts of the viral genome of various isolates or all molecularly cloned and sequenced. Chu et al., 5cienc E, 244:359-362 (1989), Chu et al., Proc, Natl.

Acad, Sci, USA, 88:2451-2455 (1991), Takami Zawa et al., J. Virol, 65:1105-1113 (1991), Kado et al. , Proc, Natl, Acad, Sci, USA, 87: 9524-952 8 (1990) and Takeuchi et al. Nucl, Ac1ds Res, 18:462 6 (1990). Nucleotide sequence similarity between different isolates of NANBV The nature suggests that they are part of a related virus family. talent moto et al., Japan J, Exp, Med, 60+183-177 (1990) and Ogata et al. Proc, Nat 1. Acad, Scf, USA, 88: 3 392-3396 (1991). The nature of the NANBV genome is that NANBV is This suggests that it may be a very distant relative of the Raviwisis lineage. however , similarities in both structural protein size and hydrotherapeutic properties suggest that the NANB virus This suggests that it may be distantly related to the Vestiuisis phylogenetic group. mirror -ra, Proc, Natl, Acad, Sci, 87:2057-20 61 (1990) and Saiki Moto et al., Japan J, Exp, Med, 60 :163-177 (1990).

NANBV isolates are difficult to characterize taxonomically, and the NANBV genome Due to the lack of information regarding the protein encoded by and to identify related gene products useful in the production of NANBV vaccines. It has become difficult.

The NANBV genome consists of positive lRNA molecules encoding a single polyprotein. Ru. The gene products of NANBV are homologous to related viruses that have been characterized. Based on their nature, they are thought to include both structural and nonstructural proteins. these phases Because of its homogeneity, NANBV produces a single polyprotein gene product from the complete viral genome. expression, which is then cleaved into functionally distinct structural and nonstructural proteins. It is expected that This form of viral morphogenesis allows individual mature viral proteins to Active identification of these qualities is precluded until they are physically isolated and characterized. Ru. An in vitro culture system for propagating the virus has not been developed for NANBV. NANBV structure or non-structural gene products (proteins) are not known in biology. It has not yet been isolated from clinical specimens or NANBV-infected cells. Therefore, NANB Identification of the V protein, its role in the viral life cycle, and its role in disease. The role of this organization remains to be determined. Especially regarding NANBV-induced diseases. The antigenic markers of the present invention remain to be fully characterized.

One NANBV gene product, nonstructural named NS3 and NS4 Antigen C100-3 derived from a part of the gene is expressed as a fusion protein, Detecting anti-C-100-3 antibodies in patients with various forms of NANB hepatitis is used for. For example, Risla, 5science, 244:362-3 64 (1989) and International Application No. PCT/US 88104125. can do. A diagnostic assay based on the C100-3 antigen is It is commercially available from Tyx Inc. (Raritan, NJ). This CI 00-3 test is , represents the current state of the art in detecting NANB V infection. But long et al., an immunoassay based on the C100-3 antigen detects antibodies in serum from chronically infected patients. It has been reported that it is preferentially detected. The seroconversion of C100-3 is Generally occurs 4 to 6 months after the onset of hepatitis, and in some cases NANBV infection is present. However, depending on C100-3, it may not be possible to detect any antibodies. Alta - et al., New Eng, J. Med, 321:l538-39 (1989) , Alter et al., New Eng, J. Med, 321:1494-1500. (1989) and Uniinar et al., Lancet, 335: 1-3 (19 90), 77-Len et al., Lancet, 335: 754-757 (+ 990)l:I;! , C100-3 in patients with autoimmune chronically active hepatitis. False-positive results have been described when antibodies were measured using an immunoassay based on Ru. Using a C100-3 based immunoassay, Gray et al., Lancet, 335 :609-610 (1990) describes the effects of various conditions other than NANBV. False-positive results for serum from patients with similar liver disease have been described.

Can accurately detect early seroconversion after infection, but cannot identify acute NANBV infection. A NANBV immunoassay is not currently available.

The HCV strain HCV is the Donn strain (HCV-1> is an isolate of clinical interest compared to HCV-H is extremely high in patients This is because it grows to the titration value.

Summary of the invention A non-A, non-B hepatitis virus designated Hatsuchi e59 isolate (or HCV-HC59). One Hutch strain (HCV-H) of Rus (NANBV) was propagated via the route in animals. The entire viral genome was cloned and sequenced. "Subgroup" When using this terminology, the present invention refers to blood cells produced by certain strains, such as Hatsuchi strain C59. This refers to a group of NANBV that has been scientifically identified. Previously reported NA , the sequence data are at the nucleotide and amino acid level when compared to the NBV strain. There are differences between the two. You may refer to the sequences of HCV isolates below. However, here, the name of the isolate is shown in parentheses for comparison. pan J, Exp, Med, 60:183-177.1990 (HC-Jl , HC-J4), Takeuchi et al., Nue]eic Ac1ds Res, +824 626°] 990 (HCV-JH), Chu et al., Proc, Nat 1. Acad, Sc i.

USA, 88:2451 2455. 1991 (HCV-IL Kado et al., P r.

c, Nat 1. Acad, Sci, USA, 87: 9524-9528 ．． 1990 (HCV-J), Takamizawa et al., J. Virol, 65:1105 -1113°1991 (HCv-BK), U.S. Patent No. 5 to Takahashi et al. No. 032,511, Ogata et al., Proc, Nat 1. Acad, Sci, US A, 88:3392-3396.1991 (HCV-Hh), and international Application number PCT/US88104125゜ The identified sequences are shown here as encoding the structural proteins of NANBV. Ta. NANBV structural proteins are also diagnostic for antibodies immunoreactive with NANBV structural proteins. particularly useful for use in vaccines that induce neutralizing antibodies against NANBV. are shown here as containing antigenic epitopes of interest. In particular, the NANB of this invention The V antigen is the Hutch c59 isolate NANBV antigen.

Nuclease encoding the amino-terminal polyprotein portion of the structural gene of NANBV Hatsuchi strain. The nucleotide sequence is included in SEQ ID NO: l (SEQ ID No.]).

In comparison with other NANEV isolates, flaviviruses and bestiviruses. Therefore, the nucleotide sequence contained in SEQ ID NO: 1 is the structural protein of NANBV, That is, it is thought to encode capsid and envelope proteins.

The structural antigens described herein include amino acid residue positions 1-120 of SEQ ID NO:l. present in the putative capsid protein, and also at residue position 121 of SEQ ID NO: 1. Existing in the amino-terminal portion of the putative envelope protein contained in ~326 .

Nucleotide and amino acid residue sequences are expressed by starting base or amino acid residue position number. Specify here by the ending base or residue position number. All arrays of this kind are It is understood to include both starting and ending position numbers.

The complete genome sequence of the Hutch c59 isolate has also been determined and described. Therefore, the present invention NANBV heart contained in nucleotide positions 1t1 to 9416 of SEQ ID NO: 46 A DNA fragment encoding the viral genome of a C59 isolate is contemplated.

The present invention also provides NANB comprising a NANBV structural antigen, preferably a capsid antigen. DNA fragments encoding V structural proteins are also contemplated. Particularly suitable capsid antigens are Residue 1 to residue 20, residue 21 to residue 40, residue 2 to residue 40 of SEQ ID NO. contains the amino acid residue sequence represented by residues 1 to 74, and the DNA fragment is preferably Preferably, base position 1 to base position 60 of SEQ ID NO: l. Base position 61 to base position 1 20, by base position 1 to base position 120 or base position 1 to base position ft222 each containing the indicated nucleotide base sequence.

Portions of the Hutch c59 isolate polyprotein, particularly in the vlV region or Vstif region. A polynucleotide sequence consisting of a nucleotide sequence encoding a part of the sequence-specific region of c59. Nucleotides are also contemplated.

Also contemplated are vectors operably linked to the DNA fragments of the invention, preferably It is a recombinant DNA molecule consisting of an expression vector. A preferred recombinant DNA molecule is p BEG-3X-690: e91, pGEX-3X-690: 694, pGE X-3X-693: 691, PC; EX-3X-15: 17, pGEX-3X -15,18, pGEX-2T-15: 17, pGEX-2T-CAP-8, pGEX-2T-CAP-B or pC; EX-2T-CAP-AB.

NANBV structural antigen, preferably capsid antigen, more preferably SEQ ID NO: 1 residue 1 to residue 20, residue 21 to residue 40, residue 2 to residue 40 or residue 1 to residue From the amino acid residue sequences that specify those containing the amino acid residue sequences contained in A NANBV structural protein is contemplated. consisting of the NANBV structural protein of this invention. Fusion proteins are also contemplated.

This invention also shows that although the Hutch c59 isolate of NANBV is immunoreactive, NAN BV isolate HCV-1, HCV-BK, HCV-J, HC-Jl, HC-J4, HCV-JH or )((:Antibody molecules that do not immunoreact with V-Hh, i.e. c Antibodies, including 59-specific antibody molecules, are also contemplated.

Further contemplated are cells transformed with recombinant DNA molecules of this invention. Culture and method for producing NANBV structural protein of the present invention using the culture It is the law.

Also contemplated are compositions comprising NANBV structural proteins. This composition is , preferably substantially free of (a) prokaryotic antigens and (b) other NANBV-related proteins. It is characterized by not containing

It is still further contemplated that a sufficient amount of this invention to perform at least one assay is provided. NANBV structural protein compositions, polypeptides or fusion proteins in separate packages. This is a diagnostic system in the form of a kit that includes the following reagents: Preferably, the diagnostic system The membrane contains the fusion protein immobilized on a solid matrix.

Further contemplated are antibodies against at least one of the NANBV structural antigens described herein. A method of assaying a sample of body fluid for the presence of antibodies to the body, preferably in vitro It is the law. This method involves the preparation of a body fluid sample and the NANBV structural protein, protein of the present invention. By mixing (contacting) with immunological reagents such as repeptides or fusion proteins. It consists of forming an immunoreaction mixture. The immunoreaction mixture contains all the components present. is sufficient for the antibody to react with the mixed immunological reagent to form an immune reaction product. This product is maintained for a period of time of 10 minutes, and upon detection, the anti-NANBV structural protein This shall indicate the presence of quality antibodies. Preferably, when carrying out this method, immunology Target reagents are immobilized on a solid matrix.

The invention also provides methods for assaying body samples for the presence of NANBV polynucleic acids. Methods, preferably in vitro methods, are also contemplated. This method generally involves a) body samples; Aqueous hybridization can be carried out by mixing the polynucleotide of this invention with b) all NANBV polynucleic acids present in the body sample; hybridizes with mixed polynucleotides to generate hybridization aqueous hybridization for a period of time sufficient to form a compound and under hybridization conditions. maintain the hybridization mixture; C) remove any hybridization mixture formed; presence of cation products and thereby the presence of NANBV polynucleic acids in body samples. It consists of detecting.

In other aspects, the invention provides pharmaceutically acceptable carriers and/or diluents. An immunologically predominant amount of the NANBV structural protein of this invention, polypeptide, dispersed in An inoculum (or vaccine) consisting of a protein or a fusion protein is contemplated. This inoculation The substance is substantially free of (a) prokaryotic antigens and (b) other NANBV-related proteins. stomach.

Treat the infection! The method of prophylaxis consists of administering the inoculum of the invention). plan

Brief description of the drawing Figure 1 shows the HCv-Hc59 genome and the HCV-Hc59 genome numbered zero to 39. The locations of 59 cDNA clones are graphically shown. Due to flavivirus The sequence of proteins encoded by the HCVC-encoded genome is shown together with the HCVC-encoded genomic central domain. vinegar. Dengue type 2Ns3 virus and carnation spot virus (CAR) Regions of amino acid homology with Mv) are indicated by shaded and unhatched boxes, respectively. vinegar.

be. Standard polypeptide nomenclature, J. Biol. Chem. 243:355 7-59 (1989), and the omission of amino acid residues is as shown in the following correspondence table. be: Correspondence table C; Iy L-glycine Phe L-phenylalanine Met L-methionine Ala L-alanine Ser L-serine lie L-isoleucine Leu L-leucine Thr　L-threonine Val L-valine Glu L-glutamic acid Glx Gin or Glu Asp L-aspartic acid Asn L-asparagine Asx Asp or Asn Cys L-cystine Xaa unknown or other All amino acid residue sequences typically referred to herein as "residue sequences" The left-to-right direction is defined here as the conventional direction from the amino terminus to the carboxy terminus. It should be noted that this is shown by the formula:

are referred to as antigenic determinants or epitopes.

the combination of base and sugar is a nucleoside .

The nucleoside contains a phosphate group attached to the 3' or 5' position of the pentose If so, this is called a nucleotide. A functionally linked sequence of nucleotides Typically referred to here as a ``base sequence,'' and its left-to-right direction. is the conventional direction from the 5' end to the 3' end.

A double-stranded nucleic acid molecule consisting of substantially complementary polynucleotides of strands. shape base pairs nucleotide bases or deoxyribonucleotide salts Therefore, the description E duplex DNAJ refers to the combination of two DNA*( DNA-DNA duplex consisting of (dsDNA) or one DNA and Refers to an RNA-DNA duplex consisting of one RNA strand.

Demon i material (bp): adenine (A) and thymine (T) in double-stranded DNA molecules, or Cytosine (C) and guanine (G) pairing. In RNA, uracil (U) is thymine water. DNA or RNA that hybridizes specifically (non-randomly) through elementary bonds The sequence of nucleotides in a single-stranded molecule.

Hybridization: The formation of hydrogen bonds between two or more complementary base pairs. Forms duplexes, heteroduplexes or complexes containing double-stranded nucleic acids Pairing of complementary nucleotide sequences (nucleic acid #I). This is competitively inhibited. specific, i.e. non-random, interactions between complementary polynucleotides Ru.

Hybridization product: polynucleotide into single-stranded or double-stranded nucleic acid A product formed during hybridization. A polynucleotide is attached to a double-stranded nucleic acid. When hybridizing, the hybridization products formed are triple molecule or double-stranded nucleic acid molecule. Moser et al., 5science.

238:645-50 (1987).

Nucleotide analog: structurally different from A, 'r, c, C or U , a purine or a protein similar enough to replace a normal nucleotide in a nucleic acid molecule. pyrimidine nucleotide. inosine CI) other nucleotides, A, TSGS A nucleotide that can hydrogen bond with either C or U. In addition, nucleic acid hives Methylated bases that can participate in redization are known.

Deoxyribonucleic acid (DNA) sequences of structural genes that encode proteins through codes directly related to columns. Therefore, a structural gene is the amino acid residue sequence it encodes, Once upon a time, many different nucleotide sequences encoded a specific sequence of amino acid residues. obtain. Nucleotide sequences of this type are considered functionally equivalent. child This is because they can result in the production of the same amino acid residue sequence in all organisms.

In some cases, methylated variants of purines or pyrimidines are given may be incorporated into the nucleotide sequence. However, what kind of methylation is this? It does not affect the chord relationship even in the format shown below.

In one aspect, the present invention provides a method for producing antigens such as capsid antigens, envelope antigens, or both. NANBV consisting of NANBV structural antigen? Nucleo encoding the jl-building protein An isolated DNA fragment consisting of a nucleotide sequence is contemplated. Preferably, structural antigens is immunologically related to the Hutch strain of NANBV.

More preferably, the encoded NA, NBV structural antigen, is contained in SEQ ID NO: 1. The amino acid residues correspond to, preferably have the same amino acid residue sequence.

In one embodiment, the putative capsid antigen comprises residues 1 to 20 of SEQ ID NO: , contained in residues 21 to 40, residues 2 to 40, or residues 1 to 74 Contains amino acid residue sequences. In other embodiments, the capsid antigen is the remainder of SEQ ID NO:-1. Also containing the amino acid residue sequence contained in group 17G or residue 121=-residue 326 205 to base position 360, base position 1361 to base position 528 or base position It is about 1,000 bases or less.

A particularly preferred amino acid residue sequence of the NANBV structural protein is residue 1 of SEQ ID NO:2. ~residue 315, residue 1 to residue 252 of SEQ ID NO: 23, residue 1 to residue of SEQ ID NO: 4 It is included in group 252 and residues 1 to 271 of SEQ ID NO: 6.

Purified DNA fragments of this invention are specified herein for DNA fragments of this invention. DNA fragments containing substantially no other nucleic acids that do not contain the same nucleotide base sequence. in the form of compositions containing purified DNA fragments, or as suspensions in solutions or with specific treatments. It doesn't matter whether it exists as a physical object or not. "Substantially free" means that DNA fragments are At least 10% of the total nucleic acids present, preferably 5% of the total nucleic acids present by weight. It shall mean the presence of more than 0%, more preferably more than 90%. .

In other embodiments, the DNA fragments of the invention contain structural genes capable of expressing the fusion protein. Contains a specific nucleotide base sequence. “The description fusion protein J is hereby disclosed. A peptide in the second polypeptide portion that specifies the NANBV structural antigen as shown. Refers to a protein that has polypeptide moieties operably linked by a bond.

A preferred first polypeptide portion comprises from about residue 1 to about residue 221 of SEQ ID NO:2. The amino acid residue sequence corresponding to the protein glutathione-s-tra derived from sferase (GST).

A preferred second polypeptide portion that specifies the NANBV structural antigen in the fusion protein is , residues 1 to 20 of SEQ ID NO: I. residue 21 to residue 4o% residue 2 to residue 4o, Residue l-residue 74, residue 69-residue 120S residue 121-residue 176 or residue Contains the amino acid residue sequence represented by the sequence contained in residues 121 to 326 It is.

In one embodiment, the fusion protein comprises, for example, residue l to residue 120 or is shown by the amino acid residue sequence contained in residue 1 to residue 326 of SEQ ID NO. NAN as a combination of two polypeptide moieties representing different structural antigens such as It may include one or more polypeptide portions that specify a BV411 antigen.

In particularly preferred embodiments, a polypeptide moiety that specifies the NANBV capsid antigen is co-coated with The portion of the fusion protein encoding the DNA fragment of this invention is shown in SEQ ID NO: 1 residue l - residue 20, residue 21 to residue 40, residue 2 to residue 1 to 40 or residue Nucleus corresponding to a sequence encoding an amino acid residue sequence such as that contained in l residue 74 Retide base sequence, more preferably base 1 to base 60 of SEQ ID NO: 1, salt Contained in group 61 to base 120, base 4 to base +20 or base 1 to base 222 nucleotide base sequences corresponding to such base sequences.

In other embodiments, a polypeptide moiety that specifies a NANBV envelope antigen is encoded. The part of the fusion protein encoding the DNA fragment of this invention is shown in SEQ ID NO: 1, residues 121 to 176 or residues 121 to 326 of comprising a nucleotide base sequence corresponding to a sequence encoding a amino acid residue sequence; Preferably base 361 to base 528 or base 361 to base 97 of SEQ ID NO: 1 A nucleotide salt corresponding in base sequence to the nucleotide base sequence contained in 8. Each contains a base sequence.

A particularly preferred fusion protein encoding the DNA fragment of this invention is the salt of SEQ ID NO: 2. Group 1 to base 945, base 1 to base 756 of SEQ ID NO: 3, base 1 of SEQ ID NO: 4 ~ Corresponds to salts 1 & 756 and sequences included in bases 1 to 813 of SEQ ID NO. 6 It has a nucleotide base sequence that

In a preferred embodiment, a DNA fragment of the invention binds to a complementary DNA fragment, thereby Form a double-stranded DNA fragment. Moreover, the two 11 DNA fragments of this invention are preferably It should be borne in mind that a single-stranded attachment tail may have a single-stranded attachment tail on one or both of its ends. Ru.

In other embodiments, the DNA fragments of the invention co-copy the genome of a Hutch isolate of NANBV. It consists of a nucleotide base sequence coded for. Preferably, the DNA fragment is The amino acid residue sequence encoding the polyprotein produced by the genome of 59 isolates The amino acid residue sequence is the nucleotide base sequence of SEQ ID NO: 46. 1 to residue 3011. More preferably, the DNA fragment of this embodiment has the sequence It grows the nucleotide sequence shown from base 1 to base 9416 of number 46. be.

The DNA fragment encoding the c59 monoll+iI genome can be obtained using polynucleotides. Hybridization in diagnostic methods based on nucleic acid hybridization NANBV structural antigen or For the preparation of the fusion protein, 11 of the infectious NANBVc59 isolate particles were cultured. All of them are listed here.

In other aspects, the invention corresponds to a portion of the NANBV genome or comprises a NANBV structure. Fragments of the DNA fragments of this invention that encode portions of synthetic antigens are contemplated. These breaks Fragments exist in -heavy chain form or are identified in the context of one strand of a double-stranded DNA fragment When a polynucleotide is used, it is referred to herein as a polynucleotide.

Polynucleotides of NANBV structural antigen or Hutch c59 isolate polyprotein When used as encoding regions, polynucleotides are listed here. corresponds to the coding strand of the NANBV genome. NANBV polynucleotide Hybridization probes for hybridization with derivatized nucleic acids Or when used as a primer, the meaning of the strand is as well known for hybridization. may depend on the target sequence to which the reaction is directed.

Accordingly, in one aspect, the present invention provides a method for producing Hatch isolates of NANBV expressed from A nucleotide base that encodes an amino acid residue sequence that corresponds to a part of a polyprotein. A polypeptide consisting of a nucleotide base sequence comprising a sequence is contemplated. Preferably The polypeptide is a c59 isolated polypeptide shown in residues 1 to 3011 of SEQ ID NO: 46. It encodes a sequence that corresponds to a part of the amino acid residue sequence of a substance.

Particularly preferred are unique and thereby amino acid residues or nucleotide bases. Based on sequence differences, Hutch isolates, more preferably c59 isolates, and NANBV This is an area of the Hatch C59 isolate that provides a means of distinguishing it from other isolates. jlt # Regions of the genome of c59 isolates that are useful for distinguishing between nucleotide sequences nucleotides or nucleotides of the isolates to be distinguished, preferably Differences in encoded amino acid residue sequences when compared to amino acid residue sequences It is something that specifies.

A representative comparison to identify differences in Hatch isolate sequences is presented here in the Examples. , especially shown in Table 11.

Thus, in one embodiment, the polynucleotides of this invention include HCV-1, HCV -BK, HCV-J, HC-Jl, HC-J4, HCV-JH and HCV-H When compared with the nucleotide sequence of a NANBVO strain selected from the group consisting of h. polynucleotides with at least one nucleotide base difference in sequence The arrangement of the honey c59 isolate of NANBV as shown in SEQ ID NO: 46 was used to grow A nucleic acid sequence containing a nucleotide sequence encoding an amino acid residue sequence corresponding to a portion of a sequence. Consists of leotide base sequence. Preferably the nucleotide base sequence consists of nucleotides Base sequence V variable region (base 1497 to base 1574 of SEQ ID NO: 46), V of leotide base sequence, variable region (base 1077 to base 116 of SEQ ID NO: 46) 6), nucleotide base sequence V, variable region (SEQ ID NO: 46 base! 707~ Base 1787L and nucleotide base sequence V, variable region (sequence '#46 of the NANBV genome selected from the group consisting of bases 7407 to 7478) Contains sequences that specify portions of the variable region.

The sequence number and the corresponding base of the sequence are included here in parentheses after the reference to the sequence. will be briefly mentioned. For example, base 1 to base 9416 shown in SEQ ID NO:-46 The nucleotide sequence is referred to as r46:1-9416J.

Particularly suitable polynucleotides include: ■ Variable region nucleotide base sequence (46149 7-1574L V, variable region nucleotide base sequence (46:1077-116 6), V, variable region nucleotide base sequence (46:1707-1787), and and V, a group consisting of variable region nucleotide base sequences (46:7407-7478) It has a nucleotide base sequence selected from.

In other embodiments, the polynucleotide comprises residues 391 to 404 of SEQ ID NO: 46. , residues 246 to 256 of SEQ ID NO: 46, residues 461 to 256 of SEQ ID NO: 466 Residue 466, residues 473 to 482 of SEQ ID NO: 46, and SEQ ID NO: 46 The amino acid residue sequence selected from the group consisting of residues 2356 to 2379 of It consists of a nucleotide base sequence containing a nucleotide sequence coded for. Preferably, The nucleotide sequence involved corresponds to the sequence shown in SEQ ID NO: 46. Said The range of amino acid residues shown represents a large amount of sequence divergence when compared to known HCV isolates. corresponds to the part of v, y, , Vl and vj that contains the points and is therefore the most preferred .

For reasons of ease of synthesis and sequence specificity, preferred polynucleotides are of varying lengths. from about 10 to about 200 nucleotides, or the specific length is about 1,000 nucleotides. may depend on the purpose for which it is used.

Polynucleotides used in the present invention in their various aspects include primers, probes, etc. , or nucleic acids.

The term "probe," as used herein, refers to nucleic acid restriction digests from ffl about 8 to 200 nucleotides, whether manufactured or synthetically produced. length of the gene of interest, i.e., a given characteristic present in the target nucleic acid. A polynucleotide that develops a nucleotide base sequence 1 that is substantially complementary to a heterologous nucleic acid sequence Points to Chido.

Polynucleotide probes connect specific nucleic acid sequences present in the gene of interest. It must be long enough to hybridize under hybridizing conditions.

The exact length of the polynucleotide probe depends on the hybridization temperature and It may depend on many factors, including the nucleotide sequence of the probe. For example, target sequence Depending on the complexity of the polynucleotide probe, polynucleotide probes typically contain 15 to 25 or more contains nucleotides, but can contain fewer nucleotides. polynucle As few as 8 nucleotides have been reported as valid for use. It has been tell. Sudgell et al., Proc, Natl, Acad, Sci, USA .

86:6917-21 (+989). Short polynucleotide probes target In order to form sufficiently stable hybrid complexes with 5, lower temperatures are generally required. request.

In a preferred embodiment, the polynucleotide probe is about 200,000 nucleotides in length. less than 30 nucleotides, preferably less than 100 nucleotides, more preferably less than 30 nucleotides Grow less than the size.

Compare by "substantially complementary" and its grammatical equivalents with respect to the probe. The specific nucleic acid sequence present in the intended gene and the polynucleotide probe There is sufficient nucleotide sequence similarity between the probes and the hybridizing condition. A device consisting of a probe and a specific sequence that can hybridize with a specific sequence under the conditions. It means forming a ublex.

Therefore, as long as the probe contains substantial complementarity with the target sequence, The probe sequence may not reflect the exact sequence of the target sequence. For example, non- A complementary polynucleotide can be attached to one end of the probe, and this the remainder of the probe sequence is substantially complementary to the target sequence. This kind of non- Complementary polynucleotides include endonuclease restriction sites or protein binding sites. can code the part of the body. Also, if there are non-complementary bases or long sequences scattered throughout the probe, This means that the probe sequence hybridizes non-randomly with the target sequence. have sufficient complementarity to hybridize under hybridization conditions. to form a hybridization product.

Polynucleotide probes are provided in single-stranded form for maximum efficiency; In addition, it can also be double-stranded. If double-stranded, use for hybridization. The polynucleotide protein is prepared before producing hybridization products using The chain is first processed to separate it into its strands. Preferably the probe is polydeoxy Ribonucleotides.

The DNA fragments or polynucleotides of the invention can be prepared from NANB as described herein. tJR12 can be easily isolated from the isolated virus obtained from the blood of Vg-stained individuals. or can be synthesized de novo by chemical techniques.

De novo chemical synthesis of DNA fragments or polynucleotides, e.g. be carried out using any suitable method such as the phosphodiester method or the phosphodiester method. I can do it. Narang et al., Meth, EnzymoJ, 68:90 (1979) , U.S. Patent No. 4,356,270, Itakura et al., Ann, Rev, Btoch em, 53:323-56 (1989L Brown et al., Meth, Enzymo l.

68:109 (1979) and v-7 Teusi et al., J. Am. Chem. Soc.

103:3185 (1981). (The technique cited here The disclosure of the technique is incorporated herein by reference. ) Of course, the structural gene part is chemically synthesized. Simply identify the appropriate base for those that feed natural amino acid residues by Any desired modification can be made by substituting . however , DN containing the same sequence as the fragment contained in Sequence ID No. 1.2.3.4 or 6 A fragment is preferred.

Derivation of polynucleotides from nucleic acids involves cloning vectors into appropriate hosts. Cloning of nucleic acids, reproduction of vectors and thus amplification of the amount of cloned nucleic acids , and subsequent isolation of subfragments of the cloned nucleic acid. subk For a description of cloned nucleic acid fragments, see Manniais et al., [Molecular Cloning, Experimental "Laboratory Manual", Cold Spring Harbor Laboratory, No. 390~ 401 (1982) and U.S. Patent No. 4,416.98. No. 8 and No. 4,403.036.

In addition, DNA fragments can be prepared by first adding oligonucleotides corresponding to part of the DNA fragment. synthesis, and then the oligonucleotides are assembled by hybridization. It can be prepared by ligating a complete NA fragment. This kind of person The law is also well known in the art. For example, Baderin et al., Ce1l, 48:441-4 52 (1987) and Lindley et al., Proc, Nat 1. Acad, Sc. f, 85:9199-9203 (2988), in which case The recombinant peptide, neutrophil activating factor, is derived from the expression of a chemically synthesized gene. - Produced in Cori.

The DNA fragment of this invention contains the NANBV structural protein or fusion protein of this invention. in the preparation of rDNA molecules during the construction of expression vectors, or in the preparation of NA in samples. As a hybridization probe to detect the presence of NBV-specific nucleic acid sequences can be used.

When the use of DNA fragments is for protein preparation, specific amino acid residues are important. The nucleotide base sequence of a DNA fragment has the desired amino acid residue sequence. As is well known, this can vary based on overlap in the genetic code. It is.

Use of DNA fragments as hybridization probes for specific nucleic acid sequences If so, a suitable one is the Hatsuchi strain NANBV nucleo disclosed herein. This is a nucleotide base sequence corresponding to the nucleotide base sequence.

C1 recombinant DNA molecule The present invention provides a recombinant DNA comprising a DNA fragment of the present invention operably linked to a vector. (rDNA) is further contemplated. Preferred rDNAs of the invention are It is characterized by being able to directly express the NANBV structural protein or fusion protein of this invention. shall be. Preferred NA fragments for use in rDNA are those described herein above.

"Directly expressed" means two or more termini from a larger translated precursor protein. The mature polypeptide of a protein, as opposed to proteolytic cleavage of amino acid residues. This means that the chain is formed solely by translation. Preferred rDNAs of the present invention are Plasmid pGEX-3X-690: 694, pGEX-3 described in Example 1 X-693: 691. pGEX-3X-690+ 691. pGEX-3X- 15・17, pGEX-3X-15: 18, pGEX-2T-15: I 7 , pGEX-2T-CAP-ASpGEX-2T-CAP-B and pGEX- 2T-CAP-A-B.

The recombinant DNA molecule (rDNA) of the present invention can be produced by using the DNA fragment of the present invention as a vector. It can be produced by functionally linking. Exemplary rDNA molecules and and methods of their preparation are described in Example 1.

In other embodiments, the rDNA molecules of the invention are derived from the genome of a Hutch isolate of NANBV. A vector functionally linked to a DNA fragment consisting of a nucleotide base sequence encoding Consists of ta. Preferably, the rDNA is produced by the genome of a Hutch c59 isolate. containing the nucleotide base sequence encoding the amino acid residue sequence of the polyprotein , its amino acid residue sequence is shown in SEQ ID NO: 46, residues 1 to 3011. Furthermore Preferably, the rDNA molecule of this embodiment has bases 1 to 941 of SEQ ID NO: 46. It contains the nucleotide base sequence shown in 6.

As used here, the term "vector" refers to a vector that can self-replicate in a cell. refers to a DNA molecule that can be attached to other DNA fragments by functionally linking them. Replication of the attached fragment can occur. Typical vectors are plasmids, bacteria Theriophage, etc. , directs the expression of NANBV structural proteins or fusion proteins. The vector that can be used is 1, which is referred to as the F expression vector J in Koni. Therefore, recombinant D An NA molecule (rDNA) is made up of at least two molecules that are not normally found together in nature. It is a hybrid DNA molecule consisting of a single sequence.

Selection of a vector to which the DNA fragment of the present invention is functionally linked can be carried out as is well known in the art. desired functional properties, such as protein expression and direct transformation of the host cell to be transformed. These are inherent limitations in the technology of constructing recombinant DNA molecules. . However, vectors contemplated by the present invention may be operably linked to At least the replication or replication of the recombinant or fusion protein structural gene contained in the DNA fragment. Preferably, expression can also be directed.

In preferred embodiments, vectors contemplated by the present invention contain prokaryotic replicons (ori). i.e. prokaryotic host cells, e.g. IB bacteria transformed thereby. the ability to direct the self-replication and maintenance of a recombinant DNA molecule extrachromosomally in a host cell It is a DNA sequence that has power. Replicons of this type are well known in the art. Also , such embodiments that include prokaryotic replicons, typically whose expression is It also contains genes that confer drug resistance to the transformed bacterial host. These bases Typical bacterial drug resistance genes used in Kuta are ampicillin or tetracycline. Provides resistance to Klin. Typical vector plasmids of this type are BioRad pUC8, pUC9, available from Laboratories (Richmond, CA); pBR322 and pBR329.

Such vectors containing prokaryotic replicons can be used to transform E. coli encoding NANBV structural proteins or fusion proteins in bacterial host cells such as It can also include prokaryotic promoters that can direct the expression (transcription and translation) of genes that Wear. A promoter is responsible for the binding of RNA polymerase and subsequent transcriptional opening. The expression WR node element is formed by a DNA sequence that allows the beginning of the expression. Bacterial host and harmony A compatible promoter sequence typically includes convenient restriction sites for insertion of the DNA fragments of the invention. is provided in a plasmid vector containing the sequence. A typical vector is Pharmacia ( pPL-λ, available from Bispower (Tower, NJ).

A vector plasmid with a bacterial promoter that is inducible by IPTG is The pTTQ plasmid, available from Jam (Arlington High, IL), and the pKK223-3 plasmid available from Pharmacia. fusion protein Additional expression vectors for producing cloned gene products in prokaryotes in the form of known and commercially available.

Expression vectors r) GEX-3X and pGEX-2T fusion proteins described herein used in the example to produce the vector, but other functionally equivalent expression vectors could be used. I can do it. Functionally equivalent vectors are suitable for expression of fusion proteins in E. coli. Contains an expression promoter inducible by rPTG, and consists of a DNA fragment into a vector. Insertion 1. It is assumed that a fusion protein is produced at this time. Vector p used here Commercially available vectors that are functionally equivalent to GEX-3X and pGEX-2T include T The fusion between the amino-terminal portion of 7 genes and 10 proteins and the cloned insert gene pGEMEX-1 plasmid vector from Promega (Madison, W+), L plasmid vector, and the enzyme glutathione-8-transferase (GS pGEX- from Pharmacia giving a fusion of T) and the cloned insert gene. 3X and pGEX-27 plasmids, respectively.

Construction and use of pGEX-3X and pGEX-2T vectors are described in Smith et al., G. ene, 67:31-40 (198g), and this reference is incorporated herein by reference.

In particularly preferred embodiments, the fusion protein is functionally compatible with the NANBV structural antigen of this invention. Concatenated added! It contains a GST-inducing polypeptide portion as a functional domain.

All inducible promoter-inducing vectors, such as the vectors pTTQ, pKK2, described above. 23-3, pGEX-3X or 7'=l; NANBV structural protein (referred to here as GST: NANBV fusion protein) ) can be expressed. Therefore, pGEX-3X and pGEX-2T vectors Although vectors are described as examples, the DNA molecules of this invention are not limited to these vectors. should not be construed as This is because one aspect of the invention merges into GST. rDNA-directed proteins for the expression of proteins with NANBV structural antigens This is because it is not derived from the vector itself.

Various methods have been developed to operably link DNA fragments to vectors via complementary cohesive ends. It is being emitted. For example, a complementary homopolymer system can be attached to the DNA fragment to be inserted and It can be added to vector DNA. Then, the vector and DNA fragments are hydrogenated. ligation between complementary homopolymer tails to form a recombinant DNA molecule. Synthetic linkers containing the above restriction sites can be used to connect other methods of joining DNA fragments to vectors. provide. The DNA fragment generated by endonuclease restriction digestion is -Remove the protruding 3' single-stranded end by the 5' engineering nucleic acid degrading activity, and its polymerization activity bacteriophage T4 DNA polypeptide, an enzyme that fills in the 3' end of the merase or E. coli DNA polymerase (■).

The combination of these activities thus produces blunt-ended DNA fragments. the Afterwards, ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase, is performed. in the presence of an enzyme capable of catalyzing the linkage, using a large molar excess of linker molecules. Incubate the slip-end fragments. In this way, the products of the reaction are deposited at their ends. This results in a DNA fragment carrying a polymeric linker sequence. These DNA fragments are then An enzyme that is cleaved by an appropriate restriction enzyme and provides ends that are compatible with those of the DNA fragment. ligate into a pre-cleaved expression vector.

Synthetic linkers containing various restriction endonuclease sites are available from the International It is commercially available from a number of sources including Bioticnoseeds, Inc., New Haven CN. ing.

Also contemplated by the invention are RNA equivalents of the recombinant DNA molecules described above. There are also things.

D. Transformed cells and cultures. The invention also relates to host cells transformed with recombinant DNA molecules of the invention. It is something. The term "host cell" includes both eukaryotic and prokaryotic hosts.

Suitable rDNA molecules for use in transformed cells are those described herein above, and preferred or rDNA capable of expressing a recombinant or fusion protein. transformation Certain preferred embodiments of transduced cells include one of the preferred NA fragments described hereinabove. contains an rDNA molecule that grows the rDNA plasmid pGEX-3X. -690:694, pGEX-3X-693+ 691, pCyEX-3X-6 90゛691, pGEX-3X-15+17, pGEX-3X-15: 1 B , pGEX-2T-15:17, pGEX-2T-CAP-A, pGEX-2T - Cells transformed with CAP-B or pGEX-2T-CAP-AB It is.

Bacterial cells are the preferred prokaryotic host cells, typically strains of E. coli, e.g. E. coli available from Suda Research Laboratories, Inc., Bethesda, MD. Stock shall be DH5. Transformation of a suitable cellular host with a recombinant DNA molecule of the invention , typically by well-known methods depending on the type of vector used. prokaryotic host Regarding cell transformation, see, for example, Cohen et al., Proc, Nat 1. Aca d, SCi, USA, 69:2110 (1972) and Samblecock et al. “Molecular Cloning, Laboratory Manual”, 2nd edition, Cold Spring Ha Harbor Laboratory, Cold Spring Harbor 1NY (+989) The second reference is -C.

Successfully (7) transformed cells, i.e. cells containing a recombinant DNA molecule of the invention. can be identified using well-known techniques (for example, introduction of the rDNA of the present invention). Cells originating from can be isolated as a single colony. A roller like this Cells from the knee were collected, lysed, and analyzed by Southern, J., Mo. 1. 8 io 1゜ 98:503 (+975) or Berendt et al., Biotech, 3:208 (1985) for the presence of rDNA. DNA content can also be tested.

In addition to directly assaying for the presence of rDNA, If protein expression can be directed, cells transformed with the appropriate rDNA can be circumscribed. It can be identified by known immunological methods. For example 1. Expression vector of the second invention Cells that have been successfully transformed by this vector contain proteins that exhibit NANBV structural protein antigenicity Produces white matter. Collect a sample of cells expected to have been transformed, and using antibodies specific for the genus I2 (such antibodies are further described herein). Assay for the presence of ANBV structural antigen.

Thus, in addition to the transformed host cells themselves, the present invention provides for the transformation of these cells in nutrient medium. cultures, preferably monoclonal (clonally identical P1) cultures, or Cultures derived from noclonal cultures are also contemplated. Preferably, this culture NANBV structural proteins also contain proteins that exhibit antigenicity.

Nutrient media useful for culturing transformed host cells are well known in the art, and several can be obtained from commercial sources.

E. Method for producing NANBV structural proteins, polypeptides and fusion proteins of the present invention Other aspects relate to methods of producing recombinant proteins and fusion proteins of the invention. .

The method includes the use of the present invention, which is capable of expressing NANBV structural proteins or fusion proteins. A host cell, preferably an E. coli cell, transformed with a recombinant DNA molecule of It involves starting a culture consisting of a nutrient medium containing cells. The transformed cells are N Culture for a period of time sufficient to express the ANBV structural protein or fusion protein. maintain. The expressed protein is then recovered from the culture.

Expression vectors and expression vector culture conditions for producing NANBV structural proteins are generally Well known in the art. These types of vectors and culture conditions do not affect the spirit of the invention. Can be changed without giving. However, preferred is NANBV Proteins not normally found in the host cells used to express structural proteins is a vector specifically designed for the production of Examples include NANBV structural proteins. There are vectors that contain an inducible promoter that directs the expression of the encoding DNA fragment.

Vectors with promoters inducible by IPTG are also well known. For example, it Plasmids pTTQ and p, available from Pharmacia and Amajam, respectively. Reference may be made to KK223-3. Particularly suitable are the pGs described here. EX vector, I) by IPTG present in GEX-3X and pGEX-2T. It is an inducible promoter.

A vector containing an inducible promoter is used to express NANBV structural proteins. , as known in the art, and as detailed in Example 2. A lag period is required at the beginning of the maintenance process.

Methods for recovering expressed proteins from cultures are well known in the art and include well-known biochemical techniques. involves fractionating the protein-containing portion using chemical techniques. For example, protein fraction Gel filtration, gel chromatography, ultrafiltration, electrophoresis as known for Using methods such as chromatography, ion exchange, and affinity chromatography, The expressed protein can be isolated. Also, using well-known methods Immunological methods such as , immunoaffinity, immunoadsorption, etc. can be performed.

Particularly suitable is the device as a means of separating fusion proteins from complex mixtures of proteins. Lutathione-3f, a polypeptide that specifies transferase (C; ST) This is an isolation method that takes advantage of the presence of the moiety. against a solid phase containing immobilized glutathione. Affinity adsorption of GST-containing fusion proteins has been described by Smith et al., Gene, 67:31 ( +988). In addition, the GS of the fusion protein T-containing polypeptide portions are described in Smith et al., Gene, 67:31 (1988). The method allows selective cleavage of the fusion protein at specific proteolytic cleavage sites. can be separated from the NANBV structural antigen. Example of an exemplary isolation method 5 and 6.

In addition to its preparation through the use of rDNA expression vectors, it consists of NANBV structural antigens. NANBV structural genes can be prepared in the form of synthetic polypeptides.

Polypeptides are synthesized by any technique known to those skilled in the polypeptide art. I can do it. Synthetic chemistry techniques, such as phase-based Merrifield-type synthesis, can improve purity, antigen It is preferred for reasons such as specificity, absence of undesired by-products, and ease of production. Merrifield et al. J. Am. Chem. Soc. 85:2149-21 54 (1963) and Houten et al., Int. J. Pept. Prot. Res. 16:311-320 (1980). Wear. An excellent summary of the many techniques available is provided by J.M. Steward and Noe. Lee D. Young, “Solid Phase Peptide Synthesis”, WHC Freeman, Inc. San Francisco, 1969, M. Bodansky et al., “Peptide Synthesis”, Jo. N. Riley and Lance, 2nd edition, 1976, and Solid Phase Peptide Synthesis. Jay Meyenhofer, “Hormonal Proteins and Peptides,” No. 2 Vol. 46, Academic Press (NY), 1983, and classical solutions. On Synthesis E. Schroeder and K. Kubke, "Peptides", 11k, Aca. Demik Press (New York), 1965, and these Each is incorporated herein by reference.

Suitable protecting groups that can be used for this type of synthesis are described in the above text and in G.F. ・W. McComiy, Protecting Groups in Organic Chemistry J, Blenheim Press New York, 1973, which is incorporated herein by reference.

The polypeptide of interest includes all of the polypeptides whose amino acid residue sequences are shown here. Contains chemical derivatives. Therefore, this polypeptide may be subjected to various modifications. and in this case, this type of modification confers a certain advantage in its use. Ru.

"Chemical derivative" means one or more chemically derived derivatives by reaction of functional side groups. Refers to a polypeptide of interest that contains residues. This type of derivatized molecules includes, for example, free radicals. A separate amino group is induced to form amine hydrochloride, p-toluenesulfonic acid group, Carbobenzoxy group, t-butyrobutyrodicarbonyl group, chloroacetyl group or Included are molecules that form lumyl groups. Inducing free carboxyl groups salts, methyl and ethyl esters or other types of esters or hydrazides can be formed. Free hydroxyl group is induced to form O-acyl or O-alkyl Derivatives can be formed. By inducing the imidazole nitrogen of histidine, N- lm-benzylhistidine can be formed. Also included as chemical derivatives. Included are naturally occurring amino acid derivatives of one or more of the 20 Enoki amino acids There are peptides that contain For example, 4-hydroxyproline replaces proline. 5-hydroxylysine can replace lysine, 3-hydroxylysine can replace lysine, and 5-hydroxylysine can replace lysine. Methylhistidine can replace histidine, and homoserine can replace serine. and ornithine can replace lysine. Also from the main The sequence of the polypeptide shown herein may be modified as long as the desired activity is maintained. It also encompasses all polypeptides that develop one or more additions to the peptide sequence. With Additional residues can label the polypeptides of this invention or attach them to a solid matrix or For the purpose of providing a "linker" that can be conveniently fixed to a carrier, It can also be added to either end. Preferably, the linker residue is a NANBV structure. Does not form antigens.

Labels, solid matrices that can be used with polypeptides of this invention and the carrier will be described later here.

Amino acid residue linkers usually have at least one residue, and can include 40 or more residues, and even Forms a NANBV epitope, although it can often be 1-10 residues shall not be done. Typical amino acid residues used for linking are tyrosine, These include sostein, lysine, glutamic acid and aspartic acid. Also, target - Polypeptides, unless specified otherwise, have a terminal NH2 acyl (!Z, e.g. By tylation or thioglycolic acid dimidation, e.g. The sequence modified by terminal carboxylation using min et al. may differ from the natural sequence of the V polyprotein.

It is conjugated with a carrier to create what is known in the industry as a carrier hub ten bond. When formed, the polypeptides of the invention induce antibodies that are immunoreactive with NANBV. can do. Therefore, due to the well-established principles of immunological cross-reactivity In light of this, the present invention contemplates antigenically related variants of the polypeptides described herein. do. "Antigenically related variants" refer to polypeptides of the invention and NANBV A polypeptide of interest that can induce antibody molecules to immunoreact with the polypeptide.

All peptides of the invention can be used in the form of pharmaceutically acceptable salts. Ru. Suitable acids capable of forming salts with the peptides of the invention include inorganic acids, e.g. Hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, propylene acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, Leic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfosic acid, sulfuric acid anilic acid and the like.

Suitable bases capable of forming salts with the peptides of the invention include inorganic bases, e.g. sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc., and organic bases , such as mono-, di- and tri-alkyls and arylamines (e.g. tri- and tri-alkyls) ethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and and as required (correspondingly substituted ethanolamines (e.g. ethanolamine, :) ethanolamine, etc.).

Generally, the solid-phase synthesis method contemplated will provide a growing peptide chain with one or more Amino acid residues or appropriately protected amino acid residues are added sequentially. : consists of and 1° Usually the amino or carboxyl group of the first amino acid residue is protected by a protective group that can be removed automatically. containing reactive side groups such as lysine. For amino acids, different selectively removable protecting groups are utilized.

For example, solid phase synthesis can be used to synthesize protected or derivatized amino acids. It is attached to an inert solid support through no carboxyl or amino group.

The amino or carboxyl protecting group is then selectively removed and properly protected. The next amino acid in the sequence with the complementary (amino or carboxyl) group suitable for mixing acids and forming amide bonds with residues already attached to the solid support. react under appropriate conditions. The amino or carboxyl protecting group is then added to this new removed from the first added amino acid residue, and then added to the next (appropriately protected) amino acid residue. Add and do the same from here on. After joining all desired amino acids into the appropriate sequence and tm , remove all remaining terminal and side group protecting groups (and solid support) sequentially or identically. to give the final polypeptide.

F. In the NANBV structural protein and fusion protein composition aspect, the present invention provides Preferably a single amino acid residue sequence that specifies the NANBV structural antigen of the invention. Compositions containing separated NANBV structural proteins are contemplated.

"Isolated" means that the NANB"V structural protein of this invention is a major protein component. typically in an amount greater than 10% of the total protein in the composition, but preferably means present in the composition in an amount greater than 90% of the total protein in the composition. Ru.

As used herein, NANBV structural antigen is encoded by the NANBV genome. are structural proteins that are specifically immunized with antibodies, such as those specified herein. It consists of the properties of an antigen that can be reacted with. NANBVllI protein is capsi NANB The V structure was partially characterized as containing an antigenic capsid and envelope.

The NANBV capsid as described here is derived from the putative Hatsuchi strain NANBV capsid. It consists of a sequence of amino acid residues that are immunologically related to the psid antigen. , whose sequence includes residues 1 to 120 of SEQ ID NO: 1.

As described herein, the NΔNBV envelope antigen is derived from the putative Hatsumi strain N. Amino acid residues that are immunologically related in sequence to the ANBV envelope antigen. The base sequence consists of a portion of the sequence contained in residues 121 to 326 of SEQ ID NO: l. Go around.

“Immunologically related” refers to antibodies specific for one protein or antibodies that are immunologically related to other proteins. Compare the reactions (cross-reactions) in the amino acid sequences of two protein sequences. This means that there is sufficient homology to Immunological cross-reactivity described here It can be measured by well-known methods including immunoassay methods.

As used herein, the term "recombinant protein" refers to at least is also 20 amino acid residues, preferably at least 50 residues, and has SEQ ID NO. Preferably identical amino acid residues corresponding to the portion of the NANBV structural protein contained Refers to a protein that contains a base sequence.

In a preferred embodiment, the NANBV structural protein comprises residue l-residue 20 of SEQ ID NO:1; Sequences contained in residues 21 to 40, residues 2 to 40, or residues 1 to 74 preferably contains the same amino acid residue sequence that is immunologically related to, and preferably identical to, the amino acid residue sequence. The array shown NANBV structural proteins having a suitable. This is because the capsid antigen contained in this is acute NANBV. This is because it has been shown to be particularly useful for detecting infection. Related NANBV structure The constructed protein has SEQ ID NO: 1, residue 1 to residue 120, residue 1 to residue 176, and [Group] ~ Contains the sequence contained in residue 326. Examples include residues l to residues of SEQ ID NO:2. 315, residue 1 of SEQ ID NO: 3 to residue 252, residue 1 to residue 25 of SEQ ID NO: 4 2 or as described herein having the sequence contained in residues 1 to 271 of SEQ ID NO: 6. There is a protein that

In other embodiments, the NANBV structural protein comprises residues 69 to 120 of SEQ ID NO:l. Immunologically (!I), preferably identical amino acid residue sequences to the sequences contained in including. Exemplary NANBV structural proteins include rDNA plasmid pGEX-3X -693:691.

immunologically related to the sequence contained in residues 121 to 176 of SEQ ID NO: l; Preferably a further comprising a NANBV envelope antigen comprising the same amino acid residue sequence. A NANBV structural protein is contemplated. Examples include rDNADNA plasmid -3X-15:17, pGEX-3X-15:18 and pGEX-2T-1 5:17. Ru.

In other embodiments, a NAN consisting of an amino acid residue sequence according to a polypeptide of the invention BV structural proteins are contemplated.

In preferred embodiments, the NANBV structural proteins are prokaryotic antigens (i.e., host cell-specific antigen) and other NANBV-related proteins. [Substantial J not included in NANBV structural antigen structural antigen versus external antigen prokaryotic antigen or other of NANBV-related proteins is at least 10:1, preferably 100+1°. This means that the ratio is preferably 200:1.

The presence and amount of contaminant proteins in NANBV structural protein preparations was determined by well-known methods. I can decide. Preferably, a sample of the composition is prepared using sodium dodecyl sulfate. The entire amount present was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). NANBVm-forming proteins are separated from protein contaminants. then present in the sample. The ratio of the amount of protein present is determined using a 1:11 degree soft laser scan, as is well known in the art. Determined by. Guylian et al., Anal, Biochem, 129:277- 287 (1983).

The NANBV structural protein can be prepared using the methods disclosed herein for producing the NANBV structural protein. more preferably prokaryotic antigen or NANBV nonstructural antigen isolated by TN418 as a protein that does not contain qualitatively! can do. Especially suitable is , a method that depends on the properties of the polypeptide region of the fusion protein; present in the fusion protein to facilitate separation of the fusion protein from host cell proteins based on do.

An example is a GST-containing synthetic protein whose amino acid sequence is SEQ ID NO: 2.3.4. or 6, in which case each GST polypeptide region functionalized drug with affinity to bind to the usual substrate for glutathione, i.e. glutathione. A fusion protein with a main is provided. Fusions involving GST polypeptide regions Protein purification is further described herein.

In a related aspect, the invention describes polypeptides that specify the NANBV antigen. It is something that Therefore, the present invention provides a method for use in serological assays as described herein. or useful as a NANBV antigen in inocula that induce anti-NANBV antiserum. A polyprotein corresponding to a region of the NANBV polyprotein that specifies antigenic determinants of the virus. A repeptide is contemplated.

The polypeptides of this invention have a length of about 7 to about 200 residues, preferably about 7 to about 200 residues in length. The polynucleotide of this invention consists of a sequence of about 20 to 150 amino acid residues. It consists of the amino acid residue sequence specified by the nucleotide sequence of leotide.

Suitable polypeptides include residues 391 to 404 of SEQ ID NO: q:46; 46 residues 246 to 256, SEQ ID NO: 46 residues 461 to 46G, the sequence Residues 473 to 482 of sequence number 46 and residues 2356 to 46 of sequence number 46 Amino acid comprising an amino acid residue sequence selected from the group of sequences consisting of the remaining 12379 It consists of a residue sequence. In a particularly preferred ti-like manner, the polypeptide has SEQ ID NO: 46. Upolybebutide having an amino acid residue sequence corresponding to the sequence shown was isolated by Hatsuchi. As long as it is useful for distinguishing between about 7 and about 200 amino acid residues, The configuration of the Hajj c59 isolate of NANBV having the length of Volibeb f t”tJ’ consisting of the amino acid residue sequence corresponding to the column part: Figure 1m At 6°: HCV-L HCV-BKSHC'V-J, HC-Jl, H C-J4.1 (NAN selected from the null group from CV-JH and HCV-Hh) When compared to the amino acid residue sequences of isolates of BV, the polypeptides differ in sequence. There is a difference in at least one amino acid residue.

preferably as determined by a standard serological immunoassay as described herein; The polypeptide is immunoreactive with anti-Hutch strain HANBV antiserum.

Further preferably, the polypeptide is a NANBV virus as specified herein. Difference in at least one amino acid residue in the variable region of the genome-encoded polyprotein Including differences. For example, ■ variable region amino acid residue sequence (residue 386 of SEQ ID NO: 46) ~residue 411), V, variable region amino acid residue sequence (residue 246 of SEQ ID NO: 46) ~residue 275), V, variable region amino acid residue sequence (residue 456 of SEQ ID NO: 46) ~ residues, H12), and V, variable region amino acid residue sequence (residues of SEQ ID NO: 46). an amino acid residue sequence selected from the group of sequences consisting of groups 2356--residues 2379); It is a column.

In other aspects, compositions comprising isolated fusion proteins are also contemplated by the invention, It is functionally attached to other polypeptides at one or both ends by peptide bonds. It consists of linked NANBV structural proteins of this invention. The added polypeptide is , all polypeptides designed to increase the number of functional domains present on the fusion protein. It can be called petit de. The added functional domains contain additional antigenic epitopes. imparts a tope, adds mass to the fusion protein, and changes the solubility of the fusion protein. It is included to provide a means of isolation based on the affinity of the fusion protein. Ru.

Examples of functional domains added include vectoring the fusion protein of interest as described here. given when produced in pGEX-3X or pGEX-2T, respectively. It is a thrombin or factor Xa specific cleavage site. Further exemplary domains are: using affinity chromatography on a solid phase containing immobilized glutathione. This is a GST-induced protein domain that allows for rapid isolation.

In one embodiment, thrombin or factor Xa cleavage site-containing domains are used herein. and enables the production of NANBV structural proteins that do not contain the GST functional domain. . An example is the amino acid residue sequence contained in residues 226 to 315 of SEQ ID NO: 2. This is the protein produced in Example 6. The factor XaM cleft site-containing domain also From the New England Biolapse described here (~(-Lee, MA) It is used in the commercially available fusion protein expression vector pMAL.

In a related aspect, proteins produced using the pGEX-2T vector, e.g. Residues 225 to 252 of SEQ ID NO: 3, residues 225 to 25 of SEQ ID NO: 4 2 or the amino acid residue sequence contained in residues 225 to 271 of SEQ ID NO: 6. The NANBV structural protein is produced by thrombin cleavage of the protein.

The fusion protein of the present invention comprises residues 1 to 20 and residues 21 to 40 of SEQ ID NO: 1. , residue 2 to residue 40, residue 1 to residue 74, residue 69 to residue 120, residue 121 to The amino acid residue sequence contained in residue 176 or residues 121 to 326 contains amino acids. Contains the corresponding amino acid residue sequence from the terminus to the carboxy terminus. Suitable fusion proteins The quality is residue 1 to residue 315 of SEQ ID NO: 2, and residue 1 to residue 252 of SEQ ID NO: 3. , residue 1 to residue 252 of SEQ ID NO: 4 or residue 1 to residue 271 of SEQ ID NO: 6 The amino acid residue sequence corresponds to, and more preferably has the same amino acid residue sequence in.

Other suitable fusion proteins include the rDNA plasmid pGET-3X-690:69 4, pGEX-3X-690:69L pGEX-3X-693:691, pG EX-3X-15: 17, pGEX-3X-15: l 8, pGEX-2T- + 5:17, pGEX-2T-CAP-AlpGEX-2T-CAP-B and amino acids of the expressed protein coding sequence present in pGEX-2T-CAP-A-B. identified by the amino acid residue sequence.

As used herein, the term "fusion protein" refers to the NANBV structure of this invention. Refers to an isolated protein as defined by the protein.

The isolated fusion protein thus preferably contains more than 10% of the total protein in the composition. or a composition having a fusion protein of this invention in an amount greater than 90% of the total protein in the composition. It is a product.

Preferred fusion proteins are heterologous fusion proteins, i.e. , proteins derived from pathogens or animals, i.e. non-NANBV proteins. It is a fusion protein containing a polypeptide portion. Preferably a heterologous fusion protein are non-NANBV polypeptides that are not immunologically related to the NANBV structural antigens of this invention. Contains petite portion.

In one embodiment, the fusion protein is an immunogenic antigen other than the NANBV structural antigen specified herein. Contains a functional domain that confers a pathogenic or antigenic epitope, preferably associated with another disease. Derived from original sources or from some pathogens. Functional domains are immunogenic Yes, in which case the domain is present in both the NANBV structural protein and the second pathogen. to form a multivalent vaccine or immunogen for the purpose of inducing antibodies immunoreactive with exist. A functional domain is antigenic, in which case the domain has at least Multivalent Antigens for Use in Diagnostic Systems and Methods to Detect Antibodies from Two Species It exists to form.

This t! ! Of particular interest are BW hepatitis virus and hepatitis A virus. designed to contain functional domains derived from other hepatitis-causing viruses such as It is a fusion protein. These viruses are used in the fusion proteins of this invention. fully characterized as containing appropriate antigenic and immunogenic determinants for The advantages of versatile biochemical reagents in both diagnostic and vaccine applications It gives Furthermore, the functional domains encompassed may be related to other pathogens, preferably In particular, amino acids derived from substances such as HTV that also infect individuals with NANBV hepatitis. and amino acid sequences.

A preferred NANBV structural protein or fusion protein comprising the NANBV structural antifreeze of the present invention. White matter is in a non-reduced form, i.e. sulfur, due to intramolecular Cys-Cys bonds. Contains substantially no hydryl groups.

In preferred compositions, the NANBV structural proteins or fusion proteins described herein are: or in isotonic preparations, such as sterile suspensions or solutions, or with suitable preservatives. present in liquid compositions such as

One such composition useful for inducing anti-NANBV structural protein antibodies in animals. One is referred to as a vaccine, and the NANBV structural protein or fusion protein of this invention It includes quality.

The candy "vaccine", in its various grammatical forms, refers to NANBV and immunity. In order to induce the production of antibodies in a responsive mammal, preferably NANBV1: A pharmaceutically acceptable excipient used to induce active immunity in a host mammal to Containing one or more NANBV structural antigens of this invention as an active ingredient in a formulation It is used here to describe the type of inoculum.

The inoculum comprises an immunogenic component, an immunologically effective amount of at least one of the invention. NANBV structural proteins, polypeptides or fusion proteins, or combinations thereof. It will be.

Because inocula are typically designed to induce specific antibodies, As described above, the inoculum consists only of NANBV structural antigens and no other functional domains. It is preferred to include NANBV structural proteins that do not contain yne. Therefore, suitable inoculation The substances are residues 1 to 20, residues 21 to 40, and residues 2 to 4 of SEQ ID NO: 1. 0, residue l to residue 74, residue 69 to residue 120, residue I21 to residue 176, or NANB of the present invention comprising the amino acid residue sequence contained in residues 21 to 326 Contains V structural proteins. Particularly suitable as active component in the inoculum are sequences I D8 No. 1 residues 1 to 2o, residues 21 to 40. residue 2 to residue 4o, residue l ~Residue 74, contained in residues 1 to 120, or residue 226 of SEQ ID NO: 2 ~Contained in residue 315, contained in residues 225 to 252 of SEQ ID NO: 3, Residues contained in residues 225 to 252 of SEQ ID NO: 4 or residues of SEQ ID NO: 6 225~? ! 1. NANBV structure having the amino acid residue sequence contained in group 271 It is a protein.

A preferred inoculum contains DNA spanning nucleotides 571-2197 of SEQ ID NO:46. Consists of the entire E1 domain and E,/NSI domain encoded by the sequence .

The inoculum may contain one or more polypeptides of this invention as an active component. Wear. This type of inoculum is particularly useful for producing antibodies immunoreactive with NANBV. It is. Because the small and therefore unique epitope of the NANBV polyprotein This is because it is possible to design a polypeptide that specifies the group. This type of antibody , as specified herein, is isolate specific.

It also cultivates more than one immunogenic functional domain and targets different antigens, i.e. The first NANBV structural antigen as described or from a different strain of HCV Antibody clones specific for the corresponding region and further antigens present on distinct pathogens A multivalent inoculum consisting of fusion proteins useful for inducing ras is contemplated. suitable change Antigens typically associated with NANBV-infected patients, such as Those derived from hepatitis B virus, human immunodeficiency virus (HIV), etc. It is.

Although the embodiment contemplates two immunogenic domains, the inoculum of Arashi is polyprotein to induce antibodies specific for the two regions of HCV encoded by are derived from different regions of HCV.

A suitable pre-injection liquid for individual P East can also be prepared. Y4 products are available "Physiologically acceptable" (physiologically acceptable) refers to In cases, typically allergic or similar undesirable symptoms such as gastric disorders, glare, etc. Refers to molecular entities and compositions that do not cause reactions. Suitable excipients are can take a wide variety of forms depending on the intended use, e.g. containing salts. aqueous solution, phosphate buffered saline (PBS), dextrose, glycerin, Tanol etc. and combinations thereof. Also, if desired, the inoculum can be small amounts of auxiliary substances that enhance the effectiveness of the product, such as wetting or emulsifying agents, pH buffers, It may contain mineral oil, carriers or adjuvants. A preferred embodiment is At least about 0.0% as a sex component. OI%~99% of NANBV structural proteins or or fusion, typically at about 10-200 μg of active component per ml of excipient. It is present at a concentration of

3. Carrier The vaccine is used to promote the generation of an immune response in the immunized mammal. a polypeptide or NAN of the invention conjugated to a carrier or an antigenic carrier Consists of BV structural proteins.

If not already present at that position, add one or more additional amino acid residues to NANB. It is added to the amino- or carboxy-terminus of V-structure proteins and It can assist in white matter binding. attached to the amino- or carboxy-terminus Cystine residues are particularly useful for forming polymers through disulfide bonds. It was found to be useful. However, it is well known in the art to prepare conjugates. Other methods of can also be used. Exemplary addition bonding procedures include Michael addition Reaction products, dialdehydes such as glutaraldehyde, Krivstein et al., J. .

Infect, Dis, +47+318-326 (1983), etc., and used a water-soluble carbodiimide to form an amide bond to the carrier. This includes the use of carbodiimide technology.

Useful carriers are well known in the art and are generally the protein itself. this kind of Examples of carriers include keyhole limpet hemocyanin (KLH), edestin, Brine, albumin, such as bovine serum albumin (BSA) or human serum albumin Bumin (H3A), red blood cells such as sheep red blood cells (SRBC), tetanus toxin Cholera toxin and polyamino acids such as poly(D-lysine, D-glutamine) acid), the intermediate linking group is preferably n]-maleimidobenzoic acid N-human O If a chemical is to be used, use a chemical that does not cause undesirable reactions in that particular animal. carrier should be selected.

4. Administration The preparations are conveniently administered parenterally by injection, eg, subcutaneously or intramuscularly.

Additional formulations suitable for other modes of administration include suppositories and in some cases oral administration. It is included. For suppositories, traditional binders and carriers are e.g. This type of locus can contain polyalkylene glycols or triglycerides. The drug contains active constituents in the range 0.5% to 10%, preferably 1 to 2% It can be formed from a mixture. Oral formulations contain commonly used excipients such as: Excipients, such as pharmaceutical grade mannitol, lactose, starch, and magnesium stearate. Contains sium, sodium saccharin, cellulose, magnesium carbonate, etc. set The composition may be in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation or powder. and contains 10% to 95%, preferably 25 to 70%, of active constituents.

NANBV structural proteins can be formulated into inocula in neutral or salt form . Pharmaceutically acceptable salts include acid addition salts (formed by free amino groups of the antigen). ), which include inorganic salts such as hydrochloric or phosphoric acid, or organic acids such as vinegar Formed by acids, oxalic acid, tartaric acid, mandelic acid, etc. free carboxyl group Salts formed with inorganic bases such as sodium hydroxide, potassium, ammonia um, calcium or ferric iron, and organic bases such as isopropylamine, It can also be derived from trimethylamine, histidine, proline, etc.

The inoculum is administered in a manner compatible with the dosage formulation and is immunized to induce an immune response. The amount should be sufficient to be both genic and effective. Desired full protection when used as a vaccine The amount of inoculum to be administered to achieve immunity depends on the subject being immunized and the amount of antibody synthesized. the ability of the subject's immune system to induce cell-mediated responses, and the degree of protection desired. Dependent. The precise amount of active component required to be administered depends on the judgment of the treating physician. Although each individual is unique, it is generally appropriate to Dosage can be specified. Appropriate dosage ranges for adult humans are For a single immunization dose of approximately 10 micrograms (μg) to several milligrams (m gL preferably about 10 to 500 micrograms, more preferably about 100 micrograms of the order of gram active constituents. for the first dose and additional doses. Appropriate therapy may also vary, but subsequent injections or Other administrations are typically performed.

The inoculum can also include an adjuvant as part of the excipients. laboratory suckling Complete Freund's adjuvant (CFA) used in mammals, incomplete Freund's adjuvant Adjuvants such as adjuvants (IFA) are well known in the art. Alum Pharmaceutically acceptable adjuvants such as drugs can also be used. Therefore example Exemplary inocula include about 0.5 mg to about 2.5 mg alum or 0.1% to 1 Approximately 50-200 μg of NANBV structural protein adsorbed on top of % AI (OH) from 1 ml of phosphate buffered saline (PBS) containing protein or polypeptide. Ru. A preferred inoculum is 100μ adsorbed onto a 2.5mg alum carrier. 1 ml of PBS containing g of NANBV structural protein.

After administration of the inoculum, the mammal's immune system An immunogen is a substance sufficient to produce an immunological response by producing immunoreactive antibodies. It is maintained for a period of time (typically on the order of 2-8 weeks, as is well known).

H1 antibody composition Antibodies of the invention are directed to NANBV structural antigens and to single isolates of NANBV, preferably or c59 isolates, and NANBV structural proteins, polypeptides or polypeptides of the invention. contains an antibody molecule that immunoreacts with the fusion protein (anti-NANBV structural protein antibody molecule). It is a composition having Suitable antibodies include residues 1 to 326 of SEQ ID NO: 1. Epitopes present on polypeptides with amino acid residue sequences and immune reactions , preferably residue I to residue 20, residue 21 to residue 40, residue of SEQ ID NO: 1 2 to residue 40, residue l to residue 74, residue 49 to residue 120 or residue 121 to residue Includes antibody molecules that immunoreact with polypeptides that raise sequences contained in group 326.

In addition, the anti-NANBV structural protein antibody molecule is derived from NANBV isolate HCV-L HC V-BK, HCV-J, HC-JL HC-J4, HCV-JH also I;! H.C. V-Hh and the city listed here and available from Ortho Diagnostics It is preferred that it does not immunoreact with C-100-3, which is available in commercially available assays.

Antibodies of the invention are typically isolated from Hutch C59 isolates or NANBV constructs of the invention. immunizing a mammal with an inoculum containing a synthetic protein or polypeptide; Antibody molecules having immunospecificity for the NANBV structural antigens described herein by is produced by inducing it in a mammal. Antibody molecules are then collected from mammals. The desired to obtain an IgG fraction.

To enhance the specificity of the antibody, solid-phase immobilized immunized NANBV structural proteins were used, Antibodies can be purified by immunoaffinity chromatography. NAN Enough time for BV structural proteins to immunoreact with antibody molecules to form solid-phase immobilized immune complexes. The antibody is brought into contact with the solid-phase immobilized NANBV structural protein for a period of time of 10 minutes. join The antibody is separated from the complex by standard techniques.

C-100-,3 antigen or the NANBV isolates identified above. Immunization is performed by contacting a solid phase with one or more immobilized antigens or NANBV isolates. forming an adsorbent mixture. Preferably, undesired substances in the immunoadsorption mixture are Excess antigen or NA in the solid phase in proportion to the antibodies in the composition with high immunospecificity. Assume that NBV exists.

The immunoadsorption mixture is then subjected to immunoreaction conditions sufficient to form immune complexes on the solid phase. maintained for a period of minutes. Then, the liquid phase and solid phase are separated and immunoadsorbed onto the solid phase. Retain the liquid phase with undesired antibody molecules that were not removed.

Particularly preferred are the Hutch C59 isolates, or preferably unique to c59. having an amino acid residue sequence, preferably v, v, , Vl or V of NANBV.

of this invention selected herein as being derived from a variable region. Contains a c59 isolate-specific antiserum formed by immunization with a polypeptide This is an antibody composition. The produced antibody composition was then immunoadsorbed and described here. Antibodies immunoreactive with NANBV isolates other than c59 are removed as shown.

The antibodies thus produced are used, inter alia, in the diagnostic methods and systems of the present invention. and detect NANBV structural antigens present in body samples as described here. can do.

■ The active component of this invention used in the preparation of antibodies immunoreactive with structural antigens. The description here and the fact that the vaccine also induces antibody production (this design and are closely approximated insofar as they are illustrative of the preparation and use of inoculum.

The key difference is that, as is well known, inocula are intended for use in animals rather than humans. This is why it is prescribed.

Preferred antibodies are monoclonal antibodies, and the antibodies of the invention are not disclosed herein. What is contemplated by this invention is: 1) Ibridoma cells, and materials of this invention. Eliminate the risk of microbial contamination.

or a diagnostic system for assaying for the presence of NANBV structural antigens. BY antibody compositions. Instructions for the use of the packaged reagents are also typically included. a label capable of giving a signal for the formation of a complex containing a protein or an anti-NANBV antibody. or further includes display means.

As used herein, the terms "label" and "display means" refer to Part of this invention insofar as it is used by proteins, methods and/or systems. be able to. Suitable fluorescent labeling reagents include fluorescent dyes such as fluorescein isocyanate (FIC), fluorescein isothionanate (FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), te Tramethylrhodamine isothiocyanate (TRTTC), lissamine, rhodami 8200 sulfonyl chloride (RB2ooSC), chelated lanthanide bond ( For example, there are Eu, Tb, Sm), etc. A description of immunofluorescence analysis techniques can be found in Dell, R.I. ``Fluorescence Analysis'', Antibodies as Tools, Maruki Ronis et al., John Reilly ・Acknowledged by And Lands, pp. 189-231 (+982), with reference to this Enzymes such as monosulfur oxidase, alkaline phosphatase, etc. major label If the enzyme is an enzyme such as HRP or glucose oxidase, the antibody is 2,2'-azinodi-(3-ethylbenzthiapurine-6-sulfonic acid) Similarly, for 1, ++indium, 3H, IIs, 14C or 8! It's P's You can use eel bee. An example of a pairing is biotin:avidin, which In the case of biotin is the label and peroxidase: anti-peroxidase (PAP), in which case peroxidase is the label.

Attachment of labels, i.e., labeling of polypeptides and proteins, is well known in the art. be. For example, antibody molecules produced by hybridomas are combined with components of the culture medium. labeling by metabolic uptake of radioisotope-containing amino acids given as can be done. For example, Galfre et al., Meth, Enzymol, 73:3- 46 (1981). Proteins via activated functional groups Bonding or servicing techniques are particularly applicable. For example, Aurameas et al., 5ca nd, J., Immuno! , Volume 8 Supplement, 7:7-23 (1978 Ll:l de Well et al., Biotech, 3,889-894 (1984) and U.S. Pat. No. 4.493.795.

The diagnostic system may also include a specific binding agent, preferably as separate packaging. . A “specific binding agent” is a molecular entity that can selectively bind to a reagent species. , which can then be reacted with the products of the invention, but as such are not subject to the invention. Not a protein expression product. Exemplary specific binding agents include antibody molecules, e.g. 1gG or anti-human IgM, complementary protein or its fragment, protein A, etc. Ru. Preferably the specific binding agent is when the antibody is present as part of an immune complex; It can bind to the anti-NANBV antibody to be detected.

In a preferred embodiment, the specific binding agent will be labeled. However, the diagnostic system If the stem contains an unlabeled specific binding agent, that binding agent is typically Use as an amplification tool or reagent. In these embodiments, the amplification means contains reagent species. When bound to the complex, the labeled specific binding agent binds specifically to the amplification means. can be done.

The diagnostic kit of the present invention detects antibodies in body fluid samples such as serum, plasma or saliva. It can be used in a ``rELISA'' format to detect the presence or amount. rELI SA' refers to the antibody or antigen bound to the solid phase and the antigen or antigen present in the sample. is an enzyme linkage using an enzyme-antigen or enzyme-antibody conjugate that detects and quantifies the amount of antibody. Refers to the immunosorbent assay. The ELISA technique was first described by Lande Mede in 1982. Publications published by Ross Aldo CA ・Chapter 22 of the 4th edition of “Basic and Clinical Immunology” by P. Cyto et al., and U.S. Patent Nos. 3,654.090, 3.850.752 and 4.016 ．． No. 043, all of which are incorporated herein by reference.

Therefore, it is suitable! '1, the NANBV structural proteins, polypeptides, and fusions of the present invention Synthetic proteins or anti-NANBV antibodies can be immobilized on solid matrices and used in targeted diagnostic systems. The solid support can be packaged separately in the system.

Reagents are typically immobilized on solid matrices by adsorption from aqueous media; Other modes of fixation known to those skilled in the art may be used.

Useful solid matrices are well known in the art. This type of material is Registered trademark Cefazo So from A Fine Chemicals (Bistoway, NJ) or holes in microtiter plates, e.g. polystyrene or polyvinyl chloride. A diagnostic system for assaying the presence of NANBV nucleic acids in body samples using polynucleotide of the invention in an amount sufficient for at least one assay as a reagent A label or label capable of giving a signal for the formation of daidzecicon complexes Fusion proteins, anti-NANBV antibodies, polynucleotides, labeled specific binding agents or or amplification reagents in solution, as a liquid dispersion or, for example, in lyophilized form. It can be provided as a dry powder. If the display means is an enzyme, the The bare substrate can also be provided in separate packaging of the system. The micro tie mentioned above A solid support such as a tablate and one or more buffers may also be used in this diagnostic assay system. can be included as separately packaged elements within.

The packaging discussed here with respect to diagnostic systems is the one customarily utilized in diagnostic systems. It is something that will be done. This type of packaging is made of glass and plastic (e.g. polyethylene). polypropylene, polypropylene and polycarbonate) bottles, vials, plastic This includes paper bags and plastic foil laminated envelopes.

2. Diagnostic method The present invention provides NANBV structural proteins, polypeptides, fusion proteins or The anti-NANBV structural antigen antibody was used as an immunochemical reagent to determine the amount in the sample. Forming immune reaction products that are directly or indirectly correlated to the amount of material to be detected Detection of anti-NANBV structural protein antibodies or NANBV structural antigens in body samples We are planning all kinds of diagnostic methods that will result in the diagnosis. Those skilled in the art will be able to understand the immunochemistry of this invention. using a reagent whose amount correlates to the amount of a specific antibody or antigen in a body sample Understand that there are a number of well-known clinical diagnostic chemistry procedures that can form immune reaction products. Good morning.

Various heterologous and homologous procedures, competitive or non-competitive, can be used in this − It can be used to carry out the assay method of the invention. Therefore, the illustrative method is However, the present invention is not limited thereto.

To detect the presence of anti-NANBV structural proteins in a patient, a body sample, preferably Samples of body fluids such as blood, plasma, serum or saliva from patients can be NANBV antigenic molecules of the present invention, such as V structural proteins, and preferably contacting the polypeptide or fusion protein by mixing under biological assay conditions; Form an immunoreaction mixture. The mixture is then divided into NANBV antigenic molecules-antibody molecules. maintained for a period of time sufficient to allow the formation of immune reaction products (immune complexes) do. The presence, and preferably amount, of the complex is then detected as described herein. Ru. The presence of the complex is indicative of anti-NANBV antibodies in the sample.

In a preferred embodiment, the immune reaction formed between the NANBV antigenic molecule and the patient's antibodies The presence of reaction products can be detected by using specific binding reagents such as those discussed herein. put out For example, immunoreaction products are first mixed with a labeled specific binding agent and then labeled. Form a belling mixture. Labeled specific binding agents are specifically labeled as described herein. Consists of a foreign binding agent and a label. Then adapt the labeling mixture for specific binding All immune reaction products present bind to the labeled specific binding agent under conditions that and for a period of time sufficient to form a labeled product. Then the shape detecting the presence, and preferably the amount, of labeled product produced and generating an immune response. To indicate the presence or quantity of something.

In a preferred embodiment, the diagnostic method of the present invention comprises a diagnostic system as disclosed herein. This is done in such a way that the immune complex is formed and detected on a solid phase.

Therefore, a preferred diagnostic method involves converting NANBV structural proteins or polypeptides into solid matrices. Immobilize on Trix to form a solid phase. The specific binding agent is a NANBV structural protein. A protein that can be immunoconjugated with an anti-NANBV structural protein antibody on a solid phase. More preferably, it is Tin A or anti-human Tg, such as IgG or IgM. Ru. Most preferred is the use of a labeled specific binding agent, where the label is As described for the diagnostic system or as detailed by the references provided below. such as radioactive isotopes, enzymes, biotin or fluorescent markers, e.g. It is assumed to be a lanthanide.

This solid phase version B shows the embodiment in the fusion protein as described in Example 7. As shown in SEQ ID NO: I, residues summer to residue 20, residues 21 to residue 40, residues 2 to residues Antigen specified by the amino acid residue sequence contained in 40 or residue Natsu to residue 74 It is particularly preferred to use a recombinant protein comprising.

In other preferred diagnostic methods, the NANBV antigenic molecules of this invention are immobilized as described above. The unbound material is immobilized on the body matrix and the solid surface is contacted with a diluted sample of the sample. The dilution of the biological sample is subjected to an immunoconjugation step by removing the sample.

Multivalency of antibodies (for IgG) present in biological samples from infected individuals divalent for IgM and pentavalent for IgM), the label of this invention for this mixture Subsequent addition of NANBV structural proteins, polypeptides or fusion proteins As a cross-link between a solid-phase NANBV antigenic molecule and a soluble labeled molecule of the invention of As a result, the X sample antigen will be attached to the same phase. Presence of label in solid phase indicates the presence, and preferably the amount, of specific antibodies in the sample. Be a person skilled in the art For example, determine the range of dilution and from this determine the concentration of labeled antigen in the solid phase. You can do something like that. Biological samples and labeled NANBV antigenic components of this invention Pre- or simultaneous mixing of the biological sample and the solid phase in contact with each other By utilizing the cross-linking properties of divalent or multispecific antibodies, it is possible to As particularly useful labels characterized by the formation of heavy molecular complexes, the biotinylated molecules of this invention Labeled NANBV antigenic molecules can be labeled antigens, enzyme-strep toavidin or enzyme-avidin complex, followed by addition of the appropriate substrate. Enables subsequent detection. Horseradish peroxidase, alkaline phosphater Enzymes such as enzymes, β-galactosidase or urease are often used, Others are commercially available with some suitable substrates. The complex formed Suitable labels with markers that allow direct detection of conjugation include radioactive molecules. Isotopes such as iodine or lanthanide chelates such as europium Included.

Designed to detect the presence of NANBV structural antigens in body samples from patients In other embodiments, the sample (e.g., blood, plasma, serum, urine, or saliva) is contact by mixing with a specific anti-NANBV structural protein antibody under biological assay conditions. to form an immunoreaction mixture. After that, NAN complexed with the antibody of this invention sufficient to allow the formation of an antigen-antibody immune reaction product containing BV structural antigens. jl# the mixture for a period of time. Thereafter, the presence and preferably amount of the complex can be determined, thereby indicating the presence of an antigen in a body fluid sample.

In a preferred embodiment, the antibody will be present in a solid phase. More preferably, formed The amount of immune complexes present in the patient should be measured in a competitive immunoassay format, where The antigen in the body fluid sample is conjugated to the homologous antibody against the labeled recombinant antigen of this invention. Assume that there are conflicts regarding binding. This method requires that a body fluid sample be (1) fixed; A solid support for growing antibodies according to this invention and (2) immunoreacting with the solid phase antibody. mixed with the labeled NANBV antigenic molecule of this invention, and both liquid and solid phases were mixed. consists of forming a competitive immune reaction mixture that grows. Then label in solid phase. for a period of time sufficient to form an immune reaction product containing NANBV antigenic molecules. Maintain this mixture. The amount of label present in the solid phase is then determined and this Figure 2 shows the amount of NANBV structural antigen in body fluid samples.

Enzyme immunoassay techniques are direct or Both competitive assays have been extensively described in the art. An exemplary technique is Magis, [Enzyme Immunoassay J, CRC Breath, Cleveland, 0H (1981 ), and Chissen, “Practical and Theory of Enzyme Immunoassay J1 Elsevier, AMS This can be seen in Terdam (1988).

The bioassay conditions include NANBV antigenic molecules and anti-NAN in the immunoreaction mixture. such as to maintain the biological activity of BV structural proteins. These conditions include , about 4°C to about 45°C5 preferably about 37°C, about 5-9, preferably or about 7, and distilled water to about 1 molar sodium chloride, preferably A range of ionic strengths of saline is included. How to optimize these types of conditions Well known in the art.

Also contemplated is the detection of immune reaction product formation without the use of labels. This is an immunoassay that can be used to This type of method uses “detection means.” and this means is well known per se in clinical diagnostic chemistry, and these are particularly useful for novel polypeptides. Forms part of this invention insofar as it utilizes the peptides, methods and systems It is. Exemplary detection means include methods known as biosensors; Changes in surface reflection power (surface plasmon resonance), absorption of surface dissipated waves by optical fibers Biosensing methods based on detecting changes in the absorption or transmission of surface acoustic waves have been proposed. Included.

In other embodiments, detection of immunoreaction products using time-resolved fluorescence analysis (TR-FIA) In this case, the label used is a signal that can be detected by TR-FIA. generate. Typical labels suitable for TR-FIA are lanthanide and aromatic β- metal complexing agents such as lanthanide chelates formed with diketones; Because tantanide binds to antigens or antibodies via EDTA analogs, fluorescence A tanide complex is formed.

The principle of time-resolved fluorescence analysis is described by Soini et al., CI in Chem, 25: 35. 3-361 (1979) and has been widely applied in immunoassays. There is.

For example, Halonen et al., Current Topics in Microbiol ogy and Immunology, 104:133-146 (1985) , Suonpa et al., C11nic Chfmica Acta, 145:34f-3 48 (1985), Lovgren et al., Taanta, 31: 909-916 (1984), U.S. Patent Nos. 4,374,120, 4,569.790, and published international patent application numbers EPO139675 and WO3710270 8 can be referred to. Lanthanides suitable for use in TR-F IA are It's europium.

Reagents and systems implementing the TR-FIA technique were purchased from commercial sources (Pharmacia Das Iagnosticus, Absara, Sweden).

Particularly suitable is carried out as a typical "Western plot", herein Example 7. This is a solid-phase immunoassay described in .

This diagnostic method is based on the appearance of anti-NANBV antibodies in species infected by NANBV. It can be implemented in combination with other methods to detect. For example, this The composition of the present invention detects the presence of either or both of the two antigens and immunoreactive antibody species. In the assay to determine, commercially available C100-3 antigen (Ortho Diagnostics, LA Can be used with Litan, N, J, ).

The present invention also provides body support using the polynucleotide or DNA fragment of the present invention. of a nucleic acid hybridization method to detect the presence of NANBV nucleic acids in a sample. I also plan to use it. This method generally comprises a) a body sample and a polynucleotide of the invention; Aqueous hybridization by mixing with octides or oligonucleotides b) all NANBV polynuclei present in the body sample; The acid hybridizes with mixed polynucleotides or oligonucleotides. a period of time sufficient to form hybridization products and C) maintaining the aqueous hybridization mixture under The presence of hybridization products and thereby N in body samples consisting of detecting the presence of ANBV polynucleic acid.

The NANBV nucleic acid sequence to be detected is here used as a target nucleic acid sequence. Ru.

The target nucleic acid sequences to be hybridized in this method are tested for purity and concentration. All nuclei, as long as the sample is in a form compatible with nucleic acid hybridization reactions. Assuming that an acid is present in the sample, two things can be done. Hybridization It is generally known to isolate nucleic acids to a degree suitable for isolation, and can be done by a variety of means. I can do what I want to do. For example, body tissues such as skin, muscles, hair, etc., and body fluids, e.g. Nuclei can be extracted from a variety of nucleic acid-containing samples including, for example, blood, plasma, urine, amniotic fluid, brain fluid, etc. The acid can be isolated. For example, Maniatis et al., rMolecular Cloning: Experiments. Cold Spring Harbor Library (1982), and Ausubel et al., “Modern Procedures in Molecular Biology,” John Reilly and - Reference may be made to Song (1987).

polynucleotide or oligonucleotide probe or phase present in the sample. hybridizes with a complementary nucleic acid sequence to produce a hybridization product, i.e. hive for a period of time sufficient to form a complex containing the probe and target nucleic acid. A method for preparing a hybridizing zeta-3 reaction mixture under redizing conditions. maintain.

The description of "hybridization conditions" and its grammar", etc. are damaged during the maintenance period. When used with ma, polinuku [otido or oligonukushi/saiko dobrobu] sufficient to anneal to the target sequence and typically form a nucleic acid duplex. The concentrations of the reactants and accompanying reagents in the mixture are adjusted over time, temperature, and pH conditions. In relationships, Hypuridae)! - Indicates that a reaction mixture is provided. -1 These time, temperature and pH conditions necessary to carry out hybridization are , the polynucleotide or oligo to be hybridized, as is well known in the art. Advantages of nucleotide probes, polynucleotide or oligonucleotide probes [jl! degree of complementarity of polynucleotide or oligonucleotide 1 7 guanidine and tocin containing 11 desired hybridization@ The kinetics of hybridization zeta 9 can be influenced by Depends on the presence of salts or additional reagents in the hybridization reaction mixture. Give Hybridize zeta 3 reaction mixture Methods for optimizing conditions are well known in the art. .

Typical hybridization conditions use solutions buffered to l) H values of 4 to 9. Including, 18 degrees Celsius (18°C) -75°C1, preferably about 37°C to about at a temperature of 65°C, preferably about 54°C, for 0.5 seconds to 24 hours, preferably Perform for a 2 minute time period.

Hybridization is carried out in the form of homologous or xenogeneic origin, as is well known. be able to. The homologous nozobridization reaction occurs entirely in solution; In this case, the polynucleotide probe and the nucleic acid sequence to be hybridized (f1 ! target) is present in soluble form in solution. Heterologous reactions are involves the use of a matrix insoluble in the reaction medium to which the lobe or target nucleic acid is attached. nothing. For example, the body sample to be assayed is fixed in a solid matrix and in situ Hybridization

In situ hybridization typically typically ranges from about 0 microns, preferably about 1 micron to about 25 microns, more preferably about 1 micron. Chron - a body in the form of a section or fragment of tissue with a thickness in the range of about 10 microns (performed on a bubble). This type of sample can be prepared using a commercially available cryostat. To prepare, to be able to do two things.

Also, the widely used xenogenic form is the Southern Brosotho procedure 22), and this If so, electrophores the genomic DNA after restriction enzyme digestion, and collect the electrophoresed DNA fragments. After the pieces are first denatured, they are transferred to an insoluble -7) lix in the 1° plotting procedure. , then immobilized gene 1 containing the complementary nucleic acid (target) sequence, polynuclease 17 to the nucleic acid. 1.・Hybridize ocide or oligonucleotide 17 ocide 3, or further , a widely used heterogeneous form is the library search procedure, Form a library of nucleic acids. After that, the library that was created by brll-d is Hybridizes with nucleotide or oligonucleotide blebs and The use of glass slides, nitrocellulose sheets, etc. as solids 71-lithox used or included.

Also suitable is reverse transcription of isolated mRNA to form cDNA. Oxo-sequencing and polynucleotide or oligonucleotide hybridization For other procedures using primer extension reactions with Isaeng Yong as the first step It is a homologous hybridization reaction as performed. Particularly suitable are Allogeneic vectors that amplify specific nucleic acid sequences via polymerase chain reaction (PCR) This is an hybridization reaction.

If the nucleic acid containing the target sequence is in the double M (ds) form, hybridization The dsDNA is first denatured by heating or alkali treatment before carrying out the reaction. It is suitable for Denaturation of dsDNA is caused by hybridizing polynucleotides or before mixing with oligonucleotides, or with dsDNA. This can be done after mixing with polynucleotides or oligonucleotides.

given as a polynucleotide or oligonucleotide itself or as a double-stranded molecule If so, this can also be denatured before mixing in the hybridization reaction mixture. or simultaneously denature the target-containing dsDNA. Ru.

A method for detecting a specific target nucleic acid sequence is first the hybridization described above. conduct a reaction to form a hybridization product, and then Detects the presence of redization products and thereby detects specificity in samples containing nucleic acids. This is done by detecting the presence of a specific nucleic acid sequence.

The nucleic acid-containing sample can be a body tissue or fluid and is subject to hybridization. The reaction mixture can be prepared as described above for the reaction mixture.

Detection of hybridization products formed in hybridization reactions This can be done by various means. Detection of hybridizator 3 products Although preferred embodiments are disclosed herein, other well-known tests will be readily apparent to those skilled in the art. The means of output are appropriate for the method contemplated and for use in the accompanying diagnostic system. You should understand that.

One approach to detecting the presence of specific nucleic acid sequences is to detect the presence of a polynucleotide sequence. DNA or oligonucleotide probes exist as something that the probe can detect. a label or label that can give a hybridization product that typical Typically, this type of label contains radioactive atoms, chemically modified nucleotide salts, Including groups etc.

whether operably linked to a polynucleotide or oligonucleotide probe; The radioactive element present as a component facilitates the detection of hybridization products. provide a useful means of progress. Typical radioactive elements are those that give beta radiation It is. 'H1+4c: , , Up and ■ Elements that emit β rays such as S are β It represents a type of radiation-generating radioactive element label. radioactive polynucleotide DNA or oligonucleotide probes typically use DNA polymerase prepared by enzymatic incorporation of a radioactively labeled nucleotide into a nucleic acid The labeled nucleic acids are then denatured and radiolabeled polynucleotides or oligonucleotides are added. Form a cleotide probe.

Radioactively labeled polynucleotide or oligonucleotide probes Alternatives include chemically modified metal conjugates, biotin-containing groups, fluorescent compounds, etc. It is a polynucleotide or oligonucleotide.

One useful gold i*i combination is formed by a lanthanide and an aromatic β-diketone. is a lanthanide chelate that is to nucleic acids, polynucleotides or oligonucleotides via rate-forming compounds. bind, thereby forming a fluorescent lanthanide complex. U.S. Patent No. 4.374 ．． No. 120 and No. 4,569.790 and published patent application number EP0 139675 and number WO37102708.

biotin or acridine ester labeled oligonucleotides, and The use of in polynucleotides has been previously described. U.S. Patent No. 4.707 ．． No. 404, published patent application EPO 212951 and European Patent No. 0 Reference may be made to No. 087636. Useful fluorescent marker compounds include full Included are olecein, rhodamine, Texas Red, NBD, and the like.

Due to the labeled nucleotides present in the hybridization product, A. The hybridization product itself is labeled and therefore the sample to be assayed. It becomes distinguishable from other nucleic acids present in the nucleic acid. hybridization products Detection of the presence of a label in the medium and thus the presence of hybridization products is typically not hybridized to the hybridization product. from any labeled polynucleotide or oligonucleotide probe. including separating the hybridization products.

- heavy chain polynucleotides or oligonucleotides, e.g. unhybridized hybridize labeled polynucleotide or oligonucleotide probes. Separation techniques from soy products are well known and typically involve the separation of their chemical separating single strands from non-heavy stranded nucleic acids on the basis of their quality. Even more often, minutes Detachment techniques involve hybridization that is bound to a solid matrix, typically through washing. Heterologous hybridization to separate unhybridized probes from hybridization products Including using the zeta3 form. An example is the Southern plot technique, where In this case, the matrix is a nitrocellulose sheet and the label is "P". , J. Mo1.8jo1.98:503 (1975).

In other embodiments, the hybridization product detection step comprises amplified nucleic acid production. It consists of detecting objects. The amplified nucleic acid product is processed by polymerase chain reaction ( It is the product of an amplification method well known in the art, referred to as PCR).

Methods and systems for amplifying specific nucleic acid sequences are both described in the US patent to Mullis et al. Patent Nos. 4,683,195 and 4,683,202, and rPCR technology ”, Teachings in Erlich, Stockton Press (1989), Farona et al., M. Th.

ds in Enzymol, +55+335-50 (1987) and "Remerase Chain Reaction", edited by Errichi et al., Cold Spring Harbor Lab. Described in Latrize Press (1989).

Example The following examples are given for illustrative purposes only and may not be used in any manner. This does not limit the scope of the invention.

Example 11 Production of recombinant DNA molecules A. Isolation and sequence analysis of NANBV clones as a source of NANBV virus particles. Hutchinson (Hutchjnson) showing acute phase NANBI ( ) blood was collected from chimpanzees infected with the strain. By centrifugation and filtration The plasma was purified. HANBV was then bound to protein G Sepharose. By immunoaffinity chromatography on an IgG (Hatsuchi strain) column. NANBV virus particles were isolated from purified plasma. guanidium thio Sepharose beads are separated from NANBV RNA by immersion in cyanate. The eluted RNA was then concentrated via cesium chloride (CsCI) buffer. Shrunk. Sambrok et al., “Molecular Cloning: Laboratory Manual”, Sambroch et al. Edited by K. et al., 2nd edition, Cold Spring Harbor Laboratory Press, NY (1989).

Picogram amounts of purified NANBV RNA were added to random and oligo dT platforms. template in a primer extension reaction mixture containing primers, dNTPs, and reverse transcriptase. was used to form the first strand cDNA. The obtained first strand cDNA was of the second strand in a reaction mixture containing DNA polymerase I and RNA5eH. It was used as a template for cDNA synthesis to form double @ (ds) cDNA. (Sambrock et al., supra). Synthesized using an asymmetric synthetic primer adapter system dscDNA was amplified, but the sense and antisense primers were used interchangeably. Anneal the two strands of NANB using T4 ligase under blunt-end conditions. The ends of the VcDNA were ligated to form a cDNA-adapter molecule. cDNA-a adapter molecule and the same positive sense adapter primer, dNTP and TAQ polymer. by mixing with Lase (Promega Biotique, Madison, WT). Polymerase chain reaction (PCR) amplification was performed as described, and the amplified NANB VcDNA was prepared in m. Thereafter, the resulting amplified NANBV cDNA sequence was Subsequent amplification in PCR reactions using heterologous NANBV oligonucleotide primers Used as a mold for width.

Oligonucleotides were selected to correspond to the 5' sequence of hepatitis C (HCJI sequence). Column: Saikimoto et al., Jap, J, EXP, Med, 60:167-177.19 90).

The selected oligonucleotides were purified by Pharmacia Gene A, according to the manufacturer's instructions. Synthesized by Assembler and purified by polyacrylamide gel electrophoresis, As shown in Table 1, the nucleotide base sequence and the consecutive sequence starting with 15 and ending with 23 are shown in Table 1. It has a column number.

Table 1 synthetic oligonucleotide Oligonucleotide Putative NANBV region Oligonucleotide sequence sequence name 8 Array number 690(+) Capsid 1-21 ATGAGCACGATrCCCAAACC T 15693 (+) Capsid 146-+62 GAGGAAGACTrCC GAGC16694(-) Capsid 208-224 GTCCTGCCCCTC GGGCCG I 7691 (-) Capsid 340-359 ACCCAAA I↑χαtAccTAcG 1814 (+) Envelope 356-374 TGGGTAAGGTCATCGATAC19+5 (+) Envelope 36 1-377 AAGGTCATCGATAACCCT 2018 (-) Enve Loaf”512-529 AGATAGAGAAAGAGCMC211S(-) Engineering: zvelope 960-981 GGACCAGTrCATCATCATATAT AT 22+7 (-) Envelope 957-976 CAGTrCATCA TCATATCCCA 238 oligonucleotides are identified by number and sense and its polarity indicating the sequence corresponding to the antisense coding strand (10) and (− ) respectively. All sequences are listed in the 5' to 3' orientation.

(3) NANBVC DNA (7) PCR Amplification Example IA The plastic prepared in (1) Imma-compatible amplified cDNA sequences and synthetic oligonucleotides 690 and 690 as primers. and 694 (primer pair 690:694) to perform PCR amplification. went. The resulting PCR reaction mixture contains primer-matched amplified cDNA template, oligo Nucleotides 690 and 694, dNTPs.

It contained salts (KCI and MgClt) and TAQ polymerase. cD NAc) PCR amplification was carried out by maintaining the mixture for 30 cycles at an annealing temperature of 37 °C. This was done by Sample fractions from the first round of amplification were incubated at 55°C. Re-amplification was carried out under similar conditions for 30 cycles at a cool temperature.

(4) Preparation of vector containing PCR-amplified dsDNA The second fraction of PCR amplification was % acrylamide gel.

After separation of the PCR reaction products, the expected 690:694 amplification product of approximately 224 bp Regions of the gel containing DNA fragments corresponding to the product were excised and subjected to standard electroelution techniques. It was purified according to the technique (Sambrock et al., supra). The purified fragments were treated with Kytase. , into the pUc18 plasmid cloning vector at the Smal polylinker site. A clone containing DNA fragment 690:694 operably linked to pUc18. A sumid was formed.

pUClB and the resulting mixture containing a fragment corresponding to the 690:694 sequence was then transformed into E. coli strain JM83. X-g containing ampicillin Identification of plasmids containing inserts as Iac- (white) colonies on a1 medium did. The pUc18 plasmid containing the 690:694 DNA fragment was subjected to restriction enzyme analysis. and pUC18690, identified by subsequent electrophoresis on an agarose gel. :694rDNA molecule.

pUc18 with 690:694 DNA fragment (pUCl8 690:694) Amplify two independent colonies suspected of containing the vector and Plasmid DNA was prepared by CsC1 density gradient centrifugation according to Mbrok et al., supra). It was used to make. ssS dideoxy procedure using pUC18-specific primers Plasmids were sequenced using One plasmid on both DNA chains Sequenced independently to ensure accuracy of sequencing. The obtained sequence information is They are shown as bases 1 to 224 of D number l.

Plasmid pU018 690:694 has a length of 224 bp NANBV DN The putative capsid contains the A fragment and when compared to the HCJI prototype sequence. Two nucleotide substitutions and one amino acid in the amino-terminal region of the protein Residue differences are obvious.

(6) Preparation of NANBV clones from the 5' end of the genome To obtain the sequence of the encoding NANBV Hatsuchi genome (Saikimoto et al., supra), Ligonucleotides 693 and 691 (listed in Table 1) were used in the PCR reaction.

Virus NANBV from Hatsuchi to RNA as described in Example 1A () The cDNA was prepared using oligonucleotide pair 693:691 in Example I. A. Used in PCR amplification as described in (3). The obtained PCR-enhanced dsD The NA was then cloned into the pUc18 cloning vector and Example IA (4) The insert was retrieved and pUc18 693:691 was created as described in . So Then, clone using pUc18-specific primers as described in Example IA (5). The loan was sequenced.

Plasmid pUc18 693:691 is 57 bp long and has a sequence ID number of Contains a NANBV DNA fragment spanning nucleotide bases 203 to 360 of No. 1.

This fragment has been extended to the sequence of the 693 primer used to generate the fragment. not present. The sequence of this fragment shows three nuclei when compared with the known sequence of HCJI. leotide differences are obvious and the corresponding amino acid changes relative to the HCJI sequence. It doesn't exist at all.

The NANBV code that encodes the estimated envelope region (Saikimoto et al., supra) To obtain the sequence of the antigenome, oligonucleotide primers 14 to 18 (written in ml) were used. ) were used in various combinations with the NANBV Hatch R Hatchzamble.

As a source of NANBV RNA, Hatsuchi strain was inoculated at 4 weeks post-inoculation to induce acute sensitivity. A liver biopsy specimen from a chimpanzee showing staining was used. The biopsied sample is first was frozen and then ground. The resulting powder was treated with guanidine for RNA extraction. Treated with isothiocyanate. to phenol at 65 °C in the presence of SDS. RNA was extracted from guanidium-treated liver samples. Two liver samples and then with chloroform. Isopropanol and sodium acetate The extracted RNA was precipitated at -20°C using a microtube filter.

Purified liver was purified by primer extension reaction using oligonucleotides 1B and 16. NANBV-specific cDNA was generated using RNA derived from . To prepare cDNA for Hatsuchi strain amine-terminated protein food sequence j: d Antisense oligonucleotides were incubated at 42°C in the presence of NTP and reverse transcriptase. A separate pair of oligonucleotides 14:16 (16 primed e D used as a template for the second amplification, in which each oligonucleotide Pair 15817 and 15:18 were used as primers.

The PCR reaction products from each primer pair reaction were transferred to a low melting point agarose gel. - fragment and kinase separately and pBluescript plasmid vector. Transformed E. coli DH5 colonies were purified by restriction enzyme analysis. The DNA fragments present in the medium were each arranged as described in Example IA (5). The row has been decided.

+5:17 DNA fragment is contained in nucleotides 361-978 of SEQ ID NO:l. Ru. The sequence of the 15:18 DNA fragment is also nucleotides 361-52 of SEQ ID NO:1. given to 9. The two clones were separated by 168 bp of the 15:18 DNA fragment. Duplicate.

The sequencing results were 1569 when compared to the HC, monthly sequence (Saikimoto et al., supra). A plasmid of 0:694 DNA fragment was formed.

Mido was identified. The stationary phase bacterial culture was subjected to an alkaline lysis procedure and the crude vDNA! [uses a single Xho1 site not in pGEX-3X, 690:694D Vectors containing NA fragments were searched.

Several 690:694 DNA fragment-containing vectors were amplified, and the resulting amplified vector D NA'f:Cs was purified by Cl density gradient centrifugation. Included in sequence ID number 2 Hybridizes to the pGEX-3X sequence at nucleotide positions ft814-633 In this way, vectors containing the 690 and 694 DNA fragments containing The NANBV polypeptide is cleaved from the GST carrier by digestion using selection The product is a GST:NANBV690:694 fusion protein. and CApGEX-3X-693+ 691: iEI in Example IA (6) The prepared plasmid pUCI8 693:691 was purified as in Example IB (1). by first subjecting it to restriction enzyme digestion with EcoRr and BamHI. Plasmid pGEX-3X-693:691 was formed. SEQ ID NO: 1 base 2 The obtained free DNA fragment having a sequence included in bases 05 to 360 was used in Example Purified as done in IB (1). Purified DNA fragment and presence of T4 ligase Linearized by restriction enzyme digestion at 16°C with EcoRI and BamHI in the presence of plasmid pGEX-3X- 693: 691 was formed.

The crude cDNA preparation was digested with EcoRI and BamHI at 693:69. 693:691 as done in Example IB(2) except to release the 1 insert. A pGEX-3X plasmid containing the DNA fragment was identified by selection. NANBV 693:691 DNA with exact coding sequence for in-frame translation of structural proteins The pGEX-3X vector containing the fragment was subjected to sequence analysis as performed in Example IB(2). was identified and selected to form pGEX-3X-693:691.

The obtained vector encodes the fusion protein (GST:NANBV 693:691) This is the amine terminal corresponding to residues 1 to 221 of GST contained in SEQ ID NO: 2. Cleavage of the protease factor Xa corresponding to the terminal polypeptide portion, residues 222-225 The intermediate polypeptide portion that specifies the site, each nucleotide base sequence (SEQ ID NO. Issue: 24): Amino acid residue sequence encoded by GGG ATG CCCAAT TCA ( SEQ ID NO: 25) Residue 22 consisting of two Glylie Pro Asn Ser Iruka protein corresponding to 6-230, amino acid residue sequence 69-1 of SEQ ID NO: 1 Corresponds to residues 231-28 which specify the NANBV capsid antigen with 20 carboxy-terminal polypeptide portion, and each nucleotide base sequence (sequence Number + 26): AAT TCA TCG TGA Amino acid residue sequence encoded by (SEQ ID NO: 27): Asn Ser S er　END The carboxy-terminal linker moiety corresponding to residues 283-287 consists of It will be done.

pGEX-3X-15:I8: Plasmid blue prepared in Example IA (6) - Script 15:18 was adapted for restriction enzyme digestion with EcoRV and BamHI. Plasmid pGEX-3X-15:18 was formed by first applying Bam The H■ cohesive ends were filled in as in Example IB(1). Base 3 of SEQ ID NO: 1 The resulting free DNA fragment having a sequence containing bases 61 to 528 was used in Example IB Purification was performed as in (1). with SmaI as done in Example IB(+). pGEX-3Xl linearized by restriction enzyme digestion:? DNA fragment produced by tJ were mixed and ligated to form plasmid pGEX-3X-15:18.

15: The pGEX-3X plasmid containing the 18 DNA fragment was carried out in Example + B (2). Identified by selection as described above, the crude DNA11 product was purified with EcoRI and BamH. The 15:18 insert was released by cutting with I. NANBV structural protein frame pGEX-3X containing a 15:18 DNA fragment with the correct coding sequence of internal translation The vector was identified and selected as in Example IB(2) and pGEX-3X-1 5:18 was formed.

The obtained vector encodes the fusion protein (GST:NANBVl 5:18) This is the amino-terminal polypeptide portion corresponding to residues 1 to 221 of GST; an intermediate polypeptide corresponding to groups 222-225 and specifying the cleavage site of protease factor Xa; Peptide part, each nucleotide base sequence (SEQ ID NO: +28): GGG A TCCCCATCGM　TTCCTG　CAG　Ami coded by CCC Noic acid residue sequence (SEQ ID NO: 29) + Gly lie Pro lie Glu Phe Leu Gin Pr.

Linker protein corresponding to residues 226 to 234, consisting of amino acids of SEQ ID NO: l Residue 2 specifies the NANBV envelope antigen with residue sequence 121-176 the carboxy-terminal polypeptide portion corresponding to 35-290, and the respective nucleic acids. Reotide base sequence (SEQ ID NO: 30): TGG GGG ATCGGG MT Amino acid residue sequence encoded by TCA TCG TGA (SEQ ID NO: 31 ): Trp Gly [le Gly Asn Ser Ser END] The carboxy-terminal linker moiety corresponds to residues 291-298.

Plasmid pGEX-3X-15:17 was formed by subjecting it to The edges were filled as in Example IB(+). SEQ ID NO: 1 base 361 to base The resulting free DNA fragment containing a sequence containing 978 was used in Example IB (1). ff1l like I did! did. by Smar as done in Example IB(1). Mix the purified DNA fragment with pGEX-3X linearized by restriction enzyme digestion. and ligated to form plasmid pGEX-3X-15:I7.

The pGEX-3X plasmid containing the 15:17 DNA fragment was prepared in Example IB (2). The DNA1i product was identified by selection as described above, and the DNA1i product was purified with EcoRI and as indicated above. and BamHI. Exact copy of in-frame translation of NANBV structural proteins A pGEX-3X vector containing a 15:17 DNA fragment having a code sequence was prepared according to the example. identified and selected as done in step (2) to form pGEX-3X-15:17. Ta.

The obtained vector encodes the fusion protein (GST:NANBVl 5:17) This is the amino-terminal polypeptide portion corresponding to residues 1 to 221 of GST, the remainder an intermediate polypeptide corresponding to groups 222-225 and specifying the cleavage site of protease factor Xa; Peptide part, each nucleotide base sequence (SEQ ID NO: 32): GGG A TCCCCAAT Amino acid residue encoded by TCCTGCAGCCCT Sequence (SEQ ID NO:+33): Gly IIs Pro Asn Ser Cys Ser　Pr.

Linker protein corresponding to residues 226-233 consisting of amino acids of SEQ ID NO: l Residue 2 specifies the NANBV envelope antigen developing residue sequence 121-326 34-439, and the carboxy-terminal polypeptide portion corresponding to nucleotides 34-439, respectively. Reotide base sequence (SEQ ID NO: 34): GGG ATCGGG AAT TCA Amino acid residue sequence encoded by TCG TGA (SEQ ID NO: 35): G Residues 440~ consisting of ly lie Gly Asn Ser Ser END The carboxy-terminal linker moiety corresponds to 446.

■ The cohesive ends were filled in as in Example IB(1). Base 3 of sequence ID number l The resulting free DNA fragment having a sequence containing bases 61 to 978 was used in Example 1B. Purified as performed in (]). Small as done in Example IB(1) pGEX-2T vector (Pharmacia) linearized by restriction enzyme digestion with The purified DNA fragments were mixed and ligated to create plasmid pGEX-2T-15:1. 7 was formed.

+5　2　　7DNAArrfr fragment-containing pGEX-2T plasmid was used in Example I B(2) identified and selected as done in Example IB(2), pGEX-27-15 : I7 is the amino-terminal polypeptide corresponding to residues 1 to 221 of GST. The nucleotide base sequence (SEQ ID NO: :36): GTr　CCG　CGT　GGA　Amino encoded by TCC Acid residue sequence (SEQ ID NO: 37): From Val Pro Arg Gly Ser The intermediate polypeptide portion that specifies the cleavage site of the protease thrombin, which Respective nucleotide base sequence (SEQ ID NO: +38): CCA TCG MT TCC Amino acid residue sequence encoded by TGCAGCCCT (SEQ ID NO: 39) P ro Ser Asn Ser Cys Ser Pr.

A linker protein corresponding to residues 227-233 consisting of NANBV en bellow The callevoxy-terminal IJ peptide corresponding to residues 234-439 specifies the and the respective nucleotide base sequences (SEQ ID NO: 40): GGA A Amino acid residue sequence (sequence) encoded by Tr　CAT　CGT　GACTGA Column number: 41): Gly [le His Arg Asp END The carboxy-terminated moiety corresponding to groups 440-446 is composed of:

Oligonucleotide 690:691 was used in the PCR reaction as done in (6). did. The obtained PCR amplified dsDNA was purified as described in Example IA (4). This pUC18 cloning vector was then cloned into pUC18690:6 91 was formed. pUCl 8 primers (2) as described in Example IA(5) More clones were then sequenced to identify plasmids containing the complete sequence.

The resulting t17′ identified plasmid was selected and designated pUc18690:691. (361 bp in length and nucleotides 1 to 3601 of SEQ ID NO: 1: : Contains the NANBV DNA fragment.

Plasmid pUc18 690:691 carried out in Example IB (1) f, Z J: The sea urchin was first subjected to restriction enzyme digestion with EcoRI and BamHI. From this, plasmid pGEX-3X-490:691 was formed. ptrctsbo I] A sequence with 1 non-calg sequence j and containing base 1--base 360 of SEQ ID NO: 1 The isolated free DNA fragment with did. Lines were isolated by restriction enzyme digestion with SmaI as performed in Example IB(1). Mix and ligate the rt-prepared DNA fragments to the t-pGEX-3X vector. The lasmid pGEX-3X-69o:691 was formed.

The pGEX-3X plasmid containing the 690:691 DNA fragment was prepared in Example IB (2). Identification was made by selection as was done in . In-frame translation of NANBV structural proteins pGEX-3X vector containing the 690:691 DNA fragment with the exact coding sequence. pGEX-3X-690 was identified and selected as in Example IB(2). :691 was formed.

The obtained product 9) contains the fusion protein (GST: NANBV690:691). This is the amino-terminal polypeptide portion corresponding to residues 1-221 of GST. , an intermediate corresponding to residues 222-225 and specifying the cleavage site of protease factor Xa. PoIJ peptide portion, each nucleotide base sequence (SEQ ID NO: 42): GG G ATCCCCAAT TCG AGCTCG GTA Coded by CCC Amino acid residue sequence (SEQ ID NO: 43): Glylle Pro Asn Ser　Ser　Ser　Val　Pr.

A linker protein corresponding to residues 226-234 consisting of NANBV capsid antigen a carboxy-terminal po IJ peptide moiety corresponding to residues 235-355 specifying and nucleotide base sequence (SEQ ID NO: 44): ACG GGG A Amino acid residue sequence encoded by TCGGG MT TCA TCG TGA Column (SEQ ID NO: 45): Thr　Gly　[le　Gly　Asn　Ser　S The carboxy-terminal l-luca moiety corresponding to residues 356-363 consisting of er　END (It consists of

20 (-) to SEQ ID NO. nia and SEQ ID NO. 28 (two corresponding nucleotides), respectively. Preparation 1 as described in Example IA (2), ．． Sixty-two.

Oligonucleotides 1-20(+) and 1-20(-) were combined with EeoRI and Expression vector pGEX-2T predigested with BamHI (Pharmacia ) and kept under annealing conditions to remove the complementary oligonucleotide. double-stranded (ds) t ribonucleotides obtained by allowing hybridization The cohesive ends of the product are pGEX-2Tf with EcoRI and BamHI cohesive ends. B) It made it possible to hybridize. After ligation, pGEX-2T-CAP-A The resulting plasmid contains a single copy of the ds oligonucleotide product, and CAP having the amino acid residue sequence shown in residues 1 to 252 of SEQ ID NO: 3. -A contains a structural gene encoding a fusion protein.

The resulting fusion protein is digested using the site-specific protease thrombin (digestion 1; The pGEX-27 vector is similar to the pGEX-3X vector described above, except that it is cleavable. Similar to number two.

2l-40(-) correspond to SEQ ID NO: 9 and SEQ ID NO: 10f, respectively. As those having the nucleotide base sequence, as described in Example IA (2) + Sea urchin was prepared.

Oligonucleotides 2l-40 (10) and 2l-40 (-) were purified with EcoRI and and pGEX-2T expression vector predigested with BamHI in equal amounts. and maintained under annealing conditions to separate the complementary oligonucleotides. cohesive end forces of the resulting double-stranded oligonucleotide product. (pGEX-2Tl with EcoRI and BamHT cohesive ends) It made it possible to zoom in. After ligation, pGEX-2T-CAP-B was obtained. The plasmid contains a single copy of the ds oligonucleotide product and the sequence 7 CAP- having the amino acid residue sequence shown in residue 1 to residue 252 of fi1 number = 4 It contains a structural gene encoding the fusion protein designated as B.

4l-60(-), respectively SEQ ID NO: ll and SEQ ID NO: 12+: corresponding The nucleotide base sequence described in Example IA (2) Yo number = 11E! did.

Oligonucleotides 4l-60(+) and 4l-60(-) were purified with EcoRI and and pGEX-2T expression vector predigested with BamHI in equal amounts. The complementary oligonucleotides are then separated into two molecules by maintaining them under clear-neeling conditions. The sticky ends of the resulting double-stranded oligonucleotide product Hybridizes to pGEX-2T with EcoRI and BamHI cohesive ends. made possible. After ligation, the resulting plasmid as pGEX-27-CAP-C The code contains a single copy double-stranded oligonucleotide product, and SEQ ID NO: A fusion resulting in CAP-C that develops the amino acid residue sequence shown from residue 1 to residue 252 of 5. It contains structural genes that code for proteins.

pGEX-2T-CAP-A-B: Vector expressing CAP-AB fusion protein The oligonucleotides constituting pGEX-2T-CAP-A-B were Has a nucleotide base sequence corresponding to SEQ ID NO: 13 and SEQ ID NO: 14 It was prepared as described in Example IA (2).

Oligonucleotides according to SEQ ID NO: 13 and SEQ ID NO: 14 were prepared in Example IB Mix in equal amounts with plasmid PGEX-3X-690:694 described in (2). Ta. Combine the mixture with reagents for polymerase chain reaction (PCR) and perform the PCR reaction. The two mixed acids on pGEX-3X-690+694 mixed as template using oligonucleotides as primers containing residues 2 to 40 of SEQ ID NO: l. BamHI encodes the amino acid residue sequence at the 5' end. P consisting of a double-stranded nucleic acid molecule containing PCR addition restriction sites for EcoRI at the ends. A CR extension product was formed. The PCR extension product was then treated with restriction enzymes BamHI and and EcoRI to generate cohesive ends on the PCR extension product. Adhesive powder The resulting end-growing product was pre-digested with EcoRI and BamHI. The pGEX-27 expression vector was mixed in equal amounts with the pGEX-27 expression vector, maintained under annealing conditions, and then The sticky end of the full-strand PCR extension product is peptidized with EcoRI and BamHI sticky ends. This made it possible to hybridize with GEX-2T. After ligation, pGEX-2T The resulting plasmid named -CAP-A-B contains two single copies of 1. PcR extension containing a long product and having the amino acid residue sequence shown in residues 1 to 271 of SEQ ID NO: 6. Contains a structural gene encoding a fusion protein with a sequence of CAP-A-B It is.

Bacterial colony containing pGEX-3X-690+694 plasmid in correct orientation The properties of the fusion protein were investigated. In the presence of ampicillin (50℃g/ I) GEX-3X-690:694 bacterial culture at 37 °C (ml final concentration) was grown to the stationary stage. This culture was incubated at 37°C in the presence of ampicillin 1: Inoculate fresh LB medium at 50 dilution and maintain at 37°C with stirring at 25 Orpm. When measured using a spectrophotometer equipped with a light source detector with a wavelength of 550 nm It was assumed that the bacteria reached an optical density of 0.5. Then 1 m for bacterial cultures. Mix isopropylthio-β-D-galactoside (IPTG) at a final concentration of M. , initiates the synthesis of the fusion protein under the control of the tac promoter in the pGEX-3X vector. started (induced).

Starting at zero time after mixing of IPTG and then at 1 hour intervals for 3 hours (i.e. During the lag phase), bacterial cultures were maintained as described above to allow expression of recombinant proteins. . During this maintenance phase, the optical density of the bacterial culture was measured and 1 m removed 100% of Each resulting pellet containing crude protein lysate was , 1% β-mel in a final volume of 50 μm for 0.5 0D550 units, respectively. Resuspend in Laemmli dye mix containing captoethanol. Ta. Boil the samples for 15 min, and add 10 μm of each sample to 10% 5DS. - Electrophoresed on PAGE layer gel.

By transformation with appropriate expression vectors and induction as described above, other GS T:NANBV fusion proteins were also expressed in bacteria.

Example 33 Detection of expressed fusion protein To visualize IPTG-induced fusion proteins, coat Laemili gel with Coomassie blue. and destained in acetic acid and methanol. Derivation from separate clones The predicted proteins were tested at time zero to time three hours after IPTG treatment. Comparisons were made based on the increase in protein bands in the given size range. Development of fusion protein Currently, the increase from zero time in the intensity of the protein band corresponding to the fusion protein is Found in the indicated clones.

When analyzed on a 12.5% PAGE gel as described in Example 2, GST:NANBV fusion proteins CAP-ASCAP-B and CAP-C are approximately It exhibited an apparent molecular weight of 30,000 Daltons.

Example 4. western plot analysis Samples from IPTG derivatives containing GST:NANBV fusion proteins of this invention separated by gel electrophoresis and nitrocellulose for subsequent immunoplot analysis. Moved it above. The nitrocellulose filter was soaked in antibody blocking buffer (20mM phosphorus). Sodium acid, pH 7,5,0,5M sodium chloride, 1% bovine serum albumin and 0.05% Tween 40) for 3 to 12 hours at room temperature. NANBV structure Humans with NANBV hepatitis thought to contain antibodies immunoreactive with the protein-producing protein or Serum from chimpanzees was diluted 1:500 in antibody blocking buffer and mixed with cellulose and kept at room temperature for 12 hours to form immunoreaction products on the solid phase. I let it happen. The nitrocellulose was then washed three times in excess volume of antibody blocking buffer. did. After washing, 50 μm l1ll Protein A (Niney England Nu Clare, Boston, MA) in antibody blocking buffer at a 1:500 dilution. Mix the nitrocellulose at room temperature for an hour and add the labeled protein A to the nitrocellulose. All immunoreaction products present on the solid phase on Lulose were bound. then nitro The cellulose was washed and dried as described here, labeled and therefore nitro To visualize all immunoreaction products on the cellulose, incubate at -70°C for 1-3 hours. Exposed to X-ray film.

The results of the Western blot immunoassay are shown in Tables 2-7. p producing control GST Samples using the GEX-3X vector were also prepared as described above, Tested as a control using Western blot procedure. Fusion protein of this invention (e.g. GST:NANBV690:694 fusion protein) The expressed GST protein could not be detected.

After culturing the transformed E. coli strain W3110 culture for 3 hours, fPTG Induction treatment was performed. The cells are then centrifuged to form a bacterial cell pellet and l/ 200 culture volumes of lysis buffer (MTPBS: l 50mM NaC), +6m Resuspend the cells in MNat HPO4, 4mMNa5 PO4, pH 7,3), The cell suspension was lysed using a French press cell. Cell lysis with Triton X-100 It was mixed into the lysate to give a final concentration of 1%. 50,000×g of the mixture Centrifugation was performed for 30 minutes at 4°C. The resulting supernatant was collected and pre-expanded in MTPBS. 2 ml of moistened 50% (w/v) glutathione agarose beads (Sigma, SE) Lewis, MO). Hold the mixture at 25°C for 5 minutes to remove GST and solidify. Centrifugation of IoooXg after allowing specific affinity binding with glutathione in the phase Beads were collected by separation and washed three times in MTPBS.

2ml of 50mM trifle containing 5mM reduced glutathione, HCl, pH 8, Mix and incubate the glutathione beads for 2 min at 25 °C using The GST:NANBV690:694 fusion protein was washed with glutamine. GST:NANBV690:694 fusion eluted from on-beads and purified formed proteins.

As determined by SDS PAGE according to the affinity purification procedure described above. More than 95% pure fusion protein was produced. In other words, the purified protein is substantially containing prokaryotic antigens and nonstructural NANBV antigens, as specified herein. do not have.

In addition, the GST:NANBV690:694 fusion protein was subjected to anion exchange chromatography. Cultures were prepared as described above, and cell pellets were purified by chromatography. The fusion protein was solubilized by resuspending in M guanidine and kept at 4°C overnight. the The cell suspension was then subjected to an S-300 Sepharose chromatography column and GST :NANBV690: Collect and pool the peak fractions containing the l394 fusion protein. The fusion protein was dialyzed in 4M urea and purified by anion exchange chromatography. Formed white matter.

After its introduction by transformation into the E. coli host, the GS clone produced in Example 1 Other GST:NANBV fusion proteins described herein using the fusion protein vector was also expressed in culture of E. coli strain W3110 as described above. Culture attraction After induction and lysis, use glutathione agarose affinity chromatography The GST fusion protein was purified as described above and determined by 5DS-PAGE. The resulting fusion protein was more than 95% pure. Therefore, each pGEX-2T-CAP-A vector, pGEX-2T-CAP-B vector or Using pGEX-2T-CAP-C vector, CAP-A, CAP-B and C All AP-C fusion proteins were expressed and purified as described above, and PGEX-2T - Express and purify CAP-A-B fusion protein using CAP-A-B vector did.

Purified GsT:NANBV690:694 fusion i white produced by vR in Example 5 The GST carrier was subjected to treatment with activating factor (Xa) (Sigma) and the GST carrier was Cleaved from the NBV690:694 fusion protein (Smith et al., supra). 75 nano grams (ng) of activated enzyme, 8mM) Lis HCI (pH 8,0), 70mM by mixing with NaC1 and 8mM CaC1t and maintaining at 37°C for 5 min. Activate 7 μg of Factor X before mixing with the purified fusion protein. after that, 50mM Tris HCI, 5mM reduced glutathione, 100mM as described in Example 5. Purified cells were purified at 50 °C in elution buffer containing MNaCl and ImMCaCI*. The fusion protein was mixed with soon activated human factor Xa and incubated at 25°C for 30 minutes. maintain for a while.

The resulting cleavage reaction product was then converted into the glutathione-agarose prepared in Example 5. It was adsorbed onto beads and released from the completely cleaved NANBV structural antigen-containing protein. GST is separated by affinity binding. The liquid phase was then collected and the residue of SEQ ID NO: 2 A purified NANBV construct having an amino acid residue sequence comprised between residues 226 and 315 A solution containing the synthetic protein is formed.

(INOc) and five different diagnostic assays to determine NANBV. We analyzed the immunological responses to Chimpanzees have no acute or chronic phase of infection. It was classified based on whether it was present or not. Assays used to assess immune responses included l) alani Aminotransferase (ALT) Enzyme Detection (Alter et al., J AMA.

246:630-634.1981, and Arch et al., N., Engl., J., M. ed, 304:989-994.1981), 2) by IE electron microscopy (EM) Histological evaluation for NANBV virus particles, 3) containing C100-3 antigen Detection of anti-HCV antibodies using a commercially available kit (Ortho Diagnostics) , 4) Immunoplot as described in Example 4 using CAP-N fusion protein Detection of anti-CAP-N antibodies by analysis, and 5) PC as described in Example 1. Includes detection of viruses by R amplification.

ALT, EM, anti-HCV ( The results of anti-C100-3), anti-CAP-N and PCR assays are shown in Table 2.

Table 2 Chimpanzee 59 - week after acute NANBV hepatitis 1 vaccination.

“Plus (+)” indicates that an immune reaction was observed between the mixed serum and the fusion protein. This was designated as rcAP-Nj because it is the presumed NANBV capsid protein. This is because it corresponds to the amino terminus of white matter (Western profiling described in Example 4). using immunoassay)a The results in Table 2 demonstrate the relationship between the fusion protein and anti-NANBV structural protein antibodies in the tested serum. It shows an immune response between. Additionally, immunoassays using fusion proteins containing capsid antigens , the same serum is assayed in a C100-3-based immunoassay at an earlier time point. can detect seroconversion.

ALT against serum collected from humans with confirmed NANB hepatitis, anti-HCV (anti-C10 0-3) and the results of the anti-CAP-N assay are shown in Table 3.

Table 3 The results in Table 3 show that in human lineage 169 seroconverted serum samples, the presence of fusion proteins in NANBV-specific antibodies were detected as early as 14 weeks after vaccination using the CAP-N antigen. In contrast, in the immunoassay based on CI 00-3, anti-NAN This shows that no BV antibodies are detected.

ALT, EM, anti-H against serum from chimpanzees with self-limiting infections given The results of the CV and anti-CAP-N assays are shown in Table 4.

Table 4 1328ND - 10 16 25ND -+ 18 23ND + + 2025-++ The results in Table 4 show that sera sampled during the course of a self-limiting NANBV infection When used, the CAP-N antigen elicited anti-NANBV antibodies faster than the C100-3 antigen. Indicates that it is detected.

ALT on serum from chimpanzees that have converted from an acute mode of infection to a chronic mode of infection. , anti-HCV and anti-CAP-N assay results are shown in Table 5.

Table 5 Acute 2 223 - 10 Chronic 40 223 + 10 Chronic 42 223 + + Chronic 44 223 + + Chronic 51 223 + - The results in Table 5 show that the CAP-N antigen is NANBV! Anti-NANBV during the acute stage of infection Indicates that the body is preferentially detected.

Blood collected at various intervals from several chimpanzees with acute or chronic NANB hepatitis. ALT, EM, anti-HCV (anti-C100-3) and anti-CAP-N tests against serum The results are shown in Table 6.

Table 6 8 + 4 125 - + +5 +1 82 - + 156 +131110 + ten The results in Table 6 show that the CAP-N antigen was found in anti-NANBV antibodies in serum from acutely infected individuals. C100-3 antigen is detected more frequently than the C100-3 antigen.

The results in Tables 2-6 show that the vector pGEX-3X-690 = 69 containing CAP-N antigen The NANBV structural protein of this invention in the form of a fusion protein produced by C. 100-3 antigen earlier than can be detected by current state of the art methods. occasionally detecting specific seroconverting lineages in infected patients or chimpanzees. show. Furthermore, these results indicate that the CAP-N antigen is present in acute NANBV early in infection. Shows particular utility in detecting infection.

Together, these results demonstrate that NANBVI: immunospecific for the indicated NANBV antigens, in particular the putative CAP-N-specific Contains antibodies circulating in the blood that are shown to be immunoreactive with capsid antigens Show that. Therefore, these antibodies can be used as anti-NANBV structural protein antibodies. previously described using NANBV nonstructural protein antigen C100-3. It should be distinguished from the type of antibody detected in

Next (7) HCV capsid fusion proteins: CAP-N, CAP-A, CAP-B and In Example 4 using various chimpanzee and human sera against and CAP-C. Comparison from anti-HCV capsid fusion proteins by the basic immunoplot assay described. The results are shown in Table 7.

Table 7 Serum type "CAP-N" CAP-A' CAP-B' CAP-C"C1 0 chimpanzee 10 (A) +++++ 10-CIO chimpanzee 194 (A) ++10+++ +++ -59-16 Chimpanzee 59 (A) +++ + +++ ND59-12 Chimpanzee 59 (A) ND' ++ +++ - C9 chimpanzee 181 (A) +++ -++++2+3-18 chimpanzee -213 (A) ND + + -C2 chimpanzee 10 (C) ++ --- CI Chimpanzee 10 (C) + 10 --- C19 Chimpanzee 10 (C) +10---C4 chimpanzee 68 (C) +++++++++ ND169 -16 Human ND +++ +++ - Chimpanzee when derived from pansy – Identification number and whether the serum donor was acute (A) or is indicated by the indication (in parentheses) when indicating chronic (C) HCV infection.

b. CAP-N was produced in Example 5 containing HCV capsid protein residues 1-74. This shows the GST:NANBV690:694 fusion protein.

c, cAP-A was produced in Example 5 containing HCV capsid protein residues 1-20. The resulting GST:NANBV fusion protein is shown.

d. CAP-B was produced in Example 5 containing HCV capsid protein residues 21-40. The resulting GST:NANBV fusion protein is shown.

e, cAP-C produced in Example 5 containing HCV capsid protein residues 41-60. The resulting GST:NANBV fusion protein is shown.

f, +, ++ and +++ are anti-HC detected by Western blot assay. Indicates the relative amount of V capsid antibody immunization product, in this case XPJ! Fi ++ indicates a weak band after overnight exposure of X-ray film, and ++ indicates a strong band after overnight exposure of X-ray film. indicates a band, +++ indicates a strong band after 1-2 hours exposure of X-ray film; Also, +/- or - is faint or non-existent, respectively, after overnight exposure of the X-ray film. Indicates a new band.

g, indicates that rNDJ was not tested.

The results shown in Table 7 show that the fusion protein containing CAP-A antigen or CAP-B antigen Antibodies and immune reactions present in serum derived from HCV-infected humans or chimpanzees indicates gender. Additionally, the CAP-C antigen can be expressed in HCV-infected humans or chimpanzees. It does not react immunologically with serum derived from G.

NANBV, also referred to as hepatitis C virus (HCV), was prepared in Example IA (1). ) was inoculated with the Hatsuchi (H) strain of HCV (designated HCV-Hc59). Two tissue sources derived from an HCV-infected chimpanzee, number 59 (C59). isolated from. Chimpanzee livers were biopsied during the acute phase of infection (4 weeks after inoculation). , chimpanzee plasma was collected 13 weeks after vaccination. Ogata et al., Proc, Natl. , Acad, Sci, USA, 8B:3392-3396 (199]) and Nucleic acid extraction from liver was performed as described in Example IA(6). 10'・1~ 10’・’CIDI. HC virus as having a virus titer of /ml Particles were isolated from plasma. The immunoaffinity clone described in Example IA (1) HCV RNA was isolated from blood by matography or by isopropanol precipitation. Purified from plasma samples.

Briefly, 4.2M guanidinium isothiocyanate, 0.2M guanidinium isothiocyanate; 5% Sarcosyl and 0.025 M Tris-H (1, pH 8,0) using a water-cooled buffer containing 50 μl of plasma was diluted. Then diluted plasma and 100mM 1'Lis HCI , pH 8,0, l OmMEDTA and 50 μl extraction containing 1% SDS A buffer solution was mixed to form an extraction mixture. Stir the mixture with a portex The extraction was started by holding at 65°C for 5 minutes. Then, at 65°C, phenol/chloride Serum proteins were removed from the mixture using loloform, followed by chloroform alone. One extraction was performed. Then 2 volumes of water-cooled isopropanol and 1/10 by mixing a volume of 3M sodium acetate and keeping the mixture at -20°C overnight. HCV RNA was precipitated from the protein-free mixture. Etzbendol pelleted by centrifugation at 1400 rpm for 30 min at 4°C in a microcentrifuge. After that, HCV RNA was washed once with 70% ethanol, dried in vacuum, and then μm RN8 was resuspended in water without e.g. Below and Example IA (1 ) Purified HCV RNA samples were prepared prior to cDNA synthesis as described in Pull for 1 minute! Heated at 1$65°C.

(2) Cloning of HCV-Hc59 cDNA 5 μg of ffI liver or plasma Induced HCV RNA was used per cDNA prime reaction. Published HCV distribution using specific nucleotide primers that span the entire genome sequence derived from the sequence and reported. The reaction was started using Saiki Moto et al., Japan.

J, Exp, Med, 60:167-177 (1990), Kado et al., Pro c.

Natl, Acad, Sci, USA, 87:9524-9528 (1990 ), Han et al., Proc, Nat 1. Acad,Scot,USA,88: 1711-1715 (1991) and Houghton et al., European Patent Application [*8 Reference may be made to No. 8310922.5 and Publication No. 318218. Selection The selected target sequence is a combination of various nucleotide sequences as described in Example IA (3). Amplification was performed using a PCR-based method using primers. Prime Nucleotchie The code sequences are listed in Table 8 below, by primer number and corresponding sequence number. It has been identified.

Nucleonucleotide primers cloned from HCV-HC59 cDNA Sequence Nucleotide sequence (5'-3') ffi 8 (#) 10 number 8 Q 78 CAGAGCTTCCAGGTGGCTC+795’ 79 C to GGCTCCGTCACTGTG794 80 GTATrGCAGTCT 464 1111 GGCTkTACCGGC(, ACT' rCGk Ju 40 82 CGTrGAGTGCCGGAGACAG463 B 3　TCACC input TTGAGAC input ATCACC+788　Fi4　GTAAG AAC; GTTCTCCCCCACTC-57185ATGCCCACTTrC TATCCCAGACAj+J to C+625 E18 C (, TATTGCCT TCAACAGGC + 6 pieces 1 89 λGCGCCCAC input GGC input GT λG342 90 CCTCTTcAACATATT to GGG +84 91 CCAGG input eight CC Niheni AGCAT (:G359 92 λCO, GTGG kTAAGcTcGG +904 9:l CC, TGGTGTAGGC input TT Entering TG862 94 ATGTGI;AGTGGGACCTTCC+861 95 CTCTCCTC; TTATAT to GGAGGF4 96 to TTGAC GTCCATGCTCACTG + 4 97 TTTCCACGTCTCCA CTAGCC;-84998GT[;CGCFl to AGCλCO800GT 99 TTCCACCTCC; request GTCCC ('I' ↓50 105 people G CTTCCCCATCACGGCCAA502 106 GATCGCTrTGT ACCACGTC; +55 107 C; CACCTGCG into T into GCC AGTE152 108 GTCCCTCACCGλG input GGCT +85 pieces 109 GATTGGAGCTAGATkoAACTG4 110 TACCAC TT into GA (, CTCATAAC62in, CAAG into CACACTCC) GTCA61 1L! GCCTATTGGCCTGGAGTG to TTAGCa (+) indicates sense value (=) indicates antisense strand The amplified sequences were then isolated, blunt-ended, and prepared as described in Example IA (4). pUC or pBluescript (Stratagene) clones using standard procedures such as Inserted into loan vector.

(3) Sequence analysis of cloned HCV-Hc59 cDNA DuPont automatic sequence Cloning using the dideoxy chain termination method using the Genesys 2000 determination device was sequenced. Due to PCR artifact (misreading by Taq polymerase) To minimize sequencing error, three independent clones were run for each target sequence. The sequence was isolated and subsequently sequenced. HCV Hatsuhatsu (HCV-H) genome The resulting sequences were compared to derive the final consensus sequence represented. how many In some cases, several clones derived from independent studies share the same genomic domain. Including in. Sequencing of these clones provides further confirmatory data. Ru.

(4) Characteristics of cDNA clones and primary structure of HCV-Hc59 Above and Examples Select the primer pair as described in IA (3) and inject the HCV-Hc 59 gene. They amplified specific regions of the genome and generated overlapping clones, whose sequences were not derived from the entire genome. ). The primer pairs used in the specific PCR reactions are listed in Table 9 below. Selection The resulting 40 cDNA clones generated from the selected primer pairs are zero in the same table. The estimated map positions shown in Figure 1 are listed by numbers starting from and ending with 39. This corresponds to The calculated base pair size of each isolated cDNA The same is shown in Table 9.

Table 9 PCR-induced HCV-HC59 clone 2 6ri 0:694 224 7 71f + 567 548 a121 tHcV-Ha59'f/mu upper (:') phase n position.

5 Sense (+) and ant with the nucleotide sequences shown in Table 1 and sequence listing. Chisens〈- Primer Vs.

゛Using the primer pairs shown as described in Examples IA(3) and 8A(3) The base pair Cbp) of the cloned insert produced by the PCH used size.

Comparison of the sequences of three cDNAs independently isolated from the same genomic domain revealed that It became clear that there were almost no differences in cleotides, and the virus preservation was uniform. to show that The complete HCV-H genome sequence was sequenced, but only 9416 nucleotides were sequenced. This represents the previously isolated HCV genomes HCV-ISHCV-J and Similar in length to HCV-BK. Kado et al., supra, Chu et al., Proc, Nat] , Acad, Sci, USA, 88:2451-2455 (1991) and See Takamizawa et al., J. Virol, 65:1105-1113 (+991). 46), one corresponding to a protein of 3011 amino acid residues (SEQ ID NO: 246) Includes large oven reading frame. HCV-Hc59's performance The nucleotide sequence has been deposited with GenBank, accession number M6746. 3.

5' and 3' ends spanning 341 and 42 nucleotides, respectively The HCV-Hc59 sequence from the (NC) domain was identified. first 12 nuclei octide and the last 20 nucleotides (SEQ ID NO: 46, see characteristics ) corresponds to the nucleotide primers used in the amplification process and therefore HCV - Not confirmed as H sequence. However, the previously reported HCV genome The 5' non-cod sequence is highly conserved (>98%) and the HC The 5' end sequence of V-H is very similar, if not identical, to that shown. give the possibility of However, the large difference in F(CV-3' non-coal sequence Therefore, the HCV-Hc59 3' terminal sequence remains to be confirmed. Oligo ( dT) Using different combinations of primers after using the primers for cDNA synthesis No viral sequences were obtained when PCR amplification was performed. This result is The Rus genome contains an internal 8-rich region at the 3' end or 3' end poly bA) sequence. Indicates that the amount is missing. Similarly, two reported Japanese isolates, HCV-J and 8-rich complementary to the 3′-terminal (U-rich) nucleotide sequence of HCV-BK. The sequence was not amplified when the primers were used in the RT brining reaction. Therefore, it is suggested that there is no U-rich terminal sequence in the genome of HCV-Hc59. be done.

There are five large open reading frames in the HCV-Hc59 RNA genome. AUG codon (as shown in SEQ ID NO: 46) Base numbers I3.32.85.96 and 2+4)l: leading from HCV genome The existence of a hypothesized small open reading frame in the 5'NC region of It is confirmed. Shown in sequence ID number 46 listed as part ~ R6 in the listed characteristic part Several repeated sequences are present in the 5' and 3'NC regions and in the putative NS5 It was identified at the C-terminus of the domain. These sequences are involved in controlling viral replication. This probably corresponds to an important Cis-acting element.

The repeat sequences R1 and R1 are found to be conserved in all HCV isolates. I can't stand it. Other repeat sequences are now already recognized at the terminal end of the HCV genome. However, sequences with regulatory functions, such as Rt and R8, are present in all HCV viruses. It may be a conserved sequence. The repeating array R1 is a particularly significant stream. This is because it is localized in We still know nothing about the presumed cyclization of the HCV virus. It has not been done. These highly conserved self-complementary sequences are probably The replicase recognition site used on both the positive and negative strands of Nome is shown. There is a possibility.

As described in previous reports for other HCV isolates (Kado et al., Proc.

Natl, Acad, Sci, USA, 87:9524-9528 (1990 ), Chu et al., Proc, Natl, Acad, Sci, USA, 88:245 1-2455 (+991) and Takamizawa et al., J. Virol, 65:11 05-1113 (1991) L HCV-Hc59 genome Mataha protein is 3 All but one domain shares only limited similarity with other known viral sequences. (L) Some strands of nucleotides in the 5'NC sequence are similar to the American prototype. Type HCV-1 (SEQ ID NO: 46) was reported by Chu et al., supra. (2) putative NTP-binding helium The putative NS3 domain corresponding to the proteinase and trypsin-like serine proteases found in (nucleotide base numbers 3693-5198, SEQ ID NO: 46) A block of amino acids is conserved by flaviviruses and pestiviruses, (3) The GDD consensus sequence is an RNA-dependent sequence encoded by all viruses. native RNA polymerase (amino acid residues 2737-2739, SEQ ID NO: 46) It is preserved in. Furthermore, a total of 19 putative N-glycosylation sites , Mayer et al., Virol, 17++555-567 (1989) and Collet. Tora, Virol.

+62:167-180 (1988), bestivirus Amino acid residue 1 in a manner similar to the configuration observed for envelope proteins. It was located between 96 and 647 and was mainly clustered.

Different genomic domains of HCV-Hc59 and American type (HCV-1) or American type (HCV-1) between the American-type isolates (HC-Jl) and the Japanese-type isolates HC-J4, H CV-JH,I (comparison of CV-J and HCv-BK) summary is shown in Table 10. show. Sequence comparisons are limited by HC-Jl, HC-J4 and HC-JH.

This is because the complete genome sequences of these isolates have not yet been reported. array Presentation of a hypothetical map of HCV-encoded proteins derived from the HCV genome and flaviviruses and/or pestiviruses. , was used to make the comparison. References for compared sequences are listed at the bottom of Table 1O. Write down. Based on sequence comparisons to related viruses, the HCV genome is shown in Table 10. It is believed to encode at least eight domains as shown. In other words, From the cleocapsid (C) and two envelope (El and R2) proteins structural domains and five proteins NS2, NS3, N54a, N54b and This is an unstructured region consisting of NS5 and NS5. The domain naming is based on the associated H for comparison purposes. Based on the composition of CV strains, the current method of characterizing the domains of HCV-Hc59 The technique does not necessarily reflect the domains of HCV-Hc59.

Table 1O HCV-Hc59, H Creotide and wrapped T. Ami 7-cho rhodomain 1 41 self 5'NC ratio Mi 32 ≦ 5th ward E! ≦■　≦Work i Hiyu≦■＊bp”　99 ,7 99.1 99.1 9B, 9 9L2 98. Bbp 98.4 98 ．． 9 90.0 90. ) 91.0 90jbp 93.6 9yu, 7 67 ．． 7 65゜4 73.5 712bp 9co-8---71472,7 bp　95.4　--　--　--　80.1　713.9≦ヱ　　巳rim　≦ bmu　Tsuji Yu≦■≦Concave go out Nyo book 54a Nuku performed from J, Virol, 65:1105-1113 (1991) The two sets of repeat sequences found in 46) are conserved in all reported isolates. has been done. Two copies of the repeat sequence R1 are also present in the two American isolates of HCV- Conserved between Hc59 and HCV-1, but Japanese car isolate HCV-J and Only one copy is found in both HCV-BK and HCV-BK. of these genomes The 5'NC sequence does not extend far enough to span the second copy. Other HCV Nucleotide sequences reported for exophytes are not far enough 5' to allow comparison. It has not been extended to NC.

Regions of loose identity are found throughout the unstructured domain; in this case there are two groups (American A clear separation between isolates of type 7 (Japanese type) can be observed. HCV-Hc59 as the first When compared with the group, nucleotide identity of 93.8 to 95.9% is observed. In contrast, only 72.7 to 80.0% identity with the second group was observed ( Amino acid identity of 95.1-97.2% and 78.2-92.6% respectively ). Found in predicted NS5 (amino acid residue position It235 of SEQ ID NO: 46) 6-2379), one region called region V3 as shown in Table 11 below is This reflected the more pronounced differences between the two subgroups of HCV monomers. this The region showed 100% identity between the two American isolates (data not shown). However, only 12.5% of the stocks were from Japan Y stocks. Most changes are preserved. It is thought that this is a change in the biological properties of the protein, and therefore results in a modification of the protein function, resulting in an undesirable change. However, it remains to be seen whether this genomic region is immunologically active or not, and whether the two enzymes in HCV isolates It would be interesting to assess whether there is also antigenic variation between groups. Dew.

Table 11’ Area V (SEQ ID NO: 46 residues 386-411) Isolate 2 (Sequence number: 113) His vax Thr Gly GIY ser Ala Gly His Thr Val 5erG1y Phe Val Ser Lau Leu 1a Pro GIY included 1a Lys GlnASn Val (Sequence number: 114) His Val Thr to ly Gly Gln enter 1a enter 1a Arg Enter 1a Met 5erGly Leu Val Ser Leu PheTh r　Pro　Gly　Ala　Lys　G1n5er　xAu　Val　Ser Trp Lau Sir Gin Gly Pro sir Ginλrg Wu Val Ser Met Phe Enter a Ser Gly Pro S er GlnTyr Thr Ser Gly Gly Ala Enter Sa r　His　Thr　Thr　5erThr　Lau　Ala　Ser　Lau Phe Ser Pro G Y input a Ser Arg (Sequence number: 11 FI) 1 no iS Vako Thr Gly Gly Val Gln Gly His Val Thr 5erThr Leu Tor Ser Leu Pha Arg Pro Gly Enter a Ser GlnHis Val Thr G ly Gly ser Ala Gly enter rg Thr Thr Ayu and Gl y Leu Val Gly Leu Lau Thr Pro Gly Al a Lys GinHCV-Hl>-H2O: His Val Thr Gly Gly Ser Enter 1a Gly Arg Ser　VaL　LeuGly　Ile　entering 1a　Ser　Phe　Leu　T hr Arg Gly Pro Lys GlnASn” (Array 1: 12 1) Area V.

(SEQ ID NO: 46 residues 246-275) Lau entered 1a entered 1a Arg entered Sn Ser Ser Engineering 1a Pro Thr Thr Thrlle Ar g Arg His Val Asp Lau Lau Val Gly Al a λ yu a in yu a, mu cys ser in” Met (Array No. 1: 124 ) Leu Enter a Enter Yu a Arg Enter sn Val Thr Engineering 1a Pr o Thr Thr Thrlle Arg)srq Hls Val As p Leu LQu -, 'al GIY Ala enter 1aAla, Ph'' Cys S°″′1″Met (Sequence number: 125) HC-J4: Leu Ala enter 1a Arg enter sn Ala Ser Val Pro Thr Thr Thrlle Arg enter rg His Val enter sp L eu Leu Val Gly Enter 1a Enter 1aAla Phe C! II'5 5erAlaM" (Array No. 18: 126) Leu 1a Ala krq Asn Enter 1a Ser Val Pro Thr Thr ThrLeu A rg enter rg His Val enter sp Lau Leu Val Gly T hr Input 1a Input 1a Phe Cys Ser Input 1a Met (Sequence number: 1 27) Val enter la Thr Arg enter sp Gly Lys Lau Pro Thr　Thr　GinLeu　Arg　Arg　His　工１eλsp　Le u　Lau　Val　Gly　Sar　1aThr　Leu　Cys　Sar Input “” (Sequence number: 122>Val Input a Thr Arg Input sp Gl y Lys Leu Pro Thr Thr GyunLeu Ar(l Ar g His Engineering 1e Enter sp TAu Leu Val Gly Ser Enter 1 aTh″L″Shima゛°”A1°″″ (9th row number 21°2) Area V.

(Residues 456-482 of SEQ ID NO: 46) Lau Ala Sir Cys A rg Enter rg Lau Thr Asp Phi Ala Glr+Gly T rp Gly Pro Eyu e 5er Tyr Ala Asn Gly S er　GlyLeu　Asp　Glu (Sequence number: 128) Leu Ala ser CyS Arg Pro Lau Thr Asp Phe 8sp GinGly Trp Gly Pro Engineering 1e Ser’ ryr Enter 1a Asn Gly 5er GlyPro Asp Gln (Sequence number 129) HC-Jl: Gly Trp Gly Pro Ila Sey: His entering 1 & entering sn Gly 5ar GlyPr’ “p” (Array No. 1: 130’)°”A sp　Gl”, 1 number: 131) Gly　Trp　Gly　pro　Eyu a　 Thr Tyr Enter a Glu Ser Ser Enter r95°y Asp G lo (SEQ ID NO: 132) Gly Trp Gly Pro engineering 1e Thr Tyr Thr Glu Pro Asp 5erLeu Asp Glu ( SEQ ID NO: 128)"pGlu (SEQ ID NO: 134) region V.

(Residues 2356-2379 of SEQ ID NO: 46) (SEQ ID NO: 135) Pro Pro Asp Gin 1a 5er Asp Asp Gly Asp Lys Guy (SEQ ID NO:+136) (Sequence number +137) 1 Regions ■, V, of HCV-Hc59, along with other American and Japanese isolates. , V, and v3 sequence L2 isolate of performed amino acid sequences: HCV-Hc59: American type/chimpanzee 59, Inschausbe et al., Pro c, Natl, Acad, Sci, USA, 1991. Genbank Trustee Number Sci , USA, 88:1711-1715 (1991), Genbank accession number M 58407, HCV-1: 3' end, Halla, above, Genbank accession number M58406, H C-Jl: American type, Saiki Moto et al., Japan J, Exp, Med.

HCV-BK: Japanese type, Takamizawa et al., J. Virol, 65:1105-11 13 (1991), GenBank accession number M58335.4626 (1990) , Targeted protection under pressure and potentially subtype-specific in the case of regions vI and V (i.e. American versus Japanese isolates). Sequence heterogeneity is Viro 1.171:555-567 (1989), Colette et al., Vfrol.

By virtue of their existence, HCV is more closely related to bestiviruses than flaviviruses. The data confirm that it exhibits a unique structure and composition.

From left to right, from the 5' end to the 3' end, using the position number of the first base residue in each column. Conventionally indicated in the direction of

The three-letter code (correspondence table) for each amino acid residue identifies each residue. of the protein it encodes so that it is located just below the three bases (codons) it encodes. By placing the rendered amino acid residue sequence below the nucleotide sequence, The reading frame of the nucleotide sequence of column number 1 is shown. Also, the sequence number l is the linear amino acid residue sequence encoded by the nucleotide sequence of SEQ ID NO:1. Also included are columns, conventionally shown from left to right in the direction from amino terminus to carboxy terminus.

The position If number of every 5th amino acid residue shall be indicated below the amino acid residue sequence. Ru.

SEQ ID NO: 2 represents the linear amino acid residue sequence of a preferred fusion protein with CAP-N. and the amino acid corresponding to residue 221 of glutathione-3-)transferase. the terminal polypeptide portion, corresponding to residues 222-225 of protease factor Xa. Intermediate polypeptide portion specifying the cleavage site, phosphorus corresponding to residues 226-234 NANBV capsid having the amino acid residue sequence 1 to 74 of SEQ ID NO: 1 the polypeptide portion corresponding to residues 235-308 that specify the antigen, and residue 3 It is composed of a carboxy-terminal linker moiety corresponding to 09-315. Also array Number: 2 is the nucleus of the linear-heavy chain DNA fragment encoding the fusion protein described herein. Also includes nucleotide base sequence. Presentation of nomenclature and sequence information is for SEQ ID NO. This is the same as described above.

SEQ ID NO: 3 represents the linear amino acid residue sequence of a preferred fusion protein designated as CAP-A. and corresponds to residues 1 to 220 of glutathione-S-+-transferase. protease thrombi corresponding to the amino-terminal polypeptide portion, residues 221-226. The intermediate polypeptide portion that specifies the cleavage site of the protein, the amino acid residue sequence of SEQ ID NO: 1. Residues 227-24 identify the portion of the NANBV capsid antigen that grows columns 1-20. 6, and the carboxylic acid moiety corresponding to residues 247-252. It is composed of a terminal linker portion. Sequence number J+:3 is the fusion described here. It also includes the nucleotide base sequence of the linear-heavy chain DNA fragment encoding the synthetic protein.

The nomenclature and presentation of the sequence tfjjN is the same as described for SEQ ID NO:1. be.

SEQ ID NO: 4 represents the linear amino acid residue sequence of a preferred fusion protein designated as CAP-B. containing amino acids corresponding to residues 1 to 220 of glutathione-3-transferase. The protease thrombin corresponds to the terminal polypeptide portion, residues 221-226. Intermediate polypeptide portion specifying the cleavage site of, amino acid residue sequence of SEQ ID NO: 1 Residues 227-24 specifying the portion of the NANBV capsid antigen with 2-40 6, and the carboxylic acid moiety corresponding to residues 247-252. It is composed of a terminal linker portion. SEQ ID NO: 4 also represents the fusion described herein. It also includes the nucleotide base sequence of the linear-heavy chain DNA fragment that feeds the protein.

The nomenclature and presentation of sequence information is as described for SEQ ID NO:1.

SEQ ID NO: 5 represents the linear amino acid residue sequence of a preferred fusion protein with CAP-C. containing amino acids corresponding to residues 1 to 220 of glutathione-3-)transferase. The protease thrombin corresponds to the terminal polypeptide portion, residues 221-22G. Intermediate polypeptide portion specifying the cleavage site of, amino acid residue sequence of SEQ ID NO: 1 Residues 227-24 specify the portion of the NANBV capsid antigen that grows 41-60. 6, and the carboxylic acid moiety corresponding to residues 247-252. It is composed of a terminal linker portion. SEQ ID NO: 5 also represents the fusion described herein. It also includes the nucleotide base sequence of a linear-heavy chain DNA fragment encoding a protein.

The nomenclature and presentation of sequence information is similar to that described for SEQ ID NO:l.

SEQ ID NO: 6 is the linear amino acid residue sequence of a preferred fusion protein designated CAP-A-B. corresponding to residues 1-220 of glutathione-s-transferase. The amino-terminal polypeptide portion, corresponding to residues 221-226, Intermediate polypeptide portion specifying the cleavage site of Bin, amino acid residues of SEQ ID NO: 1 Residues 227-2 specify a portion of the NANBV capsid antigen with sequence 2-40 65, and the carboxyl group corresponding to residues 266-271. Consists of an xy-terminal linker moiety. Moreover, SEQ ID NO: 6 is the fusion described here. It also includes the nucleotide base sequence of the linear-heavy chain DNA fragment encoding the synthetic protein.

The nomenclature and presentation of sequence information is similar to that described for SEQ ID NO:1.

The foregoing description and examples are intended to be illustrative and should not be taken as limiting. You shouldn't take it. Still other variations are possible within the spirit and scope of this invention; As such, they will be readily available to those skilled in the art. Other aspects of the claims set forth herein. It is within the range.

Sequence list (+) General information: (1) Applicant, Suzanne Zebedee GENEBUEEBE INCHOSUBU Marta Nasoff alfred prince (ii) Title of the invention: Non-A, non-B hepatitis virus antigen, diagnostic method, and vaccine (iii) Number of arrays: 137 (jv) Contact information: (A) Recipient: Dressler Goldsmith Shaw Sutker & Milnamow. limited (B) Street: 180 North Statesman, Suite 4700 (C) City Nijikago (D) State: Illinois (E) Country: USA (F)ZIP:80801 (v) Computer readable format: (A) Media type: Floppy disk (B) Computer: IBM PC compatible Compatibility (C) Operation System: PC-DO3/MS-D.

(D) Software: Patent in Release #1.0, Version #1. 2 5 (vi) Application data: (A) Application number: US (B) Application date: (C) Classification: (vii) Prior application data: (A) Application number: US 07/616369 (B) Application date: November 2, 1990 1st (vii) early application data: (A) Application number: US 071573643 (B) Application date: ] August 25, 990 Day (v i i f) Agent information: (8) Name: Nidward B. Gamson (B) Registration number: 29,381 (C) Case number: PHAOO29P (ix) Communication information: (A) Telephone +312-616-5400 (B) 7yyri x: 312-616-5 480(2) Sequence number: l: Characteristics of CD arrangement: (A) Length: 978 base pairs (B) type: Nucleic acid (C) Number of strands/single strand (D) Topology: linear (ii) Sequence type: DNA (genomic) (iii) Hybothetica Rui N0 (iv) Antisense 2N0 (fx) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 978 (D) Other information: / Codon beginning = 1 / Product = “NANBV structural antigen ” /number=1 (x i) Sequence: Sequence number: l: CTCCTG TCT CCCCCT GGCTCT CGG CCT AGC TGG GGCCCCACA GACCCCko36(2) Sequence number, 2: (1) Characteristics of array: (A) Length, 948 base pairs (B) type: Nucleic acid (C) Number of chains - heavy chain (D) Topology; linear <i i) Sequence type: DNA (genomic) (ii) Hybocete Calni N0 (iV) antisense=NO (ix) Features: (A) Name/Cow: CD5 (B) Existence position ft: 1. , 945(xi) Sequence: Sequence number 22: 85 ta 0 95 (2) Sequence number・3゜ (i) Properties of sequences.

(A) Length, 759 base pairs (B) type/nucleic acid (C) Number of strands - single strand (D) Toporono, straight neck shape (ii) Sequence type: DNA (geno+aic) (ii) hypose Teikal N.

(iv) Antisense, No. (ix) Features (A) Name/Key CD5 (B) Existence location 1. ．． 756 (xl) Array Sequence number 3. (2) Sequence number 4゜ (1) Array characteristics (A) Length: 759 base pairs (B) type/nucleic acid (C) Number of chains, - heavyweights (D) Topology straight line (11) Sequence species 1j: DNA (genomie) (1st order) Hypothetica Le No. (iv) Antisense No. (ix) Features (A) Name/Key CD5 (B) Existence position 10.756 (xl) Array Sequence number 4 (2) Sequence number, 5° (i) Characteristics of array: (A) Length near 59 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topolono: linear (i) Type of sequence: DNA (geno@1c) (i) Hypothetical :N0 (iv) Antisense=NO (ix) Features: (A) Name/Key: CD5 (B) Existence position, 1. ．． 7s6 (xi) Sequence: Sequence number, 5: τAT GAG CGCGAT GAA GGT GAT Input πGCGλ Entering AC Entering A Entering＾G’! 7T GAA TTG 144Tyr Glu A rg Ajp Glu Gly Asp LYII Trp Arg Asn Lys Lye Ph* Glu Muko5 40 45 GGA TCCGGCCCT AGA? 'rG GGT GTG CGCGC G ACG AGG enter AG AC! ’　TCCGAG　7Q0 Gly Sar Gly Pro enter rg XAu Gly Val enter r’J Enter 1a Thr Enter rg Lys Thr Bar Gl■ 225 2 0 2 5 240 (2) Sequence number 6・ Characteristics of (vine) array: (A) Length = 816 base pairs (B) type, nucleic acid (C) Number of chains, - heavy chain CD) Topology/linear (11) Type of sequence: DNA (genomic) (ii) hyboceti Cal: N0 (1v) antisense = NO (1x) Features (A) Name/Key: CD5 (B) Location: 1. ．． 813 (Xl) Sequence, sequence number, 6° (2) Sequence number near.

(i) Characteristics of array (A) Length: 66 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: linear (ii) Types of sequences: DNA (genomic) (ii) Hypothetical :N0(IV) antisense 2N0 (xi) Sequence: Sequence number near: (2) Sequence number = 8° (j) Characteristics of array: (A) Length 266 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology, linear (ii) Type of sequence: DNA (genomie) (iii) Hybothetica Le:N0 (iv) Antisense: No (xi) Sequence: Sequence number: 8: (2) Sequence number 9 (i) Characteristics of array: (A) Length 266 base pairs (B) type, nucleic acid (C) Chain number knee zero value (D) Topology: linear (jl) Sequence type: DNA (genomic) (i i i) hypoceti Karuni N.

(iv) Antisense=NO (xi) Sequence, Sequence number: 9: GATCCGACGT CAAGTrCCCG GGπ; GCGGTCAGAT CGTrGG　TGGAGmACTTGTrGCCGC60GCAGGG　66 (2) Sequence number: 1o: (i) Characteristics of array (A) Length 266 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: linear (ii) Type of sequence: DNA (genoloie) (iii) Hypothetica Rui N.

(iv) Antisense 2N0 (xi) Sequence: Sequence number = 1o: AATTCCCTGCGCGGCAACAA GTAAAACTCCA CCAA CGATCT　GACCGCCACCCGGGGAACTTG@80 ACGTCG 66 (2) Sequence number +11: (Characteristics of D array: (A) Length = 66 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: linear (11) Sequence type: DNA (genoIlic) (iii) Hypothetica Le:N.

(1v) Antisense: No (xi) Sequence: Sequence number・ll: GATCCGGCCCCTAGATI'GGGT GTGCGCGCGA CGA GGAAGACTTCCGAGCGGGTCGCAACCTb60 (2) Sequence number = 12: (i) Characteristics of array: (A) Length, 66 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: linear (jj) Sequence type: DNA (genomic) (i i i) hypoceti Karuni N0 (iv) antisense = NO (xi) Sequence: Sequence number: 12: (2) Sequence number = 13° (i) Characteristics of array: (A) Length = 32 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: vertical (i) Type of sequence: DNA (genolIlic) (i) Hypo Cetical, N0 (iv) antisense °N0 (xl) Sequence: Sequence number, 13: GkATT'...^Cguchido reach A^CAAGTAAACτC32 (2) Sequence number: 14・ (i) Characteristics of array: (A) Length: 32 base pairs (B) type: Nucleic acid (C) Number of chains - heavy chain (D) Topology, linear (ii) Sequence type: DNA (genomic) (ii) Hybocete logical n.

(iv) Antisense, No. (xi) Sequence: Sequence number = 14: GCTGGATCCA GCACGATrCCCAAACCTCAA AG 3 2 (2) Sequence number: 15: (i) Characteristics of array (A) Length = 21 base pairs (B) type: Nucleic acid (C) Chain number knee zero value (D) Topology: linear (ii) Sequence type: DNA (genomie) (ii) hypocete ical: N0 (iv) antisense = NO (xi) Sequence, Sequence number: 15: ATGAGCA (GA TTCCAAAACCT 21 (2) Sequence number 16: (i) Characteristics of array: (A) Length: 17 base pairs (B) type: Nucleic acid (C) Chain number knee heavyweight (D) Topology: linear (ii) Type of sequence: DNA (genollie) (ii) Hypo Thetical: N0 (iv) Antisense = NO (xi') Sequence: Sequence number = 16, GAGGAAGACT TCCGAGC17 (2) Sequence number = 17 = (i) Characteristics of array: (A) Length = 17 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hyposeti Cal: N0 (iv) Antisense: YES (xi) Sequence: SEQ ID NO: I7; GTCCTGCCCT CGGGCCG 17 (2) Sequence number: 18: (i) Characteristics of array: (A) Length: 21 base pairs (B) type: Nucleic acid (C) Chain number knee heavyweight (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) antisense: YES (xi) Sequence: Sequence number: 18: ACCCAAATTG CGCGACCTACG 21(2) SEQ ID NO: 2I9: (i) Characteristics of the sequence; (A) Length 219 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: vertical (Yes) Type of sequence: DNA (genomic) (ii) Hybothetica Le: N0 (iv) Antisense: No (xi) Sequence: Sequence number: 19: TGGGTAAGACATCGATAC19 (2) Sequence number = 20 = (i) Characteristics of array: (A) Length: 17 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear Ci i) Sequence type: DNA (genocfc) (ifi) hypothetica Le:N0 (jv) Antisense: No (xi) Sequence: Sequence number = 20: AAGGTCATCG ATACCCT 17 (2) Sequence number = 21: (i) Characteristics of array: (A) Length: 18 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) antisense: YES (xi) Sequence: Sequence number = 21: AGATAGAGAA AGAGCAAC18 (2) Sequence number: 22: (i) Characteristics of the sequence; (A) Length = 22 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: DNA (genomic) (ii) Hyboceti Cal = NO (iv) Antisense: YES (xi) Sequence: SEQ ID NO: 22; GGACCAGTr'CATCATCATAT AT 22 (2) Sequence number = 2 3 = (i) Characteristics of array: (A) Length = 20 base pairs (B) type: Nucleic acid CC) number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: DNA (genomic) (iii) Hybothetical KoN.

(jv) Antisense: YES (xi) Sequence: Sequence number = 23: CAGTrCATCA TCATATCCCA 20(2) Sequence number = 24 = (i) Characteristics of array: (A) Length: 15 base pairs (B) type: Nucleic acid (C) Chain number knee heavyweight (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hybocete ical: N0 (iv) antisense = NO (ix) Features: (A) Name/Key: CD5 (B) Existence position +1. ．． 15 (D) Other information: /Product=“in GST-NANBV 693-691 linker protein” (xi) sequence: SEQ ID NO: 24: GGG ATCCCCAAT TCA 15Gly Ile Pro Asn Ser(2) Sequence number: 25: (i) Characteristics of array: (A) Length = 5 amino acids (B) type diamino acid (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence: Sequence number: 25: Gly Ile Pro Asn Ser (2) Sequence number = 26 = (i) Characteristics of array: (A) Length: 12 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) antisense: No (ix) Features; (A) Name/Key: CD5 (B) Location: 1. ．． 9 (D) Other information: /Product-“in GST-NANBV 693-691 carboxy-terminal linker protein” (xi) Sequence: Sequence number: 26: MT TCA TCG TGA 12 Asn　Ser　Ser (2) Sequence number: 27° (i) Characteristics of array (A) Length: 3 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: protein (xi) Sequence: Sequence number: 27: Asn　Ser　Ser (2) Sequence number=28= (i) Characteristics of array: (A) Length = 27 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology bilinear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) Antisense 2 N0 (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 27 (D) Other information: /Product = “Limit in GST-NANBV 15-18 Linker protein” (xi) sequence: SEQ ID NO: 28: GGG ATCCCCCATCGAA TrCCTG CAG CCC27Gay Ile Pro Ile Glu Phe Leu Gln Pr.

(2) Sequence number = 29° (i) Characteristics of array: (A) Length: 9 amino acids (B) type, amino acid (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence: Sequence number = 29: Gly　Pro　lie　Glu　Phe　Leu　Gin　Pr.

(2) Sequence number = 30 = (i) Characteristics of array: (A) Length: 24 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology; vertical shape (ii) Sequence type: DNA (genomfc) (ii) hypocety Cal: N0 (iv) Antisense: No (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 21 (D) Other information: /Product=“Cat in GST-NANBV 15-18 "Ruboxy-terminal linker protein" (xi) Sequence: Sequence number: 30: TGG GGG ATCGGG AAT TCA TCG TGA 24Trp Gly Ile Gly Asn Ser Ser (2) Sequence number = 31: (i) Characteristics of array: (A) Length = 7 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: protein (xi) Sequence: Sequence number: 31・ Trp Gly Ile Gly Asn Ser Ser (2) Sequence number = 3 2: (i) Characteristics of array: (A) Length = 24 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: DNA (genomie) (iii) Hypothetica Le=NO (iv) Antisense=NO (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 24 (D) Other information: /Product = “Limit in GST-NANBV 15-17 Linker protein” (xi) sequence: SEQ ID NO: 32: GGG ATCCCCCAAT TCCrGcAGCC (T 24Gly Ile 　Pro　Asn　Ser　Cys　Ser　Pr.

(2) Sequence number: 33: (1) Characteristics of array: (A) Length: 8 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: protein (xi) Sequence: Sequence number: 33° Gly　Ile　Pro　Asn　Ser　Cys　Ser　Pr.

(2) Sequence number: 34° (i) Characteristics of array: (A) Length: 21 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genoa+1c) (ii) hypothetical Tical II N0 (iv) Antisense = NO (ix) Sentence ■ Kai 2 (A) Name/Key: CD5 CB) Location: 1. ．． 18 (D) Other information: /Product = “Cat in GST-NANBV 15-17 "Ruboxy-terminal linker protein" (xi) Sequence: Sequence number: 34: GGG ATCGGG MT TCA muscle embryo A 21Gly IICGly As n Ser Ser (2) Sequence number: 35: (i) Characteristics of array: (A) Length = 6 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: protein (xi) Array: Sequence number=35= Gly lie Gly Asn Ser Ser (2) Sequence number: 36: (i) Characteristics of array: (A) Length: 15 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical:N.

(iv) Antisense=NO (ix) Features: (A) Name/Key: CD5 (B) Existence position = 18.15 (D) Other information: /Product; Thrombin cleavage site” (xi) sequence: SEQ ID NO: 36: GTr CCG CGT GGA TCC15Val Pro Arg Gly Ser (2-in sequence number: 37゜ (i) Characteristics of array: (A) Length: 5 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: protein (xi) Sequence: Sequence number: 37: Val Pro Arg Gly Ser (2) Sequence number: 38: (i) Characteristics of the sequence; (A) Length: 21 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology bilinear (ii) Sequence type: DNA (geoomic) (ii) Hypocete ical: N0 (iv) Antisense 2 N0 (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 21 (D) Other information: /Product; Linker protein” (xi) sequence: SEQ ID NO: 38: CCA TCG AAT TCCTGCAGCCCT 21Pro Ser A sn Ser Cys Ser Pr.

(2) Sequence number = 39: (i) Characteristics of array: (A) Length near amino acid (B) type diamino acid (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence: Sequence number 239: Pro Ser Asn Ser Cys Ser Pr.

(2) Sequence number = 40 = (i) Characteristics of array: (A) Length = 18 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology bilinear (i i) Sequence type: DN A (genomi c) (i i i) Ipothetical = NO (iv) Antisense = NO (ix) Features: (A) Name/Key: CD5 (B) Existence position It: 1. ．． 15 (D) Other information;/product; Ruboxy-terminal linker protein (xi) Sequence: Sequence number: 40: Hit A ATT CAT CGT GACTGA 18Gly Ile His Arg Asp (2) Sequence number: 41: (i) Characteristics of array: (A) Length: 5 amino acids (B) type diamino acid (D) Topology, linear (i) Two protein sequence types (xi) Sequence: Sequence number = 41: Gly Ile His Arg Asp (2) Sequence number: 42: (i) Characteristics of array: (A) Length: 27 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: DNA (genomic) (jii) Hypothetica Le=NO (iv) Antisense=NO (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 27 (D) Other information: /Product; “In GST-NANBV 690-691 linker protein” (xi) sequence: SEQ ID NO: 42: GGG ATCCCCAAT TCG AGCTCG GTA CCC27Gl y lie Pro Asn Ser Ser Val Pr.

(2) Sequence number: 43: (i) Characteristics of array: (A) Length: 9 amino acids (B) type diamino acid (D) Topology: vertical (i) Type of sequence: protein (xi) Sequence: Sequence number 43 Gly lie Pro Asn Ser Ser Val Pr.

(2) Sequence number 44: (i) Characteristics of array (A,) Length/24 base pairs (B) type: Nucleic acid (C) Number of chains - heavy chain (D) Topology, linear (i i’) Type of sequence: DNA (genoo+1e) (i i i) Hypo Seventh Tical: N0 (iv) Antisense, No (ix) Features: (A) Name/Key: CD5 (B) Location: 1. ．． 21 (D) Other information: /Product=“in GST-NANBV 690-691 carboxy-terminal linker protein” (xi) Sequence: Sequence number: 44: ACG GGG ATCGGG AAT TCA T-hole A 24Thr Gl y Ile Gfiy Asn Ser Ser (2) Sequence number: 45: (i) Properties of sequences.

(A) Length = 7 amino acids (B) type diamino acid (D) Topology, linear (100) Sequence type: Protein (xi) Array: Sequence number=45= Thr　GI3T　lie　Gly　Asn　Ser　Ser(2) Sequence number 4G (i) Characteristics of array: (A) Length: 9416 base pairs (B) type, nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: eDNA (1st) Hypo7tical No. (iv) Antisense=NO (ix) Features: (A) Name/Key: CD5 (B) Existence position +342. ．． 9374 (ix) Features: (A) Name/Key: Characteristics of mfse (B) Location: 1. ．． 12 (D) Other information: /Note=”Not confirmed as HCV-He59 sequence ” (ix) Features: (A) Name/Key: Characteristics of wise (B) Location: 9397. ．． 9416(D) Other news: /Note=“HCV- Not confirmed as Hc59 sequence' (ix) Features: (A) Name/key: Repeat unit (B) Existence position: Group (7,, 12, 42,, 47) (D) Other information /rp t type = “Other”/rpt group = “1” (ix) Features・ (A) Name/key/repeat unit (B) 1 group of existing positions (23,,28,38,,43,9209,,9214,9 391,,9396) (D) Other information: /rpt type = ``Others'' /rpt group ="2" (]X) Features.

(A) Name/key/repeat unit (B) Location: Group (128, 135, 315, 322 (D) Other information :/rpt type="other"/rpt group="3" (ix) Features: (A) Name/key: Repeat unit (B) Existence position 1 group (9231, 9237, 9245.

9251.9256. ．． 9262) (D) Other information: /rpt type=“other”/rpt group-″4′ (ix) Features: (A) Name/key/repeat unit (B) A group of existing positions (9248,,9253,9221,.

9226.9227. ．． 9232) (D) Other information: /rpt type=“Others”/rpt group=″5″ (xi) Sequence: Sequence number: 46: GCCjtGCCCCCTGATGGGGGCGAcACTC1'JCCATG AATCACTCCCCTGTGA GG entry CTACTG@60 CAG AGCi, 1c CAG GTG GCCCACσKG CAT G ( rj CCCACCGGCAGCGGT AAG 4049Gln 5ar I lk+e cln Val Ayu aJmi is yAu J(ili Al, M Pro Thr Gly! HeγGl凵@Lys 122’i 12311 1 no: 15 ACCAcc AAC: GTCCCG GLT GCG ThCGCA GC CAAG Gl’;C’i”8r 8AG GTG LIG@4097 Be; Tbr Lyfj Vai Pxo enter 1δ enter 1a Tyx: A, la Enter La Lys ChT qT Lyi Val X`u TCr-8AG GCCCAT GGG GTT ChT CUT A included 8 TCAGG 8CCGGG G’fr,” AGA 8 (ko■@419) 5ar　T-y:＜　人In　! (is Gly Vai person'p?rts entered sn Engineering 1+a Arg Thr Gly ν41 Arg@Thr 1270 ＾275 12R0 81T ＾cq ＼Cr CGC entered GCCC (8TC entered (Ta’i”8CTC C input rCTAC'; GCAAG T'rC (-7ゝゝ　4λS unit Ni le Thr Thr Thr Gly ser Pro Ala τf r Tyr Sar Tl)X’ Tyr C1y L”/G PllbL4) u 1225: L2'30 129s 1300! ', kG TGCr: A (TC ChCC; GAT GCCACA TCCATCTCG GGCh’rr: GL: bar arc@4] 37 GIIJC:/S H Yux Ser Titr Asp Ala Tor Se r engineering Is Sar Gly engineering in Gly Thr VaP 11λOYS　l]:l[+ CTT GACCA person rtCIA GAG ACT GGCGGG GCG AGA c’rc GTT to TG CTC (,-CC8 “s411115 7jlu A9p G-n AIJI cllj ThC included 1a Gly included 1a Arg LelJ Val Val 1lau Enter 1 = @Thr Yu335] Ko40 1ko45 Gr:'r　ACCCCCT　CCG　GGCTCCGTCAl:'T　GTG　 TCCCAT CCT AACATCGAG GAG 44R:! AlaTl’)rProProGly511! rValτhrVayoss+rH 1s? ro85nIlaGluG, 1 lecture 1 5013551360 GTT G(?T CTCTCACCACCGGA GAG Arc C(C TTT TACGGC entrance G CCT 8TC4481Val Ala Izu sir Thr Thr Gly Glu Eyue Pre+ Pha Ty r　Gly　Lys　8la　l1■ 1" 65 Yu 370 1:175 1 piece 80 pieces AG entering AG AAG TGCG ACGAG CTCGCCGCG Enter G CTG GTCGCJt TrG GCCATC4577Lye LYS LYS cys A5p Glu Ia u Ala Ala Lys Leu Val 1a Lau Gly l1 a1400 1405 i410 MT GCCGTG GCCTACTACCGCGGT CTT CACGTG TCT CTC nest CCcG ACC4625Asn Ala Val Al a Tyr Tyr Enter rq Gly Lau Asp Val Ssr Va l　Work 1m　Pro　Thr1t15　1420　1425 A to CGGCGAT GTr CTCGTCGTG TCG ACCGAT G CT CTCATG ACT GGCm 467ko Sir Gly Asp V al Val Val Sar Thr Mp 1a tau Me t Thr Gly Ph scold 14) 0 14ko5 1440 ^CCC, GCCACTrCGAG TCT CTG entered T^ CAC Ding GC entered ACACG τG’T GTCACT CA 4721Thr Gly As p　Phe　Asp　Sar　Val　rl＊　Asp　cys　Ajn　T? +r　C’ys　Val　Thr　G Gekigeki f1 ACA GTCGAT TTr AGCCTr GAI: CCT Acc T TT AC (: ATlr GAG ACA ACCACG@4769 Thr Val Enter Sp Pha Sar Ijlu Asp Pro Th’ ! : Pha Dinghr Xl Glu Thr Thr ■■ 1465 14) O1475 CTCCCCAG GAT G (T GTCTeCAGG ACT C entry CGCCGG GGCAGG ACT GGC41+17Lau Pro Gi n Asp Ala Val 5erAXq Dinghr Gin Arg Enter rg Gly Arg Thr Gly1480 L485 1490 GCG TACATG AACACCCCG GGG CTT CCCGTG TGCCAG GACCAT C’ff GGA 5009＾1a T”fr MPt Asn Thr Pro Gly LaI3 Pro Val cys Gin sp His Lau G attack■ TI’T TGG GAG GGCGTCT’l’T ACG GGCCTCA CT CAT ATA CAT Gem CACTTT 5O57 Pha Trp Glu cly Val Pha Dinghr Gly Leu Thr 14is II@ Enter 1p Enter 1a His Ph■ 1560 Yu565 1570 f7A TCCCAG ACA AAG CAG input σT GGG GAG input ^c’rrr ccτ TACCTG GTA GCG T105 4AuSerGllIThrLYGGinSetGIYGluAsnPhePr oTyrLauVal enter Yufu157! i15R015a5 ：Light：：Number：：　：：　Z)、：：　）；、；z：Kai G：　3”：1. : Base: C::: 5249 62516 016 5 ACCGAC Ding TT Entering AG ACC Ding GG CTG Entering A Gce kkG CTCATG C (J CAA C'Tc CCT 63T+ sar Asp Pha Lys Thr TrP Lau L’fla 1 a Lys Leu Mat Pro cln Lau P Chichi B GGA GACGGCATT ATG CACACT eGCTGCCACTC T GGA GCT GACATCACT 6449cly Asp Gly Mat) lis Thr Ar9 eys Hls Cys Gly Enter 1a Glu Engineering 1 Ding h■ 2025203020ko5 GGA CAT GTC entry AA AACGGG ACG ATG AGG AT CGTCGGT CCT AC4Ace TGC6497Gly His Va l Lys Enter sn Gly Thr Mst Arg X1* Val Gl y Pro Arg Thr Cy*AAG AACATG TGG AGT GGG kCG TTCTTCATT Input AT GCCTAC8eCAeG G GC6545Lys Asr+ Mat Trp 5ar Gly Thr P ha Pha Xla enter sn enter 1a Tyr Thr CCC Ding GT ACT CCCCTT CCT GCG CCG AAC? A.T. AAG TTCGCG CrG TCG kGG 659j Pro Cys Ding hr pro X4* pro included 1a Pr6 Ajn TY! ” LYII Pha Aia ku Trp Ar■ 20fuO207520110 GTG TCT GCA GAG GAA TACGTG GAG ATA A GG CGG GTG GGG GAC Ding TCCAC664k val 5er Aia Glu GLu Tyr Val C1u Ila Arg Arg Val Gly Asp Ph@HisτACG Ding A TCG GGCATG ACT ACT GACAAT CTCjLAA TGCCC G TGCCAG ATC6689Tyr Val sar Gly Met Thr　Thr　Sp　Asn　Lau　Lys　Cy@Pro　Cys　 Gln ll-CCA Ding CG CCCGAA TTr TTCACA GAA 'ITG GACG to G to TG CGCC to AC to ACG 673V Pro 5er Pl:OGlu Pha Pha Thr Glu Lau Ajp Gly Val Arg X4* Hls M7 TT GCG CC e CCT Ding G (: AAG CCCTTCCTCCGG G GG^GG Ding A TCT T1’CAGA 678T Phil Ala Pro Pro eys Lyg Pro Lau Lau Arg GLu Glu Val Ser Pha Ar■ 21ko521402145 (i?A GGA CTCCACGAG TACeeG TG GGG TC G CAA TrA CCT TCCGAG CCC68koR Val Gly Lau Hls Glu Tyr Pro Van Gly 5*r Gin Lau Pro Cys Glu Pl:O GAA CCG GACGTA GCCGTG TTc, ACCTCCATCC TCACT G nest CeCTCCCAT 6881Glu Pro hsp Val Ala Val TAu Thr Sir Mat! au Thr Enter sp Pro sat H1s2165 2170 2X75 2180A ^　ACA　GCA　GAG　GCG　G(:CGGG　AGA　8GG　TT G GCG AGA GGG Chichi C CCCC (1’Chichi@6929 X1e Thr enter 1a Glu Ala Ala enter rg Arg Iau ＾1a Enter rg Gly Sar Pro Pr.

TCT ATCGCCAGCTCCTCG GCT AGCCAG CTC Ding C CGCT CCA TCT CTC entry G 697 fusar Met Ala Bar Ser Ser Enter 1a Sar Gln ku Sir Ala P ro Sir IAu LysGAG GAA CAA Entry A cTc cc c Enter Te Enter CGCA CT AGe Me TCG TTG CTA CGC76X7 nilu Glu Gin Lyg Lau Pro Xla AJn Ala -u Sar kin 5*r Lau Iau Ar9CAT CAC included AT CTCGTG Input T DingCCAce ACT T (J eGc Input GT C CT’! 'GCCAA AGG 7V45 His HLx Asn X4* Val Tyr Sar Thr Thr saw Ar9 5str in 1a CYII in nitrogen ■ AAG AAG AAA GTCAGA TTT GACAGA CTG CA A c’rrcτG GACAGCCAT TAC fu9ko Lys Lys L ys Val ding hr Pha Asp enter rg ku Gin Val La u Asp 5ar Hls Tyr (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hyboceti Karuni N0 (iv) Antisense 2N0 (xi) Sequence: Sequence number: 52: CGAGGAAGACTrCCGAGC (2) Sequence number = 53: (i) Characteristics of array: (A) Length: 20 base pairs (B) type: Nucleic acid (C) Chain number knee heavyweight (D) Topology: linear (ii) Type of sequence 2 DNA (genomic) (ii) Hyposeti Karuni N0 (iv) antisense: YES Cxi') Sequence: SEQ ID NO: 53; ACCCAAATrG CGCGACCTAC (2) Sequence number: 54: (i) Characteristics of array: (A) Length = 18 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (11) Type of sequence: DNA (genomic) (iii) Hybothetical :N0 (iv) Antisense=NO (xi) Sequence: Sequence number: 54: TMGGTCATCGATACCCT 1 (2) Sequence number = 55: (Characteristics of D array: (A) Length = 20 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (i D sequence type: DNA (genomjc) (i i i) hypocety Cal: N0 (iv) Antisense: YES (xi) Sequence: Sequence number: 55: CAc mouth’CATCA TCATATCCCA 2 (2) Sequence number: 56: (i) Characteristics of array: (A) Length: 18 base pairs (B) type; nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (i) Type of D sequence: DNA (genomic) (iii) Hypothetical :N0 (iv) Antisense: YES (xi) Sequence: Sequence number: 56: AGATAGAGAA AGAGCAACII (2) Sequence number = 57: (i) Characteristics of array: (A) Length: 20 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (xD array: array number=62= (i) Characteristics of array: (A) Length: 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) antisense: YES (xi) Sequence: Sequence number: 63: CTGTCGGTCG TTCCACCA (2) Sequence number: 64: U) Array properties: (A) Length: 20 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical 2 N0 (iv) antisense=NO (xi) Sequence: Sequence number: 64: CCGCGAAGAG TGTGπm sword T (2) Sequence number: 65: (i) Characteristics of array: (A) Length = 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: DNA (genomic) (iii) Hypothetica Le:N0 (iv) Antisense: YES (xi) Sequence: Sequence number: 65: CAATGTTCTG GTGGAGGTG 19 (2) Sequence number = 66 = (i) Characteristics of array: (A) Length: 28 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear 19 C11) Sequence type: DNA (genornic) (ii) high Pothetical: N0 (jv) antisense = NO (xi) Sequence: Sequence number 266: GCCATrAAGT GGGAGTACGT CGTrCTCC28(2) arrangement Column number 267: (i) Characteristics of array: (A) Length = 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear 20 (ii) Type of sequence: DNA (genomic) (iii) Hypocete ical:N0 (4v) Antisense: YES (xi) Sequence: Sequence number: 67: CGAGGAAGGA TACAAGACC19 (2) Sequence number: 68: (Characteristics of D array: (D) Topology: linear (ji) Type of sequence: DNA (genotnic) (i i i) hypocete ical: N0 (iv) antisense: YES (xi) Sequence: Sequence number = 73: To CTAGG8GGC CCTTGTCTGC (2) Sequence number near 4 (i) Characteristics of array: (A) Length: 18 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hyposeti Cal: N0 (iv) Antisense 2 N0 (xi) Sequence: Sequence number near 4: CTCGGGCCAG CCGATGGA (2) Sequence number = 75: (Characteristics of D array: (A) Length = 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (10 types of sequences: DNA (genomic) (ii) hypothetica Le: N0 (iv) Antisense: YES (xi) Sequence: Sequence number = 75: GGGGACCTCA TGGTrGTCT (2) Sequence number = 76: (i) Characteristics of array: (A) Length = 18 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology bilinear 20 (i4) Sequence type: DNA (genomfc) (iii) hypocete ical:N0 (iv) Antisense=NO (xi) Sequence: Sequence number near 6: CCCGTGGAGTGGCTMGG 11(2) Sequence number near 7: (i) Characteristics of array: (A) Length: 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: vertical 1g (ii) Type of sequence: DNA (genomic) (jii) Hypocete ical:N0 (iv) Antisense: YES (xi) Sequence: Sequence number near 7: CTCCTCGATG 'ITGGGATGG 1' (2) Sequence number near 8: (i) Characteristics of array: (A) Length: 19 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear 19 (ii) Type of sequence: DNA (genomic) (ii) Hibo Cetical: NO (2) Sequence number 284: (i) Characteristics of array: (A) Length = 22 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hybocete ical: N0 (iv) antisense: YES (xi) Sequence: Sequence number: 84: GTAAGGAAGG”rTcrccccAc TC(2) Sequence number=85= (i) Characteristics of array: (A) Length: 27 base pairs (B) type; nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: DNA (genomic) (ii) Hypocete ical: N0 (iv) antisense = N.

(xi) Sequence: Sequence number: 85: ATGCCCACTr TCTATCCCAG ACAAAGC (2) Sequence number 286: (i) Characteristics of array: (A) Length: 17 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Sequence type: D NA (genomic) (ii) Hibose Tikal: N.

(iv) Antisense: YES (xi) Sequence: Sequence number 286: TGCATGTCAT GA″TGTAT (2) Sequence number: 87: (i) Characteristics of array: (A) Length: 18 base pairs (B) type: Nucleic acid (C) I number knee heavy chain (D) Topology: linear 22 (ii) Type of sequence: DNA (genotnic) (ii) High Pothetical: N.

(iv) Antisense: YES (xi) Sequence: Sequence number = 87: GGACAAGACG ACC (TrCC (2) sequence number = 88 = (i) Characteristics of array: (A) Length = 20 base pairs (B) type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology m: direct enemy 27(ii) Sequence type: DNA (genomic) (fii) Hyposeti Cal:N.

(iv) Antisense IIN.

(xi) Sequence: Sequence number = 88 = CGTATrGcCT GTCAAACAGGC2+ (2) Sequence number = 89 = (i) Characteristics of sequence: (A) Length: 20 base pairs (ji) Type of sequence: DNA (genomjc) (ii i) hypotheti Cal: N0 (iv) Antisense = NO (xi) Sequence: SEQ ID NO: 94: ATGTGGAGTG GGACCTrCC (2) SEQ ID NO: 95 (i) Sequence characteristics: (A) Length: 20 base pairs (B) Type : Nucleic acid (C) Number of strands Knee heavy chain (D) Topology: Linear (i i) Type of sequence: DNA (genotnic) (i i i) Hybothetical: N0 (iv) Antisense: YES (xi) Sequence: SEQ ID NO: 95: CTCTGCTGTr ATATGGGAGG (2) SEQ ID NO: 96: (i) Characteristics of the sequence: (A) Length = 20 base pairs (B) Type: Nucleic acid (C) Number of strands Knee heavy chain (D ) Topology: Linear (ii) Sequence type: DNA (genomic) (j i) Hibocete logical: N0 (iv) antisense = NO (xi) Sequence: SEQ ID NO: 96: GTrGACGTCCATGCTCACTG (2) SEQ ID NO: 97 = (i) Sequence characteristics: (A) Length: 20 base pairs (B) Type: Nucleic acid (C) Number of chains Knee heavy chain (D) Topology: Linear 19 (ii) Type of sequence: DNA (genomfc) (i i i) Hypothetical: N0 (iv) Antisense: YES (xi) Sequence : SEQ ID NO: 97: TTrCCACGTCTCCACTAGCG 21(2) SEQ ID NO: 98= (i) Characteristics of the sequence: (A) Length: I8 base pairs (B) Type: Nucleic acid (C) Number of strands knee heavy chain (D) Topology: Linear 20 (ii) Sequence type: DNA (genomic) (i ii) Hypothetical: N0 (iv) Antisense: YES (x i) Sequence: SEQ ID NO: 298: GTGAGGACCA CCGTCCGC18 (2) Sequence Number: 99: (i) Characteristics of the sequence: (A) Length = 20 base pairs (B) Type: Nucleic acid (C) Number of chains knee heavy chain (D) Topology: Linear 20 (ii) Type of sequence : DNA (genomic) (itD Hypotheti) Cal: No (iv) Antisense 2 NO (i) Characteristics of the sequence: (A) Length: 19 base pairs (B) Type: Nucleic acid (C) Number of strands knee heavy chain (D) Topology: Linear ( i i) Type of sequence: DNA (genomic) (iii) Hypothetica (iv) Antisense: YES (xi) Sequence: SEQ ID NO: IO2: AGCTTCCCCAT CACGGCCAA (2) SEQ ID NO: IO2: (Characteristics of D sequence: (A) Length = 19 base pairs (B) Type: Nucleic acid (C) Number of chains (D) Topology: Linear (i i) Sequence type: DNA (genomic) (i i i) Hypocete (i) Sequence characteristics: (A) Length = 20 base pairs (B) Type: Nucleic acid (C) Number of chains (D) Topology: Linear (ii) Sequence type: DNA (genomic) (i ii) Hypostheti Cal: N0 (iv) Antisense: YES (xi) Sequence: SEQ ID NO: 107: GCACCTGCGA TAGCCGCAGT 20 (2) SEQ ID NO: + 108: (i) Sequence characteristics: (A) Length = 18 base pairs (B) Type : Nucleic acid (C) Number of strands: heavy chain (D) Topology: linear 19 (ii) Type of sequence: DNA (genomic) (i i i) Hypothetical 2N0 (iv) Antisense 2N0 (xi) Sequence: SEQ ID NO: 108: GTCCCTCACCGAGAGGCT 1g(2) SEQ ID NO: 109: (i) Characteristics of the sequence: (A) Length: 20 base pairs (B) Type: Nucleic acid (C) Number of strands (D) Topology: Linear 19 (if) Sequence type: DNA (genomic) (i j D hypothetical: No (iv) Antisense: YES (xi) Sequence: SEQ ID NO: 109: GATrGGAGGT AGATCAA (TG 20 (2) SEQ ID NO: 110: (i) Sequence characteristics: (A) Length = 20 base pairs (B) Type: Nucleic acid His Val Thr Gly Gly Gln Ala Ala Arg Ala Met Ser Gly Leu Vat Ser@Leu Phel 5 10 15 Thr Pro Gly Ala Lys Gln Asn lie (2) Sequence number: 116: (i) Sequence characteristics: (A) Length = 26 amino acids (E3) diamino acid (D) Topology: Linear (ii) Sequence Type: Peptide (xi) Sequence: SEQ ID NO: 1162 H3s VaI Thr Gly Gly Arg Val Ala Ser Ser Thr Gln Ser Leu Val Ser@Trp Leul 5 10 15 Ser Gin Gly Pro Ser G in Lys Ile (2) Sequence number :117: (i) Sequence characteristics: (A) Length: 26 amino acids (B) Type diamino acids (D) Topology: Linear (i i) Sequence type: Peptide (xi) Sequence: SEQ ID NO: lI7 : )1is Val Thr Gly Gly Ala Gin Ala Lys Thr Thr Asn Arg Leu Val Se Chichi@Met Phe l 5 10 15 Ala Ser Gly Pro Ser Gin Lys Ile (2) Sequence Number 21】8: (Characteristics of D array: (A) Length: 26 amino acids (B) Type diamino acid (D) Topology: Linear (i i) Sequence type: Peptide (xi) Sequence: SEQ ID NO: +118+ Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr Leu Ala Ser@Leu Phel 5 1θ 15 Ser Pro Gly Ala Ser Arg Asn lie (2) Sequence number + 119: (i) Sequence characteristics: (A) Length: 26 amino acids (B) Type diamino acid (D ) Topology: Direct chain (II) Semorable type: Peptide (XI) Semorable: L19: L19: L19: HIS VAL THR THR GLN GLN GLN HIS VAL THR THR THR THR THR THR SER@LEU PHE L 5 1 10 15 ARG PRO Gly Ala Ser Gin Lys Ile (2) Sequence number: I20: (i) Characteristics of the sequence; (A) Length: 26 amino acids (B) Type diamino acids (D) Topology: Linear (if) Type of sequence: Peptide (xi) Sequence: SEQ ID NO: 120: His Val Thr Gly Gly Ser Ala Gly Arg Thr Thr Ala Gly Leu Val Gly@Leu Leu l 5 10 15 Thr Pro Gly Ala Lys Gln Asn Ile (2) sequence number: 121: (i) Sequence characteristics: (A) Length: 26 amino acids (B) Type diamino acids (D) Topology: Linear (Portrait) Sequence type: Peptide (x i) Sequence: SEQ ID NO: 121: His Val Thr Gly Gly Ser Ala Gly Arg Ser Val Leu Gly lie Ala Ser@Phe Leu i s to 1s Thr Arg Gly Pro Lys Gln Asn Tie (2) Sequence number: 122: (i) Sequence number: Characteristics: (A) Length: 30 amino acids (B) Type diamino acid (D) Topology: Linear (ii) Sequence type: Peptide (X]) Sequence: SEQ ID NO: 122: VaI Aha Thr Arg Asp Gly Lys Leu Pro Thr Thr Thr Gln Leu krg Arg His@Ile Aspl 5 10 15 Leu Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu (2) SEQ ID NO: 123: (i) Sequence characteristics: (A) Length = 30 amino acids (B) Type diamino acid (D) Topology: Linear (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: +123: Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr Gin Leu Arg Arg His@Ile Aspl 5 10 15 Leu Leu val Gly Ser Ala Thr Leu Cys Ser Ala Leu (2) SEQ ID NO: 124: (i) Sequence characteristics: (A) Length: 30 amino acids (B) Type diamino acid (D) Topology: Linear (ii) ) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 124: Leu Ala Ala Arg Asn Ser Ser lie Pro Thr Thr Thr lie Arg Arg His@Val Aspl 5 10, 15 Leu Leu Val Gly A la Ala Ala Leu Cys Ser Ala Met (2) SEQ ID NO: 1252 (i) Sequence characteristics: (A) Length: 30 amino acids (B) Type diamino acids (D) Topology: Straight-shaped (ii) Sequence type: Peptide (xi ) Sequence: SEQ ID NO: 125: Leu Ala Ala Arg Asn Val Thr Ile Pro Thr Thr Thr Ile Arg Arg His@Val Aspl 5 10 15 Leu Leu val Gly Ala Ala Ala Phe Cys Ser Ala Met (2) SEQ ID NO: 71262 (Characteristics of D sequence: (A) Length: 30 amino acids (B) Type diamino acids (D) Topology: Linear (i i) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 126: Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Lie Arg Arg His@Val Aspl 5 10 15 Leu Leu Val Gly Ala Ala Ala Ala Phe Cys Ser Ala Met(2) Sequence Number: I27: (i) Characteristics of the sequence: (A) Length: 30 amino acids (B) Type diamino acids (D) Topology: Linear (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 127: Leu Ala^Ia Arg Asn Ala Ser Val Pro T hr Thr Thr Thr Leu Arg Arg His ual Aspl 5 10 15 Leu Leu Val Gly Thr Ala Ala Phe Cys Ser Ala Met (2) SEQ ID NO: +128: (i) Sequence characteristics: (A) Length: 27 amino acids (B) Type 2 Amino Acid (D) Topology: Direct chain (II) Semorable Type: Peptide (XI) Semorable: I28: LEU Aha SERG ARG ARG ARG ARG ASP PHE ALA GLN GLY TRP GAY PRO@Lie 5 ER TYR ALA ASN GLY Ser Gly Leu Asp Gin (2) SEQ ID NO: 129: (i) Sequence characteristics: (A) Length: 27 amino acids (B) Type diamino acids (D) Topology: Linear (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: I29: Tyr Ala Asn Gly Ser Gly Pro Asp Gln (2) SEQ ID NO: +130+ (i) Sequence characteristics: (A) Length: 27 amino acids (B) Type diamino acid (D) Topology : Linear (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 130: Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Asp Gln Gly Trp Gly Pro@Ile 5er His^Ia Asn Gly Ser G ly Pro Asp Gln(2) Sequence number: I31: Ci) Sequence characteristics: (A) Length = 27 amino acids (B) Type diamino acids (D) Topology: Linear (ii) Sequence type: Peptide (xi) Sequence : SEQ ID NO: I31: &t Ala Ser Cys＾rg Pro Ile Asp Glu Ph e＾Ia Gln Gly Trp Gly Pro Il@'rhr )1is Asp Met ProGlu Ser Ser Ser Asp Gln( 2 ) Sequence number: 132: (D array Characteristics: (A) Length, 27 amino acids (B) Type diamino acids (D) Topology: Linear (i i) Sequence type: Peptide (xi) Sequence: SEQ ID NO: +132: Met Ala Gln Cys Arg Thr lie Asp Lys Phe Asp Gin Gly Trp Gly Pro@Ile Thr t s to 1s Tyr Ala Glu Ser Ser Arg Ser Asp Gln (2) SEQ ID NO: 133: (f) Sequence characteristics: (A) Length: 27 amino acids (B) Type diamino acid (D) Topology: Linear (i i) Sequence species M: Peptide (x i) Sequence: SEQ ID NO: 133: Met Ala Ser Cys Arg Pro Ile Gin Trp Phe Ala Gin Gly Trp Gly Pro@Ile Thr (2) SEQ ID NO: +134: (i) Sequence characteristics: (A) Length: 227 amino acids (B) Type diamino acids (D) Topology: Linear (i i) Sequence type: Peptide (xi) Sequence : SEQ ID NO: 134: Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Asp Gin Gly Trp Gly Pro@lie 5er Tyr Ala Asn Gly Ser Gly Pro Asp Glu (2) Sequence Number +135: (i) Characteristics of the array: (A ) Length: 24 amino acids (B) Type diamino acids (D) Topology: Linear (i i) Type of sequence: Peptide (xD sequence: SEQ ID NO: 135: Ser Thr Ser Gly IJe Thr Gay Asp Asn Thr Thr Thr Ser Ser Glu Pro@Ala Pr.

Ser Gly Cys Pro Pro Asp (2) Sequence number 2136: (i) Characteristics of array: (A) Length = 24 amino acids (B) type diamino acid (D) Topology: linear (ii) Type of sequence: peptide (xi) Sequence: Sequence number: 136: Gly Ser Ser Ala val Asp Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln@Ala Ser Asp Asp Gly Asp Lys Gly (2) Sequence number: 137: (i) Characteristics of array: (A) Length: 24 amino acids (B) type diamino acid (D) Topology: linear (i) Type of sequence: peptide (xi) Sequence; Sequence number: 137: Glu Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Leu Pro Asp Gin@Ala 5er Asp Asp Gly Asp Lys Gly field's reprimand report m-1--Shun 10-11p Roa+lQ Q, t,,,,,,.

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DK, ES, FR, GB, GR, IT, LU, NL, SE), AU , C.A., J.P., K.R. (72) Inventor Zebedee Susan California, United States 92122 San Diego Charmant Drive 7544 (72) Inventor Inchosub Genevieve United States New York East Sixty-Third Street, New York, 10021, Kentucky 504 (72) Inventor: Mark Nith, California, United States of America 92131 San Diego Mira Largoway 11734 (72) Inventor Prince Alfred M. New York, United States of America State 10,576 pounds Ridge Stone Gill Road 154

Claims (1)

[Claims] 1. Genome of non-A, non-B hepatitis virus (NANBV) belonging to the Hatch subgroup A DNA sequence encoding the following DNA sequence: (a) SEQ ID NO: 46 The hatched c59 DNA sequence shown, (b) the same polypeptide as the DNA sequence (a). However, as a result of the degeneracy of the genetic code, the said DNA sequence (a) a different DNA sequence, (c) hybridized to said DNA sequence (a) or (b); and a mutation of the NANBV hatch c59 strain showing mainly the same specific immunological properties. a DNA sequence representing a plant or variant, and (d) hybridizes to the DNA sequence (a) or (b) and produces a hatch subgroup. selected from the group of DNA sequences representing NANBV strains possessing immunological properties of The said DNA sequence. 2. Approximately 10 to 200 nuclei corresponding to a portion of the DNA sequence set forth in claim 1. A DNA sequence having a length of 1 oz, the DNA sequence comprising HCV-1, HCV-1, CV-BK, HCV-J, HC-Jl, HC-J4, HCV-JH and HCV - compared with the nucleotide sequence of a strain of NANBV selected from the group consisting of Hh. have at least one nucleotide difference in their sequences; Differences in amino acids may result in silent mutations or differences in at least one amino acid. A DNA sequence representing the mutation that occurs. 3. A DNA sequence encoding the variable region of the NANBV genome or a part thereof. and the area or a part thereof is (a) V variable region: [There is an array] (46:386-411), (b) Part of V: [There is an array] (46:391-404), (c) V1 variable region: [There is an array] (46:246-275), (d) Part of V1: [There is an array] (46:246-265), (e) V2 variable region: [There is an array] (46:456-482), (f) Part of V2: [There is an array] (46:461-466), (9) Part of V■: [There is an array] (46:473-482), and (h) V3 variable region [There is an array] (46:2356-2379) Amino acid sequences selected from the group consisting of DNA sequence with (shown in arc) 4. A DNA sequence derived from the NANBV genome and characterized in claim 3. The variable region or portion thereof that corresponds to the variable region or part thereof coded by any of the columns. or a part of the DNA sequence. 5. Approximately 18 to 200 DNA sequences comprising the DNA sequence according to claim 3 or 4. A DNA sequence having the length of a cleotide. 6. According to claim 3 or 5, which corresponds to the sequence shown in SEQ ID NO: 46. DNA sequence. 7. NANBV having the amino acid sequence contained in residues 1 to 120 of SEQ ID NO: 1 encoding a structural capsid protein or an immunologically active portion of said protein; DNA sequence encoding. 8. Hybridizes with the DNA sequence according to claim 7, and generates a NANBV structural model. A DNA sequence encoding a psid protein or an immunologically active portion thereof. 9. NANB having the amino acid sequence contained in residues 1 to 20 of SEQ ID NO: 1 A DNA sequence encoding a portion of a V-structure capsid protein. 10. NAN having the amino acid sequence contained in residues 1 to 74 of SEQ ID NO: 1 DNA sequence encoding a portion of the BV structural capsid protein. 11. NA having the amino acid sequence contained in residues 21 to 40 of SEQ ID NO: 1 A DNA sequence encoding a portion of the NBV structural capsid protein. 12. NAN having the amino acid sequence contained in residues 2 to 40 of SEQ ID NO: 1 DNA sequence encoding a portion of the BV structural capsid protein. 13. N having the amino acid sequence contained in residues 69 to 120 of SEQ ID NO: 1 DNA sequence encoding part of the ANBV structural capsid protein. 14. Having the amino acid sequence contained in residues 121 to 326 of SEQ ID NO: 1 Encoding a portion of the NANBV structural capsid protein or immunization of said protein. A DNA sequence encoding a biologically active portion. 15. hybridizes to the DNA sequence according to claim 14 and produces a NANBV structure. a DNA sequence encoding a synthetic envelope protein or an immunologically active portion thereof . 16. NA having the amino acid sequence contained in residues 121 to 176 of SEQ ID NO: 1 A DNA sequence encoding a portion of the NBV structural envelope protein. Engineering 7. Encodes the amino acid sequence contained in residues 1 to 326 of SEQ ID NO: 1 or a DNA sequence encoding an immunologically active portion of said amino acid sequence. 18. A DNA sequence according to any one of claims 1 to 17. DNA sequence. 19. The DNA sequence according to any one of claims 1 to 18 has a function. A recombinant DNA molecule consisting of a vector linked together. 20, said vector is an expression vector, said molecule is capable of expressing said protein in a compatible host; can be expressed, and the NANBV structural protein comprises residues 1 to 2 of SEQ ID NO: 2. The recombinant according to claim 19, which has the amino acid residue sequence shown in group 315. DNA molecule. 21. said vector is an expression vector and said molecule is capable of expressing said protein in a compatible host. can be expressed, and the NANBV structural protein comprises residues 1 to 3 of SEQ ID NO: 3. The recombinant according to claim 19, which has an amino acid residue sequence included in group 252. DNA molecule. 22. said vector is an expression vector and said molecule is capable of expressing said protein in a compatible host. The NANBV structural protein comprises residues 1 to 4 of SEQ ID NO: 4. The recombinant according to claim 19, which has an amino acid residue sequence included in group 252. DNA molecule. 23. said vector is an expression vector and said molecule is capable of expressing said protein in a compatible host. The NANBV structural protein consists of residues 1 to 6 of SEQ ID NO: 6. The recombinant according to claim 19, which has an amino acid residue sequence containing group 27H. DNA molecule. 24. The DNA sequence according to any one of claims 1 to 18, or A recombinant DNA molecule according to any one of claims 19 to 23. transformed host cells. 25. The DNA sequence according to any one of claims 1 to 18, or by the recombinant DNA molecule according to any one of claims 19 to 23. polypeptide or peptide encoded by 26. 26. The polyester of claim 25 having a length of about 7 to about 200 amino acid residues. Ripeptide or peptide. 27. Claims to be a NANBV structural protein having a length of at least 20 amino acids The polypeptide or peptide according to item 25. 28. At least one according to any one of claims 25 to 27 A composition comprising a polypeptide or peptide. 29. The polypeptide or the polypeptide according to any one of claims 25 to 27 or peptides, but NANBV isolates HCV-1, HCV-BK , HCV-J, HC-J1, HC-J4, HCV-JH or HCV-Hh Antibodies that are not immunoreactive. 30. immunoreactive with Hatch c59 isolates of NANBV or portions thereof; ANBV isolate HCV-1, HCV-BK, HCV-J, HC-J1, HC-J 4. Antibodies that do not immunoreact with HCV-JH or HCV-Hh. 31. Claims 25 through 2 in an amount sufficient to perform at least one test. containing at least one polypeptide or peptide as described in any one of the above clauses. A diagnostic test that assays body fluid samples for the presence of antibodies to NANBV structural antigens. Cutting kit. 32. The claimed polypeptide or peptide is immobilized on a solid matrix. The diagnostic kit according to scope item 31. 33. of (i) (a) NANBV in sufficient quantity to perform at least one assay; hatch. 59 isolate, (b) any one of claims 25 to 27. (ii) (c) NANBV Isolates HCV-1, HCV-BK, HCV-J, HC-J1, HC-J4, HC No immunoreactivity with V-JH or HCV-Hh, or (d) C-100 antigen , Body fluid samples for the presence of NANBV structural antigens, including anti-NANBV structural protein antibodies. Diagnostic kit for testing samples. 34. Diagnosis according to claim 33, wherein the antibody is immobilized on a solid matrix. kit. 35. Assaying body fluid samples for the presence of antibodies against NANBV structural antigens A method, (a) The body fluid sample and any one of claims 25 to 27. form an aqueous immunoreaction mixture by mixing with a polypeptide or peptide of accomplished, (b) all said antibodies present immunoreact with said polypeptide or peptide; the aqueous immunoreaction mixture for a sufficient period of time to form immunoreaction products. maintain, (c) the presence of all said immune reaction products formed, and thereby said antibodies; detect the presence of Methods that include. 36. The claimed polypeptide or peptide is immobilized on a solid matrix. The method according to scope item 35. 37. The detection in step (c) comprises: (i) the immunity formed in step (c); The reaction product and labeled specific binding agent are mixed to form a labeling mixture (previously The labeled specific binding agent consists of a specific binding agent and a label), (ii) all said immune reaction products present are bound to said labeled specific binding agent; the labeling mixture for a sufficient period of time to combine to form a labeled product. maintain, (iii) the presence of any said labeled product formed, and thereby 37. The method of claim 36, comprising the step of detecting the presence of the immune reaction product. Law. 38. The specific binding agent is protein A and antibodies anti-human IgG and anti-human The method according to claim 37, wherein the method is selected from the group consisting of at least one of IgM. . 39. The label can be combined with lanthanide chelates, biotin, enzymes and radioactive molecules. 38. The method of claim 37, wherein the isotope is selected from the group consisting of: 40. 1. A method of assaying a body fluid sample for the presence of NANBV polynucleic acid, comprising: , (a) having a DNA sequence according to any one of claims 1 to 18; mixing the polynucleotide or oligonucleotide and the body fluid sample; (b) forming an aqueous hybridization mixture by The NANBV polynucleic acid is a hybrid of the polynucleotide or oligonucleotide. a period of time sufficient to hybridize and form a hybridization product the aqueous hybridization mixture during and under hybridization conditions. maintain, (c) the presence of any said hybridization products formed; detecting the presence of the NANBV polynucleic acid by. 41. 1. A method of assaying a body fluid sample for the presence of NANBV structural antigen, comprising: , reacting the sample with the antibody according to claim 29 or 30. Methods that include. 42. An immunologically effective amount of any one of claims 25 to 27. Inocula containing the polypeptides or peptides described (said polypeptides or peptides) Tide can be used alone or in a pharmaceutically acceptable excipient in the presence of an antigenic carrier. scatter). 43. An immunologically effective amount of any one of claims 25 to 27. Vaccines containing the polypeptides or peptides described (said polypeptides or peptides) peptides, alone or conjugated to antigenic carriers and in pharmaceutically acceptable excipients. fraction). 44. A method for producing a NANBV structural protein, comprising: (a) claim 24; Starting a culture containing a nutrient medium containing the transformed host cells as described in Section 3. (b) when the transformed host cell is sufficient to express NANBV structural proteins; maintaining the culture for an intervening period; (c) recovering NANBV structural proteins from the culture. 45. 1. A method of inducing antibody production in a mammal, wherein the antibody is associated with NANBV. (a) administering the contact according to claim 42 to the mammal; (b) said mammal mammal for a period of time sufficient to immunologically respond and produce anti-NANBV antibodies. A method that involves maintaining things.