WO1992018532A1 - Arn, adn et proteine antigenique virale du virus de l'hepatite non-a, non-b - Google Patents

Arn, adn et proteine antigenique virale du virus de l'hepatite non-a, non-b Download PDF

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Publication number
WO1992018532A1
WO1992018532A1 PCT/JP1992/000464 JP9200464W WO9218532A1 WO 1992018532 A1 WO1992018532 A1 WO 1992018532A1 JP 9200464 W JP9200464 W JP 9200464W WO 9218532 A1 WO9218532 A1 WO 9218532A1
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ser
leu
ala
dna
hepatitis virus
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Japanese (ja)
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Terukatsu Arima
Takashi Ohara
Junichi Tomatsu
Takashi Sawada
Tetsuya Hosoda
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Eisai Co Ltd
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Eisai Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to an antigen polypeptide related to non-A non-B hepatitis virus, a DNA fragment encoding the same, and an anti-non-A non-B hepatitis virus antibody. Furthermore, the present invention relates to a method for detecting an anti-non-A non-B hepatitis virus antibody, a non-A non-B hepatitis virus antigen and a non-A non-B hepatitis virus gene.
  • Non-A and non-B hepatitis is currently thought to account for about 95% of post-transfusion hepatitis.Therefore, it is necessary to detect the virus-infected person and exclude it from blood for blood circulation, and to develop a vaccine for prevention. It is strongly desired. Recently, Houghton et al. Announced that the virus gene was isolated (Japanese Patent Application Laid-Open No. 2-500880), and have sold an antibody detection reagent by EIA using the virus antigen. The application of this antibody detection reagent is expected to prevent non-A, non-B hepatitis after blood transfusion, but the incidence of hepatitis is 5 to 7% even if positive donor blood is excluded. Yes, it has not yet disappeared.
  • HCV hepatitis C virus
  • An object of the present invention is to provide a novel non-A non-B hepatitis virus antigen polypeptide and a DNA encoding the same, and to provide a method for diagnosing non-A non-B hepatitis using the same and a diagnostic reagent. I do.
  • non-A non-B hepatitis virus As a result of intensive studies using blood donor plasma in Japan showing a high s-GPT level as a raw material, isolation and structural confirmation of DNA derived from a new non-A non-B non-B hepatitis virus different from the virus reported so far succeeded in. Furthermore, they have found that non-A, non-B hepatitis can be diagnosed, prevented, and treated using these DNAs and the polypeptides encoded thereby, thereby completing the present invention.
  • the present invention relates to partial DNAs (SEQ ID NOS: 1, 2 and 3) homologous to non-A non-B hepatitis virus gene RNA, and to the non-A non-B hepatitis virus antigen polypeptides encoded by them (SEQ ID NOs: 1, 2 and 3). 3) and a polyclonal or monoclonal antibody using these antigenic polypeptides as antigens. That is, the present invention is a polypeptide comprising all or a part of the non-A non-hepatitis B virus antigen polypeptide of the following sequences 1, 2 and 3.
  • Array 1
  • the present invention provides a DNA encoding the above-mentioned polypeptide, which comprises all or a part of the DNAs of the above-mentioned sequences 1, 2 and 3, a polyclonal antibody or a monoclonal antibody having the above-mentioned polypeptide as an antigen,
  • a method for detecting a non-A non-B hepatitis virus gene which comprises amplifying and detecting a non-A non-B hepatitis virus-derived DNA using the above DNA as a primer as a constituent of a reagent.
  • An anti-non-A non-B hepatitis virus antibody detection method for confirming the presence of an anti-non-A non-B hepatitis virus antibody in a sample by an immunological technique using the above-mentioned antigen polypeptide;
  • a non-A non-B hepatitis virus antigen detection method for confirming the presence of a non-A non-B hepatitis virus antigen in a sample by an immunological technique, and a non-A non-A non-B virus produced using the antigen polypeptide described above.
  • Hepatitis B Also provides Kuching.
  • the present invention also provides a method for detecting a non-A non-B hepatitis virus gene by amplifying and detecting non-A non-B hepatitis virus-derived DNA using a partial oligonucleotide of the DNA of the present invention as a primer. included.
  • the present invention also includes RNA homologous to the DNA of the present invention.
  • oligonucleotides and polypeptides of the present invention contain at least 10 bases or amino acids (length).
  • the configuration of the present invention is as follows.
  • RNA As a raw material for extracting non-A non-B hepatitis virus RNA, blood plasma of a donor whose S-GTP value is high can be used.
  • the virus surface is collected by centrifugation after addition of polyethylene glycol according to a known method.
  • the extraction and purification of RNA from the virus surface is performed, for example, by extracting from the virus surface with a mixed solution of guanidine thiosinate, a surfactant, a chelating agent and a reducing agent, followed by phenol extraction and organic solvent extraction.
  • Surface (Chamezynski et al, Anal, Biochem., 162 ⁇ 156, 1987), followed by density gradient ultracentrifugation to obtain purified RNA.
  • RNA as type ⁇ RNA as type ⁇
  • a conventional method such as cDNA synthesis using random primers, reverse transcriptase, DNA polymerase, etc. (Gubler, ⁇ . Et al. Gene, 25.263, 1983) Two pieces of DNA can be prepared.
  • the double-stranded DNA thus obtained is integrated into a pacteriophage such as Azap or Agtll according to a conventional method, and then cultured and screened using Escherichia coli as a supporting bacterium. Obtainable.
  • Examples of a method for incorporating the double-stranded DNA into a phage vector include the method described in Hyunh, T. V. et al., DNA Cloning, a practical approach, _1, 49 (1985).
  • the screening method of the target clone may be in accordance with a known method.
  • a double-stranded DNA is incorporated into a ⁇ gt11 phage vector
  • the vector is infected with B. coli Y1090 and contained in the formed plaque.
  • the polypeptide to be transferred is transferred to a nitrocellulose membrane, and the transferred membrane is then screened by immunological techniques using serum of a non-A, non-B virus hepatitis patient as the antibody source for the virus. For example, a method of providing for ninging is used.
  • RNA is prepared from fresh plasma of a patient confirmed to be infected with non-A non-B hepatitis virus by the above method.
  • the DNA was amplified by a conventional PCR method and subcloned to obtain non-A non-B hepatitis.
  • DNA from virus can be obtained.
  • Primers can be used as primers by synthesizing oligonucleotides by appropriately selecting well conserved portions based on known non-A non-B hepatitis virus DNA sequences. A fragment consisting of a base sequence is used as a primer.
  • the nucleotide sequence structure can be determined by the Maxam, Gilbert method (Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA, 74, 560. 1977) or the dideoxy method (Messing, J. et al, Nucl. Acids). Res., _9, 309, 1981).
  • the partial nucleotide sequences of the non-A non-B hepatitis virus DNA of the present invention are shown in SEQ ID NOs: 1 and 2, and the sequences having homology thereto are described by SDC GENETYX (SDC Software Development Co., Tokyo). Searched using As a result, no sequence with more than 60% homology was found in the GenBank database. In comparison with the previously reported nucleotide sequence of non-A non-B hepatitis virus DNA, it was 69.5% at the base level and 69.5% at the amino acid level with the sequence reported by Houghton et al. 79.7%, the sequence reported by Kato et al. (HC VJ) (Kato et. Al., Proc. Natl. Acad. Sci. USA, 87, 9524-9528, 1990) had a homology of 71.8% at the nucleic acid level and 83.1% at the amino acid level.
  • non-A non-B hepatitis virus antigen polypeptide or a part thereof of the present invention the presence or absence of anti-non-A non-B hepatitis virus antibody in a biological sample, for example, the serum of a patient, is assayed by an immunological technique. It is possible to diagnose the virus infection.
  • a biological sample for example, the serum of a patient
  • an immunological technique It is possible to diagnose the virus infection.
  • known methods can be applied to the immunological method, and examples of the known method include enzyme immunization, radioimmunization, western blotting, active agglutination, latex agglutination, and chemiluminescence immunization. .
  • Antibodies can be prepared using the non-A non-B hepatitis virus polypeptide of the present invention or a part thereof as an antigen.
  • Polyclonal anti-non-A, non-B hepatitis virus polypeptide polyclonal antibody can be obtained by subcutaneously, intramuscularly, intraperitoneally, or intravenously inoculating the antigen multiple times in animals such as mice, guinea pigs, and egrets according to standard methods. After immunization, it can be obtained by collecting blood from the animal and separating serum.
  • Monoclonal antibodies can also be prepared by known methods.
  • the culture supernatant of the hybridoma or the bipridoma is intraperitoneally
  • Monoclonal antibodies to non-A, non-B hepatitis virus polypeptides can be prepared from the ascites of the administered mouse.
  • commercially available adjuvants can also be used.
  • These antibodies are used for non-A non-B hepatitis in biological samples by known immunological techniques. It enables identification and quantification of virus antigens. That is, these antibodies can be used as diagnostic reagents for non-A, non-B hepatitis virus antigens.
  • non-A, non-B hepatitis virus reports include Houghton et al. (HCV-US), Kato et al. (HCV-J), Okamoto et al. (HCV-J6), and As is clear from the four reports including the virus of the invention, it is known that there are several subtypes of non-A non-B hepatitis virus.
  • an immunoassay for an antibody against the virus of the present invention in a biological sample or the virus antigen voriveptide is useful as a method for diagnosing virus infection, but identifies and determines each virus subtype. Difficult to do.
  • the DNA of SEQ ID NO: 1 corresponds to a part of a region encoding a non-structural protein of a virus
  • the DNA of SEQ ID NO: 2 corresponds to a part of a region encoding a structural protein.
  • RNA prepared from a biological sample such as a sample plasma is used as a category type, and the DNA is amplified by an RT-PCR method using an appropriate antisense primer, a sense primer and a reverse transcriptase, and the DNA is amplified.
  • the primer may be selected appropriately based on the DNA base sequence of each subtype virus.
  • the type of the virus can be determined by examining whether a DNA of a predetermined size has been amplified by polyacrylamide gel or agarose gel electrophoresis.
  • the confirmation can be performed by using, as a probe, a labeled synthetic oligonucleotide of each sub-Eve virus located in the middle of the primer.
  • the DNA When analyzing the structure of the amplified DNA, it is preferable to incorporate the DNA into an appropriate vector if necessary, and to introduce and replicate the DNA into an appropriate host. This makes it possible to analyze the nucleotide sequence.
  • a vaccine can be prepared by using the non-A non-B hepatitis virus subtilis dipeptide, and the vaccine can be used as a therapeutic agent for non-A non-B hepatitis.
  • RNA was prepared according to the procedure described above.
  • c-DNA Synthesis System (8267SA) of BR Shisha (Bethesda Research Laboratories Life Technologies Inc. Gait her-sbuog)
  • c-DNA synthesis was performed according to the manual, and BRL c-DNA Cloning System AgtlO a nd; using igtll (8285SA) according to the manual; double-stranded c-DNA was incorporated into one of the Igtll vectors.
  • In Vitro Packaging was performed using Gigapack TM Gold II Packaging Extract from Stratagene (Stratagene. La Jolla). Specifically, first, random primers were added to type I RNA, and single-stranded c-DNA was synthesized using reverse transcriptase.
  • double-stranded c-DNA was synthesized using RNase H, E. coli DNA Polymerase I, and E. coli DNA Ligase. Obtained double-stranded c-DNA was treated with EcoRI Methylase to methylate the EcoRI cleavage site inside the c-DNA, and then the ends were blunted using T4 DNA Polymerase. This blunt end was connected with an EcoRI linker phosphorylated using T4 DNA Ligase. After cutting excess linker with EcoRI, the EcoRI linker was removed by gel filtration. The resulting double stranded c-DNA with EcoRi-cut ends was connected to the arm DNA of the Igtll phage using T4 DNA Ligase. The obtained recombinant phage DNA was mixed with In Vitro Packaging Extract at room temperature to obtain a recombinant phage.
  • a blackout of igtll recombinant phage was formed according to a conventional method.
  • a nitrocellulose filter (Schleicher & SchueIL BA85) immersed in lOmM IPTG (Sigma) was placed on the plaque, and the cells were further cultured at 37 ° C for 3 hours.
  • the nitrocellulose filter was washed four times with TBS (pH 7.5) (10 mM Tris-HCl. 150 mM NaCl), then immersed in a 5% skim milk solution (TBS containing 5% skim milk, 0.05% Tween) for 4 e. Shake slightly with C overnight.
  • the serum of 10 patients with non-A and non-B hepatitis was mixed in equal volumes, an equal volume of E. coli Y1090 lysate was added thereto, shaken at room temperature for 60 minutes, and then centrifuged at 3,000 rpm for 30 minutes. The supernatant was diluted 10-fold with a 5% skim milk solution and used as a primary antibody solution. 3. Add the primary antibody solution to the nitrocellulose filter onto which the plaque has been transferred. Shake slightly with C overnight.
  • This nitrocellulose filter was washed twice with TBS-0.05% Tween at room temperature for 5 minutes.After that, as an enzyme-labeled secondary antibody solution, Anti human IgG (Fc) (Goat) -Peroxidase conj ugateCCappel) was diluted 500-fold with TBS-0. 05% Tween. A nitrocellulose filter was placed in this, and the mixture was slightly shaken at room temperature for 2 hours.
  • nitrocellulose filter After washing the nitrocellulose filter twice using TBS-0.05% Tween and washing at room temperature for 5 minutes twice, the substrate solution (TBS (pH 6.5) was washed with 10 ⁇ , 3 ⁇ - A nitrocellulose filter was placed in a mixture of Rho-1 naphthol nomethanol solution and a 2-3% aqueous solution of hydrogen peroxide (hydrogen peroxide solution) in a ratio of 40_ ⁇ , and the mixture was shaken at room temperature. Color development was stopped by washing the filter with distilled water, and 10 positive clones were selected.
  • TBS pH 6.5
  • the nucleotide sequence was determined by the Dideoxy Termination method using M13 phage as usual. Actually, the measurement was performed according to the manual using USB Company (United States Biochemical Corporation, Cleve-land) ⁇ Sequenase version 2.0 Labeled dCTP Edition.
  • the DNA was a partial DNA of a base encoding a novel non-structural protein of non-A non-B hepatitis virus.
  • the amino acid sequence encoded from this base sequence was also deduced as described in the same SEQ ID NO.
  • Escherichia coli 2-22-igtll in which the phage ⁇ incorporating the c-DNA was introduced into E. coli Y1088 strain, was obtained from ⁇ 305 Deposited on March 24, 1992 under No. 12899 (FERM P-12899), accession number: 1-13-1, Higashi, Tsukuba, Ibaraki, Japan .
  • RNA was prepared from a plasma specimen which was found to have the virus genome according to Example 3. Based on the nucleotide sequence of HCV-J reported by Kato et al. (Kato et al, Proc, Natl. Acad. Sci. USA, 87, 9524, 1990), primers are 1289-1309 as sense primers and as antisense primers. Positions 1572 to 1592 were selected, and PCR was performed using each synthetic oligonucleotide by a conventional method.
  • a restriction enzyme recognition sequence that can be cloned into M13mpl8 and M13mpl9 that is, a Smal and BainHi recognition sequence for a sense primer, and a Pstl and Sail recognition sequence for an antisense primer. was used.
  • nucleus was found to be a region coding for a new non-A non-B hepatitis virus structural protein, as shown in SEQ ID NO: 2. Part of the DNA. Also, the amino acid sequence encoded from this nucleotide sequence was deduced as described in the same SEQ ID NO.
  • the nitrocellulose filter was washed twice with TBS-0.05% Tween at room temperature for 5 minutes.After that, as a secondary antibody solution, Anti-human IgG (Fc) (Goat) -Peroxidase conjugate ( Cappel) was diluted 500-fold with TBS-0.05% Tween to prepare a nitrocellulose filter, and the mixture was slightly shaken at room temperature for 2 hours. Nitrocellulose filter with TBS-0.05%
  • the substrate solution (10 mg of TBS (pH 6.5), 3 mg / 4-chloro-11-naphthol / methanol solution, 2 m £, 3% A mixture of hydrogen peroxide at a ratio of 40% ⁇ ) was added to the nitrocellulose filter, and shaken at room temperature. Color development was stopped by washing the filter with purified water. Recombination with the plaque site of wild-type Agtll phage; Positive when there was a difference in the color development of the plaque site of Igtll phage (2-22), and negative when there was no difference. Table 1 shows the results.
  • C100-3 is the result according to the method of Houghton et al. (JP-A-2-500880).
  • C100-3 8 out of 12 sera of non-A non-B hepatitis patients are positive, but 3 out of 5 sera of hepatitis B patients are positive, and the specificity is not clear .
  • the method 2 to 22 of the present invention 7 out of 12 serum samples of non-A non-B hepatitis patients showed positive, serum of hepatitis B patient showed negative, and the method according to the present invention in terms of specificity. The method is much better.
  • Example 5 incorporates the DNA of SEQ ID NO: 1; since it uses Igtll, it is a measurement of an antiviral antibody using a polypeptide containing the entire structure of SEQ ID NO: 1 as an antigen. However, rather than using a polypeptide containing the entire structure, it is better to select a peptide portion with high antigenicity in that structure and detect the antibody by ELISA using the synthetic peptide of that portion as an antigen. The decision of Epitope was made because it is simple and practical.
  • peptides corresponding to positions 1-120, 11-30, 21-40, 31-50, and 41-59 were synthesized and used to determine the epitope site. Provided. Detection of the antiviral antibody was performed by a conventional ELISA method. That is, the serum coated with each synthetic peptide was added with a sample serum that was determined to be positive in Example 5 and reacted (1 hour at 37). After washing, an alkaline phosphatase-labeled anti-human IgG antibody was reacted (37%). After washing, then adding the enzyme substrate P-ditrophenyl phosphate and reacting at 37 for 30 minutes, the absorbance at a wavelength of 405 nm was measured.
  • the synthetic peptide at position 11-30 showed the highest antigen activity. From this, it is possible to test anti-non-A non-B hepatitis virus by using a synthetic peptide containing 11-130 or a synthetic peptide containing the same as an antigen.
  • RNA was prepared by the method of Example 1 from human plasma 1; ⁇ of the non-A non-B hepatitis virus infection high-risk group. RT-PCR was performed according to a conventional method using 1/6 amount of the obtained RNA.
  • c-DNA synthesis is called lOmM Tris-HW (pH8.3), 50 mM KC £, 2 mM MgC, 200 mM dNTPs, 0.01% gelatin ⁇ 20 units Placental RNase inhibitor, lOOmM antisense primer, 100 units Murine reverse transcriptase
  • the reaction was performed at 37 ° C for 30 minutes. After c-DNA synthesis, it was heat denatured at 95 ° C for 5 minutes, followed by PCR.
  • the PCR reaction composition was lOra Tris-HC (pH8.3), 50mKC, 1.5mM MgC £ 2 , 200ra dNTPs, 0.01% gelatin, lOOraM sense primer, lOOraM antisense primer, 1 unit Taq I-polymerase After 60 cycles of 94 ° (1 minute heat denaturation, 55 ° C, 2 minute annealing, 72 ° C, 1 minute 30 seconds DNA extension reaction. One tenth of the solution was subjected to 6% polyacrylamide gel electrophoresis, stained with bromide titanium, and photographed under ultraviolet irradiation. Of the 30 C100-3 antibody-positive plasma samples in Japan, 14 were positive.
  • Houghton et al., HCV-US, Kato et al., HCV-J, and the 2-22 virus of the present invention were discriminated.
  • an oligonucleotide corresponding to the same position as the oligonucleotide nucleotide primer in the nucleotide sequence of SEQ ID NO: 1 used in Example 6 was synthesized and used as a primer.
  • Table 3 shows their nucleotide sequences and position numbers.
  • the C100-3 antibody was strongly positive (2.000 ⁇ ), which was determined to be strongly positive by the antibody detection reagent using the so-called Houghton et al. Virus antigen.
  • the non-A, non-B hepatitis virus RNA prepared according to the method the obtained RNA was assumed to be plasma type 100 equivalent to type II, 200 nM of each of the above-mentioned primers was added, and normal RT-PCR was performed. By performing ⁇ 60 cycles, the genome was amplified and detected.
  • the annealing temperature in the PCR reaction was set to (at a Tni value of 15) based on the Tm value calculated from the base sequence of the primer.
  • HCV-US 5 '-GAGGTGG GGAATACACCAAAnGTGGTGACGTAGCAACG-3 * HCV-US; 7687nt-7726nt
  • non-A non-B hepatitis can be diagnosed, and infection of the hepatitis virus by blood transfusion or the like can be prevented, and vaccines and antiviral agents can be developed.
  • Sequence type nucleic acid
  • Fragment type middle fragment
  • Sequence type nucleic acid
  • Fragment type middle fragment
  • Organism name non-A non-B hepatitis virus
  • Sequence type nucleic acid
  • Fragment type middle fragment
  • Organism name non-A non-B hepatitis virus

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Abstract

L'invention se rapporte à un ARN génomique du virus de l'hépatite non-A, non-B, à un ADN homologue de cet ARN, et à un polypeptide du virus de l'hépatite non-A, non-B. On identifie un nouvel ADN du virus de l'hépatite non-A, non-B, en procédant à l'extraction d'un ARN viral contenu dans le plasma d'un patient atteint d'hépatite virale non-A, non-B, en purifiant cet ARN, et en préparant un ADN bicaténaire avec cet ARN purifié utilisé comme modèle, en introduisant l'ADN ainsi obtenu dans un micro-organisme Escherichia coli, et en recherchant par triage un clône qui exprime la protéine virale. L'utilisation de cet ARN, de cet ADN et de ce polypeptide permet de diagnostiquer et de soigner l'hépatite virale non-A, non-B.
PCT/JP1992/000464 1991-04-17 1992-04-13 Arn, adn et proteine antigenique virale du virus de l'hepatite non-a, non-b Ceased WO1992018532A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000874A1 (fr) * 2000-06-26 2002-01-03 Ajinomoto Co., Inc. Polypeptides, leur utilisation ainsi que leur procede de production

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02500880A (ja) * 1987-11-18 1990-03-29 カイロン　コーポレイション Ｎａｎｂｖの診断用薬およびワクチン

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02500880A (ja) * 1987-11-18 1990-03-29 カイロン　コーポレイション Ｎａｎｂｖの診断用薬およびワクチン

Non-Patent Citations (8)

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Title
CELL TECHNOLOGY, Volume 10, No. 11, 1 November 1991, NOBUYUKI KATO et al.: "Development of a Structure of the C-hepatitis Virus Ch(v) Gene", see p. 835-844. *
CELL TECHNOLOGY, Volume 10, No. 11, 1 November 1991, NORIO HAYASHI et al.: "A Method for Detecting the C-Hepatitis Virus Infection", see p. 853-859. *
CELL TECHNOLOGY, Volume 10, No. 11, 1 November 1991, YOSHIHARU MATSUURA et al.: "Development of the C-Hepatitis Virus Protein", see p. 845-852. *
J. VIROLOGY, Volume 65, 1991, A. TAKAMIZAWA et al., see p. 1105-1113. *
PROC. NATL. ACAD. SCI. USA, Volume 87, No. 24, December 1990 (12.90), N. KATO et al.: "Moleculan Cloning of the Human Hepatitis C Virus Genome from Japanese Patients with Non-A, Non-B Hepatitis", see p. 9524-9528. *
PROC. NATL. ACAD. SCI. USA, Volume 88, No. 5, 1 March 1991, J.H. HAN et al.: "Characterization of the Terminal Regions of Hepatitis C Viral RNA: Identification of Conserued Sequences in the 5' Untranslated Region and Poly (A) Tails at the 3' End", see p. 1711-1715. *
PROC. NATL. ACAD. SCI. USA, Volume 88, No. 6, 15 March 1991, Q-L. CHOO et al.: "Genetic Organization and Diversity of the Hepatitis C Virus", see p. 2451-2455. *
SCIENCE, Volume 244, 21 April 1989, G. KUS et al., "An assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A Non-B Hepatitis", see p. 362-364. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000874A1 (fr) * 2000-06-26 2002-01-03 Ajinomoto Co., Inc. Polypeptides, leur utilisation ainsi que leur procede de production
US7189810B2 (en) 2000-06-26 2007-03-13 Ajinomoto Co., Inc. Polypeptides, use thereof and process for producing the same

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