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WO1992001714A1 - Antigene du virus de l'hepatite non a, non b - Google Patents

Antigene du virus de l'hepatite non a, non b Download PDF

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Publication number
WO1992001714A1
WO1992001714A1 PCT/JP1991/000964 JP9100964W WO9201714A1 WO 1992001714 A1 WO1992001714 A1 WO 1992001714A1 JP 9100964 W JP9100964 W JP 9100964W WO 9201714 A1 WO9201714 A1 WO 9201714A1
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Prior art keywords
antigen
arg
pro
leu
asp
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PCT/JP1991/000964
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English (en)
Japanese (ja)
Inventor
Yasuaki Shimizu
Takeshi Imai
Junko Ishida
Emiko Yano
Syuhei Yasuda
Tetsuo Morinaga
Terukatsu Arima
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Yamanouchi Pharmaceutical Co., Ltd.
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Publication of WO1992001714A1 publication Critical patent/WO1992001714A1/fr

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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 a novel non-A non-B hepatitis virus antigen.
  • an antigen polypeptide that specifically shows an antigen-antibody reaction with serum of a non-A non-B hepatitis patient, a polynucleotide encoding the antigen polypeptide, and an antigen polypeptide using the polynucleotide
  • a method for detecting and diagnosing a non-A non-B hepatitis virus antibody using the antigen polypeptide is a method for detecting and diagnosing a non-A non-B hepatitis virus antibody using the antigen polypeptide.
  • Non-A, non-B hepatitis is the hepatitis A virus (HAV), hepatitis B virus (HBV), Hepatitis caused by hepatitis hepatitis virus (HDV) or cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, etc.
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HDV hepatitis hepatitis virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • adenovirus etc.
  • Epidemiological evidence suggests that there are three types of non-A, non-B hepatitis: Water-borne epidemics prevalent in developing countries, post-transfusion-type transmission via blood, and sporadic (population-acquisition) types found in developed countries.
  • non-A non-B hepatitis means the one caused by the remaining post-ring blood type and sporadic type, excluding this water-borne type.
  • hepatitis C virus HCV
  • HCV hepatitis C virus
  • Both C100 antigen and clone 14 antigen encoded by these genes specifically react with 60-80% of post-transfusion non-A non-B chronic hepatitis sera and sporadic non-A non-B chronic hepatitis sera.
  • HCVcore antigens [Kunitada Shimotono et al., The 38th Annual Meeting of the Japanese Society for Virology, abstract 112; Izumi Saito, et al., Abstract 104; Shizuko Harada et al., 105, 1990 (1990)], including its epitope Peptide fragments CP9, CP10 [Okamoto, H., et al., Japan J. Exp. Med., 60, 223 (1990)] and non-A non-B hepatitis-related antigen GOR derived from cellular genes [K. Akabane et al.
  • HCV diversity HCV is known to be a highly mutated virus, [Enomoto. N., et al., BBRC, 170, 1021 (1990); Shuichi Kaneko et al., Liver, 31 suppl. 213 (1990); Nobuyuki Kato et al., Liver, 31 suppl.
  • the present inventors have 1) an antigen having a high reaction rate to a serum of a non-A non-B hepatitis patient after blood transfusion, 2) an antigen having a high reaction rate to a serum of a patient with sporadic non-A non-B hepatitis, 3) acute Antigens that can also react with the serum of patients with non-A and non-B hepatitis in the early stage (antigens that can detect viral antibodies that appear earlier in infection), and 4) antigens that can detect viral antibodies that are highly correlated with viremia.
  • a novel non-A, non-B hepatitis virus antigen different from the existing antigen was obtained from Japanese human plasma.
  • nucleotide sequence of the gene When the nucleotide sequence of the gene was determined, it was found that the nucleotide sequence at positions 4138 to 4521 and 6757 to 6909 of the previously reported nucleotide sequence of HCV (the nucleotide sequence shown in FIG. 17 of European Patent Application Publication No. 388232). It was shown to be a novel non-A, non-B hepatitis virus antigen with 10% and 22% homology to the corresponding amino acid sequence, respectively, but containing different amino acid sequences. By using these antigens alone or in combination, or in combination with other antigens as appropriate, non-A, non-B hepatitis virus antibodies can be detected with a much higher detection rate than existing HCV antibody measurement kits. Knowing that it is possible, they completed the present invention.
  • the present invention relates to an antigen polypeptide Y19 group, an antigen polypeptide Y22 group or a fragment group thereof, and a composite antigen polypeptide group in which two or more antigens selected from these polypeptide groups are fused.
  • antigen polypeptides A single antigen selected from the group of antigenic polypeptides having an epitope that can be regarded as immunologically identical to the epitope contained in the Y19 group or the Y22 group, or a mixed antigen obtained by mixing two or more antigens Reacts with non-A, non-B hepatitis virus antibodies present in biological samples.
  • the present invention relates to a method for detecting a non-A non-B hepatitis virus antibody, wherein the immunological conjugate formed is measured to confirm the presence of the non-A non-B hepatitis virus antibody in a biological sample. Also, the present invention relates to a non-A non-B hepatitis virus antibody detection kit for performing this detection method. Further, the present invention provides an antigen polypeptide group used for the method and kit for detecting the non-A non-B hepatitis virus antibody, a polynucleotide encoding the polypeptide of the polypeptide group, and The present invention relates to a method for producing a polypeptide contained in the polypeptide group.
  • non-A non-B hepatitis virus antigen polypeptide Y19 group of the present invention is represented by the following general formula (I).
  • non-A non-B hepatitis virus antigen polypeptide Y22 group of the present invention is represented by the following general formula ( ⁇ ).
  • Fragments of the polypeptide Y22 group include groups represented by the following formulas (III) to (VII).
  • a non-A non-Hepatitis B virus antigen polypeptide selected from the group consisting of:
  • the composite antigen polypeptide group is a composite antigen obtained by fusing two or more types of antigens selected from the above-mentioned group of Y19, Y22, and Y22 groups using genetic recombination technology. It is a polypeptide. N which may be mentioned as its typical, for example, under Symbol of Poribe petit de is fused included in Y19 group and Y22 group of (VIII) of the polypeptide (Y22- 19) (VIII)
  • non-A non-B type Representative examples of the hepatitis virus polynucleotide include, for example, the polynucleotide Y19 of the following formula (IX), the polynucleotide Y22 of the formula (X), and the polynucleotide Y22 of the formula (XI). And those represented by the following nucleotide sequence.
  • GCT GTA GCA TAC TAC CGG GGT CTT GAC G TG
  • the present invention relates to a polynucleotide probe for detecting a polynucleotide derived from a non-A non-B hepatitis virus, comprising a continuous sequence derived from the sequence of the complementary strand of the polynucleotide.
  • Invention relating to a polynucleotide probe containing a sequence consisting of 8 bases or more, and reverse transcription-PCR method using a polynucleotide derived from a non-A non-B hepatitis virus (polymerase-chain reaction method)
  • an invention involving a pair of polynucleotide primers containing a sequence having a longer length, and an epitope which can be regarded as immunologically identical to an epitope contained in a polypeptide of the Y19 group or the Y22 group.
  • polynucleotide refers to a polymer of a nucleotide (ribonucleotide or deoxyribonucleotide) having any length.
  • the term also includes both single and double stranded forms.
  • epitope refers to an antigenic determinant of a polypeptide.
  • Epitope can also be formed with three amino acids located within the conformational hormone that is unique to epitope, but generally consists of at least 5 to 10 amino acids.
  • non-A non-B hepatitis-related antigen means an antigen other than the antigen of the present invention and capable of diagnosing non-A non-B hepatitis, and includes both antigens derived from viral genomes and cell genes. For example, C100 antigen, Core antigen, Clone 14 antigen, GOR antigen, etc.)
  • non-A non-B hepatitis virus antigen means a non-A non-B hepatitis-related antigen derived from the virus genome.
  • the “fusion antigen” refers to a non-A non-hepatitis B virus antigen of the present invention, which contains N-terminal and Z- or C-terminal other proteins (eg, ⁇ -gal, Protein A, T7 gene 10, maltose-binding protein, Glutathione S-transferase) or a part or all of them.
  • N-terminal and Z- or C-terminal other proteins eg, ⁇ -gal, Protein A, T7 gene 10, maltose-binding protein, Glutathione S-transferase
  • conventional techniques of molecular biology, microbiology, genetic engineering, and immunology known in the art are employed. Please refer to the following experiment for such a method. Experiment 1: Sambrook ⁇ , Fritsch. E.F., and Maniatis, T. Molecular Cloning 2nd ed .; Cold Spring Harbor (1989)
  • Experiment 2 DNA cloning 1 roll (Glover, DM) IRL Press.
  • Cloning policy for non-A non-B hepatitis virus antigen gene cloning is based on 1) RNA from starting material that is thought to contain the viral genome. 2) Preparation of ⁇ gtll cDNA library, 3) Immunoscreening with serum from patients with non-A and non-B hepatitis.
  • HCV is known to be rich in mutations, but multiple (10 or more) individuals are required to reliably and efficiently clone highly comprehensive antigens (epitopes) that react with any virus antibody.
  • RNA derived from Escherichia coli It is desirable to prepare cDNA based on RNA derived from Escherichia coli and perform immunoscreening on the mixed serum of multiple (10 or more) non-A non-B hepatitis patients. This makes it easier to obtain antigens that react with antibodies shared among non-A, non-B hepatitis patients.
  • HBs antigen-negative, HBV-DNA-negative, human plasma with high ALT (GPT) activity (> 35 IU / L) or non-A non-B hepatitis patient serum is pooled and used as starting material for viral RNA preparation .
  • the virus particles are concentrated and recovered as a precipitate by polyethylene glycol precipitation. Sinking Extraction of RNA from the precipitate is performed by the AGPC method using guanidine * thiosyanate [Chomczynski, P. and Sacchi, N .: Analytical Biochemistry 162, 159 (1987)] and the like.
  • chimpanzee serum infected with non-A, non-B hepatitis virus can be used as a starting material in place of human plasma or serum, but in general, when the virus is passaged in a heterologous animal, a mutation in the virus genome occurs. It is desirable to use human plasma or serum as a starting material when cloning the antigen, as they may occur or sometimes alter the pathogenicity of the virus. Because HCV is transmitted through the blood or in a collective manner, certain virus strains may be endemic in certain areas. Therefore, it is preferable to use virus antigens derived from Japanese for diagnosis in Japan.
  • RNA Preparation of RNA from liver tissue of patients Patients with liver cancer considered to have had infection with non-A, non-B hepatitis in the past can be obtained from liver tissue resected from patients with liver cancer using standard methods [Exp. 1,7]. RNA can be prepared.
  • RNA RNA as type III
  • a double-stranded cDNA is synthesized by the Gubler-Hoffman method [Gene, 25, 263 (1983)] using a random hexamer as a primer.
  • the cDNA is inserted into a gtll phage vector by a conventional method.
  • Agtll is a vector that has been developed and cloned for cloning the gene of an antigen corresponding to a specific antibody [Young, RA and Davis, RW, Proc. Natl. Acad. Sci. USA, 80, 1194 (1983) ), Experiment 2, p49-78] 0 or vectors other than ⁇ gtll such as ZAP, ⁇ ⁇ , pUC19, pUEXl may be used.
  • a recombinant infected with Escherichia coli (eg, strain Y1090); gtll grows under certain conditions according to the growth mechanism of pacteriophage, Along with this, the previously integrated cDNA is also amplified. Proliferated bacteriophages lyse host E. coli under certain conditions and form plaques that are visually recognizable. Since the cDNA inserted into the gtl phage vector is translated as a fusion antigen with ⁇ -galactosidase ( ⁇ -gal), a large number of recombinant antisera raised against specific antigens By screening the body gtll, cDNAs encoding specific antigens can be identified by immunochemical methods.
  • the cDNA library prepared in (3) above is screened with pooled sera derived from non-A non-B hepatitis patients. Specifically, the fusion antigen expressed from the culture dish forming the plaque was transferred to a ditrocellulose membrane, and the pooled serum described above was reacted as a primary antibody, and the peroxidase-labeled goat anti- Human IgG) IgG is reacted as a secondary antibody, and plaques that are positive for color reaction are isolated.
  • nucleotide sequence of the antigen gene of the present invention can be obtained by subjecting the recombinant DNA and the subcloned plasmid DNA to type III by the Sanger method [Sanger, F., et aL. Proc. Natl. Acad. USA., 74,5463 (1977), Experiments 1, 13], and can be determined using a commercially available sequence kit.
  • Polynucleotides containing a continuous sequence of 8 bases or more in length derived from the complementary strand of the non-A non-B hepatitis virus polynucleotide of the present invention hybridize with the virus genome of the present invention, so that serum And useful as a probe for detecting the virus genome in tissues.
  • These polynucleotides can be synthesized by a phosphoramidite method [Hunkapiller, M. et al. Nature 310, 105-111 (1984)] or a commercially available DNA synthesizer (for example, Applied Biosystems (ABI), 380AM DNA synthesizer).
  • non-A non-B hepatitis virus polynucleotide of the present invention can be synthesized by using the polynucleotide as a type II and subjecting it to a double translation method, a random primer labeling method, and the like.
  • the polynucleotide is used for diagnosis, it is used as a labeling probe such as a radioactive probe, a biotin fluorescent probe, and a chemiluminescent probe in a usual manner, but the hybridization method described above is used.
  • the method of labeling, and the method of signal amplification and detection are all known, and can be carried out with reference to, for example, Toyozo Takatachika.
  • the base K columns of the present invention One part or two or more primer pairs for PCR (polymerase-chain-reaction) are created using a part of the primers, and reverse transcription-PCR method [Kawasaki, ES, et al "Proc. Natl. Acad. Sci. USA, 85, 5698 (1988); Garson. JA et al., Lancet, 335, 1419 (1990); Enomoto, N “et al” BBRC, 170, 1021 (1990); Okamoto, H., et al., Japan J. Exp. Med., 60, 215 (1990)] to amplify the target viral genome and then detect it.
  • the antigen polypeptide and the polypeptide constituting the epitope and the fusion antigen polypeptide containing the sequence thereof can be expressed using a commercially available expression vector.
  • a commercially available expression vector for example, for expression in E. coli, pKK233-12, pKK223-3, pPL-lambda, pRIT5, pRIT2T, pMC1871 (Pharmacia), pMAL-c, pMAL-p (New England Biolabs), pEX 1-3 (BM), pGEX (AMRAD) or the like can be used.
  • the antigen polypeptide of the present invention is expressed in Escherichia coli, Met derived from the initiation codon is added to the N-terminus, and the linker and adapter used in constructing the expression vector, as well as the multicloning site, are used. In some cases, about 1 to 20 amino acids derived from the base sequence of the amino acid may be added to the N-terminal of the polypeptide (for example, in the examples of the present specification, the N-terminal of the Y19 antigen is Met-Ala However, Met-Ala-Ala-Ala is added to the N-terminal of the Y22-19 antigen).
  • Expressed recombinant antigen polypeptides and recombinant fusion antigen polypeptides can be purified from lysed cells or medium by protein purification methods known in the art (ultrafiltration, centrifugation, dialysis, ion exchange chromatography). , Hydrophobic chromatography, Gel 'neglect, Electrophoresis, Affinity Purification can be carried out by using chromatography, HPLC or the like; see, for example, Methods in Enzymology, Vol. 182 (Edited by Deutscher, MP), Academic Press (1990).
  • the antigen polypeptide can be prepared by chemical synthesis based on the amino acid sequence determined in the present invention. Preferably, the entire sequence is divided into fragments (10 to 50 residues) that are relatively easy to synthesize, and further divided so that adjacent fragments overlap by about 5 to 10 residues, and synthesized. After baking, it is preferable to use only the fragments containing the epitope alone or in combination.
  • polypeptides containing the amino acid sequence of the antigen polypeptide are chemically synthesized manually by a liquid phase method or a solid phase method, or by an automatic synthesizer (for example, ABI 430A type peptide synthesizer). be able to.
  • Polypeptides synthesized by the solid-phase method according to the t-Boc method or the f-moc method are cut out of the resin using trifluormethanesulfonic acid, hydrogen fluoride, or trifluoroacetic acid.
  • These synthetic peptides can be purified by preparative reversed-phase high-performance liquid chromatography (HPLC) to increase the degree of purification.
  • the non-A, non-B hepatitis virus antigen polypeptide obtained by the present invention can be used to prepare, for example, a biological sample such as blood, plasma, serum, cerebrospinal fluid, and a biological product such as a human immunoglobulin preparation by using Imnoassay. It can be used to detect the presence of non-A, non-B hepatitis virus antibodies contained in E. coli.
  • the antigen polypeptide and the sample are subjected to conditions under which an immunological conjugate (antigen antibody conjugate) can be formed between the antigen polypeptide and the non-A non-B hepatitis virus antibody in the sample. Make contact.
  • any antigen-antibody conjugate is formed, the presence of non-A non-B hepatitis virus antibody in the sample can be detected and measured by appropriate means.
  • detection methods And immunoprecipitation method, agglutination method, Radioimnoassy (RIA), EIA, ELISA. ETijssen, P. Eiji Ishikawa's translation, Enzymimnoattsie, Biochemical Experiment Vol.ll, Tokyo Chemical Dojin (1989)] , CEDIA [Henderson; DR et al., Clin. Chem., 32, 1637 (1986)], and Western blot atsey or binding imnoatsay.
  • the antigen obtained according to the present invention was expressed as a fusion protein with, for example, 3-gal, and the nitrocellulose membrane-produced antigen was immobilized on a solid phase to obtain an antigen.
  • Methods for detecting non-A, non-B hepatitis virus antibodies eg, immunoplaque assay, western plot assay, etc.
  • Examples 11, 12, and 13 show a method for detecting non-A non-B hepatitis virus antibody by ELISA.
  • Example 13 a method was described in which a titer plate was coated with a mixture of Y19, # 22-3 and # 22-16 antigens, and the virus antibody was measured at a high rate.
  • Y19 antigen and ⁇ 22 antigen increase the detection rate by combining Y19 antigen and / or ⁇ 22 antigen with one or more non-A non-B hepatitis-related antigens such as HCVcore antigen, clone 14 antigen, and GOR antigen. It is also possible.
  • a complex antigen with a non-A non-B hepatitis-related antigen polypeptide such as Y19-core, Y19-core.
  • Y19-core Y19-core.
  • Y19-core Y19-core.
  • Y19—Y22—core Y19—Y22-CP9 / CP10-GOR
  • Y19-GOR-core complex antigen with a complex antigen with a non-A non-B hepatitis-related antigen polypeptide
  • FIG. 1 shows a histogram of absorbance distribution in a healthy human sample by the Y19 ELISA method.
  • FIG. 2 shows the absorbance distribution of the serum of a non-A non-B chronic hepatitis patient by the Y19 ELISA method and the C100 ELISA method.
  • FIG. 3 shows the results obtained by examining the reactivity of various synthetic polypeptide fragments containing the sequence of the Y22 antigen of the present invention with pooled sera of non-A and non-B type patients by ELISA.
  • FIG. 4 shows a histogram of absorbance distribution in a healthy human sample by the Y19 + 22 ELISA method.
  • FIG. 5 shows the absorbance distribution of the serum of a non-A non-B chronic hepatitis patient by Y19 + 22 ELISA and C100 ELISA.
  • Example 1 Preparation of plasma-derived RNA
  • RNA from the precipitate was performed according to the AGPC method using guanidine 'thiosineate [Chomczynski. P. and Sacchi.
  • RNA was precipitated again. After centrifugation, the sediment was washed with 75% ethanol and dried. The precipitate was dissolved in 501 sterile distilled water to obtain an RNA solution.
  • Double-stranded cDNA was prepared from RNA using a commercially available cDNA synthesis system, Brass (Amersham, RPN-1256Z). The conversion rate from RNA to single-stranded cDNA was about 2%, and the conversion rate from single-stranded cDNA to double-stranded cDNA was about 100%.
  • the synthesized double-stranded cDNA was subjected to ethanol precipitation, and then dissolved in a 100 1 TE solution (10 mM Tris-CI, pH 8.0, ImM EDTA). Next, using a commercially available cDNA synthesis kit (Pharmacia LKB, 27-9260-01), The reaction was performed.
  • the buffer was exchanged with the 1X T4 ligase solution by passing the cDNA solution through the Sephacryl S-300 span column attached to the kit.
  • an EcoRI adapter addition reaction and a T4 kinase reaction were performed according to the instructions.
  • the peptide that did not bind to the cDNA was removed using a Sephacryl S-300 span column.
  • the cDNA in the eluate was ethanol precipitated with 5 ⁇ g of carrier tRNA. The precipitate was washed with 75% ethanol and air-dried.
  • Example 3 Cloning of non-A non-B hepatitis virus antigen gene
  • the reaction between plaques in the cDNA library (Lot.B) and the primary and secondary antibodies was determined by using a commercially available screen 'Immunsk Cleaning System (Amersham). , RPN 1281Z).
  • the primary antibody used for screening was bull serum from patients with non-A and non-B hepatitis (5 acute hepatitis recovery periods and 5 chronic hepatitis) in E.
  • IgtllY19 Lysogen is named Escherichia coli Y1089 ( ⁇ gtllY19), and the strain has been deposited as follows.
  • Example 5 Determination of the nucleotide sequence of cDNA derived from clone Y19 and the sequence of the antigen polypeptide encoded in the sequence
  • the clone Y19 was propagated by the plate lysate method described in 2.118 of Experiment 1, and then phage DNA was prepared. 5 ⁇ g fur After the DNA was digested with EcoRI, the DNA was purified by phenol extraction and ethanol precipitation. 3 after the g of EcoRI digest was treated alkaline phosphatase, were performed 5 'end labeled with T4 kinase and 7 32 P- ATP. After purifying the DNA fragment by phenol extraction and ethanol precipitation, 5.0% polyacrylamide gel electrophoresis was performed, and the length of the cDNA excised by EcoRI digestion was examined by autoradiography. As a result, the size of the cDNA was found to be about 400 base pairs.
  • a ⁇ 4 ligase reaction was performed with the above-mentioned EcoRI digest and pTZ19 (Pharmacia LKB, 27-4986-01) dephosphorylated after EcoRI digestion.
  • the reaction solution was transfused into Escherichia coli JM109 (Takara Shuzo, 9052), and a plasmid having an insert of about 400 base pairs was isolated from the transformants (designated ⁇ 19-Y19).
  • the nucleotide sequence of the cDNA incorporated into ⁇ 19-Y19 was determined by performing a sequence reaction using a DyeDeoxy Terminator Taq Sequencing Kit (ABI, 401070) and running with a DNA sequencer (ABI.373A).
  • the sequence was determined using both M13 primer M4 (Takara Shuzo, 3832), M13 primer RV (Takara Shuzo, 3830) and synthetic primer E 1 (5'-GGAGACGTCGTTGTTGT-3 '), E2 (5'-GCCTGTCATCAGAGCGT-1 3').
  • the chains were read and determined.
  • synthetic oligonucleotides were prepared using a 380A DNA synthesizer (ABI) and purified according to the attached instructions.
  • the base sequence was determined by directly transforming the phage DNA into a ⁇ type using the primers and the reverse primers (New England Biolabsl218, 1222).
  • the nucleotide sequence encoding the antigen that specifically reacts with the serum of non-A non-B hepatitis patients is shown in Formula (IX)
  • the amino acid sequence of the antigen is shown in Formula (I).
  • the amino acid sequence is obtained by translating the base sequence of formula (IX) from the first position.
  • Example 6 Antigen specificity test of clone Y22
  • a mixture of clone Y22 and negative clones at a ratio of 2: 8 was plated, and non-A non-B chronic chronic hepatitis (previous ring blood) serum (5 blood samples) was applied using the immunoscreening method described above. ), Non-A non-B acute hepatitis patients (no transfusion history) Serum (6 patients), non-A non-B chronic hepatitis patients (no transfusion history) sera (5 patients), normal control serum (26 patients) ), Sera from chronic hepatitis B (9 patients) and sera from acute hepatitis A (2 patients) were examined. Table 2 shows the results. The results showed that clone Y22 specifically reacted only with non-A, non-B hepatitis patient serum.
  • clone Y22 encodes an antigen (epitope) that is immunologically recognized by serum from a non-A non-B hepatitis patient.
  • ⁇ gtll (parent strain) lysogen and recombinant ⁇ gtll Y22 lysogen preparation were described in Experiment 3, p . Performed according to the method.
  • the ⁇ gtllY22 lysogen was named Escherichia coli Y 1089 ( ⁇ gtllY22), and Deposited as follows.
  • Example 7 Determination of nucleotide sequence of cDNA derived from clone Y22 and antigen polypeptide sequence encoded in the sequence
  • the nucleotide sequence was determined according to the method of Example 5. For determination, both strands were read using M13 Primer 1-4 (Takara Shuzo, 3832) and M13 Primer RV (Takara Shuzo, 3830) to determine.
  • a pair of PCR primers was prepared that hybridize to the N-terminal and C-terminal [19- ⁇ : 5 '-1c: 5'-AAAAGCTTGGATCCTTAGCGTTCCCCTGGAGTCACATACCT] 19- ⁇ Primers have BamHI and Ncol sites, and the 19-c primer has stop codons, BamHI and Hindlll sites.
  • Heat treatment 95 minutes, 10 minutes
  • PCR was performed using GeneAmp DNA amplification reagent kit (PERKIN ELMER CETUS, N801-0055) with 40 pmol of each primer for PCR added to the aqueous solution of IgtllY19 phage (94 °). C, 1 minute, 45 ° C. 2 minutes, 72. C, 3 minutes 35 cycles).
  • the reaction mixture was extracted with phenol-X-form, and ethanol was added to the aqueous phase to sediment DNA fragments.
  • the PCR product obtained in (1) was treated with ⁇ 4 kinase by a conventional method, and then ligated using ⁇ 4 ligase.
  • the reaction solution was extracted with a phenol micropore form, and ethanol was added to the aqueous phase to precipitate DNA fragments.
  • the DNA was double-digested with Ncol and Hindlll, and the digest was electrophoresed on 1.2% agarose (SeaKem GTG Agarose: Takara Shuzo, 5160). A band of about 400 base pairs was cut out, and DNA was recovered using SUPREC-01 (Takara Shuzo 9040).
  • the lysate containing 10 zg of protein and a molecular weight marker were electrophoresed on two sets of SDS-PAGE plates 10Z20 (Daiichi Kagaku, 120483). One was stained with CBB by a conventional method, and the other was Western-blotted to a nitrocellulose filter (Schleicher & Schuell, BA85) using an ISS semi-dry electroplotter (Daiichi Kagaku, SS110201). The ditrocellulose filter was reacted with a pool serum of a non-A non-B hepatitis patient according to the method described in Example 3.
  • Peroxidase-labeled goat anti-human (IgG) IgG was used as the secondary antibody.
  • IgG immunoglobulin G
  • Kappel 3201-0081
  • CBB staining a new band, which was not observed in the Kc strain, was detected at a site with a molecular weight of 17,000 in the K19 strain. That is, the K19 strain expressed an antigen (Y19 antigen) that retained reactivity with patient serum.
  • the Y19 antigen was converted to pED3d (Experiment 6, p60-89) using the T7 promoter, pGEMEX-11 (Promega, P2211), and pMAL-c (New England Biolabs, # 800) can be used to express in E. coli cells.
  • Example 9 Expression of Y22-19 complex antigen in E. coli
  • the 22-n primer has a BamHI site and a Pstl site, and the 22-c primer has an Ncol site.
  • the heat-treated (95 ° C, 10 minutes) ⁇ gtllY22 phage aqueous solution 40 pmol of each primer for PCR was added, and PCR was performed using the GeneAmp DNA amplification reagent kit (94 ° C, 1 minute, 45 ° C, 35 cycles of 2 minutes, 72 ° C, 3 minutes).
  • the reaction solution was extracted with phenol / chloroform, and ethanol was added to the aqueous phase to precipitate DNA fragments.
  • the reaction solution was extracted with form of funinol in the mouth, and ethanol was added to the aqueous phase to precipitate DNA fragments.
  • the reaction solution was extracted with phenol Z chloroform, and ethanol was added to the aqueous phase to precipitate fragments.
  • the reaction solution was extracted with phenol Z chloroform, and ethanol was added to the aqueous phase to precipitate the ligated product.
  • the ligated product was subjected to double digestion with Pstl and Hindlll, and the digest was electrophoresed on 1.2% agarose (SeaKem GTG Agarose: Takara Shuzo, 5160). A band of about 550 base pairs was cut out, and DNA was recovered using SUPREC-01 (Takara Shuzo, 9040). Next, this 550 bp DNA fragment was double digested with Pstl and Hindlll to obtain a pKK233-2 expression vector (Pharmacia, 27-5005). Ligation was performed using T4 ligase. Escherichia coli JM109 strain was transformed using this ligation product. A clone containing ⁇ ⁇ 233 ( ⁇ 22-19) with an insert of 550 bases was obtained from the transformants that appeared on the ampicillin plate (JM109 / pKY22-19: hereinafter referred to as strain K2219).
  • the lysate containing 10 / g of protein and the molecular weight marker were electrophoresed on two sets of SDS-PAGE plates 10/20 (Daiichi Shigaku, 120483).
  • color development was observed only at the K2219 strain at a site with a molecular weight of 26,000.
  • CBB staining a new band was detected at a site with a molecular weight of 26,000 in the K2219 strain, which was not observed in the Kc strain. That is, the K2219 strain expressed an antigen (Y22-19 antigen) that retained the reactivity with the patient's serum.
  • the Y22-19 antigen can be expressed using pED3d, pGEMEX-11, pMAL-c, and the like.
  • Example 10 Purification of Y19 antigen from E. coli
  • the precipitate fraction was dissolved in 50 mM Tris-HC1, ⁇ 8.4, ImM EDTA, and 6 M guanidine hydrochloride, and the centrifuged supernatant (200,000 ⁇ g, 30 minutes) was added to the C solution (0.1). M Tris-HC1, pH 8.4, ImM EDTA, 2M Urea). After dialysis, the mixture was centrifuged again, and the supernatant was applied to a Q Sepharose column (30 ml gel volume). The unbound fraction was then applied to an S Sepharose column (30 ml gel volume) and eluted with a 0.0 to 0.5 M salt gradient.
  • the peak fraction eluted at about 0.2M salt concentration was collected, and ammonium sulfate was added to a final concentration of 1M. This solution was then poured onto a Phenyl Sepharose column. The adsorbed fraction eluted with a C solution containing 0.05M ammonium sulfate. The eluted fraction was dialyzed against the C solution and concentrated on a S Sepharose column. About 7 mg of purified Y19 antigen was obtained from 6 L of culture. The purity of the purified protein was estimated to be 98% or more based on the results of SDS-PAGE CBB staining.
  • Example 11 Detection of non-A non-B hepatitis virus antibody by Y19 ELISA
  • a non-A, non-B hepatitis virus antibody (Y19 antibody) in the serum was detected by a Y19 ELISA method (Y19 ELISA method) using the Y19 antigen obtained in Example 10.
  • Coating mouthwash containing Y19 antigen (1 Z g / ml) [0. 1M sodium bicarbonate (PH9.5)] was added at 200 / zl to the immobilized plate (Nunc, 439454) overnight 4. After incubating with C, the solution was removed.
  • Add 250 1 of blocking buffer [PBS (10 mM phosphate buffer, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4), 1% bovine serum albumin (BSA)] to the gel, and add 4% Placed.
  • PBS 10 mM phosphate buffer, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4
  • BSA bovine serum albumin
  • the solution was removed and washed three times with a washing buffer [PBS, 0.05% Tween 20]. 37. To the washed well, add the sample 200 1 diluted 20-fold with the sample diluent [5.5 volumes PBS, 0.5% BSA, 1% Triton X-100]. C, incubated for 1 hour. Next, the gel washed 5 times with the washing buffer was washed with 10,000 labeled antibody solution (peroxidase-labeled goat anti-human (IgG) IgG using a labeled antibody diluent [wash buffer containing 0.5% BSA] in 10,000 buffer). (Diluted 1: 2 solution) at 37 for 1 hour.
  • a washing buffer [PBS, 0.05% Tween 20]. 37.
  • substrate reaction solution [0.4mgr / ml 0-phenylene Renjiamin'ni hydrochloride and 0.01% H 2 0 2 containing phosphoric acid-Kuen acid lOOmM slow ⁇ , PH5. 0] 200 1 was added, and the mixture was incubated at room temperature for 30 minutes in the dark. The reaction was stopped by adding 501 of 4.5 M sulfuric acid, and the absorbance was measured at 492 nm.
  • Specimens obtained from healthy subjects with normal liver function were examined using the Y19 ELISA method described above. The results obtained with these samples are shown in a histogram showing the absorbance distribution (Fig. 1). As can be seen from FIG. 1, all samples showed absorbances between 0.1 and 0.5. The average value of the absorbance of the 27 healthy subjects was 0.274 and the standard deviation was 0.086. All of these samples were included between the mean ⁇ 3-fold standard deviation.
  • the Y19 antigen showed a strong reactivity (mean 1.639, standard deviation 0.206) with all non-A non-B liver disease samples.
  • the C100 antigen did not react with two sera of patients with non-A non-B liver disease (one serum of non-A non-B acute hepatitis patients and one serum of non-A non-B chronic hepatitis patients).
  • Measurement of non-A, non-B hepatitis virus antibodies using the Y19 antigen resulted in a higher detection rate than the C100 antigen.
  • Example 7 Based on the amino acid sequence determined in Example 7 (see formula (II)), the following formulas ( ⁇ ) (VII) and (XII) selected to include a relatively hydrophilic region and Example 7
  • the control polypeptide represented by the formula (II) not containing the amino acid sequence (see formula (II)) determined in the above was synthesized as follows. (III)
  • Polypeptides of the formulas (III) and (XIII) are prepared by using triammonium phenylacetamidomethyl (Pam) resin obtained by force-pulping t-Boc-Lys and t-Boc-Gly, respectively.
  • the removal of the t-Boc group, the activation of t-Boc amino acid to be linked with ⁇ , ⁇ -dicyclohexylcarbodiimide, and the coupling were sequentially repeated to synthesize.
  • t-Boc-Arg ⁇ , N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were used in equal amounts.
  • the peptides of the formulas (IV) to (XII) were synthesized in the same manner using a Pam resin obtained by force-pulping t-Boc-Asp as a starting material. However, a mixed solution of trifluoracetic acid: triethylamine: dichloromethane (35: 1: 9) was used to remove the t-Boc group. Each polypeptide was released from the resin with trifluorosulfonic acid methanesulfonic acid according to the method described in the user's manual. The released polypeptide was freeze-dried to obtain a white or pale yellow-white solid.
  • polypeptides were analyzed by high performance liquid chromatography. Using an R— ODS—5 column (4.6 ⁇ X 250 mm, Yamamura Chemical), from solution A (3 ⁇ 40, 0.1% trifluorosulfonic acid) 0% to solution B [acetonitrinol: 0 (70:30) The elution time of the main peaks of the polypeptides of formulas (III) to (VII) and ( ⁇ ) and (XIII) was increased by linear gradient elution of 35% over 100% of 0.1% trifluoroacetic acid. Approximately 26.2 minutes, 30.7 minutes, 29.2 minutes, 33.0 minutes, 29.7 minutes, 28.4 minutes, and 29.1 minutes, respectively.
  • Non-A, non-B hepatitis virus antibodies were detected in the serum using an ELISA method using the synthetic polypeptide as an antigen.
  • Coating buffer [0.1 M sodium bicarbonate (pH 9.5)] containing each synthetic polypeptide (10 g / ml) was added to immobilized plate (Nunc, 439454) in 100/1 ratio. Then 4 overnight. After incubation with C, the solution was removed by suction.
  • Add 250 // 1 blocking buffer [PBS (l OmM Phosphate buffer, 137 mM sodium chloride, 2.7 mM chloride, pH 7.4) and 1% bovine serum albumin] were added, and the mixture was kept at room temperature for 3 hours.
  • the solution was removed and washed three times with a washing buffer (PBS, 0.05% Tween 20).
  • a washing buffer PBS, 0.05% Tween 20
  • bovine serum from a non-A, non-B hepatitis patient diluted 30 times with a washing buffer containing 0.25% BSA or 100/1 bull serum from a healthy person as a control at 4 ° C overnight. Incubated. Then, the gel washed 5 times with the washing buffer was added to a 100/1 secondary antibody solution (a solution of peroxidase-labeled goat anti-human (IgG) IgG diluted 3000-fold with the washing buffer). Treated at room temperature for hours.
  • IgG peroxidase-labeled goat anti-human
  • substrate reaction solution (0.4 mg / ml 0-Fuwenirenjiami Nni hydrochloride and phosphoric acid-Kuen acid buffer l OOmM containing 3 ⁇ 40 2 of 0.01%, pH 5.0 ) 200/1 was added and incubated at room temperature in the dark for 10 minutes. The reaction was stopped by adding 501 of 4.5 M sulfuric acid, and the absorbance was measured at 492 nm.
  • Specimens obtained from healthy subjects with normal liver function were examined using the Y19 + 22 ELISA method described above. The results obtained with these samples are shown in histograms showing the distribution of OD values (Fig. 4). As can be seen in FIG. 4, all samples showed absorbances between 0.1 and 0.4. The average value of the absorbance of the 29 healthy subjects was 0.201, and the standard deviation was 0.072. All of these samples were included between the mean ⁇ 3-fold standard deviation.
  • the Y19 + 22 antigen showed strong reactivity (mean 1.763, standard deviation 0.061) with all non-A non-B liver disease samples.
  • the C100 antigen did not react with two sera of patients with non-A non-B liver disease (one serum of non-A non-B acute hepatitis patients and one serum of non-A non-B chronic hepatitis patients).
  • Measurement of HCV antibody using Y19 + 22 showed a higher detection rate than C100 antibody. That is, it is useful for the detection of non-A non-B hepatitis virus antibodies that could not be detected conventionally.
  • the Y19 + 22 antibody positive rate was 89.3%, and the CP9 + 10 antibody positive rate was 79.5%. ⁇
  • the HCV antibody positive rate was higher in the 19 + 22 ELISA method than in the CP9 + 10 ELISA method.
  • the agreement between the Y19 + 22 antibody measurement and the CP9 + 10 antibody measurement was + Z + 93 cases (76.2%). Some specimens were positive only for Y19 + 22 or only CP9 + 10. The discordant cases were +/- 16 cases (13.1%) and one Z + 4 cases (3.3%).
  • the above results indicate that the Y19 + 22 antigen is useful for specifically detecting serum antibodies of non-A non-B chronic hepatitis patients. In addition, it shows that the detection rate can be further increased by further mixing the CP9 + 10 antigen with the Y19 + 22 antigen.
  • the positive rate of the Y19 + 22 antibody was 73.7%, and the positive rate of the C100 antibody was 68.4%.
  • Y19 + 22 antibody measurement has been shown to be useful in diagnosing non-A non-B cirrhosis. Industrial applicability
  • the antigen polypeptides of the present invention and the method for measuring non-A non-B hepatitis virus antibodies using a mixture or conjugate thereof have a higher detection rate than the conventional antigen-based test method, and the existing antigens have a higher detection rate. It is useful for diagnosing non-A, non-B hepatitis virus-related diseases that could not be diagnosed (acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer, etc.), and helps determine treatment policies.
  • the antigen polypeptide can supplement non-A non-B hepatitis virus healthy human carriers, which could not be detected with the existing antigen, and is useful for preventing the development of post-ring blood hepatitis.
  • polyclonal antibodies and monoclonal antibodies that can be used for the detection of the virus antigen and the treatment of non-A, non-B hepatitis can be prepared.
  • the antigen polypeptide can be used for production of a vaccine for prevention of virus infection and treatment after infection.
  • the non-A, non-B hepatitis virus polynucleotide of the present invention is useful as a gene for producing the antigen.
  • the presence or absence of the virus genome (particles) can be examined using the nucleotide sequence.

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Abstract

L'invention se rapporte aux groupes de polypeptides Y19 et Y22 de l'antigène du virus de l'hépatite non A, non B ou à des fragments de ces groupes, à des polypeptides d'antigènes complexes préparés par fusion de deux antigènes ou davantage choisis parmi lesdits polypeptides; et à des polypeptides d'antigènes ayant une épitope contenue dans un polypeptide de groupe Y19 ou Y22. L'invention se rapporte également à un procédé de détection d'un antigène du virus de l'hépatite non A, non B en déterminant un conjugué immunologique obtenu par la réaction de l'anticorps du virus de l'hápatite non A, non B, avec l'un de ces antigènes ou avec un mélange de ces antigènes.
PCT/JP1991/000964 1990-07-24 1991-07-19 Antigene du virus de l'hepatite non a, non b WO1992001714A1 (fr)

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JP2/195735 1990-07-24
JP19573590 1990-07-24
JP2/195736 1990-07-24
JP19573690 1990-07-24
JP2/286901 1990-10-24
JP28690190 1990-10-24
JP3/155158 1991-05-30
JP15515891 1991-05-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0644202A1 (fr) * 1990-12-14 1995-03-22 N.V. Innogenetics S.A. Antigènes synthétiques pour la détection des anticorps contre le virus de l'hépatite C
US5910404A (en) * 1990-12-14 1999-06-08 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US6007982A (en) * 1990-12-14 1999-12-28 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
EP0769954B2 (fr) † 1995-05-08 2011-03-09 Baxter Aktiengesellschaft Medicament de qualite garantie contenant un ou plusieurs derives plasmatiques

Citations (3)

* 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
WO1990014436A1 (fr) * 1989-05-18 1990-11-29 Chiron Corporation Diagnostic du nanbv (virus de l'hepatite non-a et non-b): polynucleotides utiles pour depister le virus de l'hepatite c
WO1991004262A1 (fr) * 1989-09-15 1991-04-04 National Institute Of Health Of Japan Nouveaux isolats du virus de l'hepatite c

Patent Citations (3)

* 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
WO1990014436A1 (fr) * 1989-05-18 1990-11-29 Chiron Corporation Diagnostic du nanbv (virus de l'hepatite non-a et non-b): polynucleotides utiles pour depister le virus de l'hepatite c
WO1991004262A1 (fr) * 1989-09-15 1991-04-04 National Institute Of Health Of Japan Nouveaux isolats du virus de l'hepatite c

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCIENCE, Vol. 244, (1989), QUI LIM CHOO et al., "Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome", p. 359-362. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0644202A1 (fr) * 1990-12-14 1995-03-22 N.V. Innogenetics S.A. Antigènes synthétiques pour la détection des anticorps contre le virus de l'hépatite C
US5910404A (en) * 1990-12-14 1999-06-08 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US5922532A (en) * 1990-12-14 1999-07-13 Innogenetics, N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US6007982A (en) * 1990-12-14 1999-12-28 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US6287761B1 (en) 1990-12-14 2001-09-11 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US6576417B2 (en) 1990-12-14 2003-06-10 Innogenetics, N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
US6872520B2 (en) 1990-12-14 2005-03-29 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis C virus
EP0769954B2 (fr) † 1995-05-08 2011-03-09 Baxter Aktiengesellschaft Medicament de qualite garantie contenant un ou plusieurs derives plasmatiques

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