WO1992018532A1 - Rna, dna and virus antigen protein of non-a non-b hepatitis virus - Google Patents

Rna, dna and virus antigen protein of non-a non-b hepatitis virus Download PDF

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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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PCT/JP1992/000464
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French (fr)
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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Publication of WO1992018532A1 publication Critical patent/WO1992018532A1/en

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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

A non-A non-B hepatitis virus genome RNA, a DNA homologous thereto, and a non-A non-B hepatitis virus polypeptide. A novel non-A non-B hepatitis virus DNA is identified by extracting a virus RNA from the plasma of a patient with non-A non-B viral hepatitis, purifying the same, preparing a double-stranded DNA with the purified RNA as a template, introducing the obtained DNA into Escherichia coli, and screening a clone which expresses the virus protein. The use of the RNA, DNA and polypeptide of the invention makes it possible to diagnose and cure non-A non-B viral hepatitis.

Description

明 細 書 非 A非 B型肝炎ウィルスの RNA、 DNA及びウィルス抗原蛋白質 〔産業上の利用分野〕  Description RNA, DNA and viral antigen proteins of non-A non-B hepatitis virus [Industrial applications]
本発明は非 A非 B型肝炎ウィルスに関連する抗原ポリべプチドおよびそれ をコードする DNA断片並びに抗非 A非 B型肝炎ウィルス抗体に関する。 さら には、 抗非 A非 B型肝炎ウィルス抗体、 非 A非 B型肝炎ウィルス抗原および 非 A非 B型肝炎ウィルス遺伝子の検出方法に関する。  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.
〔従来の技術〕  [Conventional technology]
非 A非 B型肝炎は、 現在、 輸血後肝炎の約 95%を占めると考えられており 、 該ウィルス感染者を検出して輪血用血液から除外ずることや、 予防用ワク チンの開発が強く望まれている。 近年、 Houghtonらは、 該ウィルス遺伝子を 分離したと発表し （特開平 2— 500880号） 、 該ウィルス抗原を用いた E I A による抗体検出試薬を販売するに至っている。 この抗体検出試薬の適用によ り、 輸血後非 A非 B型肝炎予防に対する期待が寄せられているが、 陽性の供 血者血液を除外しても、 その肝炎発症率は 5〜7 %であり、 未だ消滅を見る に至っていない。  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.
その原因として考えられることは、 該ゥィルス感染と抗体産生の関係がま だ明らかになっていないということもさることながら、 該ウィルスゲノムの 核酸配列にかなりの異質性があるらしいということがある。 この点について は、 Houghtonらの発表した核酸配列を元に、 P C R法やブライマーェクステ ンション法などにより該ウィルスゲノム c-DNA をクローニングした多数の実 験の結果 (Kubo et. al. , Nucleic Acid Research 17, 10367 一 10372， 1989 、 Kato et. al. . Proc. Japan Acad. , 65B, 219-223, 1989、 Maeno et. al. , Nuc leic Acid Research, 18, 2685-2689, 1990、 Kato et. al. , Proc. Natl. Acad . Sci. USA, 87. 9524-9528, 1990) から明らかである。 このように、 非 A非 B型肝炎の診断、 並びに輪血による非 A非 B型肝炎発症の予防は未だ完全と 言えず、 遺伝子診断を含めた新しレ、診断法の開発が望まれている。 A possible cause for this is that the nucleic acid sequence of the viral genome seems to be quite heterogeneous, not only that the relationship between the virus infection and antibody production has not yet been elucidated. Regarding this point, based on the nucleic acid sequence published by Houghton et al., The results of a number of experiments in which the viral genomic c-DNA was cloned by PCR, Brimer Extension, etc. (Kubo et. Al., Nucleic Acid Research 17, 10367-1 10372, 1989, Kato et. Al. Proc. Japan Acad., 65B, 219-223, 1989, Maeno et. Al., Nuc leic Acid Research, 18, 2685-2689, 1990, Kato et. al., Proc. Natl. Acad. Sci. USA, 87. 9524-9528, 1990). Thus, the diagnosis of non-A non-B hepatitis and the prevention of non-A non-B hepatitis due to ring blood are not yet complete, and the development of new diagnostic methods, including genetic diagnosis, is desired. I have.
また、 本発明者のひとりである有馬暉勝は、 Houghtonらとは独立して同時 期に、 ヒト患者血清を RNAソースおよび抗体ソースとして、 ィムノスクリ一 ニング法で HCV (肝炎 C型ウィルス） の core領域のェピトーブのひとつをク ローン化している（Arima et al. , Gastroenterologia Japonica, 25, 218-22 2, 1990)。  Also, Terumika Arima, one of the present inventors, reported that at the same time and independently of Houghton et al., HCV (hepatitis C virus) core was determined by immunoscreening using human patient serum as an RNA source and an antibody source. One of the epitopes in the area has been cloned (Arima et al., Gastroenterologia Japonica, 25, 218-22, 1990).
〔本発明の開示〕  (Disclosure of the present invention)
本発明は、 新規な非 A非 B型肝炎ウィルスの抗原ポリペプチドおよびそれ をコードする DNA の提拱と、 さらにこれらを用いる非 A非 B型肝炎の診断方 法、 診断試薬の提供を目的とする。  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.
本発明者らは、 上記の現状を鑑み、 非 A非 B型肝炎ウィルスの研究を開始 した。 s-GPT高値を示す日本国内の供血者血漿を原料として鋭意研究の結果 、 これまで報告されたウィルスとは異なる新しい夕イブの非 A非 B型肝炎ゥ ィルス由来の DNA の単離、 構造確認に成功した。 さらに、 これら DNA および それがコードするボリペプチドを用いて非 A非 B型肝炎の診断、 予防、 治療 を行ない得ることを見い出し、 本発明を完成させるに至った。  In view of the above situation, the present inventors have started research on 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.
本発明は非 A非 B型肝炎ウィルス遺伝子 RNA に相同的な部分 DNA (配列番号 1 , 2および 3 ) 、 それらがコードする非 A非 B型肝炎ウィルス抗原ボリべ プチド （配列番号 1 , 2および 3 ) およびこれら抗原ポリペプチドを抗原と するポリクローナル抗体またはモノクローナル抗体を提供するものである。 即ち、 本発明は、 下記の配列 1 , 2および 3の非 A非 B型肝炎ウィルス抗 原ポリべプチドの、 全部または一部を含有してなるポリべプチドである。 配列 1 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
GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47 GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47
Val l ie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu l ieVal lie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu lie
1 5 10 15 1 5 10 15
ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95
Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro l ie Asn Pro Leu Ser Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser
20 25 30  20 25 30
AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143
Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg
35 40 45  35 40 45
AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181
Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp
50 55  50 55
配列 2 Array 2
ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48 ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48
Thr Thr Leu Thr Met l ie Leu Ala Tyr Ala Ala Arg Val Pro Glu LeuThr Thr Leu Thr Met lie Leu Ala Tyr Ala Ala Arg Val Pro Glu Leu
1 5 10 15 GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val lie Phe Gly Gly His Trp Gly Val Val Phe Gly Leu 1 5 10 15 GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val lie Phe Gly Gly His Trp Gly Val Val Phe Gly Leu
20 25 30  20 25 30
GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val lie Ala He Leu GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val lie Ala He Leu
35 40 45  35 40 45
CTC CTT GTC GCA GGA GTA GAT GCA GAC ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg 50 55 60 CTC CTT GTC GCA GGA GTA GAT GCA GAC ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg 50 55 60
GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80 GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80
CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His lie Asp Leu lie CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His lie Asp Leu lie
85  85
配列 3 Array 3
CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15 AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe His Asn Lys Val Tyr Ser Thr T r Ser Lys Ser CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15 AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe His Asn Lys Val Tyr Ser Thr Tr Ser Lys Ser
20 25  20 25
さらに本発明は、 上記のポリペプチドをコードする、 上記配列 1， 2およ び 3の DNA の全部または一部を含有してなる DNA、 上記ポリペプチドを抗原 とするボリクローナル抗体またはモノクローナル抗体、 試薬の構成成分とし て、 上記の DNA をブライマーとして用い、 非 A非 B型肝炎ウィルス由来の DN A を増幅させ検出することを特徴とする非 A非 B型肝炎ウィルス ¾伝子の検 出方法、 上記の抗原ポリペプチドを用い、 免疫学的手法により、 試料中の抗 非 A非 B型肝炎ウィルス抗体の存在を確認する抗非 A非 B型肝炎ウィルス抗 体検出方法、 上記の抗体を用い、 免疫学的手法により、 試料中の非 A非 B型 肝炎ウィルス抗原の存在を確認する非 A非 B型肝炎ウィルス抗原検出方法及 び上記の抗原ポリべプチドを用いて製造された非 A非 B型肝炎ワクチンをも 提供する。  Further, 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.
従って、 本発明には、 本発明の DNA の部分オリゴヌクレオチドをプライマ 一として用い、 非 A非 B型肝炎ウィルス由来の DNA を増幅させ検出すること による非 A非 B型肝炎ウィルス遺伝子の検出方法も含まれる。  Accordingly, 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.
また、 本発明には、 本発明の DNA に相同的な RNA も含まれる。  The present invention also includes RNA homologous to the DNA of the present invention.
なお、 本発明オリゴヌクレオチドおよびポリペプチドは、 少なくとも 10個 の塩基またはアミノ酸 （長さ） を含む。  The oligonucleotides and polypeptides of the present invention contain at least 10 bases or amino acids (length).
本発明の構成は以下の通りである。  The configuration of the present invention is as follows.
(1) 非 A非 B型肝炎ウィルスの RNA の調製 非 A非 B型肝炎ウィルス RNA の抽出原料としては、 S- GTP値が高値を示す 供血者の血漿を用いることができる。 ウィルス面分は、 公知の方法に従い、 ボリエチレングリコール添加後遠心分離操作により採取する。 このウィルス 面分からの RNA の抽出、 精製は、 例えばグァニジンチオシァネート、 界面活 性剤、 キレート剤および還元剤の混合溶液にてウィルス面分からの抽出を行 つた後、 フエノール抽出、 有機溶媒分面 (Chamezynski et al, Anal, Bioc hem. , 162^ 156, 1987) 、 次いで密度勾配超遠心操作により精製 RNA を得る 方法で行うことができる。 (1) Preparation of non-A non-B hepatitis virus 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.
(2) 2本鎖 DNA の調製とクローニング  (2) Preparation and cloning of double-stranded DNA
得られた RNA を铸型として用い、 ランダムブライマー、 逆転写酵素、 DNA ボリメラ一ゼ等を用いる cDNA合成法 (Gubler, ϋ. et al. Gene, 25. 263, 19 83) などの常法により、 2本鏆 DNA を調製することができる。  Using the obtained RNA as type 、, a conventional method such as cDNA synthesis using random primers, reverse transcriptase, DNA polymerase, etc. (Gubler, ϋ. Et al. Gene, 25.263, 1983) Two pieces of DNA can be prepared.
このようにして得られた 2本鎖 DNA を、 常法に従い、 パクテリオファージ 、 例えば A zap 、 A gtllなどに組み込んだ後、 大腸菌を支持菌として培養、 スクリーニングすることにより、 目的とするクローンを得ることができる。  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.
2本鏆 DNA をファージベクターに組み込む方法としては、 例えば Hyunh, T. V . らの DNA Cloning, a practical approach, _ 1, 49 (1985) に記載の方法な どが挙げられる。  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).
目的とするクローンのスクリーニング法は、 公知の方法に準ずればよく、 例えば λ gt 11ファージベクターに 2本鎖を組み込んだ場合、 そのベクターを B. coliY1090 に感染させ、 形成されたプラーク中に含まれるポリペプチドを ニトロセルロース膜に転写し、 次いで、 該転写膜を、 非 A非 B型ウィルス肝 炎患者血清を該ウイルスの抗体源として用レ、る免疫学的手法によるスクリー 二ングに供する方法が挙げられる。 The screening method of the target clone may be in accordance with a known method.For example, when a double-stranded DNA is incorporated into a λgt11 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.
ただし、 この方法では、 非 A非 B型肝炎ウィルスの全 DNA を解析すること は難しい。 それゆえに、 以下の方法を適用することもできる。  However, it is difficult to analyze the total DNA of non-A non-B hepatitis virus using this method. Therefore, the following method can be applied.
すなわち、 まず、 前記の方法で非 A非 B型肝炎ウィルスに感染しているこ とが確認された患者の新しい血漿から RNA を調製する。 次に、 これを铸型と して作成した 1本鎖 DNA と既知の構造を有する適切なブライマーを用い、 常 法の PCR 法により DNA を増幅させ、 サブクローニングすることにより、 非 A 非 B型肝炎ウィルス由来の DNA を得ることができる。 プライマーは、 既知の 非 A非 B型肝炎ウィルス DNA配列をもとにして、 よく保存されている部分を 適宜選択してォリゴヌクレオチドを合成し、 プライマーとして用いることが でき、 通常は 10〜30個の塩基配列からなる断片がプライマーとして用いられ る。  That is, first, RNA is prepared from fresh plasma of a patient confirmed to be infected with non-A non-B hepatitis virus by the above method. Next, using single-stranded DNA prepared as type III and an appropriate primer having a known structure, 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.
塩基配列の構造は、 マキサム，ギルバート法 (Maxam, A. M. and Gilbert , W.， Proc. Natl. Acad. Sci. USA, 74, 560. 1977)あるいはジデォキシ法 (Messing, J. et al, Nucl. Acids Res. , _9, 309, 1981) によって決められ る。  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).
本発明の非 A非 B型肝炎ウィルス DNA の部分塩基配列は、 配列番号 1およ び 2に示されるが、 それらにホモロジ一のある配列について、 SDC GENETYX (SDCソフトウェア開発株式会社、 東京） を用いて検索した。 その結果、 GenB ank データベースで、 それらに 60%以上のホモロジ一のある配列は見つから なかった。 既に報告されている非 A非 B型肝炎ウィルス DNA の塩基配列との 比較では、 Houghtonらの報告した配列 （HCV-US) (特開平 2— 500880号） と 塩基レベルで 69.5%、 アミノ酸レベルで 79. 7%、 加藤らの報告した配列 （HC V-J) (Kato et. al. , Proc.Natl. Acad. Sci. USA, 87, 9524— 9528, 1990) とは 核酸レベルで 71.8%、 ァミノ酸レベルで 83. 1%のホモロジ一があった。 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.
(4) 抗非 A非 B型肝炎ウィルス抗体の検出  (4) Detection of anti-non-A, non-B hepatitis virus antibody
本発明の非 A非 B型肝炎ウィルス抗原ポリべプチドまたはその一部を用い て、 生体試料、 例えば患者血清中の抗非 A非 B型肝炎ウィルス抗体の有無を 免疫学的手法により検定することができ、 該ウィルス感染診断が可能である。 免疫学的手法としては、 公知の方法が適用でき、 公知の方法として、 例えば 酵素免疫法、 放射性免疫法、 ウェスタンプロッ ト法、 能動凝集法、 ラテック ス凝集法、 化学発光免疫法などが挙げられる。  Using the 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. 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. .
(5) 抗非 A非 B型肝炎ウィルスボリペプチド抗体の作成  (5) Preparation of anti-non-A non-B hepatitis virus polypeptide antibody
本発明の非 A非 B型肝炎ウィルスポリべプチド、 またはその一部を抗原と して抗体を作製することができる。 抗非 A非 B型肝炎ウィルスボリぺプチド のボリクローナル抗体は、 常法に従い、 マウス、 モルモッ ト、 ゥサギなどの 動物の皮下、 筋肉内、 腹腔内、 静脈に抗原を複数回接種し、 十分に免疫した 後、 該動物から採血、 血清を分雜することにより得ることができる。 モノク ローナル抗体についても公知の方法により作製することができる。 例えば、 該ボリべプチドまたはべプチド断片で免疫したマウスの脾細胞と市販のマウ スミエローマ細胞との細胞融合により得られるハイプリ ドーマを作製後、 こ のハイプリ ドーマ培養上清またはこのバイプリ ドーマを腹腔内投与したマウ スの腹水から、 非 A非 B型肝炎ウィルスポリペプチドのモノクローナル抗体 を調製することができる。 なお、 動物に免疫する際には、 市販のアジュバン トも使用できる。  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. For example, after preparing a hybridoma obtained by cell fusion of spleen cells of a mouse immunized with the polypeptide or a fragment thereof and a commercially available mouse myeloma cell, 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. When immunizing animals, commercially available adjuvants can also be used.
これら抗体は、 公知の免疫学的手法による生体試料中の非 A非 B型肝炎ゥ ィルス抗原の同定、 定量を可能とする。 すなわち、 これら抗体は、 非 A非 B 型肝炎ウィルス抗原の診断試薬として利用できる。 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.
(6) 非 A非 B型肝炎ウィルスの遺伝子解析 (6) Gene analysis of non-A non-B hepatitis virus
これまでに、 非 A非 B型肝炎ウィルスに関する報告として、 前記の Hought onらの報告 (HCV-US) 、 加藤らの報告 (HCV-J ) 、 岡本らの報告 (HCV-J6) 、 さらに本発明のウイルスを含めて 4種類の報告があることからも明らかな ように、 非 A非 B型肝炎ウィルスには、 幾つかのサブタイプが存在すること が知られている。  So far, reports on non-A, non-B hepatitis virus 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.
前記した如く、 生体試料中の本発明のウィルスに対する抗体または該ゥィ ルス抗原ボリべプチドの免疫学的検定法は、 該ウィルス感染診断方法として 有用ではあるが、 各ウィルスサブタイプを同定、 判定することは難しい。 配列番号 1の DNA は、 ウィルスの非構造蛋白質をコードする領域の一部に 相当し、 配列番号 2の DNA は、 構造蛋白質をコードする領域の一部に相当す る。 非 A非 B型肝炎ウィルスの各サブタイプの DNA塩基配列を本発明の DNA の塩基配列と比較すると、 ホモロジ一は 60〜70%とかなり低い。 この事は、 非 A非 B型肝炎ウィルスのこの部分の DNA塩基配列をもとにして、 適切なォ リゴヌクレオチドをブライマーとして用いる遺伝子解析法を行うことにより 、 非 A非 B型肝炎ウィルスサブタイプの型別判定が可能であることを示す。 具体的には、 常法に従い、 生体試料例えば検体血漿より調製した RNA を畴 型として用い、 適切なアンチセンスプライマー、 センスプライマ一および逆 転写酵素を用いる RT— PCR 法により DNA を増幅させ、 その DNA を解析するこ とにより、 非 A非 B型肝炎ウィルスサブタイプの型別判定ができる。 プライ マ—は、 各サブタイプウィルスの DNA塩基配列に基づいて適宜選択すればよ く、 所定のサイズの DNAが増幅されたかどうかをポリアクリルアミ ドゲルま たはァガロースゲル電気泳動で調べることにより、 該ウィルスの型別判定が できる。 また、 ブライマーの中間に位置する各サブ夕イブウィルスの標識合 成オリゴヌクレオチドをプローブとして使用することにより、 その確認をす ることもできる。 As described above, 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, and the DNA of SEQ ID NO: 2 corresponds to a part of a region encoding a structural protein. When the nucleotide sequence of each subtype of non-A non-B hepatitis virus is compared with the nucleotide sequence of the DNA of the present invention, the homology is considerably low at 60 to 70%. This can be achieved by performing a genetic analysis using the appropriate oligonucleotides as primers based on the DNA base sequence of this part of the non-A non-B hepatitis virus to obtain the non-A non-B hepatitis virus subtype. Indicates that type determination of is possible. Specifically, according to a conventional method, 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. By analyzing the DNA, non-A and non-B hepatitis virus subtypes can be typed. 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. In addition, 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.
また、 増幅させた DNA の構造を解析する際は、 必要に応じて適切なベクタ —に該 DNA を組み込み、 それを適切な宿主に導入、 複製させるとよい。 これ により、 塩基配列の解析が可能である。  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.
さらに、 本発明の非 A非 B型肝炎ゥィルスボリぺブチドを用いることで、 ワクチンの作製が可能であり、 該ワクチンは、 非 A非 B型肝炎治療剤として 使用できる。  Furthermore, 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.
〔実施例〕  〔Example〕
以下、 実施例により本発明を詳細に且つ具体的に説明するが、 本発明はこ れらの実施例に限定されるものではない。  Hereinafter, the present invention will be described in detail and specifically with reference to examples, but the present invention is not limited to these examples.
実施例 1 Example 1
血漿からの非 A非 B型肝炎ウィルス RNA の調製 Preparation of non-A non-B hepatitis virus RNA from plasma
S-GPT高値を示す日本国内の供血者血漿 9. 85リッ トルを用い、 終濃度 4 % となるように PEG4000 を加え、 室温で 30分〜 40分撹拌溶解した。 4てで 1夜 静置した後、 3, 300rpmで 30分間遠心し、 沈渣としてウィルスを濃縮した。 次いで、 AGPC法 (Chamezynski and Sacci, Anal. Biochem. , 162, 156 - 15 9, 1987)及び、 Cs-TFA超遠心法 (Okayama et. al. , Method Enz. , 154E， 3 -28, 1987) に従い、 RNA を調製した。 即ち、 得られた沈渣に対して 2容 の 6 M D溶液 （6 M Guanidine isothiocyanate, 0. 75% Sarcosy 1， 37. 5πΛί クェン酸ナトリウム （PH7.0)) と終濃度 0.7%となるように 2—メルカプト エタノールを加え、 よく混和した。 これに 0.1 容の 2Μ酢酸ナトリウム （ρΗ4 ) . 1容の水飽和フエノール、 0.2容のクロ口ホルム一イソアミルアルコー ル （49 : 1 ) を加えて混和し、 氷上に 15分間静置した後、 4でで 8000rpm、 20分間の遠心をし、 水層を別の容器に移すというフエノール抽出操作を 3度 行った。 この水層に 1容のイソブロバノールを加え、 一 20°Cに数時間以上放 置した後、 4でで 8000rpm、 20分間の遠心をし、 沈澱を回収するというイソ プロパノール沈澱を行った。 沈殿を少量の 4 M D溶液一 0.7 % 2—メルカプ トエタノールに溶解し、 それを 1.02gZmlトリフルォロ醉酸セシウム一 0.1 M EDTA溶液に重層し、 25でで 35, OOOrpm、 12時間の遠心をして沈澱を得た。 この沈澱を少量の 4M D溶液一 0.7 %2—メルカブトエタノールに溶解し、 イソプロパノール沈藪を行い、 RNAを調製した。 Using 9.85 liters of Japanese donor blood plasma showing a high S-GPT level, PEG4000 was added to a final concentration of 4%, and the mixture was stirred and dissolved at room temperature for 30 to 40 minutes. After standing at 4 for 1 night, the mixture was centrifuged at 3,300 rpm for 30 minutes to concentrate the virus as a sediment. Subsequently, the AGPC method (Chamezynski and Sacci, Anal. Biochem., 162, 156-159, 1987) and the Cs-TFA ultracentrifugation method (Okayama et. Al., Method Enz., 154E, 3-28, 1987) RNA was prepared according to the procedure described above. That is, 2 volumes of 6 MD solution (6 M Guanidine isothiocyanate, 0.75% Sarcosy 1, 37.5πΛί) Sodium citrate (PH7.0)) and 2-mercaptoethanol were added to a final concentration of 0.7%, and mixed well. Add 0.1 volume of sodium diacetate (ρΗ4). 1 volume of water-saturated phenol and 0.2 volume of chloroform-form-isoamyl alcohol (49: 1), mix and let stand on ice for 15 minutes. The mixture was centrifuged at 8000 rpm for 20 minutes at, and the phenol extraction operation of transferring the aqueous layer to another container was performed three times. To this aqueous layer was added 1 volume of isobrovanol, left at 120 ° C for several hours or more, and then centrifuged at 8,000 rpm for 20 minutes at 4 to perform isopropanol precipitation in which the precipitate was collected. Dissolve the precipitate in a small amount of 4 MD solution-0.7% 2-mercaptoethanol, overlay it on 1.02 g Zml cesium trifluorofluorosulfate-0.1 M EDTA solution, and centrifuge at 25, 35, OOOrpm for 12 hours at 25. A precipitate was obtained. This precipitate was dissolved in a small amount of a 4M D solution-0.7% 2-mercaptoethanol and subjected to isopropanol precipitation to prepare RNA.
実施例 2 Example 2
c-DNA ライブラリーの作 Creating a c-DNA library
BRし社 (Bethesda Research Laboratories Life Technologies Inc. Gait her-sbuog)の c-DNA Synthesis System (8267SA)を用い、 そのマニュアルに 従って c-DNA合成を行い、 同じく BRL社の c-DNA Cloning System AgtlO a nd ;igtll(8285SA)を用い、 そのマニュアルに従って； I gtllベクタ一に 2本 鎖 c-DNA を組み込んだ。 In Vitro Packagingは、 Stratagene社 (Stratagene .La Jolla)の GigapackTM Gold II Packaging Extractを用いて行った。 具体 的には、 まず、 铸型 RNA にランダムプライマーを加え、 逆転写酵素を用いて 1本鎖 c-DNA を合成した。 次に、 RNase H、 大腸菌 DNA Polymerase I、 大 腸菌 DNA Ligase を用いて 2本鎖 c-DNA を合成した。 得られた 2本鎖 c-DNA を EcoRI Methylaseで処理して、 c-DNA内部の EcoRI 切断部位をメチル化し た後、 T4 DNA Polymerase を用いて末端を平滑化した。 この平滑末端に、 T4 DNA Ligase を用いてリン酸化された EcoRI リンカーを接続した。 EcoRI で 余分なリンカ一を切断した後、 ゲルろ過で EcoRI リンカ一を除去した。 得ら れた EcoRi切断末端を持つ 2本鎖 c-DNAを、 T4 DNA Ligase を用いて； I gtll ファージのアーム DNA に接続した。 得られた組換えファージ DNA を、 In Vit ro Packaging Extract と室温で混合することにより、 組換え体ファージを 得た。 Using 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 ™ 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. Next, 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.
実施例 3  Example 3
ィムノスクリーニング  Imno screening
支持菌として E. coli Y1090を用い、 常法に従い、 ；i gtll組換えファージの ブラ一クを形成させた。 lOmM IPTG (Sigma社） に浸しておいたニトロセル口 ースフィルタ一（Schleicher & SchueIL BA85) をプラークの上にのせ、 37°C でさらに 3時間培養した。 ニトロセルロースフィルターを TBS(pH7. 5 ) (10 mM Tris-HCl. 150mM NaCl)で 4回洗浄した後、 5 %スキムミルク溶液 （ 5 % スキムミルク， 0. 05% Tweenを含む TBS)に浸して 4 eCで 1夜弱く振gした。 非 A非 B型肝炎患者 10名の血清を等量ずつ混合し、 そこにその総量と等量の 大腸菌 Y1090溶菌液を加えて室温で 60分間振盪した後、 3, 000rpm. 30分間遠 心して得た上清を 5 %スキムミルク溶液で 10倍希釈したものを 1次抗体液と して用いた。 プラークを転写したニトロセルロースフィルタ一に 1次抗体液 を加え、 4。Cで 1夜弱く振盪した。 このニトロセルロースフィルターを、 TB S-0. 05% Tweenを用いて室温で 5分間洗浄する操作を 2回繰り返した後、 酵 素標識した 2次抗体液として、 Anti human IgG(Fc) (Goat)-Peroxidase conj ugateCCappel社） を TBS-0. 05% Tweenで 500 倍希釈したものを用意し、 ここ にニトロセルロースフィルターを入れ、 室温で 2時間弱く振盪した。 この二 トロセルロースフィルターを TBS-0.05% Tweenを用レ、て室温で 5分間洗浄す る操作を 2回繰り返した後、 基質液 （TBS(pH6.5)を 10^、 3呢 ^ 4—クロ ロー 1一ナフトールノメタノール液を 2 、 3 %過酸化水素水溶液 （過酸化 水素水） を 40 _βの比率で混合したもの） にニトロセルロースフィルターを 入れ、 室温で振盪した。 発色の停止は、 フィルターを蒸留水で洗浄すること によって行い、 陽性クローンを 10個選択した。 Using E. coli Y1090 as a supporting bacterium, 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. 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_β, 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.
塩基配列の決定 Determination of nucleotide sequence
塩基配列の決定は、 常法通り、 M13ファージを用いた Dideoxy Terminatio n 法で行った。 実際には、 USB社 (Uni ted States Biochemical Corporatio n, Cleve-land) ©Sequenase version 2. 0 Labeled dCTP Editionを用い、 同 マニュアルに従って行った。  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.
その結果、 配列番号 1に示す通り、 新規な非 A非 B型肝炎ウィルスの非構 造蛋白質をコードする塩基の部分 DNA であることを確認した。 また、 この塩 基配列からコードされたァミノ酸配列も、 同配列番号に記載した如く演繹さ れた。  As a result, as shown in SEQ ID NO: 1, it was confirmed that 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.
なお、 該 c-DNA を組み込んだファージ λ ΕίΙΙを E. col i Y1088株に導入した Escherichia coli 2-22- i gtllは、 通商産業省工業技術院微生物工業技術研 究所特許微生物寄託センタ— （〶 3 0 5日本国茨城県つくば市東 1丁目 1番 3号） に、 受託番号：微ェ研菌寄第 12899号 （FERM P-12899) として、 平成 4年 3月 24日付で寄託されている。  In addition, Escherichia coli 2-22-igtll, in which the phage λΕίΙΙ 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 .
実施例 4 ブライマーを用いる非 A非 B型肝炎ウィルス DNA のクローニング Example 4 Cloning of non-A non-B hepatitis virus DNA using primers
実施例 3により該ウィルスゲノ厶を有することが明らかになつた血漿検体 より RNA を調製した。 ブライマーは、 加藤らの報告 （Kato et al, Proc, Na tl. Acad. Sci. USA, 87, 9524, 1990) の HCV-J の塩基配列から、 センスブ ライマーとして 1289— 1309位、 アンチセンスプライマーとして 1572— 1592位 を選択し、 各々の合成オリゴヌクレオチドを用いて常法により PCR を行なつ た。 この際、 各ブライマーの 5 ' 端には、 M13mpl8および M13mpl9にクロー 二ング可能な制限酵素認識配列、 すなわちセンスブライマーには Smalおよび BainHi の認識配列、 アンチセンスブライマーには Pstlおよび Sai lの認識配列 を付加して使用した。  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. At this time, at the 5 'end of each primer, 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.
PCR で増幅した DNA を各制限酵素で切断した後、 ポリアクリルアミ ドゲル 電気泳動にかけ、 当該バンドを切り出し、 DNAを抽出した。 抽出した DNA を 、 常法に従い、 M13mpl8および M13mpl9にサブクローニングし、 塩基配列を 解析した結果、 核飄 は、 配列番号 2に示す如く、 新しい非 A非 B型肝炎ゥ ィルスの構造蛋白質をコードする領域の部分 DNAであった。 また、 この塩基 配列からコードされるアミノ酸配列も、 同配列番号に記載した如く演繹され た。  After the DNA amplified by PCR was digested with each restriction enzyme, it was subjected to polyacrylamide gel electrophoresis, the band was cut out, and the DNA was extracted. The extracted DNA was subcloned into M13mpl8 and M13mpl9 according to a conventional method, and the nucleotide sequence was analyzed. As a result, 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.
該 c-DNA を含む M13mpl8を制限酵素 Sma〖および BaraHI にて処理し、 該 c-DN A部分を切り出し、 ブラスミ ド pUEX3 に組み込んだ。 このブラスミ ドで E. co l i 皿 5 α株を形質転換させた形質転換体 Escherichia col i env-pUEX3は、 通 商産業省工業技術院微生物工業技術研究所特許微生物寄託センター （〶3 0 5日本国茨城県つくば市東 1丁目 1番 3号） に、 受託番号：微ェ研菌寄第 12 900号 （FERM P-12900) として、 平成 4年 3月 24日付で寄託されている。 実施例 5 M13mpl8 containing the c-DNA was treated with restriction enzymes Sma 〖and BaraHI, the c-DNA portion was cut out, and incorporated into brassmid pUEX3. The transformant Escherichia coli env-pUEX3, which was obtained by transforming the E. coli dish 5α strain with this plasmid, was obtained from the Patent Microorganisms Depositary Center of the Institute of Microorganisms and Industrial Technology of the Ministry of International Trade and Industry (〶2005 Japan). Deposit No. 1900-3, Higashi 1-chome, Tsukuba, Ibaraki Pref., Japan, deposited under the accession number: No. 12900 (FERM P-12900) on March 24, 1992. Example 5
非 A非 B型肝炎ウィルス抗原に対する抗体検出による該ウィルス感染の診断 本発明で得られた C-DNA を組み込んだ; I gtl lファージ （2— 22) と野生型 の λ 8ΐ11ファージを約 1 ： 2の比率で混ぜ、 支持菌としては E. col i Y1090を 用い、 常法に従いプラークを形成させた。 lOmM IPTG (Sigma社） に浸してお いたニトロセルロースフィルター（Schleicher & Schuel l, BA85) をブラーク の上にのせ、 37ででさらに 3時間培養した。 ニトロセルロースフィルターをDiagnosis of virus infection by detection of antibody against non-A non-B hepatitis virus antigen The C-DNA obtained in the present invention was incorporated; Igtl phage (2-22) and wild-type λ8ΐ11 phage were mixed at about 1: The mixture was mixed at a ratio of 2, and E. coli Y1090 was used as a supporting bacterium to form plaque according to a conventional method. A nitrocellulose filter (Schleicher & Schuel, BA85) immersed in lOmM IPTG (Sigma) was placed on the black plaque and incubated at 37 with an additional 3 hours. Nitrocellulose filter
TBS (pH7. 5)で 4回洗浄した後、 それを 5 %スキムミルク溶液に浸して 4 eC で 1夜弱く振盪した。 非 A非 B型肝炎患者、 B型肝炎患者、 健常人の各々の 血清検体に各々等量の大腸菌 Y1090溶菌液を加え、 室温で 60分間振盪し、 3, OOOrpm, 30分間遠心して得た上清を 5 %スキムミルク溶液で 10倍希釈したも のを 1次抗体液として用いた。 プラークを転写したニトロセルロースフィル ターを 3麵幅のストリップにしたものに 1次抗体液を加え、 4でで 1夜弱く 振盪した。 このニトロセルロースフィルターを TBS-0. 05% Tweenを用いて室 温で 5分間洗浄する操作を 2回繰り返した後、 2次抗体液として、 Anti hu man IgG(Fc) (Goat)-Peroxidase conjugate (Cappel 社） を TBS-0. 05% Twe enで 500 倍希釈したものを調製し、 ここにニトロセルロースフィルタ一を入 れ、 室温で 2時間弱く振盪した。 ニトロセルロースフィルターを TBS- 0. 05%After washing four times with TBS (pH 7.5), it was immersed in a 5% skim milk solution and shaken slightly at 4 e C overnight. Equal amounts of Escherichia coli Y1090 lysate were added to serum samples of non-A non-B hepatitis patients, hepatitis B patients, and healthy individuals, respectively, shaken at room temperature for 60 minutes, and centrifuged at 3, OOOrpm for 30 minutes. A solution obtained by diluting the supernatant 10-fold with a 5% skim milk solution was used as a primary antibody solution. The primary antibody solution was added to a 3 mm-wide strip of the nitrocellulose filter on which the plaque had been transferred, and the mixture was slightly shaken at 4 with overnight. 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%
Tweenを用いて室温で 5分間洗浄する操作を 2回繰り返した後、 基質液 （TB S(pH6. 5)を 10 、 3 mg/ 4—クロロー 1一ナフトール/メタノール溶液を 2 m£、 3 %過酸化水素を 40〃 ^の比率で混合したもの） にニトロセルロース フィル夕一を入れ、 室温で振盪した。 発色の停止は、 フィルターを精製水で 洗净することによって行った。 野生型 A gtllファージのプラーク部位と組換え； I gtllファージ （2— 22) のプラーク部位の発色に差がある場合を陽性、 差のない場合を陰性と判定し た。 その結果を表 1に示した。 表中 2— 22が本発明による方法であり、 C100 -3が Houghtonら （特開平 2— 500880号） の方法による結果である。 C100-3は 、 非 A非 B型肝炎患者血清 12検体中 8検体が陽性を示しているが、 B型肝炎 患者血清に対しても 5検体中 3検体が陽性を示し、 特異性が明確でない。一 方、 本発明の方法 2 - 22では、 非 A非 B型肝炎患者血清 12検体中 7検体が陽 性を示し、 B型肝炎患者血清には陰性を示し、 特異性の点で本発明による方 法がはるかに優れている。 After washing twice with Tween at room temperature for 5 minutes, 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. In the table, 2-22 is the method according to the present invention, and C100-3 is the result according to the method of Houghton et al. (JP-A-2-500880). For 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 . On the other hand, in 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.
Figure imgf000019_0001
実施例 6
Figure imgf000019_0001
Example 6
合成ぺブチドによる抗非 A非 B型肝炎ウィルス抗体の検出  Detection of anti-non-A and non-B hepatitis virus antibodies with synthetic ぺ butide
実施例 5は配列番号 1の DNA を組み込んだ； I gtllを使用しているので、 配 列番号 1の全構造を含むポリべプチドを抗原とする抗ウィルス抗体の測定で ある。 しかし、 全構造を含むポリペプチドを使用するより、 その構造の中で 抗原性の高いぺプチド部分を選択し、 その部分の合成べプチドを抗原とする ELISA法によって抗体を検出する方法の方が、 簡便かつ実用的であるので、 ェピトーブの決定を行なつた。  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.
配列番号 1のポリペプチドアミノ酸配列をもとに、 1一 20位、 11一 30位、 21— 40位、 31— 50位および 41一 59位に相当するペプチドを合成し、 ェピトー ブ部位決定に供した。 抗ウィルス抗体の検出は、 常法の ELISA法にて行なつ た。 すなわち、 各合成ペプチドでコートしたゥエルに、 実施例 5で陽性と判 定された検体血清を加えて反応 （37で、 1時間） 、 洗浄後、 アルカリフォス ファターゼ標識抗ヒト IgG抗体を反応 （37で、 1時間） 、 洗浄、 次いで酵素 基質 P-二トロフヱニルフォスフヱートを加えて 37で 30分間反応させた後、 波 長 405nm における吸光度を測定した。  Based on the amino acid sequence of the polypeptide of SEQ ID NO: 1, 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.
その結果、 表 2に示すごとく、 11一 30位合成ペプチドが最も高い抗原活性 を示した。 このことから、 11一 30位またはそれを含む合成ペプチドを抗原と して用いることにより、 抗非 A非 B型肝炎ゥィルス抗体の検定が可能である。 As a result, as shown in Table 2, 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.
表 2 Table 2
Figure imgf000021_0001
Figure imgf000021_0001
実施例 7 Example 7
非 A非 B型肝炎ウィルスゲノムの検出 Detection of non-A non-B hepatitis virus genome
プライマーとして配列番号 1記載の塩基配列 39〜60位のセンスコドン部と 149 -172位のアンチセンスコドン部の合成オリゴヌクレオチドを用いた。 非 A非 B型肝炎ウィルス感染高危険群のヒト血漿 1; ^より、 実施例 1の方 法により RNA を調製した。 得られた RNA の 1/6量を用い、 常法に従い RT-PCR を行った。 c-DNA合成は、 lOmM Tris-HW (pH8.3) 、 50mM KC£, 2mM MgC 、 200mM dNTPs、 0.01% gelatin^ 20 units Placental RNase inhibitor, lOOmMアンチセンスブライマ一、 100 units Murine逆転写酵素という反応組 成で、 37°Cにて、 30分間行った。 c-DNA合成後、 それを 95°C、 5分間の保温 で熱変性させ、 続いて PCR を行った。 PCR の反応組成は、 lOra Tris-HC （ pH8.3)、 50m KC、 1.5mM MgC£2 、 200ra dNTPs、 0.01% gelatin, lOOraM センスプライマ一、 lOOraM アンチセンスプライマ一、 1 unit Taq Iポリメラ —ゼであり、 94° ( 、 1分間熱変性、 55°C、 2分間アニーリング、 72°C、 1分 30秒間 DNA 伸長反応というサイクルを 60サイクル行った。 PCR 反応後、 その 1/10量を 6 %ポリアクリルアミ ドゲル電気泳動にかけ、 臭化工チジゥムで染 色し、 紫外線照射下で写真をとつた。 日本国内の C100- 3抗体陽性血漿 30検 体のうち、 14検体が陽性であった。 As a primer, a synthetic oligonucleotide having a sense codon portion at positions 39 to 60 and an antisense codon portion at positions 149 to 172 described in SEQ ID NO: 1 was used. 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.
実施例 8  Example 8
非 A非 B型肝炎ウィルスサブタイブの識別  Identification of non-A non-B hepatitis virus subtypes
非 A非 B型肝炎ウィルスサブタイプのうち、 Houghtonらの HCV-US, Katoら の HCV-J および本発明の 2— 22ウィルスの識別検定を行なった。  Among the non-A and non-B hepatitis virus subtypes, Houghton et al., HCV-US, Kato et al., HCV-J, and the 2-22 virus of the present invention were discriminated.
各サブタイプウィルスの塩基配列に基づいて、 実施例 6で使用した配列番 号 1の塩基配列中のォリゴヌクレオチドプライマ一と同じ位置に相当するォ リゴヌクレオチドをそれぞれ合成し、 ブライマーとして使用した。 それぞれ の塩基配列と位置番号は表 3に示した。  Based on the nucleotide sequence of each subtype virus, 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.
C100-3抗体強陽性 (2. 000≥) 、 いわゆる Houghtonらのウィルス抗原を用い た抗体検出試薬で強陽性と判定された供血者血漿及び各種肝疾患患者血漿最 低 300 ^より実施例 1の方法に従って調製した非 A非 B型肝炎ウィルス RN A のうち、 得られた RNA の血漿 100 ^相当量を铸型とし、 上述の各ブライ マー各々 200nMを添加し、 通常の RT-PCR法を 40〜60サイクル行うことにより 、 ゲノムを増幅して検出した。  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. Of 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.
PCR反応においてのァニーリング温度はブライマーの塩基配列より算出さ れた Tm値をもとに （Tni値一 5で） に設定した。  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.
また、 この他、 反応条件、 増幅産物の検出方法等は、 実施例 7に記載した 通り、 またはこれに準じたものとした。 表 3 サブタイプ l Sij用プライマー ウィルスタイプ 驢删 In addition, the reaction conditions, the method for detecting the amplification product, and the like were the same as those described in Example 7 or those based thereon. Table 3 Primers for subtype l Sij Virus type Donkey
2 -22  2 -22
sense primer +5' -GCCCTCATAACACCATGTGGGC-3' 2 -22； 39nt- 60nt anti-sense primer -5' -GTCACCnCTTCGCCCTCAGAGAA-3' 2 -22； 149nt- 172nt  sense primer +5 '-GCCCTCATAACACCATGTGGGC-3' 2 -22; 39nt-60nt anti-sense primer -5 '-GTCACCnCTTCGCCCTCAGAGAA-3' 2-22; 149nt-172nt
HCV-J HCV-J
sense primer +5' -GCCTTGATCACGCCATGCGCTG-3' HCV-J ； 7611nt-7632nt anti-sense primer -5' -GTGACCTTCTTCTGCCGCAGACTT-3' HCV-J ； 7721nt-7744nt  sense primer +5 '-GCCTTGATCACGCCATGCGCTG-3' HCV-J; 7611nt-7632nt anti-sense primer -5 '-GTGACCTTCTTCTGCCGCAGACTT-3' HCV-J; 7721nt-7744nt
HCV-US HCV-US
sense primer +5" -GCACTCGTCACCCCGTGCGCCG-3' HCV-US； 7626nt-7647nt anti-sense primer - 5' -GTGACnTCTTCTGCCTrTGGCAA-3' HCV-US； 7736nt-7759nt 次いで、 増幅させた DNA をポリアクリルアミ ドゲル電気泳動にかけた後、 プライマー設定領域内中央付近に設定されたアンチセンス3 2 P 標識合成オリ ゴヌクレオチドをプローブとして反応させる、 いわゆるサザンハイブリダイ ゼーションを行なうことにより識別した。 使用したプローブは表 4に示した c この方法による非 A非 B型肝炎ウィルスサブタイプの検出結果は以下の通 りであった。 すなわち、 表 5に示す如く、 C100- 3抗体強陽性 （2. 000 ≥) で ある供血者血漿 30例においては、 各サブタイプの検出は、 該ウィルスゲノム ( 2 - 22) 型、 HCV-J 型、 HCV-US型の順に 13例 (43. 0% ) ， 21例 (70. 0%) , 13例 （43. 0%) であった。 また各種肝疾患患者血漿においては、 同順に C 型急性肝炎 3例中 0例 （0 %) , 2例 （66.7%) , 0例 （0 %) 、 C型慢性 肝炎 26例中 11例 （42. 3%) , 15例 （57. 7%)、 0例 （0 %)、 C型肝硬変 10 例中 4例 （40. 0%) , 3例 （30, 0%)， 0例 （0 %)、 Β型慢性肝炎 14例中 5例 （35. 1%)、 2例 （14.3%) , 0例 （ 0 9 であった。 この他に自己免 疫性肝炎においても 2例中 1例に該ウィルスゲノム型の非 Α非 Β型肝炎ウイ ルスサブタイプが検出された。 sense primer +5 "-GCACTCGTCACCCCGTGCGCCG-3 'HCV-US; 7626nt-7647nt anti-sense primer-5' -GTGACnTCTTCTGCCTrTGGCAA-3 'HCV-US; 7736nt-7759nt Then, the amplified DNA is subjected to polyacrylamide gel electrophoresis. after, the reaction of set antisense 3 2 P-labeled synthetic oligonucleotide near the center primer set area as probes, identified by performing a so-called Southern hybridization. probe used are shown in Table 4 c The results of detection of non-A, non-B hepatitis virus subtypes by this method were as follows: blood plasma of a donor with strong C100-3 antibody positive (2.000 ≥), as shown in Table 5. In 30 cases, each subtype was detected in the order of the virus genome (2-22), HCV-J, and HCV-US in 13 cases (43.0%) and 21 cases (70.0%). And 13 cases (43.0%) and various liver diseases In patient plasma, C Acute hepatitis type 0 out of 3 cases (0%), 2 cases (66.7%), 0 cases (0%), chronic hepatitis C 11 cases out of 26 cases (42.3%), 15 cases (57.7% ), 0 (0%), 4 out of 10 cirrhosis C (40.0%), 3 (30, 0%), 0 (0%), 5 out of 14 chronic hepatitis Β ( 35.1%), 2 cases (14.3%), 0 cases (09.) In addition, in 1 of 2 cases of self-immune hepatitis, the virus genomic non- 肝 non-Β type hepatitis virus Loose subtype was detected.
さらに 2— 22型として識別された DNA のうち 4個を選択し、 常法に従い M 13mpl8および M13mpl9に組み込み、 次いで大腸菌 E. col i DH5 a F'または JM 107 を支持菌として増殖させて組み換えファージ DNA を調製し、 両方向から それぞれの塩基配列を解析した結果、 その中の 1個では、 配列番号 3に示し た如く、 配列番号 1記載の 2— 22型と比較して一部に変異が認められた。  Furthermore, four of the DNAs identified as type 2-22 were selected, incorporated into M13mpl8 and M13mpl9 according to a conventional method, and then propagated with E. coli E. coli DH5a F 'or JM107 as a supportive bacterium. As a result of preparing the DNA and analyzing the base sequences in both directions, one of them was found to have a partial mutation as shown in SEQ ID NO: 3 compared to the 2-22 type described in SEQ ID NO: 1. Was done.
表 4 サブタイ 佣プローブ ウィルスタイプ 塩基 S^ll 塩 Sfi置 Table 4 Subtype 佣 Probe Virus type Base S ^ ll Salt Sfi
2 -22 5' -GAGGTTGTGGAGTACACCT GTTATGGAATCGCATGAGCG-3' 2 -22； 100nt-139nt HCV-J 5' -GATGTTGTGGAGTAGACCATAGTGTGGTGACGCAGCAMG-3' HCV-J ； 7672nt-7711nt 2 -22 5'-GAGGTTGTGGAGTACACCT GTTATGGAATCGCATGAGCG-3 '2 -22; 100nt-139nt HCV-J 5'-GATGTTGTGGAGTAGACCATAGTGTGGTGACGCAGCAMG-3' HCV-J; 7672nt-7711nt
HCV-US 5' -GAGGTGG GGAATACACCAAAnGTGGTGACGTAGCAACG-3* HCV-US； 7687nt-7726nt HCV-US 5 '-GAGGTGG GGAATACACCAAAnGTGGTGACGTAGCAACG-3 * HCV-US; 7687nt-7726nt
表 5 臨床検体中非 A非 B型肝炎ウィルスサブタイプの検出結果 Table 5 Results of detection of non-A and non-B hepatitis virus subtypes in clinical samples
Figure imgf000025_0001
Figure imgf000025_0001
本発明の DNA、 RNA または抗原ポリペプチドを用いることにより、 非 A非 B型肝炎の診断が可能となり、 輸血等による該肝炎ゥィルスの感染の予防、 ワクチン、 抗ウィルス剤の開発が可能となる。 By using the DNA, RNA or antigen polypeptide of the present invention, 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.
〔配列表〕 (Sequence list)
配列番号： 1  SEQ ID NO: 1
配列の長さ ： 181  Sequence length: 181
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖  Number of chains: 2 chains
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント  Fragment type: middle fragment
起源：非 A非 B型肝炎ウィルス Origin: non-A non-B hepatitis virus
配列の特徴 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 181  Location: 1.. 181
特徵を決定した方法： E  How to determine the features: E
配列の特徵 Array features
特徴を表す記号： peptide  Characteristic symbol: peptide
存在位置： 3 . . 179  Location: 3. .179
特徵を決定した方法： S  How to determine the features: S
配列 Array
GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47 Val lie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu lie 1 5 10 15  GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47 Val lie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu lie 1 5 10 15
ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95 Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143 Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95 Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143 Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg
35 40 45  35 40 45
AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181 Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181 Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp
50 55  50 55
配列番号： 2 SEQ ID NO: 2
配列の長さ ： 261 Sequence length: 261
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント Fragment type: middle fragment
起源 Origin
生物名：非 A非 B型肝炎ウィルス  Organism name: non-A non-B hepatitis virus
配列の特徵 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 261  Location: 1.. 261
特徴を決定した方法： E  How the features were determined: E
配列の特徴 Array features
特徴を表す記号： peptide  Characteristic symbol: peptide
存在位置： 1 . . 261  Location: 1.. 261
特徵を決定した方法： S 配列 How to determine the features: S Array
ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48 ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48
Thr Thr Leu Thr Met lie Leu Ala Tyr Ala Ala Arg Val Pro Glu Leu 1 5 10 15 Thr Thr Leu Thr Met lie Leu Ala Tyr Ala Ala Arg Val Pro Glu Leu 1 5 10 15
GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val He Phe Gly Gly His Trp Gly Val Val Phe Gly Leu GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val He Phe Gly Gly His Trp Gly Val Val Phe Gly Leu
20 25 30  20 25 30
GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val lie Ala lie Leu GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val lie Ala lie Leu
35 40 45  35 40 45
CTC CTT GTC GCA GGA GTA GAT GCA GAG ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg CTC CTT GTC GCA GGA GTA GAT GCA GAG ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg
50 55 60  50 55 60
GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80 GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80
CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His He Asp Leu lie CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His He Asp Leu lie
85 配列番号： 3 85 SEQ ID NO: 3
配列の長さ ： 88 Array length: 88
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント Fragment type: middle fragment
起源 Origin
生物名：非 A非 B型肝炎ウィルス  Organism name: non-A non-B hepatitis virus
配列の特徵 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 88  Location: 1.. 88
特徴を決定した方法： E  How the features were determined: E
配列の特徴 Array features
特徴を表す記号： peptide  Characteristic symbol: peptide
存在位置： 3 . . 86  Location: 3.. 86
特徴を決定した方法： S  How the features were determined: S
配列 Array
CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro l ie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15  CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15
AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Lys Ser AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Lys Ser
20 25  20 25

Claims

請求の範囲 The scope of the claims
1 . 下記の配列番号 1， 2および 3の非 A非 B型肝炎ウィルス抗原ボリぺブ チドの、 全部または一部を含有してなるボリペプチド。 1. A polypeptide comprising all or part of the non-A non-hepatitis B virus antigen polypeptide of SEQ ID NOS: 1, 2 and 3 below.
配列番号： 1  SEQ ID NO: 1
配列の長さ ： 181  Sequence length: 181
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー：直鏆状  Topology: straight
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント  Fragment type: middle fragment
起源：非 A非 B型肝炎ウィルス Origin: non-A non-B hepatitis virus
配列の特徵 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 181  Location: 1.. 181
特徴を決定した方法： E  How the features were determined: E
配列の特徵 Array features
特徵を表す記号： peptide  Symbol indicating special features: peptide
存在位置： 3 . . 179  Location: 3. .179
特徴を決定した方法： S  How the features were determined: S
配列 Array
GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47 Val lie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu He 1 5 10 15 ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95 Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser GG GTT ATC TGC TGC TCC ATG TCA TAC TCC TGG ACG GGG GCC CTC ATA 47 Val lie Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu He 1 5 10 15 ACA CCA TGT GGG CCT GAG GAG GAG AAG TTA CCG ATC AAC CCT CTG AGC 95 Thr Pro Cys Gly Pro Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser
20 25 30  20 25 30
AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143 Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg AAC TCG CTC ATG CGA TTC CAT AAC AAG GTG TAC TCC ACA ACC TCG AGG 143 Asn Ser Leu Met Arg Phe His Asn Lys Val Tyr Ser Thr Thr Ser Arg
35 40 45  35 40 45
AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181 Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp AGT GCT TCT CTG AGG GCG AAG AAG GTG ACC TTT GAC CC 181 Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp
50 55  50 55
配列番号： 2 SEQ ID NO: 2
配列の長さ ： 261 Sequence length: 261
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント Fragment type: middle fragment
起源 Origin
生物名 ：非 A非 B型肝炎ウィルス  Organism name: Non-A non-B hepatitis virus
配列の特徵 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 261  Location: 1.. 261
特徴を決定した方法： E 配列の特徴 How the features were determined: E Array features
特徵を表す記号： peptide  Symbol indicating special features: peptide
存在位置： 1 . . 261  Location: 1.. 261
特徵を決定した方法： S  How to determine the features: S
配列  Array
ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48 Thr Thr Leu Thr Met He Leu Ala Tyr Ala Ala Arg Val Pro Glu Leu 1 5 10 15  ACA ACT CTT ACC ATG ATC CTC GCC TAT GCT GCT CGT GTT CCT GAG CTG 48 Thr Thr Leu Thr Met He Leu Ala Tyr Ala Ala Arg Val Pro Glu Leu 1 5 10 15
GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val lie Phe Gly Gly His Trp Gly Val Val Phe Gly Leu GTC CTT GAA GTC ATC TTC GGC GGT CAT TGG GGT GTG GTG TTC GGC TTG 96 Val Leu Glu Val lie Phe Gly Gly His Trp Gly Val Val Phe Gly Leu
20 25 30  20 25 30
GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val He Ala He Leu GCC TAC TTC TCC ATG CAG GGA GCG TGG GCC AAG GTC ATC GCC ATC CTC 144 Ala Tyr Phe Ser Met Gin Gly Ala Trp Ala Lys Val He Ala He Leu
35 40 45  35 40 45
CTC CTT GTC GCA GGA GTA GAT GCA GAC ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg 50 55 60 CTC CTT GTC GCA GGA GTA GAT GCA GAC ACC TAT ACC ACC GGC GGA CGA 192 Leu Leu Val Ala Gly Val Asp Ala Asp Thr Tyr Thr Thr Gly Gly Arg 50 55 60
GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80 CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His l ie Asp Leu l ie GCG GGT TCT GAC ATG TAC TCG CTT GCT AGC CTT TTC AGC TCT GGT CCC 240 Ala Gly Ser Asp Met Tyr Ser Leu Ala Ser Leu Phe Ser Ser Gly Pro 65 70 75 80 CGG CAG CAC ATT GAC CTA ATC 261 Arg Gin His lie Asp Leu lie
85  85
配列番号： 3 SEQ ID NO: 3
配列の長さ ： 88 Array length: 88
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA to genomic RNA Sequence type: cDNA to genomic RNA
フラグメント型：中間部フラグメント Fragment type: middle fragment
起源 Origin
生物名：非 A非 B型肝炎ウィルス  Organism name: non-A non-B hepatitis virus
配列の特徵 Array features
特徴を表す記号： CDS  Characteristic symbol: CDS
存在位置： 1 . . 88  Location: 1.. 88
特徴を決定した方法： E  How the features were determined: E
配列の特徴 Array features
特徵を表す記号： peptide  Symbol indicating special features: peptide
存在位置： 3 . . 86  Location: 3.. 86
特徴を決定した方法： S  How the features were determined: S
配列 Array
CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro l ie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15 AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe Hi s Asn Lys Val Tyr Ser T r T r Ser Lys Ser CC GAG GAG GAG AAG TTG CCA ATC AAT CCT TTG AGT AAT TCG CTC GTG 47 Glu Glu Glu Lys Leu Pro lie Asn Pro Leu Ser Asn Ser Leu Val 1 5 10 15 AGG TTT CAC AAC AAG GTG TAC TCC ACA ACC TCA AAG AGC GC 88 Arg Phe His s Asn Lys Val Tyr Ser Tr Tr Ser Ser Lys Ser
20 25  20 25
2 . 請求項 1記載のボリペプチドをコードする、 配列番号 1 , 2および 3の DNA の全部または一部を含有してなる DNA。  2. A DNA encoding the polypeptide according to claim 1, which comprises all or a part of the DNAs of SEQ ID NOs: 1, 2, and 3.
3 . 請求項 1記載のボリべプチドを抗原とするポリクローナル抗体またはモ ノクローナル抗体。  3. A polyclonal antibody or a monoclonal antibody that uses the polypeptide of claim 1 as an antigen.
4 . 試薬の構成成分として、 請求項 2記載の DNA をプライマーとして用い、 非 A非 B型肝炎ウィルス由来の DNAを増幅させ検出することを特徵とする非 A非 B型肝炎ゥィルス ¾伝子の検出方法。  4. A non-A non-B hepatitis virus gene which is characterized in that the DNA according to claim 2 is used as a primer as a component of the reagent and the DNA derived from the non-A non-B hepatitis virus is amplified and detected. Detection method.
5 . 請求項 1記載の抗原ポリペプチドを用い、 免疫学的手法により、 試料中 の抗非 A非 B型肝炎ゥィルス抗体の存在を確認する抗非 A非 B型肝炎ウィル ス抗体検出方法。  5. A method for detecting an anti-non-A non-B hepatitis virus antibody, which comprises confirming the presence of an anti-non-A non-B hepatitis virus antibody in a sample by an immunological technique using the antigen polypeptide according to claim 1.
6 . 請求項 3記載の抗体を用い、 免疫学的手法により、 試料中の非 A非 B型 肝炎ウィルス抗原の存在を確認する非 A非 B型肝炎ウィルス抗原検出方法。 6. A method for detecting a non-A non-B hepatitis virus antigen, which comprises confirming the presence of a non-A non-B hepatitis virus antigen in a sample by an immunological technique using the antibody according to claim 3.
7 . 請求項 1記載の抗原ボリペプチドを用いて製造された非 A非 B型肝炎ヮ クチン。 7. A non-A, non-B hepatitis actin produced using the antigen polypeptide according to claim 1.
PCT/JP1992/000464 1991-04-17 1992-04-13 Rna, dna and virus antigen protein of non-a non-b hepatitis virus WO1992018532A1 (en)

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JP8503391 1991-04-17

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

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