WO2015133467A1 - Vaccine against hepatitis c virus, and diagnostic hbc/hcv e2 peptide chimeric protein - Google Patents

Vaccine against hepatitis c virus, and diagnostic hbc/hcv e2 peptide chimeric protein Download PDF

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WO2015133467A1
WO2015133467A1 PCT/JP2015/056194 JP2015056194W WO2015133467A1 WO 2015133467 A1 WO2015133467 A1 WO 2015133467A1 JP 2015056194 W JP2015056194 W JP 2015056194W WO 2015133467 A1 WO2015133467 A1 WO 2015133467A1
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hcv
hbc
protein
amino acid
virus
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健吾 下木場
中村　雄樹
克郎 小池
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株式会社シノテスト
一般社団法人 バイオ医薬研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • 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
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a recombinant protein that can be used for prophylactic / therapeutic immune response and diagnosis corresponding to infection by various genotypes of hepatitis C virus.
  • Hepatitis C virus (HCV) carriers are said to be 170 million worldwide, that is, 3% of the total population is said to be carriers, and the number of carriers in Japan is estimated to be 1.5 to 2 million . It is said that when infected with HCV, about 30% of healthy adults with normal immunity heal in the acute course, and the rest are transferred to chronic infection. In chronic hepatitis due to HCV, fibrosis is induced by persisting inflammation and progresses to cirrhosis and liver cancer.
  • Chronic hepatitis C progresses to cirrhosis 10 to 20 years after infection and eventually develops primary liver cancer at a high rate. Every year in Japan, 20,000 people die from primary liver cancer caused by HCV, which accounts for about 80% of primary liver cancer.
  • HCV is an RNA virus belonging to the Flaviviridae family, and consists of capsid, two envelope proteins E1, E2, four structural proteins p7, and six nonstructural proteins.
  • HCV is an RNA virus and is easily mutated.
  • a region called a hypervariable region (HVR) exists at the amino terminus of the envelope protein. This region has many amino acid mutations and is thought to be involved in the escape mechanism from the neutralizing antibody.
  • HCV is an RNA virus that is easily mutated is a cause of chronicity and is a major obstacle to the development of an effective vaccine.
  • HCV is known to have more than 10 types of genotypes such as 1a, 1b, 2a, 2b. It is known that depending on the genotype, the infectivity of the virus, the sensitivity to treatment such as interferon, etc. are different, and the therapeutic effect is different. Regarding neutralizing antibodies, neutralizing antibodies that recognize regions of amino acid sequences that differ between genotypes cannot prevent co-infection with other genotype viruses. The existence of many genotypes is also an obstacle to vaccine development.
  • HCV is difficult to detect in the early stages of infection and there are times when it is difficult to diagnose, and the therapeutic effect varies depending on the virus genotype as described above. ing. In terms of prevention, effective vaccines have not yet been developed due to the existence of escape mechanisms and many genotypes, and there is no major preventive method.
  • hepatitis B virus is a virus that induces hepatitis and liver cancer in humans along with HCV.
  • HBV like HCV, has been shown to be a virus that induces chronic hepatitis, cirrhosis, and liver cancer.
  • HBV is a DNA virus of the Hepadnaviridae family, and its replication mechanism and immune response are completely different from HCV.
  • HBV core protein self-assembles to form spherical core particles.
  • This core particle is known to be very immunogenic.
  • the fusion polypeptide obtained by inserting a desired epitope in the vicinity of amino acid residues 75 to 80 of the HBc protein or linking the desired epitope to the end of the HBc antigen protein forms a core particle by self-assembly.
  • An inserted or linked epitope is presented on the surface of the formed particle.
  • Patent Documents 1 to 3 attempts have been made so far to induce antibody production against an antigen that is difficult to be recognized by the immune system by using the HBc antigen protein as a vaccine platform.
  • an antigen protein that can be detected with high sensitivity corresponding to any HCV regardless of mutation or genotype. If there is an effective antigen protein, it is possible to detect an antibody in patient serum if an HCV virus is infected in a human and an antibody is produced. It is also possible to immunize animals such as rabbits and goats to produce antibodies by immunizing antigen proteins and to produce antibody reagents that detect viral antigens in patient blood, and to detect viral infections in patients.
  • An object of the present invention is to provide a recombinant protein for developing an effective vaccine and diagnostic tool for HCV.
  • HBV core protein HBc
  • 13 to 15 amino acids including the amino acid sequence of SEQ ID NOs: 129 to 141 are used instead of the vicinity of the amino acid sequence 80 of HBc.
  • the HCV E2 epitope consisting of the same number of amino acids was inserted into the region.
  • the HBc amino acid sequence near 129 to 141 is highly antigenic. By removing the region, it is possible to suppress the antigenicity of HBc and produce a recombinant protein having a high antigenicity in the HCV E2 epitope.
  • the present invention is characterized in that the HCV E2 epitope is any one region of amino acid sequence numbers 409 to 423 or sequence numbers 523 to 537 of the E2 protein.
  • HCV HCV-binding protein
  • This region of HCV is highly conserved among envelope proteins and is conserved regardless of the HCV genotype.
  • antibodies were produced in experiments using rabbits. Therefore, it is a suitable sequence for use in vaccines and diagnosis because gene mutation does not occur much.
  • the present invention is a vaccine for preventing HCV, wherein the recombinant protein is used as an antigen.
  • the antigen of the present invention produces an antibody that reacts with the envelope protein of HCV, it is considered to function as an effective vaccine.
  • the present invention is an antibody that recognizes an envelope protein of HCV, wherein the recombinant protein is obtained as an antigen.
  • An antibody that recognizes the conserved amino acid region of the HCV envelope can be obtained by obtaining an antibody using the antigen of the present invention. Therefore, antibodies useful for diagnosis and treatment can be obtained.
  • the figure which shows the produced recombinant protein typically.
  • the present invention is to produce a recombinant using HBc as a platform.
  • the site where the peptide was inserted was the region of amino acid residues 75 to 80 of HBc, but in the present invention, it was inserted into the region of amino acids 129 to 141. It is known that the 129th to 141st amino acid regions of HBc are highly antigenic.
  • the inserted peptide is presented on the surface of the HBc molecule and recognized as an epitope.
  • epitopes of E2 of HCV three conserved sites were selected and inserted regardless of genotype (FIG. 1).
  • 129th to 141st amino acids of HBc straddle both major epitopes 134th to 140th and 138th to 154th amino acid regions. Therefore, when the recombinant protein of the present invention is used, it is considered that an antibody against HBc is difficult to be produced. Therefore, when an HCV peptide is inserted into this region, it is expected that antibodies against the HBc region will not be produced, but antibodies that recognize the HCV envelope will be produced.
  • Example 1 Construction of recombinant protein expression plasmid A plasmid pGHBc expressing the HBc antigen was prepared. pGHBc was obtained by inserting HBc into the NcoI / HindIII site of pGd1.
  • the E2 epitope is highly conserved in all genotypes of HCV, and is a region reactive with human neutralizing antibodies. The area including the location was selected.
  • Amino acid sequence number 409-423 QRI QLINTNG SWHIN (2) Amino acid sequence numbers 434-446 QTGFLAALFYAHR (3) Amino acid sequence number 523-537 LGNPTYSWGENENDTDV
  • the amino acid sequence number indicates the number when the N-terminus of the polyprotein HCV core protein is represented as 1.
  • the sequence is GenBank Accession No. It is based on AF1395.94.2.
  • HCV 433-447 amino acids were inserted in place of HBc 128-142 amino acids, and protein purification was attempted. However, although RNA expression was confirmed but protein expression was not observed, amino acids 128 and 142 of HBc were left as they were, and HCV E2 peptide was used by recombining 13 amino acids from 434 to 446 ( (See FIG. 1).
  • chimeric proteins incorporating the 409-423 amino acid region of HCV E2 or the 523-537 amino acid region instead of the 15th to 142nd amino acids of HBc were replaced with HBc-E2 (aa409-423) and HBc, respectively.
  • -E2 aa 523-537
  • a chimeric protein in which the 129th to 141st amino acids of HBc are removed and the 434-446 amino acid region of HCV E2 is incorporated instead is referred to as HBc-E2 (aa 434-446).
  • E. coli JM109 (DE3) is transformed with the prepared plasmid to express the HBc-E2 chimeric protein.
  • the chimeric protein was obtained by purifying the inclusion body fraction of E. coli.
  • E. coli was cultured in LB medium for 24 hours, centrifuged, suspended in TE (50 mM TrisHCL (pH 7.5), 10 mM EDTA), and disrupted in ice with an ultrasonic disrupter for 30 minutes. did. PMSF (phenylmethylsulfonyl fluoride) is added to a final concentration of 1 mM, and the supernatant is discarded by centrifugation (15,000 rpm, 4 ° C, 15 minutes) to collect the precipitate. The resulting precipitate is suspended in TE and treated in ice for 4 minutes with an ultrasonic crusher. Washing by centrifugation is repeated twice to obtain an inclusion body fraction.
  • PMSF phenylmethylsulfonyl fluoride
  • the obtained inclusion body fraction is suspended in an 8M urea solution (8M urea, 20 mM Tris-HCL (pH 8.5), 100 mM DTT) and left to stand at 4 ° C. overnight.
  • Centrifugation (15,000 rpm, 4 ° C, 15 minutes) is performed, and after collecting the supernatant, the protein is separated and purified by S-300 gel filtration column equilibrated with 8 M urea solution after filtration through a 0.8 ⁇ m pore size filter. .
  • SDS and DTT are added to a final concentration of 1% and 100 mM, respectively, and boiled for 5 minutes. After boiling, leave it to return to room temperature and filter through a filter with a pore size of 0.8 ⁇ m.
  • the filtered protein is separated and purified by an S-300 gel filtration column equilibrated in advance with a solution of 1% SDS in 8M urea solution.
  • the obtained protein fraction was dialyzed by reducing urea stepwise, and finally a chimeric protein solution dissolved in TE was obtained.
  • the product was sterilized by filtration with a 0.2 ⁇ m pore size filter to obtain a final purified product.
  • FIG. 2A shows the purified recombinant protein separated by SDS-PAGE and stained with Coomassie brilliant blue.
  • the display of each lane in the figure represents the following.
  • Markers are protein size markers
  • HBc is purified HBc protein
  • 409, 434, and 523 are HCV E2 chimeric proteins HBc-E2 (aa409-423), HBc-E2 (aa434-446), HBc-E2 (aa523- 537).
  • the arrow indicates the molecular weight of the chimeric protein deduced from the sequence.
  • the presence of the chimeric protein is confirmed as a main band in each lane where the recombinant protein was migrated.
  • 409, 434, and 523 indicate HBc-E2 (aa409-423), HBc-E2 (aa434-446), and HBc-E2 (aa523-537), which are HCV E2 chimeric proteins. All the chimeric proteins have reactivity with anti-HBc antibodies. Therefore, it is shown that a chimeric protein in which the HCV E2 protein is incorporated into the HBc platform is expressed.
  • Administration week was 0, 2, 4, and 6 weeks, initial immunization was 0.6 mg / animal, and subsequent doses were 0.3 mg / animal.
  • the antibody titer was measured by ELISA using a synthetic peptide conjugated to BSA as an antigen.
  • HBc-E2 The reason why antigenicity was not observed in HBc-E2 (aa434-446) is that the 434-446 amino acid synthetic peptide of HCV E2 is insoluble, and even when it is a chimeric protein with HBc, the HCV peptide It is possible that the region did not appear on the surface of the chimeric protein and did not have antigenicity.
  • the 434-446 amino acid region of HCV E2 is a conformational epitope, and the three-dimensional structure may not be well formed in the environment of the chimeric protein, or the formation of HBc particles by the 434-446 amino acid region of HCV E2. There is a possibility that was prevented.
  • the present invention is the first to show that a chimeric protein recombining the amino acid sequences of 409-423 and 523-537 of HCV E2 with the major epitope part of HBc protein has immunogenicity. If HBc particles presenting the HCV E2 peptide can be prepared by examining the conditions, an antigen having higher immunogenicity can be obtained.
  • amino acid sequences 409-423 and 523-537 which can act as neutralizing antibodies as HCV E2 peptide, show high immunogenicity as a chimeric protein with HBc.
  • HCV neutralizing antibody can be produced by immunizing humans with this antigen, it can be expected to be used as a vaccine.
  • an antibody that reacts with all HCV can be obtained, so that a highly versatile diagnostic tool can be obtained.

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Abstract

　The present invention addresses the problem of providing an effective vaccine against the hepatitis C virus (HCV), and providing a recombinant protein for developing a diagnostic tool.　A recombinant protein having high immunogenicity was successfully obtained by preparing a recombinant protein in which an HCV E2 epitope is inserted into a major epitope region of a hepatitis B virus core protein.

Description

Ｃ型肝炎ウイルスのワクチン及び診断に用いるためのＨＢｃ／ＨＣＶ　Ｅ２ペプチドキメラタンパク質HBc / HCV E2 peptide chimeric protein for use in vaccines and diagnosis of hepatitis C virus
　本発明は、様々な遺伝子型のＣ型肝炎ウイルスによる感染に対応する予防的／及び治療的免疫応答、診断に使用可能な組換えタンパク質に関する。 The present invention relates to a recombinant protein that can be used for prophylactic / therapeutic immune response and diagnosis corresponding to infection by various genotypes of hepatitis C virus.
　Ｃ型肝炎ウイルス（ＨＣＶ）キャリアは全世界で１億７０００万人、すなわち全人口の３％がキャリアであると言われており、わが国のキャリア数は１５０万～２００万人と推定されている。ＨＣＶに感染すると、免疫力の正常な健康成人への感染であっても、急性の経過で治癒するものは約３０％であり、残りは慢性感染へ移行すると言われている。ＨＣＶによる慢性肝炎では炎症が持続することにより肝線維化が誘発され、肝硬変、肝癌へと進展する。 Hepatitis C virus (HCV) carriers are said to be 170 million worldwide, that is, 3% of the total population is said to be carriers, and the number of carriers in Japan is estimated to be 1.5 to 2 million . It is said that when infected with HCV, about 30% of healthy adults with normal immunity heal in the acute course, and the rest are transferred to chronic infection. In chronic hepatitis due to HCV, fibrosis is induced by persisting inflammation and progresses to cirrhosis and liver cancer.
　慢性Ｃ型肝炎は感染から１０年～２０年を経て肝硬変へと進展して、やがて高率に原発性肝癌を発症する。わが国では毎年２万数千人がＨＣＶによる原発性肝癌によって死亡しており、これは原発性肝癌の約８０％に該当する。 Chronic hepatitis C progresses to cirrhosis 10 to 20 years after infection and eventually develops primary liver cancer at a high rate. Every year in Japan, 20,000 people die from primary liver cancer caused by HCV, which accounts for about 80% of primary liver cancer.
　また、わが国では慢性肝炎、肝硬変、肝癌患者の７５％がＨＣＶ感染者であるとも言われており、ＨＣＶの診断、治療、予防は重要な問題となっている。 In Japan, it is also said that 75% of patients with chronic hepatitis, cirrhosis and liver cancer are HCV infected, and diagnosis, treatment and prevention of HCV are important problems.
　ＨＣＶはフラビウイルス科（Flaviviridae）に属するＲＮＡウイルスであり、カプシド、Ｅ１、Ｅ２の２つのエンベロープタンパク質、ｐ７の４つの構造タンパク質、及び６つの非構造タンパク質からなっている。 HCV is an RNA virus belonging to the Flaviviridae family, and consists of capsid, two envelope proteins E1, E2, four structural proteins p7, and six nonstructural proteins.
　ＨＣＶはＲＮＡウイルスであり変異しやすく、特にエンベロープタンパク質のアミノ末端には超可変領域（Hypervariable Region : HVR）と呼ばれる領域が存在する。この領域はアミノ酸変異が多く、中和抗体からのエスケープ機序に関与していると考えられている。ＨＣＶが変異しやすいＲＮＡウイルスであることが、慢性化の要因でもあり、また、有効なワクチン開発の大きな障害となっている。 HCV is an RNA virus and is easily mutated. In particular, a region called a hypervariable region (HVR) exists at the amino terminus of the envelope protein. This region has many amino acid mutations and is thought to be involved in the escape mechanism from the neutralizing antibody. The fact that HCV is an RNA virus that is easily mutated is a cause of chronicity and is a major obstacle to the development of an effective vaccine.
　また、ＨＣＶには１ａ、１ｂ、２ａ、２ｂなど１０種類以上のジェノタイプが存在することが知られている。ジェノタイプにより、ウイルスの感染性、インターフェロン等の治療への感受性が異なり、治療効果が異なることが知られている。中和抗体に関しても、ジェノタイプ間で異なるアミノ酸配列の領域を認識する中和抗体は他のジェノタイプのウイルスの重感染を防ぐことができない。多くのジェノタイプが存在することもワクチン開発の障害となっている。 HCV is known to have more than 10 types of genotypes such as 1a, 1b, 2a, 2b. It is known that depending on the genotype, the infectivity of the virus, the sensitivity to treatment such as interferon, etc. are different, and the therapeutic effect is different. Regarding neutralizing antibodies, neutralizing antibodies that recognize regions of amino acid sequences that differ between genotypes cannot prevent co-infection with other genotype viruses. The existence of many genotypes is also an obstacle to vaccine development.
　ＨＣＶは、感染初期では検出が難しく、診断が困難な時期があることや、上述のようにウイルスの遺伝子型によって治療効果が異なることから、いかに精度、感度の高い診断を行うかが問題となっている。また、予防の面では、エスケープ機序や多くのジェノタイプが存在するために有効なワクチンはまだ開発されておらず、積極的な予防法がないことが大きな問題となっている。 HCV is difficult to detect in the early stages of infection and there are times when it is difficult to diagnose, and the therapeutic effect varies depending on the virus genotype as described above. ing. In terms of prevention, effective vaccines have not yet been developed due to the existence of escape mechanisms and many genotypes, and there is no major preventive method.
　一方、ＨＣＶと並んでヒトに肝炎、肝癌を誘発するウイルスとして、Ｂ型肝炎ウイルス（ＨＢＶ）がある。ＨＢＶもＨＣＶ同様、慢性肝炎、肝硬変、肝癌を誘発するウイルスであることが明らかにされている。ＨＢＶはヘパドナウイルス科のＤＮＡウイルスであり、その複製機構や免疫応答はＨＣＶとは全く異なるものである。 On the other hand, hepatitis B virus (HBV) is a virus that induces hepatitis and liver cancer in humans along with HCV. HBV, like HCV, has been shown to be a virus that induces chronic hepatitis, cirrhosis, and liver cancer. HBV is a DNA virus of the Hepadnaviridae family, and its replication mechanism and immune response are completely different from HCV.
　ＨＢＶコアタンパク質（ＨＢｃ）は、自己集合して球状のコア粒子を形成する。このコア粒子は非常に免疫原性が高いことが知られている。このＨＢｃタンパク質のアミノ酸残基７５～８０付近に所望のエピトープを挿入するか、ＨＢｃ抗原タンパク質の末端に所望のエピトープを連結することにより得られる融合ポリペプチドは自己集合によりコア粒子を形成する。形成された粒子の表面には、挿入、あるいは連結したエピトープが提示される。この組換え抗原を用いると、エピトープが免疫系に認識されやすくなり、抗体産生を効率的に誘導することができる（非特許文献１）。 HBV core protein (HBc) self-assembles to form spherical core particles. This core particle is known to be very immunogenic. The fusion polypeptide obtained by inserting a desired epitope in the vicinity of amino acid residues 75 to 80 of the HBc protein or linking the desired epitope to the end of the HBc antigen protein forms a core particle by self-assembly. An inserted or linked epitope is presented on the surface of the formed particle. When this recombinant antigen is used, epitopes are easily recognized by the immune system, and antibody production can be efficiently induced (Non-patent Document 1).
　そこでＨＢｃ抗原タンパク質をワクチンのプラットフォームとして利用し、免疫系に認識されにくい抗原に対する抗体産生を誘導する試みが今までになされている（特許文献１～３）。 Therefore, attempts have been made so far to induce antibody production against an antigen that is difficult to be recognized by the immune system by using the HBc antigen protein as a vaccine platform (Patent Documents 1 to 3).
特許第３２２８７３７号公報Japanese Patent No. 3228737 特開平６－２７９５００号公報JP-A-6-279500 国際公開第２０１２／１４１２８０号International Publication No. 2012/141280
　ＨＣＶワクチンを開発する試みは、ＨＣＶウイルスが発見された１９８９年以来続いているもののいまだ有効なワクチンは開発されていない。したがって、ＨＣＶの積極的な予防法は現在のところないため、ワクチンの開発が望まれている。 Although attempts to develop HCV vaccines have continued since 1989 when the HCV virus was discovered, no effective vaccines have yet been developed. Therefore, there is no active preventive method for HCV at present, and therefore the development of a vaccine is desired.
　また、変異やジェノタイプによらず、あらゆるＨＣＶに対応して感度良く検出可能な抗原タンパク質の開発も望まれている。有効な抗原タンパク質があれば、ヒトにＨＣＶウイルスが感染し、抗体が産生していれば患者血清中の抗体を検出することも可能である。また、ウサギやヤギ等の動物に抗原タンパク質を免疫して抗体を産生させ、患者血液中のウイルス抗原を検出する抗体試薬を作成し、患者のウイルス感染を検出することも可能となる。 Also, it is desired to develop an antigen protein that can be detected with high sensitivity corresponding to any HCV regardless of mutation or genotype. If there is an effective antigen protein, it is possible to detect an antibody in patient serum if an HCV virus is infected in a human and an antibody is produced. It is also possible to immunize animals such as rabbits and goats to produce antibodies by immunizing antigen proteins and to produce antibody reagents that detect viral antigens in patient blood, and to detect viral infections in patients.
　本発明は、ＨＣＶの有効なワクチンや診断ツールを開発するための組換えタンパク質を提供することを課題とする。 An object of the present invention is to provide a recombinant protein for developing an effective vaccine and diagnostic tool for HCV.
　本発明の組換えタンパク質は、Ｂ型肝炎ウイルスのコアタンパク質のアミノ酸配列番号１２９～１４１のアミノ酸配列を含む１３～１５アミノ酸を除去し、代わりにＨＣＶ　Ｅ２エピトープを含む同数のアミノ酸配列を組み込んだことを特徴とする。 In the recombinant protein of the present invention, 13 to 15 amino acids including the amino acid sequence of amino acid sequence numbers 129 to 141 of the hepatitis B virus core protein are removed, and the same number of amino acid sequences including the HCV E2 epitope are incorporated instead. It is characterized by.
　本発明者らも、非特許文献１の方法と同様に、ＨＢＶのコアタンパク質（ＨＢｃ）をプラットフォームとして用いている。しかしながら、これまでのＨＢｃタンパク質をワクチン等、抗体産生を誘導する際のプラットフォームとして用いる場合とは異なり、ＨＢｃのアミノ酸配列８０付近ではなく、配列番号１２９～１４１のアミノ酸配列を含む１３～１５アミノ酸を除去し当該領域に、同数のアミノ酸からなるＨＣＶのＥ２エピトープを挿入した。ＨＢｃのアミノ酸配列１２９～１４１付近は、抗原性が高い。当該領域を除去することにより、ＨＢｃの抗原性を抑えて、ＨＣＶ　Ｅ２エピトープに高い抗原性を備えた組換えタンパク質を産生することができる。 The present inventors have also used HBV core protein (HBc) as a platform, as in the method of Non-Patent Document 1. However, unlike the case where the conventional HBc protein is used as a platform for inducing antibody production, such as a vaccine, 13 to 15 amino acids including the amino acid sequence of SEQ ID NOs: 129 to 141 are used instead of the vicinity of the amino acid sequence 80 of HBc. The HCV E2 epitope consisting of the same number of amino acids was inserted into the region. The HBc amino acid sequence near 129 to 141 is highly antigenic. By removing the region, it is possible to suppress the antigenicity of HBc and produce a recombinant protein having a high antigenicity in the HCV E2 epitope.
　さらに、本発明はＨＣＶ　Ｅ２エピトープがＥ２タンパク質のアミノ酸配列番号４０９～４２３又は配列番号５２３～５３７のいずれか一つの領域であることを特徴とする。 Furthermore, the present invention is characterized in that the HCV E2 epitope is any one region of amino acid sequence numbers 409 to 423 or sequence numbers 523 to 537 of the E2 protein.
　ＨＣＶのこの領域は、エンベロープタンパク質の中でも、保存性が高く、ＨＣＶの遺伝子型によらず保存されている。また、ウサギを使った実験で抗体が産生することを確認した。したがって、遺伝子変異があまり起こらないことからワクチンや診断に用いるのには適した配列である。 This region of HCV is highly conserved among envelope proteins and is conserved regardless of the HCV genotype. In addition, it was confirmed that antibodies were produced in experiments using rabbits. Therefore, it is a suitable sequence for use in vaccines and diagnosis because gene mutation does not occur much.
　本発明は、ＨＣＶを予防するためのワクチンであって、前記組換えタンパク質を抗原とすることを特徴とする。 The present invention is a vaccine for preventing HCV, wherein the recombinant protein is used as an antigen.
　本発明の抗原はＨＣＶのエンベロープタンパク質と反応する抗体を産生することが確認されたことから、有効なワクチンとして機能すると考えられる。 Since it was confirmed that the antigen of the present invention produces an antibody that reacts with the envelope protein of HCV, it is considered to function as an effective vaccine.
　本発明は、ＨＣＶのエンベロープタンパク質を認識する抗体であって、前記組換えタンパク質を抗原として得ることを特徴とする。 The present invention is an antibody that recognizes an envelope protein of HCV, wherein the recombinant protein is obtained as an antigen.
　本発明の抗原を用いて抗体を得ることによって、ＨＣＶエンベロープの保存されたアミノ酸領域を認識する抗体を得ることができる。そのため、診断、治療に有用な抗体を得ることができる。 An antibody that recognizes the conserved amino acid region of the HCV envelope can be obtained by obtaining an antibody using the antigen of the present invention. Therefore, antibodies useful for diagnosis and treatment can be obtained.
作製した組換えタンパク質を模式的に示す図。The figure which shows the produced recombinant protein typically. キメラタンパク質のＳＤＳ－ＰＡＧＥ，ウェスタンブロット解析を示す図。The figure which shows SDS-PAGE of a chimeric protein and a Western blot analysis. ＥＬＩＳＡによりキメラタンパク質のＨＣＶ　Ｅ２ペプチドに対する免疫原性を確認した図。The figure which confirmed the immunogenicity with respect to HCV E2 peptide of a chimeric protein by ELISA.
　本発明は、ＨＢｃをプラットフォームとして用い組換え体を作製するものである。従来ペプチドを挿入していた部位は、ＨＢｃのアミノ酸残基７５～８０の領域であったが、本発明では１２９～１４１番目のアミノ酸の領域に挿入した。ＨＢｃの１２９～１４１番目のアミノ酸領域は抗原性が高いことが知られている。挿入するペプチドの物理的性質にもよるが、当該領域を除去し代わりに所望のペプチドを挿入することにより、挿入したペプチドがＨＢｃ分子表面に提示され、エピトープとして認識されることが期待される。ＨＣＶのＥ２のエピトープとしては、遺伝子型によらず保存されている３箇所を選び、挿入した（図１）。 The present invention is to produce a recombinant using HBc as a platform. Conventionally, the site where the peptide was inserted was the region of amino acid residues 75 to 80 of HBc, but in the present invention, it was inserted into the region of amino acids 129 to 141. It is known that the 129th to 141st amino acid regions of HBc are highly antigenic. Depending on the physical properties of the peptide to be inserted, it is expected that by inserting the desired peptide instead of removing the region, the inserted peptide is presented on the surface of the HBc molecule and recognized as an epitope. As epitopes of E2 of HCV, three conserved sites were selected and inserted regardless of genotype (FIG. 1).
　ＨＢｃの１２９～１４１番目のアミノ酸は、メジャーエピトープである１３４～１４０、１３８～１５４番目のアミノ酸領域の両者に跨っている。そのため、本発明の組換えタンパク質を用いた場合には、ＨＢｃに対する抗体ができにくいものと考えられる。したがって、この領域にＨＣＶペプチドを挿入した場合には、ＨＢｃの当該領域に対する抗体が産生されず、ＨＣＶのエンベロープを認識する抗体が産生されることが期待される。 129th to 141st amino acids of HBc straddle both major epitopes 134th to 140th and 138th to 154th amino acid regions. Therefore, when the recombinant protein of the present invention is used, it is considered that an antibody against HBc is difficult to be produced. Therefore, when an HCV peptide is inserted into this region, it is expected that antibodies against the HBc region will not be produced, but antibodies that recognize the HCV envelope will be produced.
　［実施例］
１．組換えタンパク質発現プラスミドの構築
　ＨＢｃ抗原を発現するプラスミドｐＧＨＢｃを作製した。ｐＧｄ１のＮｃｏＩ/ＨｉｎｄＩＩＩ部位にＨＢｃを挿入したｐＧＨＢｃを得た。 [Example]
1. Construction of recombinant protein expression plasmid A plasmid pGHBc expressing the HBc antigen was prepared. pGHBc was obtained by inserting HBc into the NcoI / HindIII site of pGd1.
　次に、ＰＣＲを用い、定法によりＨＢｃの１２９～１４１、又は１２８～１４２番目までの１３又は１５アミノ酸を除去し、代わりにＨＣＶ　Ｅ２のエピトープ候補領域を挿入した。 Next, 13 or 15 amino acids from HBc 129 to 141 or 128 to 142 were removed by PCR using a conventional method, and an epitope candidate region of HCV E2 was inserted instead.
　Ｅ２エピトープは全てのジェノタイプのＨＣＶで保存性が高く、かつヒトの中和抗体と反応性のある領域として非特許文献２に報告されている下線で示した下記（１）～（３）の箇所を含む領域を選定した。
（１）アミノ酸配列番号４０９－４２３
　　ＱＲＩＱＬＩＮＴＮＧＳＷＨＩＮ
（２）アミノ酸配列番号４３４－４４６
　　ＱＴＧＦＬＡＡＬＦＹＡＨＲ
（３）アミノ酸配列番号５２３－５３７
　　ＬＧＮＰＴＹＳＷＧＥＮＤＴＤＶ
　なお、アミノ酸の配列番号は、ポリプロテインによるＨＣＶコアタンパク質のＮ末端を１として表したときの番号を示す。また、配列はＧｅｎＢａｎｋアクセッションＮｏ．ＡＦ１３９５９４．２をもとにしている。 The E2 epitope is highly conserved in all genotypes of HCV, and is a region reactive with human neutralizing antibodies. The area including the location was selected.
(1) Amino acid sequence number 409-423
QRI QLINTNG SWHIN
(2) Amino acid sequence numbers 434-446
QTGFLAALFYAHR
(3) Amino acid sequence number 523-537
LGNPTYSWGENENDTDV
The amino acid sequence number indicates the number when the N-terminus of the polyprotein HCV core protein is represented as 1. The sequence is GenBank Accession No. It is based on AF1395.94.2.
　当初ＨＣＶ　４３３～４４７番目のアミノ酸をＨＢｃの１２８～１４２番目のアミノ酸の代わりに挿入し、タンパク質精製を試みた。しかしながら、ＲＮＡ発現は確認されたもののタンパク質の発現が見られなかったため、ＨＢｃの１２８番目と１４２番目のアミノ酸をそのまま残し、ＨＣＶ　Ｅ２ペプチドは４３４～４４６番目までの１３アミノ酸を組換えて用いた（図１参照）。 Initially, HCV 433-447 amino acids were inserted in place of HBc 128-142 amino acids, and protein purification was attempted. However, although RNA expression was confirmed but protein expression was not observed, amino acids 128 and 142 of HBc were left as they were, and HCV E2 peptide was used by recombining 13 amino acids from 434 to 446 ( (See FIG. 1).
　以下、ＨＢｃの１２８～１４２番目までの１５アミノ酸を除去し、代わりにＨＣＶ　Ｅ２の４０９－４２３アミノ酸領域、あるいは５２３－５３７アミノ酸領域を組み込んだキメラタンパク質を夫々ＨＢｃ－Ｅ２（ａａ４０９－４２３）、ＨＢｃ－Ｅ２（ａａ５２３－５３７）、ＨＢｃの１２９～１４１番目までの１３アミノ酸を除去し、代わりにＨＣＶ　Ｅ２の４３４－４４６アミノ酸領域を組み込んだキメラタンパク質をＨＢｃ－Ｅ２（ａａ４３４－４４６）と称する。 Hereinafter, chimeric proteins incorporating the 409-423 amino acid region of HCV E2 or the 523-537 amino acid region instead of the 15th to 142nd amino acids of HBc were replaced with HBc-E2 (aa409-423) and HBc, respectively. -E2 (aa 523-537), a chimeric protein in which the 129th to 141st amino acids of HBc are removed and the 434-446 amino acid region of HCV E2 is incorporated instead is referred to as HBc-E2 (aa 434-446).
２．ＨＢｃ-Ｅ２キメラタンパク質の発現と精製
　作製したプラスミドで大腸菌ＪＭ１０９（ＤＥ３）を形質転換し、ＨＢｃ-Ｅ２キメラタンパク質を発現させる。キメラタンパク質は大腸菌の封入体画分を精製することによって得た。 2. Expression and purification of HBc-E2 chimeric protein E. coli JM109 (DE3) is transformed with the prepared plasmid to express the HBc-E2 chimeric protein. The chimeric protein was obtained by purifying the inclusion body fraction of E. coli.
　具体的には、ＬＢ培地中で２４時間大腸菌を培養し、遠心分離後、ＴＥ（５０ｍＭ　ＴｒｉｓＨＣＬ（ｐＨ　７．５），　１０　ｍＭ　ＥＤＴＡ）に懸濁し、氷中で超音波破砕機により３０分間破砕した。ＰＭＳＦ（phenylmethylsulfonyl fluoride）を終濃度１ｍＭになるように添加し、遠心分離（１５，０００ｒｐｍ, ４℃、１５分）により上清を捨て沈殿を回収する。得られた沈殿はＴＥで懸濁し、超音波破砕機により氷中で４分間処理する。遠心分離による洗浄を２度繰り返し、封入体画分を得る。 Specifically, E. coli was cultured in LB medium for 24 hours, centrifuged, suspended in TE (50 mM TrisHCL (pH 7.5), 10 mM EDTA), and disrupted in ice with an ultrasonic disrupter for 30 minutes. did. PMSF (phenylmethylsulfonyl fluoride) is added to a final concentration of 1 mM, and the supernatant is discarded by centrifugation (15,000 rpm, 4 ° C, 15 minutes) to collect the precipitate. The resulting precipitate is suspended in TE and treated in ice for 4 minutes with an ultrasonic crusher. Washing by centrifugation is repeated twice to obtain an inclusion body fraction.
　得られた封入体画分は、８Ｍ尿素溶液（８Ｍ　尿素、２０ｍＭ　Ｔｒｉｓ－ＨＣＬ（ｐＨ８．５）、１００ｍＭ　ＤＴＴ）に懸濁し、４℃で一晩静置する。 The obtained inclusion body fraction is suspended in an 8M urea solution (8M urea, 20 mM Tris-HCL (pH 8.5), 100 mM DTT) and left to stand at 4 ° C. overnight.
　遠心分離（１５，０００ｒｐｍ, ４℃、１５分）を行い、上清を回収後、孔径０．８μｍフィルターで濾過し、８Ｍ尿素溶液で平衡化したＳ－３００ゲル濾過カラムでタンパク質を分離精製する。 Centrifugation (15,000 rpm, 4 ° C, 15 minutes) is performed, and after collecting the supernatant, the protein is separated and purified by S-300 gel filtration column equilibrated with 8 M urea solution after filtration through a 0.8 μm pore size filter. .
　得られたタンパク質画分は限外濾過を用いて濃縮後、ＳＤＳ、ＤＴＴを夫々終濃度１％、１００ｍＭになるように加え、５分間煮沸する。煮沸後、室温に戻るまで静置し、孔径０．８μｍのフィルターで濾過する。 After concentrating the obtained protein fraction using ultrafiltration, SDS and DTT are added to a final concentration of 1% and 100 mM, respectively, and boiled for 5 minutes. After boiling, leave it to return to room temperature and filter through a filter with a pore size of 0.8 μm.
　濾過後のタンパク質は予め８Ｍ尿素溶液に１％ＳＤＳを加えた溶液で平衡化したＳ－３００ゲル濾過カラムで分離精製する。得られたタンパク質画分は尿素を段階的に減らして透析を行い、最終的にＴＥに溶解したキメラタンパク質溶液を得た。透析終了後孔径０．２μｍフィルターで濾過滅菌し、最終精製品とした。 The filtered protein is separated and purified by an S-300 gel filtration column equilibrated in advance with a solution of 1% SDS in 8M urea solution. The obtained protein fraction was dialyzed by reducing urea stepwise, and finally a chimeric protein solution dissolved in TE was obtained. After completion of dialysis, the product was sterilized by filtration with a 0.2 μm pore size filter to obtain a final purified product.
　図２Ａは精製した組換えタンパク質をＳＤＳ－ＰＡＧＥで分離し、クマシーブリリアントブルー染色したものである。図中の各レーンの表示は、以下のものを表す。マーカーはタンパク質のサイズマーカー、ＨＢｃは精製したＨＢｃタンパク質、４０９、４３４、５２３は夫々ＨＣＶ　Ｅ２キメラタンパク質のＨＢｃ－Ｅ２（ａａ４０９－４２３）、ＨＢｃ－Ｅ２（ａａ４３４－４４６）、ＨＢｃ－Ｅ２（ａａ５２３－５３７）を示す。また、矢印は、配列より推定されるキメラタンパク質の分子量を示す。 FIG. 2A shows the purified recombinant protein separated by SDS-PAGE and stained with Coomassie brilliant blue. The display of each lane in the figure represents the following. Markers are protein size markers, HBc is purified HBc protein, 409, 434, and 523 are HCV E2 chimeric proteins HBc-E2 (aa409-423), HBc-E2 (aa434-446), HBc-E2 (aa523- 537). The arrow indicates the molecular weight of the chimeric protein deduced from the sequence.
　図２Ａに示すように、組換えタンパク質を泳動した各レーンでは、主要なバンドとしてキメラタンパク質の存在が確認される。 As shown in FIG. 2A, the presence of the chimeric protein is confirmed as a main band in each lane where the recombinant protein was migrated.
　さらに、ウェスタンブロット法により、キメラタンパク質が発現していることを確認した。一次抗体としてＨＢｃの２～１０番目のアミノ酸配列を認識するマウスモノクローナル抗体、二次抗体として西洋ワサビペルオキシダーゼ（ＨＲＰ）標識された抗マウス抗体を用い、ジアミノベンジジン（ＤＡＢ）法により発色させた（図２Ｂ）。 Furthermore, it was confirmed by Western blotting that the chimeric protein was expressed. Using a mouse monoclonal antibody that recognizes the 2-10th amino acid sequence of HBc as the primary antibody and an anti-mouse antibody labeled with horseradish peroxidase (HRP) as the secondary antibody, color was developed by the diaminobenzidine (DAB) method (Fig. 2B).
　図２Ｂ中、４０９、４３４、５２３は、ＨＣＶ　Ｅ２キメラタンパク質であるＨＢｃ－Ｅ２（ａａ４０９－４２３）、ＨＢｃ－Ｅ２（ａａ４３４－４４６）、ＨＢｃ－Ｅ２（ａａ５２３－５３７）を示す。いずれのキメラタンパク質も抗ＨＢｃ抗体との反応性を有している。したがって、ＨＢｃのプラットフォームにＨＣＶ　Ｅ２タンパク質が組み込まれたキメラタンパク質が発現していることが示されている。 In FIG. 2B, 409, 434, and 523 indicate HBc-E2 (aa409-423), HBc-E2 (aa434-446), and HBc-E2 (aa523-537), which are HCV E2 chimeric proteins. All the chimeric proteins have reactivity with anti-HBc antibodies. Therefore, it is shown that a chimeric protein in which the HCV E2 protein is incorporated into the HBc platform is expressed.
３．免疫原性の確認
　上記で発現精製した３種の精製抗原をウサギ（各２匹）に免疫し、抗血清を採取した。抗原溶液は、アジュバントと等量混合し、エマルジョンの状態でウサギの背部皮下に投与した。なお、初回免疫にはフロイント完全アジュバントを用い、２回目以降の免疫にはフロイント不完全アジュバントを用いた。 3. Confirmation of immunogenicity Rabbits (2 each) were immunized with the three purified antigens expressed and purified above, and antiserum was collected. The antigen solution was mixed with an adjuvant in an equal amount and administered subcutaneously to the back of the rabbit in the form of an emulsion. In addition, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and subsequent immunizations.
　投与週は０、２、４、６週、初回免疫０．６ｍｇ／匹、２回目以降は０．３ｍｇ／匹で行った。抗体価は、ＢＳＡに結合させた合成ペプチドを抗原として用い、ＥＬＩＳＡにより測定した。 Administration week was 0, 2, 4, and 6 weeks, initial immunization was 0.6 mg / animal, and subsequent doses were 0.3 mg / animal. The antibody titer was measured by ELISA using a synthetic peptide conjugated to BSA as an antigen.
　ＨＢｃ－Ｅ２（ａａ４３４－４４６）を免疫したウサギでは、抗原に対する反応は見られなかったが、ＨＢｃ－Ｅ２（ａａ４０９－４２３）、ＨＢｃ－Ｅ２（ａａ５２３－５３７）では、抗体が産生していることが確認された。ＥＬＩＳＡによる抗体産生確認の結果を図３に示す。図中、４０９は、キメラタンパク質ＨＢｃ－Ｅ２（ａａ４０９－４２３）を、５２３はＨＢｃ－Ｅ２（ａａ５２３－５３７）を免疫したウサギの抗体価を示す。 In rabbits immunized with HBc-E2 (aa434-446), no response to antigen was observed, but in HBc-E2 (aa409-423) and HBc-E2 (aa523-537), antibodies were produced. Was confirmed. The results of antibody production confirmation by ELISA are shown in FIG. In the figure, 409 indicates the antibody titer of the rabbit immunized with the chimeric protein HBc-E2 (aa409-423) and 523 the HBc-E2 (aa523-537).
　なお、ＨＢｃ－Ｅ２（ａａ４３４－４４６）に抗原性が見られなかった理由としては、ＨＣＶ　Ｅ２の４３４－４４６アミノ酸の合成ペプチドが不溶性であり、ＨＢｃとのキメラタンパク質とした場合にも、ＨＣＶペプチド領域がキメラタンパク質表面に表出せず、抗原性を持たなかった可能性が考えられる。 The reason why antigenicity was not observed in HBc-E2 (aa434-446) is that the 434-446 amino acid synthetic peptide of HCV E2 is insoluble, and even when it is a chimeric protein with HBc, the HCV peptide It is possible that the region did not appear on the surface of the chimeric protein and did not have antigenicity.
　あるいは、ＨＣＶ　Ｅ２の４３４－４４６アミノ酸領域が、コンフォメーショナルエピトープであり、キメラタンパク質の環境では立体構造がうまく形成さなかった可能性や、ＨＣＶ　Ｅ２の４３４－４４６アミノ酸領域によって、ＨＢｃの粒子形成が妨げられた可能性が挙げられる。 Alternatively, the 434-446 amino acid region of HCV E2 is a conformational epitope, and the three-dimensional structure may not be well formed in the environment of the chimeric protein, or the formation of HBc particles by the 434-446 amino acid region of HCV E2. There is a possibility that was prevented.
　本発明は、ＨＣＶ　Ｅ２の４０９－４２３番目のアミノ酸、及び５２３－５３７番目のアミノ酸配列をＨＢｃタンパク質のメジャーエピトープの部分と組み換えたキメラタンパク質が免疫原性を有することを初めて示したものである。条件を検討し、ＨＣＶ　Ｅ２ペプチドを提示したＨＢｃ粒子を作成することができれば、さらに免疫原性の高い抗原とすることができる。 The present invention is the first to show that a chimeric protein recombining the amino acid sequences of 409-423 and 523-537 of HCV E2 with the major epitope part of HBc protein has immunogenicity. If HBc particles presenting the HCV E2 peptide can be prepared by examining the conditions, an antigen having higher immunogenicity can be obtained.
　以上、示したように、ＨＣＶ　Ｅ２ペプチドとして、中和抗体として作用し得るアミノ酸配列４０９－４２３、５２３－５３７番目は、ＨＢｃとのキメラタンパク質として高い免疫原性を示す。 As described above, amino acid sequences 409-423 and 523-537, which can act as neutralizing antibodies as HCV E2 peptide, show high immunogenicity as a chimeric protein with HBc.
　したがって、この抗原を用いてヒトに免疫することにより、ＨＣＶの中和抗体を産生し得ることから、ワクチンとしての利用が期待できる。また、本発明の組換え抗原を用いれば、すべてのＨＣＶに対して反応する抗体を得ることができることから、汎用性の高い診断ツールを得ることが可能となる。 Therefore, since HCV neutralizing antibody can be produced by immunizing humans with this antigen, it can be expected to be used as a vaccine. In addition, when the recombinant antigen of the present invention is used, an antibody that reacts with all HCV can be obtained, so that a highly versatile diagnostic tool can be obtained.

Claims (4)

  1. 　Ｂ型肝炎ウイルスのコアタンパク質のアミノ酸配列番号１２９～１４１のアミノ酸配列を含む１３～１５アミノ酸を除去し、代わりにＨＣＶ　Ｅ２エピトープを含む同数のアミノ酸配列を組み込んだことを特徴とする組換えタンパク質。 A recombinant protein characterized by removing 13 to 15 amino acids including the amino acid sequence of amino acid sequence numbers 129 to 141 of the hepatitis B virus core protein, and incorporating the same number of amino acid sequences including the HCV E2 epitope instead.
  2. 　請求項１に記載の組換えタンパク質において、
    　前記ＨＣＶ　Ｅ２エピトープがＥ２タンパク質のアミノ酸配列番号４０９～４２３又は配列番号５２３～５３７のいずれか一つの領域であることを特徴とする組換えタンパク質。     The recombinant protein according to claim 1, wherein
    A recombinant protein, wherein the HCV E2 epitope is any one region of amino acid sequence numbers 409 to 423 or sequence numbers 523 to 537 of the E2 protein.
  3. 　ＨＣＶを予防するためのワクチンであって、
    　請求項１又は２記載の組換えタンパク質を抗原とすることを特徴とするワクチン。     A vaccine for preventing HCV,
    A vaccine comprising the recombinant protein according to claim 1 or 2 as an antigen.
  4. 　ＨＣＶのエンベロープタンパク質を認識する抗体であって、
    　請求項１又は２記載の組換えタンパク質を抗原として得ることを特徴とする抗体。     An antibody that recognizes the envelope protein of HCV,
    An antibody characterized in that the recombinant protein according to claim 1 or 2 is obtained as an antigen.
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