HK1159650B - Antibody binding to envelope protein 2 of hepatitis c virus and method for identifying genotype of hepatitis c virus using the same - Google Patents

Description

Antibody binding to envelope protein 2 of hepatitis c virus and method for genotyping hepatitis c virus using same

Technical Field

The present invention relates to an antibody that binds to envelope protein 2 of hepatitis c virus, and a method for genotyping hepatitis c virus using the same.

Background

Hepatitis C virus (which may also be referred to hereinafter as "HCV") is a virus of major etiology, non-A and non-B hepatitis, which is infected mainly by blood transfusion and sexual contact (Choo et al, Science, Vol. 244: 359-362, 1989). It is estimated that there are 2,000,000 or more HCV carriers in japan, including those (virus carriers) that do not show hepatitis symptoms, and 170,000,000 or more HCV carriers in the world. The main reason for the increasing number of HCV carriers is the fact that the chronic rate of hepatitis due to HCV infection is as high as 70% to 80%, and the fact that there is no effective antiviral agent other than interferon.

The pathological conditions exhibited by half or more chronic hepatitis c patients will almost certainly go on every day and are known to progress to cirrhosis or liver cancer. Thus, it can be said that hepatitis c is a serious infectious disease with a poor prognosis. Therefore, research involving the treatment of hepatitis c and the detection of HCV is medically important, and the development of new therapies and therapeutic drugs is desired.

HCV is a single-stranded (+) RNA virus, approximately 9.6 kb in genome length, where the genome encodes a precursor protein that is converted to 10 viral proteins (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins) by post-translational cleavage by a host-derived signal peptidase or HCV-derived protease. HCV is classified into 10 or more genotypes (e.g., 1a, 1b, 2a, 2b, 3a and 3 b) based on phylogenetic analysis of the nucleotide sequence of the genome (Choo et al, Science, 1989, Vol.244, p.359-362; Simmonds et al, Hepatology, 1994, Vol.10, p.1321-1324; Okamoto et al, J.Gen. Virol., 1992, Vol.73, p.73-679; and Mori et al, biochem. Biophys. Res. Commun. 1992, Vol.183, p.334-342).

Recently, it has become known that the effects of interferons differ significantly depending on HCV genotype. It has been shown that the antiviral action of interferons is difficult to exert on HCV genotypes 1a or 1b (Fried et al., N. Engl. J. Med., 2002, Vol. 347, p. 975-.

Furthermore, it has become known that the antiviral action of interferon exerts differently on HCV of genotype 2a and HCV of genotype 2b, which have relatively good effects on interferon. It has been proposed that interferons exert their antiviral effects more significantly on HCV of genotype 2a than on HCV of genotype 2b (Murakami et al, Hepatology, 1999, vol. 30, p. 1045-1053).

An HCV antibody test by which anti-HCV antibodies in serum are detected using a C100-3 antigen is known as a HCV diagnostic method, because anti-HCV antibodies recognizing the NS4 region (C100-3 antigen) as a non-structural region of HCV are present at a rate of 70% to 80% in the serum of a hepatitis C patient (Choo et al, Science, 1989, Vol. 244, p. 359-362). Also, as a variation of this method, a second generation antibody assay system in which the detection sensitivity is improved using a combination of the C100-3 antigen, the core antigen and the antigen derived from the NS3 region, and a third generation antibody assay system containing the antigen derived from the NS5 region in addition to the above antigens have been developed. HCV antibody tests using these assay systems have been used (Aucella et al, Blood purify, 2000, Vol. 18, p. 110-.

Also, in addition to the aforementioned HCV antibody test, an HCV core antigen test (Fabrizi et al, j. clin. microbiol, 2005, vol. 43, p. 414-.

However, the HCV antibody test is problematic in that when a subject has experienced HCV infection in the past, the subject will inevitably become positive for the hepatitis c test, even after complete recovery. HCV antibody testing is also problematic because anti-HCV antibodies in the blood are only detected 1 to 3 months after infection. If the test is performed before this time, HCV cannot be detected and the subject will be negative for the hepatitis C test.

Also, the HCV core antigen test requires treatment by disrupting the envelope with SDS to allow the release of the core protein, because the core protein (target molecule) is present within the HCV particle. Depending on the processing time of SDS, the core protein may be denatured, or a substance inhibiting the antigen-antibody reaction may be released, thereby affecting the detection sensitivity.

Furthermore, even when the subject is positive for the HCV test in the HCV antibody test and the HCV core antigen test, it is currently not possible to identify the HCV genotype. In order to perform interferon therapy, further tests, for example, nucleic acid amplification tests, must be performed to identify the HCV genotype. This is because the antiviral effects of interferons differ significantly depending on HCV genotype. In particular, HCV genotype 1a and HCV genotype 1b do not produce effective antiviral effects, and the patient suffers from the side effects of interferon instead.

Meanwhile, the nucleic acid amplification test is problematic in that it uses serum RNA of a subject as a target molecule with respect to insufficient preservation quality and stability of a test sample. Nucleic acid amplification tests also present various problems and are essential for the use of RT-PCR methods with preventive measures. For example, PCR may be performed after transcription of RNA as a target molecule into DNA, resulting in false negative results due to RNA degradation or inactivation and/or inhibition of reverse transcriptase, or false positive results due to cross-contamination of the reaction system. Therefore, the nucleic acid amplification test is considered to be inferior to the HCV antibody test or the HCV core antigen test using a protein as a target molecule in terms of accuracy.

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide antibodies that bind to the envelope on the HCV surface and can be used for identifying HCV of genotype 1a, HCV of genotype 1b and HCV of genotype 2a, and to provide a method for identifying HCV genotype using such antibodies.

Means for solving the problems

The present inventors have conducted intensive studies to achieve the above object. They obtained hybridomas that produce monoclonal antibodies that specifically bind to only envelope protein 2 of HCV genotype 2a, antibodies that bind to only envelope protein 2 of HCV genotype 2a and envelope protein 2 of HCV genotype 1b, and antibodies that bind to envelope protein 2 of HCV genotype 2a, envelope protein 2 of HCV genotype 1b, and envelope protein 2 of HCV genotype 1a, from these hybridomas, and they completed the present invention.

In particular, the invention provides antibodies that specifically bind envelope protein 2 of HCV of genotype 2a, but do not immunoreact with envelope protein 2 of HCV of genotype 1 a.

The above antibody is preferably a peptide represented by SEQ ID NO: 1 as an epitope. An example of such an antibody is an antibody produced by the hybridoma cell line having accession number FERM BP-11181.

Preferably, the antibody specifically binds to envelope protein 2 of HCV of genotype 2a, but does not immunoreact with envelope protein 2 of HCV of genotype 1a and envelope protein 2 of HCV of genotype 1 b.

The above antibody is more preferably a peptide represented by SEQ ID NO: 2 or 3 as an epitope. Examples of such antibodies are antibodies produced by hybridoma cell lines having the accession numbers FERM BP-11180 or FERM BP-11179.

The antibody preferably binds specifically to the envelope protein 2 of the J6CF strain, but does not immunologically react with the envelope protein 2 of the JFH1 strain. Examples of such antibodies include the antibodies of SEQ ID NOs: 4, in particular an antibody produced by a hybridoma cell line having the accession number FERM BP-11183.

Also, the present invention provides a method for identifying HCV genotype, wherein: the genotype of the HCV is determined to be genotype 1b if the virus binds to an antibody produced by the hybridoma cell line having accession number FERM BP-11181, but does not bind to any of the antibodies produced by the hybridoma cell lines having accession numbers FERM BP-11180 and FERM BP-11179; the genotype of the HCV is determined to be genotype 2a if the virus binds to antibodies produced by the hybridoma cell line having accession numbers FERM BP-11181 and to antibodies produced by the hybridoma cell lines having accession numbers FERM BP-11180 and FERM BP-11179; and the genotype of HCV is determined to be genotype 1a if the virus binds to an antibody produced by the hybridoma cell line having accession number FERM BP-11182, but does not bind to any of the antibodies produced by the hybridoma cell lines having accession numbers FERM BP-11181, FERM BP-11180, and FERM BP-11179.

The present specification contains the contents of the specification and/or drawings of japanese patent application No. 2008-254338, the priority of which is claimed.

Effects of the invention

According to the present invention, simple and very accurate identification of HCV genotype 1a, HCV genotype 1b and HCV genotype 2a becomes possible, and hepatitis C patients for which interferon therapy is appropriate can be effectively selected. In particular, adverse reactions can be alleviated and opportunities for selection of new therapeutic approaches can be provided for hepatitis c patients infected with HCV of genotype 1a or 1b for which no therapeutic effect from interferon therapy is expected.

Drawings

FIG. 1 is a schematic diagram showing HCV precursor proteins. The black boxes represent the transmembrane regions.

FIG. 2 is a schematic diagram showing a fusion protein of 3XFLAG protein and antigen E2 protein.

FIG. 3 is a schematic diagram showing a fusion protein of antigen E2 protein and human immunoglobulin Fc domain.

FIG. 4 shows the SDS-PAGE results of each fraction obtained in the step of purifying the 3XFLAGJ6E2dTM protein. Expression and purification of 3 XFLAG-J6E 2dTM in COS1 cells is shown. The 3 × FLAGJ6E2dTM protein was detected in the eluted fractions. The lanes of the electropherogram in FIG. 4 represent the following, respectively: 1, a molecular weight marker; 2, culture supernatant; 3, anti-FLAG antibody column pore fraction; 4, anti-FLAG antibody column elution fraction 1; 5, anti-FLAG antibody column elution fraction 2; 6, anti-FLAG antibody column elution fraction 3; 7, anti-FLAG antibody column elution fraction 4; and 8, a molecular weight marker.

FIG. 5 shows THE SDS-PAGE results for J6E2-Fc, JFH1E2-Fc, THE2-Fc, Con1E2-Fc, J1E2-Fc and H77E2-Fc proteins. Each type of E2Fc protein was about 97 kDa under reducing conditions. Purified fusion proteins of antigen E2 protein derived from each HCV strain with the human immunoglobulin Fc domain are shown. The lanes of the electropherogram in FIG. 5 represent the following, respectively: 1, a molecular weight marker; 2, J6E 2-Fc; 3, JFH1E 2-Fc; 4, THE THE 2-Fc; 5, Con1E 2-Fc; 6, J1E 2-Fc; 7, H77E 2-Fc; and 8, a molecular weight marker.

FIG. 6 shows the binding properties of the E2 protein and antibody subtypes for the various HCV genotypes/strains of each monoclonal antibody. The binding strength of the antibody to the antigen E2 protein is expressed as-to +++ (-OD 450 nm < 0.1; +, 0.1. ltoreq. OD450 nm < 0.25; +, 0.25. ltoreq. OD450 nm < 0.4; +++, 0.4. ltoreq. OD450 nm). Figure 6 shows: 8D10-3 is an antibody that binds to antigen E2 protein of genotype 1a HCV, genotype 1b HCV and genotype 2a HCV; 1G2-32 and 2F2-7 are antibodies that bind the antigen E2 protein of HCV of genotype 2 a; 4E8-8 is an antibody that binds to the antigen E2 protein of HCV of genotype 1b and HCV of genotype 2 a; M1E12-1 is a monoclonal antibody that binds to the antigen E2 protein of strain J6 CF; and 9a5-4 is a monoclonal antibody that binds to antigen E2 proteins of J6CF strain and H77 strain.

FIG. 7 shows the binding strength of the 8D10-3 monoclonal antibody (FIG. 7A), the 1G2-32 monoclonal antibody (FIG. 7B), the 4E8-8 monoclonal antibody (FIG. 7C), the2F 2-7 monoclonal antibody (FIG. 7D), and the M1E12-1 monoclonal antibody (FIG. 7E) to peptides having the amino acid sequence of the antigen E2 protein derived from HCV J6CF strain.

FIG. 8 shows the sensitivity of detection of antigen E2 protein from various HCV genotypes/strains as determined by sandwich ELISA using the 1G2-32 and 8D10-3 monoclonal antibodies. In FIG. 8, black circles represent J6E2-Fc, white circles represent JFH1E2-Fc, black squares represent THE2-Fc, white squares represent Con1E2-Fc, black diamonds represent J1E2-Fc, and white diamonds represent H77E 2-Fc.

FIG. 9 shows the presence or absence of HCVE2 protein of various genotypes/strain as detected by Western blotting using monoclonal antibody 8D 10-3.

Detailed Description

Preferred embodiments for carrying out the present invention are described below.

The antibody of the present invention is characterized by specifically binding to envelope protein 2 (hereinafter referred to as E2 protein) of HCV of genotype 2a (hereinafter referred to as HCV2 a), but not immunoreactive with E2 protein of HCV of genotype 1a (hereinafter referred to as HCV1 a). In a preferred embodiment, such antibodies do not immunoreact with both the E2 protein of HCV1a and the E2 protein of HCV of genotype 1b (hereinafter HCV1 b).

The above antibody can be prepared by: an animal is immunized with an antigenic protein consisting of a region free from the transmembrane region (also referred to as transmembrane domain) of the E2 protein of HCV or a partial peptide of the antigenic protein as an antigen, hybridomas producing a monoclonal antibody against the E2 protein are prepared, and then hybridomas producing an antibody that specifically binds to the E2 protein of HCV2a, but does not immunoreact with the E2 protein of HCV1a, and further preferably does not immunoreact with both the E2 protein of HCV1a and the E2 protein of HCV1b are screened.

Herein, the "E2 protein" is a functional viral protein produced by cleaving HCV precursor protein with a signal peptidase derived from host cells and 2 proteases encoded by HCV itself. This is explained using the J6CF strain of HCV2a as an example, and when the methionine located at the N-terminal of the precursor protein is determined to be the 1 st amino acid, the E2 protein is a protein of 367 amino acid residues from amino acid positions 384 to 750 of the precursor protein. The region from amino acid positions 722 to 750 in the E2 protein is the transmembrane domain (Cocquerel et al, J. Virol., 2000, Vol. 74, p. 3623-3633). FIG. 1 is a schematic diagram showing HCV precursor proteins.

Hereinafter, techniques for obtaining the above-described antibodies will be described in order.

1) Selection of E2 protein-derived proteins or peptides as antigens

As an antigen for immunizing an animal in order to obtain the above-mentioned antibody, a protein composed of a region free from the transmembrane region of HCV2a E2 protein (hereinafter referred to as antigen E2 protein), or a partial peptide of the protein (antigen E2 peptide) may be used. The antigen E2 peptide must consist of a region with low homology to the E2 protein of HCV of a genotype other than 2 a.

As the antigen E2 protein, a protein comprising amino acids 384 to 720 of the precursor protein of HCV2a (e.g., SEQ ID NO: 5) can be selected. Preferably, a protein is selected comprising the amino acid sequence of amino acid positions 530 to 562 of the precursor protein, and more preferably a protein is selected comprising one or more amino acid sequences selected from the group consisting of: an amino acid sequence comprising amino acids 465 to 484 of the precursor protein; an amino acid sequence comprising amino acids 559 to 584 of the precursor protein; and an amino acid sequence comprising amino acids 683 to 719 of the precursor protein.

Also, as the antigen E2 peptide, a peptide comprising amino acids 530 to 562 (more preferably, amino acids 531 to 549, further preferably, amino acids 531 to 540) of a precursor protein of HCV2a (e.g., SEQ ID NO: 5) and having a peptide length of 10 to 19 amino acids (more preferably, 10 amino acids) can be selected. More preferably, a peptide comprising amino acids 465 to 484 (more preferably, amino acids 465 to 477, further preferably, amino acids 468 to 477) of the precursor protein and having a peptide length of 10 to 13 amino acids (more preferably, 10 amino acids) is selected; a peptide comprising amino acids 559 to 584 (more preferably, amino acids 564 to 576, further preferably, amino acid residues at positions 567 to 576) of the precursor protein and having a peptide length of 10 to 13 amino acids (more preferably, 10 amino acids); or a peptide comprising amino acids 683 to 719 (preferably, amino acids 704 to 719, more preferably, amino acids 709 to 719) of the precursor protein and having a peptide length of 10 to 19 amino acids (more preferably, 10 amino acids).

Furthermore, the nucleotide sequence of the HCV2a genome has been shown in many viral strains (Yanagi et al, Virology, 1999, Vol.262, p.250-263) and is available from GenBank. For example, the nucleotide sequence of the genome of JFH1 strain of HCV2a is disclosed in GenBank under accession number AB047639, and the nucleotide sequence of the genome of J6CF strain is disclosed in GenBank under accession number AF 177036.

2) Preparation of antigen E2 peptide

The antigen E2 peptide selected above can be chemically synthesized directly based on the amino acid sequence information of the precursor protein of HCV2 a. For example, such antigens that can be used for immunization of animals can be easily prepared in large quantities by using a peptide synthesizer.

3) Preparation of antigen E2 protein

The above selected antigen E2 protein can be prepared in large quantities as an antigen that can be used for immunization of animals by synthesizing a DNA fragment encoding the antigen E2 protein based on the nucleotide sequence information on the region encoding the precursor protein of HCV2a and then translating the antigen E2 protein from the thus obtained DNA fragment in cells. The details are described below.

The antigen E2 protein can be produced in each cell by constructing an expression vector into which a DNA fragment encoding the antigen E2 protein is inserted, and then transducing into mammalian cells, insect cells, yeast, Escherichia coli (Escherichia coli), or the like. Preferably, the protein is produced by secretory expression in a mammalian cell. In this case, a DNA fragment encoding the antigen E2 peptide is ligated in-frame downstream of the signal peptide sequence such that the frames of the codons are matched, a stop codon is added to the 3' end, and the product can then be inserted into an expression vector.

Examples of mammalian cells for secretory expression of antigen E2 protein include COS-1, COS-7, Vero, CV-1, CHO, dhfr gene-deficient CHO, hamster cell BHK, rat GH3, rat pheochromocytoma PC12, mouse L cell, mouse C127 cell, mouse myeloma cell SP2/0, NSO and NS-1, mouse lymphoma cell EL4, mouse fibroblast NIH3T3 and 10T1/2, mouse myoblast C2C12, mouse stromal cell PA6, ST2, 9 and Tst-4, human megakaryocyte CMK, human T cell Jurkat, human renal epithelial cell 293, human hepatoma cell Huh7, HepG2 and IMY-N9, human osteosarcoma cell-63, human FL cell, white fat cell, egg cell and MG cell.

The DNA encoding the protein is inserted under the control of a promoter and then used for recombinant expression of the antigen E2 protein in the cell. Examples of such promoters that can be used for recombinant expression of the antigen E2 protein in mammalian cells include the SR α promoter, the SV40 promoter, the LTR promoter, the CMV promoter, the actin promoter, the EF-1 α (elongation factor-1 α) promoter, the ubiquitin promoter, and the PGK (phosphoglycerate kinase) promoter.

Examples of expression vectors for secretory expression of antigen E2 protein in mammalian cells include pSecTag/FRT/V5-His (Invitrogen corporation), p3 XFLAG-CMV-9 (Sigma), p3 XFLAG-CMV 13 (Sigma), pFUSE-Fc2 (InvivoGen), and pTriEx-7 (Novagen). The signal peptide sequence incorporated into the expression vector is preferably the signal peptide of preproglobrypsin. Examples of vectors with the signal peptide sequence of preprotrypsin include p3 XFLAG-CMV-9 (Sigma) and p3 XFLAG-CMV-13 (Sigma). Furthermore, since the signal peptide is removed when the protein containing the signal peptide is expressed in mammalian cells, the use of such a signal peptide for the antigen E2 protein does not pose a problem.

When the antigen E2 protein is secreted and expressed in mammalian cells, the antigen of interest E2 protein is expressed as a fusion protein with a marker protein (e.g., Tag), and then the antigen E2 protein can be detected and purified using an antibody or molecule directed against or specifically binding to the marker protein. Examples of tag proteins include FLAG peptide, 3XFLAG peptide, HA peptide, 3 XHA peptide, myc peptide, 6XHis peptide, GST polypeptide, MBP polypeptide, PDZ domain polypeptide, alkaline phosphatase, immunoglobulin Fc domain, and avidin. As a marker protein for preparing the antigen E2 protein, FLAG peptide, HA peptide and immunoglobulin Fc domain are suitable, and immunoglobulin Fc domain is more suitable.

FIG. 2 is a schematic diagram showing a fusion protein of antigen E2 protein and 3XFLAG protein. FIG. 3 is a schematic diagram showing a fusion protein of antigen E2 protein and an immunoglobulin Fc domain.

As such an immunoglobulin Fc domain, a human-derived, monkey-derived, mouse-derived, rat-derived, rabbit-derived, hamster-derived, or chicken-derived immunoglobulin Fc domain can be used, and a human-derived immunoglobulin Fc domain is preferable. Furthermore, the class of immunoglobulin heavy chains of the immunoglobulin Fc domain may be IgM, IgG1, IgG2, IgG3, or IgG 4.

The amino acid sequence of human immunoglobulins is reported by Edelman et al (Proc. Natl. Acad. Sci. U.S.A., 1969, Vol. 63, p.78-85). Also, nucleotide sequence information of cDNA of human immunoglobulin heavy chain can be obtained from GenBank (e.g., heavy chain: accession No. BX 640627). PCR primers are designed based on the obtained nucleotide sequence, and then PCR is performed using a cDNA library of human spleen cells or human genomic DNA as a template, whereby cDNA of an immunoglobulin Fc domain can be cloned.

The HCV E2 proteins may be linked directly to the immunoglobulin Fc domain at a linking site therebetween, or via a linker peptide inserted therein. Examples of linker peptides include Ser-Gly, Asp-Pro-Glu, Gly-Gly-Gly-Ser, and (Gly-Gly-Gly-Ser). times.3.

In addition, for example, when the antigen E2 protein is expressed by secretion from insect cells such as Sf21, Sf9 and High Five^TMTransduction was performed using an expression vector such as polyhedrin (polyhedron) promoter, p10 promoter, and the like. Then, the antigen E2 protein or the fusion protein of the antigen E2 protein and the marker protein can be expressed.

In addition, when the antigen E2 protein is secreted and expressed by yeast, for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), or Pichia pastoris (Pichia pastoris) is transduced with an expression vector using a gal1 promoter, a gal10 promoter, a heat shock protein promoter, an MF α 1 promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, an AOX1 promoter, or the like, and then the antigen E2 protein or a fusion protein of the antigen E2 protein and a marker protein can be expressed.

When the antigen E2 protein is secreted and expressed by Escherichia coli, for example, an Escherichia coli strain such as XL1-Blue strain, BL-21 strain, JM107 strain, TB1 strain, JM109 strain, C600 strain or HB101 strain is transformed with an expression vector using trp promoter, lac promoter, PL promoter, T7 promoter, tac promoter or the like, and then the antigen E2 protein or a fusion protein of the antigen E2 protein with a marker protein can be expressed.

Transduction with expression vectors to elicit resistance in mammalian and insect cellsExamples of the method for secretory expression of the pro-E2 protein include lipofection method, calcium phosphate method, electroporation method, DEAE-dextran method, and microinjection method. More specifically, transduction may be according to Molecular Cloning 3^rdThe method described in ed.16.1-16.62 (Cold Spring Harbor Laboratory, New York, 2001).

The method for introducing the expression vector into E.coli is not particularly limited as long as it is a method for introducing DNA into E.coli. Examples of such methods include a method using calcium ions (Cohen et al, proc. natl. acad. sci., u.s.a., 1972, vol. 69, p. 2110-2114) and an electroporation method.

The method for introducing the expression vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. Examples of such methods include the electroporation method (Becker et al, methods. enzymol., 1990, Vol. 194, p. 182-.

The transduced cells can be cultured by methods known per se. A medium for culturing mammalian cells is, for example, MEM medium containing about 5% to 20% Fetal Bovine Serum (FBS), DMEM medium, RPMI1640 medium, 199 medium (proceedings of the Society for the Biological Medicine, 1950, Vol.73, p.1) and the like. The pH is preferably about 6 to 8. As the serum-free medium, CD-CHO, 293 SFM-II and Hybridoma-SFM (all of which are manufactured by Invitrogen Corporation) can be used, to which serum or supplements can be added as required. The cells may be cultured at 30 ℃ to 40 ℃ for 15 hours to 60 hours, preferably with aeration or agitation as required.

After completion of the cell culture, the cells are removed from the culture solution by centrifugation or the like, and then the antigen E2 protein or the fusion protein of the antigen E2 protein and the marker protein can be purified from the culture supernatant thus obtained. The antigen E2 protein or the fusion protein of the antigen E2 protein and the marker protein can be purified according to protein isolation and purification techniques known to those skilled in the art. For example, the protein may be isolated and purified by any combination of ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, and the like.

For example, the antigen E2 protein in the culture solution can be easily purified using a heparin column or a lectin column. In the case of the fusion protein with 3 × FLAG peptide, the antigen E2 protein can be efficiently purified using an anti-FLAG antibody column, in the case of the fusion protein with 6 × his peptide, the antigen E2 protein can be efficiently purified using a nickel column, a zinc column, or a cobalt column, in the case of the fusion protein with immunoglobulin Fc domain, the antigen E2 protein can be efficiently purified using a protein a column, or a protein G column, and in the case of the chimeric protein containing HA peptide, the antigen E2 protein can be efficiently purified using an anti-HA antibody column.

The thus purified antigen E2 protein or fusion protein of antigen E2 protein with a marker protein can be detected by Coomassie brilliant blue staining or silver staining after fractionation by SDS-PAGE. In the case of fusion proteins, the fusion protein can be detected by western blotting using an antibody against the fused marker protein.

4) Immunization with antigen E2 peptide or antigen E2 protein

In order to obtain an antibody that specifically binds to E2 protein of HCV2a, but does not immunologically react with E2 protein of HCV1a, and more preferably does not immunologically react with both E2 protein of HCV1a and E2 protein of HCV1b, immunization of an animal using the above-mentioned antigen E2 peptide or antigen E2 protein should be performed to obtain a polyclonal antibody or to screen for a hybridoma producing a monoclonal antibody of interest.

The immunized animal can be a non-human animal having spleen cells that can be used to produce hybridoma cells. Examples of such animals include mice, rats, hamsters, rabbits, and chickens. Mice may be more preferably used.

Examples of the immunization method include several times of subcutaneous or intraperitoneal administration of the above antigen E2 peptide or antigen E2 protein together with an adjuvant to 4 to 10-week-old mice, confirmation of an increase in blood antibody titer, boosting by intravenous or intraperitoneal administration of the antigen E2 peptide or antigen E2 protein alone, and then collecting blood or spleen cells on days 3 to 10 (preferably on day 4). The antibody titer of serum obtained from the collected blood was measured. In this case, if it specifically recognizes the target antigen, it can be used as a polyclonal antibody.

Examples of adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, a mixture of aluminum hydroxide gel and pertussis vaccine, Titer Max Gold (Vaxel), and GERBU adjuvant (GERBU Biotechnik).

The antibody titer in blood was measured by collecting blood from the immunized animal via the fundus venous plexus or tail vein, and then examining the obtained blood for the presence or absence of antibodies reactive with the antigen E2 peptide or the antigen E2 protein by Enzyme Immunoassay (EIA).

5) Preparation of hybridoma cells

Spleen cells collected from immunized animals, in which an increased antibody titer in blood has been confirmed, 3 to 10 days after the boosting are fused to myeloma cells, so that hybridoma cells having autonomous replication can be prepared. Monoclonal antibodies can be produced in large quantities by screening hybridoma cells that produce antibodies with the desired specificity.

Myeloma cells for cell fusion may be used, for example, mouse-derived established cell lines P3-X63Ag8-U1 (P3-U1), SP2/0-Ag14 (SP 2/0), P3-X63-Ag8653 (653), P3-X63-Ag8 (X63), P3/NS1/1-Ag4-1 (NS 1), and the like. These cell lines can be obtained from RIKEN BioResource Center, ATCC (American type culture Collection) or ECACC (European Collection of cell cultures).

Cell fusion of spleen cells and myeloma cells by washing two cells, 1: 1-10, and then adding polyethylene glycol or polyvinyl alcohol having an average molecular weight of 1000-.

After the treatment for cell fusion is completed, the fused cells are suspended in a medium and washed with the medium, and then cloned in a methylcellulose medium by a limiting dilution or colony formation method. An example of limiting dilution is a process comprising dilution to 10³To 10⁷Each cell/mL, the cells were added at 10%²To 10⁶Individual cells/well were seeded into 96-well cell culture microplates, and then cells were cultured.

When cloning of hybridoma cells is performed, it is preferable to add HAT supplement to the culture medium to enable selective obtainment of individual target fusion cells. More specifically, according to Antibodies: hybridoma cells of interest were obtained and cloned by the Methods described in A Laboratory Manual (Cold Spring Harbor Laboratory, 1988) or Selected Methods in Cellular Immunology (W.H. Freeman and Company, 1980).

6) Screening of hybridoma cells

For example, hybridoma cells of interest are screened by the EIA method as described below.

Specifically, first, an antigen E2 peptide or an antigen E2 protein is immobilized on a carrier, an antibody produced by each cloned hybridoma cell is added, and a reaction is performed at 4 ℃ to 37 ℃ for a time sufficient to form an antibody-antigen complex.

Next, a secondary antibody labeled with an enzyme, a dye, a radioisotope or the like, capable of specifically binding to the antibody portion of the antibody-antigen complex thus formed, is contacted with the formed antibody-antigen complex and reacted at 4 ℃ to 37 ℃ for a time sufficient to form an antibody-antigen-secondary antibody complex.

Finally, the presence or absence of the thus-formed antibody-antigen-secondary antibody complex is detected using a signal from an enzyme, dye or radioisotope for labeling the secondary antibody as an index, thereby determining whether or not it is an antibody having a target characteristic.

7) Preparation of monoclonal antibodies

Hybridoma cells selected by the above-described method are adapted to a serum-free medium, for example, Hybridoma-sfm (invitrogen corporation), and then monoclonal antibodies can be prepared from the supernatant of the culture in the serum-free medium. For culturing cells, flasks, petri dishes, spinner flasks, roller bottles, or high density culture flasks CELLine (Becton, Dickinson and Company) may be used.

In addition, for mass production of monoclonal antibodies, for example, 6 to 8-week-old nude mice or SCID mice can be fed with 0.5 mL of isostearyl (2, 6,10, 14-tetramethylpentadecane) intraperitoneally for 2 weeks, followed by 5X 10 intraperitoneally⁶To 2X 10⁷Individual cells/mouse hybridoma cells were raised for 10 to 21 days, so that monoclonal antibodies could be prepared from the resulting ascites fluid.

Cells and cellular debris are removed from the peritoneal fluid thus collected by centrifugation. Purification methods, for example, salting out with 40% to 50% saturated ammonium sulfate, caprylic acid precipitation, DEAE-agarose column, protein a column, protein G column, HiTrap IgM purification HP-column (GE Healthcare), mannan-binding protein column (Pierce), and gel filtration column are used alone or in combination, to collect IgG or IgM fractions, which can then be used as purified monoclonal antibodies.

8) Analysis of epitopes of monoclonal antibodies

Linear epitopes of monoclonal antibodies can be analyzed by synthesizing peptides having amino acid sequences of 8 to 12 consecutive amino acids designed to be shifted by one to several amino acids in the antigen E2 protein, examining the binding of the monoclonal antibody to the peptide when the peptide is used as an antigen, and then determining the epitope of the antibody.

Specifically, the thus-synthesized peptide is immobilized on a plate and reacted with a purified antibody. Labeled secondary antibody was added and the plate was allowed to stand. Binding capacity was measured by enzyme immunoassay (ELISA) or Radioimmunoassay (RIA).

In some cases it may not be possible to determine the epitope by this method. In this case, the epitope of the monoclonal antibody may be a conformational epitope, and thus the antibody may recognize the conformation of the antigen.

An example of an antibody that specifically binds to the E2 protein of HCV2a, but does not immunoreact with the E2 protein of HCV1a is a monoclonal antibody that binds specifically to the E2 protein of HCV2 a: 1 as an epitope. A specific example of such an antibody is an antibody produced from a hybridoma cell line having the accession number FERM BP-11181.

In addition, an example of an antibody that specifically binds to the E2 protein of HCV2a, but does not immunoreact with both the E2 protein of HCV1a and the E2 protein of HCV1b is a fusion protein of SEQ ID NOs: 2 or 3 as an epitope. Specific examples of such antibodies are those produced by hybridoma cell lines having the accession numbers FERM BP-11180 or FERM BP-11179.

Further, an example of an antibody that specifically binds to envelope protein 2 of J6CF strain of HCV2a, but does not immunologically react with envelope protein 2 of JFH1 strain is a fusion protein of SEQ ID NO: 4 as an epitope. A specific example of such an antibody is an antibody produced by the hybridoma cell line having the accession number FERM BP-11183. This antibody can discriminate the J6CF strain from HCV genotype 2a, and therefore it can be used to identify the J6CF strain.

Furthermore, the above-mentioned hybridoma cell lines having the registration numbers FERM BP-11181, FERM BP-11180, FERM BP-11179, FERM BP-11183 and FERM BP-11182 have been deposited at International depository, national institute of advanced Industrial science and technology International patent organism depositary (postal code: 305-. These cell lines were originally each deposited at the same depository (original depository date: 19.9.2008), registration numbers FERM P-21677 (provisional registration number FERM AP-21677), FERM P-21676 (provisional registration number FERM AP-21676), FERM P-21675 (provisional registration number FERM AP-21675), FERM P-21679 (provisional registration number FERM AP-21679) and FERM P-21678 (provisional registration number FERM AP-21678), and then transferred to international depository under the Budapest treaty.

In addition, the method for identifying HCV genotype of the present invention comprises: determining that the genotype of HCV is genotype 1b if HCV binds to an antibody produced by the hybridoma cell line having accession number FERM BP-11181, but does not bind to any of the antibodies produced by the hybridoma cell lines having accession numbers FERM BP-11180 and FERM BP-11179; determining that the genotype of HCV is genotype 2a if HCV binds to an antibody produced by the hybridoma cell line having accession numbers FERM BP-11181 and binds to an antibody produced by the hybridoma cell line having accession numbers FERM BP-11180 and FERM BP-11179; and determining that the genotype of HCV is genotype 1a if HCV binds to an antibody produced by the hybridoma cell line having accession number FERM BP-11182, but does not bind to any of the antibodies produced by the hybridoma cell lines having accession numbers FERM BP-11181, FERM BP-11180, and FERM BP-11179.

Whether or not HCV of unknown genotype binds to an antibody produced by a hybridoma cell line having the accession numbers FERM BP-11181, FERM BP-11180, FERM BP-11179 or FERM BP-11182 can be determined using any assay system without particular limitation so long as it can detect the presence or absence of an antigen-antibody reaction. Examples of such methods are immunoassay and western blotting methods as described below.

(immunoassay)

First, a test sample containing HCV of unknown genotype is contacted with a carrier or plate on which the above-mentioned antibody to be examined for the presence or absence of binding has been immobilized as a primary antibody, and then reacted at 4 ℃ to 37 ℃ for a time sufficient to form an antibody-antigen complex.

Next, a secondary antibody labeled with an enzyme, a dye, a radioisotope or the like, which binds HCV in a non-genotype specific manner, is contacted with the antibody-antigen complex, and then reacted at 4 ℃ to 37 ℃ for a time sufficient to form an antibody-antigen-secondary antibody complex.

Finally, the presence or absence of the formed antibody-antigen-secondary antibody complex is detected by an indicator signal derived from an enzyme, dye or radioisotope used for labeling the secondary antibody, whereby the presence or absence of binding to the above-mentioned antibody can be determined.

(Western blotting method)

First, a test sample containing HCV of unknown genotype is spotted on a membrane such as a nitrocellulose membrane or a PVDF membrane, and then the protein contained in the test sample is immobilized.

Then, the membrane is immersed in 5% skim milk, 1% BSA solution, or a commercial blocking reagent for blocking, washed sufficiently with a buffer, and then transferred to a buffer containing the above-mentioned antibody labeled with an enzyme, a dye, a radioisotope, or the like to check for the presence or absence of binding. The reaction is carried out at 4 ℃ to 37 ℃ for a time sufficient to form an antibody-antigen complex.

Subsequently, the membrane is sufficiently washed, and then a signal from an enzyme, a dye or a radioisotope for labeling the above antibody is detected for checking the presence or absence of binding, thereby determining the presence or absence of binding to the above antibody.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Examples

The present invention will be more specifically explained with reference to the following examples. However, these examples are merely illustrative, and the scope of the present invention is not limited by these examples.

Example 1 preparation of a vector for expressing a fusion protein of antigen E2 protein of HCV strain and a marker protein.

(1) Construction of vector for expressing fusion protein of antigen E2 protein derived from J6CF strain of HCV2a and 3XFLAG tag

The antigen E2 protein derived from J6CF strain of HCV2a, i.e., a protein consisting of a region without the transmembrane region of the E2 protein of J6CF strain of HCV2a, was prepared as follows.

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 720 of a precursor protein of strain J6CF (SEQ ID NO: 5) was amplified by a PCR method using cDNA of genomic RNA of strain J6CF of HCV2a (GenBank Accession number AF 177036) as a template, an Advantage GC2 PCR kit (Takara Bio Inc.), and J6E2dTM-s (SEQ ID NO: 6: CACAAGCTTCGCACCCATACTGTTGGGG) and J6E2dTM-as (SEQ ID NO: 7: GCTCTAGATTACCATCGGACGATGTATTTTGT) as primers, at which time the initial methionine at the N-terminus was determined to be the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, and is excised from the clone containing the correct nucleotide sequence insert. The gene fragment is inserted into p3 XFLAG-CMV-9 (Sigma) in reading frameHind III andBamh I so that the reading frames match. As a result, a CMV-3XFLAGJ6E2dTM vector expressing the antigen E2 protein to which 3 XFLAG-tag (hereinafter referred to as 3 XFLAG-J6E 2dTM protein) is linked was obtained.

(2) Construction of vector for fusion protein expression of antigen E2 protein derived from J6CF strain of HCV2a and human IgG Fc protein added to the antigen E2 protein

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 720 of a precursor protein of strain J6CF (SEQ ID NO: 5) was amplified by a PCR method using genomic RNA of cDNA of strain J6CF of HCV2a (GenBank Accession number AF 177036) as a template, an Advantage GC2 PCR kit (Takara Bio Inc.), and J6E2Fc-s (SEQ ID NO: 8: CACAAGCTTCGCACCCATACTGTTGGGG) and J6E2Fc-as (SEQ ID NO: 9: ACAGGATCCCATCGGACGATGTATTTTGTG) as primers, at which time the initial methionine at the N-terminus was determined as the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, and is thereby excised from the insert clone having the correct nucleotide sequence. The gene fragment was inserted downstream of the signal peptide sequence of p3 XFLAG-CMV-13 (Sigma)Hind III site andBamh I so that the reading frames (open reading frames) match, that is, are inserted in reading frames. The resulting vector was designated CMV-13-J6E 2.

Subsequently, CMV-13-J6E2 usedSacI andBamh I and digesting. The DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, DNA fragments encoding the above-described signal peptide sequence and antigen E2 protein were inserted in-frame (so that the frames match) into CDM-mIL7R-Ig vector (Sudo et al, Proc Natl. Acad Sci U.S.A., 1993, Vol. 90, p. 9125-one 9129), respectivelySacI site andBamh I, the vector expresses a chimeric protein comprising a mouse IL-7 receptor-human immunoglobulin Fc domain. Thus, a CDM-J6E2Fc vector expressing the antigen E2 protein to which the human immunoglobulin Fc domain is linked (hereinafter referred to as J6E2-Fc protein) was obtained）。

(3) Construction of vector for expression of fusion protein of antigen E2 protein derived from JFH1 strain of HCV2a and human IgG Fc protein

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 721 of the precursor protein of JFH1 strain was amplified by a PCR method using cDNA (GenBank Accession number AB 047639) of genomic RNA of JFH1 strain of HCV2a as a template, an Advantage GC2 PCR kit (Takara Bio Inc.), and JFE2Fc-s (SEQ ID NO: 10: CACAAGCTTGGCACCACCACCGTTGGAG) and JFE2Fc-as (SEQ ID NO: 11: ACAGGATCCTCCCATCGAACGACGTATTTTGTG) as primers, and the initial methionine at the N-terminus was determined as the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, which was excised from the clone with the correct nucleotide sequence insert and inserted in-frame downstream of the signal peptide sequence of p3 XFLAG-CMV-13 (Sigma)Hind III site andBamh I position in between. This vector was designated CMV-13-JFH1E 2.

Subsequently, CMV-13-JFH1E2 usedSacI andBamh I and digesting. The DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, DNA fragments encoding the above signal peptide sequence and antigen E2 protein were inserted in-frame into CDM-mIL7R-Ig, respectivelySacI site andBamh I position in between. Thus, a CDM-JFH1E2Fc vector expressing the antigen E2 protein to which the Fc domain of human immunoglobulin (hereinafter referred to as JFH1E2-Fc protein) is linked was obtained.

(4) Construction of vector for expression of fusion protein of antigen E2 protein derived from TH strain of HCV1b and human IgG Fc protein

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 717 of a precursor protein of THE TH strain was amplified by a PCR method using cDNA of genomic RNA of THE TH strain of HCV1b (International patent publication WO 2006/022422) as a template and Advantage GC2 PCR kit (Takara Bio Inc.) and THE2Fc-s (SEQ ID NO: 12: CAAAGCTTGCGACCTACGTGACGGGGGGGTCG), and THE2Fc-as (SEQ ID NO: 13: CCTCTAGATTATGGATCCCATTTGATTGCATAGGAGACAACCG) as primers, at which time THE initial methionine at THE N-terminus was determined as THE first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, and is thus excised from the clone having the correct nucleotide sequence insert. The gene fragment was inserted in-frame downstream of the signal peptide sequence of p3 XFLAG-CMV-13 (Sigma)Hind III site andBamh I position in between. This vector was designated CMV-13-THE 2.

Subsequently, CMV-13-THE2 usedSacI andBamh I and digesting. The DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, DNA fragments encoding a signal peptide sequence and antigen E2 protein were inserted in-frame into CDM-mIL7R-Ig, respectivelySacI site andBamh I position in between. Thus, a CDM-THE2Fc vector expressing THE antigen E2 protein to which a human immunoglobulin Fc domain (hereinafter referred to as THE2-Fc protein) is linked was obtained.

(5) Construction of vector for expression of fusion protein of antigen E2 protein derived from Con1 strain of HCV1b and human IgG Fc protein

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 716 of the precursor protein of Con1 strain was amplified by a PCR method using cDNA of genomic RNA of Con1 strain of HCV1b (GenBank Accession number AJ 238799) as a template and Advantage GC2 PCR kit (Takara Bio Inc.) and Con1E2Fc-s (SEQ ID NO: 14: CAAAGCTTGGAACCTATGTGACAGGGGGGACGAT), and Con1E2Fc-as (SEQ ID NO: 15: CCTCTAGATTATGGATCCCATTTGATTGCAAAGGAGACAAC) as primers, at which time the initial methionine at the N-terminus was determined as the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, and is thus excised from the clone having the correct nucleotide sequence insert. The gene fragment was inserted in-frame downstream of the signal peptide sequence of p3 XFLAG-CMV-13 (Sigma)Hind III site andBamh I position in between. This vector was designated CMV-13-Con1E 2.

Subsequently, CMV-13-Con1E2 was usedSacI andBamh I and digesting. The DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, DNA fragments encoding a signal peptide sequence and antigen E2 protein were inserted in-frame into CDM-mIL7R-Ig, respectivelySacI site andBamh I position in between. Thus, a CDM-Con1E2Fc vector expressing the antigen E2 protein to which a human immunoglobulin Fc domain (hereinafter referred to as Con1E2-Fc protein) was linked was obtained.

(6) Construction of vector for expression of fusion protein of antigen E2 protein derived from J1 strain of HCV1b and human IgG Fc protein

First, a gene encoding a protein consisting of a region corresponding to amino acid positions 384 to 716 of a precursor protein of strain J1 was amplified by a PCR method using cDNA derived from genomic RNA of strain J1 of HCV1b (GenBank Accession number D89815) as a template, Advantage GC2 PCR kit (Takara Bio Inc.), and J1E2Fc-s (SEQ ID NO: 16: CAAAGCTTCATACCCGCGTGACGGGGGGGGTGC), and J1E2Fc-as (SEQ ID NO: 17: CCTCTAGATTATGGATCCCACTTGATGGCAATGGAGACGACC) as primers, at which time the initial methionine at the N-terminus was determined as the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andBamh I, and is thus excised from the clone having the correct nucleotide sequence insert. The gene fragment was inserted in-frame downstream of the signal peptide sequence of p3 XFLAG-CMV-13 (Sigma)Hind III site andBamh I position in between. This vector was designated CMV-13-J1E 2.

Subsequently, CMV-13-J1E2 was digested with Tth 111I, blunted with T4 DNA polymerase, and then digested with T4 DNA polymeraseBamH I and digesting. The resulting DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, CDM-mILR7R-Ig was usedBamH I andXbai digestion to excise a DNA fragment containing the sequence encoding the Fc domain of human immunoglobulin. This fragment was then inserted downstream of the promoter region in pcDL-SR α 296 (Takebe et al, Proc Natil Acad Sci. U.S.A., 1987, Vol. 84, p. 7388-. In addition, DNA fragments encoding the signal peptide sequence and the antigen E2 protein were inserted in-frame into the EcoR V site of SR α IgG1Fc andBamh I position in between. Thus, SR α -J1E2Fc vector expressing the antigen E2 protein to which the human immunoglobulin Fc domain (hereinafter referred to as J1E2-Fc protein) is linked was obtained.

(7) Construction of vector for expression of fusion protein of antigen E2 protein derived from H77 strain of HCV1a and human IgG Fc protein

First, a region encoding a peptide consisting of amino acid positions 384 to 716 of a precursor protein of H77 strain was amplified by a PCR method using cDNA of genomic RNA of H77 strain of HCV1a (GenBank Accession number AF 011751) as a template and Advantage GC2 PCR kit (Takara Bio Inc.), and H77E2Fc-s (SEQ ID NO: 18: CAAAGCTTGAAACCCACGTCACCGGGGGAAA) and H77E2Fc-as (SEQ ID NO: 19: CCTCTAGATTATGGATCCCACTTAATGGCCCAGGACGCGAT) as primers, at which time the initial methionine at the N-terminus was determined as the first amino acid.

Then, the thus amplified DNA fragments were cloned into pCR-TOPO (Invitrogen corporation), and then 3 clones were subjected to sequence analysis. The gene segment for coding antigen E2 proteinHind III andXbai digestion, thus cutting from the clone with the correct nucleotide sequence insert. The gene fragment is inserted into the downstream of the signal peptide sequence of the p3 XFLAG-CMV-13 Xho vector in reading frameHind III site andXbabetween I sites, the vector was purified by cloning in p3 XFLAG-CMV-13 (Sigma)SacConversion of I site toXhoI site. The resulting vector was designated CMV-13-XhoH77E 2.

Subsequently, CMV-13-XhoH77E2 was usedXhoI andBamh I, and the DNA fragments encoding the signal peptide sequence and the antigen E2 protein were each separated by agarose gel electrophoresis and then purified using GeneElute (Sigma).

Thereafter, DNA fragments encoding the signal peptide sequence and the antigen E2 protein were inserted in-frame into SR α -IgG1Fc constructed in 5) above, respectivelyXhoI site andBamh I position in between. The SR α -H77E2Fc vector expressing the antigen E2 protein to which the human immunoglobulin Fc domain (hereinafter referred to as H77E2-Fc protein) is linked was obtained.

Example 2 expression of fusion protein of antigen E2 protein and marker protein

CMV-3XFLAGJ6E2dTM, CDM-J6E2Fc, CDM-JFH1E2Fc, CDM-THE2Fc, CDM-Con1E2Fc, SR α -J1E2Fc and SR α -H77E2Fc constructed in example 1 were introduced into COS1 cells from monkey kidney, and then each fusion protein was expressed as described below.

First, COS1 cells were subcultured in RPMI1640 medium (Invitrogen Corporation) containing 10% fetal bovine serum (Invitrogen Corporation), 100U/ml penicillin, and 100. mu.g/ml streptomycin. One day before gene transfer, COS1 cells were transfected at 1: 2 to a separation ratio of 150 cm²In culture flasks (Corning coast Corporation) and then in 5% CO₂The culture was carried out overnight in an incubator at 37 ℃.

Subsequently, DEAE dextran (GE Healthcare) and chloroquine (Sigma) were added to the RPMI1640 medium at final concentrations of 400. mu.g/ml and 100. mu.M, respectively. Mu.g of THE above expression vector (CMV-3 XFLAGJ6E2dTM, CDM-J6E2Fc, CDM-JFH1E2Fc, CDM-THE2Fc, CDM-Con1E2Fc, SR. alpha. -J1E2Fc or SR. alpha. -H77E2 Fc) was added at a concentration of 0.1. mu.g/. mu.l per 13 mL, and then THE cells were cultured for 3 to 4 days.

Thereafter, the supernatant of the cultured COS1 cells was aspirated. 10ml of PBS (-) (Nissui Pharmaceutical Co., Ltd.) was added, and once again, PBS (-) was aspirated to wash the cells. Subsequently, at 13 ml/150 cm²The flask was charged with DEAE dextran-DNA mixture, and the product was then incubated in the presence of 5% CO₂In the case of (2) was kept still at 37 ℃.

After four hours, the DEAE dextran-DNA mixture was aspirated, each flask was washed once with 10ml PBS, and 50 ml/flask was supplemented with Hybridoma-SFM medium (Invitrogen Corporation), and the cells were cultured in the presence of 5% CO₂Cultured at 37 ℃ for 4 days. Thereafter, the culture supernatant was collected in a 50 ml centrifuge tube (Corning Coaster Corporation), and then centrifuged at 2500 rpm at 4 ℃ for 30 minutes. The supernatant was filtered through a 0.2 μm filter (Whatman).

Example 3 purification of fusion protein of antigen E2 protein and marker protein

The culture supernatant of the cells into which CMV-3 XFLAG-J6E 2dTM had been introduced was purified using anti-FLAG M2 agarose (Sigma) as described below.

First, 1 ml of anti-FLAG M2 agarose was added to 500 ml of culture supernatant, and then reacted at 4 ℃ for 2 hours while stirring in a rotating bottle. After 2 hours, the mixture of supernatant and anti-FLAG M2 agarose was transferred to Econocolumn (Bio-Rad Laboratories Inc.), the pore fraction was removed, and anti-FLAG M2 agarose was collected.

Then, anti-FLAG M2 agarose was washed twice with 10ml TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Six fractions (anti-FLAG antibody column elution fractions 1-6) were eluted to 1 ml/fraction with 0.1M glycine-HCl (pH 3.5). Immediately after elution, 1M Tris-HCl (pH 9.5) was added to bring the pH back to neutral. 20 μ l of each fraction were fractionated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then stained with Coomassie Brilliant blue. As a result, it was confirmed that a fusion protein of antigen E2 protein derived from strain J6CF and 3XFLAG tag (3 XFLAG-J6E 2dTM protein) was purified (FIG. 4).

THE culture supernatant of cells into which CDM-J6E2Fc, CDM-JFH1E2Fc, CDM-THE2Fc, CDM-Con1E2Fc, SR α -J1E2Fc or SR α -H77E2Fc had been introduced was purified using THE protein-A-bound vector Prosep-A (Millipore) as follows.

First, Econocolumn was filled with 1 mL of Prosep-A, 500 mL of culture supernatant was passed at a flow rate of 1-1.5 mL/min, and then washed with 20 mL of PBS (-).

Then, 5 fractions were eluted with 0.1M glycine-HCl (pH 3.0) to 1 ml/fraction. Immediately after elution, 1M Tris-HCl (pH 9.5) was added to bring the pH back to neutral. 20 μ l of each fraction were fractionated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then stained with Coomassie Brilliant blue. As a result, a fusion protein of the antigen E2 protein from each HCV strain and the Fc domain of human immunoglobulin was purified, and the molecular weight was shown to be about 97 kDa under reducing conditions (FIG. 5).

Example 4 immunization of mice with antigen E2 protein of J6CF strain of HCV2a

An emulsion was prepared by mixing 0.3 ml of PBS solution containing 10. mu.g of 3 XFLAG-J6E 2dTM protein with 0.3 ml of Freund's complete adjuvant. 7-week-old Balb/c mice (female) were inoculated subcutaneously with half the amount of the emulsion.

After 2 weeks, an emulsion was prepared by mixing 0.3 ml of PBS solution containing 10. mu.g of 3 XFLAG-J6E 2dTM protein and 0.3 ml of Freund's incomplete adjuvant, and half of the emulsion was administered subcutaneously to mice. After another 2 weeks, mice were administered intraperitoneally with 0.15 ml of PBS solution containing 10. mu.g of 3 XFLAG-J6E 2dTM protein. After 3 days, spleen cells were prepared from mice.

In another experiment, 0.3 mL PBS solution containing 20. mu. g J6E2-Fc protein and 0.3 mL Alum (Pierce) were mixed to prepare a solution for administration. 7-week-old Balb/c mice (females) were inoculated intraperitoneally with the total number of emulsions.

After 2, 4 and 6 weeks, similarly, 0.3 ml of PBS solution containing 20 μ g J6E2-Fc protein and 0.3 ml of Alum were mixed to prepare a solution to be administered, and the whole amount of the emulsion was administered intraperitoneally to mice. After another 2 months, mice were administered intraperitoneally with 0.3 ml of PBS solution containing 20 μ g J6E2-Fc protein. After 3 days, spleen cells were prepared from mice.

Example 5 preparation of hybridoma cells

First, a mouse myeloma cell line SP2/0 (obtained from ECACC) was cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Corporation) containing 55. mu.M of 2-mercaptoethanol, 100U/ml of penicillin, 100. mu.g/ml of streptomycin, and 10% of fetal calf serum (FCS; Invitrogen Corporation) to obtain SP2/0 cells in logarithmic growth phase. Cells were washed 3 times with serum-free DMEM.

Then, spleen cells were prepared from mice that had been administered with 3 × FLAG-J6E2dTM protein or J6E2-Fc protein, followed by washing 3 times with serum-free DMEM. SP2/0 cells and mouse spleen cells were mixed at a ratio of 1: 5 into a 50 ml centrifuge tube, followed by centrifugation at 1,000rpm for 10 minutes. The supernatant was completely removed by aspiration. The centrifuge tube was then tapped to loosen the pellet. 1 ml of 50% polyethylene glycol-1500 solution (Roche) preheated at 37 ℃ was added for 1 minute, and reacted at 37 ℃ for 1 minute.

Subsequently, the ethylene glycol solution was diluted by adding 1 ml of serum-free DMEM for 1 minute, 1 ml of serum-free DMEM for 1 minute again, and the last 7 ml of serum-free DMEM for 3 minutes to the centrifuge tube. Thereafter, the centrifuge tube was centrifuged at 1,000rpm for 10 minutes to collect cells. Make the cells at 1X 10⁶Each cell/ml was suspended in DMEM containing 55. mu.M 2-mercaptoethanol, 100U/ml penicillin, 100. mu.g/ml streptomycin, 15% FCS and 10% hybridoma cloning factor (BioVeris).

The cell suspension thus obtained was seeded at 100. mu.l/well in each well of a 96-well plate, and then in 5% CO₂The culture was carried out in an incubator at 37 ℃. On the following day, 100. mu.l of DMEM containing 2 XHAT (Invitrogen corporation), 15% FCS and 10% hybridoma cloning factor was added to each well, and then the cells were incubated in 5% CO₂The culture was continued in an incubator at 37 ℃.

After 10 to 14 days of culture, the culture supernatant in each reaction well was collected, and the culture supernatant was screened for antibodies recognizing the antigen E2 protein as contained in example 6.

Example 6 screening of hybridoma cells producing antibody binding to antigen E2 protein

Hybridoma cells were screened by immobilizing the antigen E2 protein on a plate, and then evaluating whether antibodies in the culture supernatant of the hybridoma cells bind to the antigen E2 protein immobilized on the plate by EIA.

(1) Preparation of antigen E2 protein-immobilized plate

The 3 XFLAG-J6E 2dTM protein or the J6E2-Fc protein was diluted to 1. mu.g/ml with PBS and 50. mu.l of each product was added to each well of the immunoplate (Nunc). The immunoplates were allowed to stand overnight at 4 ℃ to allow the protein to become immobilized on the plate. The protein solution was removed from each well, 200 μ l of Blocking One solution (NACALAI TESQUE, INC.) prepared according to the included manual was added to each well, followed by Blocking at room temperature for 2 hours.

(2) Screening of hybridoma cells

The above-mentioned blocked protein-immobilized plate was used for screening for an anti-E2 protein antibody contained in the culture supernatant of hybridoma cells. The plate immobilized with J6E2-Fc protein was used to screen for monoclonal antibodies produced by hybridoma cells prepared from mice administered with 3 × flag-J6E2dTM protein. The plate immobilized with 3 XFLAG-J6E 2dTM protein was used to screen monoclonal antibodies produced by hybridoma cells prepared from mice administered with J6E2-Fc protein.

Specifically, the above protein-fixed plates were washed 4 times with PBS containing 0.1% Tween20 (Sigma). A sample of the supernatant of each hybridoma cell obtained in example 5 was added at 50. mu.l/well, and then reacted at room temperature for 1 hour. After completion of the reaction, the wells were washed 4 times with PBS containing 0.1% Tween 20. Subsequently, a5,000-fold dilution of HRP-labeled anti-mouse IgG antibody (GE Healthcare) with PBS containing 0.1% Tween20 was added at 50. mu.l/well for 1 hour at room temperature. After completion of the reaction, the wells were washed 4 times with PBS containing 0.1% Tween20, developed using a peroxidase development kit (Sumitomo Bakelite co., Ltd.), and then absorbance at 450 nm was measured to select positive clones.

As a result, 11 clones were selected with certainty from 980 wells subjected to screening for hybridoma cells prepared from mice administered with 3 XFLAG-J6E 2dTM protein. These clones were cloned by limiting dilution to obtain hybridoma cell lines 1G2-32, 2F2-7, 2F3-7, 4E8-8, 5D4-6, 9G3-2, 9A5-4, 9C4-2, 8D10-3 and 10G4-1 with good proliferation and antibody productivity.

Meanwhile, for hybridoma cells prepared from mice administered with J6E2-Fc protein, 10 clones were selected with certainty from 2064 wells of the screen. Cloning of these clones was performed by limiting dilution, thereby obtaining M1E12-1 hybridoma cell line with good proliferation and antibody production.

(3) Isoform and subtype analysis

The isotypes and subtypes of monoclonal antibodies produced by the hybridoma cells thus obtained were analyzed using the ImmunoPure monoclonal antibody isotypes kit (Pierce) according to the manual contained. As a result, the antibody subtype of each clone is shown in FIG. 6. They are all found to have kappa-immunoglobulin light chains.

(4) Purification of IgG antibodies

The obtained hybridoma cells were finally adapted to serum-free culture by gradually decreasing the FCS concentration in the medium.

The Hybridoma cells were each cultured to confluence in serum-free medium, Hybridoma SFM (Invitrogen Corporation). The culture solution was collected into a centrifuge tube and then centrifuged at 1500 rpm for 5 minutes. Culture supernatants were added to Prosep-g (millipore) and washed with 30 bed volumes (bed volume) of PBS. Subsequently, 6 fractions were eluted with 1 bed volume of 0.1M glycine-HCl (pH 3.0). Immediately after elution, 1M Tris-HCl (pH 9.5) was added to return the pH to neutral. 20 μ l of each fraction was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and non-reducing conditions for fractionation. The presence or absence of the protein was confirmed by staining with Coomassie brilliant blue. The IgG fraction was pooled and then dialyzed against PBS, or demineralized by gel filtration to prepare an antibody sample.

Example 7 HCV genotype specificity of monoclonal antibody against antigen E2 protein

It was examined whether or not the monoclonal antibody produced by each hybridoma cell prepared by immunization with the antigen E2 protein of J6CF strain of HCV2a bound to the E2 protein derived from J6CF strain of genotype 2a and JFH1 strain of genotype 2a, the E2 protein derived from TH strain of genotype 1b, J1 strain of genotype 1b and Conl strain of genotype 1b, and the E2 protein derived from H77 strain of genotype 1 a.

As antigens, THE J6E2-Fc protein, JFH1E2-Fc protein, THE2-Fc protein, J1E2-Fc protein, Con1E2-Fc protein and H77E2-Fc protein prepared in examples 1 to 3, which are fusion proteins of THE antigen E2 protein and THE Fc domain of human immunoglobulin, were used. These proteins were immobilized on plates and then used for evaluation as described in example 6.

Specifically, each of the above fusion proteins was diluted to 1. mu.g/ml with PBS, the diluted solution was added to an immunoplate at 50. mu.l/well, and then the immunoplate was left to stand at 4 ℃ overnight so that each fusion protein was immobilized on the plate. The protein solution was removed and then a Blocking One solution (NACALALI TESSQUE, INC.) prepared according to the manual included was added at 200. mu.l/well followed by Blocking at room temperature for 2 hours.

Next, the monoclonal antibody produced by each hybridoma cell was diluted to 1. mu.g/ml with PBS, added to the above protein-fixed plate at 50. mu.l/well, and then reacted at room temperature for 1 hour. After completion of the reaction, the wells were washed 4 times with PBS containing 0.05% Tween20, 5,000-fold dilution of HRP-labeled anti-mouse IgG antibody with PBS containing 0.05% Tween20 was added at 50 μ l/well, and then reacted at room temperature for 1 hour. After completion of the reaction, the wells were washed 4 times with PBS containing 0.05% Tween20, developed using a peroxidase development kit, and then the absorbance at 450 nm was measured.

FIG. 6 shows the binding of each monoclonal antibody to the antigen E2 protein of various HCV genotypes or strains. For the absorbance value, a value of less than 0.1 is represented by "-", a value of 0.1 or more and less than 0.25 is represented by "+", a value of 0.25 or more and less than 0.4 is represented by "+ +", and a value of 0.4 or more is represented by "+ + +". These values represent the strength of binding to the antigen E2 protein. As shown in FIG. 6, 8D10-3 is an antibody that binds to the antigen E2 protein of HCV genotypes 1a, 1b and 2a, 1G2-32 and 2F2-7 is an antibody that binds to the antigen E2 protein of genotype 2a, and 4E8-8 is an antibody that binds to the antigen E2 protein of genotypes 1b and 2 a. Furthermore, as shown in FIG. 6, M1E12-1 is a monoclonal antibody that binds to the antigen E2 protein of strain J6 CF.

These results indicate that the above monoclonal antibody panels can be used for identifying HCV genotypes or HCV strains.

In addition, on 19.9.2008, hybridoma cell producing monoclonal antibody 8D10-3 (8D 10-3) was deposited under accession number FERM BP-11182, hybridoma cell producing monoclonal antibody 1G2-32 (1G 2-32) was deposited under accession number FERM BP-11179, hybridoma cell producing monoclonal antibody 2F2-7 (2F 2-7) was deposited under accession number FERM BP-11180, hybridoma cell producing monoclonal antibody 4E8-8 (4E 8-8) was deposited under accession number FERM BP-11181, and hybridoma cell producing monoclonal antibody M1E12-1 (M1E 12-1) was deposited under accession number FERM BP-11183 at the national institute of advanced Industrial science and technology, International patent organism depositary (cental 6, 1-1, higashi 1, Tsukuba, Ibaraki, Japan).

(example 8) analysis of epitope of monoclonal antibody

A set of peptides (peptide Nos. 1 to 110) each having an amino acid sequence of 10 consecutive amino acids was synthesized, which was designed to be shifted by three amino acids from the N-terminus in the amino acid sequence of the antigen E2 protein corresponding to amino acid positions 384 to 720, when the initial methionine at the N-terminus of the precursor protein of strain J6CF (SEQ ID NO: 5) was determined as the first amino acid. The N-terminus of each peptide was biotinylated and glycinamide was located at the C-terminus of the peptide (synthesized by the delegated JPT).

The peptides thus synthesized were each dissolved in DMSO and then dissolved in PBS at 0.01 nmol/. mu.l. The peptide solution was added to the streptavidin-coated plate (Nunc) at 50. mu.l/well, and then reacted at room temperature for 2 hours. The peptide solution was discarded, and Blocking was performed by adding a Blocking One solution (NACALALI TESSQUE, INC.) prepared according to the manual contained at 200. mu.l/well, and then keeping the well at 4 ℃ overnight.

Subsequently, the blocking solution was discarded, and the wells were washed 4 times with PBS containing 0.05% Tween20, and then each monoclonal antibody diluted to 1. mu.g/ml with PBS containing 0.05% Tween20 was added at 50. mu.l/well, followed by reaction at room temperature for 1.5 hours. After completion of the reaction, the antibody solution was discarded, the wells were washed 4 times with PBS containing 0.05% Tween20, and a 5000-fold dilution of HRP-labeled anti-mouse IgG goat antibody (GE Healthcare) with PBS containing 0.05% Tween20 was added at 50 μ l/well, followed by reaction at room temperature for 1 hour. After the reaction, the antibody solution was discarded, and the wells were washed 5 times with PBS containing 0.05% Tween 20. After washing, the antibody bound to the peptide can be detected by developing with a peroxidase development kit and then measuring the absorbance at 450 nm.

FIGS. 7A-E show the binding strength of each monoclonal antibody to a peptide derived from the E2 protein, which is the antigen derived from strain J6 CF. High measurements at 0D 450 nm (shown on the vertical axis of FIGS. 7A-E) indicate that the binding strength of the monoclonal antibody to the relevant peptide is strong and that the antibody specifically recognizes the peptide. Each monoclonal antibody recognizes certain peptides from the antigen E2 protein of strain J6 CF.

A particularly strong epitope for monoclonal antibody 8D10-3 was DRCGAPTYTW (SEQ ID NO: 20; peptide No. 47), and GAPTYTWGEN (SEQ ID NO: 21; peptide No. 48) overlapping the epitope peptide (FIG. 7A). From this result, it was considered that the epitope may comprise an amino acid sequence of at least 10 consecutive amino acids in the amino acid sequence DRLGAPTYTWGEN (SEQ ID NO: 22). YPYRLWHYPC (SEQ ID NO: 23; peptide No. 78) is a weak epitope (FIG. 7A).

A particularly strong epitope for monoclonal antibody 4E8-8 is WGENETDVFL (SEQ ID NO: 1; peptide number 50). NETDVFLLNS (SEQ ID NO: 24; peptide No. 51), DVFLLNSTRP (SEQ ID NO: 25; peptide No. 52) and LLNSTRPPLG (SEQ ID NO: 26; peptide No. 53) overlapping with peptide No. 50 are weak epitopes (FIG. 7C). From this result, each epitope was considered to have an amino acid sequence of at least 10 consecutive amino acids in WGENETDVFLLNSTRPPLG (SEQ ID NO: 27).

A particularly strong epitope for monoclonal antibody 2F2-7 was GWGALQYEDN (SEQ ID NO: 2; peptide number 29) (FIG. 7D). FRVGWGALQY (SEQ ID NO: 28; peptide No. 28) that overlaps with peptide No. 29 is a weak epitope (FIG. 7D). From this result, it is considered that each epitope has an amino acid sequence of at least 10 consecutive amino acids in the amino acid sequence FRVGWGALQYEDN (SEQ ID NO: 29).

Particularly strong epitopes of monoclonal antibody 1G2-32 were KTCGAPPCRT (SEQ ID NO: 3; peptide No. 61) and GAPPCRTRAD (SEQ ID NO: 30; peptide No. 62) (FIG. 7B). From this result, each epitope was considered to have an amino acid sequence of at least 10 consecutive amino acids in the amino acid sequence KTCGAPPCRTRAD (SEQ ID NO: 31).

Particularly strong epitopes of monoclonal antibody M1E12-1 were NYTIFKIRMY (SEQ ID NO: 4; peptide No. 82) and IFKIRMYVGG (SEQ ID NO: 32; peptide No. 83) (FIG. 7E). From this result, each epitope was considered to have an amino acid sequence of at least 10 consecutive amino acids in the amino acid sequence NYTIFKIRMYVGG (SEQ ID NO: 33).

Example 9 detection of HCV envelope protein Using monoclonal antibody

Whether or not the antigen E2 protein derived from the H77 strain of genotype 1a, the antigen E2 protein derived from the J6CF strain of genotype 2a and the JFH1 strain of genotype 2a, and the antigen E2 protein derived from the TH strain of genotype 1b, the J1 strain of genotype 1b, and the Con1 strain of genotype 1b were detected using the monoclonal antibody prepared from the hybridoma cells prepared as described above was examined by a sandwich ELISA method and a western blot method.

(1) Sandwich ELISA

Monoclonal antibody 1G2-32 was diluted to 1. mu.g/ml with PBS. The antibody solution was added to the immunoplate (Nunc) at 50. mu.l/well, and the wells were then left to stand at room temperature for 2 hours, allowing the antibody to be immobilized on the plate. The antibody solution was removed, and Blocking One solution (NACALALI TESSQUE, INC.) prepared according to the manual included was added at 200. mu.l/well, and the well was left still at room temperature for 2 hours for Blocking.

Next, each of THE fusion proteins of THE antigen E2 protein and THE human immunoglobulin Fc domain added thereto (i.e., JFH1E2-Fc protein, J6E2-Fc protein, THE2-Fc protein, Con1E2-Fc protein, J1E2-Fc protein, or H77E2-Fc protein) was diluted with PBS, and then added to THE above protein-immobilized plate at 50. mu.l/well, followed by reaction at room temperature for 1.5 hours. After completion of the reaction, the wells were washed 3 times with PBS containing 0.05% Tween 20. Biotinylated 8D10-3 monoclonal antibody diluted to 1. mu.g/ml with PBS containing 0.05% Tween20 was added at 50. mu.l/well, followed by reaction at room temperature for 2 hours. After the reaction, the wells were washed 3 times with PBS containing 0.05% Tween 20. 50. mu.l of HRP-labeled anti-streptavidin (GE Healthcare) diluted 5,000 times with PBS containing 0.05% Tween20 was added, followed by reaction at room temperature for 1.5 hours.

After completion of the reaction, the wells were washed 4 times with PBS containing 0.05% Tween20, developed using a peroxidase development kit (Sumitomo Bakelite co., Ltd.), and then measured for absorbance at 490 nm. The results are shown in FIG. 8.

FIG. 8 shows the detection sensitivity of antigen E2 protein of various genotypes/strain determined by sandwich ELISA using monoclonal antibodies 1G2-32 and 8D 10-3. The horizontal axis indicates the amount of antigen E2 protein, and the vertical axis indicates the absorbance at 490 nm; i.e., the amount of antigen E2 protein detected. The sandwich ELISA using monoclonal antibodies 1G2-32 and 8D10-3 showed that only the antigen E2 protein of genotype 2a of HCV could be detected, not the antigen E2 protein of genotype 1a and genotype 1 b. These results indicate that HCV genotypes or strains can be identified using the panel of antibodies obtained according to the present invention.

(2) Western blotting method

One fifth volume of 5 Xsample buffer (0.3125M Tris-HCl, pH 6.8, 5% SDS 50% glycerol, 0.05% BPB 5% 2-ME) was added to 0.1. mu.g to 0.3. mu.g of each of THE fusion proteins of THE antigen E2 protein and THE Fc domain added thereto (JFH 1E2-Fc protein, J6E2-Fc protein, THE2-Fc protein, Con1E2-Fc protein, J1E2-Fc protein or H77E2-Fc protein), followed by treatment at 100 ℃ for 5 minutes. The product was used as a sample. Each sample was applied to a 4% -20% gradient gel (TEFCO), subjected to electrophoresis using a constant current of 40 mA, and then blotted onto a PVDF membrane using a semi-dry type blotting apparatus at a constant current of 120 mA.

After blotting, the PVDF membrane was immersed in Block Ace (Snow Brand Mill Products Co., Ltd.) for 1 hour at room temperature for blocking, washed with TBS containing 0.1% Tween20, immersed in 8D10-3 monoclonal antibody diluted to 1. mu.g/ml with Can Get Signal (Toyobo Co., Ltd.), and then reacted at room temperature for 1 hour. After the reaction, the membrane was washed with TBS containing 0.1% Tween20, and then immersed in HRP-labeled anti-mouse IgG antibody diluted 5,000 times with Can Get Signal, and then reacted at room temperature for 1 hour. Membranes were washed with TBS containing 0.1% Tween20 and bands were detected using the ECL kit (GE Healthcare).

FIG. 9 shows the detection or non-detection of antigen E2 protein of various genotypes/strains by Western blotting using the 8D10-3 monoclonal antibody. Antigen E2 proteins of all six plant types can be detected using the 8D10-3 monoclonal antibody.

Industrial applicability

The antibody of the present invention makes it possible to easily identify HCV genotypes 1a, 1b and 2a with high accuracy. Thus, hepatitis c patients infected with HCV of genotype 1a or 1b, for which the therapeutic effect of interferon therapy is not expected in advance, can alleviate side effects and can provide an opportunity to select a new therapeutic method.

Sequence listing

<110> Dongli corporation

National institute of infection

<120> antibody binding to envelope protein 2 of hepatitis C virus and method for genotyping hepatitis C virus using the same

<130> PH-4140-PCT

<140> PCT/JP2009/067051

<141> 2009-09-30

<150> JP 2008-254338

<151> 2008-09-30

<160> 33

<170> PatentIn version 3.1

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Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys

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Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Val Val Gly Ala Pro Leu

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Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp

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Gly Val Asn Phe Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile

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Phe Leu Leu Ala Leu Leu Ser Cys Ile Thr Thr Pro Val Ser Ala Ala

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Glu Val Lys Asn Ile Ser Thr Gly Tyr Met Val Thr Asn Asp Cys Thr

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Asn Asp Ser Ile Thr Trp Gln Leu Gln Ala Ala Val Leu His Val Pro

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Gly Cys Val Pro Cys Glu Lys Val Gly Asn Ala Ser Gln Cys Trp Ile

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Pro Val Ser Pro Asn Val Ala Val Gln Arg Pro Gly Ala Leu Thr Gln

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Gly Leu Arg Thr His Ile Asp Met Val Val Met Ser Ala Thr Leu Cys

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Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Gly Val Met Leu Ala Ala

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Gln Met Phe Ile Val Ser Pro Gln His His Trp Phe Val Gln Asp Cys

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Asn Cys Ser Ile Tyr Pro Gly Thr Ile Thr Gly His Arg Met Ala Trp

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Asp Met Met Met Asn Trp Ser Pro Thr Ala Thr Met Ile Leu Ala Tyr

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Ala Met Arg Val Pro Glu Val Ile Ile Asp Ile Ile Ser Gly Ala His

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Trp Gly Val Met Phe Gly Leu Ala Tyr Phe Ser Met Gln Gly Ala Trp

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Ala Lys Val Val Val Ile Leu Leu Leu Ala Ala Gly Val Asp Ala Arg

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Thr His Thr Val Gly Gly Ser Ala Ala Gln Thr Thr Gly Arg Leu Thr

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Ser Leu Phe Asp Met Gly Pro Arg Gln Lys Ile Gln Leu Val Asn Thr

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Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser

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Leu His Thr Gly Phe Ile Ala Ser Leu Phe Tyr Thr His Ser Phe Asn

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Ser Ser Gly Cys Pro Glu Arg Met Ser Ala Cys Arg Ser Ile Glu Ala

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Phe Arg Val Gly Trp Gly Ala Leu Gln Tyr Glu Asp Asn Val Thr Asn

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Pro Glu Asp Met Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Gln Cys

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Gly Val Val Ser Ala Lys Thr Val Cys Gly Pro Val Tyr Cys Phe Thr

500 505 510

Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Leu Gly Ala Pro Thr

515 520 525

Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr

530 535 540

Arg Pro Pro Leu Gly Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Ser

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Gly Tyr Thr Lys Thr Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp

565 570 575

Phe Asn Ala Ser Thr Asp Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys

580 585 590

His Pro Asp Thr Thr Tyr Leu Lys Cys Gly Ser Gly Pro Trp Leu Thr

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Pro Arg Cys Leu Ile Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys

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Thr Val Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val

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Glu His Arg Leu Thr Ala Ala Cys Asn Phe Thr Arg Gly Asp Arg Cys

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Asn Leu Glu Asp Arg Asp Arg Ser Gln Leu Ser Pro Leu Leu His Ser

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Thr Thr Glu Trp Ala Ile Leu Pro Cys Ser Tyr Ser Asp Leu Pro Ala

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Leu Ser Thr Gly Leu Leu His Leu His Gln Asn Ile Val Asp Val Gln

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Phe Met Tyr Gly Leu Ser Pro Ala Leu Thr Lys Tyr Ile Val Arg Trp

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Glu Trp Val Ile Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys

725 730 735

Ala Cys Leu Trp Met Leu Ile Leu Leu Gly Gln Ala Glu Ala Ala Leu

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Glu Lys Leu Val Ile Leu His Ala Ala Ser Ala Ala Ser Cys Asn Gly

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Phe Leu Tyr Phe Val Ile Phe Phe Val Ala Ala Trp Tyr Ile Lys Gly

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Arg Val Val Pro Leu Ala Thr Tyr Ser Leu Thr Gly Leu Trp Ser Phe

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Ser Leu Leu Leu Leu Ala Leu Pro Gln Gln Ala Tyr Ala Tyr Asp Ala

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Ser Val His Gly Gln Ile Gly Ala Ala Leu Leu Val Met Ile Thr Leu

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Phe Thr Leu Thr Pro Gly Tyr Lys Thr Leu Leu Ser Arg Phe Leu Trp

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Trp Leu Cys Tyr Leu Leu Thr Leu Gly Glu Ala Met Val Gln Glu Trp

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Ala Pro Pro Met Gln Val Arg Gly Gly Arg Asp Gly Ile Ile Trp Ala

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Val Ala Ile Phe Tyr Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu

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Leu Ala Val Leu Gly Pro Ala Tyr Leu Leu Lys Gly Ala Leu Thr Arg

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Val Pro Tyr Phe Val Arg Ala His Ala Leu Leu Arg Met Cys Thr Met

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Ala Arg His Leu Ala Gly Gly Arg Tyr Val Gln Met Ala Leu Leu Ala

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Leu Gly Arg Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met

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Ser Asp Trp Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu

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Pro Ile Ile Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala

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Glu Thr Ala Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala

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Arg Leu Gly Arg Glu Val Leu Leu Gly Pro Ala Asp Gly Tyr Thr

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Ser Lys Gly Trp Ser Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln

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Gln Thr Arg Gly Leu Leu Gly Thr Ile Val Val Ser Met Thr Gly

1040 1045 1050

Arg Asp Lys Thr Glu Gln Ala Gly Glu Ile Gln Val Leu Ser Thr

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Val Thr Gln Ser Phe Leu Gly Thr Ser Ile Ser Gly Val Leu Trp

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Thr Val Tyr His Gly Ala Gly Asn Lys Thr Leu Ala Gly Ser Arg

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Gly Pro Val Thr Gln Met Tyr Ser Ser Ala Glu Gly Asp Leu Val

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Gly Trp Pro Ser Pro Pro Gly Thr Lys Ser Leu Glu Pro Cys Thr

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Cys Gly Ala Val Asp Leu Tyr Leu Val Thr Arg Asn Ala Asp Val

1130 1135 1140

Ile Pro Ala Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu Ser

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Pro Arg Pro Leu Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val

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Leu Cys Pro Arg Gly His Ala Val Gly Val Phe Arg Ala Ala Val

1175 1180 1185

Cys Ser Arg Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu

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Thr Leu Asp Ile Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser

1205 1210 1215

Thr Pro Pro Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His

1220 1225 1230

Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr

1235 1240 1245

Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala

1250 1255 1260

Ala Thr Leu Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile

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Asn Pro Asn Ile Arg Thr Gly Val Arg Thr Val Thr Thr Gly Ala

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Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly

1295 1300 1305

Cys Ala Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His

1310 1315 1320

Ala Val Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp

1325 1330 1335

Gln Ala Glu Thr Ala Gly Val Arg Leu Thr Val Leu Ala Thr Ala

1340 1345 1350

Thr Pro Pro Gly Ser Val Thr Thr Pro His Pro Asn Ile Glu Glu

1355 1360 1365

Val Ala Leu Gly Gln Glu Gly Glu Ile Pro Phe Tyr Gly Arg Ala

1370 1375 1380

Ile Pro Leu Ser Tyr Ile Lys Gly Gly Arg His Leu Ile Phe Cys

1385 1390 1395

His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly

1400 1405 1410

Met Gly Leu Asn Ser Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser

1415 1420 1425

Val Ile Pro Thr Gln Gly Asp Val Val Val Val Ala Thr Asp Ala

1430 1435 1440

Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys

1445 1450 1455

Asn Val Ala Val Thr Gln Val Val Asp Phe Ser Leu Asp Pro Thr

1460 1465 1470

Phe Thr Ile Thr Thr Gln Ile Val Pro Gln Asp Ala Val Ser Arg

1475 1480 1485

Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Leu Gly Ile Tyr

1490 1495 1500

Arg Tyr Val Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser

1505 1510 1515

Val Val Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu

1520 1525 1530

Leu Thr Pro Ser Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn

1535 1540 1545

Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu

1550 1555 1560

Ala Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser

1565 1570 1575

Gln Thr Lys Gln Ser Gly Glu Asn Phe Ala Tyr Leu Thr Ala Tyr

1580 1585 1590

Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp

1595 1600 1605

Asp Val Met Trp Lys Cys Leu Thr Arg Leu Lys Pro Thr Leu Val

1610 1615 1620

Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ser Val Thr Asn Glu

1625 1630 1635

Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr Cys Met

1640 1645 1650

Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly

1655 1660 1665

Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys

1670 1675 1680

Val Cys Ile Ile Gly Arg Leu His Ile Asn Gln Arg Ala Val Val

1685 1690 1695

Ala Pro Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu

1700 1705 1710

Glu Cys Ala Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile

1715 1720 1725

Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala

1730 1735 1740

Ser Lys Gln Ala Gln Asp Ile Gln Pro Thr Val Gln Ala Ser Trp

1745 1750 1755

Pro Lys Val Glu Gln Phe Trp Ala Lys His Met Trp Asn Phe Ile

1760 1765 1770

Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn

1775 1780 1785

Pro Ala Val Ala Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser

1790 1795 1800

Pro Leu Ser Thr Ser Thr Thr Ile Leu Leu Asn Ile Leu Gly Gly

1805 1810 1815

Trp Leu Ala Ser Gln Ile Ala Pro Pro Ala Gly Ala Thr Gly Phe

1820 1825 1830

Val Val Ser Gly Leu Val Gly Ala Ala Val Gly Ser Ile Gly Leu

1835 1840 1845

Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Ile

1850 1855 1860

Ser Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys Pro

1865 1870 1875

Ser Met Glu Asp Val Val Asn Leu Leu Pro Gly Ile Leu Ser Pro

1880 1885 1890

Gly Ala Leu Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg

1895 1900 1905

His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu

1910 1915 1920

Ile Ala Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr

1925 1930 1935

Val Thr Glu Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly

1940 1945 1950

Ser Leu Thr Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile

1955 1960 1965

Thr Glu Asp Cys Pro Ile Pro Cys Gly Gly Ser Trp Leu Arg Asp

1970 1975 1980

Val Trp Asp Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp

1985 1990 1995

Leu Thr Ser Lys Leu Phe Pro Lys Met Pro Gly Leu Pro Phe Val

2000 2005 2010

Ser Cys Gln Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Ile

2015 2020 2025

Met Thr Thr Arg Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val

2030 2035 2040

Arg Leu Gly Ser Met Arg Ile Thr Gly Pro Lys Thr Cys Met Asn

2045 2050 2055

Ile Trp Gln Gly Thr Phe Pro Ile Asn Cys Tyr Thr Glu Gly Gln

2060 2065 2070

Cys Val Pro Lys Pro Ala Pro Asn Phe Lys Val Ala Ile Trp Arg

2075 2080 2085

Val Ala Ala Ser Glu Tyr Ala Glu Val Thr Gln His Gly Ser Tyr

2090 2095 2100

His Tyr Ile Thr Gly Leu Thr Thr Asp Asn Leu Lys Val Pro Cys

2105 2110 2115

Gln Leu Pro Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln

2120 2125 2130

Ile His Arg Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu

2135 2140 2145

Val Ser Phe Cys Val Gly Leu Asn Ser Phe Val Val Gly Ser Gln

2150 2155 2160

Leu Pro Cys Asp Pro Glu Pro Asp Thr Asp Val Leu Met Ser Met

2165 2170 2175

Leu Thr Asp Pro Ser His Ile Thr Ala Glu Thr Ala Ala Arg Arg

2180 2185 2190

Leu Ala Arg Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Ala Ser

2195 2200 2205

Gln Leu Ser Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Gly

2210 2215 2220

Lys Ala Tyr Asp Val Asp Met Val Asp Ala Asn Leu Phe Met Gly

2225 2230 2235

Gly Asp Val Thr Arg Ile Glu Ser Gly Ser Lys Val Val Val Leu

2240 2245 2250

Asp Ser Leu Asp Pro Met Val Glu Glu Arg Ser Asp Leu Glu Pro

2255 2260 2265

Ser Ile Pro Ser Glu Tyr Met Leu Pro Lys Lys Arg Phe Pro Pro

2270 2275 2280

Ala Leu Pro Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val

2285 2290 2295

Glu Ser Trp Lys Arg Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly

2300 2305 2310

Cys Ala Leu Pro Pro Pro Arg Lys Thr Pro Thr Pro Pro Pro Arg

2315 2320 2325

Arg Arg Arg Thr Val Gly Leu Ser Glu Asp Ser Ile Gly Asp Ala

2330 2335 2340

Leu Gln Gln Leu Ala Ile Lys Ser Phe Gly Gln Pro Pro Pro Ser

2345 2350 2355

Gly Asp Ser Gly Leu Ser Thr Gly Ala Gly Ala Ala Asp Ser Gly

2360 2365 2370

Ser Gln Thr Pro Pro Asp Glu Leu Ala Leu Ser Glu Thr Gly Ser

2375 2380 2385

Ile Ser Ser Met Pro Pro Leu Glu Gly Glu Leu Gly Asp Pro Asp

2390 2395 2400

Leu Glu Pro Glu Gln Val Glu Pro Gln Pro Pro Pro Gln Gly Gly

2405 2410 2415

Val Ala Ala Pro Gly Ser Asp Ser Gly Ser Trp Ser Thr Cys Ser

2420 2425 2430

Glu Glu Asp Asp Ser Val Val Cys Cys Ser Met Ser Tyr Ser Trp

2435 2440 2445

Thr Gly Ala Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu

2450 2455 2460

Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys

2465 2470 2475

Val Tyr Cys Thr Thr Thr Lys Ser Ala Ser Leu Arg Ala Lys Lys

2480 2485 2490

Val Thr Phe Asp Arg Met Gln Val Leu Asp Ser Tyr Tyr Asp Ser

2495 2500 2505

Val Leu Lys Asp Ile Lys Leu Ala Ala Ser Lys Val Thr Ala Arg

2510 2515 2520

Leu Leu Thr Met Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser

2525 2530 2535

Ala Arg Ser Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu

2540 2545 2550

Ser Gly Arg Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu

2555 2560 2565

Leu Glu Asp Ser Glu Thr Pro Ile Pro Thr Thr Ile Met Ala Lys

2570 2575 2580

Asn Glu Val Phe Cys Val Asp Pro Thr Lys Gly Gly Lys Lys Ala

2585 2590 2595

Ala Arg Leu Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu

2600 2605 2610

Lys Met Ala Leu Tyr Asp Ile Thr Gln Lys Leu Pro Gln Ala Val

2615 2620 2625

Met Gly Ala Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val

2630 2635 2640

Glu Phe Leu Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro Met Gly

2645 2650 2655

Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg

2660 2665 2670

Asp Ile Arg Thr Glu Glu Ser Ile Tyr Arg Ala Cys Ser Leu Pro

2675 2680 2685

Glu Glu Ala His Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr

2690 2695 2700

Val Gly Gly Pro Met Phe Asn Ser Lys Gly Gln Thr Cys Gly Tyr

2705 2710 2715

Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn

2720 2725 2730

Thr Ile Thr Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala

2735 2740 2745

Gly Ile Ile Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val

2750 2755 2760

Val Ile Ser Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu

2765 2770 2775

Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly

2780 2785 2790

Asp Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys

2795 2800 2805

Ser Ser Asn Val Ser Val Ala Leu Gly Pro Gln Gly Arg Arg Arg

2810 2815 2820

Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Ile Ala Arg Ala Ala

2825 2830 2835

Trp Glu Thr Val Arg His Ser Pro Val Asn Ser Trp Leu Gly Asn

2840 2845 2850

Ile Ile Gln Tyr Ala Pro Thr Ile Trp Ala Arg Met Val Leu Met

2855 2860 2865

Thr His Phe Phe Ser Ile Leu Met Ala Gln Asp Thr Leu Asp Gln

2870 2875 2880

Asn Leu Asn Phe Glu Met Tyr Gly Ala Val Tyr Ser Val Ser Pro

2885 2890 2895

Leu Asp Leu Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala

2900 2905 2910

Phe Ser Leu His Thr Tyr Thr Pro His Glu Leu Thr Arg Val Ala

2915 2920 2925

Ser Ala Leu Arg Lys Leu Gly Ala Pro Pro Leu Arg Ala Trp Lys

2930 2935 2940

Ser Arg Ala Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly

2945 2950 2955

Arg Ala Ala Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys

2960 2965 2970

Thr Lys Leu Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp

2975 2980 2985

Leu Ser Ser Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Tyr

2990 2995 3000

His Ser Val Ser Arg Ala Arg Pro Arg Leu Leu Leu Phe Gly Leu

3005 3010 3015

Leu Leu Leu Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg

3020 3025 3030

<210> 6

<211> 28

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 6

cacaagcttc gcacccatac tgttgggg 28

<210> 7

<211> 32

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 7

gctctagatt accatcggac gatgtatttt gt 32

<210> 8

<211> 28

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 8

cacaagcttc gcacccatac tgttgggg 28

<210> 9

<211> 30

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 9

acaggatccc atcggacgat gtattttgtg 30

<210> 10

<211> 28

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 10

cacaagcttg gcaccaccac cgttggag 28

<210> 11

<211> 33

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 11

acaggatcct cccatcgaac gacgtatttt gtg 33

<210> 12

<211> 32

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 12

caaagcttgc gacctacgtg acgggggggt cg 32

<210> 13

<211> 43

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 13

cctctagatt atggatccca tttgattgca taggagacaa ccg 43

<210> 14

<211> 34

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 14

caaagcttgg aacctatgtg acagggggga cgat 34

<210> 15

<211> 41

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 15

cctctagatt atggatccca tttgattgca aaggagacaa c 41

<210> 16

<211> 33

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 16

caaagcttca tacccgcgtg acgggggggg tgc 33

<210> 17

<211> 42

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 17

cctctagatt atggatccca cttgatggca atggagacga cc 42

<210> 18

<211> 31

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 18

caaagcttga aacccacgtc accgggggaa a 31

<210> 19

<211> 41

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 19

cctctagatt atggatccca cttaatggcc caggacgcga t 41

<210> 20

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 20

Asp Arg Leu Gly Ala Pro Thr Tyr Thr Trp

1 5 10

<210> 21

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 21

Gly Ala Pro Thr Tyr Thr Trp Gly Glu Asn

1 5 10

<210> 22

<211> 13

<212> PRT

<213> hepatitis C Virus

<400> 22

Asp Arg Leu Gly Ala Pro Thr Tyr Thr Trp Gly Glu Asn

1 5 10

<210> 23

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 23

Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys

1 5 10

<210> 24

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 24

Asn Glu Thr Asp Val Phe Leu Leu Asn Ser

1 5 10

<210> 25

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 25

Asp Val Phe Leu Leu Asn Ser Thr Arg Pro

1 5 10

<210> 26

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 26

Leu Leu Asn Ser Thr Arg Pro Pro Leu Gly

1 5 10

<210> 27

<211> 19

<212> PRT

<213> hepatitis C Virus

<400> 27

Trp Gly Glu Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr Arg Pro

1 5 10 15

Pro Leu Gly

<210> 28

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 28

Phe Arg Val Gly Trp Gly Ala Leu Gln Tyr

1 5 10

<210> 29

<211> 13

<212> PRT

<213> hepatitis C Virus

<400> 29

Phe Arg Val Gly Trp Gly Ala Leu Gln Tyr Glu Asp Asn

1 5 10

<210> 30

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 30

Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp

1 5 10

<210> 31

<211> 13

<212> PRT

<213> hepatitis C Virus

<400> 31

Lys Thr Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp

1 5 10

<210> 32

<211> 10

<212> PRT

<213> hepatitis C Virus

<400> 32

Ile Phe Lys Ile Arg Met Tyr Val Gly Gly

1 5 10

<210> 33

<211> 13

<212> PRT

<213> hepatitis C Virus

<400> 33

Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly

1 5 10

Claims (8)

1. A monoclonal antibody that specifically binds to envelope protein 2 of the hepatitis c virus of genotype 2a, but does not immunoreact with envelope protein 2 of the hepatitis c virus of genotypes 1a and 1b, and that binds SEQ ID NO: 2 is recognized as an epitope, and the monoclonal antibody is produced by a hybridoma cell line having the accession number FERM BP-11180.

2. A monoclonal antibody that specifically binds to envelope protein 2 of the hepatitis c virus of genotype 2a, but does not immunoreact with envelope protein 2 of the hepatitis c virus of genotypes 1a and 1b, and that binds SEQ ID NO: 3 as an epitope, produced by a hybridoma cell line having the accession number FERM BP-11179.

3. A monoclonal antibody that specifically binds to envelope protein 2 of hepatitis c virus of genotype 2a but does not immunoreactive with envelope protein 2 of hepatitis c virus of genotypes 1a and 1b, and specifically binds to envelope protein 2 of strain J6CF but does not immunoreactive with envelope protein 2 of strain JFH1, and which binds to SEQ ID NO: 4 as an epitope, produced by a hybridoma cell line having the accession number FERM BP-11183.

4. A method for identifying a hepatitis c virus genotype for non-diagnostic and therapeutic purposes, wherein:

the genotype of the hepatitis c virus is determined as genotype 1b if the virus binds to an antibody produced by a hybridoma cell line having accession number FERM BP-11181, but does not bind to any of the antibodies according to claims 1 and 2;

the genotype of the hepatitis c virus is determined as genotype 2a if the virus binds to an antibody produced by a hybridoma cell line with accession number FERM BP-11181 and binds to an antibody according to claims 1 and 2; and

the genotype of the hepatitis c virus is determined as genotype 1a if the virus binds to antibodies produced by the hybridoma cell line with accession number FERM BP-11182, but does not bind to any of the antibodies according to claims 1 and 2 and to antibodies produced by the hybridoma cell line with accession number FERM BP-11181.

5. Use of an antibody produced by a hybridoma cell line having accession number FERM BP-11181, an antibody according to claim 1, an antibody according to claim 2 and an antibody produced by a hybridoma cell line having accession number FERM BP-11182 in the manufacture of a medicament for identifying a hepatitis c virus genotype, wherein said identification comprises:

determining the genotype of said hepatitis c virus as genotype 1b if the virus binds to an antibody produced by a hybridoma cell line having the accession number FERM BP-11181, but does not bind to any of the antibodies according to claims 1 and 2;

determining the genotype of said hepatitis c virus as genotype 2a if the virus binds to an antibody produced by a hybridoma cell line having accession number FERM BP-11181 and binds to an antibody according to claims 1 and 2; and

genotyping said hepatitis c virus as genotype 1a if said virus binds to antibodies produced by a hybridoma cell line with accession number FERM BP-11182, but does not bind to any of the antibodies according to claims 1 and 2 and to antibodies produced by a hybridoma cell line with accession number FERM BP-11181.

6. A hybridoma cell line having the accession number FERM BP-11180.

7. A hybridoma cell line having the accession number FERM BP-11179.

8. A hybridoma cell line having the accession number FERM BP-11183.