High affinity monoclonal antibodies to genotype 3 and genotype 6 hcv core proteins and uses thereof

Abstract

The application discloses a monoclonal antibody or antigen-binding fragment thereof with high affinity to recognize core proteins of HCV genotype 3 and genotype 6. The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody or antigen-binding fragment thereof can be used for qualitative and quantitative detection of hepatitis C virus core antigen (HCV cAg).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monoclonal antibody with high affinity that recognizes the core proteins of HCV genotypes 3 and 6 and its applications. Background Technology

[0002] Based on the different detection targets, HCV test kits are mainly divided into three categories: anti-hepatitis C virus antibody (HCV-Ab) test kits, hepatitis C virus nucleic acid (HCV-RNA) test kits, and hepatitis C virus core antigen (HCV-cAg) test kits.

[0003] HCV-Ab test kits target anti-hepatitis C virus antibodies. Anti-hepatitis C virus antibodies are immunoglobulins produced by the immune system of infected individuals after HCV infection. However, HCV-Abs appear late, often more than 70 days after infection, resulting in a long window period. Furthermore, the HCV-Abs produced persist in the body for a long time, potentially for life in some patients. Therefore, while HCV-Ab testing can indicate HCV infection, it can miss cases where antibodies have not yet been produced in the early stages, and it cannot distinguish between current and past infections. In addition, immunosuppressed patients and those undergoing hemodialysis may have persistently negative HCV antibody results, increasing the likelihood of missed detection.

[0004] HCV-RNA detection reagents target hepatitis C virus nucleic acid. HCV genome replication occurs early after infection, with viremia appearing within days, and HCV-RNA can be detected in approximately 1-2 weeks. Therefore, HCV-RNA detection is the "gold standard" for diagnosing HCV infection, and also provides strong evidence of active viral replication and guides clinical treatment. Compared to HCV-Ab testing, HCV-RNA testing significantly shortens the window period from 8-11 weeks to 2 weeks, enabling early diagnosis. However, HCV-RNA detection technology places high demands on instruments, equipment, and personnel.

[0005] The HCV-cAg test kit targets the hepatitis C virus core antigen. The HCV genome encodes 10 viral proteins, with the core protein being one of the three structural proteins. Its amino acid sequence is conserved across different HCV genotypes. The mature protein lacks glycosylation sites, exhibits strong antigenicity, and is a good biomarker for immunoassay. The HCV core protein is released from a multiprotein by signal peptidase; it is a 23 kDa precursor protein containing 191 amino acids. This precursor core protein is subsequently processed by signal peptidase, which removes a portion of the C-terminal peptide, producing the mature core protein containing 177 amino acids. The HCV core antigen is an important marker of early HCV infection, appearing almost simultaneously with HCV-RNA, with an average detection time only about 1-2 days later. Similar to HCV-RNA detection, HCV-cAg detection can significantly shorten the window period, enabling early diagnosis. Because HCV-cAg test kits are both fast and convenient, and have the advantage of early diagnosis comparable to HCV-RNA, HCV-cAg testing has become the preferred alternative method for medical institutions when HCV-RNA testing is not available. However, meta-analyses of multiple studies have shown that while existing HCV-cAg detection reagents have a very high specificity of 99.4%, their sensitivity is low at 87.1%, and the positive concordance rate with HCV RNA detection is approximately 87% (Hsin-Yun Sun, Wang-Da Liu, Chih-WenWang, et al. Performance of Hepatitis C Virus (HCV) Core Antigen Assay in the Diagnosis of Recently Acquired HCV Infection among High-Risk Populations. Microbiol Spectr. 2022; 10(3): e00345-22. Published online 2022 May 17. doi:10.1128 / spectrum.00345-22). The reasons for this are as follows:

[0006] First, the distribution of HCV genotypes has changed significantly, with a significant increase in the proportion of genotype 3 and genotype 6 hepatitis C virus in my country. Previous studies have shown (Yu Xiang, Xiao-fei Lai, Pu Chen, Yang Yang. The correlation of HCV RNA and HCV core antigen in different genotypes of HCV. JClin Lab Anal. 2019 Jan; 33(1): e22632. Published online 2018 Aug 1. doi:10.1002 / jcla.22632.) that the expression level of HCV core antigen varies significantly among different genotypes. The expression level of core protein in genotype 1b (~1710 fmol / L) is significantly higher than that in genotype 3b (~60 fmol / L), genotype 6a (~780 fmol / L), and mixed genotype 1b / 3b (~460 fmol / L). For genotype 1b, HCV RNA load is positively correlated with HCV cAg expression level, while there is no correlation between HCV RNA and HCV cAg in other genotypes, resulting in a lower detection rate of HCV-cAg than that of HCV RNA. Therefore, HCV cAg detection reagents need to further improve their sensitivity to cope with the significant increase in the proportion of genotypes with low HCV cAg expression levels.

[0007] Second, HCV is an RNA virus that mutates very easily, resulting in high antigenic variability due to its high gene mutation rate. Although the core protein is the most conserved protein in HCV, there are still nearly 10% differences in the amino acid sequence of the core protein among different genotypes. HCV-cAg detection reagents are based on a double-antibody sandwich method and all use specific monoclonal antibodies. Mutations in key epitope amino acids may lead to a decrease in the antibody's ability to recognize and bind to the antigen. For example, some literature reports that mutations in the 49th amino acid of the HCV core protein reduce the sensitivity of antigen detection (Li Peng. Current status of HCV core antigen detection technology. Clinical Transfusion and Laboratory Medicine, 2011, 13(2): 191-192. DOI: 10.3969 / J.issn.1671-2587.2011.02.0420). Other studies have shown that the limit of detection of the three antigen reagents is affected by the amino acid polymorphism in the core region of HCV genotype 3 (especially subtype 3b) strains (Yang Ruifeng, Liu Ning, Bian Chengrong, et al. Multicenter performance evaluation of various HCV core antigen reagents for clinical infection screening. Chinese Journal of Laboratory Medicine, 2023, 46(12): 1305-1312). DOI: 10.3760 / cma.j.cn114452-20230718-00014). Therefore, in response to the significant increase in the proportion of genotype 3 and genotype 6 hepatitis C virus, it is necessary to increase the number of antibodies with high affinity for the core protein of genotype 3 and genotype 6 HCV, and further improve the sensitivity of existing HCV cAg antigen detection reagents.

[0008] Therefore, the present invention aims to prepare monoclonal antibodies with high affinity for the core proteins of genotype 3 and genotype 6 HCV by using the common dominant epitope sequence of the HCV core protein as an immunogen and combining it with rapid reaction screening technology, in order to further improve the sensitivity of existing hepatitis C virus core antigen (HCV cAg) detection reagents. Summary of the Invention

[0009] Therefore, the purpose of this invention is to provide a monoclonal antibody prepared using a fused hybridoma cell line that has high affinity for both HCV genotype 3 and genotype 6 core proteins. Furthermore, the monoclonal antibody obtained through experiments also has high affinity for HCV genotype 1 and genotype 2 core proteins. Based on capturing traditional HCV genotype core proteins, it enhances the detection capability of newly emerging HCV genotype core proteins, and thus can be used for the qualitative and quantitative detection of hepatitis C virus core antigen (HCV cAg).

[0010] Therefore, one aspect of the present invention relates to a monoclonal antibody or its antigen-binding fragment having high affinity for both HCV genotype 3 and genotype 6 core proteins, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein...

[0011] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence having one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.2;

[0012] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence having one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.3;

[0013] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.4;

[0014] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.6;

[0015] The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence with one conserved amino acid substitution compared to it.

[0016] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.7.

[0017] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1 and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0018] The present invention also relates to the above-mentioned monoclonal antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments are Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies or humanized antibodies. These antibodies or antigen-binding fragments, because they retain the variable regions of the light chain and heavy chain, or only retain the variable region of the heavy chain, are able to recognize HCV genotype 3 and genotype 6 core proteins, as well as HCV genotype 1 and genotype 2 core proteins with high affinity.

[0019] Furthermore, this invention relates to a nucleic acid molecule comprising a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment, and an expression vector comprising the aforementioned nucleic acid molecule, the expression vector being capable of expressing the aforementioned antibody or its antigen-binding fragment. This invention also relates to a recombinant comprising the aforementioned nucleic acid molecule or the aforementioned expression vector, which can produce the aforementioned antibody or its antigen-binding fragment. On another front, this invention relates to a monoclonal antibody hybridoma cell line with high affinity for HCV genotype 3 and genotype 6 core proteins, the monoclonal antibody hybridoma cell line secreting the aforementioned monoclonal antibody. Further, this invention relates to a monoclonal antibody hybridoma cell line with high affinity for both HCV genotype 3 and genotype 6 core proteins, the monoclonal antibody hybridoma cell line being mouse hybridoma cell line 1014, with accession number CGMCC No. 46562.

[0020] Furthermore, this invention relates to the application of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of products for detecting hepatitis C virus core antigen (HCV cAg). More specifically, this invention relates to a kit for detecting hepatitis C virus core antigen (HCV cAg), the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for high-affinity binding of HCV cAg; preferably, the kit is a double-antibody sandwich assay kit, wherein the monoclonal antibody or its antigen-binding fragment serves as a capture antibody.

[0021] Instructions for the Preservation of Biological Materials

[0022] The monoclonal antibody hybridoma cell line of this invention has been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the registration number CGMCC No. 46562, on July 16, 2025, and classified as a mouse hybridoma cell line. The address of the China General Microbiological Culture Collection Center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0023] Figure 1 This is an amino acid sequence alignment analysis of the core proteins of four HCV gene subtypes.

[0024] Figure 2 This is an SDS-PAGE electrophoresis image showing the expression of HCV core protein in prokaryotes, where each label is: M is the marker, and 1b, 2a, 3a, 3b, 6a, and 6n are HCV genotypes, respectively.

[0025] Figure 3 This is a graph showing the limit of detection (LOD) results for anti-HCV core protein monoclonal antibodies, where A represents monoclonal antibody 1014 and B represents commercially available antibodies.

[0026] Figure 4 This is a diagram showing the identification results of the 1014 subtype of the anti-HCV core protein monoclonal antibody. Detailed Implementation

[0027] The purpose of this invention is to provide a high-affinity monoclonal antibody that recognizes the core proteins of HCV genotypes 3 and 6, prepared using a fused hybridoma cell line. The specific preparation process involves first performing multiple sequence alignment (MSA) on the amino acid sequences of the core proteins of HCV genotypes 3a and 3b (which have shown an increasing prevalence in recent years) and genotypes 6a and 6n (which have shown an increasing prevalence in recent years) to identify common sequences. Subsequently, B-cell epitope analysis is used to screen for common dominant epitope sequences with predictive scores. These sequences are then used as immunogens to immunize mice to prepare monoclonal antibodies. Using the core proteins of six common HCV genotypes expressed in prokaryotes, and combining this with a rapid high-affinity monoclonal antibody screening technique, a high-affinity monoclonal antibody recognizing the HCV core proteins is obtained. This monoclonal antibody can bind not only to the core proteins of HCV genotypes 3 and 6 with high affinity, but also to the core proteins of HCV genotypes 1 and 2 with high affinity. The mouse hybridoma cell line secreting this monoclonal antibody, named 1014, exhibits very high affinity for HCV core proteins, particularly those of genotypes 3 and 6. This cell line was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 16, 2025, with accession number CGMCC No. 46562. In this invention, high affinity refers to the antibody's ability to rapidly recognize antigenic epitopes and exhibit high binding strength, typically manifested as the formation of a more stable complex and a lower limit of detection for the antigen.

[0028] Subsequently, the inventors sequenced and analyzed the immunoglobulin domain sequence of the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No. 46562, and found that its heavy chain variable region amino acid sequence was: QVQLEESGPGLVQPSQSLSIICIVSGFSLITYGEHWVCQSPGKGLEWLGVIWSGGSTDRNAAFISRLSINKDNSKSQVFFKMNSLQGNDTARYYCASIGTTGGELWTTGVKEPQSLSP (SEQ ID NO.1), of which the heavy chain CDR1 amino acid sequence was GFSLITYG (SEQ ID NO.2); the heavy chain CDR2 amino acid sequence was IWSGGST (SEQ ID NO.3); and the heavy chain CDR3 amino acid sequence was ASIGTTGGELWTT (SEQ ID NO.4). The amino acid sequence of the light chain variable region is: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGAPSWKS (SEQ ID NO.5), of which the amino acid sequence of the light chain CDR is KSVSTSGYSY (SEQ ID NO.6); the amino acid sequence of the light chain CDR2 is LVS; and the amino acid sequence of the light chain CDR3 is QHIRELTR (SEQ ID NO.7).

[0029] The inventors evaluated the binding affinity of the aforementioned monoclonal antibody to the core proteins of different HCV genotypes using enzyme-linked immunosorbent assay (ELISA). They found that this monoclonal antibody not only binds to the core proteins of HCV genotypes 3 and 6 with high affinity, but also to the core proteins of HCV genotypes 1 and 2 with high affinity. ELISA testing of clinical samples showed that the monoclonal antibody of this invention can significantly improve the positive detection rate of HCV genotype 3 and genotype 6 infected samples. This monoclonal antibody binds to the core proteins of HCV genotypes 1, 2, 3, and 6 with high affinity, enhancing the detection capability of newly emerging HCV genotype core proteins in addition to detecting core proteins of traditionally prevalent HCV genotypes, thus reducing false negative results.

[0030] As is well known in the art, the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens. Furthermore, a conserved substitution of a single amino acid in the CDR region may not alter the protein structure; therefore, a single amino acid substitution within these regions may still possess the ability to bind to the corresponding antigen. Thus, monoclonal antibodies or their antigen-binding fragments obtained by making a conserved substitution of a single amino acid in heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize the HCV core protein. In this patent application, the term "conservative substitution of amino acids" refers to the substitution of one amino acid in a protein by another chemically similar amino acid. Examples include substitutions between aromatic amino acids Phe, Trp, and Tyr; substitutions between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; substitutions between polar amino acids Gln and Asn; substitutions between basic amino acids Lys, Arg, and His; substitutions between acidic amino acids Asp and Glu; and substitutions between hydroxy amino acids Ser and Thr.

[0031] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing the HCV core protein from the monoclonal antibody of this invention. These fragments include, but are not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the region in the antibody structure that can bind to the antigen. It consists of a complete light chain and a variable region (VH) and a constant region (CH1) domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragment can be prepared, for example, by using papain enzymatic digestion, antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind the corresponding antigen, they can be used to prepare products for detecting the hepatitis C virus core protein (HCV cAg).

[0032] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. The single-chain antibody can be expressed, for example, in *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of recognizing the HCV core protein and can be applied to the detection of the hepatitis C virus core protein (HCV cAg).

[0033] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of the high-affinity monoclonal antibody that recognizes the HCV core protein, based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector capable of expressing a high-affinity monoclonal antibody or its antigen-binding fragment that recognizes the HCV core protein. Furthermore, those skilled in the art can introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, or yeast to obtain recombinant bodies, and then express the antibodies or their antigen-binding fragments of the present invention through these recombinant bodies. The expressed antibodies or their antigen-binding fragments can recognize the HCV core protein; therefore, the aforementioned nucleic acid molecules, expression vectors, and recombinant bodies are within the scope of protection of the claims of this invention. Moreover, the above-mentioned techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0034] As described above, the antibody or its antigen-binding fragment of the present invention can recognize HCV core protein with high affinity, and therefore can be used to prepare a kit for detecting hepatitis C virus core protein (HCV cAg). The kit can be any kit that utilizes the antibody or its antigen-binding fragment of the present invention to react with HCV core protein, such as, but not limited to, kits using enzyme-linked immunosorbent assay (ELISA), chemiluminescence, fluorescence immunochromatography, colloidal gold immunochromatography, immunoblotting, and immunohistochemistry.

[0035] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0036] Example 1: Screening and determination of common dominant epitopes of HCV core protein

[0037] For HCV genotypes 3a, 3b, 6a, and 6n, whose prevalence in my country has been increasing in recent years, their core protein amino acid sequences were searched in the GenBank database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ). The results were GenBank: AGN91965.1 (3a), GenBank: ADH82332.1 (3b), GenBank: AAC42193.1 (6a), and GenBank: ABB84856.1 (6n), respectively. The core protein sequences of the prevalent HCV genotype 1b (GenBank: BAT57445.1) and genotype 2a (GenBank: AAC42191.1) in China were also searched. The GenBank accession numbers and sequence summary information for these sequences are shown in Table 1 below.

[0038] Table 1. Summary of core protein amino acid sequence information for HCV genotypes 3a, 3b, 6a, and 6n.

[0039] Serial number Gene subtype GenBank accession number Core protein amino acid sequence SEQ ID NO 1 3a AGN91965.1 MSTLPKPQRKTKRKPIRRPQDVKFPGGGQIVGGVYVLPRRGPRLGVRATRKASERSQPRERRQPIPKARRSEGRSWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPNDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPVGGVARALAHGVRALEDGINFATGNLPGCSFSIFLLALLSCLIHPAAS 8 2 3b ADH82332.1 MSTLPKPQRQTKRNTPRRPQNVKFPGGGQIVGGVYVLPRRGPRLGVRAVRKTSERSQPRGRRQPIPKARPSEGRSWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPNDPRRRSRNLGKVIDTLTCGFADLMGYIPLIGAPVGGVARALAHGVRALEDGVNYATGNLPGCAFSIFLLALFSCLTCPASG 9 3 6a AAC42193.1 MSTLPKPQRKTKRNTTRRPMDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARQTQGRHWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPHWGPNDPRRRSRNLGKVIDTLTCGFADLMGYIPVVGAPLGGVAAAFAHGVRALEDGINYATGNITGCSFSIFLLALLSCLTTPASA 10 4 6n ABB84856.1 MSTLPKPQRKTKRNTNRRPMDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARQPTGRHWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPNWGPNDPRRRSRNLGKVIDTITCGLADLMGYIPVLGAPLGGVAAALAHGVRAVEDGINYATGNLPGCSFSIFLLALLSCLTTPASA 11 5 1b BAT5&445.1 MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASA 12 6 2a AAC42191.1 MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRTTRKTSERSQPRGRRQPIPKDRRSTGKPWGKPGYPWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRHRSRNVGKVIDTLTCGFGDLMGYIPVVGAPLGGVARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCITVPVSA 13

[0040] The amino acid sequences of the core proteins of four HCV genotypes (3a, 3b, 6a, and 6n) were compared and analyzed using the bioinformatics analysis software DNAMAN 6.0. The results are as follows: Figure 1 As shown, the core protein amino acid sequence homology of the four gene subtypes is 93.59%. Among them, the identical sequence segments with a length of ≥7 amino acids are: 1-9, 22-35, 37-48, 53-59, 61-69, 76-105, 107-125, 131-138, and 174-181.

[0041] The distribution of B-cell epitopes in the core protein of the major prevalent HCV genotype 1b (GenBank: BAT57445.1) in my country was analyzed using the bioinformatics analysis software BIOSUN. First, the full-length amino acid sequence was input, and then the B-cell epitopes were analyzed. The predicted B-cell epitopes and their scores are shown in Table 2. All epitopes with scores ≥3 were identified as dominant epitopes, namely epitopes 1, 3, 4, and 7. Combined with the homologous sequences identified above, only epitope 7 was found to be highly conserved across different genotypes, with completely identical sequences. Therefore, epitope 7 was identified as a common dominant epitope of the HCV core protein.

[0042] Table 2. Analysis of B-cell epitopes of HCV genotype 1b core protein.

[0043] Serial Number Epitope sequence number Epitope sequence Score SEQ ID NO 1 9-15 RKTKRNT 4.72 14 2 38-44 PRRGPRL 2.27 15 3 52-58 TSERSQP 4.39 16 4 67-73 KARRPEG 3.58 17 5 86-92 YGNEGMG 1.69 18 6 101-107 RGSRPSW 2.90 19 7 111-117 DPRRRSR 4.04 20 8 129-135 GFADLMG 0.55 21 9 145-151 GGAARAL 1.41 22 10 161-167 GVNYATG 2.07 23 11 185-191 LTIPASA 0.04 24

[0044] To prepare a high-affinity monoclonal antibody targeting the common dominant epitope of the HCV core protein, Shanghai Dechi Biotechnology Co., Ltd. was commissioned to prepare a synthetic peptide for the common dominant epitope, which was then conjugated with KLH and BSA, respectively. To ensure epitope integrity, the common dominant epitope was extended by two amino acids before and after it. Furthermore, to facilitate conjugation, a cysteine ​​residue (C) was added to the amino terminus. The final sequence of the synthetic peptide targeting the common dominant epitope of the HCV core protein is CPN. DPRRRSR NL (SEQ ID NO. 25), where the underlined sequence is the 7th epitope sequence.

[0045] Example 2: Preparation and rapid screening of high-affinity monoclonal antibodies against HCV core protein

[0046] Using the HCV core protein common dominant epitope synthetic peptide KLH conjugate prepared in Example 1 as the immunogen, 6-8 week old female BALB / c mice were immunized with 100 µg of antigen per mouse plus an equal amount of Freund's complete adjuvant. After thorough emulsification with a stirrer, the mice were immunized subcutaneously in the back and intraperitoneally, with 3 mice immunized. A second immunization was performed 4 weeks later, and a third immunization was performed 8 weeks later. For each third immunization, 50 µg of antigen per mouse plus incomplete Freund's adjuvant was emulsified with a stirrer and then injected subcutaneously in the back and intraperitoneally. One week after the third immunization, blood was collected from the tail vein of the mice to detect the immune serum titer. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection (50 µg per mouse). Spleen cells were harvested 3 days later for fusion. SP20 myeloma cells were resuscitated and cultured until they were in the logarithmic growth phase. Spleen cells were prepared from the spleens of the immunized BALB / c mice. The above-mentioned spleen cells and myeloma cells were mixed in serum-free DMEM medium at a ratio of 9:1, centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were gently shaken to disperse them. The cells were then fused in a 37°C water bath. 1 mL of preheated 50% PEG fusion cells was added within 1 minute, while gently shaking to mix. After the addition was complete, the cells were allowed to stand for 90 seconds, and serum-free DMEM medium was added to terminate the fusion. The cells were then allowed to stand at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, and the pellet was resuspended in HAT medium. The pellet was then aliquoted into 96-well cell culture plates containing feeder cells and cultured in a 37°C, 5% CO2 cell culture incubator for 5 days. The medium was changed once with HAT medium. The medium was changed again on day 10. When the fusion cells covered about 60% of the bottom of the wells, the cell culture supernatant was collected, and high-affinity positive clones were screened using a rapid reaction screening method. The specific method is as follows: Dilute the HCV core protein common dominant epitope synthetic peptide BSA conjugate with carbonate coating buffer to a concentration of 2.0 μg / ml, coat 150 μl per well, and incubate overnight at 4°C; wash the plate twice with washing buffer; add 200 μl / well blocking buffer and block at room temperature for 6 hours; wash the plate 5 times with washing buffer. Add 100 μl of sample dilution buffer to each well, then add 10 μl of cell culture supernatant, and incubate with shaking at room temperature for 5-15 min, then discard the supernatant. Wash the plate 5 times, invert the washed ELISA plate on absorbent paper to dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate with shaking at room temperature for 15 min. Wash the plate 5 times. Add 50 μl each of TMB chromogenic solution A and B to each well, and incubate at room temperature in the dark. Continuously observe during this process, and select the positive clone that first shows a significant color change as the high-affinity monoclonal antibody clone, which is named 1014. Hybridoma cell line 1014 was cultured in 1640 medium containing 10% fetal bovine serum. Each male BALB / c mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, cells were collected and resuspended in 10 mL of physiological saline at a cell density of 1 × 10⁻⁶ cells / mL. 70.5 mL of antibody was administered intraperitoneally to each mouse at a concentration of 1 antibody / mL. Ascites fluid was collected after 2 weeks. Antibody purification was performed using the Thermo Melon Gel Monoclonal IgG Purification Kit, and the purified antibody was aliquoted and stored at -20°C.

[0047] Example 3: Prokaryotic expression of mature HCV core proteins from six common genotypes

[0048] To identify high-affinity monoclonal antibodies against HCV core proteins, mature core proteins containing 177 amino acids from six common HCV genotypes in my country—1b (GenBank: BAT57445.1), 2a (GenBank: AAC42191.1), 3a (GenBank: AGN91965.1), 3b (GenBank: ADH82332.1), 6a (GenBank: AAC42193.1), and 6n (GenBank: ABB84856.1)—were expressed in prokaryotes. First, based on the amino acid sequences of the mature proteins and the genetic code preferences of *E. coli*, optimized nucleotide sequences suitable for expression in the *E. coli* expression system were derived for the six core proteins, and are summarized in Table 3 below.

[0049] Table 3. Optimized nucleotide sequences of the six core proteins suitable for expression in the *E. coli* expression system.

[0050] name nucleotide sequence SEQ ID NO CP-1b ATGAGCACCAACCCGAAACCACAGCGTAAGACGAAACGCAATACTAACCGTCGCCCACAAGATGTGAAATTTCCAGGCGGTGGCCAGATTGTTGGTGGCGTGTATCTGTTGCCACGTCGCGGTCCACGTCTGGGCGTCCGTGCAACCCGTAAGACTTCTGAACGTTCCCAGCCACGTGGTCGTCGCCAACCAATCCCGAAAGCTCGTCGCCCAGAGGGCCGTACGTGGGCTCAGCCAGGCTACCCGTGGCCATTATATGGTAATGAAGGCATGGGTTGGGCAGGCTGGCTGCTTTCGCCACGCGGTAGCCGTCCAAGTTGGGGCCCGACCGACCCACGTCGCCGTTCTCGCAACCTGGGTAAAGTTATTGATACTCTCACCTGCGGCTTCGCTGATCTGATGGGCTACATCCCGCTGGTAGGTGCACCGTTGGGCGGTGCTGCACGTGCTCTGGCGCATGGCGTGCGCGTTTTAGAAGACGGTGTCAATTATGCCACCGGCAACCTGCCAGGTTGTTCGTTCAGCATCTTC 26 CP-2a ATGAGCACCAACCCGAAACCACAGCGTAAGACGAAACGCAATACTAACCGTCGCCCGCAAGATGTGAAATTTCCAGGCGGTGGCCAGATTGTTGGTGGCGTGTATCTGTTGCCGCGTCGCGGTCCACGTCTGGGCGTCCGCACCACTCGTAAGACGTCTGAACGTTCCCAGCCGCGCGGTCGTCGCCAACCAATCCCGAAAGACCGTCGCTCGACCGGCAAACCGTGGGGCAAGCCAGGTTACCCGTGGCCATTATATGGCAATGAGGGTCTGGGCTGGGCGGGTTGGCTTCTGAGCCCGCGTGGCAGTCGCCCATCTTGGGGTCCGACTGATCCACGTCATCGTTCGCGCAACGTTGGCAAAGTAATTGATACCCTCACCTGCGGCTTCGGTGACCTGATGGGCTACATCCCGGTGGTTGGTGCCCCGCTGGGCGGTGTCGCGCGTGCATTGGCTCACGGCGTGCGCGTTCTGGAAGATGGTGTGAATTATGCGACGGGCAACTTACCAGGCTGTAGCTTTAGCATCTTC 27 CP-3a ATGAGCACCCTGCCGAAACCACAGCGTAAGACGAAACGCAAACCGATTCGTCGCCCACAAGATGTGAAGTTTCCAGGCGGTGGCCAGATCGTTGGTGGCGTGTATGTCTTGCCACGTCGCGGTCCACGTCTGGGCGTTCGCGCAACTCGTAAAGCCTCTGAACGTTCCCAGCCACGCGAGCGTCGCCAACCGATTCCGAAAGCGCGTCGCTCGGAAGGTCGTAGCTGGGCACAGCCAGGCTACCCGTGGCCATTATATGGCAACGAAGGTTGCGGCTGGGCTGGTTGGCTGCTTAGTCCACGTGGTTCTCGTCCATCGTGGGGTCCGAATGACCCACGTCGCCGTAGCCGCAACCTGGGCAAGGTAATCGATACCCTCACTTGTGGTTTCGCGGATCTGATGGGCTACATTCCGCTGGTGGGTGCTCCAGTTGGTGGCGTCGCACGTGCTTTGGCGCATGGTGTGCGTGCACTGGAGGACGGCATCAATTTTGCGACGGGTAACTTACCAGGCTGCAGCTTCTCTATCTTC 28 CP-3b ATGAGCACCCTGCCGAAACCACAGCGTCAAACGAAGCGCAACACTCCACGTCGCCCACAGAATGTGAAATTTCCGGGCGGTGGCCAGATTGTTGGTGGCGTGTATGTCTTGCCACGTCGCGGTCCGCGTCTGGGCGTTCGTGCAGTACGTAAAACCTCTGAACGTTCCCAACCACGCGGTCGTCGCCAGCCGATCCCGAAGGCCCGTCCATCGGAGGGTCGCAGCTGGGCTCAGCCAGGTTACCCATGGCCGTTATATGGTAACGAAGGCTGCGGTTGGGCAGGCTGGCTGCTTAGTCCACGTGGTTCTCGCCCGTCGTGGGGCCCAAATGATCCGCGTCGTCGCAGCCGTAACCTGGGTAAAGTGATTGACACTCTCACGTGTGGCTTCGCTGATCTGATGGGCTACATCCCGCTGATTGGTGCGCCAGTTGGCGGTGTCGCACGTGCATTGGCTCATGGCGTGCGTGCGCTGGAAGATGGTGTTAATTATGCCACCGGCAACTTACCGGGTTGCGCGTTTAGCATCTTC 29 CP-6a ATGAGCACTCTGCCGAAACCACAGCGTAAGACGAAACGCAACACTACCCGTCGCCCAATGGATGTGAAATTTCCAGGCGGTGGCCAAATTGTTGGTGGCGTGTATTTGCTGCCGCGTCGCGGTCCACGTTTAGGCGTCCGTGCAACTCGTAAGACGTCTGAACGTTCCCAGCCACGTGGTCGTCGCCAGCCAATCCCGAAAGCCCGTCAAACCCAGGGCCGCCATTGGGCGCAGCCGGGCTACCCATGGCCGCTGTATGGTAATGAGGGCTGCGGTTGGGCAGGCTGGCTTCTGTCGCCACGTGGTAGCCGTCCGCACTGGGGCCCAAACGACCCGCGTCGTCGCAGTCGTAATCTCGGTAAAGTTATTGATACTCTGACCTGTGGCTTCGCTGATCTGATGGGCTACATCCCAGTAGTGGGTGCACCGTTGGGCGGTGTTGCAGCAGCTTTTGCGCATGGCGTCCGCGCACTGGAAGACGGTATTAACTATGCGACCGGCAATATCACGGGTTGCTCTTTCTCGATCTTC 30 CP-6n ATGAGCACTCTGCCGAAACCACAGCGTAAGACGAAACGCAACACTAATCGTCGCCCGATGGATGTGAAATTTCCAGGCGGTGGCCAAATTGTTGGTGGCGTGTATTTGCTGCCGCGTCGCGGTCCACGTTTAGGTGTCCGTGCAACCCGTAAGACTTCTGAACGTTCCCAGCCACGCGGTCGTCGCCAGCCAATCCCGAAAGCCCGTCAACCGACGGGTCGCCATTGGGCGCAGCCAGGCTACCCGTGGCCACTGTATGGTAACGAGGGCTGCGGTTGGGCAGGCTGGCTTCTGTCTCCACGTGGTAGCCGCCCAAATTGGGGCCCGAACGACCCACGTCGTCGCAGTCGTAATCTCGGTAAAGTTATTGATACCATCACTTGTGGCCTGGCTGATCTGATGGGCTACATTCCGGTATTGGGTGCACCGCTTGGCGGTGTGGCCGCAGCTTTAGCGCACGGCGTTCGCGCCGTCGAAGACGGTATCAACTATGCTACCGGCAATCTGCCAGGTTGCTCTTTCTCGATCTTC 31

[0051] Then, using BamH I and EcoR I restriction sites, the recombinant expression plasmids pET-28a were ligated into pET-1b, pET-2a, pET-3a, pET-3b, pET-6a, and pET-6n, respectively. The correctly sequenced recombinant expression plasmids were transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and cultured overnight at 37°C with shaking in 5 mL of LB broth containing ampicillin sodium. The next day, the cells were inoculated into 250 mL of fresh LB broth and cultured until the logarithmic growth phase. The temperature was then adjusted to 16°C, and after 30 minutes, 150 μl of 1 mol / L IPTG induction solution was added. Induction was continued at 16°C for 12-14 h. Induced bacterial cells were collected by centrifugation, resuspended in 25 mmol / L Tris-HCl (pH 8.5), and sonicated. Inclusion bodies were collected by centrifugation at 12000 rpm for 20 min at 4 °C. The inclusion bodies were washed with 25 mmol / L Tris-HCl containing 2 mol / L urea, and then dissolved in 25 mmol / L Tris-HCl containing 6 mol / L urea. The supernatant was collected by centrifugation at 12000 rpm for 20 min and purified using a Ni column. The target protein was collected by elution with washing buffer containing 250 mmol / L imidazole and subjected to SDS-PAGE gel electrophoresis. The results are shown below. Figure 2 As shown, M is the marker. It can be seen that the mature core proteins of HCV gene subtypes 1b, 2a, 3a, 3b, 6a and 6n are all expressed as inclusion bodies with a molecular weight of approximately 21 kDa, which is consistent with the expected molecular weight.

[0052] Example 4: Preparation of polyclonal antibody against rabbit HCV core protein

[0053] Polyclonal antibodies against HCV core protein were prepared using the core protein of HCV genotype 1b as an immunogen. Healthy New Zealand white rabbits were selected. 1.0 mg of genotype 1b core protein was mixed with 1.0 mL of Freund's complete adjuvant, thoroughly emulsified, and then subcutaneously injected at two points along the spine of the rabbits, 0.2 mL at each point. Four weeks later, 1.0 mg of genotype 1b core protein was mixed with 1.0 mL of Freund's incomplete adjuvant, thoroughly emulsified, and then injected at different points on the same site for a second immunization. A third booster immunization was performed four weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After the blood clots and contracted, the blood was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20°C for later use. The indirect ELISA method for determining the titer of polyclonal antibodies is as follows: Enzyme-linked plates are coated with genotype 2a, 3a, 3b, 6a and 6n core proteins at a concentration of 2.0 μg / ml, 150 μL per well, and incubated overnight at 4°C; the plates are washed twice with washing buffer; 200 μL / well blocking buffer is added and the plates are blocked at room temperature for 6 hours; the plates are washed 5 times with washing buffer. Rabbit serum was diluted with PBS at dilution ratios of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000, and 1:4096000, with 100 μL added to each well. The plate was incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. HRP-labeled goat anti-rabbit secondary antibody was added and incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. Freshly prepared substrate solution was added, 100 μL per well, and the plate was incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 2 M H2SO4 to each well. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm, and the readings were taken within 10 minutes after termination. Using pre-immunization rabbit serum diluted serially as a negative control, the prepared anti-HCV core protein polyclonal antibody achieved a titer of 1:1024000 against the core proteins of gene subtypes 2a, 3a, 3b, 6a, and 6n.

[0054] Example 5: Validation of the limit of detection of HCV core protein by high-affinity monoclonal antibody

[0055] Using the high-affinity monoclonal antibody C1014 against HCV core protein prepared in this invention as the capture antibody, and the rabbit anti-HCV core protein polyclonal antibody prepared in Example 4 as the detection antibody, a specific detection method for HCV core protein based on the double-antibody sandwich method was established. Simultaneously, a purchased HCV core protein monoclonal antibody (Phipeng Biotechnology Co., Ltd., catalog number HCV-Core-McAb22) was used as a capture antibody for a control study. The specific steps were as follows: ELISA plates were coated with the anti-HCV core protein monoclonal antibody at a concentration of 2.0 μg / mL, 100 μL per well, incubated overnight at 4°C, and washed twice with washing buffer; 120 μL / well blocking buffer was added and blocked at room temperature for 6 hours, followed by washing five times with washing buffer; the mature proteins of the six common prokaryotic HCV core protein subtypes expressed in Example 3 were serially diluted with double-distilled water to concentrations of 1000, 100, 10, 1.0, 0.5, 0.1, and 0.05 pg / mL, respectively. Add 100 μL of each solution to the wells and incubate at 37°C for 60 min. Discard the solution. Wash the plate 5 times with washing buffer. Add 100 μL of horseradish peroxidase-labeled rabbit anti-HCV core protein polyclonal antibody to each well and incubate at 37°C for 45 min. Wash the plate 5 times, blot dry, and add 50 μL each of TMB chromogenic solutions A and B to each well. Incubate at room temperature in the dark for 15 min. Add 50 μL of 2 M H₂SO₄ stop solution per well to stop the reaction. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm. Read the value within 10 minutes after termination.

[0056] The results are as follows Figure 3 As shown, A represents monoclonal antibody 1014, and B represents a commercially available antibody. Using three times the detection value of the negative control as the cutoff value, the double-antibody sandwich detection method established using the high-affinity anti-HCV core protein monoclonal antibody 1014 prepared in this invention as the capture antibody achieved a limit of detection (LOD) of 0.1 pg / mL for HCV genotypes 1b, 3a, 3b, 6a, and 6n core proteins, and a LOD of 1.0 pg / mL for genotype 2a core protein. In contrast, the LOD of the commercially available control antibody was 0.5 pg / mL for genotype 1b core protein and 1.0 pg / mL for the other five HCV genotypes. The LOD of the high-affinity anti-HCV core protein monoclonal antibody 1014 prepared in this invention is significantly higher than that of the commercially available antibody, especially exhibiting high affinity for HCV genotypes 3a, 3b, 6a, and 6n core proteins.

[0057] Example 6: Isotype analysis of anti-HCV core protein monoclonal antibody 1014

[0058] The heavy and light chain isotypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. First, the antibody was diluted to 1 μg / mL with PBS. Then, 100 μl of the diluted antibody was added to each well, and the results were observed and recorded after standing for 5-10 min. The results are as follows: Figure 4 As shown, the anti-HCV core protein monoclonal antibody 1014 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0059] Example 7: Sequencing of the variable region of anti-HCV core protein monoclonal antibody 1014

[0060] Mouse hybridoma cell line 1014 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab fragment of mouse monoclonal antibodies synthesized by Beijing Qingke Biotechnology Co., Ltd. The cells were preheated at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The resulting cells were ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data were compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website's BLAST module.

[0061] Sequence analysis revealed that the heavy chain variable region consists of 118 amino acids, with the following sequence: QVQLEESGPGLVQPSQSLSIICIVS GFSLITYG EHWVCQSPGKGLEWLGV IWSGGST DRNAAFISRLSINKDNSKSQVFFKMNSLQGNDTARYYC ASIGTTGGELWTT GVKEPQSLSP (SEQ ID NO.1), where CDRs are underlined, heavy chain CDR1 is located at 26-33 aa, with the amino acid sequence GFSLITYG (SEQ ID NO.2); heavy chain CDR2 is located at 51-57 aa, with the amino acid sequence IWSGGST (SEQ ID NO.3); heavy chain CDR3 is located at 96-108 aa, with the amino acid sequence ASIGTTGGELWTT (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVMTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIREL TR SEGAPSWKS (SEQ ID NO.5), wherein CDRs are underlined, light chain CDR1 is located at 27-36 aa, and the amino acid sequence is KSVSTSGYSY (SEQ ID NO.6); light chain CDR2 is located at 54-56 aa, and the amino acid sequence is LVS; light chain CDR3 is located at 93-100 aa, and the amino acid sequence is QHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or its antigen-binding fragment for high affinity recognition of HCV genotype 3 and genotype 6 core proteins, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by mouse hybridoma cell line 1014 with accession number CGMCC No. 46562.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, or a humanized antibody.

5. A nucleic acid molecule, characterized in that, It comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A recombinant cell, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The recombinant cell as described in claim 7, characterized in that, It is a recombinant of bacteria or yeast.

9. A hybridoma cell line that secretes monoclonal antibodies with high affinity for recognizing the HCV genotype 3 and genotype 6 core proteins, characterized in that, It is mouse hybridoma cell line 1014 with accession number CGMCC No. 46562.

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