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Hepatitis C virus fusion antigen protein and application thereof

A hepatitis C virus, fusion antigen technology, applied in the direction of viral antigen components, viruses, viral peptides, etc.

Inactive Publication Date: 2017-05-31
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no clinically available hepatitis C vaccine in the prior art. This patent provides a new plant-produced hepatitis C virus fusion antigen protein. The fusion antigen protein has a strong ability to induce neutralizing antibodies and has a strong Broadly reactive, can inhibit hepatitis C virus infection in vitro, providing a new solution for hepatitis C vaccine or diagnostic kit

Method used

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  • Hepatitis C virus fusion antigen protein and application thereof
  • Hepatitis C virus fusion antigen protein and application thereof
  • Hepatitis C virus fusion antigen protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The recombinant hepatitis C virus Core of embodiment 1 tobacco expression, the strong immunogenic region of E1 and E2 protein obtains

[0036] 1.1 Artificially synthesized full-length and truncated mutant genes of Core, E1 and E2

[0037] Based on the sequence of hepatitis C virus genotype 1b (see GenBank accession number HQ639947.1 for the nucleic acid sequence, and GenBank accession number AEJ86549.1 for the protein sequence) and epitope analysis software IEDB (http: / / www.iedb.org / home_v3. php) prediction, designing Core, E1 and E2 truncation mutants containing different epitopes: Core 1-92 (encodes Core protein 1-92 amino acids), Core 93-190 (encodes amino acids 93-190 of Core protein), E1 1-54 (encodes amino acids 1-54 of E1 protein), E1 55-122 (encodes amino acids 55-122 of E1 protein), E1 123-199 (encodes amino acids 123-199 of E1 protein), E2 1-62 (encodes amino acids 1-62 of E2 protein), E2 63-218 (encoding amino acid 63-218 of E2 protein) and E2 219-344 ...

Embodiment 2

[0048] Example 2 fusion antigen Core 1-92 / E1 55-122 / E2 63-218 Preparation of (HCVles) and determination of immune characteristics

[0049] 2.1 Construction of fusion antigen HCVles expression vector

[0050] The fusion antigen HCVles gene was synthesized by overlapping PCR, and the HCVles gene encodes 316 amino acids (as shown in SEQ ID NO.1), which consists of the antigenic polypeptide Core with strong immunogenicity 1-92 , E1 55-122 and E2 63-218 Composed in series. To facilitate HCV les Gene cloning, protein expression and purification, in HCV les A ClaI restriction site and a tobacco-preferred Kozak sequence, a histidine tag (6 histidines) were introduced into the 5' end of the gene, and a stop codon and a SalI restriction site were introduced into the 3' end. 24 primers (SEQ ID NO.2-25) were designed, Sangon Bioengineering Co., Ltd. synthesized the above 24 primers, and a fusion protein gene (SEQ ID NO.26) was synthesized by one-step overlapping PCR reaction. T...

Embodiment 3

[0066] Example 3 fusion antigen Core 1-92 / E1 55-122 / E2 63-218 Determination of reactivity between (HCVles) and serum of hepatitis C patients

[0067] ELISA assay for HCVles, Core, Core 1-92 ,E1,E1 55-122 , E2 and E2 63-218 Reactivity with serum from patients with hepatitis C virus. With purified antigenic polypeptides (HCVles, Core, Core 1-92 ,E1,E1 55-122 , E2 and E2 63-218 ) was coated on a 96-well plate, and the amount of coating protein per well was 50ng. Human serum was the primary antibody, and the secondary antibody was human HRP-labeled secondary antibody (Wuhan Sanying Biotechnology Co., Ltd.). HCV-negative sera served as negative controls. HCV les Reacted with 98 out of 100 randomly selected sera of hepatitis C virus-infected patients (from Wuhan University People's Hospital, Ningbo People's Hospital and Nanchang Liver Disease Hospital), the response rate was 98%, which was significantly higher than Core, Core 1-92 ,E1,E1 55-122 , E2, and E2 63-218 T...

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Abstract

The invention discloses a recombinant hepatitis C virus fusion antigen protein. Full-length and truncated mutant expression carriers of hepatitis C virus fusion antigen proteins Core, E1 and E2, and a fusion antigen HCVles expression carrier fusing strong immunogenic regions of the three virus proteins are constructed; corresponding proteins are expressed through a tobacco host, indicating that the fusion antigen protein HCVles is remarkably enhanced on the aspects of induction of neutralizing antibodies as well as the serum reactivity and virus inhibition capability of patients infected with a hepatitis C virus compared with the full-length and truncated mutant expression carriers of the Core, the E1 and the E2, and shows a synergistic effect, so that the hepatitis C virus fusion antigen protein has a good prospect as a hepatitis C virus vaccine or a diagnostic reagent, and a new solution can be provided for preparation of the hepatitis C virus vaccine or the diagnostic reagent.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a hepatitis C virus fusion antigen protein, a hepatitis C virus genetic engineering vaccine based on the hepatitis C virus fusion antigen protein, a preparation method and application thereof. Background technique [0002] Hepatitis C virus is the pathogenic factor of hepatitis C. The total number of people infected by the virus exceeds 170 million in the world, and the number of infected people in my country exceeds 40 million. Most people with HCV infection are persistent, leading to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. According to reports, 40% of chronic liver diseases and 80% of hepatocellular carcinoma in the world are related to hepatitis C virus, and the annual global cost of hepatitis C treatment exceeds 100 billion US dollars. Existing therapeutic drugs are only effective for some hepatitis C virus-infected patien...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/82G01N33/68G01N33/569A61K39/29A61P31/14
CPCA61K39/12C07K14/005C07K2319/00C12N15/8251C12N2770/24222C12N2770/24234G01N33/56983G01N33/6803G01N2800/26
Inventor 孔令保徐晶郭韫丽
Owner JIANGXI AGRICULTURAL UNIVERSITY
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