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Truncated hepatitis C virus hcv NS3 antigen and its preparation and application

A hepatitis C virus, truncated type technology, applied in the field of molecular biology, can solve the problems of inactivation of antigen and large protein, and achieve the effect of not easy to miss detection

Active Publication Date: 2018-09-28
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application, the protein expressed by the NS3 full-length gene added by the diagnostic reagent is too large, and after being expressed in tandem with other dominant antigen epitopes, some antigens will lose their activity due to the occlusion of the spatial structure.

Method used

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  • Truncated hepatitis C virus hcv NS3 antigen and its preparation and application
  • Truncated hepatitis C virus hcv NS3 antigen and its preparation and application
  • Truncated hepatitis C virus hcv NS3 antigen and its preparation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Obtaining of truncated HCV NS3 recombinant antigenic protein sequence

[0034] According to the HCV sequences database (http: / / hepatitis.ibcp.fr.), the inventor screened out 6 NS3 antigen sequences belonging to 6 genotypes of HCV, and obtained NS31252-1526aa truncated HCV through sequence comparison and analysis For the amino acid sequence of NS3, search NCBI to obtain the corresponding nucleotide sequence, the amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2. And further select the preferred codon of Escherichia coli, design and synthesize the nucleotide sequence of the truncated NS3 antigen gene, the nucleotide sequence of the codon is shown in SEQ ID NO.3, and the amino acid encoded by it corresponds to SEQ ID NO .2 the amino acid encoded by the nucleotide sequence shown.

Embodiment 2

[0035] Example 2: Cloning and expression of truncated HCV NS3 recombinant antigen

[0036] 1. Construction of truncated HCV NS3 recombinant antigen expression plasmid

[0037] 1.1 Synthesis of truncated HCV NS3 recombinant antigen

[0038] According to the amino acid sequence of the truncated HCV NS3 antigen (its amino acid sequence is shown in SEQ ID NO.1), search the NCBI related nucleotide sequence to obtain the corresponding nucleotide sequence (its nucleotide sequence is shown in SEQ ID NO.2 shown), using Escherichia coli codon preference to modify SEQ ID NO.2 to obtain the nucleotide sequence (such as SEQ ID NO.3), entrusted by Boshang Biotechnology Co., Ltd. to synthesize truncated HCV NS3 as shown in SEQ ID NO.3 Nucleotides, and two restriction sites BamHI and Xho I were introduced at the 5' end and 3' end, respectively, to obtain the gene product.

[0039] 1.2 Construction of truncated HCV NS3 recombinant antigen expression plasmid

[0040] 1.2.1 Double digestion o...

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Abstract

The invention discloses a truncated type hepatitis c virus HCV NS3 antigen which is a nonstructural protein amino acid sequence of the NS3 full-length gene encoded 1252th-1526aath segment in an HCV genome. The amino acid sequence is shown as SEQ ID NO.1, and a corresponding nucleotide sequence is shown as SEQ ID NO.2. A preparation method of HCV NS3 recombinant antigen protein comprises the steps that an optimized escherichia coli codon for expressing the truncated type hepatitis c virus HCV NS3 antigen are established, the nucleotide sequence of the codon is shown as SEQ ID NO.3, the gene segment of the codon undergoes double enzyme digestion and then is connected with an expression vector undergoing the enzyme digestion as well to obtain a recombinant expression plasmid, the antigen can perform expression by adopting a conventional method, and the antigen protein is obtained through purification. The antigen protein has high sensitivity and good specificity and is used for detecting an HCV antibody, and the coincidence rate of a detection result of a patient with positive hepatitis c virus RNA is as high as 97%. It is indicated that the antigen can be applied to polyclonal or monoclonal antibodies and preparation of hepatitis c virus detection kits and has good prospect of clinical application.

Description

technical field [0001] The invention relates to a hepatitis C virus antigen and its preparation and application, in particular to a truncated hepatitis C virus HCV NS3 antigen and its preparation and application, belonging to the technical field of molecular biology. Background technique [0002] The most notable clinical feature of hepatitis C virus (hepatitis C virus, HCV) infection is that it causes chronic infection. About 70% of hepatitis C may develop into chronic hepatitis, 20% into liver cirrhosis, and 12% into liver cancer, which is very harmful. seriously. The global HCV infection rate is 2.8%, and about 185 million people carry HCV. my country is a low-prevalence area of ​​HCV. It is estimated that there are 10 million HCV-infected people in my country, and the number of new cases is rising every year. [0003] Since the HCV gene was cloned in 1989, people have been working on the research of HCV-related vaccines. The HCV genome is about 9.6kb long, encoding a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18C12N15/51C12N15/70C12N1/21C07K16/10G01N33/569G01N33/577
CPCC07K14/1833C07K16/109G01N33/56983G01N33/5767G01N33/577
Inventor 续薇陈振杨春云欧兰香王岩江长林李丽
Owner 山东莱博生物科技有限公司
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