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Cell line of transgene mammal with fusion antigen of S (main protein) and previous SI (large protein) of hepatitis B surface expressed in high efficiency

A hepatitis B surface antigen, surface antigen technology, applied in the direction of genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of low expression efficiency, low composition, unstable structure products, etc.

Inactive Publication Date: 2006-07-05
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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Problems solved by technology

[0010] In summary, the large proteins containing HBV surface antigens PreS1, PreS2 and S have all been expressed in CHO cells or yeast. There are mainly two structures, natural and modified, but the expression efficiency of all structures is low. Some structural products are unstable, and the composition containing PreS1 is low

Method used

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  • Cell line of transgene mammal with fusion antigen of S (main protein) and previous SI (large protein) of hepatitis B surface expressed in high efficiency
  • Cell line of transgene mammal with fusion antigen of S (main protein) and previous SI (large protein) of hepatitis B surface expressed in high efficiency
  • Cell line of transgene mammal with fusion antigen of S (main protein) and previous SI (large protein) of hepatitis B surface expressed in high efficiency

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Experimental program
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Embodiment Construction

[0012] Construction of recombinant plasmid containing HBsAg S+pre-S1:

[0013] In order to obtain high-efficiency expression in mammalian cells, the present invention is in the S+S1 structure [14] (this structure was declared for a patent in 1994, the application number is 94112096.4, and the publication number is CN1108306A and GS structure [15] (the inventor of this patent One) on the basis of the S+S1 gene, introduce the RNA addition poly-A signal AATAA, hepatitis B virus enhancer I (ENI) and enhancer II (EN2) derived from the Hepatitis B virus gene Adr type Beijing strain, and use the deletion mutation The X gene of hepatitis B virus has been transformed by technology to make it lack 16 bp from the 8th bp after ATG, which is the mx gene, thus losing the possibility of expressing X protein and reducing carcinogenicity. Another Beijing strain of hepatitis B virus Adr subtype[ 17] 1-92bp from the ATG of the S gene replaced the 1-92bp from the ATG of the original S+S1 gene [14...

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Abstract

Based on the connection of gene fragment of hepatitis B surface antigen PreS1 (21-47) to Site 223 of S gene, RNA addition polymerization signal AATAA, hepatitis B virus enhancement vector 1 (En1), enhancement vector 2 (En2) and mutational x gene (mX) are introduced in its down stream to form plasmid pCHBSS1G. Plasmid pCHBSS1G and plasmid pSV2dhfr are transferred to CHO-dhfr-cell, cloned, selected and amplified with the addition of MSX and MTX to obtain a series of genetic engineering cell lines that can efficiently express the fusion protein for hepatitis B surface antigen S and of the former S1. SS1 fusion protein displays 27KD protein band in SDS-PAGE analysis as well as 30KD protein band. Specific antibody against S and S1 can be produced by mice immunization.

Description

Technical field: [0001] High-tech bioengineering technology Background technique: [0002] my country is a high incidence area of ​​hepatitis B. 50-70% of the population has been infected with HBV, and 10% of the population (about 100 million people) are carriers. Currently there is no effective way to treat hepatitis B, and hepatitis B vaccination is an effective measure to prevent hepatitis. [0003] Blood-derived vaccines have been put into production and use at home and abroad. Because blood-derived vaccines have some problems, such as limited blood sources and certain potential dangers (such as hepatitis C, HIV, etc.), it is necessary to urgently develop hepatitis B genetic engineering vaccines. Research on hepatitis B genetically engineered vaccines at home and abroad has developed rapidly, and MSD Company in the United States has obtained a production license for producing hepatitis B genetically engineered vaccines using yeast systems. The Hepatitis B Genetic Engin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/13C12N15/62C12N15/85
Inventor 田淑芳阮力刘文军杨芙蓉宗芳李军陈红王文阮薇琴王秀平
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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