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Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion

A hepatitis C virus, mammalian technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, peptide/protein components, etc., can solve problems such as hindering HCV vaccine research

Inactive Publication Date: 2006-08-23
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

However, only the E2 protein with high glycosylation and natural spatial configuration expressed by mammalian cells has the biological function of binding to target cells and natural antigenicity, and E2 protein is a low-expression protein. Obtaining the protein expressed by mammalian cells is quite technically difficult
This is also the main reason why E2 protein is not introduced in the ELISA method at present, and it is also an important reason that hinders the research of HCV vaccine

Method used

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  • Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion
  • Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion
  • Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion

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Embodiment 1

[0025] 1. Amplification of the extracellular segment gene of HCV envelope E2 protein:

[0026] According to the nucleotide sequence of the 1b genotype HCV envelope E2 protein coding gene, primers were designed and synthesized, and the HCV envelope E2 protein extracellular segment gene was amplified by polymerase chain reaction (PCR). In order to facilitate the purification of the expression product, a nucleotide sequence encoding 6 histidine residues was introduced at the 3' end of the E2 extracellular segment gene. The template plasmid pGEM-HCJ4 amplified by PCR contains the complete sequence of HCV type 1b gene, which was constructed and provided by Dr. Yanagi M, National Institute of Health (see Virology. 1998; 244(1): 161-72). The reagents used for DNA amplification are products of Promega, USA.

[0027] Primer sequence: (downstream primer E2R contains SalI restriction site)

[0028] E2F: 5′-GAGACCCACACGACGGGGAG-3′

[0029] E2R: 5′-GTCGACAACTCAGTGGTGGTGGTGGTGGTGTTCTGACC...

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Abstract

The present invention relates to biomedicine technology. HCV envelope protein E2 mediates the combination between HCV and target cell and is key protein relates to HCV infection and one kind of low expression protein hard to obtain in gene recombination process. The present invention aims at provides high efficiency secretion method for mammal to express HCV envelope protein E2. The method constitutes one new type of mammal cell expressing plasmid, which expresses target gene E2 protein in high level while expressing glutamine synthetase as the screening marker in low level. The present invention makes it possible to batch prepare recombinant HCV envelope protein E2 with the natural biological function and antigenicity of HCV envelope protein, lays the foundation for development of serological HCV infection detecting reagent and HCV vaccine, and provides HCV molecular virological research with important material.

Description

Technical field: [0001] The invention relates to the technical field of biomedicine engineering, in particular to a method for efficiently secreting and expressing hepatitis C virus (HCV) envelope protein E2 with mammalian cells. Background technique: [0002] Hepatitis C virus (HCV) is one of the main pathogenic factors of acute and chronic hepatitis transmitted by blood. At present, there are about 170 million HCV infected people in the world, and more than 60% of HCV infections will develop into chronic infections. Chronic HCV infection is highly correlated with liver cirrhosis and hepatocellular carcinoma, and is also closely related to lymphoma and cryoglobulinemia. Although a diagnostic method for HCV infection has been established and commercialized, the most widely used serological diagnostic method, that is, the enzyme-linked immunosorbent assay (ELISA) method still has a certain miss rate, and some diagnostic methods need to be added. Viral antigen. In addition, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N15/52C12N15/85G01N33/53G01N33/535A61K38/16A61K39/29A61P1/16
Inventor 赵平廖小玲曹洁戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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