CLONING AND EXPRESSION OF SOLUBLE HCV NS5B GENE IN EUKARYOCYTES
- VernacularTitle:丙型肝炎病毒NS5B基因的克隆及在大肠杆菌的分泌表达
- Author:
Yuehong ZHANG
;
Huijuan DUAN
;
Liancai JU
- Publication Type:Journal Article
- Keywords:
hepatitis C virus;
NS5B;
Escherichia coli;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
HCV NS5B, acting as a RNA dependent replication enzyme,has emerged as an attractive protein used as a target for screenig of drugs against HCV NS5B, and plays an important role in HCV replication. In the report the gene expression of NS5B in E.coli was investigated. PCR was performed to gain the gene of HCV NS5B from plasmid pBRTM/HCV 1 which contains whole sequence of HCV, and the truncated NS5B gene containing no hydrophobic domain was cloned into pGEM Teasy vector. The gene of the truncated NS5B was cut from pGEM Teasy vector and cloned into E.coli expression plasmid pET 21b, then pET 21b NS5B was transfected into E.coli cells. The protein E.coli lysates were purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting assay. The RdRp activity of NS5B was examined by scintillation proximity assay (SPA). The truncated NS5B gene was successfully cloned into pET 21b. The results of SDS PAGE and Western blotting assay showed: ①the molecular weight of the expressed product was about 68 000 D, ②The truncated NS5B protein was existed in media of E.coli cells, ③The activity of NS5BDCT21 His from HCV 1b amounted to 6 900 cpm (total incorporation of approximately). These findings suggest that soluble NS5B can be successfully expressed in E.coli and could secret into media.
