Prokaryotic expression,identification and purification of HCV F protein
- VernacularTitle:丙型肝炎病毒F蛋白原核表达载体的构建、表达和纯化
- Author:
Yali GAO
- Publication Type:Journal Article
- Keywords:
Hepatitis Cvirus;
Fprotein;
Prokaryoti cexpression
- From:
Journal of Chongqing Medical University
1986;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a prokaryotic expression plasmid containing HCVF gene,and purify the recombinant fusion protein in E.coli system. Methods: F gene of Hepatitis C virus was amplified by PCR method from plasmid H/FL(containing full length cDNA sequence of HCV 1a subtype),cloned into pET32a(+)vector,and then transformed into E.coli JM109. After identified by restriction diges- tion and DNA sequencing,recombinant plasmid was transformed into E.coli BL21 and induced with IPTG. The fusion protein trxA- F was further confirmed by Western blot analysis and purified by affinity chromatography method. Results: Restriction digestion and PCR screening showed that HCV F gene was cloned into pET32a(+)successfully. After BL21 was transformed with recombinant vector pET32a (+)- HCVF and induced with 0.5mmol/L IPTG,a 35.4kDprotein band was found bySDS- PAGE. And this recombinant protein showed a highly specific and strong reaction with anti- His monoclonal antibody by Western blot analysis. Conclusion: The prokaryotic expression plasmid pET32a(+)- HCVF was successfully constructed,which will be helpful for further research.
