Cloning and Expression of Full-length and Truncated Hepatitis C Virus NS5B Gene and Identification of Expressed Product

Objective To construct the prokaryotic expression vectors for hepatitis C virus (HCV) NS5B-FL, NS5B-C21 and NS5B-C51 genes and identify the expressed product. Methods Amplify the NS5B-FL,NS5B-C21 and NS5B-C51 genes of HCV by PCR, identify by digestion with BamH I and Xho I and insert into prokaryotic expression vector pET-28a (+) respectively. Transform the constructed recombinant plasmids pET-28a (+)-NS5B-FL, pET-28a (+)-NS5B-C21 and pET-28a (+)-NS5B-C51 to E.coli BL21(DE3) for expression under induction of IPTG.Purify and identify the expressed protein.Results The recombinant E.coli BL21(DE3) transformed with pET-28a (+)-NS5B-FL, pET-28a (+)-NS5B-C21 and pET-28a (+)-NS5B-C51 was identified by PCR and restriction analysis, and the results showed that the target genes at expected lengths were amplified. The expressed 6His-NS5B-FL fusion protein mainly existed in a form of inclusion body.However, the solubility of expressed 6His-NS5B-C21 and 6His-NS5B-C51 increased significantly. The purified 6His-NS5B-C21 showed no degradation, while purified 6His-NS5B-FL and 6His-NS5B-C51 were degraded partially. All the 3 kinds of purified fusion proteins showed specific reactions with the sera of patients with hepatitis C.Conclusion The prokaryotic expression vectors for hepatitis C virus (HCV) NS5B-FL,NS5B-C21 and NS5B-C51 genes were successfully constructed, and 3 kinds of fusion proteins were expressed, which laid a foundation of screening of drugs against HCV.