Cloning, expression and identification of HCV ns5b gene

AIM: To construct the recombinant plasmid expressing HCV ns5b and to acquire NS5B protein so as to prepare the NS5B antibody and screen the anti HCV drug targeting the ns5b . METHODS: The coding region of HCV ns5b gene was amplified by PCR and was digested by Xho Ⅰ and Kpn Ⅰ. This fragment was cloned into plasmid pRSETA by T4 ligase and used to transform E. coli JM109. The positive recombinant plasmid was obtained, which was named pRSETA ns5b . The pRSETA ns5b was transformed into E. coli BL21 (DE3) and induced by IPTG. SDS PAGE and Western Blot electrophoresis were used to identify this expressed protein the thin layer chromatography was used to detect the quantity. RESULTS: The plasmid pRSETA ns5b expressing HCV NS5B was successfully constructed, which could distinctively express the 6His NS5B fusion protein .The production was mainly in the form of inclusion and the proportion of total bacterial protein was 25%. The result of Western Blot showed that the NS5B protein was kept active. CONCLUSION: HCV NS5B is expressed efficiently in vitro .