Cloning，Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum

Abstract：To ampUfy Hepatitis C virus(HCV)polymerase sequence and express HCV NS5b protein in order to，in the next，establish a molecular model of HCV replication．RT—PCR method was used to amplify HCV NS5b polymerase gene from a Chinese HCV(+1 patient’S serum． e polymerase fragment Was constructed in the plasmid pET一30a an d expressed in E coli．HCV NS5b protein Was identified by W lestern blot with a monoclonal an tibody against HCV NS5b．Sequence analysis showed tllat the isolate had 69％ 一95％ an d 89％一97％ homologies to the reported HCV sequences in respect to nucleotide an d amino acid respectively．HCV NS5b gene was expressed at a high level an d the molecular weight ofthe expressed prod uct was 65kDa．which was within the ran ge of expectation．This protein Was confirmed to be HCV NS5b by Western blot．HCV NS5b from a Chinese patient Can be Success—fully cloned an d expressed with pET一30a plasmid an d E coli． is protein could be used to establish a molecular mod el of HCV replication．