The Construct And Identify For A Reporter-contained HCV Replicon Expression Vector

An estimated 170 million persons worldwide are infected with hepatitis C virus(HCV), the main pathogens of chronic hepatitis C.These patients are at high risk of developing severe liver diseases,including cirrhosis(liver scarring) and hepat- ocellular carcinoma. In 1999,selectable dicistronic subgenomic HCV RNA replicon was first described by Lohmann and his colleagues in Science,which is considered to be a milestone of the study on HCV in vitro cell models.The replicon systems are useful tools for the study on HCV pathogenesis,the screening of antiviral drugs and the development of vaccines.We construct a reporter-contained HCV replicon,which can fleetly illuminate the quantity of HCV RNA in cells.This new replicon does't need neomycin(G418) resistance.It's important for rapidly evaluating compounds that seems to be a HCV inhibitor.In order to construct the new HCV replicon with EGFP as reporter gene,the fragment of EGFP was obtained by PCR amplification from pEGFP-N1,and modified at the 3'5'-end by AscI and PmeI.The pBR322-1b cDNA and the EGFP fragment were respectively digested by AscI and PmeI.Then,the EGFP was combined with the large fragment of the pBR322-1b.The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing.The function of construct was confirmed by lipofectamine mediated transient in Huh7 cells.The Huh7 cells transfected with replicon cDNA didn't express the reporter gene of EGFP while the cells transfected with transcripts could.