Objective  To investigate the effect of hepatitis C virus (HCV) NS5B gene transfection on the expression of the transfected gene and the activity of RNA polymerase in human hepatic carcinoma cell line Hep3B for studying the mechanisms of HCV replication. Methods  The recombinant vector pcDNA-5B expressing wild-type HCV NS5B gene was transfected into Hep3B cells using Lipofectamine 2000 reagent kit. Expression of the transfected NS5B gene was determined by RT-PCR and Western blot analysis. Luciferase assays were used to analyze the activity of RNA-dependent RNA polymerase (RdRp). Results  The results from both RT-PCR and Western blot analysis demonstrated that there was the expression of NS5B gene at mRNA and protein levels in the transfected Hep3B cells. Luciferase assays revealed that there was RdRp activity of expressed NS5B protein in transfected Hep3B cells. Conclusion  The enzymatically active HCV NS5B can be expressed in human hepatic carcinoma cell line Hep3B by transfection

and the expressed NS5B has RdRp activity.