Construction of recombinant eukaryotic expression plasmid containing 1.3-fold-overlength genome of HBV and its expression in Huh7 cells / 实用医学杂志

ABSTRACT

Objective To construct 1.3-fold-overlength infectious clone of hepatitis B virus isolated from Chinese patients , observe the expression of plasmid in Huh7 of liver cancer cells and establish genome of HBV in vitro. Methods HBV DNA in serum was extracted from HBV patient. SOE-PCR was performed to produce a 1.3-fold-overlength genome of HBV. The plasmid was named pHBV1.3 (C). After that,pHBV1.3 (C) was transfected into Huh7 cells, HBV related viral antigens and DNA were detected by ELISA,Western Blot and Fluorescence quantitative PCR. Furthermore, adefovir dipivoxil, a clinic anti-viral drug, was utilized to test the sensitivity of the new infectious clone. Results An infectious clone of pHBV1.3 (C) was successfully constructed. HBV gene carried in pHBV1.3 (C) could be efficiently replicated and expressed in Huh7 cells. Adefovir could inhibit HBV replication in this HBV cell model. Conclusions A recombinant plasmid containing 1.3-fold-overlength of HBV genotype C was successfully constructed. This construct is competent to support viral transcription and replication in vitro , suggesting that infectious cells are expected to be a new model of HBV infection in vitro.