Efficient replication and expression of a 1.1-ploid genome length HBV recombinant plasmid vector in Huh7 cells

Objective To construct a 1.1-ploid genome length hepatitis B virus (HBV) recombinant vector and to investigate the replication and expression of HBV DNA in human hepatoma cell line Huh7. Methods HBV DNA was extracted and two HBV DNA fragments were amplified from serum of a patient with chronic hepatitis B using polymerase chain reaction (PCR),respectively. The PCR products were digested and ligated into a replication competent plasmid pTriEx-mod. The recombinant plasmid designated as pTriEx-1.1HBV (S869-1) was confirmed by restriction enzyme digestion and DNA sequencing. Then the plasmid pTriEx-1.1HBV (S869-1) was transiently transfected into Huh7 cells by FuGENE HD transfection reagent. Expression of secreted HBsAg and HBeAg in culture supernatant were detected by ELISA and the intracellular HBV replication intermediates and HBV DNA levels were measured by real-time fluorescence quantitative PCR and Southern blotting at 72hr post-transfection,respectively. Results The restriction enzyme digestion and DNA sequencing showed that recombinant plasmid pTriEx-1.1-HBV (S869-1) was successfully constructed. High-level expression of secreted HBsAg and HBeAg was detected in culture supernatant. Furthermore,intracellular HBV replicative intermediates were successfully identified,suggesting the efficient replication of 1.1-ploid HBV genome in transfected Huh7 cells. Conclusion An in vitro transient culture system of a 1.1-ploid genome HBV vector pTriEx-1.1HBV (S869-1) was successfully constructed,and it would offer a novel strategy for studying of HBV drug resistance and screening of new antiviral drugs.