Construction of HBV replication cell model with HBsAg deletion and effect of HBsAg on HBV replication

Objective To construct HBV replication cell model with HBsAg deletion in vitro and investigate the effect of HBsAg on HBV replication.Methods The gene of S protein region in plasmid pch9 was site-directed mutated to construct HBV1.1 plasmid with HBsAg mutant,which was identified by sequencing.HepG2 cells were transiently transfected with HBV1.1 plasmid with HBsAg mutant,using plasmid pch9 without mutation as control.The expression of HBsAg in culture supernatant of HepG2 cells 72 h after transfection was determined by ELISA,while the replication of HBV DNA by Southern blot and real-time PCR.Results Sequencing result proved that HBV1.1 plasmid with HBsAg mutant was constructed correctly.The A450 value of HBsAg in culture supernatant of HepG2 cells 72 h after transfection with HBV1.1 plasmid was(0.06 ± 0.003),indicating a negative result.However,the A450 value in the cells in control groups was(1.82 ± 0.01),indicating a positive result.The concentration of HBV DNA bands in cells 72 h after transfection with HBV1.1 plasmid was significantly higher than that with plasmid pch9.The absolute copy number of HBV DNA in cells transfected with HBV1.1 plasmid in two tests were 9.33 × 106 and 5.26 × 106 respectively,which were 7.6 ~ 13.5 times of that in the cells transfected with plasmid pch9(6.91 × 105).Conclusions The HBV replication cell model with HBsAg deletion was successfully constructed,which provided an experimental basis for HBV DNA replication and the relevant studies.It indicated that HBsAg was a self-limiting factor in the formation of HBV DNA,which negatively regulated HBV DNA replication.