Construction of a 1.2-genome length genotype C HBV construct and study on its expression and replication in HepG2 cells

Objective To develop a 1.2-genome length genotype C HBV construct based on a plasmid pBlueBac4.5,and to study its expression and replication in HepG2 cells.Methods A recombinant plasmid pBB4.5HBV1.2(genotype C) was constructed using the 1.2-genome length HBV DNA sequence(genotype C) from a recombinant plasmid pWT and the pBlueBac4.5 vector sequence from a pBB4.5HBV1.3(genotype D) plasmid as templates.This recombinant plasmid was introduced into HepG2 cells by FuGENE○R HD based transient transfection method.Chemical luminescent immunoassay,Southern blot hybridization and fluorescence quantitative PCR were used for detection of HBsAg and HBeAg,HBV replication intermediates and HBV DNA levels at different time points after transfection,respectively.In addition,the amount of the recombinant plasmid used for transfection was optimized.Results The restriction digestion and plasmid sequencing showed that pBB4.5HBV1.2 recombinant plasmid was successfully constructed.HBsAg and HBeAg expression was detected in the supernatant of HepG2 cell culture after transfection.The optimized amount of the recombinant plasmid for transfection was 8~11μg pBB4.5HBV1.2 in a 60 mm cell culture plate,with the ratio of plasmid to trasfection reagent 5 μg to 9 μl.Under this condition,the persistent expression of HBsAg and HBeAg(peak expression at 3 days post transfection),persistent HBV DNA replication(106~108 copies/ml) and HBV replication intermediates were detectable throughout the experimental period(5 days).Conclusion An in vitro culture system of a 1.2-genome length genotype C HBV carried in baculovirus transfer vector pBlueBac4.5 was successfully established,and it might be used for the study on HBV drug resistance and screening new antiviral drugs.