Establishment and identification of HBV Lamivudine- YVDD resistant variant and YMDD wild-type stably expressed cell lines

Abstract

Objective To establish cell lines which stably express of hepatitis B virus(HBV) YVDD drug resistance mutations and YMDD wild type. Methods The eukaryotic expression recombinant plasmids carrying HBV YVDD resistant strain or YMDD wild strain were used to transfect into HepG2 cells.After G418 screening, stable expression cell lines were established.The expression of HBV antigen was detected by enzyme linked immunosorbent assay(ELISA)and HBV DNA load were determined by FT-PCR.FH-PCR-MC was used to verify the types of lamivudine resistance variants in cell lines.The genetic variation of HBV in cell lines was analyzed by sequencing. Results The cell lines which stably expressed HBV YVDD drug-resistant variant or YMDD wild-type were successfully established.The expression of HBsAg and HBeAg was stable in the 1st, 10th and 30th generation of the cells.In the 30th generation of the cells, the HBV DNA load was higher than 104copies/mL both intracellularly and extracellularly.The drug-resistant variant type was YVDD.Sequencing showed that the HBV gene carried by the cell genome contained multiple joint variants of A1762T/G1764A/rtM204V/rtL180M/rtV173L. Conclusions Cell lines stably expressing HBV YVDD drug-resistant variant and YMDD wild-type, namely HepG2-HBV/CV and HepG2-HBV/CM cells, were successfully established, providing an ideal cell model for screening effective drugs against HBV lamivudine resistant strains. Key words: Hepatitis B Virus; YMDD; YVDD; Stable expression cell lines