Aim: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro.
Methods: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over-length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively.
Results: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 mug/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 microg and 2.0 microg plasmid pXF3H-PAP, the levels of HBV nucleocapside-associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 mug plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.
Conclusion: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.