The Impact Of Hot-spot Mutations In HBV Precore Signal Peptide Region On The Expression Of HBeAg And HBV Genome Replication

To study the impact of A1896, T1862, A1896+A1899and A1899 mutations in HBV precore region on the expression of HBeAg and HBV genome replication. Technology of site-mutagenesis in vitro was used to construct HBV preC/C eukarotic expression vectors (EBO-preC/C) and HBV genome plasmid (P3.8II) with T1862, A1896.A1899 and A1896+A1899 mutation. All variants were transfected to HepG2 cells. HBeAg and its precursor in the supernatant and cells transfected EBO-preC/C was tested to see the difference of HBeAg expression between wild type and variants by EL1SA and Western blot. SEAP reporter system was used to monitor the transfection efficiency of P3.81I variants. And Replication changes were analysed by southern blot. The results were as follows, HBeAg was negative in the supernatant of A1896, A1896+A1899 and Tl 862 variants, however it was positive in the supernatant of wild type and A1899 variant by ELISA. By Western blot, HBeAg and its precursor were detected negative in cells transfected A1896, A1896+A 1899 variants, however it was positive in cells transfected with wild type and A1899 variant. Though HBeAg was negative in cells, its precursor in cells transfected with T1862 variant was significantly higher than any other variants and wild type. By southern blot, DNA in cells transfected with A1896 and A1896+ A1899 variants was stronger than T1862 variant and wild type, which suggested A1896 and A1896+A 1899 mutations would enhance replication of HBV genome.