Construction Of HBV Eukaryotic Expression Plasmid And Establishment Of The Stable Cell Line For HBV Secretion

Hepatitis B is a major global public health problem. About 350 million people are chronicly infected with HBV all over the world, which may lead to liver cirrhosis and even hepatocellular carcinoma(HCC). Current treatments on chronic HBV infected patients comprise type-I Interferons (IFNs) and the nucleoside analogs(NAs), which have been widely used for clinical application and slow down the aggravation of the diseases to some extent. However, the relatively high rate of HBV mutation and the limited efficacy of the drugs make it impossible to always work on inhibition of virus infection and viral replication in the human liver cells. In order to find out new treantments and improved antivirals, the mechanism of HBV infection should be further elucidated, which is dependend on the establishment of the ideal model of HBV infection. Studies about in vivo model of HBV infection made slow progress in the past 20 years, while in vitro model of HBV infection had a significant development. One such cell model, the stably HBV-expressing cell lines has become a hot subject research by the foreign and domestic scholars, because they have stable level of viral expression and essentially all cells are productive.In this study, we constructed the eukaryotic expression plasmid pneo-CH9/3091 containing 1.1-unit-length HBV and CMV promoter, which could express the anti-neomycin proteins. Following stably transfected into HepG2 cells and G418 screening, cell clones were picked out. Through multiple molecular analysis, such as ELISA was used to monitor HBsAg and HBeAg secretion in the supernatant of the cells; Western blot was to detect the expression of HBV core protein in the cells, and Southern blot was to check the situation of HBV DNA replication in the cells. One stable cell line HepG2-H7 integrated with HBV genome is successfully established. We compared this cell line with HepG2.2.15 on the level of HBV DNA replication and expression of HBV proteins, etc. The data demonstrated that the stable cell line HepG2-H7 may be valuable in antivirals screening and studying the mechanism of HBV infection.