Construction Of A Stable Expression Of Hbx Antigen In Human Hepatoma Cells By Use Of Knock-in Technique

Hepatitis B virus (HBV) infection is a worldwide health problem, particularly in China. HBV infection can cause acute, chronic and severe hepatitis. Moreover, these carriers have an elevated risk for cirrhosis and hepatocellular carcinoma (HCC). Several lines of epidemiological and experimental evidence have demonstrated a link between the HBV infection and the development of HCC. The incidence rate of HCC in patients with HBV was 200 times higher than those who were not infected. The crucial role of HBV in hepatocarcinogenesis is beyond doubt, although the mechanism of carcinogenesis by HBV is unknown.HBV is a member of the hepadnaviridae family of viruses. The natural host for HBV is humans. This virus consists of a partially double-stranded DNA genome of 3.2 kb. The genome is highly compact, such that over 50% of the coding capacity lies in at least two open reading frames. Viral mRNAs encode the viral core (HBcAg), envelope (HBsAg), polymerase (pol), and HBx polypeptides. HBx is the smallest ORF of HBV genome and distributes between 1374-1836nt, has numerous activities that are relevant to HBV-associated hepatocarcinogenesis. However, the role of HBx in the HCC is not yet well understood.Objective: Thus, establishment of an in vitro cell line with stable and effective HBx expression would greatly facilitate analysis of the function of HBx gene in the HBV-associated hapatocarcinogenesis. Here we established a hepatoma cell line stably expressing HBx antigen by means of trapping vector to integrate HBx gene into cell chromosome DNA.Methods: After the electroporation, gene trapping vector pU17 was randomly integrated into chromosome DNA of human hepatoma cells SMMC7721. The transfected cells were screened with G418. The highly expressing X-gal cells were identified by X-gal staining. With Cre-LoxP exchangeable system, HBx full length DNA was exchanged with the X-gal report gene in the highly expressing X-gal cell lines. The integration of HBx DNA and expression of HBx protein was determined by PCR checking and dot blot hybridization.Results: A stable cell line highly expressing X-gal report gene was established. With Cre-LoxP exchangeable system, HBx full length DNA was partially exchanged with the X gal report gene in the stable cell line. The integration and expression of HBx gene in the stable cell line was detectable by PCR analysis and dot blot hybridization protein detection.Conclusion: This HBx stably expressing cell line might be a useful experimental system for functional studies of the HBV X gene in the development of HCC. At the same time, it could be an experimental material for the preparation of HBx antigen.