Study On The Transactivating Function Of HBV E Antigen

Hepatitis B virus (HBV) is the prototypic member of the hepadnavirus family, which is the main cause of acute and chonic liver diseases, including chonic active hepatitis, as well as cirrhosis and hepatocellular carcinoma in our country. HBeAg is an ungranular secretory protein which encoded by pre-C and C genes of HBV DNA, and it increases with the replication of HBV. So it is one of the markers which means there is active replication of HBV in patients.To explore the possible function of HBeAg, the recombined expression plasmid pcDNA3.1(-)-HBeAg was constructed, and HepG2 cells were transfected, and pcDNA3.1(-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-HBeAg and pcDNA3.1 (-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained product was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. At the same time, Microarray was employed for detecting and analyzing of both mRNA from the HepG2 cells transfected. The obtained sequences may be target genes transactivated by HBeAg, amongwhich some genes coding proteins involved in cell cycle regulation, signal transduction, translation and synthesis of protein, correlated with tumor and nervous system , and metabolism. These may explain the possible mechanism of HBeAg in vivo. A gene whose function is unknown was named HBeAg transactivated protein (HBeAgTP).We investigated the biological functions of HBeAgTP. The whole HBeAgTP gene sequence was amplified by PCR from human hepatocarcinoma cells. Green fluorescent protein (GFP) expression vector pEGFP-HBeAgTP was established, which makes HBeAgTP can fuse with green fluorescent protein with pEGFP-Cl vector in cells. The result suggest that HBeAgTP were subcellularly located in cell plasma. Advanced research was to screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) using yeast-two hybrid technique. The obtained protein genes involve in translation of protein, immunoregulation, material and energy metabolism in vivo. It is the first step to investigated the biological functions of HBeAgTP, and pave a way for the following investigation of the more exact biological functions and the modulation of the gene expression.