Cloning And Identification Of Genes Of The HBeAg Interacting Protein In Hepatocytes And Preliminary Study The Function Of One Of Those Novel Genes

The molecular mechanism of hapetocellular damage of HBV infection is strongly associated with the interaction between virus proteins and hapetocellular proteins. HBeAg is an ungranular secretory protein, which encoded by pre-C gene of HBV DNA, and it increases with the replication of HBV. So it is one of the markers of active replication of HBV in clinical diagnosis. Investigation on these possible proteins interaction with HBeAg can partly explain the molecular mechanism of HBV infection, and may provide some new clues for the prevention and cure of hepatitis B.The yeast two hybrid system originally developed for studying protein-protein interaction in vivo. It is a powerful, reliable assay, which can be used for cDNA library screening. The yeast two hybrid system-3 is an improved system. Compared with its initial version, it contains three reporter genes and has higher positive ratio. Based on the principle of the co-expression protein can interact in the diploid yeast cell, system-3 type a and type a yeast cell were used respectively and mated together. Using this approach, the low efficiency of co-transfection bait plasmid and library plasmid has been avoided. Based on this technique, PCR was performed to amplify the genes of HBeAg from the serum of the patients of HBV and cloned into pGEM-T vector. After identified by DNA sequence, the gene of HBeAg was sub-cloned into yeast expression plasmid pGBKT7 to construct the bait plasmid. The bait plasmids were transformed into yeast AH 109 (a type) and express. The maxipreparation of Diploid yeast cells was followed by mating transformed yeast cells with yeast cells Y187( a type) containing liver cDNA library plasmid pCAT2 in 2 X YPDA medium so that diploid yeast cellswere plated for screening two times onto synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x- a -gal. 245 blue clones containing HBeAg binding protein genes were selected. Plasmids were transformed into competent Escherichia coli and analyzed by DNA sequence. Through sequences alignment in GenBank, 6 novel genes and 35 recorded genes were screened.With the intensive sequence, we selected a novel protein AK026018 and amplified the genes of AK026018 and CD81 from the mRNA of HepG2 cell by RT-PCR and cloned into pGADT7 vector, the recombination plasmids was translated in reticulocyte lysate and analyzed by immunoprecipitation in vitro with HBeAg respectively to further prove their interaction.Finally, some preliminarily research jobs have been performed to investigate the biological functions of the HBeAg binding protein AK026018. Imaged with confocol microscope, protein of AK026018 distributes mainly in cytoplast. Under the cDNA microarray analysis, we found that the expression product of AK026018 may have multiple functions and regulate the expression of other genes through different ways. Overexpression and siRNA knock down assays proved positive correlation between gene AK026018 expression and the quantity of HBeAg secretion in L2.2.15 cell. CD81 overexpression showed that the quantity of HBeAg secretion was decreasing with the increasing of CD81 mRNA.