Cloning And Identification Of Genes Of Protein Interacting With HBV In Hepatocytes And Preliminary Study Of The Function Of A Novel Gene

The molecular mechanism responsible for hapetocellular damage caused by HBV infection are strongly associated with the interaction between virus proteins and hapetocellular proteins. The investigation on these possible protein-protein interaction with each peptides of HBV can partly explain the 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 a improved system. Compared with its initial version, it contains three report gene and has higher true positive ratio. Based on the co-expression protein can interact in the diploid yeast cell, system-3 type a and type a yeast cell were used respectively for and mated together. Using this approach, the low efficiency of co-transfection of bait plasmid and library plasmid has been avoided. Based on this technique, PCR was performed to amplify the genes of HBV antigen from the plasmid pCP10(HBV ayw strain containing the whole fragment of HBV) and ligated into pGEM-T vector. After identified by DNA sequencing. The genes of HBV antigen was cut from pGEM-T clone vector by enzyme digesting and cloned into yeast expression plasmid pGBKT7 to construct the bait plasmid. The bait plasmid were transformed into yeast AH109(a type) and expressed in it. 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 + YPDA medium so that diploid yeast cells were 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. Tirty-nine blue clones containing HBeAg binding protein gene, twenty-six blue clones of PreS2, sixteen blue clones of HBcAg, forty-one blue clones of HBxAg were obtained. After extracting plasmid from blue colonies , plasmid was transformed into competence Escherichia coli and analysed by DNA sequencing. Sequences were analysed in GenBank, 13 novel genes and a lot of realized genes were screened.After the integrity sequence of the genes of metallothionein, a novel mutant o f a sialoglycoprotein r eceptor 2 a nd s ome n ovel p roteins w ere amplified from the mRNA of HepG2 cell by RT-PCR and cloned into pGADT7 vector, the recombinational plasmid was translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro together with HBV antigens respectively to further prove the interaction between them.At the last part, some preliminarily research works have been performed to investgate the biological functions of a novel HBcAg binding protein C-12. Using fluorescence protein co-expression method, we located the protein of C-12 at cell plasma. It does not influence growth of the liver cells by MTT assay. Under the cDNA microarray technology and yeast two hybrid system analysis, we found that the express product of C-12 may have complex functions and can adjust or influence the expression of other genes through different ways.