Preliminary Study In Hepatitis B Virus On Polymorphism Of X Gene And Binding Proteins Of X Protein

OBJECTIVE:To investigate a novel X gene mutation pattern in patients with chronic HBV infection and to screen for candidate proteins that interact with X protein of hepatitis B virus in hepatocytes.METHODS:A pair of primers was extracted on the basis of nucleotide sequences of X gene.Polymerase chain reaction(PCR) method was used to amplify the target region from HBV DNA samples in serum of patients from Xiamen city.After the electrophoresis of the PCR products in 9 g/L agarose gel,the target regions were cut and re-purified and TA-cloned into pMD19 T vector.The inserted regions in positive clones were sequenced.Sequence comparison with HBV genome submitted in GenBank was made to find the mutation sites.Bait plasmid expressing X gene was re-constructed by cloning the target region into pDEST32 and named as pDEST32-X. Cotransform pDEST32-X and prey plasmids which express liver cDNA library into the yeast cell MaV203 by Liac-mediated transformation.Diploid yeast was plated on synthetic dropout nutrient medium for selection.Prey plasmids were extracted from positive colonies and the inserted cDNA were sequenced for bioinformatics analysis. To validate the interaction between the candidate proteins and X protein,the bait plasmids expressing TCP1 protein were constructed.The prey plasmids for reverse yeast two-hybrid system expressing X protein was constructed as well.The reconstituted bait and prey plasmids were cotransform into the yeast cell MaV203 by Liac-mediated transformation to testify their interaction.RESULTS:Totally 74 strains from 21 patients with chronic HBV infection were sequenced.The result of comparing the sequenced strains with known HBV genome showed that there is a characteristic deletion region,length 234 nt in 54 clones,at nt 1601-1834.The HBV X gene was successfully obtained from patients with chronic HBV infection.The pDEST32-X vector and the pDEST22-liver cDNA libray vectors were successfully constructed.18 positive clones were obtained from the screen,and 3 were successfully sequenced.The TCP1 protein was also obtained.The interaction between TCP1 and HBV-X was certificated by reverse yeast two-hybrid.CONCLUSION:A novel deletion mutation in X gene was observed in 19 patients with chronic HBV infection.The successful screening and cloning of TCP1,the HBV X interacting protein genes from the hepatocytes,were very important for further investigation about the function of HBV X protein in the genesis and progress of hepatic cellular carcinoma.