Study On Candidate Interacting Proteins With Whole S Protein Of Hepatitis B Virus

OBJECTIVE:To investigate the prevalence of pre-pre-S region of hepatitis B virus (HBV) in Xiamen city and to screen for candidate proteins that interact with whole S protein of hepatitis B virus in hepatocytes.METHODS:The whole S region of HBV was amplified by polymerase chain reaction (PCR) method, and the PCR products were cloned into pMD19 T vector and sequenced. Vector 8.0 software was emploied to analyze the sequences. Bait plasmid expressing whole S gene was re-constructed by cloning the target region into pDEST32 and named pDEST32-w216. Cotransform pDEST32-w216 and prey plasmids which expressing 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 whole S protein, the bait plasmids expressing fibrinogen alpha, centaurin-gamma-like family member 6, enoyl coenzyme A hydratase, fructose-bisphosphate protein were constructed. The prey plasmids for reverse yeast two-hybrid system expressing 4 whole S protein mutants were 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:After sequencing, 18 out of 20 cases who were infected by with C2 subgenotype HBV were found coding pre-pre-S region. By yeast two-hybrid method, 25 colonies were screened out as positive colonies and their inserted cDNA were sequenced. Sequences of positive colonies code fructose-bisphosphate (ALDOB), serum albumin , jumping translocation breakpoint,fibrinogen alpha-chain,centaurin-gamma-like family member 6, enoyl coenzyme a hydratase,two vascular epithelium-cadherin and three new genes with unknown function. Reverse yeast two-hybrid results show that ALDOB, fibrinogen alpha-chain, centaurin-gamma-like family member 6, enoyl coenzyme a hydratase might be interact with four kinds of whole S protein mutants. The binding domain of whole S protein might be the leading 268 aa.CONCLUSION:The pre-pre S gene exists in HBV strains from Xiamen. According to yeast two-hybrid results, 4 human polypeptides/proteins were proved to be candidate interacting proteins with whole S protein. This research indicates that HBV envelope protein may involve in the metabolism of glucose and fat, cell proliferation, fibrinogen secretion. However, the roles of HBV envelope protein may be comprehensive and remain to be elucidated. Nevertheless, our work offers an opportunity to probe into the role of whole S protein in the pathogenesis of HBV-associated liver fibrosis and hepatic celluar carcinoma.