Screening and identification of a novel gene coding for hepatitis B virus core antigen interacting protein C-12 in hepatocytes

AIM: Hepatitis B virus(HBV) core protein (HBcAg) is present in the nucleus and cytoplasm of infected hepatocytes. Phosphorylation of HBcAg was a prerequisite for pregenomic RNA encapsidation into viral capsids. HBcAg capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors and HBcAg-specific CD4(+) T-cell responses are believed to play an important role in the control of human HBV infection. To investigate the complex biological functions of HBcAg, we employed yeast-two hybrid technique to screen proteins in hepatocytes interacting with HBcAg. METHODS: HBcAg gene was amplified by polymerase chain reaction (PCR). The pGBKT7-HBcAg bait plasmid was constructed by using yeast-two hybrid system 3 and transformed into yeast cells AH109, then mated with yeast cells Y187 containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection two times. After extracting and sequencing of plasmid from blue colonies, we conducted bioinformatics analysis. Primers of new gene were designed according to the information in Genbank and used to amplify the complete sequence of new gene C-12#. Gene of C-12# was ligated into another yeast expression vector pGADT7 and transformed into yeast cell Y187 and mated with yeast cell AH109 containing pGBKT7-HBcAg bait plasmid to further verify the interaction between HBcAg and the novel protein coded by the new gene C-12. RESULTS: Sixteen colonies were sequenced. Among them, there were four new genes with unknown function. The complete sequence of new gene C-12 was successfully amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction(RT-PCR). The interaction between HBcAg and the novel protein coded by the new gene C-12 was further confirmed by re-mating. CONCLUSION: Genes of HBcAg interacting proteins in hepatocytes were successfully cloned. The findings of new genes coding for HBcAg associated proteins pave the way for studying the biological functions of HBcAg.