Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system

摘要

The function of middle hepatitis B surface protein C-terminally truncated at amino acid position 167 (MHBst¹⁶⁷) is not currently clear. This study aimed to screen and identify the proteins that interact with MHBst¹⁶⁷ in hepatocytes using a yeast two-hybrid system, and to explore the effects of MHBst¹⁶⁷ in the development of hepatocellular carcinoma and precancerous diseases of the liver. The MHBst¹⁶⁷ gene was amplified by polymerase chain reaction (PCR) and cloned into a pGEM-T vector. The target region was sequenced and the constructed bait plasmid, pGBKT7-MHBst¹⁶⁷, was transformed into AH109 yeast cells. The transformed AH109 cells were then mated with Y187 yeast cells containing the fetal liver cDNA library plasmid using a yeast two-hybrid system. The false positives were eliminated and the true positive clones were selected by PCR and sequencing analysis. The pGBKT7-MHBst¹⁶⁷ bait plasmid was successfully constructed and 66 clones grew in the selective synthetic defined media lacking leucine, tryptophan, histidine and adenine. Fifty-two clones were identified following X-α-Gal selection and segregation analysis. Seven proteins were found to be expressed that could interact with MHBst¹⁶⁷ in hepatocytes by the yeast two-hybrid system. These results have provided novel insights into the biological functions of MHBst¹⁶⁷.