[Screening hepatitis B virus X-interactive proteins by yeast two-hybrid system].

BACKGROUND & OBJECTIVE: Hepatitis B virus-encoded X protein is a promiscuous transactivator and contributes to the development of hepatocellular carcinoma. Protein-protein interaction seems to be crucial for HBx transactivation. The aim of this study was to screen and identify the proteins which interact with hepatitis B virus (HBV) X protein by yeast two-hybrid system. METHODS: HBV X gene was amplified by polymerase chain reaction (PCR). HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system 3 and verified by sequencing. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was confirmed by Western blot analysis. Yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were cultured in selective SC/-trp-leu-his-ade medium. The second screening was performed with beta-gal activity detection. The false positive clones were eliminated by segregation analysis and mating experiment. The real positive clones were amplified, sequenced, and analyzed with bioinformatics. RESULTS: Bait plasmid pAS2-1-X was successfully constructed. The result of Western blot analysis confirmed that pAS2-1-X correctly expressed X-BD fusion protein in the transformed yeast AH109. Ninety-seven clones grew in the selective SC/-trp-leu-his-ade medium; however, only one clone past through the beta-gal activity detection, segregation analysis, and mating experiment. The inserted cDNA fragment of positive clone showed high homology with Fis gene. CONCLUSION: Fis protein is a novel protein which can interact with X protein in vivo by yeast two-hybrid system.