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


CONCLUSIONS
Fis protein is a novel protein which can interact with X protein in vivo by yeast two-hybrid system.
Li
Dan
D
Department of Gastroenterology, Renmin Hospital, Wuhan University Medicine School, Wuhan, Hubei, 430071, PR China. lidan2006@hotmail.com
Wang
Xiao-Zhong
XZ
Chen
Zhi-Xin
ZX
Huang
Yue-Hong
YH
Yu
Jie-Ping
JP
Tao
Qi-Min
QM
D015723:Gene Library; D006509:Hepatitis B / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques; D054334:Viral Regulatory and Accessory Proteins
C476525:FIS1 protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein