酵母双杂合体系筛选乙型肝炎病毒X蛋白结合蛋白

Screening Hepatitis B Virus X interactive Proteins by Yeast Two hybrid System

背景与目的:乙型肝炎病毒(hepatitisBvirus,HBV)X蛋白是一个广义反式作用因子,同原发性肝癌的发生发展密切相关,由于其功能同蛋白-蛋白间相互作用有关,故本研究利用酵母双杂合体系筛选并鉴定HBVX蛋白结合蛋白。方法:聚合酶链反应(polymerasechainreaction,PCR)法扩增HBVX基因序列,酵母双杂合体系3构建饵质粒pAS2-1-X,进行序列测定后转化入酵母菌AH109,以Westernblot法验证X-BD融合蛋白在酵母细胞中的正确表达。pAS2-1-X及正常成人肝细胞cDNA文库共转化酵母细胞,通过选择性培养、β-半乳糖苷酶活性测定筛选阳性菌落,分离实验及交合实验排除假阳性,扩增阳性克隆的目的基因并进行序列测定,检索其同源序列。结果:成功构建了pAS2-1-X饵质粒,Westernblot证实转化的酵母细胞能正确表达X-BD融合蛋白,pAS2-1-X及正常成人肝细胞cDNA文库共转化酵母细胞后,选择性培养共长出97个菌落,β-半乳糖苷酶活性测定、分离实验及交合实验后筛选出1个阳性克隆,其cDNA插入片段与Fis基因高度同源。结论:通过酵母双杂合体系筛选出一个在酵母细胞内能与X蛋白结合的Fis蛋白。

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 β 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 β 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.