酵母双杂交筛选人白细胞文库中与A型流感病毒PB1-F2相互作用的蛋白

Screening of Proteins Interacting with PB1-F2 of Influenza A Virus Using Yeast Two-hybrid System from Human Leukocyte cDNA Library

利用酵母双杂交技术筛选白细胞文库中与A型流感病毒PB1-F2相互作用的宿主蛋白,为研究PB1-F2蛋白的功能提供依据。构建pGBKT7-PB1-F2诱饵重组载体,在证实诱饵蛋白不具有自激活作用的前提下,以酵母双杂交系统筛选人白细胞文库中与PB1-F2相互作用的细胞蛋白。对阳性克隆进行测序和生物信息学分析,并在酵母细胞内验证蛋白的相互作用。Western blot结果表明,酵母表达载体pGBKT7-PB1-F2在酵母中成功表达PB1-F2融合蛋白。酵母双杂交筛选并验证,获得了8个与流感病毒PB1-F2蛋白相互作用的宿主蛋白。本研究有助于在分子水平上探索PB1-F2蛋白的新功能,为揭示流感病毒的致病机理奠定基础。

The research aimed to screen the proteins that interact with PB1-F2 of influenza A virus from human leukocyte cDNA library using yeast two-hybrid system,which will provide the basis for the research of PB1-F2 function.The bait plasmid pGBKT7-PB1-F2 was constructed and transformed into yeast AHl09.After exclusion of self-activation capacities of bait protein,a screening was performed using a pre-transformed human leukocyte cDNA library to screen the cellular proteins interacting with PB1-F2.The positive clones were sequenced and analyzed by bioinformatic methods,and verification of positive clones was also performed to further confirm the interactions in AH109 yeast cells.The result of Western blot showed that PB1-F2 fusion protein was expressed successfully in yeast by pGBKT7-PB1-F2 vector.Eight positive clones interacting with PB1-F2 were obtained via yeast two-hybrid screening and identification.The study will contribute to explore the PB1-F2 function at molecular level,which could provide evidence to explain the pathogenic mechanism of influenza A virus.