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目的运用酵母双杂交技术从人肝cDNA文库中筛选出与埃博拉病毒(EBOV)GP、VP30、NP、L蛋白存在相互作用的宿主蛋白,用于研究GP、VP30、NP、L蛋白在EBOV传染病中的生物学功能。方法利用重组PCR技术构建诱饵质粒pGBKT7-GP、pGBKT7-VP30、pGBKT7-NP、pGBKT7-L,将诱饵菌株与人肝cDNA文库菌株进行杂交筛选,对筛选到的阳性克隆进行DNA测序和生物信息学分析,然后再利用酵母回复性实验进一步验证,排除假阳性结果。结果成功构建了诱饵质粒pGBKT7-GP、pGBKT7-VP30、pGBKT7-NP、pGBKT7-L,筛选出6个可能与GP、VP30、NP、L蛋白有相互作用的宿主蛋白,分别是COMMD1、ALB、PSMD8、APOA2、CYP2E1、HP。酵母回复性实验进一步说明了COMMD1和APOA2与NP蛋白可能存在相互作用。结论酵母双杂交筛选出多个可能与EBOV的GP、VP30、NP、L蛋白存在相互作用的捕获蛋白,为研究EBOV的致病机制提供了参考。
OBJECTIVE To screen out the host proteins interacting with EBV GP, VP30, NP and L proteins from human liver cDNA library by yeast two-hybrid technique and to study the effects of GP, VP30, NP and L protein on EBOV Biological function in infectious diseases. Methods The bait plasmids pGBKT7-GP, pGBKT7-VP30, pGBKT7-NP and pGBKT7-L were constructed by recombinant PCR technique. The bait strains were screened by hybridization with human liver cDNA library. The positive clones were sequenced and bioinformatics Analysis, and then use yeast recovery test to further verify, eliminate false positive results. Results Six bait plasmids, pGBKT7-VP30, pGBKT7-VP30, pGBKT7-NP and pGBKT7-L, were successfully constructed. Six host proteins that could interact with GP, VP30, NP and L proteins were selected and identified as COMMD1, ALB and PSMD8 , APOA2, CYP2E1, HP. Yeast recovery experiments further illustrate the possible interaction of COMMD1 and APOA2 with the NP protein. Conclusion A large number of proteins that may interact with GP, VP30, NP and L proteins of EBOVS were screened by yeast two - hybrid system, which provided a reference for studying the pathogenesis of EBOV.