酵母双杂交系统筛选与狂犬病病毒M蛋白相互作用的宿主蛋白

Screening Host Proteins Interacting with Rabies Virus Matrix Protein by Yeast Two-hybrid System

为筛选人脑cDNA文库中与狂犬病病毒基质蛋白(M蛋白)具有相互作用的蛋白基因,并对阳性克隆进行初步的分析和鉴定。首先构建了含有M基因的诱饵质粒pGBKT7-CVS11-M,并对其进行自激活和毒性检测,然后采用酵母双杂交技术初步筛选出与M蛋白具有相互作用的12个阳性克隆,对阳性克隆进行PCR鉴定及测序分析,并通过酵母回复性试验最终获得3个无重复性克隆,编码的蛋白质分别为TAB2、P4HA2和RCN1。为研究狂犬病病毒M蛋白在致病机制中的作用提供了参考。

In this study,a human brain cDNA library was screened to identify rabies virus matrix protein（M protein）interaction partners,and the positive clones were preliminarily identified.The bait plasmid pGBKT7-CVS11-M was constructed successfully and tested for auto-activation and toxicity and then was used to screen the proteins interacting with M protein in a yeast two-hybrid assay.Twelve positive clones were obtained.Subsequently,they were verified by PCR and sequencing analysis,and further conformed by retransformation assay.Three final positive proteins were verified as interacting proteins with M protein through retransformation assay in yeast cells,which were TAB2,P4HA2 and RCN1.The research will contribute to explore the function of M protein at molecular level,which would provide important clues for studying the pathogenic mechanism of rabies virus.