脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证  

Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System

作　　者：谢晶莹[1,2] 冯若飞[1,3] 徐雷[2] 张海霞 李向茸 侯兰新[2] 马忠仁 

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730030 [3]甘肃省动物细胞工程技术研究中心,兰州730030

出　　处：《生物技术通报》2015年第1期198-202,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31460665;31160033);教育部"长江学者和创新团队发展计划"项目(IRT13091);西北民族大学研究生科研创新项目(ycx13180)

摘　　要：为筛选与脑心肌炎病毒VP1蛋白相互作用的靶细胞cDNA文库蛋白,构建VP1蛋白的诱饵载体pDHB1-VP1。扩增EMCV的VP1基因并克隆至pMD18-T载体中,经测序验证正确后定向克隆至酵母双杂交诱饵载体pDHB1。将重组pDHB1-VP1载体进行酶切验证和测序分析,并转化酵母报告菌株NMY51,检测其在酵母细胞中有无表达和自激活作用。结果表明,构建的p DHB1-VP1基因可以在酵母细胞中正确表达,产物大小约66 kD,而且可以与兔抗EMCV血清发生特异性结合,有较好免疫原性。成功构建了诱饵载体pDHB1-VP1,可以在酵母细胞中表达且其对报告基因无自激活作用,可以应用于酵母双杂交筛选试验中。Bait vector pDHB1-VP1 was constructed for screening cellular proteins interacting with VP1 protein of encephalomyocarditis virus from yeast two-hybrid cDNA library of target cells in this study. VP1 gene was amplified and cloned into pMD18-T vector. After being verified by sequencing, it was directional cloning into bait vector pDHB1 of yeast two-hybrid system. Then the recombinant plasmid was identified by enzyme digestion and sequencing and transformed into yeast cells NMY51.The bait vectors’ expression and self-activation to reporter genes were tested.The results showed that the bait plasmid pDHB1-VP1 could express in yeast cells, and product size was 66 kD. It was specific binding with rabbit anti EMCV serum, which showed better immunogenicity. Bait plasmid pDHB1-VP1 was successfully constructed, could express in yeast cells and proved to be no self-activation to reporter genes.It could be used in the yeast two-hybrid system screening test.

关 键 词：脑心肌炎病毒 VP1蛋白 酵母双杂交 诱饵蛋白 

分 类 号：S852.65[农业科学—基础兽医学]