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.
出处
《生物技术通报》
CAS
CSCD
北大核心
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)
