牛源犬新孢子虫NcAMA1基因酵母双杂交诱饵载体构建及鉴定  

Construction and indentification the Neospora caninum AMA1 bait vector in yeast two-hybrid system

作　　者：李积旭 张守发[1] 李男礼[1] 贾立军[1] 

机构地区：[1]延边大学农学院,吉林延吉133002

出　　处：《中国兽医学报》2016年第1期66-69,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31160501;31360605);吉林省重点科技攻关资助项目(20140204078NY)

摘　　要：为筛选牛源犬新孢子虫AMA1与虫体互作的靶蛋白,构建NcAMA1基因酵母双杂交诱饵载体pGBKT7-AMA1。本试验应用RT-PCR技术从虫体总RNA中扩增了去除跨膜螺旋的AMA1基因,定向克隆到酵母双杂交诱饵载体pGBKT7中,将重组诱饵载体转化酵母Y2H,并验证其在酵母细胞中毒性作用和有无自激活现象。结果显示:本试验成功构建了诱饵载体pGBKT7-AMA1,并证明其对酵母细胞无毒性,且对报告基因无自激活。结果表明:诱饵载体pGBKT7-AMA1可用于酵母双杂交系统筛选与虫体互作的宿主蛋白。The recombinant bait vector pGBKT7-AMA1 was constructed for screening the proteins which could interact with Neospora caninum AMA1 of the host target protein and be used for yeast two-hybrid screening technology. The AMA1 gene of removal transmembrane helices was amplified from total RNA which Neospora caninum by the RT-PCR technology, then subcloned into the bait vector pGBKT7 of yeast two-hybrid system, and the vector was transformed into yeast cells Y2H. The toxic action and self-activation of the bait vector pGBKTT-AMA1 were test- ed. The results indicated that pGBKTT-AMA1 was successfully constructed and with no toxic and self-activation to the host yeast. The pGBKTT-AMA1 could be used for screening the target pro- teins interacting with Neospora caninurn in the yeast two-hybrid system.

关 键 词：犬新孢子虫 AMA1 酵母双杂交 诱饵载体 

分 类 号：S852.72[农业科学—基础兽医学]