柔嫩艾美耳球虫钙依赖蛋白激酶4酵母双杂交诱饵质粒的构建与检测  

DETECTION AND CONSTRUCTION OF EIMERIA TENELLA CDPK4 BAIT PLASMID FOR YEAST TWO-HYBRID SYSTEM

作　　者：王自文[1] 董辉[1] 赵其平[1] 夏伟丽 朱顺海[1] 门启婓 李聪[1] 朱雪龙[1] 韩红玉[1] 黄兵[1,3] 

机构地区：[1]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室,上海200241 [2]上海师范大学生命与环境科学学院,上海200234 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2016年第1期62-69,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31572266);中央级公益性科研院所基本科研业务费专项资金项目(2014JB03)

摘　　要：为了筛选出柔嫩艾美耳球虫钙依赖蛋白激酶(Et CDPK4)在虫体内发挥作用时的作用受体即相互作用蛋白,构建了能够应用于筛选柔嫩艾美耳球虫子孢子酵母双杂交c DNA文库的诱饵重组质粒p GBKT7-Et CDPK4,本研究以柔嫩艾美耳球虫子孢子为材料,提取了总RNA,经反转录得到c DNA第一链,利用PCR的方法扩增获得了该基因大小为1660 bp的Et CDPK4的功能区片段,并将其连接于酵母BD诱饵p GBKT7载体上,经双酶切鉴定和序列分析,以及检测了p GBKT7-Et CDPK4在酵母双杂交系统中的自激活、细胞毒性和表达情况。结果显示,成功构建了酵母双杂交诱饵重组质粒p GBKT7-Et CDPK4,在酵母双杂交系统中没有自激活活性和细胞毒性,且在酵母细胞Y2HGold中能够进行表达。表明所构建的诱饵质粒p GBKT7-Et CDPK4可以应用于酵母双杂交系统中,用于筛选柔嫩艾美耳球虫子孢子酵母双杂交c DNA文库,获得可能与Et CDPK4具有相互作用的蛋白质。In order to screen for the interacting protein with CDPK4 of Eimeria tenella, the recombinant bait plasmid pGBKT7-EtCDPK4 was constructed, which can be used to screen for Eimeria tenella yeast two-hybrid c DNA library. The total RNA of E. tenella sporozoite was extracted, then the fi rst strand of c DNA was obtained by reverse transcriptase. The 1660 bp fragment of EtCDPK4 was amplifi ed by PCR and cloned into yeast BD bait vector pGBKT7, then the recombinant plasmid pGBKT7-EtCDPK4 was identifi ed by double enzyme digestion and sequenced. The self-activation, toxic action and protein expression of the recombination bait plasmid pGBKT7-EtCDPK4 were tested, respectively. These results show that the recombinant pGBKT7-EtCDPK4 plasmid was constructed successfully and proved to be no self-activation and toxic action. Moreover, it was expressed in the Y2 HGold yeast cell. So the recombinant plasmid pGBKT7-EtCDPK4 can be used to screen for Eimeria tenella sporozoites yeast two-hybrid c DNA library.

关 键 词：柔嫩艾美耳球虫 钙依赖蛋白激酶 诱饵质粒 自激活作用 细胞毒性 

分 类 号：S852.723[农业科学—基础兽医学]