中文摘要：

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。

英文摘要：

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.