放线杆菌外膜蛋白酵母双杂交诱饵质粒的构建及自激活与毒性检测

Construction of Actinobacillus pleuropneumoniae Omp bait plasmid for yeast two-hybrid system and detection of its self-activation and toxic effect

为了筛选胸膜肺炎放线杆菌外膜蛋白在肺泡上皮细胞上的作用受体,应用酵母双杂交技术构建诱饵质粒载体pGBKT7-omp(胸膜肺炎放线杆菌外膜蛋白),从胸膜肺炎放线杆菌提取基因组DNA,用PCR方法获得omp,克隆于pGBKT7载体上,进行酶切鉴定及序列分析,并检测pGBKT7-omp在酵母双杂交系统中的自激活和毒性作用。结果表明,成功构建了诱饵质粒载体pGBKT7-omp,并证明其在酵母双杂交系统中无自激活及毒性作用。表明pGBKT7-omp可应用在酵母双杂交系统中,用于寻找猪肺泡上皮细胞cDNA文库中与omp相互作用的蛋白质。

To construct the bait vector pGBKT7-omp of Matchmaker GAL4 two-hybrid system,omp fragment was amplified by PCR from A.pleuropneumoniae and cloned into pGBKT7.Self-activation and toxic action of the recombination vector pGBKT7-omp were tested.The pGBKT7-omp vector was successfully constructed and proved to be no self-activation and toxic action.The pGBKT7-omp could be applied for the screening of proteins interacting with the omp in porcine alveolar epithelial cell cDNA library in the yeast two-hybrid system.