羊布鲁氏菌16M Omp25蛋白酵母双杂交诱饵表达载体的构建及其毒性和自激活效应检测  

Construction of a Bait Plasmid Containing Brucella melitensis Omp25 Gene in a Yeast Two-hybrid System and Evaluation of Its Toxicity and Self-activation

作　　者：屈海龙[1,2] 王海浪[3] 李晓燕[1,2] 张瑞[1,2] 王秀然[1,2] 陈思[1,2] 钱晶[1,2] 王兴龙[1,2] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130122 [2]吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]北京奶牛中心,北京100192

出　　处：《安徽农业科学》2012年第19期10156-10159,共4页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金(30972198/C180503);国家科技支撑计划(2010BAD04B03)

摘　　要：[目的]构建含羊布鲁氏菌16M Omp25基因的酵母双杂交诱饵质粒,检测诱饵质粒表达产物对cdc25H酵母细胞有无毒性作用以及对报告基因有无激活作用。[方法]聚合酶链反应(PCR)扩增布鲁氏菌Omp25基因的编码序列,定向克隆到酵母表达载体pSos上,构建诱饵重组质粒pSos-Omp25,经测序正确后,将其将转化到酵母菌cdc25H感受态细胞,检测其表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。[结果]序列测定证明重组诱饵质粒pSos-Omp25构建成功;重组质粒表达产物对cdc25H酵母细胞无毒性,对报告基因无激活作用。[结论]利用SOS恢复系统(SRS)成功构建了羊布鲁氏菌16M Omp25蛋白酵母双杂交诱饵质粒,为筛选与羊布鲁氏菌16M Omp25蛋白相互作用的蛋白创造了条件。[Objective] To construct a yeast expression vector of Brucella melitensis Omp25 gene using the Sos-recruitment system（SRS）,and evaluate the effect of the expression product on the growth of yeast cells and activation of the reporter gene.[Method] The coding sequence of Brucella melitensis Omp25 was amplified by PCR and cloned into the yeast expression plasmid pSos.The recombinant bait plasmid pSos-OMP25 was verified by sequencing before being transformed into competent yeast cells.The effects of the expression product on the yeast cell growth and activation of the reporter gene were evaluated.[Result] The yeast expression vector of Brucella melitensis Omp25 gene was constructed successfully.The recombinant bait plasmid showed no toxic effect on yeast cdc25H cells without a self-activation of the reporter gene.[Conclusion] The SRS can be used to study the proteins interacting with Brucella melitensis Omp25 protein and provides a means for obtaining insight into the pathogenic mechanism of Brucella melitensis.

关 键 词：Omp25蛋白 诱饵质粒 自激活作用 

分 类 号：S811.6[农业科学—畜牧学]