利用Sos招募系统(SRS),通过聚合酶链反应(PCR)扩增羊布鲁菌16MVjbR基因的编码序列,定向克隆到酵母表达载体pSos中,构建诱饵重组质粒pSos-VjbR,经测序正确后其将转入酵母菌cdc25H(α)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自激活作用。结果表明,经序列测定证实重组诱饵质粒pSos-VjbR构建成功。重组质粒转化入酵母细胞后,经检测其表达产物对cdc25H(α)酵母细胞无毒性作用,对报告基因亦无自激活作用。这为利用SRS来研究与羊布鲁菌16MVjbR蛋白相互作用的蛋白奠定了基础。
To construct a yeast expression vector of Brucella melitensis 16M VjbR gene using the Sos-recruitment system (SRS), the coding sequence of Brucella rnelitensis 16M VjbR was amplified by PCR and cloned into the yeast expression plasmid pSos. The recombinant bait plasmid pSos-VjbR was verified by sequencing before transformation into competent yeast cells. The effects of the expression product on the yeast cell growth and activation of the report- er gene were evaluated. The results show the yeast expression vector of Brucella melitensis 16M VjbR gene was con- structed sucessfully. The recombinant bait plasmid showed no toxic effect on yeast cdc25H cells without a self-acti vation of the reporter gene. The SRS can be used to study the proteins interacting with Brucella melitensis 16M VjbR protein and provides a means for obtaining insight into the pathogenic mechanism of Brucella melitensis 16M.