酵母双杂交诱饵载体pGBKT7_pprⅠ的构建及自激活检测  

Construction and identification of yeast two-hybrid bait vector pGBKTT-pprⅠ

作　　者：廖永强[1] 何淑雅[1] 蒋亮[1] 杜邱[1] 马云[1] 

机构地区：[1]南华大学生物化学与分子生物学教研室,衡阳421001

出　　处：《国际遗传学杂志》2010年第1期1-4,15,共5页International Journal of Genetics

基　　金：基金项只：国家自然科学基金（30770647）;湖南省自然科学基金（06JJ2023）

摘　　要：目的构建耐辐射球菌（Deinococcvz radiodurans）R1 pprⅠ蛋白的诱饵表达载体，为应用酵母双杂交系统筛选与pprI相互作用的蛋白建立实验基础。方法PCR扩增pprⅠ基因片段，克隆入诱饵载体pGBKT7中，经测序鉴定后，将构建好的诱饵载体pGBKT7_pprⅠ转化到酵母细胞AH09中，检测诱饵蛋白有无毒性、渗漏和自激活作用。结果成功扩增了pprⅠ基因片段，并克隆入pGBKT7中，测序结果正确。诱饵载体成功转化到酵母细胞AH109中，诱饵蛋白无毒性、渗漏和自激活作用。结论成功构建了pprⅠ的酵母诱饵表达载体，为进一步运用酵母双杂交技术筛选与之相互作用的蛋白奠定了基础。Objective To construct the bait expression vector pGBKT7-pprⅠ of Deinococcus radiodurans R1, for screening the target proteins interacting with the bait protein through the yeast two-hybrid system. Methods The fragments of pprⅠ was amplified by PCR,and then was cloned into the bait expression vector pGBKTT. After being verified by sequencing, the bait vector pGBKTT-pprⅠ was transformed into AH109 yeast cells. Then the toxicity, leakage and self-activation of the bait protein were detected. Results the ppr I was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109. No toxicity , leakage and self-activation were found. Conclusion The bait expression vector of ppr I was constructed successfully,which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.

关 键 词：耐辐射球菌 pprⅠ蛋白 载体构建 诱饵蛋向 

分 类 号：Q78[生物学—分子生物学]