酵母双杂交诱饵载体pGBKT7-UL49的构建、表达与鉴定  

Construction and identification of yeast two-hybrid bait vector pGBKT7-UL49

作　　者：朱峰[1,2] 李月琴[1,2] 周天鸿[1,2] 何智强[1,2] 李实骞[1,2] 邹奕[1,2] 李弘剑[1,2] 

机构地区：[1]暨南大学广东省生物工程药物重点实验室,广东广州510600 [2]广州市职业病防治院,广东广州510620

出　　处：《中国现代医学杂志》2009年第22期3374-3377,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金重大计划面上项目(No:90608024);广东省科技计划项目(No:2006B35502002)

摘　　要：目的构建人巨细胞病毒UL49基因的诱饵表达载体,以利于筛选与pUL49相互作用的蛋白。方法PCR扩增UL49全序列,克隆入pGBKT7,将构建好的pGBKT7-UL49转化到酵母细胞AH109;用蛋白印迹法分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应。结果克隆成功pGBKT7-UL49;pG-BKT7-UL49成功转化到AH109,转化细胞AH109[pGBKT7-UL49]表达诱饵蛋白pUL49,pUL49对转化细胞无细胞毒性、无自激活。结论成功构建了酵母诱饵表达载体pGBKT7-UL49,为进一步筛选与pUL49相互作用的蛋白提供了实验基础。[ Objective ] To construct the bait expression vector pGBKT7-UL49 of human cytomegalovirus UL49 gene coding protein for screening the target proteins interacting with the bait protein pUL49 through the yeast twohybrid system. [Methods] The fragments of UIA9 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKTT-UL49, being verified by sequencing, was transformed into AH109 yeast cells. Then the bait protein pUL49 was analyzed by Western Blotting. Toxicity and self-activation of the bait protein were detected by cultured in different SD. [ Results ] UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH109 as well and those cells exhibited neither toxicity nor self-activation. The expression of the bait protein pUL49 was detected by Western Blotting. [ Conclusion ] The bait expression vector of UL49 was constructed successfully, which layed the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique.

关 键 词：酵母双杂交 人巨细胞病毒 UL49 质粒构建 诱饵蛋白 

分 类 号：Q733[生物学—分子生物学]