酵母双杂交诱饵载体pGBKT7-PML-V的构建、表达及鉴定  

Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V

作　　者：钟梁[1] 王翀 刘北忠[1] 王东生[1] 郝坡[1] 刘畅[1] 金丹婷[1] 王春光[1] 

机构地区：[1]重庆医科大学临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2008年第3期257-260,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金项目(No.30300449);国家中医药管理局基金项目(No.02-03ZP52);重庆医科大学校级课题(No.XBYB2007104;No.XBYB2007 108)。

摘　　要：目的:构建早幼粒细胞白血病基因变异蛋白(Promyelocytic leukemia,PML-V)的诱饵表达载体,为应用酵母双杂交系统(Yeast two-hybrid system)筛选与PML-V相互作用的蛋白建立实验基础。方法:PCR扩增PML-V,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-PML-V转化到酵母细胞AH109中,并利用蛋白印迹法(Western Blotting)分析诱饵蛋白的表达情况。同时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PML-V基因片断,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,Western Blotting分析证实酵母细胞表达诱饵蛋白。结论:成功构建了PML-V结合域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-PML-V of retinoic acid receptor variant protein,for screening the target proteins interacting with the bait protein through the yeast two-hybrid system. Methods：The fragments of PML-V binding domain was amplified by PCR,and then was cloned into the bait expression vector pGBKT7. After being verified by sequencing,the bait vector pGBKT7-PML-V was transformed into AH109 yeast cells. Then the expression of the bait protein was analyzed by Western Blotting. Toxicity,leakage and self-activation of the bait protein were detected. Results： PML-V was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 as well and no toxicity and self-activation were found. The expression of the bait protein was confirmed by Western Blotting. Conchtsion：The bait expression vector of PML-V 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：PML变异蛋白 质粒构建 诱饵蛋白 

分 类 号：R733.71[医药卫生—肿瘤]