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

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.