【摘要】 目的构建维甲酸受体变异蛋白(RARα-V)的诱饵表达载体,为应用酵母双杂交系统筛选与RARα-V相互作用的蛋白建立实验基础。方法PCR扩增RARα-V,克隆入诱饵载体pGBKT7中,将构建好的诱饵载体pG-BKT7-RARα-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RARα-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,无自激活和渗漏,Western Blotting分析证实酵母细胞表达诱饵蛋白。结论成功构建了RARα-V结合域的酵母诱饵表达载体,为进一步筛选与之相互作用的蛋白提供基础依据。更多还原

【Abstract】 Objective To construct the bait expression vector pGBKT7RARα-V of retinoic acid receptor variant protein for screening the target proteins interacting with the bait protein through the yeast two-hybrid technique.Methods The fragments of RARα-V binding domain was amplified by PCR,and then cloned into the bait expression vector pGBKT7.After being verified by sequencing,the bait vector pGBKT7-RARα-V was transformed into AH109 yeast cells.Then the expression of the bait protein was analyzed by Western Blotting.Toxicity and self-activation of the bait protein were detected.Results RARα-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.Conclusion The bait expression vector of RARα-V was constructed successfully,which layed the foundation for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.更多还原