Stra8酵母双杂交诱饵载体的构建及自激活活性分析

The construction of yeast two-hybrid bait vector pGBKT7-Stra8 and identification of its toxicity and self-activation

目的构建小鼠Stra8的诱饵表达载体,为应用酵母双杂交系统筛选与Stra8相互作用的蛋白建立实验基础。方法应用PCR扩增Stra8编码序列的不同长度片段,将其先克隆入pGBKT7载体内,经序列测定确认无误后,将构建好的系列诱饵载体pGBKT7-Stra8转化到酵母细胞AH109及Y187中,进行诱饵载体的毒性及自激活活性分析。结果构建了Stra8的诱饵载体共8种,经过自激活活性的分析发现,其中7种诱饵载体均具有自激活活性,仅有一种诱饵载体没有自激活活性,可用于后续的酵母双杂交筛选实验。结论成功构建了Stra8的酵母双杂交诱饵表达载体,为进一步筛选与之相互作用的蛋白提供实验基础。

[Objective]To construct the bait expression vector pGBKT7-Stra8 for screening the target proteins interacting with the bait protein through the yeast two-hybrid technique.[Methods]The fragments of Stra8 were amplified by PCR,and then were cloned into the bait expression vector pGBKT7.After being verified by sequencing, these bait vectors were transformed into AH109 and Y187 yeast cells.Then toxicity and self-activation of these bait proteins were detected.[Results]The fragments of Stra8 were amplified and cloned into pGBKT7 successfully.The 8 bait vectors were transformed into AH109 and Y187.Among eight recombinant plasmids pGBKT7-Stra8,seven of them have self-activation.Only one of them has no self-activation and toxicity to AH 109 and Y187 yeast cells. [Conclusion]The bait expression vector of Stra8 was constructed successfully,which was suitable for the further study