人HL-60细胞GR-αLBD诱饵表达载体的构建和鉴定

Construction and identification of the bait expression vector of GRα-LBD of human HL-60 cell

目的:构建糖皮质激素(Glucocorticoid,GC)受体α配体结合域(GRα-LBD)的诱饵表达载体,为小分子配体酵母三杂交系统的建立奠定基础。方法:RT-PCR扩增HL-60细胞的GRα-LBD,克隆入诱饵载体pGBKT7中,测序正确后,再把构建好的诱饵载体pGBKT7-GRα-LBD转化到酵母AH109细胞中,提取酵母蛋白,并用Western blot分析诱饵蛋白的表达情况。同时检测诱饵蛋白的毒性和自激活作用。结果:成功扩增了GRα-LBD,并成功亚克隆到pGBKT7中,测序结果正确。诱饵载体成功转化到酵母AH109细胞中,无毒性和自激活作用,Western blot分析也证实了酵母细胞高表达诱饵蛋白。结论:成功构建了GRα-LBD酵母诱饵表达载体,为建立小分子配体酵母三杂交系统奠定了基础。

Objective：To construct the bait expression plasmid pGBKT7-GRα-LBD of glucocorticoid receptor ligand binding domain in order to lay the foundation for constructing small molecule ligand yeast three-hybrid system. Methods：A fragment of GRα-LBD of human HL-60 cell was amplified by RT-PCR,and then was subcloned into the bait expression vector pGBKT7. After verified by sequencing, the bait vector pGBKT7-GRα-LBD was transfected into AH109 yeast cells. And the expression of the bait protein was analyzed by Western-blotting. Toxicity and self-activation of the bait protein were detected simultaneously. Results ：GR α-LBD was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 yeast cells successfully, and no toxicity and self-activation were found. The expression of the bait protein was confirmed by western blot. Conclusion：The bait expression vector pGBKT7-GRα-LBD was constructed successfully,which lays the foundation for constructing small molecule ligand yeast three-hybrid system.