Construction and identification of the bait expression vector of GR?-LBD of human HL-60 cell / 重庆医科大学学报

ABSTRACT

Objective:To construct the bait expression plasmid pGBKT7-GR?-LBD of glucocorticoid receptor 1igand binding domain in order to lay the foundation for constructing small molecule ligand yeast three-hybrid system.Methods:A fragment of GR?-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-GR?-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?-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-GR?-LBD was constructed successfully,which lays the foundation for constructing small molecule ligand yeast three-hybrid system.