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

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.

Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V / 重庆医科大学学报

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 serf-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.Conclusion: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.

Construction and identification of bait expression vector of glucocorticoid receptor binding domain / 第三军医大学学报

Objective To construct the bait expression plasmid pGBKT7-GR of glucocorticoid receptor(GR)binding domain.Methods The fragments of GR binding domain was amplified by RT-PCR,and then was cloned into pMD18-T.After being verified by sequencing,it was subcloned into the bait expression vector pGBKT7.Then the bait vector pGBKT7-GR was transformed into AH109 yeast cells and the expression of the bait protein was analyzed by Western blot.Toxicity and self-activation of the bait protein were detected.Results GR binding domain was amplified and cloned into pMD18-T and pGBKT7 successfully.The bait vector was transformed into AH109 yeast cells successfully,without toxicity or self-activation.The expression of the bait protein was confirmed by Western blot.Conclusion The successful construction of bait expression vector of glucocorticoid receptor binding domain lays the foundation for constructing small molecule ligand yeast three-hybrid system.