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

ABSTRACT

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.