The construction of yeast two-hybrid bait vector pGBKT7-RARα-V

Objective To construct the bait ex pression vector pGBKT 7-RARA-V of retinoic acid receptor variant protein for screening the target proteins interacting with the bait protein through the yeast two-hybrid technique.Methods The frag ments of RARA-Vbinding domain was amplified by PCR,and then cloned into the bait expression vector pGBK T7.After being verified by sequencing,the bait vector pGBKT 7-RARA-V was transformed into AH109 yeast cells.Then the expression of the bait protein was analyzed by Western Blotting.Toxicity and self-activation of the bait protein were detected.Results RARA-V was amplified and cloned into pGBKT 7 successfully.The bait vector was transfor med into AH109 as well and no tox icity and self-activation were found.The expression of the bait protein was confirmed by Western Blotting.Conclusion The bait expression vector of RARA-V was constructed successfully,which layed the foundation for screening tar get proteins interacting with the bait protein using the yeast two-hybrid technique.