协同激活分子CBP诱饵表达质粒的构建和鉴定

构建协同激活分子CBP（CREB（cAMP response element binding）binding protein）的诱饵表达质粒pGBKT7-CBP并检测其蛋白表达、毒性和自激活作用.PCR扩增小鼠CBP的基因编码序列（CDS（coding sequence）序列）并克隆入诱饵表达载体pGBKT7中,通过酶切和测序鉴定后,把构建好的诱饵表达质粒pGBKT7-CBP转化到酵母AH109细胞中,Western blot检测诱饵蛋白表达情况,同时检测诱饵蛋白的毒性和自激活作用.结果成功扩增了小鼠CBP基因的CDS序列,并成功克隆到酵母诱饵表达载体pGBKT7中,测序结果正确.诱饵表达质粒成功转化到酵母AH109细胞中,Western blot分析结果证实酵母细胞高表达诱饵蛋白CBP,但诱饵蛋白有自激活作用.提示pGBKT7-CBP不能用于酵母双杂交或三杂交系统检测CBP与其他蛋白质或小分子的相互作用,对其他研究CBP生物学功能试验的方法选取具有一定的借鉴意义.

Construction and Identification of Bait Expression Plasmid of Co-activator CBP

To construct the bait expression plasmid pGBKT7-CBP(CREB(cAMP response element binding)binding protein)of co-activator CBP and detect its protein expression,toxicity and self-activation,the CDS (coding sequence)sequence of mouse CBP was amplified by PCR,and then was cloned into pGBKT7.After being verified by cutting and sequencing,it was transformed into yeast cells AH109 and the expression of the bait protein was analyzed by Western blot.Toxicity and self-activation of the bait protein were detected.It Was demonstrated that the CDS sequence of motlse CBP Was amplified and cloned into pGBKT7 successfully,the bait expression plasmid Waa transformed into yeast cells AH109 successfully and was verified self-activated,the expression of the bait protein was confirmed by Western blot.This indicate that the full-length CDS sequence of CBP couldn't be used as bait in yeast two or three hybrid system,and is of important significance for other study related to the interaction of CBP with other proteins and small moleculars.