HLH缺失型Id2在酵母双杂交系统中的表达及自激活检测

Expression of HLH domain-deleted Id2 protein in yeast cells and detection of its self-activation

目的:构建HLH结构域缺失的DNA结合抑制因子(Id2)诱饵质粒,并检测其在AH109酵母中的表达及表达产物对酵母的有无毒性作用和对报告基因的有无转激活作用。方法:用重叠延伸PCR方法将Id2中的HLH结构域缺失,并插入到pGBKT7载体,构建BD:Id2-DBM-δHLH融合诱饵质粒,将构建成功的诱饵质粒转化AH109感受态酵母菌中,检测其是否具有自激活作用及对酵母的毒性作用,用Western blot方法检测其在酵母中表达的融合蛋白。结果:成功构建Id2的HLH缺失体,Id2的HLH缺失体在酵母中能够正确表达,对AH109酵母无毒性作用,对报告基因无自激活作用。结论:HLH结构域缺失型Id2蛋白适用于采用酵母双杂交系统筛选其相互作用蛋白。

Objective：To construct the bait vector of helix-loop-helix（HLH）-deleted Id2 for yeast two-hybrid system and to detect its effect on the growth of yeast cells and the self-activation of reporter genes. Methods： HLH-deleted Id2 was amplified by overlap extension PCR and subsequently cloned into pGBKT7 to construct the bait plasmid for yeast two-hybrid system. These recombinant plasmids were transfected into AHI09 yeast cells and their effect on reporter genes and the growth of yeast calls were measured. Results：The fragment encoding HLH-deleted Id2 was successfully amplified and sequenced to confirm its correct ex- pression frame in pGBKT7 plasmid. HLH-deleted Id2 was correctly expressed in yeast cells, both the toxicity and self-activation of which were not observed. Conclusion ：The HLH-deleted Id2 interaction proteins can be screened in yeast two-hybrid system.