用于酵母双杂交的EBP50诱饵载体的构建和鉴定

Construction and Identification of Bait Expression Plasmid pGBKT7-EBP50 in Yeast Two-hybrid System

构建用于酵母双杂交系统的结合域(binding domain,BD)诱饵载体pGBKT7-EBPS0,并检测其是否具有自身激活作用。方法:用PCR方法从pBK-CMV-HA-EBP50质粒中扩增出EBP50全长,引入酶切位点EcoR I、BamH I,与pGBKT7载体连接起来,并检测pGBKT7-EBP50在酵母双杂交系统中的自激活作用。结果:成功构建了BD诱饵载体pGBKT7-EBP50,并证明其在酵母双杂交系统中无自激活作用。结论:pGBKT7-EBP50可用于筛选和鉴定新的与其相互作用的蛋白,从而有助于进一步研究EBP50在体内的功能。

Objective:To construct the BD bait vector in yeast two-hybrid system by using pBK-CMV-HA-EBP50plasmid.Methods:Coding region was obtained from the EBP50 plasmid by PCR.EcoR I/ BamH I-digested PCR products and bait vector pGBKT7 were ligated,and then transformed into E.coli DH5α to obtain the transformants.The positive clones were screened and identified by restriction endonuclease analysis and DNA sequencing.The correct recombinant plasmid pGBKT7- EBP50 was transformed into the competent yeast AH109.The activation of pGBKT7- EBP50 on the reporter genes and its toxicity on AH109 were also determined.Results:The size of the inserted fragment and recombinant plasmid was consistent with theoretically predicted value.There were no activation of pGBKT7- EBP50 on the reporter genes and no toxicity on AH109.Conclusion:The bait plasmid pGBKT7- EBP50 is successfully constructed for the yeast two-hybrid system.,may be used to screen for the novel protein-protein interactions and further contribute to the study of EBP50 roles in regulation of cellular functional activities.