顺序转化法进行酵母双杂交筛选相互作用蛋白质  

Screening Interacted Protein by Yeast Two-Hybrid Sequencing Transformation

作　　者：吴怡[1] 王岚[1] 刘兰[1] 韩清 

机构地区：[1]沈阳医学院基础医学院病原生物学教研室 [2]解放军95979部队卫生教研室

出　　处：《沈阳医学院学报》2011年第3期132-134,共3页Journal of Shenyang Medical College

基　　金：沈阳医学院优秀人才启动基金项目(No.20103052)

摘　　要：目的:利用顺序转化法进行酵母双杂交从人胎脑cDNA文库中筛选PAK4新的相互作用蛋白。方法:将PAK4激酶域序列克隆到酵母表达载体pGBKT7构建诱饵质粒。提取质粒并转化酵母菌AH109,验证蛋白表达。用顺序转化法将质粒和人胎脑cDNA文库转化酵母菌AH109,在SD/-Trp/-Leu/-His/-Ade和SD/-Trp/-Leu/-His/-Ade/x-α-gal平板上筛选,对阳性克隆质粒进行酶切鉴定。结果:利用顺序转化酵母双杂交方法筛选出了多个与PAK4蛋白质相互作用的Ade+/His+/LacZ+阳性转化子。结论:利用顺序转化酵母双杂交方法成功筛选了与PAK4相互作用的蛋白质。Objective：To screen the p21-activated kinase 4（PAK4） novel interaction proteins from the human fetal brain cDNA library by yeast two-hybrid sequencing transformation.Methods：The kinase domain sequence of PAK4（PAK4 KI） constructed to yeast-expressing vector pGBKT7 （pGBKT7-PAK4 KI） as ＂bait＂ was transformed into the yeast competent cells AH109.Protein expression of the ＂bait＂ in yeast cells was identified by Western blot.Plasmids and the human fetal brain cDNA library were transformed into the yeast cells AH109.The transformants were selected on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/x-α-gal plates.The constructions of the positive clones were digested with BglⅡ.Results：Several Ade＋/His＋/LacZ＋ positive colonies which could interact with PAK4 were screened by yeast two-hybrid sequencing transformation.Conclusion：The proteins interacting with PAK4 can be screened by yeast two-hybrid sequencing transformation.

关 键 词：PAK4 酵母双杂交 信号转导 

分 类 号：R37[医药卫生—病原生物学]