酵母双杂交系统筛选GATA-1相互作用蛋白质及功能验证

目的 利用酵母双杂交技术从人脑cDNA文库中筛选与人GATA-1相互作用的蛋白质.方法 从人K562细胞中扩增出全长GATA1基因,设计引物将其3段截断体亚克隆入酵母表达载体pDBLeu中,转化至AH109感受态酵母中,利用酵母双杂交技术筛选人脑cDNA文库中与其相互作用的蛋白质,阳性克隆通过回转及免疫共沉淀试验进行验证,利用3xGATA荧光素酶报告基因对相互作用蛋白质进行功能验证.结果 成功构建出酵母诱饵蛋白表达质粒pDBLeu-GATA1（1）,pDBLeu-GATA1（2）,pDBLeu-GATA1（3）,筛到34个阳性克隆,用生物信息学分析及回转验证得到5个与GATA-1相互作用的候选蛋白,通过免疫共沉淀试验进一步验证,获得3个蛋白质能与GATA-1相互作用,分别是ECSIT,EFEMP1和GPS2.荧光素酶试验表明这3个蛋白质均能对GATA1的转录活性产生影响,证实它们之间的相互作用具有影响GATA1转录的功能.结论 应用酵母双杂交技术及免疫共沉淀试验,从人脑cDNA文库中成功获得3个与GATA-1相互作用并对其转录活性具有调节作用的蛋白质,为研究GATA1蛋白质的功能提供了新的线索.

Screening and Identification of GATA-1 Interacting Proteins by Use of Yeast Two-Hybrid System

Objective To screen proteins interacting with GATA-1 via yeast two-hybrid system. Methods The full length of GATA-1 was amplified from K562 cells. Three fragment deletions were amplified and cloned into pDBLeu vector as bait plasmids. The plasmids were transformed into yeast competent cells AH109. Then yeast two-hybrid assay was performed to screen the proteins that interact with GATA-1 from human brain cDNA library. Yeast two-hybrid re-transformation, co-immunoprecipitation assay （Co-IP） and lueiferase activity assay were applied to confirm the interactions. Result pDBLeu-GATA1 （ 1 ）, pDBLeu-GATA1 （2）, pDBLeu-GATA1 （3） were successfully cloned, and 34 positive clones were obtained via yeast two-hybrid screening. After further examining with re-transformation and co-immunoprecipitation assay （Co-IP）, 3 binding proteins of GATA-1 were identified, including ECSIT, EFEMP and GPS2. Luciferase activity assay suggested that these binding proteins regulated transactivation activity of GATA-1. Conclusion Three binding proteins of GATA-1 were identified in human brain cDNA library, which provided new clues for further studying biological functions of GATA-1.