摘要
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.
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.
出处
《医学分子生物学杂志》
CAS
CSCD
2010年第3期189-194,共6页
Journal of Medical Molecular Biology
基金
国家自然科学基金(No.30630035)
关键词
GATA-1
酵母双杂交
蛋白质相互作用
免疫共沉淀
荧光素酶试验
GATA-1
yeast two-hybrid system
protein-protein interaction
co-immunopre-cipitation assay (Co-IP)
luciferase activity assay