酵母双杂交技术筛选NECL1胞内区的结合蛋白  

Screening of proteins interacting with the cytoplasmic tail of NECL1 by yeast dual-hybrid system

作　　者：李晨[1] 陈涛[1] 阴彬[1] 龚燕华[1] 

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2009年第2期119-123,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30571039;30430200);国家高技术研究发展计划("863"项目)(2006AA02Z137)

摘　　要：目的筛选人源膜表面黏附分子NECL1蛋白胞内区相互作用蛋白。方法构建含人NECL1蛋白胞内区氨基酸编码序列的诱饵质粒pGBKT7N-ECL1C,对人胎脑cDNA文库进行酵母双杂交筛选。用GST pu ll down实验进行体外蛋白相互作用的验证。结果酵母双杂交阳性克隆测序后显示共存在9段不同序列(存在重复克隆)。比对氨基酸序列得到5个可能相互作用蛋白。通过GST pu ll down实验验证了其中两个蛋白与NECL1胞内区的相互作用。结论应用酵母双杂交系统,获得了一些候选的NECL1胞内区相互作用蛋白。Objective To screen the proteins interact with cytoplasmic tail of NECL1. Methods The cytoplasmic tail sequence of human NECL1 gene was inserted into the ＂bait＂ vector pGBKT7 in frame. The recombinant pGBKT7-NECLI1 was then transformed into yeast cells and followed by screening of human fetal brain eDNA library. Then the GST pull down assay was used to confirm the interactions between the proteins. Results Nine different sequences by sequencing the positive clones and five alternative proteins were obtained by the amino acid sequence blast. Using the GST pull down analysis, we confirmed that two of them can interact with the cytoplasmic tail of NECL1 in vitro. Conclusion The alternative proteins obtained with the yeast dual-system may interact with the cytoplasmic tail of NECL1.

关 键 词：酵母双杂交 NECL1 相互作用蛋白 

分 类 号：Q78[生物学—分子生物学]