酵母双杂交系统3筛选人源核不均一核糖核蛋白E1的功能伙伴  

Screening of interactional partners of human heterogeneous ribonucleo protein E1 using yeast two-hybrid system 3

作　　者：霍丽蓉[1,2] 邹俊华[3] 钟南[3] 

机构地区：[1]首都医科大学基础医学院生理学系,北京市100069 [2]山西医科大学基础医学院微生物与免疫教研室,山西省太原市030001 [3]北京大学医学部基础医学院遗传系,北京市100083

出　　处：《中国组织工程研究与临床康复》2010年第20期3714-3717,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：山西医科大学青年基金项目(02200805)~~

摘　　要：背景:酵母双杂交系统3是目前最常用的筛选相互作用蛋白质的技术平台。目的:运用酵母双杂交系统3筛选人源核不均一核糖核蛋白E1的相互作用子。方法:构建酵母细胞表达载体hnRNPE1-pGBKT7/c-myc,转化酵母菌AH109,Western blot证实蛋白表达,进行毒性和自激活检验,与转化了cDNA文库的酵母菌Y187接合,经过营养删除、滤膜反应以及回交试验筛选酵母文库中与hnRNP E1相互作用的蛋白。结果与结论:hnRNP E1蛋白能在酵母中正常表达,对酵母细胞无毒性,不存在自激活现象。经缺陷培养基3次传代及滤膜反应初步筛选出了16个候选基因,再经NCBI基因比对(BLASTN和BLASTP)及酵母细胞中的免疫沉淀反应最终获得了8个候选蛋白。提示hnRNP E1可能参与特定细胞内的复制,转录,mRNA运输、细胞周期、细胞自噬及免疫应答的过程,并且可能参与某些遗传病的发病过程。BACKGROUND： Now yeast two-hybrid system 3 is a common platform applied in screening interactive proteins. OBJECTIVE： To screen interactional partners with human heterogeneous ribonucleo protein E1 （hnRNP E1） using yeast two-hybrid system 3. METHODS： The carrier of hnRNP E1-pGBKT7/c-myc were constructed. Then AH109 yeast cells were transformed with it, and the expression of hnRNP E1 in yeast cells was identified by Western blot. After toxicity and self-activation assays, yeast-mating was utilized to screen interactional proteins with hnRNP E1 in human cDNA library including nutritional deletion, filtration membrane reaction and yeast mating. RESULTS AND CONCLUSION： hnRNP E1 protein was expressed normally and there was no self-activation and toxicity in AH109. Sixteen candidate genes were screened after 3 passages and filter assay. And eight candidate proteins were gained at last after BLASTN/P from NCBI. All results revealed that hnRNP E1 maybe participate in the process of replication, transcription, transport for mRNA, cell cycle, cell autophagy and immune response, and it can participate in the pathogenesis of some genetic disorders.

关 键 词：核不均一核糖核蛋白 酵母双杂交 蛋白质 相互作用子 质粒 

分 类 号：R318[医药卫生—生物医学工程]