酵母单杂交体系筛选胃cDNA文库中IRX1的核心启动子结合蛋白  

Screening of interacting proteins with IRX1 core promoter in gastric cDNA library by yeast one-hybrid method

作　　者：张荣娟[1] 于颖彦[1] 霍克克[2] 张迪[2] 李璞[2] 计骏[1] 张佳年[1] 陈雪华[1] 刘炳亚[1] 朱正纲[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院消化外科研究所,200025 [2]复旦大学生命科学院遗传学研究所

出　　处：《中华实验外科杂志》2011年第3期346-348,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家863计划重大项目（2006AA02A402、2006AA02A301）;国家自然科学基金项目（30572127、30770961、30973486）;上海市科委重大及重点研究项目（09DZ1950100、09JC1409600）

摘　　要：目的 以IRX1基因启动子为诱饵,在胃cDNA文库内钓取与之相互作用的蛋白质.方法 构建IRX1启动子诱饵报告基因质粒并转化酵母单杂交（Y1H）Gold酵母获得诱饵基因酵母菌;抽提人胃组织总RNA,逆转得到cDNA文库,文库片段为0.3～2.5 Kb,作为猎物与线性化pGADT7-Rec文库质粒共转染诱饵酵母菌.结果 对胃cDNA文库的筛选效率达到3×106,从人胃组织cDNA文库共钓取到8个阳性克隆,测序分析获得3个具有完整开放读码框（ORF）的基因,其编码蛋白均含锌指结构域.结论 以酵母单杂交筛选文库方法得到与IRXI基因启动子序列相互作用蛋白质,为探讨IRX1基因的转录调控机制奠定了基础.Objective To construct bait yeast reporter plasmid with IRXI core promoter sequence and screen gastric cDNA library for characterizing the binding proteins in IRX1 transcriptional regulation.Methods Bait yeast reporter plasmid with IRX1 promoter was constructed and transfected into Gold Y1H yeast.Meanwhile,gastric cDNA library with size range of 0.3 to 2.5 kb was constructed by total RNAs extracted from normal gastric tissues.The interacting proteins were screened by yeast one-hybrid （Y1H） system.Results With screening efficiency of 3 × 106,we got eight positive colonies.By sequence analysis,3 candidate genes with complete opening reading frame were confirmed.Conclusion Characterizing the interacting proteins with IRXI core promoter by YIH system facilitates the understanding of regulatory mechanism for IRX1.

关 键 词：抑癌基因 IRX1 胃 CDNA文库 

分 类 号：R735.2[医药卫生—肿瘤]