GW112 mRNA 3’端非翻译区特异性结合蛋白研究  

Specific binding proteins interacting with 3’ untranslated region of GW112 mRNA

作　　者：吴莉雅[1] 安娜[1] 蒋雪[1] 刘先俊[1] 黄轶[2] 

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心.重庆400016 [2]重庆医科大学附属儿童医院临床分子中心,重庆400010

出　　处：《重庆医科大学学报》2013年第4期369-373,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金项目资助项目(编号:81001017)

摘　　要：目的:通过体外实验筛选出与GW112 mRNA 3’非翻译区临床特异性结合蛋白,并研究蛋白性质。方法:以稳定表达GW112基因的胃癌细胞SGC-7901为实验对象,体外转录法获得GW112 mRNA 3’非翻译区片段并用生物素标记,结合亲和素磁珠特异性吸附原理,从细胞总蛋白中纯化出与GW112 mRNA 3’非翻译区特异性结合蛋白。经过质谱鉴定得到蛋白信息,并用Western blot验证结果。结果:用磁珠纯化的蛋白样品经SDS-PAGE电泳后,取目的条带进行质谱分析发现存在核仁素蛋白,以核仁素单克隆抗体进行Western blot验证后发现与质谱结果一致。结论:核仁素是GW112 mRNA 3’非翻译区特异性结合蛋白的一种,并很可能对GW112 mRNA稳定性有直接影响,这为进一步揭示GW112在胃癌细胞抗凋亡机制中的作用奠定基础。Objective：To filter the special RNA binding proteins which interact with 3’ untranslated region（UTR） of GW112 mRNA and to analyze the characteristics of the proteins.Methods：By transcribing and marking the fragments of GW112 mRNA 3’UTR,we used the technology of magnetic bead adsorption to filter the GW112 mRNA 3’UTR binding proteins from the total proteins of the gastric cancer cell（SGC-7901） in vitro.Proteins were identified by mass spectrometry（MS） and Western blot.Results：Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAG） of proteins filtered out by magnetic bead adsorption and MS of the bands revealed that the identity of the protein was nucleolin.After detection by Western blot with monoclonal antibody of nucleolin,the target band appeared on SDS-PAG was in consistent with MS results.Conclusions：Result demonstrates that nucleolin can specifically bind to GW112 mRNA 3’UTR and may influence the stability of GW112 mRNA.This result will provide good basic for functional research of GW112 in gastric cancer cell apoptosis.

关 键 词：GW112基因 GW112 MRNA 3’非翻译区 体外转录 磁珠吸附 核仁素 

分 类 号：R735.2[医药卫生—肿瘤]