高迁移率族蛋白B1与活化T细胞核因子2之间存在直接相互作用  

High mobility group box-1 protein could directly bind to nuclear factor of activated T cells-2

作　　者：刘辉[1] 姚咏明[2] 宋青[1] 丁丽华[3] 王晓辉[3] 袁斌[3] 叶棋浓[3] 盛志勇[2] 

机构地区：[1]解放军总医院外科重症医学科,北京100853 [2]解放军总医院第一附属医院烧伤研究所 [3]解放军军事医学科学院生物工程研究所

出　　处：《临床急诊杂志》2011年第6期364-368,共5页Journal of Clinical Emergency

基　　金：国家重点基础研究发展规划项目(No:2012CB518102);国家自然科学基金(No:30801187;81071545)

摘　　要：目的:探讨高迁移率蛋白1(high mobility protein box1,HMGB1)与活化T细胞核因子2(nuclearfactor of activating T cells2,NFAT2)之间的直接相互作用。方法:首先观察HMGB1与NFAT2在细胞外的相互作用,构建pET28a(+)-HMGB1、pGEX-KG-NFAT2质粒,应用网织红细胞系统进行体外翻译得到带有放射性硫同位素35S的His-HMGB1融合蛋白。应用蛋白纯化技术得到的GST-NFAT2融合蛋白,利用GST-pulldown实验检验二者在体外是否可直接结合;然后观察HMGB1与NFAT2在细胞内能否直接结合,构建HMGB1和NFAT2的真核表达质粒,共同转染人胚胎肾293T细胞系,刺激之后裂解细胞,应用相应的抗体进行免疫共沉淀检测,观察二者在细胞内直接结合的情况。结果:利用GST-pull down实验及免疫共沉淀实验证实,HMGB1全长蛋白与NFAT2全长蛋白在细胞内、外有直接结合的条带,而对照组没有结合条带,说明HMGB1可与NFAT2特异性结合。结论:HMGB1与NFAT2之间存在直接相互作用。Objective：To investigate the direct interaction between HMGB1 and NFAT2.Method：Firstly,the direct interaction between HMGB1 and NFAT2 in vitro was examined. pET28a（＋） -HMGB1 and pGEX-KG-NFAT2 plasmid were constructed. pET28a（＋） -HMGB1 was used for in vitro transcription and translation in TNT Reticulocyte Lysate System to get 35S-labeled His-HMGB1 fusion protein. GST-NFAT2 fusion protein was isolated and purified for GST-pull down examination with His-HMGB1 protein. Secondly,their direct interaction in vivo was examined. HMGB1 and NFAT2 eukaryotic expression plasmid were transfected into 293T cells simultaneously. After 24 h,cell lysate was collected for co-immune precipitation（co-IP） measurement. Proteins were precipitated with anti-Flag antibody and subsequently detected with anti-HMGB1 antibody.Result：Result showed that HMGB1 specifically bound to GST-NFAT2 in vitro. Co-IP showed that HMGB1 protein could bind to NFAT2 protein directly in vivo.Conclusion：HMGB1 protein could directly bind to NFAT2 protein in vivo and in vitro.

关 键 词：高迁移率蛋白B1 活化T细胞核因子 蛋白纯化 免疫共沉淀 

分 类 号：R392[医药卫生—免疫学]