重组人肿瘤坏死因子蛋白TNF-α的纯化与功能鉴定  

Purification and functional analysis of human recombinant tumor necrosis factor-α

作　　者：孔超[1,2] 李丹[2] 袁晓君[1] 李斌[2] 

机构地区：[1]上海大学生命科学学院上海市能源作物育种及应用重点实验室,上海200444 [2]中国科学院上海巴斯德研究所分子病毒与免疫重点实验室,上海200031

出　　处：《科技导报》2017年第8期70-74,共5页Science & Technology Review

基　　金：国家重点基础研究计划(973计划)项目(2014CB541803);中国博士后科学基金项目(2012M520946);国家自然科学基金重点项目(81330072)

摘　　要：串联亲和纯化系统(TAP)是近年来广泛使用的一种可在接近于生理条件下探究蛋白间相互作用的生化纯化方法。目前许多关于TAP的研究报道均使用细胞内转录因子为目标蛋白,探索细胞内与其相互作用的蛋白分子。本文尝试建立一种细胞外配体-受体刺激介导的信号转导初始复合体的纯化体系,为此选择了一种多功能的免疫分子配体蛋白——肿瘤坏死因子蛋白超家族(TNFSF)中的核心成员TNF-α。TNF-α是一个强效促炎因子,可介导炎症反应、细胞凋亡和激活等生理效应,是至今研究最多的TNFSF成员。利用原核表达系统纯化出了一种带有多个标签的重组人源TNF-α蛋白,通过检测其受体下游信号通路鉴定该蛋白与天然TNF-α蛋白的生理活性相似,从而证明该蛋白可以用于胞外TAP系统探究TNFR下游信号通路,为配体-受体刺激介导的信号转导初始复合体的胞外串联亲和纯化系统的建立奠定了基础。The Tandem Affinity Purification （TAP）is a powerful tool for the investigation of the protein-protein interactions under physiological or similar conditions. Many previous studies including our group＇s identified massive protein-protein interactions by manipulating the TAP system. However, almost all of these studies were focused on the interactions between the endocellular transcription factors, not so much on the extracellular ligand-transmembrane receptor mediated signal transduction process. This paper establishes an EX-TAP system for analyzing the extracellular ligand-transmembrane receptor mediated signal initial transduction process, constructs a prokaryotic recombinant protein expressing system and purifies a multi-tagged recombinant TNF-α from bacteria. The TNF-α is a pleiotropic pro-inflammatory factor which triggers the initial inflammation in vivo and is well defined in the past few decades, which is helpful for the identification of the feasibility of our EX-TAP system. By testing the phosphorylation of the inhibitor of the transcription factor KB, under the TNFR pathway, our recombinant TNFα is determined biologically similar to the natural TNF-α and is functionally available to the EX-TAP system.

关 键 词：串联亲和纯化 肿瘤坏死因子 蛋白纯化 

分 类 号：Q51[生物学—生物化学]