免疫刺激诱导草鱼肿瘤坏死因子TNF-α的体外表达特征研究  

IN VITRO EXPRESSION PATTERN OF GRASS CARP TUMOR NECROSIS FACTOR(TNF-α) IN RESPONSE TO IMMUNE STIMULATION

作　　者：卢翠玉[1,2] 李炎[1] 刘庆慧[2] 吕利群[1] 

机构地区：[1]上海海洋大学水产与生命学院,农业部淡水种质资源重点实验室,上海201306 [2]中国水产科学研究院黄海水产研究所,青岛266071

出　　处：《水生生物学报》2016年第3期481-487,共7页Acta Hydrobiologica Sinica

基　　金：现代农业产业技术体系建设专项资金(CARS-46-12);国家自然科学基金(31372561)资助~~

摘　　要：为了阐明免疫刺激诱导草鱼肿瘤坏死因子TNF-α的体外表达特征,克隆了草鱼TNF-α的c DNA,构建了原核表达载体p ET-32-TNF-α,并在大肠杆菌DH5α中表达了His6-TNF-α。利用亲和层析镍柱纯化表达重组蛋白后将其作为免疫原免疫小鼠制备了抗草鱼TNF-α多克隆抗体。免疫印迹实验显示,制备的抗体能特异性识别细胞内源性TNF-α。在此基础上,分别研究了GCRV(草鱼呼肠孤病毒)感染、免疫刺激物Poly(I:C)及LPS处理下不同时间点草鱼肾细胞CIK中TNF-α的表达情况,结果表明,TNF-α在草鱼肾细胞内翻译水平的表达量基本保持稳定。研究显示经典的TNF信号通路激活因子不能引起CIK细胞内TNF-α蛋白水平的显著变化。Tumor necrosis factor alpha （TNF-a） is a multi-functional cytokine that plays important role in immune response, the homeostasis of the immune system, apoptosis, cell proliferation, and differentiation. Whether pathogen and immune stimulation can enhance the protein level of TNF-a is unclear. In this study, the cDNA of grass carp TNF-a was cloned into the prokaryotic expression vector pET-32 for expression of His6-tagged TNF-a. After purification through Ni2＋ -affinity chromotopraphy column, purified His6- TNF-a was subjected to immunize mouse to get poly- clonal antibody, anti- TNF-a. Western blot analysis showed that the anti- TNF-ct could specifically recognize endogenic grass carp TNF-aas well as His6- TNF-a. Furthermore, our results indicated that the CIK TNF-a level remain constant when challenged with GCRV or treated with immune stimulators in vitro. Since CIK cells seemed to be not a good cell line in investigating the response of TNF alpha signal pathway to stress, an in vivo experiment might be required to monitor the protein expression of TNF-a in specific immune organs or cells.

关 键 词：草鱼呼肠孤病毒 肿瘤坏死因子TNF-a 原核表达 多克隆抗体 

分 类 号：S941.4[农业科学—水产养殖]