中文摘要：

为了阐明免疫刺激诱导草鱼肿瘤坏死因子TNF-a的体外表达特征，克隆了草鱼TNF-a的cDNA，构建了原核表达载体pET．32．TNF-a，并在大肠杆菌DH5a中表达了His6．TNF-a。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-a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-a。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-a的表达情况，结果表明，TNF-a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-a蛋白水平的显著变化。

英文摘要：

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.