中文摘要：

肿瘤坏死因子（tumornecrosisfactor,TNF）与肿瘤坏死因子受体（TNFreceptor,TNFR）超家族在机体的先天免疫和获得性免疫方面都发挥重要作用。本研究从本实验室构建的大黄鱼（Larimichthyscrocea）EST（expressedsequencetag）数据库中筛选出TNFR（I~TNFR）基因片段，利用SMART-RACE技术克隆出大黄鱼缸孙f职基因cDNA全序列，全长2016bp，其中5q乍翻译区（untranslatedregion，UTR）64bp，3’非翻译区602bp，开放阅读框（openreadingframe，ORF）1350bp。ORF编码449个氨基酸（GenBank登录号：KF432416）。预测的蛋白质等电点为5．47，分子量为49．7kD。对LcTNFR蛋白三维结构进行分析发现，其含有6个伐螺旋，不含B折叠。Blast比对分析显示，与斑马宫丽鱼（Maylandiazebra）的TNFR10B．1ike氨基酸序列一致性最高，达74％；与斑马鱼Daniorerio）卵巢型TNFR一致性较高，达51％；与鲫鱼（Carassiusauratus）TNFRI的一致性为47％。鱼类TNFR进化树分析进一步表明，LcTNFR与TNFR 10B—like、TNFRI及卵巢型TNFR聚为一大支。荧光定量PCR结果显示，TNFR基因在大黄鱼各组织中均有表达，在卵巢的表达水平最高，显著高于精巢及其它组织；其次在免疫相关组织脾脏也有较高的表达。副溶血弧菌（Vibrioparahaemolyticus）感染后的第2天和第4天，LcTNFR基因在大黄鱼脾脏组织中的表达量达到最高，显著高于对照组fP〈0．05），到第8天逐渐回落到对照组水平。本结果可为大黄鱼免疫学研究及抗病相关分子标记的开发提供参考依据。

英文摘要：

The tumor necrosis factor （TNF） and tumor necrosis factor receptor （TNFR） superfamilies play crucial roles in both innate and adaptive immunity. In this study, the fragment of TNFR （LcTNFR） was identified from the EST（expressed sequence tag）database of large yellow croaker （Larimichthys crocea）, and the full length cDNA of LcTNFR of 2 016 bp was obtained using SMART-RACE method, consisting of a 5＇ UTR of 64 bp, an ORF of 1 350 bp and a 3＇ UTR of 602 bp（GenBank accession No KF432416）. The deduced protein was composed of 449 amino acids, with a predicted pI of 5.47 and an estimated molecular mass of 49.7 kD. Protein structure prediction by SWISS-MODEL found that LcTNFR contained 6 ~t-helix and 0 13- fold. Blast analysis results revealed that LcTNFR had the highest identity with Maylandia zebra 10B-like （74%）, followed with Danio rerio ovarian TNFR （51%） and with Carassi~ auratus TNFR I （47%）. Phylogenetic tree of the TNFR amino acid sequences analysis furtherly indicated that LcTNFR together with TNFR 10 B - like, TNFR type I and ovarian TNFR formed a branch. The results of quantitative Real-time PCR （qRT-PCR） showed that LcTNFR was expressed ubiquitously in examined tissues of male and female fishes. The expression level of LcTNFR was significantly higher in ovary than that in other tissues including testis, and the expression level of LcTNFR in spleen, which is an organ involved in immune function, was also higher than that in other examined tissues. In the spleen, the expression level of LcTNFR was up-regulated significantly （P 〈 0.05） at the 2 day and 4 day after Vibrio parahaemolyticus challenge, and then it restored to the control level at 8 day, The results may provide insight into the immunology research and the development of disease-resistance genetic markers for large yellow croaker.