中文摘要：

肿瘤坏死因子-α（tumor necrosis factor-alpha,TNF-α）是TNF超家族成员之一,在动物机体抵抗细菌和病毒入侵时发挥重要作用。本研究基于香鱼（Plecoglossus altivelis）单核/巨噬细胞转录组数据库,获得了香鱼TNF-α基因（Pa TNF-α）全长c DNA序列。Pa TNF-α由1932个核苷酸组成,包含一个大的开放阅读框,编码235个氨基酸,预测分子量为26.4 k Da。氨基酸序列多重比对分析表明,Pa TNF-α具有TNF超家族的特征序列、1个TACE酶切位点和1个保守的二硫键,与虹鳟（Oncorhynchus mykiss）TNF-α同源性最高,为53.4%;系统进化树分析表明,鱼类TNF-α形成一个大簇,Pa TNF-α与虹鳟TNF-α进化相关性最高。实时荧光定量PCR（quantitative real-time PCR,q RT-PCR）结果显示,Pa TNF-αm RNA在头肾中表达量最高;鳗弧菌（Vibrio anguillarum）感染后香鱼肝、脾、头肾和外周血细胞中Pa TNF-αm RNA表达量显著上调;香鱼单核/巨噬细胞经鳗弧菌、LPS和poly（I：C）处理后,Pa TNF-αm RNA表达量显著上调。原核表达了Pa TNF-α成熟肽,并制备了抗血清。Western blot分析表明,鳗弧菌感染后香鱼血清和单核/巨噬细胞上清中的Pa TNF-α表达量含量也显著增加。综上,香鱼TNF-αm RNA及蛋白的表达与病原体感染密切相关,为深入研究鱼类TNF-α的生物学功能及其在病原体感染中的作用机制提供理论基础。

英文摘要：

Tumor necrosis factor alpha（TNF-α） is a member of TNF superfamily, playing an important role in fighting against bacteria and viruses infection in animals. We obtained and studied that of ayu Plecoglossus altivelis, known as TNF-α（Pa TNF-α） gene, with de novo transcriptome sequencing of ayu monocytes/macrophages. The full-length of c DNA sequence of the gene was 1932 nucleotides, containing a large open reading frame that encoded 235 amino acids, and the molecular mass was deduced for 26.4 k Da. The multiple sequence alignment of complete amino acid sequences indicates that Pa TNF-α possessed the TNF family signature motifs, a protease cleavage site, and a conserved disulfide bond. Pa TNF-α shared the highest amino acid sequence identity（53.4%） with that of rainbow trout（Oncorhynchus mykiss）. Phylogenetic tree analysis also confirmed that Pa TNF-α fell into the fish TNF-α cluster and was most closely related to that of rainbow trout. Quantitative real-time PCR（q RT-PCR） analysis showed that Pa TNF-α m RNA had the highest expression level in head kidney of healthy ayu. Upon Vibrio anguillarum infection, Pa TNF-α transcripts upregulated significantly in the liver, spleen, head kidney, and peripheral blood leucocytes（PBLs）. Upon lipopolysaccharide（LPS）, poly（I： C）, or V. anguillarum infection, Pa TNF-α transcripts upregulated remarkably in ayu monocytes/macrophages. The mature peptide of Pa TNF-α was overexpressed and the antiserum against Pa TNF-α was prepared. The Western blot assay demonstrated that Pa TNF-α increased obviously in ayu serum and monocyte/macrophage culture supernatant that infected with V. anguillarum. Therefore, the relationship between Pa TNF-α expression and pathogen infection is close, which may provide a theoretical basis for studying the functions of fish TNF-α and mechanisms of regulating fish immune response to pathogens.