| 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(PaTNF-α)全长cDNA 序列。PaTNF-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 kDa.氨基酸序列多重比对分析表明, PaTNF-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, PaTNF-α 与虹鳟TNF-α 进化相关性最高。实时荧光定量PCR (quantitative real-time PCR, qRT-PCR) 结果显示, PaTNF-α mRNA 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PaTNF-α mRNA 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, PaTNF-α mRNA 表达量显著上调。原核表达了PaTNF-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PaTNF-α 表达量含量也显著增加。 综上, 香鱼TNF-α mRNA 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。 |
| 关键词: TNF-α 香鱼 鳗弧菌 单核/巨噬细胞 表达 |
| DOI：10.11693/hyhz20150600166 |
| 分类号: |
| 基金项目:国家自然科学基金项目, 31372555 号; 浙江省自然科学基金项目, LZ13C190001 号; 高等学校博士学科点专项科研基金, 20133305120008 号。 |
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| MOLECULAR CLONING, CHARACTERISATION AND IMMUNE-RELATED EXPRESSION OF TUMOR NECROSIS FACTOR α (TNF-α) GENE IN AYU PLECOGLOSSUS ALTIVELIS |
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YANG Zhi-Jing, LI Chang-Hong, ZHANG Hao, MIAO Liang, CHEN Jiong
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Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China
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| Abstract: |
| 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 (PaTNF-α) gene, with de novo transcriptome sequencing of ayu monocytes/macrophages. The full-length of cDNA 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 kDa. The multiple sequence alignment of complete amino acid sequences indicates that PaTNF-α possessed the TNF family signature motifs, a protease cleavage site, and a conserved disulfide bond. PaTNF-α shared the highest amino acid sequence identity (53.4%) with that of rainbow trout (Oncorhynchus mykiss). Phylogenetic tree analysis also confirmed that PaTNF-α fell into the fish TNF-α cluster and was most closely related to that of rainbow trout. Quantitative real-time PCR (qRT-PCR) analysis showed that PaTNF-α mRNA had the highest expression level in head kidney of healthy ayu. Upon Vibrio anguillarum infection, PaTNF-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, PaTNF-α transcripts upregulated remarkably in ayu monocytes/macrophages. The mature peptide of PaTNF-α was overexpressed and the antiserum against PaTNF-α was prepared. The Western blot assay demonstrated that PaTNF-α increased obviously in ayu serum and monocyte/macrophage culture supernatant that infected with V. anguillarum. Therefore, the relationship between PaTNF-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. |
| Key words: TNF-α Plecoglossus altivelis Vibrio anguillarum monocyte/macrophage expression |
