Kadowaki Takeshi, Harada Hideaki, Sawada Yoshifumi, Kohchi Chie, Soma Gen-Ichiro, Takahashi Yukinori, Inagawa Hiroyuki
National Fisheries University, Applied Aquabiology, 2-7-1 Nagata-Honmachi Shimonoseki, Yamaguchi 759-6595, Japan.
Fish Shellfish Immunol. 2009 Nov;27(5):585-94. doi: 10.1016/j.fsi.2008.12.006. Epub 2008 Dec 31.
Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator and has also the potential as a prominent biomarker of innate immunity. In this study, we identified and characterized TNF-alpha from bluefin tuna, which is an important cultured species. Two types of TNF-alpha were also cloned incidentally (TNF1 and TNF2). The open reading frame of TNF1 and TNF2 cDNA encoded 247 and 245 amino acids, respectively. The amino acid sequence identity among sea perch, red sea bream, and tiger puffer was 73, 70, 59% for TNF1 and 49, 51, 45% for TNF2, respectively. The identity between TNF1 and TNF2 amino acid sequences of the bluefin tuna was only 43%. The positions of cysteine residues, transmembrane sequence, and protease cleavage site in bluefin tuna TNFs were similar with other reported fish and mammalian TNF-alpha. In a phylogenetic analysis, TNF1 is grouped with other reported Perciformes TNF-alpha. On the other hand, TNF2 is grouped with ayu TNF and is quite distant from the fish TNF-alpha group and lymphotoxin-beta group. While TNF1 mRNA showed no significant difference in all tissues, TNF2 mRNA was expressed significantly higher in the blood than in the gill, intestine, head kidney, spleen, heart, and ovary. In peripheral blood leucocytes (PBL), expressions of TNF2 mRNA were significantly increased by stimulation with lipopolysaccharide, phytohemagglutinin, concanavalin A, pokeweed mitogen, phorbol myristate acetate in vitro, but those of TNF1 were not. Recombinant mature TNF1 and TNF2 proteins significantly enhanced phagocytic activity of PBL. Our results suggest that bluefin tuna possess two types of TNF-alpha homologue, and TNF2 is a potential biomarker for innate immunity.
肿瘤坏死因子-α（TNF-α）是一种关键的炎症介质，也有潜力成为先天性免疫的重要生物标志物。在本研究中，我们从重要的养殖鱼类蓝鳍金枪鱼中鉴定并表征了TNF-α。同时还意外克隆出了两种类型的TNF-α（TNF1和TNF2）。TNF1和TNF2 cDNA的开放阅读框分别编码247和245个氨基酸。鲈鱼、真鲷和虎河豚中TNF1的氨基酸序列同一性分别为73%、70%、59%，TNF2的分别为49%、51%、45%。蓝鳍金枪鱼TNF1和TNF2氨基酸序列之间的同一性仅为43%。蓝鳍金枪鱼TNFs中半胱氨酸残基的位置、跨膜序列和蛋白酶切割位点与其他已报道的鱼类和哺乳动物TNF-α相似。在系统发育分析中，TNF1与其他已报道的鲈形目TNF-α归为一组。另一方面，TNF2与香鱼TNF归为一组，与鱼类TNF-α组和淋巴毒素-β组相距甚远。虽然TNF1 mRNA在所有组织中均无显著差异，但TNF2 mRNA在血液中的表达明显高于鳃、肠、头肾、脾脏、心脏和卵巢。在外周血白细胞（PBL）中，脂多糖、植物血凝素、伴刀豆球蛋白A、商陆有丝分裂原、佛波酯体外刺激可显著增加TNF2 mRNA的表达，但TNF1的表达无明显变化。重组成熟TNF1和TNF2蛋白显著增强了PBL的吞噬活性。我们的结果表明，蓝鳍金枪鱼拥有两种类型的TNF-α同源物，且TNF2是先天性免疫的潜在生物标志物。
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