Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha
Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha
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DOI：
10.1016/j.dci.2007.09.009
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发表时间：
2008-01-01
影响因子：
2.9
通讯作者：
Belosevic, Miodrag
中科院分区：
文献类型：
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作者：
Grayfer, Leon;Walsh, John G.;Belosevic, Miodrag
我们从金鱼中鉴定并鉴定了肿瘤坏死因子α（TNF α）的两种亚型，TNFo α-1和TNF α-2。在蛋白质水平上，金鱼TNF α-1和TNF α-2分别与鲤鱼TNF α-1和TNF α-2同源性最高。在植物遗传学上，这两种金鱼亚型与鲤鱼TNF α亚型和其他鲤科鱼类的TNF种类最为接近。实时PCR分析显示金鱼TNF α-1和TNF α-2在所有组织中的组成型表达，在所有检查的组织中TNF α-2 mRNA水平高于TNF α-1。金鱼TNF α-1和TNF α-2在肾源性单核细胞中的表达适度上调，成熟巨噬细胞中两种亚型的表达显著增加，这是对巨噬细胞激活因子激活的反应。随后使用原核表达系统表达TNF α-2，并对重组分子（rTNF α-2）进行功能表征。rTNF α-2诱导剂量依赖性趋化反应并增强原代金鱼巨噬细胞的吞噬作用。此外，rTNF α-2引发单核细胞的呼吸爆发，并诱导初级金鱼巨噬细胞产生一氧化氮。我们的研究结果表明，金鱼TNF α是金鱼的炎症和抗菌反应的中央调节和效应细胞因子。(c)2007爱思唯尔有限公司版权所有。
We identified and characterized two isoforms of tumor necrosis factor-alpha (TNF alpha) from the goldfish, TNFo alpha-1 and TNF alpha-2. At the protein level, goldfish TNF alpha-1 and TNF alpha-2 were most homologous to carp TNF alpha-1 and TNF alpha-2, respectively. Phytogenetically, the two goldfish isoforms grouped most closely with the carp TNF alpha soforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNF alpha-1 and TNF alpha-2 in all tissues with TNF alpha-2 mRNA levels higher than TNF alpha-1 in all tissues examined. A modest up-regutation in expressions of goldfish TNF alpha-1 and TNF alpha-2 in kidney-derived monocytes and significant increase in expression of both isoforms in mature macrophages were observed in response to activation with macrophage-activating factors. TNF alpha-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNF alpha-2) was functionally characterized. The rTNF alpha-2 induce a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNF alpha-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNF alpha is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish. (c) 2007 Elsevier Ltd. All rights reserved.