Ordás M C, Costa M M, Roca F J, López-Castejón G, Mulero V, Meseguer J, Figueras A, Novoa B
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello, 6, 36208 Vigo, Spain.
Mol Immunol. 2007 Jan;44(4):389-400. doi: 10.1016/j.molimm.2006.02.028. Epub 2006 Apr 17.
The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNFalpha), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNFalpha family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNFalpha cluster. Therefore, the turbot TNF here studied was identified as TNFalpha. The complete TNFalpha gene was obtained by gene walking, and, similarly to the other known fish TNFalpha genes, presented three introns and four exons. A PCR was designed to study the turbot TNFalpha expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNFalpha expression, but this response was shorter in time than that induced by bacteria. In addition, TNFalpha expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNFalpha (rTNFalpha) was obtained by IPTG induction of bacteria transformed with the pET15b-TNFalpha construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNFalpha neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNFalpha was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNFalpha.
肿瘤坏死因子（TNF）超家族由几种结构和功能相似的蛋白质组成。该家族的主要代表之一是TNF-α（TNFα），它是一种促炎细胞因子，由不同的免疫细胞产生，并具有多种活性。利用RACE技术，我们克隆并测序了大菱鲆TNF cDNA。对其序列的分析显示了TNFα家族成员特有的几个保守基序。用鱼类和哺乳动物的不同TNF构建的系统发育树将我们的序列归入鱼类TNFα簇。因此，这里研究的大菱鲆TNF被鉴定为TNFα。通过基因步移获得了完整的TNFα基因，与其他已知的鱼类TNFα基因类似，它有三个内含子和四个外显子。设计了一个PCR来研究大菱鲆TNFα在体内的表达，使用海弧菌菌株Hq222和病毒VHSV作为刺激物。细胞因子的表达在注射后早期发生，并且它取决于注射的病原体和分析的器官。病毒诱导了更高的TNFα表达，但这种反应在时间上比细菌诱导的反应更短。此外，正如预期的那样，由于肾脏是鱼类的造血器官，TNFα的表达通常在肾脏中比在肝脏中更高。通过IPTG诱导用pET15b-TNFα构建体转化的细菌获得了大菱鲆重组TNFα（rTNFα），并在天然条件下进行了纯化。重组蛋白大小约为20 kDa，并在体外评估了其生物学活性。在任何测试时间，均未观察到rTNFα单独或与LPS联合对大菱鲆巨噬细胞的化学发光活性有影响。然而，在添加处理72小时后，重组蛋白单独或与LPS一起增强了NO的产生。最后，大菱鲆rTNFα注射到金头鲷中时能够募集和激活炎症细胞，尽管程度比金头鲷rTNFα小。
