大菱鲆1个CXCL10样趋化因子的克隆、鉴定与表达分析

Cloning,characterization and expression analysis of a CXCL10-like chemokine from turbot Scophthalmus maximus

趋化因子是一类趋化性细胞因子的总称,它们在白细胞的趋化反应中起着重要作用。本研究从构建的大菱鲆(Scophthalmus maximus)脾脏cDNA文库中筛选到一种大菱鲆CXC趋化因子,经过5′-RACE和3′-RACE扩增得到了它的全长cDNA。该趋化因子的全长cDNA含有1个79 bp的5′UTR,1个372 bp的开放阅读框(ORF)和1个227 bp的3′UTR。开放阅读框编码123个氨基酸残基。此CXC趋化因子基因内含有4个外显子和3个内含子,3个外显子的碱基长度分别为591 bp、86 bp和372 bp。Blast分析和系统发生分析都表明这个趋化因子和已知的脊椎动物的CXCL10相近。用鳗弧菌(Vibrio anguillarum)感染大菱鲆后12 h,该趋化因子在肝脏和脾脏中表达水平达到最高,在头肾中则是在感染后5 h表达水平最高。在肝脏中,从感染后12 h开始该趋化因子的表达水平逐渐下降并且在72 h后恢复到正常水平。在脾脏中,一直到感染后24 h该趋化因子保持高水平表达,随后表达水平减弱至正常水平。在头肾中,从感染后5 h开始一直到24 h,表达水平逐渐升高,随后表达水平,立刻降低至与正常水平一致。在正常的大菱鲆组织内没有检测到该趋化因子的表达,表明它仅在受到鳗弧菌感染的时候才被诱导表达。结论认为该趋化因子在免疫反应中可能起着重要作用。

Chemokines represent a superfamily of small secreted protein （6 - 14 kD）, which is an essential part of both innate and adaptive immune system. They have been implicated in several immune mediated responses, such as inflammation, intercellular, communication, antigen presentation, blood cell development, metastatis, viral infection and wound healing. Based on the arrangement of the first two cysteine residues of amino acid sequences, chemokines were divided into four groups： CXC, CC, C and CX3C subfamilies. A CXC chemokine gene, corresponding to clone 950, was identified from this turbot spleen cDNA library （GenBank accession number DQ339043）. The CXC chemokine gene has a partial sequence of 510 bp with unknown 5′ and 3′ ends, which was obtained by using the methods of 5＇ and 3＇-RACE, respectively. The complete cD- NA contains a 79 bp 5′ UTR,a 372 bp open reading frame （ORF） encoding 123 amino acids and a 227 bp 3′UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. The three introns were 591 bp, 86 bp and 125 bp, respectively. A condensed phylogenetic tree was constructed based on the amino acid sequences of turbot CXC chemokine and other well-defined vertebrate CXC chemokines. The overall topology of the tree showed the turbot CXC chemokine clusters with all CXCL10. Alignment of amino acid residues of the turbot CXCL10 genes with those from mammals and fish indicated high level conservation of amino acid sequence. The turbot CXC10-1ike chemokine was not expressed in normal turbot tissues. Interestingly, the highest expression levels of the turbot CXC10-1ike chemokine were detected at 12 h in liver and spleen after challenging with V. anguillarum and at 5 h in head kidney. The expression in liver decreased gradually after 12 h and came back to the level of background after 72 h. In spleen, the expression kept high levels till the 24th hour and then became faintly consistent with background. The expression of the CXC chemokine was not detected in normal tissues of turbot, which suggested that it was only induced when challenged with V. anguillarum. These results indicate that the turbot CXC10-1ike chemokine plays an important role in turbot immune response.[ Journal of Fishery Sciences of China, 2007,14（4）：554-562]