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趋化因子家族是一类能诱导白细胞激活和迁移的趋化性细胞因子家族,是鱼类先天免疫的重要组成部分。CC类家族是趋化因子家族中最大的家族之一。为研究鲤CC类家族成员的组织表达和进化模式,以及进一步研究鲤CC类趋化因子的免疫调控机制,本研究选择该家族其中的一个基因,CCL-C5a样基因,根据EST序列结合RACE方法从鲤(Cyprinus carpio)脾脏中克隆了该基因的全长cDNA序列(GenBank登录号:JQ026408)。该序列全长830bp,5端非翻译区174bp,3端非翻译区296bp,开放阅读框360bp,编码一个由119个氨基酸组成的蛋白。结构域分析发现,该蛋白质含有分泌信号肽和保守的趋化因子结构域;Gene Ontology注释该基因参与免疫细胞迁移的生物学过程。CCL-C5a基因在鲤10个组织中表达,其中在脾和肝的表达量最高。脂多糖刺激鲤外周血白细胞和头肾白细胞后,该基因表达量出现显著上升。氨基酸序列保守性分析表明,鲤CCL-C5a样趋化因子与斑马鱼(Danio rerio)的同源性最高,为61%,与蓝鲶(Ictalurus furcatus)、沟鲶(Ictalurus punctatus)和大西洋鲑(Salmosalar)的同源性分别为47%、47%和42%;与人(Homo sapiens)CCL19蛋白的同源性为37%。适应性进化分析发现,鲤与斑马鱼CCL-C5a的Ka/Ks小于1,说明CCL-C5a基因在鱼类的进化过程受到负选择压作用。本研究结果表明,鲤CCL-C5a主要在免疫器官上高表达,并受到脂多糖诱导表达上升;同时该基因在鱼类中受到负选择作用。这些结果提示CCL-C5a可能是一个与免疫相关的基因。
The chemokine family is a family of chemotactic cytokines that induce leukocyte activation and migration and is an important part of innate immunity in fish. The CC family is one of the largest families of chemokine families. In order to study the tissue expression and evolution pattern of CC family members of Cyprinus carpio and to further study the immune regulatory mechanism of CC chemokines in Cyprinus carpio, one gene of this family, CCL-C5a-like gene, The full-length cDNA sequence of this gene was cloned from the spleen of Cyprinus carpio (GenBank accession number: JQ026408). The full length of this sequence is 830bp, 174bp in the 5 untranslated region and 296bp in the 3 untranslated region. The open reading frame is 360bp and encodes a protein consisting of 119 amino acids. Domain analysis revealed that the protein contains secretory signal peptide and conserved chemokine domain; Gene Ontology annotated the gene involved in the biological process of immune cell migration. The CCL-C5a gene was expressed in 10 tissues of common carp, with the highest expression in spleen and liver. Lipopolysaccharide stimulation of peripheral blood leucocytes and head kidney white blood cells, the gene expression increased significantly. Conservative analysis of the amino acid sequence showed that CCL-C5a-like chemokines shared the highest homology with Danio rerio (61%) and were closely related to Ictalurus furcatus, Ictalurus punctatus and Atlantic salmon Salmosalar) were 47%, 47%, and 42%, respectively. The homology with CCL19 protein of Homo sapiens was 37%. The results of adaptive evolution analysis showed that the Ka / Ks of CCL-C5a in common carp and zebrafish was less than 1, indicating that CCL-C5a gene is under negative selection pressure during fish evolution. The results of this study showed that the common carp CCL-C5a is mainly expressed on immune organs and is up-regulated by lipopolysaccharide, meanwhile, the gene is negatively-selected in fish. These results suggest that CCL-C5a may be an immune-related gene.