鲤CC型趋化因子CCL-C5a样基因的克隆、组织表达与进化分析

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)脾脏中克降了该基因的全长cDNA序列(GenBank登录号:JQ026408).该序列全长830 bp,5端非翻译区174 bp,3端非翻译区296 bp,开放阅读框360 bp,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弓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 futrcatus)、沟鲶(Ictalurusp unctatus)和大西洋鲑(Salmo salar)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.

Cloning, Tissue Expressions and Evolutional Analysis of Cyprinus carpio CC chemokine CCL-C5a-like Gene

Chemokines belong to a family of chemotactic cytokines which are involved in activation and migration of leukocytes. They play important roles in the innate immune system of fish species. The CC chemokines are one of the biggest superfamilies of chemokines. To understand tissue expression, evolution models and immune regulation mechanism of Cyprinus carpio CC chemokines, we selected a member of CC chemokines, CCL-C5a, for further study. In this study, a full-length cDNA of CC chemokine CCL-C5a-like gene was amplified in C. carpio spleen by RACE method (GenBank accession No. JQ026408). The full-length cDNA was 830 bp in size, with 174 bp 5’UTR, 296 bp 3’UTR and 360 bp open reading frame(ORF). The ORF encoded a protein of 119 amino acids. Functional domain analysis showed that this protein contained signal peptide and conserved chemokine structure. Gene Ontology annotation indicated that this protein was involved in the biological process of immune cell migration. CCL-C5a was widely expressed in brain, skin, muscle, head kidney, body kidney, intestine, gill and heart and mainly in spleen and liver. After lipopolysaccharide stimulation, the expression of CCL-C5a showed significantly increase in peripheral blood leukocytes and head kidney leukocytes. The deduced amino acid sequence of C. carpio CCL-C5a shared high identity with other seven species, which was from 37% to 61%, and the highest was 61% with Danio rerio. Adaptive evolution analysis revealed that the Ka/Ks of CCL-C5a bewteen C. carpio and Danio rerio was less than 1, indicating that CCL-C5a was under negative selection pressure in the evolutionary process of fish species. We conclude that C. carpio CCL-C5a are principally expressed in immune organs and up-regulated after lipopolysaccharide stimulation and that this gene is under negative selection pressure in fish species. These results indicate that C. carpio CCL-C5a may be a immune-relevant gene.