Abstract：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.
