Gene Cloning And Expression Of Intertleukin-8(IL-8) From Orange-Spotted Grouper (Epinephelus Coioides)

Cytokines are a group of small-molecule peptide. They mainly play fundamental roles in the development, inflammation, and function of the immune system. Inertleukin-8 (IL-8) is a inflammatory cytokine, which is produced by monocyte or macrophages, in response to inflammation, infection and other physiological challenges.Orange-spotted grouper (Epinephelus coioides) is an important aquacultured fish species. Culture and some physiological factors of orange-spotted grouper are most reported, but up to now, few researches of immunity of this species are available. In the present study, in order to study triggering mechanism of immune defense of E. coioides, we used RT-PCR and RACE-PCR to obtain the full-length cDNA of IL-8 and used RT-PCR to exam the expression IL-8 in various tissues.The cDNA sequence of E. coioides IL-8 obtained in this study contained 235bp 5'UTR, 438 bp 3'UTR and 288 bp open reading frame (ORF) which could translate a putative peptide of 95 amino acids including two cysteines. It also contained a CXC family signature and the ELR motif. According to the three-dimensional structure prediction of the putative peptide, there were 3 pleated sheets in the N-terminal and a spiral structure in the C-side.Phylogenetic analysis of amino acid sequences encoded by IL-8 of orange-spotted grouper and other animals was performed by a program-MEGE4. The phylogenetic tree based on IL-8 showed that orange-spotted grouper IL-8 belonged to CXCa Chemokine family, and it was closest to black spotfed bass, more evolutionarily related to haddock, but less related to those of other two species in Chimaeriformes and Salmoniformes.The tissue expression analysis using RT-PCR showed that the unstimulated IL-8 gene is stronger expressed in the heart, head-kidney, spleen and liver of of E. coioides; there was a little IL-8 mRNA expressed in gills and intestines, but little in the stomach, muscles and skins.