Gene Cloning And Expression Of Interleukin-8 And Interleukin-8 Receptors Of Epinephelus Coioides

In this paper, the cDNAs of Interleukin-8 (IL-8) and Interleukin-8 receptor 1 (IL-8R1) were isolated and analyzed from the head kidney SMART cDNA library of orange-spotted grouper (Epinephelus Coioides). Results showed that IL-8 of orange-spotted grouper (Epin-IL-8) contained 1143 nucleotides, including 5'-UTR 345nt, 3'-UTR 507nt, and a coding region of 291nt. The deduced coding protein of Epin-IL-8 had 96 amino acids peptide, including 23 amino acids signal peptide and 73 amino acids mature peptide, which shared a highest amino acid identity of 95% with Acanthopagrus schlegelii, 93%, 91% followed with Lateolabrax japonicus and Dicentrarchus labrax, and with Takifugu rubripes, Paralichthys olivaceus, Cyprinus carpio, and Oncorhynchus mykiss, the amino acid identities were 86%, 77%, 69% and 59% respectively. It shared the lowest amino acids identity of 53% with Triakis scyllium. The cloned IL-8R1 cDNA of Epinephelus coioides (Epin-IL-8R1) encompassed 1505nt, including 5'-UTR 37nt, 3'-UTR 394nt, and a coding region 1074nt. The cloned Epin-IL-8R1 coded 357 amino acids peptide, including 62 amino acids signal peptide and 295 amino acids mature peptide. The Epin-IL-8R1 shared a highest amino acid identity of 82% with Siniperca chuatsi, and with Takifugu rubripes, Paralichthys olivaceus, Oncorhynchus mykiss and Cyprinus carpio, the amino acid identities were 70%, 68%, 65% and 55%. The IL-8R2 of Epinephelus coioides (Epin-IL-8R2) was cloned using designed degenerated primers, and 531nt Polypeptide was got, encoding 177 amino acids. The Epin-IL-8R1 shared a highest amino acid identity of 81% with Siniperca chuatsi, and with Oncorhynchus mykiss the amino acid identity was 54%.Prokaryotic expression systems of the PET21a/ Epin-IL-8 and Epin-IL-8R1 were constructed and used to express Epin-IL-8 and Epin-IL-8R1. The results of Tricine SDS-PAGE and SDS-PAGE showed that the Epin-IL-8 and Epin-IL-8R1 had a molecular weight of about 9.1kDa and 34.34kDa respectively.Real-time PCR of Epin-IL-8 demonstrated that gene expression was detected in head kidney, spleen, liver, kidney, intestine, heart and gill. The Epin-IL-8 had the highest expression in head kidney, while the Epin-IL-8R1 and Epin-IL-8R2 had the highest expression in spleen and liver respectively. The Epin-IL-8 was up-regulated after the Vibro alginolyticus infection, and 10ppm carbolic acid, 50ppm CdCl₂, 2ppm CuSO₄ bath.