Cloning and tissue expression analysis of the interleukin 8 gene from crucian carp (Carassius auratus) in Qihe River

Abstract

Interleukin 8(IL-8),belonging to CXC family,is the first discovered chemokine,and is closely associated w ith non-specific immunity of fish.In this study,a full-length cDNA sequence of the IL-8 gene w as cloned from crucian carp(Carassius auratus) in Qihe River using RT-PCR and RACE techniques.The IL-8 cDNA sequence consisted of 641 bp nucleotides,including 102 bp at 5'UTR,242 bp at 3'UTR and an open reading frame w ith 297 bp nucleotides.This cDNA sequence encoded a precursor protein of 98 amino acids and the former 22 amino acid residues formed a signal peptide.The putative peptide contained four cysteine residues,and the former tw o cysteine residues w ere separated by an arginine residue and consequently formed a CXC structure.Furthermore,similar to the most fishes,IL-8 of crucian carp lacked the completed ELR(Glu-Leu-Arg) motif in front of the CXC structure,w hich w as commonly present in mammalian and chicken IL-8 molecules.Therefore,DPR motif w as substituted for ELR motif in IL-8 of crucian carp.Comparison of the amino acid sequences show ed that the crucian carp IL-8 shared the highest similarity w ith Cyprinus carpio(94%).Phylogenetic analysis also demonstrated that the crucian carp IL-8 had the close relationship w ith Cyprinidae species such as Cyprinus carpio(94%),Hypophthalmichthys nobilis(81%),Ctenopharyngodon idella(81%) and Hypophthalmichthys molitrix(80%).IL-8 mRNA expression in eight different tissues of crucian carp w as determined by Real-time Quantitative PCR technique.The results displayed that the level of IL-8 mRNA w as highest in muscle,follow ed by spleen,head kidney and brain.The research w ould be beneficial to the regulation of non-specific immunity in crucian carp,and also provides a certain reference value for improving the health breeding level of crucian carp.