Cloning and tissue expression analysis of the interleukin 8 gene from crucian carp(Carassius auratus)in Qihe River
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Graphical Abstract
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Abstract
Interleukin 8(IL-8),belonging to CXC family,is the first discovered chemokine,and is closely associated with non-specific immunity of fish.In this study,a full-length cDNA sequence of the IL-8 gene was 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 with 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 two cysteine residues were 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,which was commonly present in mammalian and chicken IL-8 molecules.Therefore,DPR motif was substituted for ELR motif in IL-8 of crucian carp.Comparison of the amino acid sequences showed that the crucian carp IL-8 shared the highest similarity with Cyprinus carpio(94%).Phylogenetic analysis also demonstrated that the crucian carp IL-8 had the close relationship with 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 was determined by Real-time Quantitative PCR technique.The results displayed that the level of IL-8 mRNA was highest in muscle,followed by spleen,head kidney and brain.The research would 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.
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