淇河鲫IL-8 cDNA克隆及组织表达分析

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

  • 摘要: IL-8是最早发现的趋化因子,属于CXC亚家族,与鱼类非特异性免疫水平关系密切。实验采用同源克隆和cDNA末端快速扩增(RACE)方法,从淇河鲫总RNA反转录产物中获得了641 bp IL-8 cDNA序列,其中包含102 bp的5′非编码区,242 bp的3′非编码区和297 bp的开放阅读框。该基因编码由98个氨基酸残基组成的前体蛋白,前22个氨基酸残基为信号肽。肽链中含有4个保守半胱氨酸,前2个半胱氨酸被1个精氨酸分隔,形成CXC结构。第一个半胱氨酸前缺乏ELR基序,其组成是DPR。由氨基酸同源性分析和进化分析可知,淇河鲫IL-8与鲤、鳙、草鱼、鲢等鲤科鱼类的进化关系较近,与鲤IL-8的同源性最高(94%)。通过实时荧光定量PCR检测,淇河鲫IL-8在被检测的8个组织中都有表达,其中在肌肉中的表达量最高,其次为脾脏、头肾和脑等。研究结果对调控淇河鲫非特异性免疫能力,提高淇河鲫健康养殖水平具有一定参考价值。

     

    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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