淇河鲫IL-8 cDNA克隆及组织表达分析
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河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学水产学院,河南师范大学水产学院,河南师范大学水产学院,河南师范大学水产学院,河南师范大学水产学院

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国家自然科学基金项目(30972252);河南省重点科技攻关项目(122102110049)


Cloning and tissue expression analysis of the interleukin 8 gene from crucian carp(Carassius auratus)in Qihe River
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College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Fisheries,Henan Normal University,College of Fisheries,Henan Normal University,College of Fisheries,Henan Normal University,College of Fisheries,Henan Normal University,College of Fisheries,Henan Normal University

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    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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王俊丽,雒燕婷,郝 光,孙君君,段艳艳,卢荣华,李学军,孔祥会,聂国兴.淇河鲫IL-8 cDNA克隆及组织表达分析[J].水产学报,2013,37(9):1281~1289

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  • 收稿日期:2013-03-20
  • 最后修改日期:2013-04-30
  • 录用日期:2013-08-10
  • 在线发布日期: 2013-09-13
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