半滑舌鳎CD40基因克隆及其免疫功能分析

发布时间：2018-11-04 18:34

【摘要】：本研究通过RACE技术在半滑舌鳎(Cynoglossus semilaevis)中克隆得到1个CD40同源基因。该基因c DNA全长为2098 bp,开放阅读框为1011 bp,可编码由336个氨基酸组成的蛋白质。推导得到的CD40氨基酸序列包含1个信号肽、1个跨膜结构区、2个N-糖基化位点以及含4个富含半胱氨酸的保守结构区。不同物种氨基酸序列同源分析结果显示,该基因与哺乳类、两栖类以及硬骨鱼类相似性为28%~47%,其中与硬骨鱼类相似性较高。系统进化分析结果显示,半滑舌鳎CD40与其他硬骨鱼类CD40聚类为一支,哺乳类与两栖类CD40聚类为另一支。荧光定量PCR结果显示,CD40在健康半滑舌鳎不同组织中均有表达,其中在鳃、肝和脾等组织中表达量较高,在肠、肌肉和皮肤中表达量较低,在脑和肾中微量表达。经哈维氏弧菌(Vibrio harveyi)感染刺激后,与PBS处理的对照组相比,该基因m RNA在半滑舌鳎3个主要免疫组织(肝、脾和肾)中表达量均呈显著性升高,在肝中12 h到达峰值,在脾和肾中6 h达到峰值。上述结果表明CD40基因参与了半滑舌鳎抵抗哈维氏弧菌的免疫应答反应。
[Abstract]:In this study, a CD40 homologous gene was cloned from (Cynoglossus semilaevis) of Cynoglossus semilaevis by RACE technique. The full length of c DNA is 2098 bp, open reading frame and 1011 bp, can encode a 336 amino acid protein. The deduced amino acid sequence of CD40 consists of a signal peptide, a transmembrane domain, two N-glycosylation sites and four conserved domains rich in cysteine. The results of amino acid sequence homology analysis of different species showed that the similarity between the gene and mammals amphibians and bony fishes was 2847 and the similarity between this gene and bony fish was high. Phylogenetic analysis showed that CD40 and other bony fish CD40 were clustered into one branch, and mammalian and amphibian CD40 cluster was another branch. The results of fluorescence quantitative PCR showed that CD40 was expressed in different tissues of healthy tongue sole, including Gill, liver and spleen, lower in intestine, muscle and skin, and slightly expressed in brain and kidney. Compared with the control group treated with PBS, the expression of m RNA was significantly increased in three major immune tissues (liver, spleen and kidney) of Cynoglossus semilaevis, and reached its peak at 12 h after (Vibrio harveyi) infection by Vibrio Harvey. The peak value was reached in spleen and kidney at 6 h. These results suggest that CD40 gene is involved in the immune response of Cynoglossus semilaevis against Vibrio Harvey.
【作者单位】：中国水产科学研究院黄海水产研究所农业部海洋与渔业资源重点实验室;上海海洋大学水产与生命学院;青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室;
【基金】：国家自然科学基金重点项目(31530078) 山东省泰山学者攀登计划专项资助项目
【分类号】：S917.4

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