尼罗罗非鱼CD209基因的克隆、表达及功能鉴定

Cloning,expression and functional identification of CD209 gene in Nile tilapia Oreochromis niloticus

为揭示尼罗罗非鱼模式识别受体CD209的分子结构、组织表达特性并初步了解其免疫功能,利用聚合酶链式反应(PCR)获得尼罗罗非鱼Oreochromis niloticus(体质量为100 g±5 g)CD209基因的开放阅读框片段(open reading frame,ORF),命名为OnCD209;使用生物信息学软件对其氨基酸序列进行分析,采用RT-qPCR法对健康尼罗罗非鱼的各组织及无乳链球菌刺激后尼罗罗非鱼肝脏、脾脏、鳃和肠道的CD209基因表达量进行分析,并构建了CD209-pEGFP-N1及CD209-pcDNA3.1载体,研究了CD209基因在293T细胞中的定位及在细胞中过表达时对NF-κB酶活性的影响。结果表明:OnCD209的ORF区片段长度为1383 bp,可编码460个氨基酸,预测该蛋白具有一个C型凝集素结构域;多重氨基酸序列比对及系统进化分析显示,CD209氨基酸序列在各物种间相对不保守,尼罗罗非鱼与田纹狮子鱼Liparis tanakae的CD209氨基酸序列聚为一支,亲缘关系最近;RT-qPCR结果显示,所有组织(脑、血液、皮肤、肌肉、头肾、肠道、鳃、肝脏、脾脏和胸腺)中均能检测到OnCD209的mRNA表达,且在血液中表达量最高;尼罗罗非鱼在受到无乳链球菌感染后,OnCD209的表达在各组织中呈现相似的变化趋势,大致呈先在6 h时上调,后在12、24 h时下调,再在48 h后上调的表达模式;亚细胞定位分析显示,OnCD209定位于细胞膜上;双荧光素酶报告基因系统检测发现,OnCD209对NF-κB通路的活性显著性激活。研究表明,OnCD209可能参与尼罗罗非鱼对细菌的免疫应答过程及炎症反应过程。

To further understand the molecular structure,expression characteristics and immune function of CD209 in Nile tilapia Oreochromis niloticus(OnCD209)with body mass of(100±5)g,bioinformatics software was used to analyze OnCD209 amino acid sequence of the open reading frame(ORF)in Nile tilapia,and the relative expression of OnCD209 in healthy tilapia tissues including liver,spleen,gill and intestine post Streptococcus agalactiae infection was detected by real-time fluorescent quantitative PCR(RT-qPCR),and CD209-pEGFP-N1 and CD209-pcDNA3.1 vectors were constructed to study the localization of OnCD209 molecular in 293T cells and its influence on NF-κB enzyme activity.The results showed that the ORF region of OnCD209 was found to be 1383 bp,encoding 460 amino acids,and to have a carbohydrate recognition domain,accordance with the C-type lectin family.Multiple sequence alignment showed that OnCD209 was note conserved among species.Phylogenetic tree analysis revealed that OnCD209 were closely related to grassfish Liparis tanakae.Tissue expression showed that OnCD209 was detected in all tissues(brain,blood,skin,muscle,head kidney,intestine,gill,liver,spleen and thymus)with the maximal expression level in blood.The up-regulated expression of OnCD209 was observed in all detected tissues of Nile tilapia exposed to S.agalactiae infection in 6 h,then down-regulated at 12 h and 24 h,and then up-regulated at 48 h(P<0.05).Subcellular localization analysis showed that OnCD209 was locatzed on the cytomembrane and the dual-luciferase reporter system indicated that OnCD209 significantly enhanced the NF-κB enzyme activity.The findings indicated that OnCD209 was involved in the immune response to bacteria and inflammatory response in Nile tilapia,which provides a theoretical basis for further study on the biological function of OnCD209.