鲤鱼MAVS基因的克隆、表达及免疫功能分析  

Cloning,Expression and Immune Function Analysis of MAVS Gene in Carp

作　　者：左明忠 张美娜 刘雨晴 陈孟娟 刘变枝 李明[1] 于光晴 ZUO Mingzhong;ZHANG Meina;LIU Yuqing;CHEN Mengjuan;LIU Bianzhi;LI Ming;YU Guangqing(College of Animal Science and Technology,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物科技学院,郑州450046

出　　处：《中国畜牧兽医》2024年第11期4653-4664,共12页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年项目(32102833);中国博士后科学基金第70批面上项目(2021M701107);河南农业大学大学生创新创业项目(2022DC051)。

摘　　要：【目的】获取鲤鱼线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)基因CDS区,对其进行生物信息学分析,并探讨MAVS基因在免疫反应中的功能。【方法】参考NCBI中公布的鲤鱼MAVS基因预测序列(GenBank登录号:XM_019072216.2)设计引物对MAVS基因进行克隆和测序,使用生物信息学软件分析鲤鱼MAVS蛋白的性质和功能。对鲤鱼进行嗜水气单胞菌攻毒试验,使用实时荧光定量PCR技术检测MAVS基因在攻毒前后鲤鱼各组织中的表达水平。通过免疫荧光检测试验进行MAVS和线粒体的亚细胞定位;使用双荧光素酶报告基因系统检测MAVS基因调控下游基因的表达情况。【结果】克隆得到鲤鱼MAVS基因CDS区序列全长1749 bp,共编码582个氨基酸。鲤鱼MAVS基因核苷酸序列与鲫鱼、多磷白甲鱼、虎皮鱼、野鲮、草鱼、团头鲂、拉氏鱥、金线鲃、胖头鱥的相似性分别为92.3%、89.5%、85.1%、79.1%、73.6%、73.1%、72.0%、70.1%和69.8%。系统进化树结果显示,鲤鱼与鲫鱼、野鲮、多磷白甲鱼等聚为一支。鲤鱼MAVS蛋白为酸性、不稳定蛋白质,具有较强的亲水性,含有133个磷酸化位点及1个跨膜螺旋,属于跨膜蛋白;不含有信号肽,不属于分泌蛋白。蛋白保守性分析显示,MAVS蛋白的N-端CARD结构域、中间富含脯氨酸结构域(PRR)和C-端跨膜结构域(TM)在不同物种之间高度保守。鲤鱼MAVS蛋白的二级结构和三级结构显示,该蛋白主要由无规则卷曲(58.93%)和α-螺旋(23.37%)构成,延伸链(13.92%)和β-转角(3.78%)含量较低,表明其为一种混合性蛋白。蛋白互作结果显示,MAVS蛋白与NLRP3、TRAF6、ATG5、DDX58、TBK1等免疫蛋白之间存在相互作用。实时荧光定量PCR结果表明,与对照组相比,攻毒后鲤鱼MAVS基因在心脏、脑、脾脏、肝脏、肠道、肌肉、鳃组织中表达量显著或极显著升高(P<0.05;P<0.01),在肾脏中表达量极显著降低(P<0.01),在眼组织中表�【Objective】This study was aimed to obtain the CDS region of mitochondrial antiviral signaling protein(MAVS)gene in carp,perform bioinformatics analysis on it,and explore the function of MAVS gene in immune response.【Method】Referring to the predicted sequence of MAVS gene in Cyprinus carpio published in NCBI(GenBank accession No.:XM_019072216.2),primers were designed for its cloning and sequencing,the property and function of MAVS protein in carp were analyzed using bioinformatics softwares.Carp was subjected to Aeromonas hydrophila challenge experiment,and Real-time quantitative PCR was used to detect the expression of MAVS gene in various tissues of carp before and after challenge.Experimental subcellular localization of MAVS and mitochondria was performed by immunofluorescence assay.The expression of MAVS-regulated downstream genes was detected using dual-luciferase reporter gene system.【Result】The CDS region of MAVS gene in carp was cloned to a length of 1749 bp,encoding a total of 582 amino acids.The similarities of the nucleotide sequences of MAVS gene in carp with those of Carassius auratus,Onychostoma macrolepis,Puntigrus tetrazona,Labeo rohita,Ctenopharyngodon idella,Megalobrama amblycephala,Rhinichthys klamathensis goyatoka,Sinocyclocheilus anshuiensis and Pimephales promelas were 92.3%,89.5%,85.1%,79.1%,73.6%,73.1%,72.0%,70.1%and 69.8%,respectively.The results of phylogenetic tree showed that carp clustered into a single unit with Carassius auratus,Labeo rohita,Puntigrus tetrazona,etc.MAVS protein of carp was an acidic,unstable protein with strong hydrophilicity,containing 133 phosphorylation sites and 1 transmembrane helix,which belonged to transmembrane proteins.MAVS protein did not contain a signal peptide,and did not belong to secretory proteins.Protein conservation analysis results showed that the N-terminal CARD structural domain,the middle proline-rich structural domain(PRR)and the C-terminal transmembrane structural domain(TM)of MAVS were highly conserved among different species.The

关 键 词：鲤鱼 MAVS基因 克隆 嗜水气单胞菌 免疫 

分 类 号：S917[农业科学—水产科学]