欧洲鳗鲡RIG-I基因的克隆、序列特征分析与原核表达  

Cloning,sequence characterization analysis and prokaryotic expression of Anguilla anguilla RIG-I gene

作　　者：李英英[1,2] 陈曦 杨金先[1] 陈强[1] 宋铁英[1] 葛均青[1] LI Yingying;CHEN Xi;YANG Jinxian;CHEN Qiang;SONG Tieying;GE Junqing(Institute of Biotechnology,Fujian Academy of Agricultural Science,Fuzhou 350003,China;Fujian Province Engineering Technology Research Center of Breeding Animal Nutrition and New Type of Feed Enterprise,Fuzhou 350003,China)

机构地区：[1]福建省农业科学院生物技术研究所,福州350003 [2]福建省养殖动物营养与新型饲料企业工程技术研究中心,福州350003

出　　处：《海洋渔业》2022年第5期513-520,共8页Marine Fisheries

基　　金：福建省人民政府、中国农业科学院农业高质量发展超越“5511”协同创新工程(XTCXGC2021013);福建省公益类科研院所专项(2021R10270011,2018R1019-4);福建省养殖动物营养与新型饲料企业工程技术研究中心开放课题(HLJT2021-04)。

摘　　要：为研究欧洲鳗鲡(Anguilla anguilla)RIG-I基因的特性,通过设计引物,扩增其序列,克隆至pCE2-TA/Blunt-Zero载体,测序验证后对所获得欧洲鳗鲡RIG-I基因序列进行生物信息学分析及原核表达。结果表明,欧洲鳗鲡RIG-I基因ORF序列有2823 bp碱基,编码940个氨基酸,与日本鳗鲡(A.japonica)的序列同源性为96.71%;RIG-I蛋白属酸性亲水性蛋白,不存在跨膜结构,无信号肽,较不稳定,主要分布于细胞质中,有6个保守功能区;SDS-PAGE和western blot结果显示,实现了RIG-I基因在大肠杆菌中的高效表达,为后期制备RIG-I多克隆抗体和进一步探索RIG-I介导的天然免疫在鳗鲡抵御鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)侵染过程中的作用机制提供了研究基础。Retinoic acid-inducible gene I(RIG-I)is one of RIG-I like receptors(RLRs),a member of DExD/H Box RNA helicase family.There are two cascades of caspase enrichment domains(CARD)at its N-terminal,which are cytoplasmic sensors of viral RNA.They can recognize viral mRNA and activate RLRs to bind to specific ligands during viral invasion,inducing the production of interferon(IFN)and inflammatory factors to resist viral invasion.RIG-I is inclined to recognize short dsRNA and 5′PPP dsRNA,and keeps self-inhibition state without stimulation by dsRNA.RIG-I can recognize both RNA virus and DNA virus,thus RIG-I and its mediated immune response play an important role in the host antiviral immune response during the infection of DNA viruses.Compared with mammals,the acquired immune system of fish is not fully differentiated,and its resistance to pathogen invasion is more dependent on its natural immune system.A large number of studies have shown that the natural immunity mediated by RIG-I plays an important role in the process of fish resisting virus invasion during virus infection.RIG-I is activated and specifically mediates the production of type II IFN-I of zebrafish during the infection by neuronecrosis virus(NNV);RIG-I and regulatory factors in its mediated signaling pathway will be activated in silver crucian carp after carp herpesvirus type II(cyprinid herpesvirus 2,CyHV-2)infection.Previous studies have shown that RIG-I gene and several genes in the RLRs signaling pathway of eel skin mucus are activated after infection by Anguillid herpesvirus(AngHV).In order to study the characteristics of RIG-I gene of Anguilla anguilla,primers were designed to amplify its sequence.Then the target fragment was cloned into pCE2-TA/Blunt Zero vector and verified by sequencing.The sequence characteristics of A.anguilla RIG-I were analyzed.NCBI BLAST online tool was used to analyze the homology of the amplified sequences.The composition and physicochemical properties of nucleotide and amino acid sequences were analyzed by Protparam.Prot

关 键 词：欧洲鳗鲡 RIG-I 克隆 序列特征分析 原核表达 

分 类 号：S917.4[农业科学—水产科学]