基因Ⅰ型日本脑炎病毒NS1蛋白的多克隆抗体制备及鉴定  

Preparation and identification of polyclonal antibodies against NS1 protein of genotype Ⅰ Japanese encephalitis virus

作　　者：柴风雪 田占成[1] 杨吉飞[1] 张鸿歌 牛庆丽[1] 独军政[1] 关贵全[1] 殷宏[1,2] CHAI Fengxue;TIAN Zhancheng;YANG Jifei;ZHANG Hongge;NIU Qingli;DU Junzheng;GUAN Guiquan;YIN Hong(State Key Laboratory for A nimal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州730046 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2025年第1期26-32,共7页Chinese Veterinary Science

基　　金：甘肃省自然科学基金项目(22JR5RA029);国家肉牛牦牛产业技术体系专项(CARS-37);甘肃省基础研究创新群体项目(22JR5RA024);中国农业科学院科技创新工程项目(CAAS-ASTIP-2016-LVRI)。

摘　　要：乙型脑炎(Japanese encephalitis,JE)是由三带喙库蚊媒介传播的日本脑炎病毒(Japanese encephalitis virus,JEV)感染引起的,临床表现以神经症状为主。NS1蛋白作为JEV编码的非结构蛋白,能够诱导机体产生非中和作用的保护性抗体,是JE诊断和疫苗研发的主要靶标。本研究以基因Ⅰ型JEV的NS1蛋白为靶标,将构建的重组质粒p ET30a-His-NS1转化Rosetta(DE3)表达菌,以0.5 mmol/L IPTG诱导表达。结果显示,与预期蛋白分子量大小一致的重组NS1蛋白以包涵体形式表达。经亲和层析柱纯化获得浓度为2.568 mg/mL的重组His-NS1蛋白。免疫印迹和间接免疫荧光结果表明,制备的高效价抗NS1蛋白多克隆抗体能够特异性识别蛋白分子量分别为46和55 ku的天然NS1蛋白。此外,本研究首次发现制备的抗NS1蛋白多克隆抗体与JEV预孵育能够剂量依赖性的显著降低JEV感染BHK-21细胞的病毒产量。将抗NS1蛋白多克隆抗体作为免疫沉淀抗体,用于JEV感染PK-15细胞的免疫沉淀质谱分析。结果表明,NS1蛋白能够与囊膜E蛋白相互作用。总之,本研究为深入了解NS1蛋白的生物学功能、JE诊断方法的建立和疫苗研制提供了基础材料。Japanese encephalitis(JE)is caused by Japanese encephalitis virus(JEV)that is transmitted by the vector of Culex tritaeniorhynchus.The clinical manifestations are mainly neurological symptoms.NS1 protein,as a nonstructural protein encoded by JEV,can induce host producing the nonneutalizing protective antibodies and is the main target for JE diagnosis and vaccine development.In this study,NS1 protein of genotype Ⅰ JEV was used as the target,and the constructed recombinant plasmid p ET30a-His-NS1 was transformed into Rosetta(DE3)expression bacteria,which was induced by 0.5 mmol/L IPTG.The results showed that the recombinant NS1 protein,which was consistent with the expected protein molecular weight,was expressed in the form of inclusion bodies.The recombinant His-NS1 protein was purified by nickel column affinity chromatography,with a concentration of 2.568 mg/mL.The results of western-blot and indirect immunofluorescence assay(IFA)indicated that the prepared anti-NS1 polyclonal antibody can specifically recognize natural NS1 proteins with molecular weights of 46 and 55 ku,respectively.In addition,this study found for the first time that the anti-NS1 polyclonal antibody can significantly decrease the virus production of JEV infected BHK-21 cells in a dose-dependent manner after pre-incubated with JEV.Anti-NS1 polyclonal antibody was used as an immunoprecipitation antibody for immunoprecipitation mass spectrometry analysis of JEV infected PK-15 cells.The results showed that NS1 protein can interact with envelop E protein.In summary,this study provides a fundamental material for further understanding of the biological functions of NS1,the establishment of JE diagnostic methods and vaccine development.

关 键 词：日本脑炎病毒 NS1蛋白 多克隆抗体 制备 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]