中国兽医科学2025，Vol.55Issue(1)：26-32,7.DOI:10.16656/j.issn.1673-4696.2025.0009
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
摘要
Abstract
Japanese encephalitis(JE)is caused by Japanese encephalitis virus(JEV)that is trans-mitted 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 non-neutalizing 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 pET30a-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 pro-tein 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 poly-clonal 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 ma-terial 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Key words
Japanese encephalitis virus/NS1 protein/polyclonal antibody/preparation/identification分类
畜牧业引用本文复制引用
柴风雪,田占成,杨吉飞,张鸿歌,牛庆丽,独军政,关贵全,殷宏..基因Ⅰ型日本脑炎病毒NS1蛋白的多克隆抗体制备及鉴定[J].中国兽医科学,2025,55(1):26-32,7.基金项目
甘肃省自然科学基金项目(22JR5RA029) （22JR5RA029）
国家肉牛牦牛产业技术体系专项(CARS-37) （CARS-37）
甘肃省基础研究创新群体项目(22JR5RA024) （22JR5RA024）
中国农业科学院科技创新工程项目(CAAS-ASTIP-2016-LVRI) （CAAS-ASTIP-2016-LVRI）
