基因Ⅰ型日本脑炎病毒NS1蛋白的多克隆抗体制备及鉴定OA北大核心

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.