Detection Of JEV Antibody Blocking ELISA And The Study Of Interaction Between JEV NS1’ Protein And Vimentin

Epidemic Encephalitis B,also known as Japanese encephalitis B,is a natural zoonotic infectious disease caused by Japanese Encephalitis Virus(JEV).The disease is a mosquito-borne central nervous system infectious disease,which occurs mainly in the south of Asia and the Pacific coast.JEV can cause encephalitis symptoms in humans and horses and reproductive disorders in pigs.So far,in addition to vaccination and mosquito control measures that can be used to prevent JEV infection,there is no direct effective treatment.With the warming of the global climate,the epidemic of JE has been expanding.Therefore,it is particularly important to monitor the epidemic situation of JE.The NS1’ protein is produced by a procedural frameshift of the JEV genome during the translation process,it includes all NS1 protein and the 9 amino acids of NS2A protein N-terminal,which is associated with the virulence and neurotoxicity of the virus.In the process of virus replication,NS1’ can function as NS1.It also can interact with NS1 protein and NS5 protein,but there has been no report about its interaction with host protein.Vimentin,a type of cytoskeleton protein type Ⅲ intermediate filament,has been shown to play an important role in the entry and replication of many viruses.In DENV and CSFV infected cells,Vimentin’s structural can rearrangement through phosphorylation and play a positive role in virus replication.In JEV infected cells,it’s only reported that Vimentin as a cell receptor mediates JEV cell entry.In this study,an epitope recognized by JEV EDⅢ monoclonal antibody(1B10)was identified and analyzed conservatively.On the basement,a purified recombinant JEV EDⅢprotein and HRP-labeled 1B10 monoclonal antibody were used to establish a blocking ELISA for the detection of JEV antibodies,and were preliminary applied to detecting JEV antibodies in clinical swine,bovine and sheep serums.This ELISA has a good application prospect.In addition,the present study confirmed the existence of interactions between JEV NS1’ protein and Vimentin in cells,and we found the obvious structural rearrangement of Vimentin in JEV-infected A549 cells.The specific research content is the following three parts:1.Identification of JEV EDⅢ monoclonal antibody epitopesIn order to identify the antigenic epitope recognized by a monoclonal antibody(1B10)of the JEV EDⅢ protein.In this study,firstly,western blot showed that 1B10 monoclonal antibody recognizes the first part of EDⅢ protein,and then designs and synthesizes 6 epitope peptides JE1-JE6 as the coating antigen according to the amino acid sequence.The ELISA result shows that 1B10 monoclonal antibody recognizes JE6,and finally This segment of the sequence construction of expression vector pGEX-6P-1-JE61/JE62 and successfully expressed the protein,and finally identified the 1B10 monoclonal antibody recognition epitope is 356LNDMTPVGR365.After comparative analysis,the epitope was in the predominant antigenic region of EDⅢ protein and was highly conserved in the 5 JEV genotypes,So,it taken the foundation for the development of JE vaccines and the establishment of JEV diagnostic methods.2.Detection of JEV antibody blocking ELISAIn order to establish an ELISA method that can be used for detection of various animal JEV antibodies,a blocking ELISA was established using the purified JEV EDⅢ protein as coated antigen and an HRP conjugated JEV monoclonal antibody(1B10)as detection antibody.The reaction step of the blocking ELISA was determined by optimizing the reaction conditions.The determination rate of the blocking rate was determined by the detection results of 100 JEV antibody negative pig serums:PI>34%was determined as positive,and PI≤25%was determined as negative.25%<PI<34%is considered suspicious.Compared with the commercial kit showed that the sensitivity of the blocking ELISA method was 98%and the specificity was 94%,the coincidence rate of the two methods was 97%;and no cross reaction with other common swine virus antibody positive serum.Intra-and inter-assay replicate variation less than 5%.The application of the blocking ELISA method was used to detect 514 clinical swine,cattle and sheep serum samples,the positive rate was 65%,12%and 9%respectively.The SNT results of 9 serum samples verified the reliability of the blocking ELISA,and it indicated that this blocking ELISA is suitable for the simultaneous detection of JEV antibodies in swine,cattle and sheep serums.It provided the possibility for the development of JEV antibody detection blocking ELISA kits.3.The interaction between JEV NS1’ protein and vimentinTo researching the relationship between JEV NS1’ protein and host protein Vimentin.In this study,the human eukaryotic expression plasmid of Vimentin was successfully constructed.It was verified by CO-IP and confocal microscopy that Vimentin interacts with NS1’protein in 293T cells that over-expressing Vimentin and NS1’ protein and in JEV infected A549 cells.Then,it was further explored that over-expression of NS1 and NS1’proteins had no effect on the endogenous Vimentin content in 293T cells,and the over-expression of Vimentin and NS1’ proteins did not significantly promote the proliferation of JEV in 293T cells.In JEV infected A549 cells,the structural rearrangement of Vimentin was observed,with the phenomenon of nuclear condensation,and its co-localization ratio with NS1’ protein was more significant than E protein.In conclusion,this study confirmed the interaction of Vimentin and NS1’ proteins in cells and explored the role of Vimentin in JEV-infected cells.However,the rearrangement of Vimentin in JEV infected A549 cells needs further investigation.