| Japanese encephalitis, JE for short, is common viral encephalitis caused by Japanese encephalitis virus. The epidemic is characterized as high lethality in human beings (about25-40%), ever expanding infectious ranges and sever threaten for public health and husbandry due to the main reason that leads to reproductive hindrance in pregnant sows. JEV-MAbs with neutralizing activity posses capabilities of reducing the viral pathogenecity, plus with the higher specificity and biologic activity, JEV-MAbs can be applied for further development in the diagnosis, detection, prevention and treatment of JEV.Here we obtained7strains of MAbs against JEV (strain NJ2008, GQ918133, all the mentioned JEV hereafter within this paper are strain NJ2008) by following the standard preparative technique of hybridomas. The virus was inoculated in BHK-21cells and stored at-20℃. After3times’frost thawings, centrifugation and sucrose density gradient centrifugation (SDGC) were used for the virus purification. Then female BALB/c mice were immunized subcutaneously three times with100μg purified JEV and protein EDIII was used in the forth strengthened immunization, Spleen cells were fused with SP2/0myeloma cells. ELISA was performed for screening with purified JEV or EDⅢas antigen respectively. Finally7positive clones that secreted high-titer JEV-specific or EDⅢ-specific antibodies in indirect ELISA were obtained and named as1H5F9,2D7E4,2B4B4,1H4,2E3,2B4and4D4. After generating ascites with hybridoma cells, isotypes of MAbs were determined and purified by HiTrap affinity columns for further identification.The bionomics of MAbs were determined by ELISA, Western Blot, immunofluorescence assay (IFA), flow cytometry analysis and neutralization test, etc. Results showed that1H5F9,2D7E4,2B4B4could identify proteins of BHK-21cells,1H4and2B4could only recogonize EDIII while2E3and4D4could identify both. So1H5F9,2D7E4,2B4B4were not included in the following experiments due to their non-specificities, but2E3and4D4were still taken as controls.1H4,2E3,2B4and4D4showed certain neutralizing activities in the neutralization test:2B4>1H4～4D4>2E3, especially2B4, which enjoyed highest neutralizing activity and biological activity, was used in the clinical application. Results proved that2B4could detect the virus and its level in the cerebrospinal fluid of the mice infected with JEV.In accordance with the characteristics of MAbs:all4MAbs can react with both EDIII and MEP, and exactly with the same protein, which concluded that joint recognition sites of the4MAbs located within the consensus sequence of these2proteins. In addition to this, M13phage display was used to screen the epitopes for further verification. Based on the results of MegAlignand Blast,"HH-H"(E394-E397) might be the most probable target binding site. According to this,2consensus sequence and one peptide were constructed: TVN PFV A (E356-E362)(marked as P1), EME PPF GDS YIV VGR GDK QIN HHW HKA (E373-E399)(marked as P2) and NHH WHK (E394-E398)(marked as P3)which included "HH-H", ELISA analysis revealed that1H4could react with all3peptides while2B4could only identify P3, which could detect positive serum of JEV-infected swine as an antigen. Therefore, E394HHWHE398is a neutralizing linear epitope that can effectively inducing antibodies, and E396-M may probably the key site deciding the neutralizing activity. The spatial conformation and physical and chemical analysis revealed that E394HHWHE398located on the erminatio of folding. With relatively more amino-acid residue with positive charge (H), E34HHWHE397presented high hydrophilicity, which enabled it be a epitope capable of inducing MAbs in immune response. Thus, the neutralizing epitope will definitely conducive to the further investigations of JEV, and1H4and2B4are hopeful for being applied in the diagnosis of JEV and the development of related vaccines. |
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