Generation And Biological Characteristics Analysis Of The Monoclonal Antibodies Against NS5and E Protein Of Japanese Encephalitis Virus

Japanese encephalitis is a serious zoonoses caused by the Japanese encephalitis virus (JEV) which is a vector-borne pathogen. JEV is mainly spread by pigs and mosquitoes. The infection of JEV causes terrible consequences in the sow, such as miscarriage, stillbirth, and boar testicular inflammation, which results in great losses to the pig industry and threatens public health.In this study, mice were immunized with prokaryotic expressed NS5protein purified JEV virus respectively. Six specific monoclonal antibodies against NS5and five monoclonal antibodies against JEV was generated. The biological characteristics of these monoclonal antibodies were verified.The content of this thesis is as follows:1. Preparation of monoclonal antibodies of JEV NS5protein and analysis of target antigen epitopesIn this study, mice were immunized with prokaryotic expressed JEV NS5protein. Six specific monoclonal antibodies was prepared after cell fusion, which respectively named:1G6,1F12,2A5,3C4,3H7,3F10. Subtypes are:IgG2b, IgG2aIgG2b, IgGl, IgG2a and IgG2a, respectively. The light chains of these antibodies are all Kappa chain. In indirect immunofluorescence and Western blot analysis, six monoclonal antibodies showed high reactivity and specificity to NS5protein. By using sub-fragment proteins and polypeptides of NS5, the target epitope of3C4mAbs were identified to locate in the432amino acid residues of NS5.3C4mAb could specific bind with NS5when this residue is arginine but not leucine. Therefore,3C4strains of monoclonal antibody can be applied to distinguish JEV P3, SA14-14-2and SX09S-1strain.2. Preparation of monoclonal antibodies against JEV and neutralizing activity analysisIn this study, mice were immunized with JEV Vaccine strain SA14-14-2.5specific monoclonal antibodies were prepared, which are respectively named:2A5,2H12,3H10,4B4,4E1. The subtypes are:IgGl, IgG2b, IgG2b, IgG2b and IgG2b. Light chains: Kappa chain. Through Indirect immunofluorescence and Western blot analysis,5monoclonal antibody were observed to specific react with JEV. But3H10and4B4mAbs were not able to react with prokaryotic expression E protein. Neutralizing activity of these mAbs have been examined.4of them exhibite neutralizing activity against JEV virus.2H12strains showed a significant antibody dependent enhancement (ADE) effect, which increases the infection ability of the JEV...