Stury On The Immune Response Of Jev Vaccine Mutant Strains On Mice And Epitope Identification On Envelope Protein

The JEV envelope protein contains three domains,including I,II,III,respectively.Which is constructed with 500 Amino acid residues.Specially,domain III can fold into a immunoglobulin-like structure,independently located on the viral surface,closely related to binding to the receptor.Studies had shown that the JEV EDIII contain neutralizing epitope of the virus,could stimulate to produce neutralizing antibodies in animals.ED? protein can be an important antigen used to detect the immnue effect of the vaccineIn this study,we mainly utilize the JEV EDIII protein,to detect JEV antibody level in the serum of mice which are immunized with Japanese encephalitis attenuated vaccine strains from three different corporations,and explore the causes of the different levels of antibody.Besides,identify a linear epitope of EDIII via the neutralizing monoclonal antibodies 1c4 which was preparaed in our laboratory.The purpose of this research is to provide valued references for the safely and effectively production of the JEV vaccine,and to offer practical information of new JEV vaccines' preparation.1.Study on immune response in mice of the swine encephalitis attenuated vaccine from different corporations.Via plaque formation assay on hamster kidney cells(BHK-21),we find that the size of the plaque is different,of which are from three different swine JEV attenuated vaccine corporations.Then we immunize ICR mice with the attenuated vaccine,respectly,and detect the antibody levels,we find that the strain,which forms significantly smaller plaque morphology,could produce a higher antibody response after immunization.Then,we purify two kinds of strain which only displayed large plaque or small plaque,via the method of plaque purification,respectly.Besides,whole genome sequencing analysis shows that the strain which form small plaques have a acid mutations on the nucleotide sequence positon of 1813,causes a substitute of the amino acids on the position of E279,from the original methionine(M)to lysine(K).Utilizing these two kinds of purificated strains,we develop mice immunication as before,the trend of the production of the antibodys show a consistency patttern as before,which reveal the mutation on nucleotide sequence positon 1813 can lead to a stronger immune response on mice.The current research provides a reference for the preparation and evaluation of JEV attenuated vaccines.2.Identifing a linear epitope of JEV protein EDIII by a neutralizing monoclonal antibody(Mab)Since the monoclonal antibody can specifically identify a single epitope,we utilize a Mab,which is prepared in our laboratory,named lc4,to identify an epitope of JEV EDIII protein.According to the published sequence of JEV E protein,gene primers were designed.Then,we construct prokaryotic expression vectors which can express the fusion protein of EDIII and GST-tag.Via Western Blotting,the Mab lc4 capture one of the fusion protein which could bound to it.Then,we design the peptides,further narrow the scope of the epitope through the method of ELISA.At last,we design gene primers and contruct prokaryotic expression vectors and determine the epitope via Western blotting.In conclusion,we utilize the Mab 1c4,succeed located a linear epitope of JEV EDIII protein,the sequence is 375EPPFGDS381.Our research lays a foundation for further study of the biological characteristics of JEV,meanwhile,providing a theoretical basis for genetic engineering vaccines and Immune colloidal gold technique et al.