Baculovirus Surface Display Of E Envelope Protein Of Janpanese Encephalitis Virus And Its Immunogenicity

Janpanese encephalitis(JE)caused by Janpanese encephalitis virus(JEV), is a zoonotic severe acute infectious disease. There is no effective medicine for JE, and inoculate vaccine is the most important measure to control JE. Inactivated JEV vaccine prepared from infected mouse brain is widely used over the world. In China, attenuated JEVvaccine SA14-14-2, produced from primary hamster kidney cells, plays an important role against JE. However, allergic reactions of the inactivated vaccine, high cost of production, need for multiple immunizations and lack of long term immunity necessitate the need to develop less expensive, more effective and safer vaccines for JE.It has been proved that envelope protein of JEV combines with the cell receptors, and plays the most important role in JEV invading into the target cells. It also closely related with adsorption, intrusive, pathogenicity and organizational eosinophilic, blood clots, serum specificity and specific immue response of JEV. In this study, recobinant baculovirus expressing E protein of JEV was constructed, and its immunogenicity was investigated. The main results are follows:1 Construction of recombinant baculovirus expressing E protein of Janpanese encephalitis virusSynthesis specificity primers P1 and P2, obtin purpose gene E, inserted it into the transfer vector BacSC, and constructed the recombinant transfer plasmid pBacSC-E. Transfer plasmid was transformed into E.coli DH10Bac containing baculovirus shuttle vecttor(bacmid) and helper vector. Recombinant shuttle vector BacSC-E was obtained by resisitance and blue/white selection. Recombinant bacmid DNA was transfected into Sf9 cells to yield recombinant baculovirus. With the western blot analysis, laser confocal microscopy analysis and immune electron microscope analysis, it was proved that E protein expressed on the membrane of insect cells and the envelope of recombinant baculovirus.2 The immunogenicity of recombinant baculovitus20 Balb/C mice were divided into 4 groups. Treatment group were injected with recombinant baculovirus, positive control group were injected with attenuated JEV vaccine and negative group were injected with PBS, the fouth group was injected with BacSC. To investigate the antigen-specific immune response mediated by recombinant baculovirus, indirected ELISA, antibody neutralization test and lymphocyte proliferation test were adopted. Antibody titer of treated mice was 1:5000, the titer of positive was 1:4000. Neutralization activity was 1:32 and 1:16 separately. Stimulation index was 5.20 and 2.13. The results show that recombinant baculovirus developed higher JEV-specific ELISA and neutralizing antibodies compared with mice vacciinated with attenuaeted vaccine. Recombinant baculovirus has good immunogenicity, and may be used as a strategy to develop a new genetration of vaccine against JEV in the nearly future.