The Study On Construction And Immunogenicity Of Japanese Encephalitis Virus DNA Vaccine

Japanese encephalitis virus (JEV) is responsible for epidemics of Japanese encephalitis in human beings and animals.The swine serve as an intermediate amplifier from which anthropophilic mosquitoes become infected, spreading virus to humans.Therefore, mass vaccination of swine can prevent disease in swine and help to prevent JE epidemics in humans.Current JE vaccines for use in swine consist of attenuated or inactivated virus, and there are concerns about the safety and cost of producingand using these products. Though there are attempts to develop vaccines against it. Design and development of safer and more efficacious vaccines against Japanese encephalitis virus is a high-priority target in the world. In this study, we proposed novel genetically engineered DNA vaccines against the JEV.which fused secretory signal sequence derived from tissue plasminogen activator (TPA).1. Cloning of JEV prMAE gene, secretory signal sequence derived from tissue plasminogen activator (TPA) and the eukaryotic expression plasmids constructionThe fragment,of the JEV SA14-14-2 strain prMâ–³E gene was amplified by RT-PCR and cloned into pMD-19T vector; the recombinant was designated as 19T-prMâ–³E. Nucleotide sequencing and analysis showed that the amplified prMâ–³E gene was 1695 bp in length, which respectively encode 565 amino acids. The TPA signal sequence was amplified by overlap PCR and cloned into pMD-19T vector; the recombinant was designated as 19T-TPA. Nucleotide sequencing and analysis showed that the amplified prMâ–³E gene was 69 bp in length, which respectively encode 23 amino acids.The eukaryotic expression plasmids PVAX-prMâ–³E and PVAX-TPA-prMâ–³E, which expressing or fusion expressing JEV prMâ–³E and TPA signal sequence, were constructed using pVAXl as vector. PVAX-TPA-prMâ–³E and PVAX-prMâ–³E were transiently tranfected into COS-7 cells and the expression of recombinant plasmids were confirmed by indirect immunofluorscence assay. The results showed that the eukaryotic expression plasmids were constructed correctly and the transfected COS-7 cells displayed specific immunofluorscence.The successful construction of the PVAX-prMAE and PVAX-TPA-prMAE provide foundation for further research of JEV DNA vaccines.2. Study on immunogenicity of JEV DNA vaccineSix-week old KunMing mice were immunized intramuscularly three times at two weeks interval with JEV DNA vaccine of PVAX-TPA-prMAE and PVAX-prMAE. The anti-JEV IgG antibodies in murine serum were detected by indirect ELISA at 14,35 and 42 days post immunization. The results showed that specific anti-JEV antibodies were induced in all test groups. The antibody level of PVAX-TPA-prMAE group was higher than PVAX-prMAE groups, the difference was extremely significant (P<0.01) at 35 and 42 days post immunization. So, the results show that it is an effective route to enhance the efficacy of DNA vaccine by fusing secretory signal sequence derived from tissue plasminogen activator (TPA) to the plasmid. Vaccinated animals were challenged against CZ-1 in intracerebral inoculations, PVAX-TPA-prMâ–³E-immunized mice were protected. Overall, these results provide further support for the use of such a plasmid in a possible approach for the development of a vaccine against JEV.