Prokaryotic Expression Of Major Antigenic Segment Of Japanese Encephalitis Virus And The Immune Efficiency Study Of Its DNA Vaccine Delivered By Salmonella Choleraesuis C500

Japanese encephalitis virus E protein is the major antigen protein, the experiment will be the major antigenic segment of JEV E protein gene in series, including the E protein on the two major antigenic domain of 73aa～88aa,292aa～402aa. Then the fragment was cloned into the prokaryotic expression vector pET-32a (+) and named pET-EAB. After confirmed positive by enzyme digestion, the recombinant plasmid was transformed into E.coli Rosetta (DE3), induced using IPTG for the expression of EAB protein. The expressed protein was detected in the form of inclusion body and secretory protein. Detected by Western Blot, the EAB protein possesses specific reactionogenicity. The pET-EAB plasmid was digested with EcoR I and Not I to obtain EAB gene fragment, then the fragment was subcloned into the the eukaryotic expression vector pCI-neo and named pCI-EAB. The recombinant plasmid pCI-EAB was transformed into Vero cell and the expression of recombinant plasmid was identified by RT-PCR and indirect immunofluorscence assay.6-week-old BALB/c mice were inoculated orally with S.C500/pCI-EAB at dosage of 1×10'CFU, 1×108 CFU and 1×109 CFU respectively. The immunized mice showed no clinic symptom. and recombinant plasmid pCI-EAB was stable with in the host strain in vitro and in vivo as show n by restriction enzymes analysis identification of the EAB gene; the recombinant bacteria S.C500/pCI-EAB in vitro cultured, OD600nm around 0.8 plasmid is relatively stable. Set up S.C500 harboring empty plasmid vaccinated group, recombinant plasmid (pCI-EAB) vaccinated group, swine attenuated Japanese encephalitis immunization group, plasmid vaccinated group and phosphate buffered saline inoculated group as compared with the control reference. Anti-JEV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method and the dynamic monitoring of the T-lymphocyte subsets respectly. BALB/c mice were immunized orally with S.C500/pCI-EAB harboring recombinant plasmid at the dosage of 108 and boosted one week later with the same dose, for a total of three times The S.C500/pCI-EAB could induce significant humoral immune response in mice compared with the control (P<0.01) at 1 w post-boosting and 1 w post-three immunization; spleen lymphocyte proliferative response showed that recombinant bacteria on ConA reactivity significantly, and the significant differences between the control group; by flow cytometry, it was found CD3+,CD4+,CD8+ T ratio in the mice of S.C500/pCI-EAB group were significantly higher than the control group (P<0.01). The results indicate that the attenuated Salmonella choleraesuis C500 as an oral delivery vector for DNA vaccine is of safety and stability and immunogenicity. S.C500/pCI-EAB vaccine strain could probably serve as a vaccine against Japanese encephalitis virus.