3.2.1 Identification of W541 DNA vaccine and W540 recombinant protein
The sequencing findings indicate that the DNA sequences inserted in the W541 vaccine and W540 plasmid are congruent with the design. Upon induction with IPTG, the transformed E. coli BL21(DE3) with W540 plasmid expressed protein with an approximate molecular weight of 70kDa on SDS-PAGE (figure 6).
3.2.2 Antibody production induced by W541 DNA vaccine
After three immunizations, there was no significant difference in the antibody IgG, IgG1, and IgG2a levels among the W541 vaccine group, Control group, and pVAX1 group (P>0.05) (figure 7).
3.2.3Detect number of splenocytessecreting IFN-γ using ELISPOT assay
The ELISPOT assay results showed that the number of spots produced by splenocytes secreting IFN-γ in the vaccine group (26±10) was significantly higher than that in the normal control group (1.2±0.6, P<0.01) and the pVAX1 vector control group (2±1, P<0.01) (figure 8). These findings indicate that the W541 DNA vaccine effectively activated the splenic lymphocytes in mice; upon stimulation with the W540 protein, the activated splenocytes exhibited a rapid and powerful secondary immune response.
3.2.4 Analyses of Th1, Th2, and Th17 cytokines in splenocyte culture supernatants of mice in each group
For Th1-type and Th17-type cytokines, the IFN-γ and IL17 levels were significantly higher in the supernatants of splenocyte cultures from mice immunized with the W541 DNA vaccine compared to the normal control and pVAX1 vector groups (P<0.05, figure 9A&F). However, there were no significant differences in IL-2 levels among the vaccine and control groups (P>0.05, figure 9B). For Th2-type cytokines, comparing the W541 vaccine group to the normal control group revealed that the W541 vaccine decreased expression of IL-4 and IL-6 by murine splenocytes (P<0.05, figure 9C&D). There were no significant differences in IL-10 expression among the three groups(P>0.05, figure 9E). These results demonstrate that the W541 DNA vaccine effectively activated a Th1-type immune response in the host.