The amounts of IL-2, IL-4, IL-10 and IFN-γ were determined by indirect ELISA according to the manufacturer’s instructions (Jiamay Biotech, Beijing, China). Fourteen days after the final vaccination, eight BALB/c mice were selected randomly from each group and challenged intraperitoneally with 500 tachyzoites of the Autophagy signaling pathway inhibitors highly virulent T. gondii RH strain. All mice were observed twice daily, and the survival times were recorded. Those mice that were alive 2 weeks after the challenge
were considered to have survived. Statistical analysis was performed using SPSS 14·0 software for variance (anova) and Duncan’s multiple ranges. P < 0·05 was considered to be statistically significant. The coding region of TgCyP was amplified by RT-PCR and combined with the eukaryotic expression vector pVAX1. The constructed plasmid pVAX1-TgCyP, which carried the TgCyP insert, was verified by sequencing. Forty-eight hours after HeLa cells were transfected with the recombinant plasmid pVAX1-TgCyP, the recombinant CyP protein (green fluorescence) was found to be significantly expressed by immune-fluorescence staining. There was no signal in the pVAX1 vector-transfected cells. These results indicated that the recombinant plasmid was successfully
constructed and expressed in vitro (Figure 1). A specific antibody response against Opaganib in vitro T. gondii tachyzoites was detected in the pVAX1-TgCyP vaccinated BALB/c mice. Two weeks after the final immunization, the antibody level of the pVAX1-TgCyP group was significantly higher than control groups, which were immunized with pVAX1 or PBS (P < 0·05). This result was shown in Figure 2. Splenocytes collected 2 Enzalutamide weeks after the final vaccination were stimulated with TLA, and a significant increase in splenocyte proliferation was detected in the pVAX1-TgCyP group (Table 1) (P < 0·05). The production of IFN-γ and IL-2 was highly elevated in splenocytes after stimulation with TgCyP in the pVAX1-TgCyP-vaccinated BALB/c mice (Figure 3) (P < 0·05). Nevertheless, a slight difference was observed
in PBS- and pVAX1-immunized mice. No significant difference was observed in IL-4 or IL-10 release among all of the study groups. Two weeks after the last vaccination, all of the mice were challenged intraperitoneally with 500 tachyzoites of the T. gondii RH strain. There was no significant difference in the protection levels between the pVAX1- and PBS-immunized groups (P > 0·05). In comparison to the control groups, significantly higher protection was observed in the pVAX1–TgCyP vaccinated group with a survival rate of 37·5% (P < 0·05) (Figure 4). Overall, the TgCyP DNA vaccine produced significant protection in BALB/c mice. In this study, the protection efficacy of the T. gondii vaccine candidate TgCyP was determined in BALB/c mice.