Evaluation Of Protective Immune Responses Induced By DNA Vaccines Encoding Toxoplasma Gondii Rhoptry Protein 5 And Rhoptry Protein 7 In BALB/c Mice

Toxoplasma gondii is an apicomplexan and obligate intracellular parasitie with a worldwide distribution, which can lead to the occurrence of zoonotic parasitic diseases. Most people usually have no symptoms when infected with Toxoplasma gondii, but in immunosuppressed and immunocompromised individuals, including AIDS patients, organ transplant, cancer patients undergoing long-term chemotherapy may lead to serious consequences. Infection during pregnant women can cause miscarriage, neonatal malformations, stillbirth and fetal blindness. Livestock animals infected with T. gondii can also bring considerable economic loss. Considered there is no effective therapy for the treatment of toxoplasmosis, thus, the research about development of an effective vaccine against Toxoplasma gondii is particularly important. In this study, we selected ROP5 and ROP7 two kinds of candidate genes to construct a complex DNA vaccine.Objective:The aim of this study was to explore the immune protection of ROP5/ROP7 gene against Toxoplasma gondii in BALB/c mice.Methods:PCR primers were designed based on the ROP5 and ROP7 sequences of Toxoplasma gondii in Genbank. The ROP5 and ROP7 genes were amplified by PCR. The two genes were subcloned into the eukaryotic expression vector pBudCE4.1 to produce pBudCE4.1-ROP5(pROP5), pBudCE4.1-ROP7(pROP7) and pBudCE4.1-ROP5/ROP7(pROP5/ROP7), and they are identified by restriction enzyme. The recombinant plasmids were transfected into HEK293-T cells and analyzed by Western blot. After identification of the expression of the plasmids, a large number of the recombinant plasmids were extracted to immunize mice. The special serum IgG level and cytokines were detected by ELISA and the survival time of mice infected with Toxoplasma gondii was observed to evaluate its protective immunity.Results:PCR amplified 1,650 bp and 1,728 bp target gene fragments. The recombinant eukaryotic expression vectors were successfully constructed and expressed in HEK293-T cells. ROP5 and ROP7 protein were recognized by Western blot analysis. The mice immunized with complex gene vaccine produced higher level of serum antibodies. In animal experiment, the mice immunized by the complex gene vaccine had a longer survival time compared with the other groups. The levels of IgG and IgG2a in mice immunized with complex gene vaccine were higher than the other mice (P<0.05). In cytokine detection, compared to other group, the mice vaccinated with complex gene vaccine showed higher level of IFN-y (P<0.05), but no significant differences were found in IL-4 and IL-10(P>0.05).Conclusion:Our findings suggested that the constructed gene vaccines had part protection against to Toxoplasma gondii infection and multi-gene vaccine was better than single-gene DNA vaccines. This study provided a reference for the development of vaccine against to Toxoplasma gondii.