| Toxoplasma gondii,an obligatory intracellular parasite,can infect virtually all warm-blooded animals and huamns,and cause economic losses worldwide and serious public health problems.Currently,no available drugs could effectively control the growth of T.gondii in infected animals.In our study,we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice.Eukaryotic expression plasmids pVAX-GRA17 and pVAX-GRA23 were constructed and transfected into HEK 293-T cell.The expression of pVAX-GRA17 and pVAX-GRA23 in cells was analyzed.Then,BABL/c mice were intramuscularly immunized with pVAX-GRA17,pVAX-GRA23 and pVAX-GRA17 + pVAX-GRA23 followed by infection with the highly virulent RH strain of T.gondii.The specific immune responses and protective efficacy against T.gondii were analyzed by measurements of cytokines and serum antibodies,lymphocyte proliferation assays,flow cytometric on lymphocytes and the survival time after challenge.We also created a RH?GRA17 deletion mutant by using the CRISPR-Cas9 technique.Then,we tested whether RH?GRA17 deletion mutant can be used as an attenuated live vaccine against T.gondii infection.The results of vaccinations with GRA17 and GRA23 DNA vaccines showed that pVAX-GRA17,pVAX-GRA23 and pVAX-GRA17 + pVAX-GRA23 induced specific humoral and cellular responses in BALB/c mice,with higher level of IgG antibody,and increased levels of Th1-type cytokines IFN-? and CD3+CD4+CD8-and CD3+CD8+CD4-T cells,as well as prolonged survival time compared to control mice.Our study proved that pVAX-GRA17 and pVAX-GRA23 can produce certain level of immune protection against T.gondii acute infection and the combined gene vaccine is better than signle gene vaccine.The results of vaccination with RH?GRA17 attenuated live vaccine showed thatRH?GRA17 deletion mutant induced specific humoral and cellular responses in Kuming mice and can produce strong immune protection against T.gondii infection. |
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