Enhancement Of Protective Immune Responses Induced By Toxoplasma Gondii Dense Granule Antigen7(GRA7) Against Toxoplasmosis In Mice Using A Prime-boost Vaccination Strategy

Toxoplasma gondii is an obligate intracellular parasite that infects any nucleated cell of warm blooded vertebrates including humans. It is responsible for the worldwide antropozoonotic disease called toxoplasmosis. So far, there are no drug treatments available to eliminate the parasite. Development of effective vaccines against Toxoplasma gondii may therefore contribute to preventing and controlling the spread of toxoplasmosis, which has critical significance for humans and animals. In the present study, for further exploration of its vaccine effect, a heterologous prime-boost vaccination strategy basing on GRA7was performed in BALB/c mice.Objective:Constructed a eukaryotic plasmid pEGFP-GRA7and a prokaryotic plasmid pET30-GRA7, both expressing GRA7of T. gondii, and then examined the immunogenicity and protective effect of DNA vaccine and protein vaccine in BALB/c mice against lethal challenge infection with the highly virulent RH strain of T. gondii. The feasibility of the heterologous prime-boost vaccination strategy with DNA vaccine or protein vaccine was also investigated.Methods:Tachyzoites of T. gondii RH strain were harvested from mice peritoneal cavity, with a genomic DNA Extraction Kit estracted genomic DNA. GRA7gene fragment was amplified by PCR. The GRA7gene fragment was cloned into pEASY-T1vector in order to constructed cloned plasmid pEASY-GRA7, which identified by PCR, restriction enzymes and sequencing. The target gene GRA7was subcloned into a eukaryotic expression plasmid pEGFP-Cl and a prokaryotic expression plasmid pET-30a(+) to produce pEGFP-GRA7and pET30-GRA7with appropriate restriction enzymes digestion, respectively. They were both identified by PCR, restriction enzymes and sequencing. HFF cells were transfected with pEGFP-GRA7or pEGFP-C1for identifying. E. coli BL21(DE3) cells were transformed by pET30-GRA7. GRA7protein was induced by IPTG and purified by NiNTA resin for proteins vaccines. The heterologous prime-boost vaccination strategy was used to inject DNA or protein vaccine.Results:Using specific primers amplified GRA7gene in vitro,708bp long gene fragment was obtained. By colony PCR, restriction enzymes and sequencing the constructing of the eukaryotic expression plasmid pEGFP-GRA7and the prokaryotic expression plasmid pET30-GRA7were identified correctly. In the eukaryotic system, the green fluorescence was observed in HFF cells transfected with pEGFP-GRA7or pEGFP-C1, whereas no fluorescence was observed in the untransfected HFF cells. A specific protein band (57kDa) was revealed in SDS-PAGE and recognized by Western blotting using mouse anti-STAg sera. In the prokaryotic system, a specific protein band (37kDa) was showed from IPTG-induced bacteria, which was absent in uninduced bacteria. Western blotting demonstrated that the expressed protein was reacted with anti-STAg and anti-histidine antibodies, respectively. The survival rate and the levels of antibody and cytokine showed that the DNA prime-protein boost vaccination regimen was effective in eliciting robust humoral and cellular immune responses against T. gondii.Conclusions:The eukaryotic expression plasmid pEGFP-GRA7and the prokaryotic expression plasmid pET30-GRA7were constructed successfully. The DNA vaccine of GRA7was prepared by E.coli DH5a and the protein vaccine of GRA7was expressed in E.coli BL21(DE3) cells. After administration of vaccine to BALB/c mice, the DNA prime-protein boost vaccination based on GRA7might be a very promising regimen for the further development of effective vaccines against T. gondii.