Evaluation Of Protective Immune Responses Induced By Toxoplasma Gondii MAPK1 Nucleic Acid Vaccine In Mice

Toxoplasma gondii is an obligate intracellular protozoan parasite which causes common zoonotic disease known as toxoplasmosis in the intermediate hosts such as humans and domestic animals.It is reported that as many as 20-30%of the world’s people are infected with T.gondii and there are no effective drug treatments available to eliminate the parasite.Considering that there is no available T.gondii vaccine and there are many side effects caused by chemotherapy,it is very important for humans and animals to develop a safe and effective vaccine against T.gondii infection.Based on the good immunogenicity and immunoreactivity of T.gondii MAPK1 antigen,we selected T.gondii MAPK1 gene to construct DNA vaccine in this study.Objective:We constructed recombinant eukaryotic expression plasmid pEGFP-MAPK1 and prepared the MAPK1 nucleic acid vaccine(DNA vaccine)of T.gondii to evaluate the immunoprotective effect elicited by TgMAPK1 protein in BALB/c mice and explore the possibility of TgMAPKI protein as a vaccine candidate antigen.Methods:The RNA of T.gondii RH strain was extracted and it was transcribed into cDNA by RT-PCR.The TgMAPK1 gene fragment was amplified by PCR from cDNA.The TgMAPKI gene fragment was cloned into pEASY-T1 vector to construct recombinant plasmid pEASY-MAPK1,which identified by PCR and restriction enzymes.The target gene TgMAPKI was subcloned into a eukaryotic expression vector pEGFP-C1 to construct recombinant plasmid pEGFP-MAPK1,which identified by PCR,restriction enzymes and sequencing.The recombinant plasmid pEGFP-MAPK1 was transfected into HEK293T cells and then fluorescence microscopy and Western blot were performed to detect the target protein expressions.Large-scale plasmid preparation and purification were performed for the preparation of DNA vaccines.BALB/c female mice were randomly divided into three groups and were immunized intramuscularly three times.ELISA was used to detect changes of antibodies in sera and cytokines in splenocyte cultures of mice.Two weeks after the last immunization,all mice were challenged intraperitoneally with 1×103 tachyzoites of T.gondii and the survival time of challenged mice was observed and recorded daily.Results:The amplified TgMAPKl gene fragment was about 1599bp by PCR.The recombinant eukaryotic expression plasmid pEGFP,MAPK1 was identified correctly by PCR,restriction enzyme digestion and DNA sequencing which had 100%homology to the MAPK1 gene sequence of T.gondii in GenBank.The green fluorescence was observed in HEK293T cells transfected with pEGFP-MAPK1 or pEGFP-C1,whereas no fluorescence was observed in the untrasfected cells.The total protein of the transfected cells was extracted and the target protein about 58kD was detected by Western blot.Mice immunized with pEGFP-MAPK1 produced higher levels of IgG,IgG2a and IFN-y than the control groups(P<0.05),while the levels of IgG1,IL-4 and IL-10 showed no significant difference between the groups(P>0.05).Compared with the control groups,the survival time of mice in pEGFP-MAPK1 experimental group was significantly prolonged after T.gondii challenge(P<0.05).Conclusion:The study suggested that T.gondii MAPK1 nucleic acid vaccine induced strong humoral immune response and Th1 cell immune response in mice and played a protective role in the process of resistance to toxoplasmosis,so MAPK1 antigen of T.gondii is a promising antigen candidate for vaccine.