| ObjectiveThe purpose of this study was to perform bioinformatics analysis to the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii. This gene was cloned, expressed and purified. The immunogenicity of TgPGAM2 and its effects intranasal immunized on protection of mice against Toxoplasma gondii were analyzed. The feasibility of TgPGAM2 to be used as candidate antigen for toxoplasmosis vaccine has been discussed in this study.MethodsThis study included four parts. First part summarized the physico-chemical property and research progression of PGAM of different source, such as vertebrate, invertebrates and protozoa. The second part was to predict the immunogenicity of the phosphoglycerate mutase 2 protein extracted from Toxoplasma gondii (TgPGAM2) encoding by TgPGAM2 gene and characterize its structure and epitope by bioinformatics approaches. The physico-chemical property, solubility, surface accessibility, flexibility, post-translational modification sites, transmembrane domain, hydrophilicity / hydrophobicity, secondary structure, and specific epitope of TgPGAM2 protein were analyzed and predicted by using the bioinformatics tools-ExPASy, including ProtParam, SOSUI, TMHMM, HNN, MotifScan, ORF finder, and Bcepred, combined with bioinformatics softwares (Gene Runner, DNAMAN, etc.).The recombinant pET30a/TgPGAM2 was constructed and expressed, and the immunogenicity of TgPGAM2 was analyzed in the third part. Tachyzoites of Toxopalsma gondii RH strain were harvested and genomic RNA was prepared. Apair of primers and restriction site Kpn I / BamH I were designed. The coding region of PGAM2 was amplified with RT-PCR and cloned into the prokaryotic expression vector pET30a. The recombinants were confirmed by Kpn I / BamH I, PCR, and DNA sequencing. And then transformed into E. coli BL21 induced by IPTG. The expressed ptotein were analyzed by SDS-PAGE. A lot of soluble rTgPGAM2 were purified with Ni-NTA affinity chromatography and analyzed by Western blotting.The mucosal and systemic immune responses after immunized with rTgPGAM2 and the effects of protecting mice against Toxoplasma gondii were studied in the fourth part. BALB/c mice were intranasally immunized with 10μg, 20μg, 30μg or 40μg rTgPGAM2 per mouse at an internal of two weeks, while mice intranasally immunized with PBS in the same means were control. Five mice per group were killed on the day 14 after immunization. Levels of IgG, IL-2, IL-4 and IFN-γin serum and sIgA in faces were detected by ELISA. Numbers of Intestinal intraepithelial lymphocytes (IEL) and spleens lymphocytes were counted. Other 35 mice were challenged intragastrically with 1×10~4 tachyzoites per mouse on the day 14 after the last immunization. The health status of mice infected was observed every day during the experiment. Mice were killed on the 30th day after challenge, the tachyzoites of their lives and brains were counted.ResultsThe results of bioinformatics analyze suggested that TgPGAM2 protein has 10 zones with surface accessibility≥1.9, 3 zones with hydrophilicity≥1.9, 3 zones with flexibility≥2, 8 post-translational modification sites, 16 potential epitopes, can be used as a candidate antigen for toxoplasmosis vaccination because of its immunogenic property.T. gondii PGAM2 gene with a molecular size of 756 bp and the recombinant pET30a/TgPGAM2 was successfully constructed. The results of SDS-PAGE revealed that the molecular weight of recombinant protein was approximately 30ku. Western blotting revealed that rTgPGAM2 can be recognized by rabbit antiserum of Toxoplasma gondii.The levels of IgG, IFN-γ, sIgA in faces of 30μg group (0.9704, 0.8456 and 0.9098) were higher than that of 20μg group (0.8213, 0.7244 and 0.6504), the differences between groups were significant (F=8.741, F=4.53, F=6.94, P<0.05); the levels of IL-4 and IL-2 in serum were not observed significant difference between various groups. Two mice in control group and one mouse in the 40μg group were died after challenged with tachyzoites. The numbers of tachyzoites load in lives and brains in 30μg group (58.3×105/g, 3.99×105/g) was lower than that of control (93.88×105/g, 5.65×105/g) and 10μg group (85.54×105/g, 4.86×105/g) and shown significantly differences (F=8.56, F=9.31, F=7.39, F=9.68, P<0.05). ConclusionThe results of bioinformatics analyze suggested that TgPGAM2 protein has 16 potential epitopes, can be used as a candidate antigen for toxoplasmosis vaccine because of its immunogenic property. The recombinant pET30a/TgPGAM2 was successfully constructed. A lot of soluble rTgPGAM2 was producted. The recombinant protein has been confirmed with immunogenicity, it may be used as candidate antigen. Compared with other groups, the mucosal and systemic immune response has been induced by 30μg rTgPGAM2 and effectively reduced the tachyzoites load in lives and brains. Taken together, these data demonstrate that TgPGAM2 had induced the mucosal and systemic immune response, can be used as candidate antigen for toxoplasmosis vaccine because of its immunogenic property. |
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