| Toxoplasma gondii is an opportunistic intracellular parasite which is capable of infecting almost all warm-blooded animals including wild mammals, birds, domestic animals and humans. Toxoplasma infection caused by toxoplasmosis is in every corner of the globe. Toxoplasmosis is a zoonosis and a serious threat to livestock development and human health. There is no effective drug for toxoplasmosis. So it’s urgent to prevent toxoplasmosis instead of treatment. In this study, the appropriate software and online databases were used to analysis the physical and chemical characteristics of homology, the two-dimensional structure, the three-dimensional structure, B cell epitopes and T cell epitopes of Toxoplasma gondii surface antigen glycoprotein 5 (SAG5). The recombinant DNA vaccine SAG5A and SAG5D were constructed to immunize mice. The using of antigenic polypeptides and a galactosylceramide (a-GalCer) enhanced the immune response of the vaccines. The authenticity of the vaccines were verified by animal experiments. This work fills the blank of knowledge in surface antigen protein 5 of Toxoplasma gondii and provides a reference for the development of an effective vaccine against Toxoplasma gondii.Objective:The basic physical and chemical properties, two-dimensional and three-dimensional spatial structure of the surface antigen protein 5 were studied in this study. The B cell epitopes and T cell epitopes of SAG5A, SAG5B, SAG5C and SAG5D were analyzed by some means. The recombinant DNA vector of SAG5A was constructed to immunize mice with a polypeptide (HAPTPSFLGLLAVVF). The brain cysts of mice were counted to evaluate the immune protection of S AG5 A gene and polypeptide. The SAG5D gene expression vector was built to immunize mice together with a-GalCer. The survival times of mice challenged with tachyzoites was recorded to evaluate the protective ability of SAG5D gene and a-GalCer.Methods:The online analysis software was used to predict physicochemical properties, transmembrane region and signal peptide sequences of SAG5A, SAG5B, SAG5C and SAG5D. The online program PSORT II was used to analyze the subcellular localization of SAG5A, SAG5B, SAG5C and SAG5D. The online program NetNGlyc1.0 was used to analyze the N-glycosylation sites of SAG5A, SAG5B, SAG5C and SAG5D. The online program was used to predict the phosphorylation sites and acylation sites of SAG5A, SAG5B, SAG5C and SAG5D. DNAStar, Gene Runner and DNAMAN software were used to analyze the B-cell epitopes of SAG5A, SAG5B, SAG5C and SAG5D. The databases of IEDB was used to predict T cell epitopes of SAG5A, SAG5B, SAG5C and SAG5D. The online tool, T-Coffee, was used to compare the sequences of the four proteins. The online software SWISS-MODEL and software VMD were used to predict the two-and three-dimensional structures. The primer design software was used to design the upstream and downstream primers of SAG5D and SAG5A. The genes of SAG5A and SAG5D were connected to pEGFP-C1 vectors to construct the SAG5A and SAG5D eukaryotic expression vector after digested with two kinds of restriction enzyme. The integrity of the SAG5A and SAG5D genes in constructed vectors was verified by sequenced. A large number of constructed ukaryotic expression vectors of SAG5A and SAG5D were extracted by kit to immunize BALB/c mice. The adjuvant, a galactosylceramide (a-GalCer) was added to SAG5D gene vaccine to improve the immunity when injected into mice. The peptide was added to SAG5A gene vaccine in a way of strategy to improve the immunity. The blood serum of mice was collected after immunized with DNA vaccine. The levels of total IgG, IgGl, IgG2a serum in serum and cell cytokines IL-4, IL-10, IFN-y in spleen were detected to assess the immune response stimulated by DNA vaccine. The experiment of the attack of tachyzoites (RH strain) to verify the immune protection of SAG5D gene and a-GalCer. The experiment of attack of cysts (PRU strain) to test the immune protection of SAG5A gene and peptides.Results:The software and online tools were used to analyze the physical and chemical properties of SAG5A, SAG5B, SAG5C and SAG5D. The prediction showed that the four proteins had similar physical and chemical parameters. SAG5A and SAG5D had similar physical and chemical parameters, while SAG5B and SAG5C had a more similar physical and chemical properties. The online program PSORT II was used to analyze the cellular localizations of the four proteins. The prediction showed that SAG5A, SAG5B, SAG5C and SAG5D were located in the plasma membrane with a large of probability. The prediction of post-translational modifications of proteins indicated that they have similar glycosylation sites, phosphorylation sites and acylation sites and the modification sites of SAG5B and SAG5C are basically the same. The IEDB database and DNASTAR software were used to analyze the B cell epitopes of SAG5A, SAG5B, SAG5C and SAG5D protein, which showed that SAG5B and SAG5C had similar B cell epitopes. The IEDB database was used to analyze their T-cell epitopes and the epitopes with hydrophilic strong, high plasticity and high antigen index were selected. The results showed that the four proteins were good protein antigen. In addition, the software and database were used to analyze the B-cell epitope and T-cell epitope of SAG1. Compared to SAG1, SAG5A and SAG5D protein had better epitopes, while SAG5B and SAG5C had similar epitopes. The analysis of sequence alignment by T-Coffee software indicated that SAG5A, SAG5B, SAG5C and SAG5D had high homology and SAG5B and SAG5C were 96% similar to each other. The prediction by DNAStar and VMD software showed that SAG5A, SAG5B, SAG5C and SAG5D had similar regions of a-helix and β-fold, while SAG5B and SAG5C had almost the same distribution of a-helix and P-fold. The prediction of VMD software showed that these proteins had similar three-dimensional structures and SAG5B and SAG5C had almost the same spatial structures.The primer design software was used to successfully design the upstream and downstream primers of SAG5A and SAG5D. The cDNA of Toxoplasma gondii was used as a template and PCR products were obtained by PCR reaction. The fragments of SAG5A and SAG5D with a size of 1,089bp were found after gel electrophoresis. A large number of target genes (SAG5A and SAG5D) were obtained with the recovery and purification by gel extraction kit. After digested, the target genes and pEGFP-Cl eukaryotic expression vectors combined together with the help of DNA ligase. After constructed, the recombinant vectors were transfected DH5a competent cells.5-10 independent bacteria strains were obtained on the plate after cultured. The strains were picked to liquid culture medium and the plasmids were extracted by a plasmid extraction kit. The digestion and agarose gel electrophoresis experiments showed that there were two bright bands at 4,700bp and 1,089bp departments. The result indicated that we had successfully constructed the eukaryotic expression vectors of pSAG5A and pSAG5D. A large number of bacteria was cultured and lots of endotoxin-free plasmids were extracted by a kit of endotoxin-free plasmid extraction. The concentrations of endotoxin-free plasmids were detected in 1,000μg/ml or more, which could be used in the following experiments. To verify the expression of the constructed vectors in the cell, the endotoxin-free plasmids were transfected into HEK 293-T cells. The cells transfected with pSAG5A and pSAG5D were detected by fluorescence microscopy. The two groups of cells could be excite green fluorescence, which indicated that the recombinant vectors were capable of expressing the protein in cells. The transfected cells were collected and lysed to extract a large number of the whole proteins of cells. The proteins were analyzed by Western blot and a 60 kD band was found, which demonstrated that the SAG5A and SAG5D recombinant vectors could express the SAG5A and SAG5D proteins.The pSAG5A vectors and the peptide were used to immunize BALB/c mice by the strategy designed before experiment. pSAG5D eukaryotic expression vectors and a-GalCer were used to immunize mice. The serum of all mice was collected two weeks after each immunization. The serum was used to detect the concentration of anti-Toxoplasma serum total IgG, IgGl and IgG2a antibody. The IgG and IgG2a antibody levels of mice immunized with pSAG5A and peptides were significantly higher than other experimental groups (P<0.05); The levels of IgG and IgG2a antibody of mice immunized with pSAG5A or peptides were significantly higher than the control groups (P<0.05). There was no difference in the levels of IgG and IgG2a antibody between pSAG5A group and peptides group (P>0.05). The levels of IgGl in experimental groups were similar to that of control groups (P>0.05). The IgG and IgG2a antibody levels in serum of mice immunized with a-GalCer and pSAG5D were significantly higher than the other groups (P<0.05); The IgG and IgG2a antibody levels of mice immunized by pSAG5D or a-GalCer were significantly higher than the control groups (.P<0.05), whereas no significant difference was found between pSAG5D group and a-GalCer group (P>0.05); The levels of IgG1 antibody in all group were similar (P>0.05).To further demonstrated immune responses in mice, the IL-4, IL-10, and IFN-y cytokines levels in mouse spleen cells were detected. The levels of IFN-y in pSAG5A and polypeptides common immune group (721.0±99.6) was significantly higher than other experimental groups and the control group (P<0.05); the levels of IFN-y in pSAG5A (505.3±71.6) and polypeptides (489.7±98.9) two groups were significantly higher than that in empty vector group (52.4±11.6) and PBS group (55.5±11.7) (P<0.05), but there was no significant difference between them (P>0.05); and the cytokines IL-4 and IL-10 levels did not significantly differ among all the groups (P>0.05). The IFN-y levels in spleen cells of mice immunized together with pSAG5D and a-GalCer (781.36±57.57) was significantly higher than other groups (P<0.05); the IFN-y levels in pSAG5D (573.43±60.2) and a-GalCer group (343.6±50.88) were significantly higher than that in group pEGFP-C1 (53.15±8.71) and PBS group (52.07±8.23) (P<0.05). The levels of IL-4 in the mouse spleen cells in pSAG5D and a-GalCer common immune group (76.73±6.08) and a-GalCer immune group (75.46±5.7) were higher than that in group pSAG5D (36.65±3.52), pEGFP-C1 group (39.24±6.43) and PBS group (36.36±5.25) (P<0.05); all mice had no significant difference of IL-10 level.Finally, in order to evaluate the gene vaccine immunity, all the mice were attacked by Toxoplasma gondii. The virulent strain (RH) tachyzoites of Toxoplasma gondii attacked SAG5D mice of all groups, the results showed that the mice survival time of pSAG5D and a-GalCer common immune group could be up to 17 days, while pSAG5D group, a-GalCer, pEGFP-C1 and PBS group, the mice survival time was 13 days,10 days,7 days and 6 days. The attenuated strain (PRU) cysts of Toxoplasma gondii attacked SAG5D mice of all groups, the results showed that the number of cysts in mouse brain in pSAG5A and polypeptides common immune group (436±174) was significantly less than the other groups (P<0.05); the number of cysts in mouse brain in pSAG5A group (815±197) and polypeptides group (732±160) were significantly less than the pEGFP-C1 group (1350±268) and PBS group (1260±241) (P<0.05), and there was no significant difference between pSAG5A group and polypeptides group.Conclusion:Bioinformatics analysis showed that SAG5A, SAG5B, SAG5C and SAG5D have high homology, they have excellent epitopes of B cell and T cell, furthermore the SAG5A and SAG5D protein antigenicity are more prominent. The eukaryotic expression vectors of pSAG5A and pSAG5D were successfully expressed in eukaryotic cells and mice; both SAG5A and SAG5D DNA vaccine could induce strong humoral immunity and cellular immunity in mice; a-GalCer could indeed enhance the immune effect of SAG5D gene vaccine; the immune strategy combining antigen polypeptides with SAG5A gene vaccine could provoke more strong immune effect; in the animal experiments, two kinds of DNA vaccines could both provide strong immunity ability to resistance Toxoplasma gondii infection for mice. |
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