| Toxoplasma gondii, the direct pathogenic factor of toxoplasmosis, is an obligate intracellular protozoan parasite of the phylum Apicomplexa which can infect all warm-blooded vertebrates, including humans, livestock and marine mammals. Although most infections are clinically asymptomatic, the parasite can cause severe disease in immunocompromised populations and congenitally infected individuals. In addition, infection in domestic animals may result in economic losses as well as brings enormous psychological troubles, since it can cause abortion, stillbirth and neonatal loss.Superoxide dismutase (SOD), an important enzyme that widely exists in many organisms including animals, plants and microorganisms, can promote the conversation of superoxide (O2-) into hydrogen peroxide and oxygen. In view of SOD can eliminate extra superoxide (O2-) anion in the cells and protect cells from oxidative damages. It has potential applications in medicine, food industry and agriculture. SOD also exists in different T. gondii strains, however, there is little information regarding the sequence variation of SOD gene. We hereby examined sequence variation of SOD gene among ten T. gondii isolates from different hosts and geographical regions, and assess SOD could be used as a new marker for genetic study or a potential vaccine candidate against T. gondii.Additionally, a recombinant eukaryotic expression plasmid pEGFP-SOD was constructed and verified by PCR, restriction enzyme digestion and sequencing. The expression protein SOD was detected by SDS-PAGE and Western blotting after transient transfection of pEGFP-SOD into HEK293T cells.Methods:The SOD gene was amplified and cloned into pEASY-C1 to produce pEASY-SOD. After identification by restriction enzymes digestion, PCR and sequencing, the SOD gene sequences were analyzed by PCR-RFLP, sequence analysis and phylogenetic reconstruction, which could provide essential information revealing the evolution and transmission of this widespread parasiteThen the recombinant plasmid was transmitted into HEK293-T cells. After Fluorescence microscopy, SDS-PAGE and Western blotting, the target protein expressions were detected.Results:The amplification of the SOD gene resulted in a single product of approximate 1700 bp in length on agarose gel for all tested T. gondii strains. There are no obvious differences in all tested T. gondii strains after digestion of the amplification SOD products with EcoR I and Xba I, revealing subtypes I, II, and III could not be differentiated in this condition. Phylogenetic analysis of the 10 examined T. gondii isolates based on the SOD gene sequences demonstrated two major clusters. TgCatBr5, TgCatBr64 and TgCgCal strains were clustered in one clade, and PRU, MAS, GT1, CTG, TgTgucan, PTG and RH were clustered in the other clade. Overall, the topologies of all trees based on nucleotide sequences inferred by two different methods were similar, with only small differences of bootstrap values. Analysis of PCR, restriction enzyme cleavage and sequencing confirmed that the recombinant eukaryotic expression plasmid of pEGFP-SOD was precisely constructed. The identification of the green color fulorescence, SDS-PAGE and western blotting showed that SOD of T. gondii expressed from HEK293T cells.Conclusion:The present study demonstrated the existence of low sequence variability in the SOD gene sequences among T. gondii isolates from different hosts and geographical regions, suggesting that SOD cannot be an appropriate genetic market for differentiation of T. gondii strains. However, it may be a potential vaccine candidate against toxoplasmosis. The results of PCR, restriction enzyme digestion and sequencing showed the correct construction of the eukaryotic expression plasmid pEGFP-SOD.The SOD of T. gondii was transiently expressed in HEK293T cell in vitro. In our laboratory, the DNA vaccine encoding T. gondii SOD gene is being investigated. |
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