Toxoplasma gondii is identified as an obligate intracellular apicomplexan parasite that infects warm blooded animals and humans worldwide. SAG5 protein includes SAG5A, -5B, -5C, -5D, and -5E five subtypes. SAG5A, -5B, -5C, and -5D are expressed on the surface of Toxoplasma gondii. In this study, we used online T-Coffee tool to analyze SAG5 proteins sequence alignment. SMART software was used to predict secondary structures of SAG5A, -5B, -5C, and -5D. The 3D models of SAG5 proteins were constructed and analyzed with SWISS-MODEL server and VMD software. Results indicated that SAG5A, -5B, -5C, and -5D are highly homologous proteins. Furthermore, liner-B cell epitopes and Th-cell epitopes of the four proteins were predicted using DNAMAN software and Epitope Database online service. The bioinformatics analysis of SAG5A, -5B, -5C, and -5D proteins could provide valuable information on prevention and treatment of toxoplasmosis. In addition, the four genes were obtained by PCR and inserted into an eukaryotic expression vector pEGFP-C1 respectively. Identified by restriction enzyme digestion, the four recombinant plasmids were transfected into HEK 293-T cells and tested by RT-PCR. Results showed that the constructed plasmids were all transfected to HEK 293-T cells successfully. © 2016, Malaysian Society for Parasitology. All rights reserved.