Bioinformatics analysis and expression of a novel protein ROP48 in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular apicomplexan parasite, and can infect warmblooded animals and humans all over the world. In the past years, ROP family genes encoding particular proteins of T. gondii had made a great contribution to toxoplasmosis. In this study, we used multiple bioinformatics approaches to predict the physical and chemical characteristics, transmembrane domain, epitope, and topological structure of the rhoptry protein 48 (ROP48). The results indicated that ROP48 protein was mainly located in the membrane and had several positive linear-B cell epitopes and Th-cell epitopes, which suggested that ROP48 is a potential DNA vaccine candidate against toxoplasmosis. Then the PCR product amplified from the ROP48 cDNA was inserted into a pEASY-T1 vector to build a recombinant cloning plasmid. After sequencing, ROP48 was subcloned into a eukaryotic expression plasmid pEGFP-C1 to obtain pEGFP-C1-ROP48 (pROP48). After identification by PCR and restriction enzyme digestion, the recombinant plasmid pROP48 was transfected into HEK 293-T cell and identified by RT-PCR. The results showed that the eukaryotic expression plasmid pROP48 was constructed and transfected to the cells of HEK 293-T successfully. Western blotting showed that the expressed proteins can be recognized by anti-STAg mouse sera.