Development and applications of a protozoan parasite vector using Toxoplasma gondii to express heterologous genes

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An improved method for the construction of recombinant Toxoplasma gondii expressing heterologous genes was developed using pyrimethamine-resistant DHFR-TS and green fluorescent protein (GFP) genes as double selection markers. This improved method allows the construction of recombinant T.gondii expressing foreign genes with great case and speed. Because the foreign genes were placed between the DHFR-TS and GFP genes, all of the drug-resistant and fluorescent clones were confirmed to be expressed foreign genes. This method was employed to construct recombinant T.gondii expressing the Neospora caninum major antigen SRS2 gene (Tg/NcSRS2) and the Cryptosporidium parvum major antigen P23 gene (Tg/CpP23). The expression levels of NcSRS2 and CpP23 in recombinant Tg/NcSRS2 and Tg/CpP23 were similar to those in N.caninum and C.parvum, respectively. The molecular weight and antigenic property of recombinant NcSRS2 and CpP23 were similar to those of native NaSRS2 and CpP23, respectively. Mice immunized with recombinant Tg/NcSRS2 were partially protected from N.caninum challenge infection. On the other hand, the mice immunized with recombinant Tg/CpP23 produced specific neutralizing antibodies against C.parvum. In addition, a bioactive molecule, such as mouse interferon gamma was successfully expressed by T.gondii vector. These results indicate that the T.gondii vector may provide a new tool not only for the production of recombinant vaccines against protozoan parasite infections in animals, but also for the production of bioactive molecules.