Design of a dual-promoter expression vector harboring Sag1 and Gra7 genes from Toxoplasma gondii (RH strain)

Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, has possible irreparable consequences in immunocompromised patients and fetuses. Finding an effective method of prevention, such as vaccination, is crucial because of the global distribution of the parasite and the lack of effective anti-toxoplasmosis drugs. The Sag1 and Gra7 antigens of T. gondii can induce strong humoral and cell-mediated immune responses. Therefore, to develop a novel DNA vaccine against toxoplasmosis, we prepared a eukaryotic construct expressing the Sag1 and Gra7 genes of T. gondii (RH strain). We then verified the ability of this construct to produce the corresponding Sag1 and Gra7 antigens in mammalian cells. Using specific primers, the complete coding sequences of Sag1 and Gra7 genes were amplified by polymerase chain reaction (PCR) from the genomic DNA of T. gondii. Then, both genes were subcloned into pVitro2-neo-mcs plasmid. The pVitro-Sag1-Gra7 construct was subjected to colony PCR, enzymatic digestion, and sequencing to confirm successful subcloning. Sag1 and Gra7 expression in HeLa cells was investigated. Sag1 and Gra7 were successfully subcloned in pVitro2-neo-mcs plasmid. The expression of Sag1 and Gra7 in HeLa cells was confirmed through Western blot analysis. The recombinant pVitro-Sag1-Gra7 construct that simultaneously produces Sag1 and Gra7 antigens in one mammalian cell may be used to develop a novel protective vaccine against toxoplasmosis.