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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.

Reviews have shown increasing number of Iranian patients with cutaneous leishmaniasis (CL) who are unresponsive to pentavalent antimonial compounds such as meglumine antimoniate (Glucantime, MA). The present investigation aims to determine the correlation between clinical responses (healing, or non-healing) with susceptibility of Leishmania parasites to glucantime. Initially, in vitro susceptibility of Leishmania parasites was carried out on 93 isolates using macrophage models. Identification of these species was also performed by molecular methods including Nested-PCR and PCR-RFLP. The f indicated that total isolated were L. major. A significant association between the clinical outcome and the in vitro effective concentration 50% (EC 50 ) values was observed. Leishmania derived from patients with non-healing lesions had EC 50 values at least 3-fold higher than parasites isolated from lesions of healing patients. By molecular methods, patterns for both sensitive and resistant samples demonstrated restriction band which is related to L. major. The obtained findings in the present study demonstrated that MA-resistant L. major field isolates are now frequent in Iran. Such studies help to find strategies for rapidly diagnosing resistance in order to improve the clinical management of CL.