Touraj Miandoabi1, Fariborz Bahrami2, Vahideh Moein Vaziri1, Soheila Ajdary2. 1. Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2. Pasteur Institute of Iran, Department of Immunology, 69 Pasteur Ave., Tehran, Iran.
Abstract
Background: Cutaneous leishmaniasis (CL) is a serious public health problem in many tropical countries. The infection is caused by a protozoan parasite of Leishmania genus transmitted by Phlebotominae sandflies. In the present study, we constructed a eukaryotic expression vector to produce a fusion protein containing LmSTI1 from Leishmania major (L. major) and PpSP42 from Phlebotomus papatasi (Ph. papatasi). In future studies we will test this construct as a DNA vaccine against zoonotic CL. Methods: The nucleotide sequences encoding the LmSTI1 protein and a fragment encoding 79% of PpSP42 were amplified using L. major and Ph. papatasi genomic DNA, respectively. The amplicons were cloned into the pcDNA3.1(+) eukaryotic expression vector. The recombinant plasmid pcDNA-LmSTI1Pp42 was propagated in Escherichia coli (E. coli) and used to transfect HEK-293T cells. The expressed fusion protein was analyzed by Western blotting using anti-LmSTI1 mouse serum. Results: Sequences encoding LmSTI1 and partial PpSP42 were cloned into pcDNA3.1(+). Production of the recombinant LmSTI1Pp42 fusion protein was confirmed by Western blotting. Conclusion: An LmSTI1Pp42 fusion protein was expressed HEK-293T cells. This construct may be an effective DNA vaccine against CL.
Background: Cutaneous leishmaniasis (CL) is a serious public health problem in many tropical countries. The infection is caused by a protozoan parasite of Leishmania genus transmitted by Phlebotominae sandflies. In the present study, we constructed a eukaryotic expression vector to produce a fusion protein containing LmSTI1 from Leishmania major (L. major) and PpSP42 from Phlebotomus papatasi (Ph. papatasi). In future studies we will test this construct as a DNA vaccine against zoonotic CL. Methods: The nucleotide sequences encoding the LmSTI1 protein and a fragment encoding 79% of PpSP42 were amplified using L. major and Ph. papatasi genomic DNA, respectively. The amplicons were cloned into the pcDNA3.1(+) eukaryotic expression vector. The recombinant plasmid pcDNA-LmSTI1Pp42 was propagated in Escherichia coli (E. coli) and used to transfect HEK-293T cells. The expressed fusion protein was analyzed by Western blotting using anti-LmSTI1 mouse serum. Results: Sequences encoding LmSTI1 and partial PpSP42 were cloned into pcDNA3.1(+). Production of the recombinant LmSTI1Pp42 fusion protein was confirmed by Western blotting. Conclusion: An LmSTI1Pp42 fusion protein was expressed HEK-293T cells. This construct may be an effective DNA vaccine against CL.
Entities:
Keywords:
Cloning; DNA vaccine; Leishmania major; LmSTI1; PpSP42
Authors: Clarissa Teixeira; Regis Gomes; Nicolas Collin; David Reynoso; Ryan Jochim; Fabiano Oliveira; Amy Seitz; Dia-Eldin Elnaiem; Arlene Caldas; Ana Paula de Souza; Cláudia I Brodskyn; Camila Indiani de Oliveira; Ivete Mendonca; Carlos H N Costa; Petr Volf; Aldina Barral; Shaden Kamhawi; Jesus G Valenzuela Journal: PLoS Negl Trop Dis Date: 2010-03-23