Cloning of Mannose-1-Phosphate Guanyltransferase Encoding Gene (Mrho/Ir/Er/75) in Leishmania Major



Zia N¹ ; Eslami G² ; Bandehpour M³ ; Salehi R⁴ ; Kazemi B³ ; Parivar K⁵ ; Hejazi H⁶
Source: Journal of Isfahan Medical School Published:2009

Abstract

Background: Leishmania is an obligate interacellular protozoa and sand fly, as a vector, transmits infectious forms of the parasite to vertebrate host. In this way it is important to find candidate antigens which could tend to prevent the disease. Methods: The gene coding mannose 1 phosphate guanyl transferase enzyme was amplified from genomic DNA isolated from the Iranian strain of L. major (MRHO/IR/75/ER) as a template. The Polymerase Chain Reaction (PCR) product was ligated into the pTZ57R plasmid and the recombinant gene was digested using restriction enzymes, BamHI and EcoRI. The fragment was ligated into the pET32a plasmid, as an expression vector. The cloned pET32a-GDP mannose was confirmed using restriction enzyme digestion method and the DNA fragment was sequenced. Findings: Electrophoresis method confirmed the PCR product is related to the enzyme mannose 1 phosphate guanyl transferase. After the ligation of the product into the pTZ57R and pET32a, and the restriction enzyme digestion by BamHI and EcoRI, the correct frame of cloned gene in vectors was confirmed. Conclusion: There was 92 percent homology between the cloned gene coding enzyme mannose 1 phosphate guanyl transferase in this study and the ones present in gene bank. It is suggested that the gene encoding mannose 1 phosphate guanyl transferase enzyme is conserved among different genera of Leishmania. © 2009, Isfahan University of Medical Sciences(IUMS). All rights reserved.