Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

Abstract

Background: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines espe­cially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression.Methods: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 μg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR–tansfected promastigotes. West­ern blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits.Results: The PTR1 protein was not expressed in pcDNA-rPTR– tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR– tansfected promastigotes.Conclusion: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Keywords

• Pteridine Reductase 1
• Antisense
• Leishmania
• Gene Regulation
• Inhibition