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

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.