Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

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. 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 micro g 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. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. 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.

[Construction of a novel recombinant vector carrying herpes simplex virus thimidine kinase and yeast cytosine deaminase suicide genes to manipulate leishmania genetically]

Leishmaniasis is a group of diseases with various clinical pictures, which is caused by Leishmania spp. This parasite causes disease in human and about one hundred animal species. The disease is widely distributed in Iran and also across the world. One of the best approaches in the development of vaccine against Leishmania is genetically modification of the parasite. Therefore, the aim of this study was to design a novel genetic construct in order to insert Herpes Simplex Virus thimidine kinase [HSV-tk] and Yeast cytosine deaminase [Yeast-cd] suicide genes into the Leishmania genome. In this work, at the first step, HSV-tk and Yeast-cd fragments along with a gene of Leishmani, alpha-tubuline were cloned into pBluescript vector and the arrangement of tk-aplha tub-cd was created. Subsequently, these fragments were cut off from the plasmid and sub cloned into the plasmid pF4X1.4.4sat. The final construct was confirmed by digestion with relevant restriction enzymes. The fragments tk-alpha tub-cd was cloned successfully in the plasmid pBluescript and its authenticity was confirmed. Subsequently, this genetic collection was inserted into the plasmid pF4X1.4sat and finally, the orientation of it was checked. The construct designed in this study was able to insert two cellular suicide genes HSV-tk and Yeast-cd into the Leishmanai genome. Thus, this is an approach in achievement of vaccine against Leishmanai in the coming up researches