Almani Pooya Ghasemi Nejad, Sharifi Iraj, Kazemi Bahram, Babaei Zahra, Bandehpour Mojgan, Salari Samira, Dezaki Ebrahim Saedi, Tohidi Farideh, Mohammadi Mohammad Ali
Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran.
Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; Leishmaniasis Research Centre, Kerman University of Medical Sciences, Kerman, Iran.
Acta Trop. 2016 Feb;154:63-72. doi: 10.1016/j.actatropica.2015.10.025. Epub 2015 Nov 10.
At present, there are no efficacious vaccines or effective drugs against leishmaniasis; therefore new and innovative control methods are urgently required. One way to achieve this important goal is through using reverse genetic engineering to evaluate important enzymes, proteins and macromolecules. One of the most important enzymes for Glycosylphosphatidylinositol (GPI) biosynthetic pathways is GlcNAc-PI-de-N-acetylase (GPI12). The molecular constructs were cloned in Escherichia coli strain Top 10 and confirmed by molecular methods and were transfected by electroporation into Leishmania major. We demonstrated that two alleles of the GPI12 gene in L. major were successfully removed and enabling the generation of a null mutant, which supports the idea that GPI12 is not an essential gene for the growth and survival of Leishmania and the homozygous knockouts of Leishmania are able to survive. We were able to produce a mutant parasite that caused no damaged to the host. Further investigations are essential to check the safety profile in laboratory animals.
目前，尚无针对利什曼病的有效疫苗或药物；因此，迫切需要新的创新性控制方法。实现这一重要目标的一种方法是利用反向基因工程来评估重要的酶、蛋白质和大分子。糖基磷脂酰肌醇（GPI）生物合成途径中最重要的酶之一是N-乙酰葡糖胺-磷脂酰肌醇脱N-乙酰酶（GPI12）。分子构建体在大肠杆菌Top 10菌株中克隆，并通过分子方法进行确认，然后通过电穿孔转染到硕大利什曼原虫中。我们证明，硕大利什曼原虫中GPI12基因的两个等位基因已成功去除，并能够产生一个无效突变体，这支持了GPI12不是利什曼原虫生长和存活所必需的基因以及利什曼原虫纯合敲除能够存活的观点。我们能够产生一种对宿主无损害的突变寄生虫。进一步的研究对于检查实验动物的安全性至关重要。
