Student Research Committee, Lorestan University of Medical Sciences, Khorramabad, Iran.
USERN Office, Lorestan University of Medical Sciences, Khorramabad, Iran.
Acta Parasitol. 2024 Jun;69(2):1107-1114. doi: 10.1007/s11686-024-00822-1. Epub 2024 Mar 27.
Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function.
In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH-hIFNγ-antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47 pg/mL.
Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH-hIFNγ-antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite.
The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania.
利什曼原虫是一种能引起人体多种临床疾病的细胞内鞭毛原生动物寄生虫。针对利什曼病的免疫抵抗基础取决于 Th1 反应，并在细胞因子功能的时间范围内。
在这项研究中，生产了人源抗白细胞介素 17（IL17）抗体和 IFNγ 产生的前鞭毛体，用于利什曼化。抗白细胞介素 17（IL17）抗体和人 IFNγ（hIFNγ）的轻链和重链基因序列从 NCBI 数据库中获得，并在质粒 pGH 的 ECORV 反应位点合成，称为 pGH-hIFNγ-antiIL17。使用限制酶 ECORV 从质粒中提取合成部分，通过电穿孔纯化后转移到伊朗蜥蜴利什曼原虫（I.L.L）中。在 DNA 和 mRNA 水平评估了在 I.L.L 基因组中的结构存在。使用 ELISA 和 Western blot 分析评估和确认 hIFNγ和抗 IL17 的表达。从培养基中收集 hIFNγ的分泌量高达 124.36±6.47 pg/mL。
首次使用 pGH-hIFNγ-antiIL17 质粒，在相同的替换过程中，成功地将靶向基因替换到 I.L.L 基因组中。稳定的重组 DNA 含有对寄生虫无毒的靶基因。
首次通过靶向基因替换，用质粒 pGH-hIFNγ-antiIL17 替换 I.L.L-CPC 基因，成功实现了重组基因的有效生产。该基因能够调节 hIFNγ和抗 IL17 的产生。这使其成为消除利什曼原虫的可行选择。
