Preparation of transgenic Iranian lizard Leishmania‏ coding HIL-12

  • Tahereh Donyavi Departement of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti, University of Medical Sciences, Tehran, Iran; Vice Chancellor for Health, Iran University of Medical Sciences, Tehran, Iran
  • Mojgan Bandehpour Departement of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti, University of Medical Sciences, Tehran, Iran; Cellular and Molecular Biology Research Center, Shahid Beheshti, University of Medical Sciences, Tehran, Iran; Departement of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  • Bahram Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti, University of Medical Sciences, Tehran, Iran; Departement of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Keywords: hIL-12, Iranian lizard Leishmania, Cysteine Peptidase C, Leishmanization‏

Abstract

Background and Objectives: Leishmania are intracellular flagellate protozoan parasites cause a wide spectrum of clinical manifestations in human. The immunological basis for resistance against leishmaniasis depends on Thl responses in the course of performance of cytokines like IL-12. In this study, a transgenic Leishmania coding human IL-12 was produced that can be used in Leishmanization.Materials and Methods: A fragment of Iranian lizard Leishmania (I.L.L) gene, named Cysteine Peptidase C (CPC), was amplified separately as two parts with PCR reaction. Then, they were attached using SOEing PCR such that the restriction site of SalI was placed in the middle of it. SOEing PCR product was purified and cloned in HindIII restriction site of pGEM-7z-f and named pKDB-CPC. After clone optimization, the hIL-12 construct was cloned in SalI restriction site of pKDB-CPC and named pKDB-IL12. Prokaryotic section of the above construct was removed and transferred into I.L.L by electroporation.Results: Production of recombinant hIL-12 in transgene parasites was proved by ELISA. rhIL-12 secreted into supernatant culture medium accumulated at concentrations up to 246.53 ± 15.92 pg.mL-1.Conclusion: Targeted gene replacement into the I.L.L genome using plasmid pKDB-cpc identical replacement process was successfully completed for the first time. Stabilized recombinant DNA consist of target gene didn’t have any toxicity for the parasite. Transgenic I.L.L produced and secreted active human interleukin 12 and can be an appropriate candidate for Leishmanization.

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Published
2017-12-11
How to Cite
1.
Donyavi T, Bandehpour M, Kazemi B. Preparation of transgenic Iranian lizard Leishmania‏ coding HIL-12. IJM. 9(5):305-11.
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Original Article(s)