Iranian Journal of Microbiology 2017. 9(5):305-311.

Preparation of transgenic Iranian lizard Leishmania‏ coding HIL-12
Tahereh Donyavi, Mojgan Bandehpour, Bahram Kazemi

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.


Keywords


hIL-12; Iranian lizard Leishmania; Cysteine Peptidase C; Leishmanization‏

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References


Antinori S, Schifanella L, Corbellino M. Leishmaniasis: new insights from an old and neglected disease. Eur J Clin Microbiol Infect Di 2012;31:109-118.

Olivier M, Badaro R, Medrano F, Moreno J. The pathogenesis of Leishmania/HIV co-infection: cellular and immunological mechanisms. Ann Trop Med Parasitol 2003;97 Suppl 1:79-98.

Dunning N. Leishmania vaccines: from leishmanization to the era of DNA technology. Bioscience Horizons 2009;2:73-82.

Jain K, Jain N. Vaccines for visceral leishmaniasis: A review. J Immunol Methods 2015;422:1-12.

Breton M, Tremblay MJ, Ouellette M, Papadopoulou B. Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis. Infect Immun 2005;73:6372-6382.

Novo SP, Leles D, Bianucci R, Araujo A. Leishmania tarentolae molecular signatures in a 300 hundred-years-old human Brazilian mummy. Parasit Vectors 2015;8:72.

Niimi T. Recombinant protein production in the eukaryotic protozoan parasite Leishmania tarentolae: a review. Methods Mol Biol 2012;824:307-315.

Shahbazi M, Zahedifard F, Taheri T, Taslimi Y, Jamshidi S, Shirian S, et al. Evaluation of live recombinant nonpathogenic Leishmania tarentolae expressing cysteine proteinase and A2 genes as a candidate vaccine against experimental canine visceral leishmaniasis. PloS one 2015;10(7):e0132794.

Murray HW, Hariprashad J. Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis. J Exp Med 1995;181:387-391.

Jalah R, Patel V, Kulkarni V, Rosati M, Alicea C, Ganneru B, et al. IL-12 DNA as molecular vaccine adjuvant increases the cytotoxic T cell responses and breadth of humoral immune responses in SIV DNA vaccinated macaques. Hum Vaccin Immunother 2012;8(11):1620-1629.

Kazemi B, Tahvidar-Bideroni GH, Hashemi Feshareki SR, Javadian E. Isolation a lizard Leishmania promastigote from its natural host in Iran. J Bio Sci 2004;4:620-623.

Klatt S, Konthur Z. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microb Cell Fact 2012;11:97.

Medina-Acosta E, Cross GA. Rapid isolation of DNA from trypanosomatid protozoa using a simple ‘mini-prep’ procedure. Mol Biochem Parasitol 1993;59:327-329.

Raymond F, Boisvert S, Roy G, Ritt J-F, Légaré D, Isnard A, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res 2012;40:1131-1147.

Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 1983;166:557-580.

Beverley SM, Clayton CE. Transfection of Leishmania and Trypanosoma brucei by Electroporation. Methods Mol Biol 1993:21:333-348.

Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin 2007; 45(2) :27-37.

Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827-845.

Singh M, Billeter MA. A recombinant measles virus expressing biologically active human interleukin-12. J Gen Virol 1999;80 (Pt 1):101-106.

Kwon T, Seo J, Kim J, Lee J, Jang Y, Yang M. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol Bioeng 2003 ;81(7):870-875.

Rahmati M, Khan AH, Razavi S, Rasaee MJ, Sadroddiny E. Cloning and expression of human bone morphogenetic protein-2 gene in Leishmania tarentolae. Biocatal Agric Biotechnol 2016;5: 192-203.

Yadegari Z, Bandehpour M, Kazemi B, Sharifi-Sarasiabi K. Expression of recombinant human amelogenin in Iranian lizard Leishmania and its biological function assay. Iran J Public Health 2015;44(7):987-996.

Basile G, Peticca M. Recombinant protein expression in Leishmania tarentolae. Mol Biotechnol 2009;43(3):273-278.


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