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J Police Med 2015, 4(2) Back to browse issues page
Cloning, Expression, Purification and Toxicity Assessment of Diphtheria Toxin- Interleukin 2 Fusion Protein
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Background: Immunotoxin is a fusion protein containing two distinct sections covalently bonded by specific linkers. It includes an immunological section with a diagnostic role to connect the immunotoxin to the specific receptors and a toxic section with a cytotoxic role that leads to the cell death. The aim of this study is development of a lymphoma cancer protein therapy method using a recombinant immunotoxin produced by connecting diphtheria toxin to human interleukin 2 (IL2). Materials and Methods: After the synthesis of the fusion protein gene sequence (IDZ: DT-IL2), subcloning was done in the pET expression system. The pET-IDZ plasmid was transformed to BL21 (DE3) bacteria and then was induced. The protein purification was accomplished by nickel affinity chromatography system and then the function of the produced recombinant fusion protein was evaluated on K-562 cancerous cell line by MTT biological assay. Results: After production of pET-IDZ plasmid, the expression of the recombinant fusion protein in BL21 (DE3) bacteria was validated using electrophoresis and Western Blot methods. The purification yield determined above 95% using densitometry method. Afterwards, K-562 cells were treated by different concentrations of the produced fusion protein. The results showed the proper function of the produced fusion protein. Also, IC50 % amount (immunotoxin concentration to remove 50% of the cells) was determined as 6×10-6 M. Conclusion: Immunotoxin or targeted toxin is a novel strategy for cancer patients. The first recombinant immunotoxin (named Ontak), in which diphtheria toxin had been connected to interleukin 2, was approved from United States Food and Drug Administration (FDA) in 1999. Considering the importance of this drug, recombinant fusion protein was attempted to be produced in this research. Experimental data confirmed effective protein function, but complementary studies will be required to be compared with Ontak.

Keywords: cloning, expression, purification immunotoxin, diphtheria, interleukin
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Article Type: Original Research | Subject: Police Medicine Related Technologies
Received: 2015/01/18 | Accepted: 2015/07/5 | Published: 2016/03/15
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Cloning, Expression, Purification and Toxicity Assessment of Diphtheria Toxin- Interleukin 2 Fusion Protein. J Police Med. 2015; 4 (2)
URL: http://jpmed.ir/article-1-238-en.html

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Volume 4, Issue 2 (9-2015) Back to browse issues page
نشریه علمی پژوهشی طب انتظامی Journal of Police Medicine
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