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Synthesis and Production of Recombinant Human Interleukin-2 via a Cell-Free Expression System
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H Radi    , E Ferdosi-Shahandashti * , Gh Kardar |
| 2.Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R.Iran. , elaheh.ferdosi@yahoo.com |
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Abstract: (237 Views) |
Background and Objective: Cell-free protein expression system is a novel method for synthesizing recombinant proteins such as interleukin-2 (IL-2), which is of great importance in immune system responses in cancer and autoimmune patients. The aim of this study is to synthesize IL-2 using cell-free expression technique with high efficiency and lower cost.
Methods: In this experimental study, the bacterial host DH5α was used to amplify the IL-2 cDNA in the pcDNA3.1 expression vector. After culturing the eukaryotic CHO cell line and preparing the cell lysate, the cell cytoplasm containing protein expression factors along with the addition of essential substances required for amino acid expression was placed in the vicinity of the expression vector containing the IL-2 gene fragment. The negative control sample included all the materials of the tested sample without the presence of the expression vector containing the gene fragment. Both samples were incubated for 20 hours at 25 °C. The accuracy of the expression of recombinant human IL-2 was examined using SDS-PAGE and Dot Blot techniques with monoclonal antibodies against IL-2.
Findings: In the assessment of the expressed protein based on SDS-PAGE technique, a band corresponding to the IL-2 protein produced in the test sample measuring 15 kDa was detected, while no band was found in the vector-free cell lysate sample. In the dot blot technique using gold nanoparticles, the desired spot was obtained from the binding of IL-2 as an antigen to the monoclonal antibody specific to IL-2. Moreover, quantitative analysis of the expression level using Nanodrop was reported to be 17.8 µg/ml.
Conclusion: The results of the techniques showed that there is the possibility to synthesize and produce IL-2 using a cell-free expression method. |
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| Keywords: Recombinant Human Interleukin-2, Cell-Free Expression System, CHO Cell Line, Synthesis of Recombinant Human Interleukin-2. |
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Full-Text [PDF 363 kb]
(34 Downloads)
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Type of Study: Applicable |
Subject:
Genetics, Cell and Molecular Biology
Received: 2024/06/18 | Accepted: 2024/10/12 | Published: 2025/08/26
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