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Synthesis and Production of Recombinant Human Interleukin-2 through 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: (23 Views) |
Background and Objective: The cell-free protein expression system represents an innovative approach for synthesizing recombinant proteins like interleukin-2, which plays a crucial role in the immune system's responses in cancer and autoimmune patients. This study, concentrating on the synthesis of interleukin-2, aims to express that with enhanced efficiency and reduced cost.
Methods: In this experimental investigation, the cDNA of interleukin-2 was amplified within the expression vector pcDNA3.1 utilizing the bacterial host DH5α. Following the culture of the eukaryotic cell line CHO and the preparation of the cell lysate, the cell cytoplasm containing protein expression factors was mixed with essential materials necessary for expression, such as amino acids, alongside the expression vector harboring the interleukin-2 gene fragment. The negative control sample comprised all the components of the test sample excluding the expression vector containing the gene fragment. Both samples were incubated for 20 hours at 25°C. The precision of human recombinant IL-2 expression was evaluated using SDS-PAGE and Dot Blot methods with a monoclonal antibody targeting interleukin-2.
Findings: In verifying the accuracy of the expressed protein through the SDS-PAGE method, the band corresponding to the generated IL-2 protein was detected in the analyzed sample, measuring 15 kilodaltons, whereas no band was found in the cell lysate sample lacking the vector. In the dot blot method utilizing gold nanoparticles, the expected spot was achieved from the interaction of IL-2 as an antigen with the specific monoclonal antibody targeting IL-2. Furthermore, the quantitative assessment of the expression level using nanodrops was recorded as 17.8 μg/ml.
Conclusion: Upon examining the outcomes derived from the methods, the capacity to synthesize and generate IL-2 via cell-free expression was validated.
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| Keywords: Interleukin-2, Cell-Free System, CHO Cells, In Vitro Techniques, Recombinant Proteins. |
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Type of Study: Applicable |
Subject:
Genetics, Cell and Molecular Biology
Received: 2024/06/18 | Accepted: 2024/10/12
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