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Designing an expression construct containing the disintegrin gene identified from the transcriptome of the scorpion Hemiscorpius lepturus and evaluation of its function in an in vitro model
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Sahar Abolghasemi   , Mahdi Behdani , Fatemeh Kazemi-Lomedasht *  |
| Laboratory of Venom and Biotherapeutics Molecules, Biotechnology Research center, Pasteur Institute of Iran, Tehran, Iran |
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Abstract: (410 Views) |
Background and Aim: Cell adhesion plays an important role in the invasion of cancer cells. Disintegrins are considered a suitable therapeutic tool for cancer. This study aims to clone and express the disintegrin gene identified from the transcriptome of Hemiscorpius lepturus scorpion venom in a bacterial host and to investigate its function in an in vitro model.
Methods: Initially, the first, second and third structures of disintegrin protein were investigated using bioinformatics tools. Then, the gene was synthesized and cloned into pET-22b vector. The resulting construct was transformed into E.coli BL-21 bacterial host. The protein expression was induced by 0.5mM isopropyl-beta-D-thiogalactopyranoside. The expression was checked and confirmed by Sodium dodecyl sulfate-polyacrylamide gel and western blot. The protein was purified using nickel affinity chromatography under denaturing conditions. Following purification, the protein was refolded by gradually removing urea through dialysis. After protein expression and purification, the inhibitory effect of disintegrin on the survival and migration of HUVEC cell lines was investigated using MTT and transwell assay, respectively.
Results: The results of structural analysis showed that the predicted disintegrin structure exhibits typical characteristics and conforms to expected structural patterns observed in disintegrin proteins. The highest level of protein expression was observed with Isopropyl-beta-D-thiogalactopyranoside concentration of 0.5mM and 5-hour incubation. The molecular weight of the expressed protein was approximately 12 kDa. The disintegrin inhibited the growth of HUVEC cells with an IC50 of 37 µg/ml after a 48-hour incubation period. Furthermore, the purified disintegrin inhibited the migration of 70% of HUVEC cells towards the vascular endothelia growth factor source, compared to the control group without disintegrin.
Conclusion: These findings demonstrate the potential of the disintegrin identified from the transcriptome of the scorpion Hemiscorpius lepturus to inhibit the growth and migration of HUVEC cells.
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| Keywords: Transcriptome, Disintegrin, Hemiscorpius lepturus scorpion. |
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Full-Text [PDF 969 kb]
(144 Downloads)
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Type of Study: Original Research |
Subject:
Neuroscience
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