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Construction and Characterization of Human Embryonic Kidney-(Hek)-293T Cell Overexpressing Truncated Α4 Integrin Publisher



Fatahi A1 ; Rahimmanesh I2 ; Mirian M3 ; Rohani F4 ; Boshtam M5 ; Gheibi A6 ; Shariati L5, 7 ; Khanahmad H2 ; Kouhpayeh S8
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Authors Affiliations
  1. 1. Department of Biology and Biochemistry, Payame Noor University, Taft, Yazd, Iran
  2. 2. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
  4. 4. Faculty of Veterinary Medicine, University of Shahrekord, Shahrekord, Iran
  5. 5. Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
  6. 6. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
  7. 7. Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
  8. 8. Isfahan Neurosciences Research Center, Alzahra Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

Source: Research in Pharmaceutical Sciences Published:2018


Abstract

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (ITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized ITGA-4 plasmid and subsequently screened for stable truncated ITGA-4 expressing cells. Chromosomal DNA of truncated ITGA-4-transfected cells was extracted and the presence of truncated ITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated ITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated ITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin. © 2018 Medknow Publications. All rights reserved.
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