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Plant Expression of Human Proinsulin Using the Plasmid Viral University of Tehran (Pvut) Vector



Kheirollahi M1, 2, 4, 5 ; Shahnejatbushehri AA1, 2, 4, 5 ; Kheirollahi M1, 2, 4, 5 ; Abooeimehrizi F1, 2, 4, 5 ; Alizade H1, 2, 4, 5
Authors
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Authors Affiliations
  1. 1. Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
  2. 2. Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
  3. 3. Pediatrics Inherited Diseases Research Center AND Research Institute for Primordial Prevention of Non-communicable Diseases AND Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  4. 4. Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
  5. 5. Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

Source: Journal of Isfahan Medical School Published:2015

Abstract

Background: Insulin is a hormone exclusively produced by pancreatic beta cells. The production of recombinant proteins in microorganisms has some disadvantages such as high cost, the possibility of contamination with toxic proteins, and costly purification steps; so, the production of recombinant proteins in plants can be investigated. Development of transient expression system based on a deleted version of Cowpea mosaic virus RNA-2, Cowpea Mosaic Virus-Hyper Translatable (CPMV-HT), has provided the extremely high-level and rapid production of proteins without viral replication. Methods: In this study, two constructions were prepared; pBI121-Proinsulin-Zera (pBI-ProZ) containing human proinsulin gene and Zera (N-terminal proline-rich domain of γ-zein) and pCAMBIA1304-Proinsulin-Extensin (pCAMBIA-ProE) containing CPMV-HT expression system for improvement of translation of human proinsulin gene and carrot extensin signal peptide. Both structures were transiently transferred in to lettuce and alfalfa leaves using Agrobacterium tumefasiens pv. C58. Statistical analysis of this study was conducted on the concentration of produced proinsulin per each gram of transgenic leaf using factorial arrangement of treatment in a complete randomized design. Gene expression was confirmed in transcription level using reverse transcription polymerase chain reaction (RT-PCR) method and in translation level using enzyme-linked immunosorbent assay (ELISA) and Dot blot assay. Findings: Protein accumulation for pCAMBIA-ProE and pBI-ProZ constructs were 6.82 and 4.32 ng/g in recombinant alfalfa leaves and 6.6 and 3.8 ng/g in recombinant lettuce leaves, respectively. Conclusion: Results showed that the expression of proinsulin in pCAMBIA-ProE contains CPMV-HT expression system was more than pBI-ProZ. © 2015, Isfahan University of Medical Sciences(IUMS). All rights reserved.
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