Transfer and Expression of Native Human Insulin-Like Growth Factor-1 in Tobacco Chloroplasts

Document Type : Research Paper

Author

Department of Biotechnology, Faculty of Agriculture, Azarbaijan Shahid Madani University, km 35 Tabriz-Maraqeh Road, Tabriz, Iran.

Abstract

Background: Insulin-like growth factor-1 (IGF-1), in addition to having insulin-like effects, has boosting effects on all cells in human body. Most of the recombinant IGF-1 required for patients suffering from its deficiencyis currently produced by bacterial and yeast systems. Plant systems, especially chloroplasts, have many benefitsfor producing human blood proteins. Production costs are low in these systems, and their side effects are less than other systems.
Objectives: In this study, the transfer and expression of mature IGF-1 protein cDNA in tobacco chloroplasts under the control of strong plastid transcription and translation elements was evaluated.
Materials and Methods: The biolistic transformation method was used to transfer the IGF-1 gene cloned into the pRB94-IGF1 chloroplast vector (1). Homoplasmic transplastomic plants were produced through four selection rounds on the selective medium. Transfer of foreign genes to chloroplast genome was confirmedby PCR, Southern blotting and seed progeny test. RT-PCR and SDS-PAGE methods were used to evaluate the expression of IGF-1 gene in transgenic line.
Results: A truly transformed line was identifiedfrom selected seedlings by PCR method. The seed progeny test of 4th-regeneration-round transgenic plants of this line showed maternal inheritance and homoplasmic level for the selectable marker gene, which confirmsthe transfer and expression of the marker gene in the chloroplast genome. The Southern blot test also confirmedthe transfer of the IGF-1 gene into the chloroplast genome. RT-PCR test showed that IGF-1 gene transcription is performed correctly in transgenic plants. Finally, accumulation of IGF-1 protein in transgenic plants was detected by SDS-PAGE.
Conclusions: Successful transfer and expression of the native human IGF-1 gene in tobacco chloroplast genome is reported.
 

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