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. Summer 2012;7(3):111-6.
Epub 2012 Aug 25.

Cloning, Transformation and Expression of Human Interferon α2b Gene in Tobacco Plant (Nicotiana Tabacum Cv. Xanthi)

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Free PMC article

Cloning, Transformation and Expression of Human Interferon α2b Gene in Tobacco Plant (Nicotiana Tabacum Cv. Xanthi)

Shahrzad Ahangarzadeh et al. Jundishapur J Nat Pharm Prod. .
Free PMC article

Abstract

Background: Molecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments.

Objectives: This study reports the transformation of INF α2b gene in tobacco plant for the first time in Iran.

Materials and methods: Interferon α2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINFα. pCAMINFα was transferred to E. coli strain DH5α and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon α2b gene was confirmed by dot blotting.

Conclusions: Since no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran.

Keywords: Agrobacterium; DNA Transformation Competence; Interferons; Molecular Farming; Tobacco.

Figures

Figure 1
Figure 1. T-DNA Region of pCAMINFα-2b. LB and RB: Left and Right Borders, HYG(R): Hygromycin Selectable Marker, CaMV35s: Cauliflower Mosaic Virus Promoter, NcoI and BstEII: Restriction Sites and NOS: Nopaline Synthase Terminator.
Figure 2
Figure 2. Confirmation of Cloning of INFα-2b Gene in E. coli by Colony PCR Technique. M: GeneRuler™ 100 bp DNA Ladder (Fermentase), Lane1: E. coli Without pCAMBIA-INFα (Negative Control), Lane 2: Positive Control (pCAMIFNα), Lane 3-8: Random Colonies Selection.
Figure 3
Figure 3. Confimation of INFα-2b Gene in Agrobacterium by Colony PCR Technique. M: GeneRuler™ 100 bp DNA Ladder (Fermentase), Lane1: Agrobacterium Without pCAMBIA-INFα (Negative Control), Lane 2: Positive Control (pCAMIFNα), Lane 3-6: Random Colonies Selection.
Figure 4
Figure 4. Regeneration of Transgenic Tobacco (Nicotiana tabacum cultivar xanthi) Plants.
Leaves Were Inoculated by Agrobacterium tumefaciens, without pCAMINFα as Control (A) and With pCAMINFα (B) on MSIII Medium (MS II + 15 mg/lit Hygromycin + 200 mg/lit Cephatoxim). Regeneration and Rooting on MSIV Medium (MS III Without Hormone) (C), Transporting to Perlite (D).
Figure 5
Figure 5. PCR Analysis of Transgenic Plants. M: 1kb Marker, Lane 1: ddH2O, Lane2: Wild Type Plant, Lane 3: Transgenic Plant, Lane 4: Positive Control (pCAMIFNα).
Figure 6
Figure 6. Dot Blot Analysis of Protein Extraction of Tobacco. Lane 1: Protein Extraction of Negative Control Plants (Wild Type), Line 2: Protein Extraction of Transgenic Plant.

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