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Showing 2 results for Agroinfiltration
M. Farsi , M. Mirzaei , J. Zolala ,
Volume 9, Issue 4 (12-2018)
Abstract
Aims: The production of recombinant proteins in transgenic plants (molecular farming) is considered a functional aspect of genetic engineering. Unlike animal and bacterial cell-based production systems, proteins produced by plants are very safe and have low production costs due to the absence of common pathogens in humans and animals. The aim of this study was the transient expression of recombinant PARS II endonuclease enzyme using agroinfiltration in tobacco.
Materials and Methods: In this experimental study, the possibility of producing a recombinant form of PARS II endonuclease was investigated, using transient expression system via Agrobacterium. The pBI-Pars expression construct (based on the binary vector pBI121) containing the full sequence of the PARS II encoder, upstream kozak, and a downstream 8x-His tag sequence, was infiltrated into Nicotiana tabacum leaves with Agroinfiltration method. After 72 hours, the expression of PARS II gene in agroinfiltrated leave samples was confirmed through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and protein Dot-blot, using Anti-His antibody at the levels of mRNA and protein, respectively.
Findings: The accuracy of the constructed expression construct was confirmed, and the results of Dot-blot by Anti-His antibody confirmed the expression of the recombinant PARS II protein, while no recombinant protein expression was observed in agroinfiltrated control plants with pBI121 construct. Significant amounts of recombinant PARS II nucleases were produced in tobacco leaves.
Conclusion: Agroinfiltration is an effective and short-term method for mass production of pure recombinant PARS II nucleases in tobacco.
Volume 22, Issue 6 (11-2020)
Abstract
Exendin-4 is a human Glucagon-Like Peptide-1 (GLP-1) analogue, resistant to DiPeptidyl Peptidase (DPP), which activates the GLP-1 receptor, increases insulin secretion, and improves glycemic control. In this study, Exendin-4 (EX4) was fused to Cholera Toxin B subunit (CTB) and transiently expressed in tobacco leaves. The sequence of the Ex4 fused to CTB subunit gene, with BamHI and SacI restriction enzymes sites at the beginning of CTB and at the end of EX4 gene. After codon optimization, the sequence was synthesized and cloned in pUC57 plasmid. The recombinant vectors were transformed into Escherichia coli strain DH5α. The pUC57-CTB-EX4 construct was digested with BamHI and SacI restriction enzymes, cloned into pBI121 expression binary vector, and transferred into tobacco leaves through agroinfiltration. Transcription of the Ex4 fused to cholera toxin B subunit gene in leaves was confirmed by RT-PCR analysis. After agroinfiltration, the protein was extracted from treated leaves, and ELISA test was performed using anti-CTB antibody. The production of recombinant protein was approved by ELISA test in transformed leaves.
