Transient Expression of CTB-Exendin Fused Genes in Nicotiana tabacum via Agrobacterium tumefaciens

Exendin-۴ is a human Glucagon-Like Peptide-۱ (GLP-۱) analogue, resistant to DiPeptidyl Peptidase (DPP), which activates the GLP-۱ receptor, increases insulin secretion, and improves glycemic control. In this study, Exendin-۴ (EX۴) was fused to Cholera Toxin B subunit (CTB) and transiently expressed in tobacco leaves. The sequence of the Ex۴ fused to CTB subunit gene, with BamHI and SacI restriction enzymes sites at the beginning of CTB and at the end of EX۴ gene. After codon optimization, the sequence was synthesized and cloned in pUC۵۷ plasmid. The recombinant vectors were transformed into Escherichia coli strain DH۵α. The pUC۵۷-CTB-EX۴ construct was digested with BamHI and SacI restriction enzymes, cloned into pBI۱۲۱ expression binary vector, and transferred into tobacco leaves through agroinfiltration. Transcription of the Ex۴ 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.

کلیدواژه ها:

Agroinfiltration ، CTB-EX۴ gene ، Restriction enzymes ، Tobacco.