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

Document Type : Original Research

Authors
S. Kokei
B. Bahramnejad
Department of Agronomy and Plant Breeding, University of Kurdistan, Sanandaj, Islamic Republic of Iran.
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.

Keywords

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Journal of Agricultural Science and Technology
Volume 22, Issue 6
November and December 2020
Pages 1603-1612