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Transient expression of etanercept therapeutic protein in tobacco (Nicotiana tabacum L.)

Bidarigh fard, Amir, Dehghan Nayeri, Fatemeh, Habibi Anbuhi, Mahdi
International journal of biological macromolecules 2019 v.130 pp. 483-490
Agrobacterium radiobacter, Nicotiana tabacum, antibodies, autoimmune diseases, drugs, enzyme-linked immunosorbent assay, genes, humans, immunoglobulin G, leaves, molecular cloning, patients, plasmids, polymerase chain reaction, protein secretion, recombinant fusion proteins, signal peptide, tobacco
Etanercept is a recombinant fusion protein of TNFR2 with the Fc portion of human IgG1. Etanercept, an anti-TNF drug, treats autoimmune diseases and improves patients' health. The main goal of the present study was to investigate the possibility of expressing recombinant protein of etanercept in a plant system. For this aim, first a modified version of pCAMBIA1305.1 plasmid with a new multiple cloning site and signal sequence of KDEL for protein secretion was constructed (pCAMBIA1305.1-linker). Then etanercept gene was cloned into the linker fragment of pCAMBIA1305.1-linker vector. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. To evaluate the transient expression of the gene, agroinfiltrated tobacco leaves were inoculated with Agrobacterium tumefaciens containing etanercept gene cassette. The recombinant etanercept protein was examined by dot blot and ELISA assays. Our results using anti-human IgG HRP-conjugated antibody confirmed a high level expression of etanercept gene in the tobacco leaves.