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Expression of anti‐tumor necrosis factor alpha (TNFα) single‐chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens
Author(s) -
Balaji Parthasarathy,
Satheeshkumar P. K.,
Venkataraman Krishnan,
Vijayalakshmi M. A.
Publication year - 2015
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1373
Subject(s) - agrobacterium , agrobacterium tumefaciens , biology , single chain variable fragment , tumor necrosis factor alpha , microbiology and biotechnology , genetically modified crops , expression vector , transformation (genetics) , immunogenicity , transgene , antibody , recombinant dna , biochemistry , gene , genetics , immunology
Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single‐chain variable fragment (scFv) can negate the limitations tagged with the anti‐TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti‐TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β‐Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.