Generation and Expression in Plants of a Single-Chain Variable Fragment Antibody Against the Immunodominant Membrane Protein of Candidatus Phytoplasma Aurantifolia

Abstract

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

Microbial approach of epitope tagged MFE-23 single fragment antibodies production

“…Detection is performed by evaluating the activity of conjugated enzyme following incubation process. 21 Use of ELISA is not limited to the full-length antibody detection. Moreover, this method can be used to identify single chain variable fragment antibody against immunodominant membrane protein (IMP) in plants.…”

“…Besides, detection of this antibody can be achieved by using simple ELISA technique which is sensitive and specific, relatively easy, low cost, and it has been extensively used in clinical setting. 21…”

Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

“…The final vector pTRAkc-thanatin was used for the transient expression of thanatin in the apoplast of tobacco leaves. The pTRAkc-thanatin vector was propagated in Escherichia coli K12 strain DH5a and transferred to Agrobacterium tumefaciens strain GV3101:pMP90RK by electroporation as previously described (Shahryari et al 2013).…”

Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis

“…ScFv can be produced using a wide range of platforms, including production in the periplasm of prokaryotes [12]; in the endoplasmic reticulum of eukaryotes [13]; and in cell-free expression systems [14]. Of these expression systems, Escherichia coli is a widely used host that is suitable for expressing small non-glycosylated recombinant antibody fragments, including scFv [15,16].…”