Expression of a single‐chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco

Artsaenko, Olga; Peisker, Martin; Nieden, Uta zur; Fiedler, Ulrike; Weiler, Elmar W.; Müntz, Klaus; Conrad, Udo

doi:10.1046/j.1365-313x.1995.08050745.x

Cited by 156 publications

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“…The monoclonal IgG1 antibody MAK33 [14] is directed against the way, plant cell metabolism can be investigated [7,8] and/or human creatine kinase-MM (CK-MM). A MAK33 scFv-encoding sequence was constructed by polymerase chain reaction (PCR) acspecifically modified to obtain or omit certain products.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…The resulting sion of functional scFv fragments in the cytoplasm [7,9], apophagemid was named pBAS2. plast [11], and ER [8,12] mating. Transformation of Nicotiana tabacum SR1 was done by leaf 14900×g for 10 min at 4°C and the supernatant was centrifuged disc infection as described by [21] and transgenic GV-AS and GVagain for 10 min for young leaves or for 10 rain and 5 min for mature SSAS plants were selected on hygromycin-containing (50 gg/ml) medleaves.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

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Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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A gene encoding a single-chain variable (scFv)signal peptide sequence was compared, lower or even no acantibody fragment was expressed as a cytoplasmic and cumulation of the cytosol-targeted protein was observed, both endoplasmic reficulum-targeted protein in transgenic tobacco in leaves [11,12] and in seeds [13]. This suggests a lower staplants. In both cases, the scFv accumulated up to 0.01% of total bility of scFv proteins in the cytoplasm. Yet, intracellular soluble protein (TSP). The same scFv fragment was also expression of an anti-artichoke mottled crinkle virus scFv in produced in the periplasm of Escherichia coll. Measurement of Nicotiana benthamiana generated plants with scFv accumulathe affinity by ELISA indicates that the affinity of the tion levels up to 0.1% of TSP, which resulted in reduced inbacterially made scFv is about 80-fold lower than that of the fection incidence and delayed symptom development [9]. parental Fab fragment. The results suggest that the affinity of the However, few data are available on the efficiency of antigenplant-produced scFv fragments is reduced to a similar extent, binding scFv fragment synthesis and on the affinity of the implying that all the plant-produced scFv fragments are antigen produced scFv fragments for the cognate antigen. binding.To investigate the properties of plant-produced scFv mole- The accumulation levels were determined and the antigenbinding properties of the plant-produced scFv fragments were compared to those of a bacterially produced scFv.

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Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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“…6C). Although some antibodies or antibody fragments that have been fused with plant signal sequences have been confirmed by immunogold electron microscopy as being secreted through the cell wall into intercellular spaces, despite limitations in cell wall porosity (8), other antibodies were retained in the endoplasmic reticulum compartment or in the cytoplasm (1,10,29). The construct without the signal sequence failed to generate any transgenic plants in this study.…”

Section: Discussionmentioning

confidence: 66%

“…When fed to animals, the compound causes hyperestrogenism with symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility (16,23). In the last 10 years, the expression of specific antibodies or antibody fragments in plants has attracted great interest (6,15,21,30,36,37), and it has shown some potential in improving plant resistance against pathogens (33) and in altering plant metabolic pathways (1,27). Recently, we developed a singlechain Fv (scFv) antibody with high affinity for zearalenone (38).…”

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Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic Arabidopsis Plants

Yuan

Pestka

et al. 2000

Appl Environ Microbiol

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The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S 2 . After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-␤-resorcylic acid lactone] is a mycotoxin produced by members of the genus Fusarium after infection of corn and small grains (14,24,25). When fed to animals, the compound causes hyperestrogenism with symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility (16,23). In the last 10 years, the expression of specific antibodies or antibody fragments in plants has attracted great interest (6,15,21,30,36,37), and it has shown some potential in improving plant resistance against pathogens (33) and in altering plant metabolic pathways (1,27). Recently, we developed a singlechain Fv (scFv) antibody with high affinity for zearalenone (38). To explore the possibility of using "plantibodies" to neutralize mycotoxin through passive immunization of animals in their feed, as a first step we used the newly cloned antizearalenone scFv DNA fragment to transform Arabidopsis plants. In this report, we demonstrate that expression of the antizearalenone scFv gene in transgenic Arabidopsis plants leads to the accumulation of a soluble scFv plantibody with high affinity for the mycotoxin zearalenone. MATERIALS AND METHODSGeneral. All chemicals and solvents were reagent grade or better. Chemicals were purchased from Sigma Chemical Company (St. Louis, Mo.) unless otherwise noted. All DNA manipulations, if not described, were carried out by standard procedures (28).Construction of scFv cloning vector. scFv, a single-chain fragment of the antibody variable region antigen-binding protein, is c...

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“…5) The expression of scFv against abscisic acid (ABA) expressed in the endoplasmic reticulum (ER) of tobacco induced a wilty phenotype of the transgenic plants, 6) and seed specific production of the scFv modulated the levels and the effect of ABA leads in a tissue-or time-specific manner.…”

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confidence: 99%

Expression of a Single-chain Antibody against Indole-3-acetic Acid inEscherichia coli

Mitani

Matsumoto

2004

Bioscience, Biotechnology, and Biochemistry

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A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.Key words: single chain variable fragment (scFv); indole-3-acetic acid (IAA); enzyme-linked immunosorbent assay (ELISA)

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Expression of a single‐chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco

Cited by 156 publications

References 0 publications

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic Arabidopsis Plants

Expression of a Single-chain Antibody against Indole-3-acetic Acid inEscherichia coli

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