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SELECTION, CHARACTERIZATION, AND APPLICATION OF A MYCOTOXIN-SPECIFIC SINGLE DOMAIN ANTIBODY FRAGMENT.

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Title: SELECTION, CHARACTERIZATION, AND APPLICATION OF A MYCOTOXIN-SPECIFIC SINGLE DOMAIN ANTIBODY FRAGMENT.
Author: Doyle, Patrick
Department: Department of Environmental Biology
Program: Environmental Biology
Advisor: Hall, J. Christopher
Abstract: 15-Acetyl-deoxynivalenol (15-AcDON) is a low molecular weight (M.W. = 338 Da) sesquiterpenoid trichothecene mycotoxin associated with Fusarium Head Blight (FHB), a global disease of small grain cereals. Estimated economic losses from the most recent Canadian and US FHB epidemic during the mid 1990s range from $1.3 to $3.0 billion USD, depending on valuations assigned to reduced crop yield and mycotoxin contamination of food and feed. The accumulation of DON and related trichothecene mycotoxins within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. A property of 15-AcDON, and structurally-similar trichothecene mycotoxins that are intimately involved in FHB pathogenesis, is the propensity to inhibit peptidyl transferase activity and thus inhibit protein synthesis within infected host-plant cells. This effect, coupled with secondary phytotoxic effects, act as mechanisms of virulence for pathogenic Fusarium species. A single-domain variable heavy chain (VHH) recombinant antibody (rAb) fragment specific to 15-AcDON was isolated from a hyper-immunized phagemid library. The dominant clone (NAT-267) was expressed and purified as a VHH monomer with a competitive binding affinity of 1.24 μM. The gene encoding the NAT-267 camelid single-domain antibody fragment (VHH) was expressed within the methylotropic yeast ii Pichia pastoris. Mycotoxin-mediated cytotoxicity was assessed by continuous measurement of cellular growth over time. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON, which, in turn, was more toxic than 3-AcDON. Intracellular expression of this toxin-specific VHH „intrabody‟ within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·mL-1, making this the first report of VHH-based sequestration of a haptenic mycotoxin. Furthermore, a biochemical transformation of DON to 15-AcDON was documented, which explained significant attenuation of the efficacy of DON at 100 and 200 μg·mL-1. This “proof of concept” model suggests that in planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby serve as a novel tool to help limit Fusarium infection of commercial agricultural crops such as wheat and barley that are engineered to express this mycotoxin-specific VHH.
URI: http://hdl.handle.net/10214/2254
Date: 2009-11-24


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