Increasing the expression of an anti-picloram scFv antibody in transgenic tobacco (Nicotiana tabacum)

Production of antibodies and antibody fragments in plants has great potential for conferring new traits to the host species, and for large-scale production of these valuable proteins. In our laboratory, a model for herbicide resistance has been developed by expressing an anti-picloram single-chain antibody variable fragment (scFv) antibody in transgenic tobacco (Nicotiana tabacum ) plants. This thesis describes research that resulted in increased levels of expression of the scFv in tobacco. To achieve this goal, four chimeric gene constructions were assembled with one of two promoters: tobacco constitutive promoter 3 (tCUP3) or a double-enhanced version of the cauliflower mosaic virus 35S promoter (CaMV35S); and one of two coding sequences: the original (O), that was of murine origin, and a modified (M) synthetic version. Both O and M sequences encoded identical polypeptides, but the M sequence was synthesized to mimic tobacco codon usage and eliminate potential problems in transcription and translation. Agrobacterium-mediated transformation was used to produce 25 transgenic plants for each of the four scFv gene constructs. The amount of active and total scFv in each plant was evaluated using quantitative enzyme-linked immunosorbent assay (ELISA) and immunoblot technologies, respectively. A picloram-resistance bioassay was used to confirm increased levels of active scFv expression among T1 offspring of the highest scFv-expressing plants. These plants with increased resistance levels are important for further development of this plant-base expression system as a bioremediation and/or herbicide resistant crop model.