, Volume 14, Issue 5, pp 785-792

Transgenic Tobacco Plants Expressing a Dimeric Single-chain Variable Fragment (scFv) Antibody Against Salmonella enterica Serotype Paratyphi B

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Abstract

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T0) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 μg of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T1 plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T0 plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T1 generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.