Engineering of a single-chain variable-fragment (scFv) antibody specific for the stolbur phytoplasma (mollicute) and its expression in Escherichia coli and tobacco plants

• Published in: Applied and Environmental Microbiology Vol. 64; no. 11; pp. 4566 - 4572
• Main Authors: Le Gall, F. (Institut National de la Recherche Agronomique, France.), Bove, J.M, Garnier, M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.11.1998
• Subjects: Agrobacterium tumefaciens; Agronomy. Soil science and plant productions; Bacteria; Biological and medical sciences; Biotechnology; DISEASE RESISTANCE; Ecology, environment; ESCHERICHIA COLI; FITOPLASMAS; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genetic engineering; GENETIC RESISTANCE; Genetic technics; Genetics and breeding of economic plants; IMMUNOGLOBULIN STRUCTURAL GENES; IMMUNOGLOBULINE; IMMUNOGLOBULINS; INMUNOGLOBULINA; Life Sciences; Methods. Procedures. Technologies; Microbiology; NICOTIANA TABACUM; Pest resistance; PHYTOPLASMAS; PHYTOPLASME; Plant Microbiology; Plant pathogens; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; RESISTANCE AUX MALADIES; RESISTANCE GENETIQUE; RESISTENCIA A LA ENFERMEDAD; RESISTENCIA GENETICA; Tobacco; Transgenic animals and transgenic plants; TRANSGENIC PLANTS; Varietal selection. Specialized plant breeding, plant breeding aims; Genetic transformation; Plant pathogen; Escherichia coli; Fv-Fragment; IgG1; Gene expression; Nicotiana tabacum; Antigen; Mollicutes; Resistance; Binding capacity; Gene; Dicotyledones; Angiospermae; Bacteria; Spermatophyta; Transgenic plant; Solanaceae; Recombinant protein; Molecular cloning; Enterobacteriaceae; Single chain antibody
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.11.4566-4572.1998

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in Escherichia coli. The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefaciens to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died