Generation of recombinant antibodies specific for Sclerotinia sclerotiorum

摘要

Sclerotinia sclerotiorum causes disease on numerous economically important crops including oilseeds, vegetables and ornamentals worldwide. Breeding cultivars with durable resistance in oilseed crops is a major challenge in modem agriculture due to the scarcity of innate resistance germ plasm. Antibodies and their fusions have been reported to create fungus-resistant plants as a novel approach developed recently. Fungal antigens prepared from S. sclerotiorum were used for immunization of chickens.Polyclonal antibodies were purified from chicken egg yolks, and their reactivity and specificity towards the corresponding fungal antigens were evaluated under optimized conditions. Chicken antibodies were shown a high specificity and reactivity. RNA from immunized chickens were isolated and subjected to cDNA synthesis, which was used for construction of phage display libraries. Several rounds of panning were performed by phage display, resulting in selection of several single-chain antibodies with high affinity. The recombinant antibodies selected were expressed in bacterium upon induction with IPTG and purified by affinity-chromatography. Fungal specific antibodies were characterized by enzyme-linked immunosorbent assays and western blot analyses. To further explore the fungal specific antibodies for plant resistance improvement, the coding sequence of a characterized antibody was used for construction of fusion proteins to anti-fungal peptides. The antibody fusions were introduced into Arabidopsis thaliana plants. Transgenic Arabidopsis plants expressing the fusions will be evaluated for their resistance response to infection with S. sclerotiorum.