Efficient Selection\nof the 2,4-Dichlorophenoxyacetic\nAcid Nanobody Gene from the Phage Library Constructed with Sorted\nSpecific Cells and Expression in Plants to Confer Herbicide Resistance

Immunomodulation in biotechnological processes requires\nan adequate\nlevel of specific, high-affinity recombinant antibodies expressed\nin the affected cells. Here, we report a new strategy to obtain a\nsensitive nanobody against the 2,4-dichlorophenoxyacetic acid (2,4-D)\nherbicide. Unlike the previous methods that used total peripheral\nblood lymphocytes, specific peripheral lymphocytes stained with a\nfluorescein isothiocyanate-labeled 2,4-D coating antigen were isolated\nvia fluorescence-activated cell sorting and used for phage display\nlibrary construction. This strategy significantly reduced interference\nof anticarrier protein nanobody phages to small-molecule nanobody\ndevelopment and required only two cycles of panning to obtain the\ndesired positive clones. Nb4–11, one of the most sensitive\nphage clones, showed good sensitivity and specificity against 2,4-D.\nCompared with previously reported 2,4-D nanobodies, the sequence of\nNb4–11 exhibited completely different complementarity-determining\nregions (CDRs). The half-maximum inhibition concentration of the Nb4–11-based\nic-ELISA was 29.3 ± 1.9 ng/mL. The Nb4–11 gene was subsequently\ntransferred into Arabidopsis thaliana to confer herbicide resistance, and homozygous transgenic T3 lines\nwere obtained. Stable expression of the 2,4-D nanobody in the model\nplant was confirmed via PCR, ic-ELISA, and Western blot analysis.\nIn the dose-response bioassay, the transgenic T3 lines were resistant\nto 2 g of 2,4-D ai/ha. This work offers a new way to sort specific\nperipheral lymphocytes, efficiently develop nanobodies against small\nmolecules, and create a novel mechanism for herbicide resistance based\non the expression of nanobodies in plants.