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It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7–CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7–CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7–CR11 was constructed by homology modeling. Molecular docking results of CR7–CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7–CR11, while CR7–CR8 was the region of CR7–CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7–CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification.

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05μg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation

The cultivation of genetically modified crops (GMCs) has greatly increased worldwide. Given the fact that over 10 Bacillus thuringiensis Cry toxins have been applied in GMCs, there is a need to develop an efficient and economically affordable detection method that simultaneously screen such compounds with similar structures in crops and foodstuff. Here we described an approach using a site-directed mutagenesis that enhances the generic specificity of single chain variable fragment (scFv). After three rounds of panning against mixed antigen (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F) from the constructed mutagenesis library, one mutant showed greater ability to bind to a range of Cry toxins. The phage mutant, named D9, was cloned into pET26b expression vector, and then induced and purified. The mutant D9 was used to develop an indirect competitive immunoassay that sucessfully recognizes five targeted Cry1 toxins with a working range from 0.20 to 2.22 μg mL −1 . The recoveries of five Cry toxins from spiked rice samples ranged from 86.67% to 96.67%, with a coefficient of variation less than 8%. The results suggest that the immunoassay based on a scFv is a promising approach for detection of Cry toxins with a broad specificity.