Dual‐receptors traction‐based directional design of a novel anti‐idiotypic insecticidal antibody for simulating Bacillus thuringiensis cry toxin against Plutella xylostella

Abstract Background Bacillus thuringiensis (Bt) Cry toxins have led to problems such as drug resistance in target pests, thereby attracting considerable attention. β ‐type anti‐idiotypic antibody (anti‐Id) has been demonstrated to possess characteristics that mimic the structure and activity of antigens, providing a theoretical foundation for designing novel insecticidal genetically engineered antibodies (GEAbs) with similar insecticidal function to Cry toxins. In this study, we innovatively devised an ‘idiotype monoclonal antibody (imAb)&dual‐receptors’‐based screening strategy to rapidly isolate insecticidal simulants ( β ‐type anti‐I‐GEAbs) of the Cry1Ah toxin against Plutella xylostella ( P. xylostella ) from the constructed phage display single‐chain variable fragment (scFv) library. Results The β ‐type anti‐I‐GEAb (3A5) was isolated from a Cry1Ah‐imAb F(ab)2 fragment immunized rabbit phage scFv library. Subsequently, a mutant (3A5‐m6) with binding activity increased 55.58%↑, was captured from the mutagenesis library targeted at the V L /V H ‐CDR1/CDR3 regions of the 3A5‐parent. The lethal virulence of the 3A5‐m6 against P. xylostella increased by 29.60% compared with that of the 3A5‐parent. The lethal virulence of the 3A5‐m6 and 3A5‐parent reached 48.55% and 37.46% of that of the Cry1Ah toxin against P. xylostella , respectively. The hotspot amino acids involved in the interactions between 3A5‐m6 and the P. xylostella midgut receptor Px‐ALP were located within V L ‐CDR1 (D31/Y32/N34). Regarding the Px‐CAD‐TBR, the hotspot amino acids were located within V L ‐CDR1 (D31/Y32), V H ‐CDR1 (Y158), and V H ‐CDR3 (Y226), respectively. Conclusion This study demonstrates an efficient directional‐design strategy for screening novel anti‐ idiotypic insecticidal antibodies that simulate CryAh toxin against P. xylostella , which broadens feasible paths for the green control of agricultural pests. © 2026 Society of Chemical Industry.