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.
Xu et al. (2026) studied this question.