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Here, we firstly reported to generate panels of anti-idiotypic singlechain variable fragments (scFvs) of Bacillus thuringiensis Cry2Aa toxin by chain shuffling method. Light-and heavy-chain-shuffled libraries based on two parental clones were constructed. The signal of the light-chain-shuffled library was 4.2-fold higher than the heavy one. Solely after one round panning, eight mutants were rapidly isolated from the chain-shuffled libraries. The apparent affinity of the best clones was 2.65 × 10 6 M −1 , a 2.59-fold improvement over the parental clone. The binding abilities between anti-idiotypic scFvs with brush border membrane vesicle of Plutella xylostella larvae were also determined. This work demonstrated that chain shuffling could rapidly generate panels of antibodies with similar binding abilities but of the distinct idiotypic composition. The serials of mutants can be used to map the binding epitope of the receptors in target insects instead of Cry2Aa toxin.