Construction and characterization of a novel single-chain variable fragment against Bacillus thuringiensis vegetative insecticidal protein

Abstract

Bacillus thuringiensis (Bt) vegetative insecticidal protein 3Aa (Vip3Aa) is widely used in transgenic crops to control Lepidopteran pests. Reliable detection methods are required for food safety or environmental monitoring. In this study, a single-chain variable fragment (scFv) gene was first constructed from a hybridoma cell line raised against Vip3Aa and cloned into the pET26b(+) vector. The functional scFv was expressed in Escherichia coli BL21(DE3) with a yield of 1.3 mg/L. Molecular docking revealed that scFv binds to the Vip3Aa interface via epitopes in Domains III and V, with two salt bridges and six key hydrogen bonds observed in the complex. A sandwich ELISA was subsequently established using the purified scFv as the detection antibody and anti-Vip3Aa pAbs as the capture antibody. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.12 and 0.50 μg/mL, respectively, with a working range of 0.50-10.00 μg/mL. The sandwich ELISA had no cross-reactivity to other commonly used insecticidal proteins (Cry1Ac, Cry1Ab, Cry1F, and Cry2Ab) in genetically modified (GM) crops. This scFv shows potential for GM crops detection and could reduce costs compared to monoclonal antibody-based detection methods; the antibody-binding information obtained in this study may also guide future antibody design.

Keywords: Bacillus thuringiensis; Molecular docking; Sandwich ELISA; Vip3Aa; single-chain variable fragment.