Construction,Expression Of Single-chain Variable Fragment (scFv) Antibody For The Determination Of Bacillus Thuringiensis (Bt) Cry1Ab/Cry1Ac Toxin

Bacillus thuringiensis(Bt),a widely distributed Gram-positive pathogenic bacteria,produces crystalline proteins with specific insecticidal activity during sporulation.As the most widely used microbial pesticide in the world,Bt plays a significant and positive role in the field of agricultural pest control,food safety and biology fields,but the ecological safety risks and safety hazards to humans and other non-target organisms it may bring about draw more and more public attention.CrylAb and Cryl Ac proteins,which are the most commonly used Bt toxins in environmental health and agricultural production,have highly similar homologous sequences and three-dimensional structures and are easily recognized by the same antibody.Therefore,it is particularly urgent to establish a reliable detection method for Cryl Ab/CrylAc.The commonly used ELISA detection methods for toxin proteins expressed in transgenic Bt crops are indirect competition ELISA and double-antibody sandwich ELISA assay methods based on monoclonal antibody,polyclonal antibody and single-chain variable fragment(scFv).Although there are many detection kits for Cryl Ab/Cry1Ac toxins currently on the market,but they are often expensive.So it is necessary to research and establish an economical and effective detection method.In this study,hybridoma cells were used as the source of antibody variable region gene sequences to construct and express scFv with activity.The important factors affecting the binding properties between scFv and Cryl Ac were analyzed through homology modeling and molecular docking techniques.A DAS-ELISA method for CrylAb and Cryl Ac were established which lay the foundation for study of economic,convenient and rapid Bt toxin detection methods.Specific contents are as follows:1.Construction of single-chain variable fragment genesAfter verifying the biological activity of the hybridoma cell line 2D10,RNA was extracted and reverse transcribed.The heavy chain variable region(VH)and light chain variable region(VL)genes of the antibody were amplified using a degenerate primer using cDNA as a template.The single-chain variable fragment gene was amplified by overlap-extension PCR(SOE-PCR).Upstream and downstream primers were designed based on the sequences of the VH and VL genes,and the gene sequences of restriction endonuclease NcoI and NotI and complementary gene sequences of linkage peptide were added.The results showed that the VH and VL gene fragments were successfully spliced by peptide(Gly4Ser)3 gene sequence,and the restriction endonucleases NocⅠ and NotⅠ sites were successfully introduced,and the scFv gene was successfully constructed.2.Prokaryotic expression,purification and identification of scFvPrimers were designed to amplify the scFv gene and the prokaryotic expression vector pET26b-scFv was constructed with genetic engineering technology.After being verified by sequence alignment,the extracted plasmid was transfered into E.coli BL21(DE3),and the scFv protein was induced by IPTG.The expressed and purified scFv showed an immune response against the His-tag murine monoclonal antibody.The molecular mass of the protein was approximately 28 kDa,which was in agreement with the expected results.It will lay the foundation for the next step of establishing a specific detection method for CrylAb/Cry1Ac toxin based on the scFv.3.Establishment of double antibody sandwich ELISA and preliminary study on the scFv’s recognition difference for CrylAa and CrylAcThe optimal concentration of capture antibody and detection antibody was determined by checkerboard titration and a double-antibody sandwich ELISA was established.The important factors affecting the binding properties between scFv and Cryl A toxins were analyzed by homology modeling and molecular docking techniques.The established ELISA could detect Cryl Ab/Cry1Ac toxins with a detection limit of 20 ng/mL and a detection range of 0.45-6.0μg/mL.Cross-reaction results showed that scFv could specifically recognize CrylAb and CrylAc.Based on the source of the scFv material,the factors affecting the recognition of the Cry1Aa and Cry1Ac toxins by the scFv were initially analyzed by homology modeling and molecular docking techniques.The results showed that the CDR regions of the heavy and light chains of the scFv were mainly involved in the binding to the toxin and hydrogen and hydrophobic bonds play an important role during the process.