Construction Of Phage Antibody Library Specific For Pyrethroids And Preparation Of Genetically Engineered Antibody

Antibodies are the core reagent of immunoassay, and its preparation is the critical factor which determines the application of immunoassay in detection of pesticide residue. Therefore, seeking new antibody preparation methods is very important for immunological detection of pesticide residues.In this study, we used phage display and genetic engineering techniques to prepare single-chain antibody (scFv) of pyrethroids (PBA). The structure and antigen-specific reactions of this scFv were also analyzed. Firstly, total RNA was extracted from a hybridoma clone2G2E7secreting monoclonal antibodies against pyrethroids. Then cDNA was obtained by reverse transcription. Using degenerate primers and the cDNA as template, the heavy chain variable region fragments (VH), about350bp, and the light chain variable region fragments (VL), about325bp, were obtained using PCR method. Overlap extension PCR was employed to amplify the scFv fragment of pyrethroids, which was about750bp in length. The scFv fragment and pCANTAB5E were digested by Sfi I and Not I, and linked to form the recombinant phagemid. After transformation into E. coli TG1, the primary antibody library of pyrethroids with capacity of2.3×107and positive rate of100%was constructed. The antibody library were panned by the antigen and the pyrethroid-specific phage antibodies were effectively enriched after five rounds of panning. The positive rate of the phages that being panned5rounds reached95%. We obtained three scFv genes through random sequencing the5rounds panned clones. The three scFv genes were reamplified by PCR, digested by EcoR I and Xho I, and inserted into pET-26b vector. The recombinant plasmids pET26b-scFv were transformed into expression strain BL21for scFv antibodies preparation, The expression conditions were optimized and SDS-PAGE results indicated that after being induced by0.1mM IPTG for12h in a25℃,200rpm incubator, three scFvs were partly expressed in soluble forms, but most of them formed inclusion bodies in the E. coli. The activity of three PBA scFvs were initially examined using IcELISA and the results showed that PBA-scFv2exhibited specific activities with pyrethroid. The inhibition rate reached about20%under conditions of10μg/mL concentration of fenvalerate. Sequence analysis showed that PBA-scFv2protein had245amino acids including123amino acids of VH,107amino acids of VL and15amino acids of linker peptide. Using SOPMA software, we found that the secondary structure of PBA-scFv2contained21α-helix,109β-folds,23β-corner and92random coils. The space structure of PBA-scFv2was predicted using CPH models3.2Server and the complex formed between PBA-scFv2and fenvalerate was simulated using Discovery Studio2.5. The results demonstrated that antigen binding region in the antibody were composed of three parts:(1) Met106, Asp107, Tyr108and Trp109residues of VH-CDR3region;(2) Thr91and Ser92residues of VL-FR3;(3) Ala41, Ser42and Pro43residues of VL-CDR2.The development of the present study lays foundation for preparation of genetically engineered antibodies of pyrethroid pesticides and its application in ELISA detection method.