Construction And Prokaryotic Expression Of A Phage-Displayed SCFV Antibody Library Against Deoxynivalenol

Deoxynivalenol (DON) is the major mycotoxin produced by fusarium fungi in grains. With the occurrence of fusarium head blight (FHB), DON occurs worldwide, and turns to be one of the most important mycotoxins. Consumption of DON contaminated feeds by livestock and food by humans has been associated with a variety of adverse health effects including feed refusal, reduced weight gain, immunotoxic, embryo toxicity and cytotoxicity. Therefore, it is important to identification and quantification of DON in food and feeds. In this study, single-chain antibody against DON were screened out from phage antibody libraries, and prokaryotic expression was progressed, which would be the basic work for its immunoassay application and development.Test I Synthesis and characterization of complete antigen for DON. Introduction of succinct acid by succinic anhydride in DON’s three hydroxyl groups as connecting arm to bound to bovine serum albumin (BSA) by the earbodiimide activationa, to preparate immune antigen DON-HS-BSA. Coating Antigen DON-OVA was preparated by DON bound to ovalbumin (OVA) directly by CDI cativation. Both of the antigens were determine by ultraviolet absorption spectroscopy (UV) and DON detection kit.Test Ⅱ Preparation identification of hybridoma cells against DON. We immunized BALB/c mice with DON-HS-BSA, and fused Immune mouse spleen cells with mouse myeloma cells Sp2/0. DON specificity hybridoma cells were screened by indirect ELISA, and cloned by limiting dilution assay. The results showed that the titers of antiserum up to1:2000, one strains of hybridoma cell lines against DON were obtained after two rounds of subcloning, which secreting IgG2a and subtype of light chain is X.Test Ⅲ Constructing, screening and prokaryotic expression of phage-displayed scfv antibody library. A total RNA was extracted from DON hybridoma cell. The heavy chain and light chain variable region genes (VH and VL) were amplified by PCR, genes of single chain antibody ScFv were obtained by linkage of VH and VL with a flexible polypeptide (G1y4Ser)3, then amplified by PCR. The ScFv genes were assembled in vector pCANTAB5E after digested by enzyme Sfi I and Not I. Recombinant phagemid vector were transformed into E.coli TG1. Rescued by M13K07helper phage, phage display scFv library was constructed. After three times biopanning enrichment and screening with DON antigen, a DON specific phage ScFv antibody were selected successfully from the library, and the library had been enriched for144times, with titer up to7.7×107cfu/mL. Bacteria E.coli HB2151was infected by the positive phage, after cultivated under the condition of25℃and induce by IPTG meanwhile which’s concentration is1.0mmol/mL, soluble antibody with the molecular weight of approximately35kDa was obtained and detected by SDS-PAGE and western blot.