Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone.
Abstract
The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses reve...Continue Reading
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