Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone.

Applied and Environmental Microbiology
Q YuanL P Hart

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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Citations

Mar 21, 2009·Analytical and Bioanalytical Chemistry·Chris M Maragos
Aug 5, 2009·Biotechnology Progress·Hong WangYongsheng He
Apr 28, 2016·Bioscience, Biotechnology, and Biochemistry·Gorawit YusakulSatoshi Morimoto
Jan 11, 2007·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Tatsuya SuzukiHiroshi Ueda
Apr 28, 2005·Journal of Agricultural and Food Chemistry·Javier L UrracaJames J Pestka
Jun 10, 2021·Protein Science : a Publication of the Protein Society·Zhaoyong XiRieko Ishima
Feb 17, 2005·Journal of Agricultural and Food Chemistry·Björn LauerThomas Reinard
Oct 12, 2021·ACS Omega·Kuntalee RangnoiMontarop Yamabhai

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