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

• Published in: Applied and Environmental Microbiology Vol. 63; no. 1; pp. 263 - 269
• Main Authors: Yuan, Q. (Michigan State University, East Lansing, MI.), Clarke, J.R, Zhou, H.R, Linz, J.E, Pestka, J.J, Hart, L.P
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-01-1997
• Subjects: ADN; Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cloning; Cloning, Molecular; Corn; DNA Primers - genetics; DNA, Complementary - genetics; ELISA; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Fusarium; Gene Expression; Genes, Immunoglobulin; Genetic engineering; Genetic technics; Genetics; Hybridomas; Methods. Procedures. Technologies; Mice; Modification of gene expression level; Molecular Sequence Data; Peptides; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; TEST ELISA; Zearalenone - immunology; Recombinant virus; Escherichia coli; Mycotoxin; Zearalenone; Fv-Fragment; Phage M13; Gene; Cross reaction; Bacteria; Recombinant protein; Molecular cloning; Fusion protein; Enterobacteriaceae; Single chain antibody; Nucleotide sequence; Antibody; Hybridoma; Corn; Gene expression; Virus; Cereal; Detection; Application; ELISA assay; Comparative study; Phage
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.63.1.263-269.1997

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-afflnity 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 revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene V(kappa)23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts