Polyclonal antibodies to glutathione S-transferase--verotoxin subunit a fusion proteins neutralize verotoxins

Clin Diagn Lab Immunol. 2002 May;9(3):687-92. doi: 10.1128/cdli.9.3.687-692.2002.

Abstract

The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% +/- 7.9% and 3.6% +/- 2.3%, respectively, and those of VT2 were 1.7% +/- 2.3% and 82.5% +/- 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bacterial / biosynthesis
  • Antibodies, Bacterial / genetics
  • Antibodies, Bacterial / immunology*
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / immunology*
  • Antigens, Bacterial / isolation & purification
  • Cloning, Molecular
  • Cytotoxicity Tests, Immunologic
  • Enzyme-Linked Immunosorbent Assay / methods
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Bacterial
  • Glutathione Transferase / genetics
  • Neutralization Tests
  • Polymerase Chain Reaction / methods
  • Rabbits
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification
  • Shiga Toxin 1 / genetics
  • Shiga Toxin 1 / immunology*
  • Shiga Toxin 1 / isolation & purification
  • Shiga Toxin 2 / genetics
  • Shiga Toxin 2 / immunology*
  • Shiga Toxin 2 / isolation & purification

Substances

  • Antibodies, Bacterial
  • Antigens, Bacterial
  • Recombinant Fusion Proteins
  • Shiga Toxin 1
  • Shiga Toxin 2
  • Glutathione Transferase