title: Antibodies against recombinant shiga toxin subunit B neutralize shiga toxin toxicity in HeLa cells.
abstract: Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin 1 (Stx) and Shiga toxin1 (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticaly-active A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti-StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.
authors.affiliation: Biotechnology Division, Defence Research & Development Establishment, Gwalior-474002, India.
authors.forename: Pallavi
authors.initials: P
authors.lastname: Gupta
authors.forename: Manglesh Kumar
authors.initials: MK
authors.lastname: Singh
authors.forename: Padma
authors.initials: P
authors.lastname: Singh
authors.forename: Mugdha
authors.initials: M
authors.lastname: Tiwari
authors.forename: Ram Kumar
authors.initials: RK
authors.lastname: Dhaked
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000494:pharmacology; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D022621:Shiga Toxin / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000906:Antibodies; D057134:Antibodies, Neutralizing; D007106:Immune Sera; D021122:Protein Subunits; D011994:Recombinant Proteins; D022621:Shiga Toxin