Document Type: Original Article
Biological Research Center, Faculty of Science, Imam Hossein University, Tehran, Iran
Human infection by Escherichia coli enterohemorhagic (EHEC) can lead to watery diarrhea, blood flow, or hemolytic uremic syndrome (HUS). This syndrome occurs in 5 to 10 % of patients with E. coli O157: H7 infection. Children under the age of 5 years old and the elderly and people with immune deficiency are the most prone to severe complications caused by this pathogen. The entry of this bacteria that has the ability to produce Stx-like toxin causes gastrointestinal symptoms including diarrhea and intestinal mucus. This toxin is a hexamer protein with a molecular weight of 70.5 kDa and is composed of A and B units. The purpose of this study is to purify the Shiga-like toxin, which can be used to provide a diagnostic kit, antibody production and vaccine studies. First, E. coli O157: H7 was confirmed by PCR technique and cultured in LB medium. After centrifugation, the cell wall of the bacteria was destroyed by a sonication. Since the toxin is secreted both in the medium and intra-cellular, to increase the concentration of toxin, the precipitate and supernatant were mixed together then the mixture was precipitated with ammonium sulfate salt, it was dialyzed against the salt in a PBS buffer. The presence of toxin was confirmed by SDS-PAGE and Western Blot techniques. In order to confirm the toxicity of protein, supernatant, lysed sediment and a mixture of both were injected into mice groups. In this experiment, the yield of toxin production was 650 μg/ml and the final purity was 90%. Our results demonstrate that Shiga-like toxin (Stx) can be purified without chromatographic methods whit an acceptable purity and yield.