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AIM:To establish the purification method for Clostridiumdifficile (C.difficile) toxin A.METHODS:C.difficile VPI 10463 filtrate was culturedanaerobically by the dialysis bag methods.And then thetoxin A was purified by precipitation with 500 g/L (NH_4)_2SO_4and acid precipitation at pH 5.5,followed by ion-exchangechromatography on DEAE-Toyopearl.RESULTS:Purified toxin A exhibited only one band on nativepolyacrylamide gel electrophoresis (native-PAGE) andOuchterlony double immunodiffusion.The molecular weightof toxin A was estimated to be 550 000.The purified toxinA had a protein concentration of 0.881 mg/mL.The minimumlethal dose was 1×10~6 MLD/mL (i.p.mice).The cytotoxictiter was 10~7 CU/mg.The haemagglutinate activity was ata concentration of 1.72 μg/mL.The ratio of fluid volume (mL)accumulated to the length (cm) of the loop was 2.46.CONCLUSION:The modified method for purification oftoxin A of C.difficile was simple and convenient.It may beeven more suitable for purification of toxin A on large scales.
AIM: To establish the purification method for Clostridium difficile (C. difficile) toxin A. METHODS: C. difficile VPI 10463 filtrate was cultured in aerobase by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g / L (NH 4) 2 SO 4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl. RESULTS: Purified toxin A drawings only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxinA had a protein concentration of 0.881 mg / mL.The minimumlethal dose was 1 × 10 ~ 6 MLD / mL (ipmice). The cytotoxictiter was 10 ~ 7 CU / mg.The haemagglutinate activity was ata concentration of 1.72 μg / mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46. CONCLUSION: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be more suitable for purification of toxin A on large scales.