PURIFICATION AND CHARACTERIZATION OF TOXIN-A AND TOXIN-B OF CLOSTRIDIUM-DIFFICILE
PURIFICATION AND CHARACTERIZATION OF TOXIN-A AND TOXIN-B OF CLOSTRIDIUM-DIFFICILE
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DOI：
10.1128/iai.35.3.1032-1040.1982
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发表时间：
1982-01-01
影响因子：
3.1
通讯作者：
WILKINS, TD
中科院分区：
文献类型：
--
作者：
SULLIVAN, NM;PELLETT, S;WILKINS, TD
通过培养C.艰难梭菌VPI菌株10463在2升脑心输注透析瓶中于37 ℃培养。C下培养3天。纯化方案的初始步骤涉及通过XM-100膜过滤器进行超滤。两个毒性活动，指定毒素A和B，通过离子交换色谱DEAE-NaCl梯度分离。通过在pH 5.5下的乙酸沉淀将毒素A纯化至均匀。其他分离技术，包括CM Sepharose CL-6 B，（NH 4）2SO 4和乙酸沉淀，和疏水相互作用色谱，试图进一步纯化毒素B。虽然这些方法不能增加毒素B的比活性，但它们提供了额外的证据，证明这2种毒素是不同的分子。该毒素对酸和热不稳定，可被胰蛋白酶和胰凝乳蛋白酶灭活，但不被淀粉酶灭活。通过凝胶过滤和梯度聚丙烯酰胺电泳估计，毒素A的分子量范围为440，000 - 500，000。毒素B的估计分子量为360，000 - 470，000。
Toxin preparations were obtained by growing C. difficile VPI strain 10463 in 2 l brain heart infusion dialysis flasks at 37.degree. C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the 2 toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The MW of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000-500,000. The estimated MW of toxin B was 360,000-470,000.