PURIFICATION AND CHARACTERIZATION OF AN ENTEROTOXIN FROM BACTEROIDES-FRAGILIS
PURIFICATION AND CHARACTERIZATION OF AN ENTEROTOXIN FROM BACTEROIDES-FRAGILIS
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DOI：
10.1128/iai.60.4.1343-1350.1992
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发表时间：
1992-04-01
影响因子：
3.1
通讯作者：
WILKINS, TD
中科院分区：
文献类型：
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作者：
VANTASSELL, RL;LYERLY, DM;WILKINS, TD
对一种由脆弱类杆菌产生的肠毒素进行了纯化，并对其生物活性和基本分子特性进行了表征。 通过生长B制备毒素制剂。将脑心浸液肉汤中的fragilis VPI 13784培养至早期稳定期，立即用70%硫酸铵沉淀培养上清液，并用蛋白酶抑制剂TPCK（甲苯磺酰基苯丙氨酰氯甲基酮）稳定沉淀。 毒素依次通过Q-Sepharose阴离子交换层析、苯基琼脂糖疏水作用层析和Mono Q高分辨率离子交换层析纯化。 通过聚丙烯酰胺凝胶电泳判断，毒素呈现均一性。 通过Superose-12上的凝胶过滤色谱法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的高度纯化毒素的估计分子量为19，000。 它的等电点约为4.5，在pH 5至10下稳定。 纯化的毒素在-20 ℃和4 ℃以及冷冻干燥时稳定，但在55 ℃以上的温度下不稳定。 它对蛋白酶K和链霉菌蛋白酶敏感，但对胰蛋白酶和糜蛋白酶有抗性。 纯化的毒素的活性被致突变株B的抗血清中和。fragilis，但不通过抗血清非致突变株。 N-末端氨基酸分析表明其序列为Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly-Ser-Thr。 高度纯化的毒素在羔羊回肠环试验中诱导强烈的液体蓄积反应，以及对HT-29结肠癌细胞的细胞毒性反应（细胞变圆）。 因此，纯化的毒素可引起肠毒性和细胞毒性活性。
An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4-degrees-C and upon freeze-drying, but it is unstable at temperatures above 55-degrees-C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly-Ser-Thr. The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.