ISOLATION AND PURIFICATION OF SUBUNIT B OF SHIGA TOXIN

志贺毒素是由单一酶组成的多聚体蛋白质。 亚基(A亚基，相对分子质量32 kDa)和对称排列的五聚体 B亚基(MW 7.7 kDa)。后者介导全毒素的结合。 以细胞表面糖脂作为其内吞作用的第一步。 Galabiose是识别毒素的最小结合受体， 末端或内部放置在糖脂中的。绑定是 较强的P血型决定簇三糖，如P~ 抗原和P1抗原。志贺(类志贺)的专用吸附剂 毒素可能有助于血清学试验的发展，以快速诊断 大肠杆菌感染和受体类似物低聚糖 抑制这些毒素与其细胞表面受体的结合可以 成为治疗此类疾病的候选药物。无毒的 亚基B可以作为合成亚基的载体 多糖-蛋白质结合物。根据报道的碳水化合物- 针对B亚基的特殊性，我们开发了一种简单有效的方法 对于该毒素片段的制备规模分离，使用 受体类似物亲和吸附剂。一种P1抗原性三糖，配备 在它的减速端有一个锚，共价连接到固体上 通过这个锚支撑。使用得到的亲和材料 用20个循环分离志贺毒素B亚单位 一株非致病性霍乱弧菌发酵液的研究 这种蛋白质的基因，以半自动的方式。由此产生的 B亚基共价结合于E。 Coli0-157及其结合物可诱导内毒素和抗毒素 小鼠体内的抗体。

The Shiga toxins are multimeric proteins consisting of a single enzymatic subunit (subunit A, mw 32 kDa) and a symmetrically arranged, pentameric subunit B (mw 7.7 kDa). The latter mediates the binding of the holotoxin to cell-surface glycolipids as the first step of its endocytosis. Galabiose is the minimum binding receptor that recognizes the toxin, placed either terminally or internally in glycolipids. The binding is stronger with P blood group determinant trisaccharides, such as the P~ antigen and the P1 antigen. Specific adsorbents of Shiga (Shiga-like) toxins may aid the development of serologic tests for rapid diagnosis of Escherichia coli infections, and receptor-analog oligosaccharides that inhibit the binding of these toxins to their cell-surface receptors can be candidates for therapeutics against such diseases. The non-toxic subunit B may be a candidate as a carrier in the synthesis of polysaccharide-protein conjugates. Based on the reported carbohydrate- specificities of subunit B, we developed a simple and efficient method for the preparative scale isolation of this toxin fragment, using a receptor-analog affinity sorbent. A P1 antigenic trisaccharide, equipped with an anchor at its reducing end, was covalently attached to a solid support through this anchor. The resulting affinity material was used over twenty cycles to isolate subunit B of Shiga toxin from the fermentation fluid of a non-virulet Vibrio cholera strain that containd the gene for this protein, in a semi-automated fashion. The resultant B-subunit was covalently bound to the 0-specific polysaccharide of E. coli 0-157 and the resultant conjugate shown to induce LPS and antitoxin antibodies in mice.