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首页> 外文期刊>Infection and immunity >Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins.
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Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins.

机译：通过组装来自I型和II型热不稳定肠毒素的A和B多肽来表征在大肠杆菌中产生的杂合毒素。

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The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-IIa and LT-IIb were cloned and tested for complementation in Escherichia coli. Each gene encoding an A polypeptide was cloned into pACYC184, and each gene encoding a B polypeptide was cloned into the compatible plasmid Bluescript KS+. In addition, operon fusions representing all combinations of A and B genes were constructed in Bluescript KS+. Extracts from strains of E. coli expressing each combination of A and B genes, either from compatible plasmids or from operon fusions, were tested for immunoreactive holotoxin by radioimmunoassays and for toxicity by Y1 adrenal cell assays. Biologically active holotoxin was detected in each case, but the toxicity of extracts containing the hybrid toxins was usually less than that of extracts containing the wild-type holotoxins. The ganglioside-binding activity of each holotoxin was tested, and in each case, the B polypeptide determined the ganglioside-binding specificity. The A and B polypeptides of the type II heat-labile enterotoxins were also shown to form holotoxin in vitro without exposure to denaturing conditions, in contrast to the polypeptides of the type I enterotoxins that failed to form holotoxin in vitro under comparable conditions. These findings suggest that type I and type II enterotoxins have conserved structural features that permit their A and B polypeptides to form hybrid holotoxins, although the B polypeptides of the type I and type II enterotoxins have very little amino acid sequence homology.
机译：克隆了编码I型肠毒素LTp-I和II型肠毒素LT-IIa和LT-IIb的单个A和B多肽的基因，并在大肠杆菌中测试其互补性。将每个编码A多肽的基因克隆到pACYC184中，然后将每个编码B多肽的基因克隆到兼容质粒Bluescript KS +中。此外，在Bluescript KS +中构建了代表A和B基因所有组合的操纵子融合体。通过放射免疫测定法测试了表达A和B基因每种组合的大肠杆菌菌株的提取物，无论是兼容质粒还是操纵子融合蛋白，均通过放射免疫测定法检测了免疫反应性全毒素，并通过Y1肾上腺细胞测定法检测了毒性。在每种情况下都检测到具有生物活性的全毒素，但是含有杂合毒素的提取物的毒性通常小于含有野生型全毒素的提取物。测试每种全毒素的神经节苷脂结合活性，并且在每种情况下，B多肽确定神经节苷脂结合特异性。与I类肠毒素的多肽在相当的条件下体外不能形成全毒素相比，II类热不稳定肠毒素的A和B多肽在体外也没有暴露于变性条件下会形成全毒素。这些发现表明，I型和II型肠毒素具有保守的结构特征，允许它们的A和B多肽形成杂合的全毒素，尽管I型和II型肠毒素的B多肽具有非常少的氨基酸序列同源性。

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