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首页> 外文期刊>Infection and immunity >Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins.
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Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins.

机译：构建针对产生热不稳定和热稳定肠毒素的大肠杆菌菌株的无毒融合肽。

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摘要

The 5' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LT-B). The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer. For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties. The LT-B-ST fusion peptide was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol. The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins. More importantly, the LT-B-ST fusion peptide was immunogenic. Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST.
机译：编码大肠杆菌（ST）热稳定肠毒素的基因的5'末端与融合了大肠杆菌（LT）的热不稳定肠毒素的结合亚基的基因的3'末端遗传融合-B）。用于这些研究的ST编码基因是通过适当的限制位点合成而构建的，以允许寡聚体在读框内向下游插入。对于这种构建，当在LT-B和ST部分之间包含含七个氨基酸的脯氨酸的连接子时，获得了ST抗原性的最大表达。 LT-B-ST融合肽通过亲和色谱法纯化，由十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳检查时的表观分子量为18,000的一条多肽链组成。当融合肽在SDS中或在二硫苏糖醇的SDS中煮沸时，没有多聚体形成的迹象，融合肽的迁移率也没有变化。 LT-B-ST融合肽是无毒的，LT和ST的免疫学决定因子均被天然毒素的抗体识别。更重要的是，LT-B-ST融合肽具有免疫原性。用含有杂种分子的粗制或纯化制剂免疫的动物产生的抗体能够在体外识别天然毒素。重要的是，这些抗体能够中和天然ST的生物活性。

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