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首页> 外文期刊>Journal of Clinical Microbiology >Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology.
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Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology.

机译：志贺样毒素II的A和B亚单位与谷胱甘肽S-转移酶融合的表达及其在血清流行病学中的潜力。

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We used the plasmid vector pGEX-2T for the expression of recombinant subunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl-beta-D-thiogalactopyranoside (IPTG) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.
机译：我们使用质粒载体pGEX-2T来表达志贺样毒素II（SLT-II）的重组亚基。将编码SLT-IIA或SLT-IIB亚基的基因的5'末端与融合了谷胱甘肽S-转移酶的基因的3'末端遗传融合，该酶在此表达系统中充当载体。亚基基因是通过聚合酶链式反应合成的，具有适当的限制性酶切位点以允许框内向下游插入基因。得到的含有A和B亚基基因的质粒分别命名为pFG1和pFG2。用异丙基-β-D-硫代半乳糖吡喃糖苷（IPTG）诱导携带pFG1的大肠杆菌实验室菌株，仅产生少量的SLT-IIA融合蛋白。由于IPTG诱导对携带pFG2的细胞具有致死性，因此我们构建了重组质粒pFG4，该质粒包含slt-IIB的亚基因片段，但没有5'信号序列。使用该构建体，我们能够表达非常大量的33.5-kDa融合蛋白，该蛋白通过固定在谷胱甘肽上的亲和层析纯化，并在免疫印迹分析中用作抗原。抗天然SLT-II的兔血清以及对SLT-II具有高中和活性的所有12种血清样品均与从大肠杆菌pFG4表达系统纯化的SLT-IIB反应，而208种人血清样品中只有3种具有低中和活性中和效价，没有SLT-II中和能力的54个血清样品中没有反应。在大多数中和活性低的人血清样品中，与SLT-IIB融合蛋白的特异性反应失败，表明在这些情况下，除免疫球蛋白外的血清因子可能是中和活性的原因。可以建议将重组SLT-IIB作为抗原的免疫印迹测定法作为一种诊断可靠的方法用于诊断，以检测针对SLT-II的特定人血清抗体。

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