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目的表达肠出血性大肠杆菌(EHEC)O157∶H7志贺毒素1型A亚基(Stx1A)重组蛋白并鉴定。方法从EHEC O157∶H7基因组中扩增编码Stx1A的基因,经测序无误后,克隆入表达载体pET-22b(+),转化大肠杆菌BL21(DE3),经诱导、纯化获得目的蛋白Stx1A,并对纯化的目的蛋白进行质谱鉴定。重组的Stx1A蛋白免疫BALB/c小鼠,观察小鼠抗血清与EHEC O157∶H7毒株特异性反应。结果 PCR扩增的Stx1A基因为945 bp,成功构建重组质粒pET22b(+)-Stx1a,重组蛋白在原核细胞中获得高效表达,通过AKTATM-His亲和层析柱获得纯化。质谱分析表明目的蛋白为Stx1A。Western blot显示鼠抗Stx1A血清可与EHEC O157∶H7毒株产生的天然毒素蛋白结合。结论成功克隆了EHEC O157∶H7 Stx1A基因,并获得重组表达,为后续研究奠定基础。
Objective To express and identify recombinant Enterotoxigenic Escherichia coli (EHEC) O157: H7 Shiga toxin type 1 A subunit (Stx1A). Methods The gene encoding Stx1A was amplified from the genome of EHEC O157:H7 and cloned into the expression vector pET-22b (+) after it was sequenced. The recombinant plasmid was transformed into E. coli BL21 (DE3). The target protein Stx1A was induced and purified The purified protein was identified by mass spectrometry. Recombinant Stx1A protein was used to immunize BALB / c mice and the mouse antiserum was observed to react specifically with the EHEC O157: H7 strain. Results The Stx1A gene amplified by PCR was 945 bp. The recombinant plasmid pET22b (+) - Stx1a was successfully constructed. The recombinant protein was highly expressed in prokaryotic cells and purified by AKTATM-His affinity chromatography. Mass spectrometry showed that the target protein was Stx1A. Western blot showed that the murine anti-Stx1A sera can bind to the native toxin protein produced by the EHEC O157: H7 strain. Conclusion The EHEC O157:H7 Stx1A gene was successfully cloned and the recombinant protein was expressed, which laid the foundation for further study.