肠出血性大肠杆菌O157∶H7志贺毒素I型A亚基的表达与鉴定

Expression and identification of enterohemorrhagic Escherichia coli O157 ∶ H7Shiga toxin1A subunit

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 enterohemorrhagic Escherichia coil （EHEC） O157：H7 Shiga toxin 1 A subunit （SODA）. Methods StxlA encoded gene was amplified from EHEC O157：H7 genome by PCR, confirmed by sequencing and cloned into vector pET-22b（＋）. The recombinant plasmid pET-22b（＋）-SODA was transformed into E. coli BL21 （DE3） which was induced by IPTG to express the target protein. After purified by AKTATU-His affinity chromatography, the recombinant protein was identified by mass spectrometry. With the recombinant protein, BALB/c mice were immunized to develop the anti-sera and evaluate its specific reaction with the natural StxtA by Western blotting. Results The SOdA gene with a size of 945 bp was amplified and cloned into prokaryotic expression vector pET22b（＋） to form pET-22b（＋）-SOdA. The recombinant protein was effectively expressed in E. co//BL21 （DE3） and purified by 6 x His-based affinity chromatography. The mass spectrometry analysis showed that the target protein was StxlA. Western blotting demonstrated that its immunized sera could react specifically with the natural Stx]A. Conclusion The EHEC O157：H7 SOdA gene was successfully cloned and expressed, which laid a solid foundation for the following researches.