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目的原核表达并纯化金黄色葡萄球菌肠毒素B(staphylococcal entertoxin B,SEB)。方法以合成的SEB全基因序列为模板,PCR扩增SEB基因片段,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-SEB,转化E.coil BL21(DE3),IPTG诱导表达。表达的重组蛋白经硫酸铵盐析、SP阳离子层析柱、Chelating金属鳌合层析铜(Cu2+)柱纯化后,用Thrombin切除组氨酸标签,再经Chelating金属鳌合层析铜(Cu2+)柱和DEAE阴离子层析柱纯化。结果重组原核表达质粒pET-28a-SEB经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为31 000,主要以可溶性形式表达,表达量约占菌体总蛋白的30%。最终纯化后的重组SEB蛋白纯度可达90%以上。结论成功构建了SEB基因重组原核表达质粒,在大肠杆菌中表达并纯化了重组SEB蛋白,为进一步研究其致病机理及防控方法奠定了基础。
Objective To express and purify staphylococcal entertoxin B (SEB) in prokaryotic cells. Methods The SEB gene fragment was amplified by polymerase chain reaction (PCR), and inserted into the prokaryotic expression vector pET-28a. The recombinant plasmid pET-28a-SEB was constructed and transformed into E.coil BL21 (DE3) for expression under IPTG. The expressed recombinant protein was purified by ammonium sulfate salting-out, SP cation chromatography and Chelating metal chelate chromatography (Cu2 +) column, then the histidine tag was excised by Thrombin, and the Cu2 + Column and DEAE anion chromatography purification. Results The recombinant plasmid pET-28a-SEB was verified by double enzyme digestion and sequencing. The recombinant protein was constructed with a molecular weight of about 31 000 and expressed mainly in soluble form, which accounted for about 30% of total bacterial proteins. The purified recombinant SEB protein can reach more than 90% purity. Conclusion The recombinant prokaryotic expression plasmid of SEB gene was successfully constructed and the recombinant SEB protein was expressed and purified in E. coli, which laid the foundation for further study on its pathogenesis and prevention and control.