金黄色葡萄球菌肠毒素B的原核表达及纯化

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。

Prokaryotic expression and purification of staphylococcal enterotoxin B

Objective To express staphylococcal enterotoxin B(SEB)in prokaryotic cells and purify the expressed product.Methods SEB gene was amplified by PCR using synthetic whole SEB gene sequence as a template,and inserted into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-SEB was transformed in to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by salting out with ammonium sulphate,SP cation exchange chromatography and copper ion chelating chromatography then,after His Tag was digested with thrombin,further purified by copper ion chelating chromatography and DEAE anion exchange chromatography.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a-SEB was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 31 000,mainly existed in a soluble form,contained about 30% of total somatic protein and reached a purity of more than 90% after purification.Conclusion Recombinant prokaryotic expression vector for SEB gene was successfully constructed,and recombinant SEB was expressed in E.coli and purified,which laid a foundation of further study on the pathogenic mechanism and methods for control of SEB.