纤溶活性重组纳豆激酶在大肠杆菌中的表达

Expression of Fibrinolytic Active Recombinant Nattokinase in Escherichia Coli

通过PCR方法扩增纳豆激酶成熟肽和纳豆激酶酶原(proNK)基因片段,并分别克隆到表达载体pET28a上,构建与His标签融合表达的重组表达质粒pET28a-NK和pET28a-proNK,转化大肠杆菌宿主菌BL21(DE3),IPTG诱导表达,SDS-PAGE电泳和纤维蛋白平板检测目的蛋白的表达及表达产物的纤溶活性。结果表明,纳豆激酶成熟肽基因在大肠杆菌中的表达产物以包涵体形式存在,表达产物没有纤溶活性;纳豆激酶酶原基因在大肠杆菌中的表达产物能自我剪切,形成有纤溶活性的纳豆激酶,但同时对宿主细胞有降解毒性。

With Genomic DNA of B.subtilis natto as the template,the gene of Nattokinase(NK)and pro-nattokinase(proNK)were amplified by PCR,and subsequent cloned into plasmid pET28a successfully by analyzing with restriction enzyme and PCR.The gene of NK and pro-NK were expressed in Escherichia coli BL21(DE3)by IPTG induction respectively.The expressed products were analysed by SDS-PAGE eletrophoresis and fibrinolytic activity assay.It showed that the expressed product of NK,which is accumulated as inclusion bodies,has no fibrinolytic activity on fibrin plate.While expressed product of pro-NK has fibrinolytic activity on fibrin plate and has toxic effect on host cell.