【摘要】 目的 获得具有高纯度、高生物活性的重组人组织型纤溶酶原激活剂改构体(瑞替普酶,reteplase,r-PA)。方法 reteplase基因克隆到表达质粒pJZ-100中,转化Escherichia cli B121(DE3),在0.05 mmol·L-1异丙基硫代-β-D-半乳糖苷(IPTG)诱导下获得高效表达,表达产物通过脉冲稀释法复性,复性后的蛋白质用ETI-Sepharose 4B亲和色谱纯化。结果 表达产物占菌体总蛋白质的20％,脉冲稀释法复性后复性收率为28％,经纯化后纯度达96％以上,比活为580 000 IU·mg-1。结论 建立了reteplase表达、复性与纯化工艺,以此工艺可获得高活性高纯度的reteplase。更多还原

【Abstract】 OBJECTIVE To obtain the recombinant human tissue-type plasminogen activator mutant(reteplase,r-PA) with high purity and high specific activity. METHODS Reteplase sequence was cloned into pJZ-100 expression vector. Being induced by IPTG, reteplase was highly expressed in Escherichia coli BL21 (DE3). The activity of expressed protein was recovered by step-wise dilution in vitro. The reteplase was purified by affinity chromatography. RESULTS Expressed reteplase constituted more than 20% of total bacterial protein. The activity recovery by step-wise dilution was 28%. After purification,the purity of reteplase was above 96% and its specific activity was 580000 IU·mg-1 . CONCLUSION The method of reteplase expression, renaturation and purification was established. The process can be a scheme to obtain reteplase with high purity and the high activity from E. coli.更多还原