天津医药

• 论著 •    

多拷贝水蛭素基因在毕赤酵母中的分泌型表达

马精彩1,曹晓娜2,熊蔚俐2,李敬华2,左爱军2,孙蓓3,梁东春4   

  1. 1. 天津医科大学代谢病医院
    2.
    3. 医科大学代谢病医院
    4. 天津医科大学内分泌研究所
  • 收稿日期:2009-11-16 修回日期:2009-12-03 出版日期:2010-11-15 发布日期:2010-11-15
  • 通讯作者: 马精彩

Construction of a multi-copy secretory exspresstion vector and hirudin expression in Pichia pastoris

  • Received:2009-11-16 Revised:2009-12-03 Published:2010-11-15 Online:2010-11-15

摘要： 摘要 目的：通过多拷贝克隆技术实现水蛭素（Hirudin）基因在巴斯德毕赤酵母中的高效分泌表达。方法：利用基因重组技术，从pPIC9-Hirudin中扩增α-facor-Hirudin插入到载体pAO815中，并构建pAO815-(α-Hirudin)n 多拷贝重组质粒，转化毕赤酵母GS115后进行诱导表达，并鉴定表达产物活性。结果：PCR证实成功构建了水蛭素多拷贝毕赤酵母表达载体pAO815-(α-Hirudin)n，经SDS-聚丙烯酰胺凝胶电泳证实能成功高效分泌重组水蛭素，活性测定表明表达的水蛭素有良好的抗凝血活性。结论：成功构建了分泌型水蛭素多拷贝质粒，并筛选出多拷贝稳定整合表达菌株，成功表达出具有抗凝血活性1600ATU/ml的重组水蛭素，为大规模表达纯化水蛭素蛋白及其临床应用奠定了基础。

关键词: 巴斯德毕赤酵母, 分泌表达, 水蛭素, 多拷贝

Abstract: Abstract Objective: To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris. Methods: The α-facor- Hirudin gene was amplified from pPIC9- Hirudin by PCR and subcloned into PAO815. and then construct multicopy recombinant plasmid pAO815-(α-Hirudin)n .The recombinant was transformed into P. pastoris strain GS115 for induction expression and then the activity of secreted products were identified. Result: A new multicopy vector pAO815-(α-Hirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently, confirmed respectively by PCR, SDS-PAGE. The products possess the activity of thrombin inhibitor. Conclusion: This result offers efficient P. pastoris strains for mass production of biologically active hirudin.

Key words: Pichia pastoris, Secrete expression, Hirudin, Multicopy