• 中国科学论文统计源期刊
  • 中国科技核心期刊
  • 美国化学文摘(CA)来源期刊
  • 日本科学技术振兴机构数据库(JST)
临床输血与检验

临床输血与检验 ›› 2019, Vol. 21 ›› Issue (3): 305-307.DOI: 10.3969/j.issn.1671-2587.2019.03.022

• 临床检验 • 上一篇    下一篇

人凝血因子Ⅸ-V107A-R338A高活性突变体在毕赤酵母中的表达及活性测定

戴永刚   

  1. 250031 山东省立第三医院（山东省交通医院）
  • 收稿日期:2017-11-01 出版日期:2019-06-20 发布日期:2019-06-17
  • 作者简介:戴永刚（1985-）,男,山东济南人,主管技师,硕士,主要从事出凝血检验、分子生物学检验工作,（Tel）15562663553 （E-mail）dg730@163.com。

Expression and Activity Assay of High Activity Mutant V107A-R338A of FⅨ in Pichia Pastoris

DAI Yonggang   

  1. Shandong Jiaotong Hospital,Shandong,Jinan,250031
  • Received:2017-11-01 Online:2019-06-20 Published:2019-06-17

PDF

817

摘要： 目的 人凝血因子Ⅸ（hFⅨ）在人类内源性凝血过程中起着非常重要的作用。hFⅨ表达量绝对或相对不足会导致B型血友病。本研究用毕赤酵母生产重组高活性突变体hFⅨ-V107A-R338A。方法 首先构建pPICZaA-hFⅨ-V107A -R338A酵母分泌表达载体,其次将其用电转化入毕赤酵母SMD1168中,再次利用G418抗药性的能力筛选表达量高的单克隆重组菌株,培养并将表达产物纯化,稀释后检测其凝血活性。结果 通过本研究获得的重组SMD1168-hFⅨ-V107A-R338A表达量达到80-120 mg/L;活力约为人血中野生型hFⅨ凝血活性的（44.06±; 2.3）%,比野生型的酵母重组hFⅨ高7.75倍,比单突变R338A高13.17%。结论 人凝血因子Ⅸ高活性突变体V107A-R338A是潜在的天然凝血因子替代品。;

关键词: 人凝血因子Ⅸ, 高活性突变体, 毕赤酵母, 蛋白表达

Abstract: Objective Human coagulation factor Ⅸ （hFⅨ） plays a very important role in the human endogenous coagulation. Absolute or relative lack of hFⅨ can lead to hemophilia B. In our study,Pichia pastoris was used to produce recombinanted high activity mutant hFⅨ-V107A-R338A. Methods First,PPICZaA-hFⅨ-V107A-R338A yeast expression vector was be constructed,and then transformed into Pichia pastoris SMD1168.The resistance ability recombinant strain was screened by G418,and the expression product was obtained and purified. Results The recombinant SMD1168-hFⅨ-R338A production was obtained at 80-120 mg/L in this study,which was about（44.06±2.3）% of the natural hFⅨ coagulation activity,7.75 times higher than that of the wild-type yeast recombinant hFⅨ and better than hFⅨ R338Aby 13.17）.Conclusions High activity mutant hFⅨV107A -R338A producted by Pichia pastoris might be the succedaneum of natural hFⅨ

Key words: Human coagulation factor Ⅸ, high activity mutant, Pichia pastoris, Protein expression

中图分类号: