人凝血因子Ⅸ-R338A高活性突变体在毕赤酵母中的表达及活性测定  

Expression and activity assay on highly active mutant R338A of FⅨ in Pichia Pastoris

作　　者：戴永刚[1] 尹胜菊[1] 程凤凤[1] 程世亮 张艳丽[1] 李萌[1] 王园[1] 刘新风[1] 

机构地区：[1]山东省交通医院检验科,山东济南250031

出　　处：《中国输血杂志》2017年第3期276-278,共3页Chinese Journal of Blood Transfusion

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-R338A。方法首先构建pPICZaA-hFⅨ-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。结果通本研究获得的重组SMD1168-hFⅨ-R338A表达量达到(90-120)mg/L;比活力约为人血中野生型hFⅨ凝血活性的(38.93±5.1)%,比野生型的酵母重组hFⅨ高6.85倍。结论人凝血因子Ⅸ高活性突变体R338A是潜在的天然凝血因子替代品。Objective Human coagulation factor Ⅸ （hFⅨ） plays a very important role in intrinsic coagulation in humans.The absolute or relative lack of hFⅨ can lead to hemophilia B.In this study,recombinant and high activity mutant hF Ⅸ-R338A produced by Pichia pastoris was analyzed.Methods First,PPICZaA-hFⅨ-R338A yeast expression vector was constructed,and then was transformed into Pichia pastoris SMD1168.The resistance ability of the recombinant strain was screened by G418,and the expression product was obtained and purified.Results The production of recombinant SMD1168-hFⅨ-R338A achieved 90-120 mg/L in this study,which was approximately 38.93% of the natural hFⅨ coagulation activity,6.85 times higher than that of the wild-type yeast recombinant hFⅨ.Conclusion High activity mutant hFⅨR338A which is produced by Pichia pastoris exhibits potential to become replacement for natural hFⅨ.

关 键 词：人凝血因子Ⅸ高活性突变体 毕赤酵母 蛋白表达 

分 类 号：R457.11[医药卫生—治疗学]