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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。
The target human factor Ⅸ (hFⅨ) plays a very important role in human endogenous coagulation. The absolute or relative lack of hFIX expression results in hemophilia B. In this study, Pichia pastoris production of recombinant high activity mutant hFIX-R338A. Methods The recombinant plasmid pPICZaA-hFIX-R338A was secreted into yeast Pichia pastoris SMD1168 by electroporation. The monoclonal antibody recombinant plasmid was screened for its ability to resist G418 again. The recombinant plasmid was cultured and purified. After dilution test its coagulation activity. Results The recombinant SMD1168-hFIX-R338A obtained in this study reached a level of (90-120) mg / L and the specific activity was about 38.93 ± 5.1% of the wild-type hFⅨ clotting activity in human blood. 6.85 times higher. Conclusion Human coagulation factor Ⅸ highly active mutant R338A is a potential substitute for natural coagulation factors.