Doering Christopher, Parker Ernest T, Healey John F, Craddock Heather N, Barrow Rachel T, Lollar Pete
Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA.
Thromb Haemost. 2002 Sep;88(3):450-8.
Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.
甲型血友病是一种遗传性出血性疾病，由凝血因子VIII（fVIII）突变引起。本文描述了一种产生重组小鼠fVIII的调控表达系统的构建。在无血清培养基中，小鼠无B结构域fVIII的表达峰值为4单位/10⁶细胞/24小时。随后，两步纯化程序实现了5300倍的富集，产率为70%。高度纯化的重组小鼠fVIII的比凝血活性为每纳摩尔660单位。它经凝血酶进行蛋白水解加工，产生一种活化的异源三聚体，其稳定性明显高于活化的人fVIII。重组小鼠fVIII被用于产生抗fVIII多克隆抗体。按照能产生高滴度人fVIII抑制性抗体的方案，将重组小鼠fVIII静脉注射到甲型血友病小鼠体内，未能诱导出显著的抗fVIII免疫反应。这可能为研究对fVIII的免疫耐受提供一个重要模型。
