新型重组膜联蛋白B1的特征及其抗血液凝固活性的比较

目的 研究毕赤酵母重组膜联蛋白B1的特征变化．比较其和原核表达产物对血细胞凝集与血栓形成过程的影响。方法 对毕赤酵母重组膜联蛋白B1行SDS-PAGE．等电聚焦．脱磷酸反应．蛋白质N端氨基酸测序．白陶土部分凝血活酶时间(KPTT)测定及大鼠下腔静脉血栓形成观察。结果 毕赤酵母重组膜联蛋白B1显示两条电泳带．其中一条带的相对分子质量比原核表达产物大．已被磷酸化．等电点较理论值低．两条带的N端均携带了来自载体的4个氯基酸．毕赤酵母重组膜联蛋白B1和大肠杆菌谷胱甘肽转移酶融合膜联蛋白B1具有明显的抗凝及抑制血栓形成作用。结论 虽然毕赤酵母重组膜联蛋白B1的分子特征较原核产物有了明显变化．但其抗凝及抑制血栓功能基本未受影响。

Characteristics of A Noval Recombinant Annexin B1 in Pichia Pastoris and its Anti-Coagulation Activity

Objective To study characteristics of the recombinant annexin B1 in Pichia pastoris and its effect on anti-coagulation activity. Methods The characteristic of recombinant annexin B1 was assayed with SDS-PAGE, isoelectric focusing(IEF), dephosphorization, sequencing of amino-N-terminal, kaolin partial thromboplastin time(KPTT) and venous thrombosis in inferior vena cava of rats. Results The expression product of the recombinant annexin B1 in Pichia pastoris showed two bands: 40 kD band was larger than that of recombinant annexin B1 in E.coli, pI was lower than the theorial pI value of annexin B1 because of phosphorization. N-terminus of two bands contained 4 amino acids from the plasmid. KPTT and venous thrombosis in inferior vena cava of rats revealed that the recombinant annexin B1 in Pichia pastoris had significant anticoagulatant and antithrombotic effects corrected with glutathione transferase(GST) combinated annexin B1 in E.coli. Conclusion Although characteristics of the recombinant annexin B1 in Pichia pastoris varied from that of in E.coli, its anticoagulation and antithrombosis were sustained.