人vWF-A1区蛋白的表达及其对血小板聚集的抑制作用  

Expression of von Willebrand factor-A1 domain in E coli and it's the inhibitory effect on platelet aggregation

作　　者：祝怀平[1] 王迎春[2] 白霞[1] 季顺东[1] 张威[1] 邵波静[1] 朱明清[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,江苏苏州215006 [2]南通医学院附院,江苏南通217004

出　　处：《中国病理生理杂志》2004年第1期47-50,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目 (No .3 0 0 70 3 2 2 )

摘　　要：目的 :进一步研究血栓形成的机制 ,开发抗血栓药物。方法 :应用基因重组技术在大肠杆菌中表达人vWF -A1区蛋白 ,经过纯化、复性 ,获得重组蛋白 (rvWF -A1) ,同时用流式细胞术检测rvWF -A1与血小板膜糖蛋白血小板膜糖蛋白 (glycoprotein ,GP)Ib的结合能力 ,应用血小板聚集仪测定rvWF -A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集抑制作用。结果 :重组表达载体pQE -3 1-vWF -A1在大肠杆菌M15中得到高效表达 ,表达的重组蛋白量占菌体总蛋白的 3 0 % ,Ni -NTAagrose柱纯化后 ,其纯度为 95% ,经复性的rvWF -A1蛋白具有良好的生物学活性。它可与血小板模糖蛋白血小板膜糖蛋白GPIb结合 ,阳性率为 78 6% ;它可以抑制ristocetin诱导的血小板聚集 ,抑制率为 84 7%。结论 :在原核细胞中可以成功地高效表达人vWF -A1区蛋白 。AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6×his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

关 键 词：血栓形成 人vWF-A1区蛋白 基因重组 抗血栓药物 血小板聚集 抑制作用 血管性血友病因子 

分 类 号：R589.6[医药卫生—内分泌]