人vW因子A2区基因表达及其生物学活性的研究  

A2 Domain of Human Von Willebrand Factor Expressed in E.Coli and Its Biological Activity

作　　者：苏健[1] 白霞[1] 高维强[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所苏州215006

出　　处：《中国实验血液学杂志》2005年第6期1090-1093,共4页Journal of Experimental Hematology

基　　金：江苏省"135工程"重点学科(SK200206);血液病学开放课题基金(135XY0404);国家自然科学基金(30470732)资助

摘　　要：血管性血友病因子(vWF)在血管性血友病(vWD)以及血栓性血小板减少性紫癜(TTP)的发病中起着重要的作用,并受血浆vWF裂解蛋白酶(ADAMTS13)的调节。本研究表达vWF中的A2区蛋白,并研究ADAMTS13对其裂解活性。首先,应用基因重组技术在大肠杆菌中表达人vWFA2区蛋白,经纯化、复性获得重组蛋白(rvWFA2),并与正常人以及TTP患者血浆进行反应,分析rvWFA2蛋白的生物学活性。结果表明:重组表达载体pQE30vWFA2在大肠杆菌M15中得到高效表达,目的蛋白表达量占菌体总蛋白的42%,NiNTAagrose柱层析纯化后,其纯度为98%,经复性的rvWFA2可作为ADAMTS13良好的酶解底物,可被枸橼酸钠抗凝的正常人血浆切割,其切割程度随时间的延长而增加,而EDTA抗凝的正常人血浆以及TTP患者血浆无法切割此蛋白。结论:在大肠杆菌中高效表达的rvWFA2具有良好的生物学活性,为建立定量测定ADAMTS13活性的方法奠定了坚实的基础。Von Willebrand factor （vWF） is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease （vWD） and thrombotic thrombocytopenic purpure （TTP）. To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 × His tag （pQE-30）, the recombinant expression vector was transformed into E. coli （strain M15 ） and induced by IPTG. The recombinant fragment comprising residues 718 -905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.

关 键 词：血管性血友病因子 蛋白表达 蛋白酶 

分 类 号：R345[医药卫生—基础医学]