血管性血友病因子vWF73和vWF114片段的表达及其在ADAMTS13活性测定中的应用

目的 构建血管性血友病因子(vWF)A2区片段vWF73和vWF114的表达质粒,在大肠杆菌中表达两种谷胱甘肽S转移酶(GST)融合蛋白,并探讨两种蛋白作为底物在测定ADAMTS13活性中的应用价值.方法 应用PCR的方法扩增vWF A2区内vWF73和vWF114的相应DNA片段,分别克隆至GST融合表达载体pGEX-6P-1进行诱导表达,Ni-NTA琼脂糖柱纯化可溶性蛋白部分.以Western blot法榆测正常人血浆、血栓性血小板减少性紫癜(TTP)患者血浆和重组ADAMTS13(rADAMTS13)水解两种重组vWF A2区片段的情况,并以此为底物用抗GST和抗His两种抗体建立酶联免疫吸附试验(ELISA)法测定血浆ADAMqS13活性的新方法.结果 成功表达和纯化了ADAMTS13的两种可溶性小分子底物GST-vWF73-H和GST-vWF114-H,均能被rADAMTS13或正常人血浆有效地水解,而遗传性和特发性TTP患者血浆则无此作用.同时,以此为底物建立了测定ADAMTS13活性的ELISA方法.结论 采用原核表达系统成功得到了vWF A2区片段vWF73和vWF114两种GST融合蛋白,可作为底物用于ADAMTS13活性的测定.

Abstract：

Objective To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E.coli, and to explore their values in measuring ADAMTS13 activity as substrates. Methods The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1 , a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 ( rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay ( ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GSTvWF114-H were used to measure plasma ADAMTS13 activity as substrates. Results Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified,which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GSTvWF73-H and GST-vWF114-H as substrates. Conclusions Two GST fusion proteins in vWF A2 domain,vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

Expression of vWF73 and VWF114 fragments of von Willebrand factor A2 domain and their utilization in detecting ADAMTS13 activity

Objective To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E.coli, and to explore their values in measuring ADAMTS13 activity as substrates. Methods The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1 , a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 ( rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay ( ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GSTvWF114-H were used to measure plasma ADAMTS13 activity as substrates. Results Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified,which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GSTvWF73-H and GST-vWF114-H as substrates. Conclusions Two GST fusion proteins in vWF A2 domain,vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.