人vWF-A1多肽的融合表达及生物学活性鉴定

Expression of von Willebrand Factor-A1 Fragment in E.coli and Identification of Its Biological Activities

血管性血友病因子 (vWF)通过与血小板膜糖蛋白结合介导血小板的粘附和聚集 ,在血栓形成过程中发挥重要作用 .通过阻断血小板与vWF的结合可抑制血栓形成 .应用RT PCR方法从人脐带内皮细胞中克隆vWF A1区基因并在原核细胞内进行表达 ,经过纯化、复性 ,获得重组蛋白(rvWF A1) .用流式细胞术检测rvWF A1与转染了糖蛋白Ib(GPⅠb)的CHO K1细胞和血小板GPⅠb的结合能力 ,血小板聚集仪测定rvWF A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集作用的影响 .重组表达载体pET 2 0b(+ ) vWF A1在大肠杆菌BL2 1(DE3)plus中得到有效表达 ,表达的重组蛋白量占菌体总蛋白 30 % .次氮基三乙酸镍琼脂糖 (Ni NTAagarose)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 ,它可与转染了GPⅠb的CHO K1细胞和血小板结合 ,阳性率分别为 96 90 %与 78 6 0 % ,且可以抑制ristocetin诱导的血小板聚集 ,其抑制效应呈剂量依赖性 .IC50 的rvWF A1浓度为 0 5 6 μmol L ,当浓度为 1 4 μmol L时抑制率最高达 84 70 % .结果表明 ,在原核细胞中表达人rvWF A1区蛋白可抑制血浆中野生型vWF与血小板的结合 。

von Willebrand factor (vWF) mediates platelet adhesion through binding to platelet glycoprotein Ⅰb(GPⅠb). vWF domain A1 (vWF\|A1) was involved in binding GPⅠb, ristocetin or botrocetin. To further investigate the mechanism of thrombosis and develop a new remedy of anti thrombosis, reverse transcription PCR was used to amplify cDNA of vWF domain A1 from endothelial cells. After sequence analysis, the amplified DNA fragment was inserted into expression vector with 6×his Tag pET20b(+). The recombinant expression vector was transformed into E.coli (strain DE3) and induced by IPTG. The recombinant fragment, comprising residues 449—728 of mature vWF subunit, was designated rvWF A1. It was purified by chromatography on Ni NTA agarose column and renatured by Tris HCl buffer containing GSH and GSSG. FACS was adopted to analyse the rvWF A1 function of binding to platelets and Chinese hamster ovary cells transfected with pcDNA3.1 GP Ⅰb, meanwhile, platelet aggregometer was applied to detect its effect on the inhibition of ristocetin induced platelet aggregation. The rvWF A1 was expressed in E.coli , accounted for 30% of total bacterial protein. Its purity was over 95% after chromatography on Ni NTA agarose. It was proved to be able to bind GPⅠb. Its rates of binding platelets and the transfected CHO K1 cells were 78 60% and 96 90%, respectively. The activity to inhibit ristocetin induced platelet aggregation was dose dependent, its maximal inhibiting rate was 84 70% at 1 4 μmol/L. The results indicated that high level expression of rvWF A1 was achieved in E.coli and rvWF A1 might be an effective antithromotic agent in preventing thrombosis.