\r吴祖泽^{1, 2}，汪 坤¹，李世崇²，董晓娜²，窦桂芳²，葛志强¹，靳继德²

AuthorsHTML:\r吴祖泽^{1, 2}，汪 坤¹，李世崇²，董晓娜²，窦桂芳²，葛志强¹，靳继德²

AuthorsListE:\rWu Zuze^{1, 2}，Wang Kun¹，Li Shichong ²，Dong Xiaona²，Dou Guifang²，Ge Zhiqiang¹，Jin Jide²


AuthorsHTMLE:\rWu Zuze^{1, 2}，Wang Kun¹，Li Shichong ²，Dong Xiaona²，Dou Guifang²，Ge Zhiqiang¹，Jin Jide²


Unit:\r1. 天津大学化工学院，天津 300072；
2. 军事科学院辐射医学研究所，北京 100850



Unit_EngLish:\r1． School of Chemical Engineering and Technology，Tianjin University，Tianjin 300072，China；
2．Institute of Radiation Medicine，Academy of Military Science，Beijing 100850，China


Abstract_Chinese:\r新蛭素(EH)具有很好的抗凝血效果，但由于半衰期短限制了其在临床上的推广和应用．为延长EH 的半衰期，制备人IgG-Fc 和EH 的融合蛋白，并对其活性和药代动力学进行初步研究．将合成的Fc-EH 和Fc-L-EH 融合基因克隆至表达载体pcDNAHC 上，再将重组表达载体转染CHO 细胞．蛋白A 亲和柱纯化融合蛋白，用SDSPAGE和Western Blot 鉴定蛋白表达情况，质谱法检测蛋白的分子质量和C 端序列，体外凝块法和大鼠颈静脉血栓模型分别验证融合蛋白的体外抗凝活性和体内抗栓效果，应用化学发光免疫法检测融合蛋白半衰期．结果表明，成功构建了pcDNAHC-Fc-EH 和pcDNAHC-Fc-L-EH 两个重组表达载体，融合蛋白Fc-EH 和Fc-L-EH 分子质量分别为68 476,u 和70 542,u，蛋白的C端序列正确，Fc-EH 和Fc-L-EH 活性分别为256,ATU/mg 和64,ATU/mg．大鼠颈静脉血栓实验表明，Fc-EH 具有抗血栓作用，体内消除半衰期为39.4,h．融合蛋白Fc-EH 能延长EH 体内半衰期，为融合蛋白的进一步研究奠定了基础．


Abstract_English:\rNeorudin(EH)has a good anticoagulant effect．However，the short half-life of EH limits its clinical popularization and application．To prolong the half-life of EH，fusion proteins of Fc segment of human IgG and EH were prepared and the anticoagulant activity and pharmacokinetics of the fusion proteins were studied．The fusion genes Fc-EH and Fc-L-EH were cloned into the expression vector pcDNAHC and expressed in CHO cells．The fusion proteins were purified by protein A affinity chromatography and identified by SDS-PAGE and Western Blot．The molecular mass and C-terminal amino acid sequences of the fusion proteins were detected using mass spectrometry．The anticoagulant activities of fusion proteins in vitro and in vivo were detected with clot methods and jugular vein thrombosis rat models respectively．The half-life of the fusion protein was tested in rats by chemiluminescence immunoassay．The results of restriction enzyme reaction indicated that the two recombinant expression vectors pcDNAHC-Fc-EH and pcDNAHC-Fc-L-EH were constructed，and the molecular masses of two targeting proteins were measured 68,476,u and 70,542,u，respectively．The correct C-terminal amino acid sequences of the fusion proteins were obtained．The anticoagulant activity of the fusion protein Fc-EH is 256 ATU/mg，whereas that of Fc-L-EH is 64 ATU/mg．The results of jugular vein thrombosis rat models indicated that the fusion proteins of Fc-EH dosedependently inhibited thrombosis．In vivo pharmacokinetic，studies showed that the elimination half-life of Fc-EH spanned 39.4,h．These results demonstrate that the Fc-EH fusion protein prolongs the half-life of EH，paving the way for the further studies of its anticoagulant effects.



Keyword_Chinese:水蛭素；新蛭素；抗凝活性；半衰期；Fc 融合蛋白


Keywords_English:hirudin(HV)；neorudin(EH)；anticoagulant activity；half-life；Fc fusion protein



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