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The Antiplatelet Action of Glutathione S-transferase-KGDX Fusion Protein
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- Author:
- No author available
- Journal Title:
- JOURNAL OF MODERN CLINICAL MEDICAL BIOENG INEERING
- Issue:
- 4
- DOI:
- 10.3760/cma.j.issn.1674-1927.2001.04.003
- Key Word:
- 基因重组;表达及纯化;大肠杆菌;GPIIb/IIIa;拮抗剂;GST-KGDX融合蛋白;抗血小板作用
Abstract： 目的观察重组GST-KGDX(谷胱甘肽S-转移酶-赖-甘-天冬-X)融合蛋白对血小板聚集的抑制作用.方法设计合成了两条编码KGDX(赖-甘-天冬-X)的寡核苷酸链,长度为36个碱基对,两端分别为BamHI酶和XcoI酶切位点.将该基因插入原核高效表达载体PGEX-4T-1的tac启动子下游,获得重组质粒PGEX-4T-1KGDX,转化大肠杆菌DH5a,37℃诱导使重组子表达.采用谷胱甘肽-琼脂糖悬珠亲和层析法获得纯化蛋白.用血小板凝聚仪检测活性.结果 GST-KGDX融合蛋白具有可溶性,产量为35mg/升培养基,表达量占菌体总蛋白48.02%,容易从裂解菌中纯化得到.体外实验:GST、GST-KGDX融合蛋白均能抑制ADP诱导的人血小板聚集, GST-KGDX强于GST(p＜0.05或＜0.01),其IC50值分别为4μmol/L、6μmol/L.结论 (1)GST、GST-KGDX融合蛋白在体外均能抑制ADP诱导的人血小板聚集.(2)大肠杆菌中能够表达出较高产量的GST-KGDX融合蛋白,为抗血小板因子提供了一个很好的蛋白质来源.
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