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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 48.0 2 % ,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 .0 5或 <0 .0 1) ,其IC50 值分别为 4μmol/L、6 μmol/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 .
Objective To observe the inhibitory effect of recombinant GST-KGDX on platelet aggregation.Methods Two recombinant plasmids encoding KGDX (L-Gly-Asp-Asp-X) X), a 36 - base pair long terminus of BamHI and XcoI at both ends, was inserted into the downstream of tac promoter of prokaryotic expression vector PGEX - 4T - 1 to obtain Recombinant plasmid PGEX - 4T - 1KGDX was transformed into E.coli DH5a to induce recombinant expression at 37 ℃ .The purified protein was detected by glutathione - Sepharose beads affinity chromatography.The activity of GST - KGDX fusion protein was soluble in 35mg / L medium, the total amount of which was 48.0 2%, which was easily purified from the lysing bacteria.In vitro experiments: Both GST and GST-KGDX fusion proteins could inhibit ADP-induced human GST-GGDX was stronger than GST (p <0.05 or <0.01), and its IC50 values were 4μmol / L and 6μmol / L respectively.Conclusion (1) GST and GST- In vitro can inhibit ADP-induced human platelet aggregation. (2) in E. coli can express higher Amount of a fusion protein GST -KGDX, provides a good source of protein is an anti-platelet factor.