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利用定点突变及DNA重组技术 ,构建了在尿激酶原K区C 端的β 发夹区插入了精氨酸 甘氨酸 天冬氨酸 丝氨酸 (RGDS)片段的尿激酶原嵌合体基因 ,并利用昆虫杆状病毒表达系统通过感染Sf9细胞对嵌合体尿激酶原进行了高效表达 .用尿激酶原单抗亲和柱纯化表达产物 ,获得初步纯化的嵌合体蛋白 .对嵌合体蛋白进行血小板膜结合实验表明 ,此嵌合体具有依赖于钙离子的结合活化血小板膜的活性 .用生色底物Chromozym U测定嵌合体的酰胺解活性 ,结果显示 ,纤溶酶激活后的嵌合体比活为 62 0 0 0IU/mg,与文献报道的纤溶酶激活的尿激酶原比活 65 3 5 5IU/mg相近 .纤溶酶激活后的嵌合体激活纤溶酶原的反应符合米氏方程 ,其Km 值为 0 .97μmol/L ,与天然尿激酶的Km 值 1.64μmol/L相近 .嵌合体还显示了较强的体外抑制血小板聚集活性 .这些结果表明 ,此尿激酶原 RGDS嵌合体有可能成为一种新的双功能溶栓药物 .
By using site-directed mutagenesis and DNA recombination technology, we constructed a pro-urokinase chimeric gene with arginine glycine aspartic acid (RGDS) fragment inserted in the β hairpin region of the C-terminal of pro-urokinase K, The virus expression system efficiently expressed chimera urokinase by infecting Sf9 cells, and the purified product was purified by pro-Urokinase affinity column to obtain the initially purified chimeric protein.The platelet membrane binding assay on the chimeric protein showed that this The chimera has a calcium ion dependent binding activity to the activated platelet membrane.The amidolytic activity of the chimera was measured with a chromogenic substrate Chromozym U and the specific activity of the chimera after activation by plasmin was 62,000 IU / mg , Which was similar to the reported specific activity of fibrinolytic activated urokinase (65 35-5 IU / mg) in the literature.The response of chimera-activated plasminogen after plasmin activation was in accordance with the Mie equation with a Km value of 0.97 μmol / L, similar to the Km value of native urokinase of 1.64 μmol / L. Chimeras also showed a stronger in vitro inhibition of platelet aggregation activity.These results suggest that this pro-urokinase RGDS chimera may become a Bifunctional thrombolytic drugs.