【摘要】 为赋予单链尿激酶型纤溶酶原激活剂 (singlechainurokinase typeplasminogenactivator,scu PA ,尿激酶原 )以抗血小板聚集的功能 ,在scu PA的kringle区 118位Gly与 119位Leu之间插入PRGDWR序列 (insertmutantB ,InB) .利用甲醇酵母 (Pichiapastoris)进行分泌表达 ,经金属离子螯合亲和层析与S强阳离子交换层析 ,得到纯蛋白 .实验测定InB对人工合成底物S 2 44 4的酰胺解活性为 5 90 0IU/mg ,动力学常数为 :KInBm ,S 2 4 44 =5 6 8μmol·L-1,kInBcat,S 2 4 44 =0 33s-1;水解天然底物 plasminogen的动力学常数为 :KInBm ,plg=0 397μmol·L-1,kInBcat,plg=0 0 16 4s-1.InB激活 plasminogen的反应在有fibrin存在条件下InB的活性为无fibrin条件下的 46 3% .该突变体在体外激活 plasminogen的活性与同一系统表达的野生型scu PA基本相同 .该突变体表现出较强的抗血小板聚集活性 ,IC50 =12 7μmol·L-1,而野生型scu PA无此功能 .实验表明scu PA的K区插入突变体InB是一种极具潜力的双功能溶栓分子 .更多还原

【Abstract】 In order to obtain the bifunctional chimeric molecule of single chain urokinase type plasminogen activator (scu PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly 118 and Leu 119 (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris . The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: K InB m,plg = 56 8 μmol·L -1 , k InB cat,plg =0 33 s -1 . The kinetic constants of plasminogen activation reaction is: K InB m,plg = 0 397 μmol·L -1 , k InB cat,plg =0 0164 s -1 . Fibrin inhibit the catalytiv ability of InB during plasminogen activation, the influence factor is 0 463(means InB remain 46 3% of the catalytic ability when fibrin was involved in the reaction system). The mutant not only has almost the same catalytic ability as wild type scu PA, but also has strong ability of anti platelet aggregation(compared with scu PA), IC 50 of InB is 12 7 μmol·L -1 .更多还原