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双链尿激酶(tcu-PA)作为最早进入临床使用的溶栓药物,由于它对纤维蛋白(fibrin)没有选择性,大剂量静脉注射时会使血液中纤维蛋白原(Fibrinogen)降解,从而引发出血,因此在临床应用上受到限制.单链尿激酶(scu-PA,又称尿激酶原)是tuc-PA的前体,但它不同于一般的酶原,具有一定的内在纤溶酶原激活剂(PA)活性,而且能选择性地在Fibrin表面活化纤溶酶原,从而选择性地溶解血栓凝块.scu-PA经纤溶酶(Plasmin)的作用在Lys158-Ile159肽键断裂,转变成tcu-PA,由Cys148和Cys279间形成的一对二硫键将两条钛链连接、我们以前的工作证明尿激酶A链的Lys151和Arg154氨基酸残基是导致双链尿激酶对纤维蛋白低选择性的结构基础.本文通过定点突变手段构建了变体尿激酶原(Glu151-Gly154-rscu-PA)基因(以下简称mscu-PA),并在E.coli中获得表达、测定了表达产物的双链形式mtcu-PA及未突变的重组尿激酶原(rscu-PA)及其双链形式(rtcu-PA)对Fibrin的亲和性以及对血浆中纤维蛋白的选择性,结果证明所得变体双链尿激酶对Fibrin具有较高的选择性.
As the earliest clinical use of thrombolytic drugs, double-stranded urokinase (tcu-PA), due to its lack of selectivity for fibrin, can cause fibrin (Fibrinogen) in blood to degrade upon high dose intravenous injection Bleeding, and therefore limited in clinical application.Single chain urokinase (scu-PA, also known as pro-urokinase) is a precursor of tuc-PA, but it is different from the general zymogen, with some intrinsic plasminogen Activator (PA) activity, but also selective activation of plasminogen on the Fibrin surface, thereby selectively dissolving thrombotic clots.scu-PA plasmin under the action of Lys158-Ile159 peptide bond cleavage, Into tcu-PA, a pair of disulfide bonds formed between Cys148 and Cys279 link the two titanium chains. Our previous work demonstrated that the Lys151 and Arg154 amino acid residues of urokinase A chain are responsible for the effect of double-stranded urokinase on fibrin Low selective structural basis.In this paper, a variant urokinase (Glu151-Gly154-rscu-PA) gene (hereinafter referred to as mscu-PA) was constructed by site-directed mutagenesis and expressed in E.coli, and the expression product Double-stranded form of mtcu-PA and unmutated recombinant prourokinase (rscu-PA) and Double-stranded form (rtcu-PA) Fibrin affinity and selectivity for fibrin in plasma, urokinase results demonstrate duplexes resulting variant has a higher selectivity to Fibrin.