半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重级及定位突变技术成功地构建了ｔ－ＰＡ的Ｋ１区缺失突变体ｔ－ＰＡｄｅｌＫ１、ＰＡＩ－１结合位点缺失突变体ｔ－ＰＡｄｅｌ（２９６－３０２）及两的组合突变全ｔ－ＰＡｄｅｌ（Ｋ１，２９６－３０２），并在ＣＯＳ－７细胞中实现三的暂时性表达，在ＣＨＯ细胞中实现了ｔ－ＰＡｄｅｌ（Ｋ１，２９６－３０２）的稳定性表达。对表达产物的生物学特性分析表明，ｔ－ＰＡｄｅｌ（２９６－３０２）及ｔ－ＰＡｄｅｌ（Ｋ１

Construction, Expression and Characterization of t-PA Mutants with Increased Plasma Half-life and Resistance to Inhibition by PA1-1

In this paper, three t-PA mutants, t-PA del K1 (with deletion of Kl domain), t-PA del (296 - 302) (with deletion of,PAI-1 binding site and their combination mutant t-PA del (K1, 296 - 302), were constructed by DNA recombination and site-directed mutagenesis techniques. Then the three t-PA mutants were transiently expressed in COS-7 cells, and the combination mutant t-PA del (K1296-302) was stably expressed in CHO cells. The biological analyses of the expression products demonstrated that t-PA del (296 - 302) and t-PA del (K1, 296 - 302) had obtained the resistance to inhibition by PAI-1. In addition , the half-life of t-PA del (K1, 296 - 302) in rat plasma was increased 5 times while the mutant affinity for fibrin was just a little affected. Therefore, it was reasonable to consider that the mutant t-PA del (K1, 296 - 302) may become a potent candidate of new thrombolytic agent.