人组织型纤溶酶原激活剂（t－PA）突变体FrGGI的构建、表达及特性分析

为了获得半衰期延长，特异活性提高及具有ＰＡＬ－１抗性的新型ｔ－ＰＡ溶栓剂，利用基因重组及定位突变技术构建了ｔ－ＰＡ的Ｋ１、Ｋ２区糖基化位点消除，ＰＡＩ－１结合位点缺失，Ｆ与Ｅ区连接序列Ｈｉｓ４４～Ｓｅｒ５０置换为纤粘蛋白Ⅰ型Ｆ区间连接序列ＧｌｕＳｅｒＬｙｓＰｒｏＧｌｕＡｌａＧｌｕＧｌｕ的ｔ－ＰＡ组合突变体ＦｒＧＧＩ，并在中国仓鼠卵巢细胞中获得了高效表达。对表达产物的生物学特性分析表明，ＦｒＧＧＩ在大鼠血浆中的半衰期延长了１５倍，并获得了ＰＡＩ－１抗性，是一株很有希望的新型溶栓剂候选株。

Construction,Expression and Characterization of a Combination Mutant of Recombinant Tissue-type Plasminogen Activator(t-PA)

A recombinant tissue-type plasminogen activator mutant (designated as FrGGI)with mutations of deglycosylation of K1 and K2 domain,deletion of PAI-1 binding site(residues Lys 296 to Gly302) and substitution of residues His44 to Ser50 (the linking sequence between F and E domain) for Glu Ser Lys Pro Glu Ala Glu Glu (the linking sequence between finger 1 and finger 2 domains in fibronectin),was constructed by DNA recombinant and site-directed mutagenesis techniques. The mutant was expressed in Chinese Hamster Ovary (CHO) cells with expression level reached to 1 500 IU/10￣6 cells/24 h through coamplification of the mutant and DHFR cDNA. The biological analyses of the mutant product displayed that the clearance of it in rat plasma prolonged 15 fold,its specific fibrinolytic activity increased and its resistance to inhibition by PAI-1 significantly enhanced. This novel mutant was reasonable to be considered as a potent candidate for thrombolytic therapy.