凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究  

Construction,Expression,Purification and Characterization of a Thrombin activated Single chain Urokinase type Plasminogen Activator

作　　者：张彦斌[1] 徐长法[1] 

机构地区：[1]北京大学蛋白质工程及植物基因工程国家重点实验室

出　　处：《中国生物化学与分子生物学报》1999年第3期403-408,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：家863计划资助项目

摘　　要：用Ｋｕｎｋｅｌ突变法，将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变，并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中，与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右，与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活，但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明，激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明，ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块。The sequence encoding Pro 155 Lys 158 in scu PA cDNA gene was deleted by site directed mutagenesis.The mutant was expected to be activated by thrombin but not by plasmin in order to distinguish a forming thrombus from an established hemostatic plug and reduce the risk of haemorrhagic strokes and other bleeding episodes.The mutant cDNA(tscu PA)was cloned in pCM β neo expression vector and co transformed CHO/DHFR - cells with pCM dhfr.The stable expression cell line was selected.tscu PA was purified from CHO expression supernatant by Zn 2+ Sepharose affinity chromatography.The apparent molecular weight of tscu PA according to the reduced SDS PAGE analysis was 53 kD.As expected,tscu PA was activated by thrombin but not by plasmin.The activated tscu PA(ttcu PA)immigrated as two distinct bands of 33 kD and 20 kD by the reduced SDS PAGE analysis.The results of kinetic constants study showed that when S 2444 was lysed,the K m and k cat of ttcu PA were similar to those of high molecular weight urokinase.And tscu PA kept the fibrin affinity of scu PA. In vitro plasma clot lysis assays showed that when incorporated as part of a thrombin induced clot,tscu PA was able to lyse the clot,while scu PA was ineffective.These results suggested that tscu PA was a thrombin activated plasminogen activator that could slelectively lyse the thrombin rich clots and spare the established clot which was depleted in thrombin.

关 键 词：凝血酶 构建 表达 血栓选择性 纤溶酶原激活剂 

分 类 号：R973.2[医药卫生—药品]