人尿激酶原突变体的构建及其特性的研究

由于人尿激酶原（pro-UK）存在凝血酶和纤溶酶原激活剂抑制剂（PAI-1）的作用位点，在生产和治疗过程中容易失去活性。采用PCR介导的定点突变技术对pro-UK基因进行了突变，构建了3种人pro-UK突变体， 分别将蛋白序列中的凝血酶作用位点Arg¹⁵⁶突变成His¹⁵⁶，构建成pro-UKM1；将PAI-1的作用位点Arg¹⁷⁸、Arg¹⁷⁹、Arg¹⁸¹突变为Lys¹⁷⁸、Lys¹⁷⁹、His¹⁸¹构建成pro-UKM2；同时将凝血酶和PAI-1的作用位点突变构建成pro-UKM3。分别在CHO细胞中获得稳定表达。并对3种突变体进行了SDS-PAGE纤维蛋白自显影和Western blot分析，证明3种细胞表达的pro-UKM与天然尿激酶原带型一致，绝大多数为单链。体外溶栓试验表明，pro-UKM1对凝血酶有抵抗性，pro-UKM2对PAI有抵抗性，pro-Ukm3能有效抵抗凝血酶和PAI的活性抑制。特别是pro-UKM3具有开发成新型溶血栓药物的潜力

The Construction and Characterization of Human Prourokinase Mutant

It is very easy for the pro-UK to lose it's biological activity because of the digestion of pro-UK by the thrombin or the inhibition of pro-UK by the PAI-I.So three pro-UK mutant (pro-UK) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg~ 156in pro-UK was mutated into His~ 156, and named as pro-UKM1; PAI binding sites Arg~ 178, Arg~ 179,Arg~ 181 were mutated into Lys~ 178, Lys~ 179, His~ 181, named as pro-UKM2; The mutant containing His~ 156, Lys~ 178, Lys~ 179, His~ 181 as pro-UKM3. Three mutants were expressed in CHO cells respectively and analyzed with SDS-PAGE fibrin plate assay and western blot. The results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK2 could resist against PAI inhibition; while pro-UK3 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK3 can be a new medicine of dissolving thrombus.