RGDS-尿激酶原嵌合体的构建与表达

Construction and Expression of RGDS Prourokinase

利用定点突变及ＤＮＡ重组技术，构建了在尿激酶原Ｋ区Ｃ端的β发夹区插入了精氨酸－甘氨酸－天冬氨酸－丝氨酸〔ＲＧＤＳ〕片段的尿激酶原嵌合体基因，并利用昆虫杆状病毒表达系统通过感染Ｓｆ９细胞对野生型及嵌合体尿激酶原进行了高效表达，表达量分别为１２００～１８００ＩＵ／（１０６细胞·ｍｌ）和１８００～２４００ＩＵ／（１０６细胞·ｍｌ）．经过ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ离子交换层析、ＳｅｐｈａｄｅｘＧ－７５凝胶过滤层析及超滤浓缩对野生型及突变型进行了部分纯化，并对其性质进行了初步研究．表明突变体尿激酶原保留了全部尿激酶原的纤溶酶原激活活性，并具有很强的抗血小板聚集活性．

Despite advantages of plasminogen activators demonstrated in vitro, significant shortcomings have been observed in terms of system haemorrhage and platelet mediated reocclusion.Therefore,it has been suggested that thrombolytic therapy can not be considered as a monotherapy but that potent and fibrin specific plasminogen activators should be combined with anti platelet and/or anti thrombin agents or strategies.Many researches on synthetic petides or some snake venom proteins containing the RGD sequence were capable of inhibiting fibrinogen mediated platelet aggregation by competitively inhibiting the binding of fibrinogen to activated platelets receptor GPⅡb/Ⅲa.In order to obtain bifunctional molecules which combined thrombolytic and anti platelet aggregation activity,based on the structure analysis and computer modelling,a prourokinase chimera which was inserted a RGDS sequence into the β hairpin near the C terminal of its kringle domain was constructed by using site directed mutagenesis and DNA recombination methods.The wild type and chimerical prourokinase were highly expressed in BacPAK6 Bacu lovirus expression system and secreted about 1 200～1 800 IU/(10 6 cells·ml)(wild type) or 1 800 ～2 400 IU/(10 6 cells·ml)(mutant) of the supernatant.The wild type and mutant prourokinase were partially purified by combination of CM Sepharose FF ion exchange chromatography,Sephadex G 75 gel filtration and ultra filtration.Specific fibrinolytic activities of wild type proUK(rproUK) and proUKIn1 measured on the human fibrin plates were 120 000 IU/mg and 128 000～136 000 IU/mg,respectively.PRP aggregation was inhibited at 66 4% and 85 3% when 1 4 μmol and 2 525 μmol proUKIn1 were incubated with PRP,respectively.Therefore,RGDS proUK has full thrombolytic activity and strong anti platelet aggregation activity.