【摘要】 利用重组DNA技术和原核表达,得到了人体组织型纤溶酶原激活剂t-PA的缺失型突变体r-PA和Kringle2功能区,并在变性条件下通过金属螯合层析纯化得到纯度较高的r-PA和Kringle2重组蛋白.用纤维蛋白活性平板检测到复性的r-PA和Kringle2都具有体外纤溶活性,并且β2糖蛋白Ⅰ(β2-glycoprotein Ⅰ,β2GPⅠ)对r-PA的体外纤溶活性有显著的促进作用:在β2GPⅠ浓度为1μmol/L和4μmol/L时,r-PA的纤溶活性分别提高1.8和2.1倍.ELISA方法检测发现,β2GPⅠ与r-PA,Kringle2均有特异性结合,并且随着β2GPⅠ浓度的增加,它与包被在酶标板上的重组蛋白r-PA和Kringle2的结合都有趋于饱和的趋势,但其饱和浓度却均远高于β2GPⅠ与t-PA结合的饱和浓度.更多还原

【Abstract】 The recombinant t-PA mutants, r-PA and Kringle2, were expressed in E. coli and purified by immobilized metal-chelated affinity chromatography (IMAC). Both of the renatured r-PA and Kringle2 were found to possess the fibrinolytic activities in vitro by fibrin agarose plate assay. In contrast to Kringle2, the catalytic activity of r-PA corresponding to the ring area of lysis, was enhanced by 1.8 and 2.1 folds as the β2-glycoprotein Ⅰ (β2GPⅠ) concentration was increased to 1 μmol/L and 4 μmol/L, respectively. Both mutants were observed to specifically bind β2GPⅠ in enzyme-linked immunosorbent assays(ELISA), and the saturated concentrations for β2GPⅠ-binding were significantly higher than that of wild type t-PA.更多还原