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rPA作为溶栓药物之一,仍存在血浆半衰期短和颅内出血等问题.将rPA分子中PAI-1的水解部位突变,并与具有不同溶栓机制的NR3进行融合,再引入RGD序列,可能延长半衰期、增强溶栓作用、减轻出血副作用.据此,本研究克隆、表达和纯化了两个新型蛋白mrPA和mrPA-NR3,并进行活性评价.通过PCR扩增编码mrPA和mrPA-NR3的DNA片段,并克隆到pET-30a中构建重组质粒pET30a-mrPA和pET30a-mrPA-NR3,随后转化大肠杆菌E.coli BL21(DE3).通过异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)诱导重组蛋白的包涵体表达,使用镍(Ni)亲和层析纯化,经过复性后用BCA法进行蛋白定量.分别对mrPA和mrPA-NR3的体内和体外溶栓活性及出血副作用进行了评价,结果发现:mrPA和mrPA-NR3均有明显的体外溶栓活性,在高浓度时,mrPA-NR3溶栓效果强于mrPA.mrPA和mrPA-NR3也具有明显的体内溶栓活性,在高剂量时,mrPA-NR3具有比mrPA更强的溶栓效果;二者的尾出血时间明显低于模型组,接近正常组.这些结果可能为提高溶栓药的药效和减轻出血副作用提供了新的思路,为进一步研究奠定了基础.“,”Recombinant tissue plasminogen activator (rPA) has been used as a thrombolytic agent.However,considerable improvements have been done to prolong its plasma half-life (tl/2) and reduce its side effects,such as intracranial hemorrhage.Based on these improvements,a mutant of rPA,mrPA,was designed by mutating its PAⅠ-1 binding site to extend its t1/2.Furthermore,a fusion protein conjugating mrPA with NR3 was designed,which was a rAcAp5 mutant with a platelet GPⅡb/Ⅲa-binding RGD motif,to enhance the ability of targeted-thrombus and thrombolysis.The synthesized DNA sequences coding the two proteins were amplified by PCR,cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3,and transformed into E.coli BL21 (DE3).The two proteins were expressed in inclusion bodies induced by isopropyl ｀-D-1-thiogalactopyranoside.After purified to qualified purity using one-step Ni affinity chromatography,the denatured proteins were refolded by dialysis.Their thrombolytic effects in vitro and in vivo were evaluated.In vitro 3.5 and 7 μmol/L of mrPA significantly reduced thrombus weight;1.75,3.5 and 7 μmol/L of mrPA-NR3 also significantly reduced the thrombus weight,and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 μmol/L.In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 μmol/kg in thrombolytic model of inferior vena cava.Importantly,mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA (positive control) at 240 μmol/kg.In addition,these two novel proteins did not increase bleeding time while they exerted thrombolytic effect.In conclusion,we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein,and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect of rPA and alleviated hemorrhage side reaction.This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.