Design, preparation and activity evaluation of novel recombinant thrombolytic proteins  

新型溶栓蛋白的设计、制备及其活性评价(英文)

作　　者：胡桂珍 刘晓岩[1] 朱元军[1] 王银叶[1] 

机构地区：[1]北京大学医学部药学院分子与细胞药理学系,北京100191

出　　处：《Journal of Chinese Pharmaceutical Sciences》2017年第11期819-826,共8页中国药学（英文版）

基　　金：National Natural Science Foundation of China(Grant No.81573333 and 81503060)

摘　　要：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 ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/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 isopropy113-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 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/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 p.mol/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 ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents rP A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。

关 键 词：Recombined thrombolytic protein mrPA mrPA-NR3 

分 类 号：R91[医药卫生—药学] R96