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为了验证溶栓剂联合神经保护剂治疗可能比单纯溶栓治疗产生更好的治疗效果的假设,通过融合NR6和BH4设计了一种具有溶栓和神经保护双重功能的蛋白质TBN.NR6是通过将两个RGD序列引入溶栓蛋白AcAP5获得的,BH4是抗凋亡蛋白Bcl-xL的关键结构域.编码TBN和NR6的DNA合成后分别克隆到pET30a和pET 16b载体中,两种蛋白都在大肠杆菌BL21(DE3)中以包涵体的形式表达.NR6带有His标签,通过镍亲和层析纯化,而TBN通过离子交换层析纯化,纯化后的蛋白通过透析使其复性.通过大鼠动静脉旁路模型评价两种蛋白质的溶栓活性,在低剂量(6 nmol/kg)时,NR6和TBN具有一定的降低血栓重量的作用(P＜0.05),高剂量(24 nmol/kg)时二者可显著降低血栓重量.并且TBN表现出与NR6相同的溶栓作用,表明NR6在融合蛋白TBN中的功能未受影响.通过小鼠血栓栓塞性大脑中动脉堵塞(eMCAO)模型评价两种蛋白质对脑卒中的治疗效果,结果显示TBN降低梗死体积和抑制细胞凋亡的效果优于NR6.通过小鼠尾出血实验评价目的蛋白的出血副作用,结果发现与tPA相比,NR6和TBN的出血时间较短.这些结果证实了我们的假设:溶栓联合神经保护治疗缺血性脑卒中的效果优于单独溶栓治疗.这两个蛋白具有作为新型溶栓候选药进一步开发的潜力.“,”We hypothesized that thrombolysis in combination with neuroprotection might have better therapeutic effects for ischemic stroke compared with thrombolysis alone.In order to verify such hypothesis,we designed a protein TBN by fusing NR6 and BH4,which possibly had dual functions of thrombolysis and neuroprotection.NR6 was obtained by introducing two RGD motifs to thrombolytic protein AcAP5 to target thrombus.BH4 is the key domain of anti-apoptotic protein Bcl-xL.The DNA fragments encoding TBN and NR6 were synthesized and cloned into pET30a and pET16b vectors,respectively.Both proteins were expressed in E.coli.BL21 (DE3),mainly in the form of inclusion bodies.With His-tag,NR6 was purified by nickel affinity chromatography,while TBN was purified by ion exchange chromatography.Purified proteins were refolded by dialysis assay.The thrombolytic activity of both proteins was evaluated by the rat arteriovenous bypass model.Both NR6 and TBN significantly reduced thrombus weight at higher dose (24 nmol/kg),TBN showed similar effect to NR6.These results suggested that NR6 was a thrombolytic protein,and fusion protein TBN reserved the thrombolytic activation of NR6.The effects of both proteins were also evaluated in thromboembolic middle cerebral artery occlusion (eMCAO) in mice.TBN exhibited better effect on reducing infarction volume and inhibiting apoptosis of cells than NR6,indicating that the introduction of BH4 increased the protective effect of NR6.The hemorrhagic side effects of the two proteins were evaluated by tail bleeding in mice,and it was found that NR6 and TBN showed shorter bleeding time compared with tPA.In conclusion,we designed and prepared the two novel proteins,and testified that they had significant thrombolytic effect and protective effect on cerebral IR injury.The protective effect of TBN was more potent than NR6.Their bleeding side reaction might be weaker than tPA.These results suggested that these two novel proteins deserved to be further investigated as new thrombolytic candidate agents.