具有溶栓和脑保护作用的融合蛋白的表达、纯化及活性研究

doi:10.5246/jcps.2018.11.079

摘要/Abstract

摘要：

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pET16b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。

关键词: 缺血性脑卒中, 溶栓, 神经保护, 融合蛋白

Abstract:

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 introducingtwo 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.

Key words: Ischemic stroke, Thrombolysis, Neuroprotection, Fusion protein

中图分类号:

R962

Supporting:

陈欢, 郑丹萍, 刘晓岩, 朱元军, 王银叶. 具有溶栓和脑保护作用的融合蛋白的表达、纯化及活性研究[J]. 中国药学(英文版), 2018, 27(11): 787-798.

Huan Chen, Danping Zheng, Xiaoyan Liu, Yuanjun Zhu, Yinye Wang. Design, preparation and activity evaluation of two novel proteins with thrombolysis or/and neuroprotection[J]. Journal of Chinese Pharmaceutical Sciences, 2018, 27(11): 787-798.

参考文献

[1] Grupke, S.; Hall, J.; Dobbs, M.; Bix, G.J. Understanding history, and not repeating it. Neuroprotection for acute ischemic stroke: From review to preview. Clin. Neurol. Neurosurg. 2015, 129, 1–9.

[2] Powers, W.J.; Rabinstein, A.A.; Ackerson, T.; Ackerson, T.; Adeoye, O.M.; Bambakidis, N.C.; Becker, K.; Biller, J.; Brown, M.; Demaerschalk, B.M.; Hoh, B.; Jauch, E.C.; Kidwell, C.S.; LeslieMazwi, T.M.; Ovbiagele, B.; Scott, P.A.; Sheth, K.N.; Southerland, A.M.; Summers, D.V.; Tirschwell, D.L. 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2018, 49, e46–e110.

[3] Eltzschig, H.K.; Eckle, T. Ischemia and reperfusion—from mechanism to translation. Nat Med. 2011, 17, 1391–1401.

[4] Akpan, N.; Troy, C.M. Caspase inhibitors: prospective therapies for stroke. Neuroscientist. 2013, 19, 129–136.

[5] Cao, G.; Pei, W.; Ge, H.; Liang, Q.; Luo, Y.; Sharp, F.R.; Lu, A.; Ran, R.; Graham, S.H.; Chen, J. In Vivo Delivery of a Bcl-xL Fusion Protein Containing the TAT Protein Transduction Domain Protects against Ischemic Brain Injury and Neuronal Apoptosis. J Neurosci. 2002, 22, 5423–5431.

[6] Gabellini, C.; Trisciuoglio, D.; Del, B.D. Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain. Carcinogenesis. 2017, 38, 579–587.

[7] Shimizu, S.; Konishi, A.; Kodama, T.; Tsujimoto, Y. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc. Natl. Acad. Sci. USA. 2000, 97, 3100–3105.

[8] Miljić, P.; Heylen, E.; Willemse, J.; Djordjević, V.; Radojković, D.; Colović, M.; Elezović, I.; Hendriks, D. Thrombin activatable fibrinolysis inhibitor (TAFI): a molecular link between coagulation and fibrinolysis. Srp. Arh. Celokupno. Lek. 2010, 138, 74–78.

[9] Nagashima, M.; Werner, M.; Wang, M.; Zhao, L.; Light, D.R.; Pagila, R.; Morser, J.; Verhallen, P. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb. Res. 2000, 98, 333–342.

[10] Wang, Y.X.; Da, C.V.; Vincelette, J.; Zhao, L.; Nagashima, M.; Kawai, K.; Yuan, S.; Emayan, K.; Islam, I.; Hosoya, J.; Sullivan, M.E.; Dole, W.P.; Morser, J.; Buckman, B.O.; Vergona, R. A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis. Thromb. Haemost. 2007, 97, 54–61.

[11] Cappello, M.; Vlasuk, G.P; Bergum, P.W.; Huang, S.; Hotez, P.J. Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa. Proc. Natl. Acad. Sci. USA. 1995, 92, 6152–6156.

[12] Ding, S.; Liu, X.Y.; Zhu, Y.J.; Wang, Y.Y. Thrombolytic effect of rAcAP5 and its mechanism. J. Chin. Pharm. Sci. 2012, 21, 70–75.

[13] Bledzka, K.; Smyth, S.S.; Plow, E.F. Integrin alpha IIb beta 3: From Discovery to Efficacious Therapeutic Target. Circ. Res. 2013, 112, 1189–1200.

[14] Guo, S.; Shen, S.; Wang, J.; Wang, H.; Li, M.; Liu, Y.; Hou, F.; Liao, Y.; Bin, J. Detection of High-Risk Atherosclerotic Plaques with Ultrasound Molecular Imaging of Glycoprotein IIb/IIIa Receptor on Activated Platelets. Theranostics. 2015, 5, 418–430.

[15] Zhang, L.; Jing, W.; Yu, M.; Ru, B. Functional properties of a recombinant chimeric plasminogen activator with platelet-targeted fibrinolytic and anticoagulant potential. Mol. Genet. Metab. 2004, 82, 304–311.

[16] Yan, H.L.; Sun, S.H.; Wang, W.T.; Ding, F.X.; Mei, Q.; Xue, G. Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency. Biotechnol. Appl. Biochem. 2007, 46, 115–125.

[17] Yang, M.; Cui, G.; Zhao, M.; Wang, C.; Wang, L.; Liu, H.; Peng, S. The effect of complexation of Cu (II) with P6A peptide and its analogs on their thrombolytic activities. Int. J. Pharm. 2008, 362, 81–87.

[18] Zhang, Z.; Chopp, M.; Zhang, R.L.; Goussev, A. A Mouse Model of Embolic Focal Cerebral Ischemia. J. Cereb. Blood Flow Metab. 1997, 17, 1081–1088.

[19] Yin, J.; Bao, L.; Tian, H.; Gao, X.; Yao, W. Quantitative relationship between the mRNA secondary structure of translational initiation region and the expression level of heterologous protein in Escherichia coli. J. Ind. Microbiol. Biotechnol. 2016, 43, 97–102.

[20] Bai, C.; Wang, X.; Zhang, J.; Sun, A.; Wei, D.; Yang, S. Optimisation of the mRNA secondary structure to improve the expression of interleukin-24 (IL-24) in Escherichia coli. Biotechnol. Lett. 2014, 36, 1711–1716.

[21] Hirsh, A.G.; Tsonev, L.I. Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 2: Computer controlled pH gradients in the presence of urea provide improved separation of proteins: Stability influenced anion and cation exchange chromatography. J. Chromatogr. A. 2017, 1495, 22–30.

[22] Bunnage, M.E.; Blagg, J.; Steele, J.; Owen, D.R.; Allerton, C.; McElroy, A.B.; Miller, D.; Ringer, T.; Butcher, K.; Beaumont, K.; Evans, K.; Gray, A.J.; Holland, S.J.; Feeder, N.; Moore, R.S.; Brown, D.G. Discovery of potent & selective inhibitors of activated thrombin-activatable fibrinolysis inhibitor for the treatment of thrombosis. J. Med. Chem. 2007, 50, 6095–6103.

[23] Hendrickx, M.L.; Zatloukalova, M.; Hassanzadeh-Ghassabeh, G.; Muyldermans, S.; Gils, A.; Declerck, P.J. In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody. Thromb. Haemost. 2014, 111, 824–832.

[24] Durand, A.; Chauveau, F.; Cho, T.H.; Kallus, C.; Wagner, M.; Boutitie, F.; Maucort-Boulch, D.; Berthezène, Y.; Wiart, M.; Nighoghossian, N. Effects of a TAFI-Inhibitor Combined with a Suboptimal Dose of rtPA in a Murine Thromboembolic Model of Stroke. Cerebrovasc. Dis. 2014, 38, 268–275.

[25] Del-Zoppo, G.J. The neurovascular unit in the setting of stroke. J. Intern. Med. 2010, 267, 156–171.

[26] Romanos, E.; Planas, A.M.; Amaro, S.; Chamorro, A. Uric acid reduces brain damage and improves the benefits of rt-PA in a rat model of thromboembolic stroke. J. Cereb. Blood Flow Metab. 2007, 27, 14–20.

[27] Amaro, S.; Soy, D.; Obach, V.; Cervera, A.; Planas, A.M.; Chamorro, A. A pilot study of dual treatment with recombinant tissue plasminogen activator and uric acid in acute ischemic stroke. Stroke. 2007, 38, 2173–2175.