Journal of Chinese Pharmaceutical Sciences ›› 2018, Vol. 27 ›› Issue (11): 787-798.DOI: 10.5246/jcps.2018.11.079

• Original articles • Previous Articles     Next Articles

Design, preparation and activity evaluation of two novel proteins with thrombolysis or/and neuroprotection

Huan Chen, Danping Zheng, Xiaoyan Liu, Yuanjun Zhu*, Yinye Wang*   

  1. Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
  • Received:2018-04-27 Revised:2018-07-23 Online:2018-11-28 Published:2018-10-10
  • Contact: Tel.: +86-010-82802652, E-mail:;
  • Supported by:

    National Natural Science Foundation of China  (Grant No. 8157333 and 81503060).


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

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