rAcAP5高表达工程菌株的筛选、多肽的制备、纯化及对栓塞性脑缺血的溶栓作用

doi:10.5246/jcps.2014.01.003

中国药学(英文版) ›› 2014, Vol. 23 ›› Issue (1): 22-27.DOI: 10.5246/jcps.2014.01.003

rAcAP5高表达工程菌株的筛选、多肽的制备、纯化及对栓塞性脑缺血的溶栓作用

朱亚楠, 朱元军, 卜琦鑫, 刘晓岩, 王银叶^*

北京大学医学部药学院分子与细胞药理学系, 北京 100191

收稿日期:2013-04-24 修回日期:2013-05-10 出版日期:2014-01-23 发布日期:2014-01-22
通讯作者: 王银叶*
作者简介:*Corresponding author. Tel.: 86-10-82802652; Fax: 86-10-62015584; E-mail: wangyinye@bjmu.edu.cn
基金资助:
National Technology Graveness Special Purpose Fund (Grant No. 2009ZX09301-010).

rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

Yanan Zhu, Yuanjun Zhu, Qixin Bu, Xiaoyan Liu, Yinye Wang^*

Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2013-04-24 Revised:2013-05-10 Online:2014-01-23 Published:2014-01-22
Contact: Yinye Wang*
About author:*Corresponding author. Tel.: 86-10-82802652; Fax: 86-10-62015584; E-mail: wangyinye@bjmu.edu.cn
Supported by:
National Technology Graveness Special Purpose Fund (Grant No. 2009ZX09301-010).

摘要/Abstract

摘要：

犬钩虫抗凝肽5 (AcAP5) 是一种具有抗凝活性的多肽。本实验室发现重组AcAP5 (rAcAP5) 具有TAFIa抑制活性, 并在体内、体外实验中均具有溶栓活性。本研究拟筛选rAcAP5高表达菌株, 并用大鼠栓塞性MCAO模型来评价rAcAP5的溶栓作用。根据大肠杆菌偏爱密码子对编码AcAP5的目的基因进行了优化; 用具有不同特点的6种表达质粒和11种大肠杆菌菌株构建了66种重组菌株, 从中选出rAcAP5可溶性表达量最高的菌株; 采用阴离子交换色谱和阳离子交换色谱来纯化目的蛋白; 用高效液相色谱法测定纯化过的rAcAP5的纯度; 通过飞行质谱测定所表达的rAcAP5的分子量; 用大鼠栓塞性大脑中动脉阻塞 (MCAO) 模型评价溶栓作用。rAcAP5表达量最高的菌株是C2566H/pTYB1-rAcAP5, 经过两步离子交换层析纯化之后, 纯度达到90%, 纯蛋白产量为28 mg/L。激光多普勒血流仪测定rCBF评价溶栓效果, 给生理盐水 (溶媒)后rCBF无明显变化, 而给予rAcAP5 (50–200 µg/kg, i.v.) 后, rCBF显著增加, 剂量有依赖性, 表明rAcAP5具有明显的溶栓作用。综上所述, 我们筛选到rAcAP5的高表达菌株, 表达、纯化并鉴定了该多肽, 发现它在栓塞性MCAO模型上有明显的溶栓作用, 提示它有可能作为溶栓剂或辅助溶栓剂应用。

关键词: 犬钩虫抗凝肽5, 蛋白表达, 纯化, 溶栓

Abstract:

Recombinant ancylostoma caninum anticoagulant peptide-5 (rAcAP5) has been reported to inhibit thrombin-activatable fibrinolysis inhibitor (TAFIa) activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion (MCAO) model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow (rCBF) was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB1-rAcAP5. AcAP5 (28 mg) with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle (normal saline) treatment did not change the rCBF, while treatment with rAcAP5 (50–200 µg/kg, i.v.) increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.

Key words: rAcAP5, Over-expression, Purification, Thrombolysis

中图分类号:

R965

Supporting: National Technology Graveness Special Purpose Fund (Grant No. 2009ZX09301-010).

朱亚楠, 朱元军, 卜琦鑫, 刘晓岩, 王银叶*. rAcAP5高表达工程菌株的筛选、多肽的制备、纯化及对栓塞性脑缺血的溶栓作用[J]. 中国药学(英文版), 2014, 23(1): 22-27.

Yanan Zhu, Yuanjun Zhu, Qixin Bu, Xiaoyan Liu, Yinye Wang*. rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model [J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(1): 22-27.

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rAcAP5高表达工程菌株的筛选、多肽的制备、纯化及对栓塞性脑缺血的溶栓作用

rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

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