PEG定点修饰重组人睫状神经营养因子及其生物活性评价

doi:10.13523/j.cb.20150503

中国生物工程杂志

2015, Vol. 35

Issue (5): 15-21 DOI: 10.13523/j.cb.20150503

研究报告

PEG定点修饰重组人睫状神经营养因子及其生物活性评价

冯翠¹, 王祺², 张纯², 秦培勇¹, 郑秀玉², 王健³, 刘永东², 苏志国²

1. 北京化工大学生命科学与技术学院北京 100029;
2. 中国科学院过程工程研究所生化工程国家重点实验室北京 100190;
3. 北京生物制品研究所有限责任公司北京 100024

Preparation of mPEG-MAL-modified Recombinant Human Ciliary Neurotrophic Factor and Evaluation of Biological Activity

FENG Cui¹, WANG Qi², ZHANG Chun², QIN Pei-yong¹, ZHANG Xiu-yu², WANG Jian³, LIU Yong-dong², SU Zhi-guo²

1. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;
2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
3. National Vaccine & Serum Institute, Beijing 100024, China

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摘要：

采用分子质量为40kDa的马来酰亚胺聚乙二醇(mPEG-MAL),对重组人睫状神经营养因子突变体CNTF-C¹⁷的第17位半胱氨酸巯基进行定点修饰,通过离子交换层析获得单修饰产物Mono-PEG-CNTF-C¹⁷,并对其结构及体内外活性进行评价。实验结果表明,在pH 7.5的Tris-HCl缓冲液体中,蛋白质与修饰剂的为1:3,4℃下反应12h,修饰率可达到90%以上,修饰混合物通过一步阴离子交换层析可获得纯度98%以上的单修饰产物。荧光光谱(FL)及圆二色(CD)图谱显示Mono-PEG-CNTF-C¹⁷与原蛋白二、三级结构一致。TF-1.CN5a.1细胞增殖活性检测表明,Mono-PEG-CNTF-C¹⁷的比活达到了6.51×10⁵ IU/mg,体内循环半衰期相对原蛋白显著提高了30.3倍。该研究可为开发CNTF长效产品提供基础。

关键词： 马来酰亚胺聚乙二醇; CNTF-C¹²单修饰物; 重组人睫状神经营养因子; 分离纯化

Abstract:

The site-direct modification conditions for mutant CNTF-C¹⁷ with a molecular weight of 40kDa amaleimide-PEG (mPEG-MAL) was optimized. The mono-PEG-CNTF-C¹⁷was obtained through ion-exchange chromatography and characterized by CD and FL. Experimental data showed that the optimal ratio of protein to PEG was 1:3 and reaction condition was at 4℃ for 12h in Tris-HCl buffer. At the optimized condition, the PEGylatoin yield of mono-PEG-CNTF-C¹⁷ can easily reach more than 90%. By anion exchange chromatography mPEG_40K-MAL-CNTF-C¹⁷ could efficiently separated from the modified mixture with a purity of 98% and a recovery of 78.1%. FL and CD spectra illustrated that mono-PEG-CNTF-C¹⁷has almost the same second and tertiary structure as the natural protein. After PEGylation, the specific activity of CNTF-C¹⁷ reduced to 6.51 × 10⁵IU/mg measured by TF-1.CN5a.1 cell. But the half-life in vivo was prolonged 30 times. This study provided a foundation for developing new long-acting CNTF product.

Key words: mPEG-MAL Mono-PEG-CNTF-C¹⁷ Recombinant human ciliary neurotrophic factor Separation and purification

收稿日期: 2014-11-11 出版日期: 2015-05-25

ZTFLH:

Q-31

通讯作者: 王健, 刘永东 E-mail: wangjian.bj@cnbg.com.cn;ydliu@home.ipe.ac.cn

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引用本文:

冯翠, 王祺, 张纯, 秦培勇, 郑秀玉, 王健, 刘永东, 苏志国. PEG定点修饰重组人睫状神经营养因子及其生物活性评价[J]. 中国生物工程杂志, 2015, 35(5): 15-21.

FENG Cui, WANG Qi, ZHANG Chun, QIN Pei-yong, ZHANG Xiu-yu, WANG Jian, LIU Yong-dong, SU Zhi-guo. Preparation of mPEG-MAL-modified Recombinant Human Ciliary Neurotrophic Factor and Evaluation of Biological Activity. China Biotechnology, 2015, 35(5): 15-21.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150503 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I5/15

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