PEG定点修饰重组人睫状神经营养因子及其生物活性评价  

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

作　　者：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] 

机构地区：[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

出　　处：《中国生物工程杂志》2015年第5期15-21,共7页China Biotechnology

摘　　要：采用分子质量为40k Da的马来酰亚胺聚乙二醇(m PEG-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p H 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,MonoPEG-CNTF-C17的比活达到了6.51×105IU/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。The site-direct modification conditions for mutant CNTF-C^17 with a molecular weight of 40kDa amaleimide-PEG （mPEG-MAL） was optimized. The mono-PEG-CNTF-C^17was 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-HC1 buffer. At the optimized condition, the PEGylatoin yield of mono-PEG-CNTF-C^17 can easily reach more than 90%. By anion exchange chromatography mPEG4oK-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-C^17has almost the same second and tertiary structure as the natural protein. After PEGylation, the specific activity of CNTF-C^17 reduced to 6.51 × 10^5IU/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.

关 键 词：马来酰亚胺聚乙二醇 CNTF-C^12 单修饰物 重组人睫状神经营养因子 分离纯化 

分 类 号：Q-31[生物学]