PEG定点修饰重组人睫状神经营养因子及其生物活性评价
发布时间：2019-04-21 17:14
【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。
[Abstract]:The 17th cysteine sulfhydryl group of recombinant human ciliary neurotrophic factor mutant CNTF-C17 was modified by maleimide polyethylene glycol (m PEG-MAL) with molecular weight of 40 k Da. The single modified product Mono-PEG-CNTF-C17, was obtained by ion exchange chromatography and its structure and activity in vitro and in vivo were evaluated. The results showed that in pH 7.5 Tris-HCl buffer solution, the ratio of protein and modifier was 1? 3, and the modification rate was more than 90% at 4 鈩，
本文编号：2462396
[Abstract]:The 17th cysteine sulfhydryl group of recombinant human ciliary neurotrophic factor mutant CNTF-C17 was modified by maleimide polyethylene glycol (m PEG-MAL) with molecular weight of 40 k Da. The single modified product Mono-PEG-CNTF-C17, was obtained by ion exchange chromatography and its structure and activity in vitro and in vivo were evaluated. The results showed that in pH 7.5 Tris-HCl buffer solution, the ratio of protein and modifier was 1? 3, and the modification rate was more than 90% at 4 鈩，
本文编号：2462396
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