转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

doi:10.13523/j.cb.20160407

中国生物工程杂志

2016, Vol. 36

Issue (4): 43-49 DOI: 10.13523/j.cb.20160407

研究报告

转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

殷爽¹, 冯翠², 张纯², 王祺², 王健³, 余蓉¹, 刘永东², 苏志国²

1. 四川大学华西药学院靶向药物及释药系统教育部重点实验室成都 610041;
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北京 100024

Preparation of Transferrin Conjugated Ciliary Neurotrophic Factor and Evaluation of Biological Activity

YIN Shuang¹, FENG Cui², ZHANG Chun², WANG Qi², WANG Jian³, YU Rong¹, LIU Yong-dong², SU Zhi-guo²

1. Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China;
2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
3. National Vaccine and Serum Institute, Beijing 100024, China

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摘要：

为了实现在体内更持久的药效作用,根据睫状神经营养因子CNTF第17位为游离半胱氨酸残基,而转铁蛋白Tf无游离半胱氨酸的特点,采用N-羟基琥珀酰亚胺-聚乙二醇5K-马来酰亚胺(NHS-PEG5k-MAL)作为偶联剂,实现了两者定点偶联,然后结合蛋白自身特性制定纯化方法,制备获得纯度高于90%的转铁蛋白-聚乙二醇5k-睫状神经营养因子(Tf-PEG5k-CNTF)耦合物。高效凝胶色谱和动态光散射分析显示耦合物的表观分子体积大于两蛋白之和。细胞试验结果显示耦合物的活性下降至原蛋白的65.8%。大鼠药代动力学试验显示Tf-PEG5k-CNTF耦合物在体内的代谢半衰期延长至8.2小时,与CNTF原蛋白相比提高了约17倍。小鼠动物试验显示在每周2次的给药频率,每次1.0 mg/kg的剂量下,Tf-PEG5k-CNTF能更为显著地影响小鼠对食物摄入量和减轻体重。因此,转铁蛋白偶联技术可用于脑部靶向蛋白药物的长效递送。

关键词： 转铁蛋白; 睫状神经营养因子; 减肥; 蛋白偶联; 长效

Abstract:

To achieve a persistent in vivo efficacy, based on the characteristic that ciliary neurotrophic factor has a free cysteine residue while transferrin does not, transferrin was coupled to ciliary neurotrophic factor by using NHS-PEG5k-MAL. Combining with the purification method of each module, transferrin-PEG5k-CNTF conjugate was successfully prepared with a purity of above 90%. High performance size exclusion chromatography and dynamic light scattering analysis showed the apparent molecular volume of Tf-PEG5k-CNTF was larger than CNTF and transferrin together. The survival activity of TF-1(CN5α-1) cells showed that the bioactivity of Tf-PEG5k-CNTF was decreased to 65.8% of the CNTF. Pharmacokinetics study showed that the in vivo half-life of Tf-PEG5k-CNTF conjugate extended from about 28 minutes to 8.2 hours, increasing by about 17 times compared with CNTF. Pharmacodynamics showed that Tf-PEG5k-CNTF impacted more significantly on reducing food intake and losing weight in mice with twice weekly administration of 1.0 mg/kg drugs. Thus, transferrin coupling technique could be used for long-acting protein drugs delivery to the brain.

Key words: Anti-obesity Ciliary neurotrophic factor Protein conjugates Long acting Transferrin

收稿日期: 2015-10-12 出版日期: 2015-12-09

ZTFLH:

Q819

基金资助:

教育部博士点基金(120120181110036)、国家自然科学基金(21576267)资助项目

通讯作者: 余蓉, 刘永东 E-mail: yurong@scu.edu.cn;ydliu@ipe.ac.cn

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引用本文:

殷爽, 冯翠, 张纯, 王祺, 王健, 余蓉, 刘永东, 苏志国. 转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价[J]. 中国生物工程杂志, 2016, 36(4): 43-49.

YIN Shuang, FENG Cui, ZHANG Chun, WANG Qi, WANG Jian, YU Rong, LIU Yong-dong, SU Zhi-guo. Preparation of Transferrin Conjugated Ciliary Neurotrophic Factor and Evaluation of Biological Activity. China Biotechnology, 2016, 36(4): 43-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160407 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I4/43

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