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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (12): 59-66    DOI: 10.13523/j.cb.20171209
技术与方法     
聚乙二醇20k修饰与转铁蛋白偶联睫状神经营养因子的生物活性对比研究 *
迟胜男1,2,李增兰1,2,张纯2(),殷爽2,冯翠2,王祺1,2,刘永东1,2(),苏志国1,2
1 中国科学院大学 北京 100049
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
Comparison of the Biological Activity of mPEG20k Modified and Transferrin Coupled Ciliary Neurotrophic Factor
Sheng-nan CHI1,2,Zeng-lan LI1,2,Chun ZHANG2(),Shuang YIN2,Cui FENG2,Qi WANG1,2,Yong-dong LIU1,2(),Zhi-guo SU1,2
1 University of Chinese Academy of Sciences, Beijing 100049, China
2 State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing　100190,China
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摘要：

为了延长重组睫状神经营养因子在体内的保留半衰期,基于CNTF中天然的游离半胱氨酸残基,在前期工作中采用聚乙二醇修饰和转铁蛋白偶联的两种方式对CNTF进行了改造。此后又采用常规分析手段对PEG20k-CNTF和Tf-PEG5k-CNTF进行对比表征。高效凝胶过滤和动态光散射分析结果显示两者拥有相近的表观分子体积。细胞试验结果显示两种耦合物的活性分别下降至未修饰CNTF的50.6%和65.8%。抗体CNTF抗体亲和力结果显示PEG20k修饰后亲合力下降至原蛋白的3.8%,转铁蛋白偶联后保留89.9%原蛋白亲合力。药代动力学结果显示PEG20k-CNTF和Tf-PEG5k-CNTF在SD大鼠血液中的保留半衰期分别为5.34 ± 0.26和8.65 ± 0.60小时,与未修饰rhCNTF相比延长了约21.4倍和34.6倍。药效学结果显示在每周两次每次1.0 mg/kg (rhCNTF等量)的给药频率和剂量下,PEG20k-CNTF比Tf-PEG5k-CNTF更显著地降低实验小鼠体重。
关键词： 聚乙二醇修饰;  转铁蛋白偶联;  睫状神经营养因子;  减轻体重    
Abstract:

In order to prolong the blood half-life of the recombinant ciliary neurotrophic factor in vivo, CNTF was previously formulated by mPEG20k modification and transferrin coupling based on the natural free thiol of Cys17 residue. These two conjugates of PEG20k-CNTF and Tf-PEG5k-CNTF were charicterized and compared using conventional analysis. The HP-SEC and DLS results showed that the conjugates exhibited a similar apparent molecular size. TF-1(CN5α-1) cell survival test showed significant decreased activity of 50.6% (mPEG20k-CNTF) and 65.8% (Tf-PEG5k-CNTF) compared with the unmodified rhCNTF, respectively. Antibody binding affinity revealed that PEG20k-CNTF decreased to 3.8% of the rhCNTF, and Tf-PEG5k-CNTF retained 89.9% of the rhCNTF. Pharmacokinetics study showed that the blood half-lives of PEG20k-CNTF and Tf-PEG5k-CNTF conjugates in vivo were 5.34 ± 0.26 and 8.65 ± 0.60 hours, respectively, which were about 21.4 times and 34.6 times higher than that of rhCNTF. Pharmacodynamics study showed twice weekly administration of 1.0 mg/kg (rhCNTF equivalent) both conjugates led to significant weight-losing, and the efficacy of PEG20k-CNTF group was more potent than that of Tf-PEG5k-CNTF group.
Key words: PEGylation    Transferrin coupling    Ciliary neurotrophic factor    Losing weight
收稿日期: 2017-07-14 出版日期: 2017-12-16
ZTFLH:  Q819  
基金资助: 生化工程国家重点实验室开放基金(2014KF-05)、国家自然科学基金(21576267)、北京市自然科学基金(2162041)资助项目
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迟胜男
李增兰
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殷爽
冯翠
王祺
刘永东
苏志国

引用本文:

迟胜男,李增兰,张纯,殷爽,冯翠,王祺,刘永东,苏志国. 聚乙二醇20k修饰与转铁蛋白偶联睫状神经营养因子的生物活性对比研究 *[J]. 中国生物工程杂志, 2017, 37(12): 59-66.

Sheng-nan CHI,Zeng-lan LI,Chun ZHANG,Shuang YIN,Cui FENG,Qi WANG,Yong-dong LIU,Zhi-guo SU. Comparison of the Biological Activity of mPEG20k Modified and Transferrin Coupled Ciliary Neurotrophic Factor. China Biotechnology, 2017, 37(12): 59-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171209    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I12/59

图1  高效液相色谱分析rhCNTF,转铁蛋白,mPEG20k-CNTF和Tf-PEG5k-CNTF耦合物纯度(a)与12%电泳分析结果(b)1: CNTF原蛋白; 2:mPEG20k-CNTF; 3:转铁蛋白;4:Tf-PEG5k-CNTF
图2  高效凝胶过滤色谱(a)和动态光散射(b)分析耦合物
图3  细胞活性测定和抗CNTF抗体亲合力大小比较
图4  雄性SD大鼠体内药代动力学
图5  rhCNTF和耦合物与对雄性C57小鼠体重的影响
图6  Cy5.5标记rhCNTF和耦合物在对雄性Balb/c裸小鼠体内的荧光发光强度比较
图7  mPEG20k-CNTF与Tf-PEG5k-CNTF空间构象差异示意图(a)PEG链包裹在rhCNTF表面 (b)CNTF与转铁蛋白相对独立,呈现哑铃状构象
图8  mPEG20k-CNTF和Tf-PEG5k-CNTF通过血脑屏障的转运方式对比示意图路线A,Tf受体介导转运途径;路线B,CNTF自身转运途径;路线A’,CNTF自身转运途径;路线B’,非特异性PEG辅助转运途径
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