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  

Comparison of the Biological Activity of m PEG20k Modified and Transferrin Coupled Ciliary Neurotrophic Factor

作　　者：迟胜男 李增兰 张纯[2] 殷爽 冯翠 王祺 刘永东[2] 苏志国[2] 

机构地区：[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

出　　处：《中国生物工程杂志》2017年第12期59-66,共8页China Biotechnology

基　　金：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

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rh CNTF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(rh CNTF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。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 Cysl7 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.

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 

分 类 号：Q819[生物学—生物工程]