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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (4): 43-49    DOI: 10.13523/j.cb.20160407
    
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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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     
Received: 12 October 2015      Published: 09 December 2015
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YIN Shuang
FENG Cui
ZHANG Quan
YU Qi
YU Jian
YU Rong
LIU Yong-Dong
SU Zhi-Guo
Cite this article:

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.

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

[1] Barbin G, Manthorpe M, Varon S. Purification of the chick eye ciliary neuronotrophic factor. Journal of Neurochemistry, 1984,43(5): 1468-1478.
[2] Lambert P D, Anderson K D, Sleeman M W, et al. Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity. Proceedings of the National Academy of Sciences, USA, 2001, 98:4652-4657.
[3] Joseph F K, Carol F E, Charlotte E L. Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes, 2004, 53:911-920.
[4] McDonald N Q, Panayotatos N, Hendrickson W A.Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing. The EMBO Journal, 1995, 14 ( 12 ) : 2689-2699.
[5] 杨光,李祎,项光亚. 重组人睫状神经营养因子肽图分析.生物技术通报,2010,11: 162-165. Yang G, Li W, Xiang G Y. Peptide mapping analysis of recombinant human ciliary neurotrophic factor. Biotechnology Bulletin, 2010,11: 162-165.
[6] Lam A, Fuller F, Miller J, et al. Sequence and structural organization of the human gene encoding ciliary neurotrophic factor. Gene, 1991, 102(2):271-276.
[7] Dittrich F, Thoenen H, Sendtner M. Ciliary neurotrophic factor: pharmacokinetics and acute-phase response in rat. Annals of Neurology, 1994, 35:151-163.
[8] Shannon M, Maria B. Ciliary neurotrophic factor variants. US Patent, A1, 20050064555, 2005.
[9] Hopkins C R, Trowbridge I S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. The Journal of Cell Biology, 1983, 97:508-521.
[10] Gomme P T, McCann K B, Bertolini J. Transferrin: structure, function and potential therapeutic actions. Drug Discovery Today, 2005, 10:267-273.
[11] 冯翠,赵大伟 ,张纯,等. 一种重组人睫状神经营养因子突变体的分离纯化及结构鉴定.中国生物工程杂志, 2013,33(10):21-27. Feng C, Zhao D W, Zhang C, et al. Purification and characterization of a new recombinant ciliary neurotrophic factor mutant expressed in soluble form by E.coli. China Biotechnology, 2013, 33(10):21-27.
[12] Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, 1951, 193 (1): 265-275.
[13] 周虎,汪怡,朱俊铭.定量测定重组人睫状神经营养因子活性的新方法.中国生物工程杂志,2008,28( 2) : 86-89. Zhou H, Wang Y, Zhu M J. A new quantitative determination method for the biological activity of recombinant human ciliary neurotrophic factor. China Biotechnology, 2008, 28(2):86-89.
[14] 毕华,袁力勇,史新昌,等.睫状神经营养因子突变体蛋白的活性研究.中国生物工程杂志,2007,27( 1) : 1-5. Bi H, Yuan L Y, Shi X C, et al. Activity study of ciliary neurotrophic factor (CNTF) mutant. China Biotechnology, 2007, 27(1):1-5.
[15] Kluger R, Alagic A. Chemical cross-linking and protein-protein interactions-a review with illustrative protocols. Bioorganic Chemistry, 2004, 32:451-472.
[16] 冯翠,王祺,张纯,等.PEG 定点修饰重组人睫状神经营养因子及其生物活性评价.中国生物工程杂志,2015,35(5):15-21. Feng C, Wang Q, Zhang C, et al. Preparation of mPEG-MAL-modified recombinant human ciliary neurotrophic factor and evaluation of biological activity. China Biotechnology, 2015, 35(5):15-21.
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