Expression, purification and PEGylation of recombinant human ciliary neurotrophic factor

王彦; 范开; 夏永鹏; 张继兰; 梅翔; 陈海容; 李秀梅; 邱宗荫

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Expression, purification and PEGylation of recombinant human ciliary neurotrophic factor

更新时间：2026-01-09

- Expression, purification and PEGylation of recombinant human ciliary neurotrophic factor
- Journal of Army Medical University Vol. 31, Issue 12, Pages: 1181-1184(2009)
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  CLC： R914.5;R977.6
- Published：2009
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王彦,范开,夏永鹏,张继兰,梅翔,陈海容,李秀梅,邱宗荫.重组人睫状神经因子的表达、纯化以及聚乙二醇修饰[J].陆军军医大学学报(原第三军医大学学报),2009,31(12):1181-1184.

　WANG Yan,FAN Kai,XIA Yong-peng,ZHANG Ji-lan,MEI Xiang,CHEN Hai-rong,LI Xiu-mei,QIU Zong-yin.Expression, purification and PEGylation of recombinant human ciliary neurotrophic factor[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(12):1181-1184.
王彦,范开,夏永鹏,张继兰,梅翔,陈海容,李秀梅,邱宗荫.重组人睫状神经因子的表达、纯化以及聚乙二醇修饰[J].陆军军医大学学报(原第三军医大学学报),2009,31(12):1181-1184. DOI：

　WANG Yan,FAN Kai,XIA Yong-peng,ZHANG Ji-lan,MEI Xiang,CHEN Hai-rong,LI Xiu-mei,QIU Zong-yin.Expression, purification and PEGylation of recombinant human ciliary neurotrophic factor[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(12):1181-1184. DOI：

摘要

目的研究重组人睫状神经因子（rh-CNTF）的表达、纯化及聚乙二醇化修饰、纯化方法。方法重组人睫状神经因子（rh-CNTF）工程菌经37 ℃培养至对数生长期，加入IPTG诱导4 h，经Q Sepharose F.F凝胶柱纯化和复性后得到纯化的rh-CNTF。以体外培养鸡胚背根神经节（DRG）方法测定重组蛋白rh-CNTF的神经营养活性。用单甲氧基聚乙二醇丁醛（mPEG-ButyrALD-20 k）对rh-CNTF进行修饰，所得修饰产物经DEAE Sepharose F.F和Superdex 75 凝胶分离纯化后获得mPEG-rh-CNTF偶联物。结果目的蛋白占总蛋白30%左右，为包涵体，经Q Sepharose F.F凝胶柱纯化和复性，获得纯度达90%的rh-CNTF。表达的rh-CNTF能促进培养的鸡胚背根神经节神经突起的生长。修饰后的单聚PEG-rhCNTF经DEAE Sepharose F.F和Superdex 75凝胶纯化后纯度达到95%。结论确立了rh-CNTF蛋白表达纯化及PEG修饰的方法。

Abstract

Objective To investigate the method for the expression

purification

and PEGylation of the recombinant human ciliary neurotrophic factor (rh-CNTF). Methods rh-CNTF strains were cultured at 37 ℃ to logarithmic phase. After induction with IPTG for 4 h

the target protein was harvested as the inclusion body and then refolded and purified with Q Sepharose FF to gain highly purified rh-CNTF. The in vitro bioactivity of rh-CNTF was evaluated by chicken embryo dorsal root ganglia (DRG) method. rh-CNTF was modified with mPEG-ButyrALD-20 k

the product of which was modified and purified by DEAE Sepharose FF and Superdex 75 to obtain mPEG-rh-CNTF conjugates. Results The interesting protein expression level as the inclusion body in E. coli accounted for 30% of total protein and 90% purity of rh-CNTF was obtained by one step refolding and purification of Q Sepharose FF column. rh-CNTF could promote the growth of DRG tubercles. The purity of monomer PEG-rhCNTF purified by DEAE Sepharose FF and Superdex 75 reached as high as 95%. Conclusion The method for the expression

purification

and PEG-modification of rh-CNTF has been established.

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