人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化

doi:10.3969/j.issn.0438-1157.2013.05.034

化工学报 ›› 2013, Vol. 64 ›› Issue (5): 1750-1756.DOI: 10.3969/j.issn.0438-1157.2013.05.034

人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化

唐思远, 林东强, 童红飞, 姚善泾

化学工程联合国家重点实验室, 浙江大学化学工程与生物工程学系, 浙江杭州 310027

收稿日期:2012-09-05 修回日期:2012-12-05 出版日期:2013-05-05 发布日期:2013-05-05
通讯作者: 林东强
作者简介:唐思远(1987-),男,硕士研究生。
基金资助:
国家自然科学基金项目;浙江省自然科学基金项目。

Preparation and separation of Fab and Fc fragments from human immunoglobulin G with papain digestion

TANG Siyuan, LIN Dongqiang, TONG Hongfei, YAO Shanjing

State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China

Received:2012-09-05 Revised:2012-12-05 Online:2013-05-05 Published:2013-05-05
Supported by:
supported by the National Natural Science Foundation of China and Zhejiang Provincial Natural Science Foundation.

摘要/Abstract

摘要： 为了制备抗体Fab和Fc片段,采用木瓜蛋白酶酶解人免疫球蛋白G(IgG),通过优化酶解条件和色谱法分离过程,得到了纯度较高的Fab片段和Fc片段。考察了酶解pH、酶加入量、添加半胱氨酸和酶解时间等对IgG酶解过程的影响,优化了酶解条件,提高酶解效率,IgG转化率大于98%。酶解产物通过Protein A亲和色谱法和DEAE阴离子交换色谱法进行了纯化,分离得到Fc片段和Fab片段,收率分别为72.6%和40.1%。经SEC-HPLC分析,Fc片段纯度达95.7%,Fab片段纯度达96.6%。

关键词: 人免疫球蛋白G, Fc片段, Fab片段, 木瓜蛋白酶, 亲和色谱法, 离子交换色谱法

Abstract: For the preparation of Fab and Fc fragments with high purity,human immunoglobulin G (hIgG) was treated with papain and its purification with chromatographic techniques was explored.The concentration of cystein,papain addition,pH and incubation time were optimized to improve digestion efficiency with papain,and conversion of IgG could reach more than 98%.Then Protein A affinity chromatography and DEAE anion-exchange chromatography were used to separate the Fab and Fc fragments from the digestion mixture of hIgG.Based on the SEC-HPLC analysis,the purity of Fab fragment was 96.6% with the yield of 40.1%,while the purity of Fc fragment was 95.7% with the yield of 72.6%.

Key words: human immunoglobulin G, Fc fragment, Fab fragment, papain, affinity chromatography, ion exchange chromatography

中图分类号:

TQ028

唐思远, 林东强, 童红飞, 姚善泾. 人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化[J]. 化工学报, 2013, 64(5): 1750-1756.

TANG Siyuan, LIN Dongqiang, TONG Hongfei, YAO Shanjing. Preparation and separation of Fab and Fc fragments from human immunoglobulin G with papain digestion[J]. CIESC Journal, 2013, 64(5): 1750-1756.

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人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化

Preparation and separation of Fab and Fc fragments from human immunoglobulin G with papain digestion

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