色谱

色谱 ›› 2012, Vol. 30 ›› Issue (08): 851-855.DOI: 10.3724/SP.J.1123.2012.04002

• 技术与应用 • 上一篇    

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

秦宗华, 陈婷, 李任强*   

  1. 暨南大学生物工程学系 广东省分子免疫与抗体工程重点实验室, 广东 广州 510632
  • 收稿日期:2012-04-05 修回日期:2012-06-04 出版日期:2012-08-28 发布日期:2012-08-17
  • 通讯作者: 李任强,博士,教授,研究方向为蛋白质工程. E-mail: trqli@jnu.edu.cn.
  • 基金资助:

    暨南大学自然科学基金项目(109057, 640073).

Purification of immunoglobulin and serum albumin from serum via strong anion exchange chromatography coupled with molecular exclusion chromatography

QIN Zonghua, CHEN Ting, LI Renqiang*   

  1. Department of Biotechnology, Jinan University, Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Guangzhou 510632, China
  • Received:2012-04-05 Revised:2012-06-04 Online:2012-08-28 Published:2012-08-17

PDF

536

被引次数

16

摘要： 动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。

关键词: 白蛋白, 纯化, 动物血清, 分子排阻色谱, 免疫球蛋白, 阴离子交换色谱

Abstract: The isoelectric points of immunoglobulin (Ig) and serum albumin (SA) in animal serum are about 7.8 and 4.8, respectively. Based on their larger difference of isoelectric points, Q SepharoseTM-XL strong anion exchange chromatography coupled with molecular exclusion chromatography was used to purify Ig and SA simultaneously from the high immune rabbit serum. After the Q SepharoseTM-XL strong anion exchange column was equilibrated with 0.02 mol/L Tris-HCl buffer of pH 8.0, a 10-fold dilution sample of rabbit serum was loaded onto the column and the pH gradient elution was performed. With the low flow rate of elution of 0.3 mL/min, the high-purity Ig was obtained when the elution pH was at 6.0. Continuously eluted at pH 4.0 with the same flow rate of elution, the SA was obtained and its purity was greater than 95% after molecular exclusion chromatography through Sephadex G-75. The purified Ig and SA were demonstrated to maintain normal activities by activity analysis. The results of protein content showed that the purification recoveries of Ig and SA were over 95% and 90%, respectively. The method has the advantages of simple operation and rapidity, and the Ig and SA purified simultaneously from the animal serum could maintain normal activities.

Key words: albumin, animal serum, immunoglobulin, molecular exclusion chromatography, purification, anion exchange chromatography