色谱

色谱 ›› 2012, Vol. 30 ›› Issue (01): 86-90.DOI: 10.3724/SP.J.1123.2011.08018

• 技术与应用 • 上一篇    下一篇

重组蛋白A亲和填料的制备及其性能评价

郭明明1,2, 王俊玲2, 吴彦卓2, 徐明波2*, 高向东1   

  1. 1. 中国药科大学生命科学与技术学院, 江苏 南京 210009; 2.
  • 收稿日期:2011-08-15 修回日期:2011-09-26 出版日期:2012-01-28 发布日期:2012-03-01
  • 通讯作者: 徐明波,博士,教授级高级工程师,研究方向为生物制药. Tel: (010)68727127,
  • 基金资助:
    国家重大新药创制专项(2009ZX09401-006).

Preparation and characterization of recombinant protein A affinity packing

GUO Mingming1,2, WANG Junling2, WU Yanzhuo2, XU Mingbo2*, GAO Xiangdong1   

  1. 1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China; 2. Beijing SL Pharmaceutical Co., Ltd., Beijing 100041, China
  • Received:2011-08-15 Revised:2011-09-26 Online:2012-01-28 Published:2012-03-01

PDF

377

被引次数

10

摘要： 为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。

关键词: 纯化, 抗体, 亲和填料, 制备, 重组金黄色葡萄球菌蛋白A

Abstract: To obtain an excellent antibody purification medium, affinity chromatographic packing with recombinant staphylococcal protein A (rProtein A) was synthesized and verified. With E. coli cells harboring the recombinant plasmid, the rProtein A was expressed and purified, then was conjugated to epichlorohydrin-activated Sepharose 4 Fast Flow to prepare an affinity chromatographic packing. The performances of the packing were validated with rabbit anti-urate oxidase. After the reaction, the concentration of rProtein A coupled to Sepharose 4 Fast Flow was 1.5×10~4 mol/L. Scatchard analysis of the binding isotherm for IgG showed excellent binding capacity on the adsorbent, giving a dissociation constant (Kd) of 2.28×10~7 mol/L and a theoretical maximum adsorption capacity of 20.697 g/L. The identification showed the packing was stable in 0.1 mol/L NaOH solution at 1 h. By using the packing, the pure antibody exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from rabbit serum after one-step elution, with 96.1% of yield and 19 mg IgG for one milliliter of gel. The research laid the foundation of the localization of rProtein A affinity packing.

Key words: affinity packing, antibody, preparation, purification, recombinant staphylococcal protein A (rProtein A)