重组人源性抗HBsAg Fab抗体纯化方法的比较研究  

Study on the Methods of Purification of Recombinant Humanized anti-HBsAg Fab Antibody

作　　者：邓宁[1] 向军俭[1] 饶桂荣[2] 陈文吟[2] 

机构地区：[1]暨南大学生命科学技术学院分子免疫学与抗体工程实验室,广州510632 [2]人民解放军第458医院全军传染病研究中心,广州510601

出　　处：《生物工程学报》2004年第6期884-889,共6页Chinese Journal of Biotechnology

基　　金：广州市科技局重点项目 (No .2 0 0 3J1 C0 171);国家自然科学基金项目 (No .40 3 760 3 0 )~~

摘　　要：抗HBsAgFab抗体被认为在预防和治疗HBV引起的肝病中具有重要的作用。为了建立稳定的、适于生产应用的重组人源性抗HBsAgFab抗体的纯化工艺 ,本实验对不同纯化方法进行了比较研究。比较了抗Fab抗体亲和层析、ScFv单克隆抗体亲和层析和离子交换层析等 3套纯化工艺在酵母发酵生产的重组人源性抗HBsAgFab抗体纯化中的效率。结果显示 ,抗Fab抗体亲和层析柱纯化酵母表达的重组Fab ,纯度为 96 8% ,但回收率偏低 ,只有30 %～ 4 0 %。ScFv单克隆抗体亲和层析纯化的重组Fab ,纯度为 97 5 % ,回收率达 75 %～ 85 %。该工艺能很好的适应较小规模的生产应用。离子交换层析纯化的重组Fab ,纯度为 97% ,回收率为 75 %～ 85 %。该工艺能很好的适应较大规模的生产应用。以上结果表明 ,应用ScFv单克隆抗体亲和层析和离子交换层析纯化技术均能很好的纯化出重组人源性抗HBsAgFab抗体 ,这两种纯化工艺不仅大大节约纯化成本 ,且纯化效率和回收率有很大提高。Anti-HBsAg Fab antibody is useful in prevention if it binds to and block surface regions of the virus, which is essential for the infection of hepatocytes. In order to establish steady and high effective methods to purify the recombinant humanized anti-HBsAg Fab antibody from fermentation supernatant of Pichia pastoris for industrial production,anti-Fab chromatography, anti-ScFv Mab chromatography and ion exchange chromatography were used. Results showed that the purity of recombinant anti-HBsAg Fab antibody purified by affinity chromatography of anti-Fab antibody column is 96.8%, and the recovery rate of the Fab antibody is about 30%～40% and much of Fab fragment was lost. The purity of recombinant anti-HBsAg Fab antibody purified by affinity chromatography of anti-ScFv Mab column is 97.5% and the recovery rate of the Fab antibody is about 75%～85%. This method can be used in smaller scale purification of recombinant Fab antibody from fermentation supernatant of recombinant yeast. The purity of recombinant Fab fragment purified by ion exchange chromatography is 97%, and the recovery rate of the Fab antibody is about 75%～85%. The method of ion exchange chromatography can be used to produce the recombinant Fab antibody in large scale for industrial production. The results demonstrated that the recombinant humanized anti-HBsAg Fab antibody can be purified high effectively by the methods of anti-ScFv Mab chromatography and ion exchange chromatography from fermentation supernatant of recombinant yeast, and the methods of anti-ScFv Mab chromatography and ion exchange chromatography can be used effectively in industrial production.

关 键 词：HBSAG 重组Fab 纯化方法 毕赤酵母 

分 类 号：R392[医药卫生—免疫学]