基于层析技术探究金环蛇蛇毒多克隆抗体F(ab′)_(2)的制备工艺  

A study on the preparation technology of F(ab')_(2) fragments from polyclonal antibodies against Bungarus fasciatus venoms by chromatography

作　　者：李爽[1] 王炜杰 赵伟睿 胡升[2] 王磊 范泉水 吕常江 黄俊 梅乐和[1,2,5] LI Shuang;WANG Weijie;ZHAO Weirui;HU Sheng;WANG Lei;FAN Quanshui;LÜChangjiang;HUANG Jun;MEI Lehe(College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China;School of Biological and Chemical Engineering,NingboTech University,Ningbo 315100,China;Zhejiang Jianbo Biotechnology Co.,Ltd.,Ningbo 315602,China;School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China;Jinhua Advanced Research Institute,Jinhua 321019,China)

机构地区：[1]浙江大学化学工程与生物工程学院,杭州310027 [2]浙大宁波理工学院生物与化学工程学院,宁波315100 [3]浙江健博生物科技股份有限公司,宁波315602 [4]浙江科技大学生物与化学工程学院,杭州310023 [5]金华高等研究院,浙江金华321019

出　　处：《浙江理工大学学报(自然科学版)》2025年第2期276-282,共7页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：宁波市“科技创新2025”重大专项(2021Z078);国家自然科学基金项目(31971372,32371542)。

摘　　要：为制备高纯度马源金环蛇毒多克隆抗体F(ab′)_(2),以金环蛇毒高免马血浆为原料,采用Protein G亲和层析捕获马血浆中的IgG,经胃蛋白酶酶解后制得F(ab′)_(2),分别通过亲和层析、凝胶过滤层析和混合模式层析对F(ab′)_(2)进行精制纯化,并以间接ELISA法测定F(ab′)_(2)对金环蛇毒的中和活性。结果表明:Protein G亲和层析捕获的IgG纯度为83.6%,较传统的盐析纯化法有明显提高;在3种层析精制方法中,通过凝胶过滤层析精制的F(ab′)_(2)纯度最高,为89.3%,远高于《中华人民共和国药典》标准(60%以上),且收率为56.0%;间接ELISA法测定结果显示,F(ab′)_(2)的效价高于1∶4096。该研究为马源抗毒素多克隆抗体F(ab′)_(2)的工业化制备提供了工艺参考。To prepare high-purity horse-derived F(ab′)_(2) fragments of polyclonal antibodies against Bungarus fasciatus venoms,IgG was captured from hyperimmuned horse plasma with Bungarus fasciatus venoms as the raw material by Protein G affinity chromatography,and then the prepared IgG was enzymatically cleaved by pepsin into F(ab′)_(2) fragments,which were purified by affinity chromatography,gel filtration chromatography,and mixed-mode chromatography,respectively.The neutralizing activity of the prepared F(ab′)_(2) against Bungarus fasciatus venoms was determined by indirect ELISA.The results showed that IgG captured by Protein G affinity chromatography attained a purity of 83.6%, which wassignificantly higher than that of the traditional salting-out method. Among the three chromatographicrefinement methods we tested, gel filtration chromatography refined F(ab′)_(2) had the highest purity of89.3%, which was much higher than the standard of Pharmacopoeia of the People’s of Republic ofChina (more than 60%), and the recovery was 56.0%. The titer of the F(ab′)_(2) was higher than 1:4096by indirect ELISA. The study provides reference for the industrialized preparation of horse-derivedantitoxin polyclonal antibody F(ab′)_(2).

关 键 词：金环蛇 多克隆抗体 胃蛋白酶 F(ab′)_(2)片段 层析纯化 

分 类 号：R392-33[医药卫生—免疫学] R392.7[医药卫生—基础医学]