全氟辛酸特异性单域重链抗体的制备

发布时间：2018-06-15 06:51

本文选题：全氟辛酸 + 完全抗原　；参考：《环境与职业医学》2015年08期

【摘要】：[目的]基于抗原抗体特异性结合的原理,制备一种针对全氟辛酸(PFOA)小分子的特异性抗体,以用于PFOA的现场快速检测。[方法]采用碳二亚胺法将PFOA与牛血清白蛋白(BSA)偶联,将此完全抗原作为包被抗原,用重链抗体噬菌体文库进行筛选获得阳性克隆,聚合酶链反应扩增重链抗体片段并与含有人抗体Fc段的质粒连接,采用原核表达系统进行抗体蛋白表达和纯化,间接竞争酶联免疫吸附试验(ELISA)进行验证。[结果]成功合成完全抗原PFOA-BSA,偶联比为8。采用重链抗体库进行4轮筛选,获得7株阳性克隆,经进一步的原核表达系统进行蛋白表达和纯化,得到5种与预期大小相符的、含有人抗体Fc段的抗体蛋白。经间接竞争ELISA方法初步检测验证,获得1个可与PFOA特异结合的单域重链抗体。[结论]最终获得针对PFOA的特异性抗体,有望用于建立快速检测不同介质中PFOA的免疫学检测方法。
[Abstract]:[objective] based on the principle of specific binding of antigen and antibody, a specific antibody against PFOA-perfluorooctanoate (PFOAA) was prepared for rapid in situ detection of PFOA. [methods] the PFOA was coupled with bovine serum albumin (BSA) by carbodiimide method, the complete antigen was used as the coated antigen, and the positive clones were obtained by the screening of heavy chain antibody phage library. The heavy chain antibody fragment was amplified by polymerase chain reaction (PCR) and ligated with plasmid containing FC fragment of human antibody. The antibody protein was expressed and purified by prokaryotic expression system and verified by indirect competitive enzyme linked immunosorbent assay (Elisa). [results] complete antigen PFOA-BSA was successfully synthesized with a coupling ratio of 8. Using the heavy chain antibody library for 4 rounds, 7 positive clones were obtained. After further prokaryotic expression system, five kinds of antibody proteins containing FC segment of human antibody were obtained according to the expected size. A single domain heavy chain antibody which can specifically bind to PFOA was obtained by indirect competitive Elisa. [conclusion] the specific antibody against PFOA was obtained, which could be used to establish an immunological method for rapid detection of PFOA in different media.
【作者单位】：上海交通大学公共卫生学院;上海交通大学基础医学院;南方人类基因组研究中心;
【基金】：科技部十二五支撑计划(编号:2012BAK17B10-5)
【分类号】：R114

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