TopBP1多克隆抗体的制备及初步鉴定  

Preparation of polyclonal antibody and identification of TopBP1

作　　者：郭莲娣[1] 李佳川[1] 曾鸣[2] 

机构地区：[1]西南民族大学民族医药研究院,四川成都610041 [2]四川大学华西附二院,四川成都610041

出　　处：《西南民族大学学报（自然科学版）》2016年第1期34-38,共5页Journal of Southwest Minzu University(Natural Science Edition)

基　　金：国家自然科学基金面上项目(30970950;31171319);中央高校基本科研业务费专项基金项目(2015NZYQN63)

摘　　要：研究以人cDNA文库为模板,构建重组质粒pET41a-TopBP1,在原核表达菌株BL21中通过IPTG诱导,表达并经GST亲和纯化重组蛋白TopBP1-GST,免疫家兔后制备多抗血清,并采用Western-Blot检测其效价和特异性,为TopBP1在DNA损伤修复中的研究奠定基础.结果显示在表达受体菌株BL21中成功表达并纯化了TopBP1-GST重组蛋白,免疫家兔并获得了高效价的多抗血清,利用Western-Blot也证实了抗体特异性,并能识别出经CPT处理后的特异性磷酸化条带.以上表明,高效价的兔抗人TopBP1血清抗体制备成功,为TopBP1相关领域的研究提供了技术支撑.The aim of the study was to produce the polyclonal antibody against TopBP1 by expressing and purifying the protein of TopBPI in prokaryocytes. TopBP1 gene was amplified from human cDNA library, and constructed into the recombination plas- mid pET41a- TopBP1. Fusion protein TopBP1-GST was expressed in E. coli BL21 after being induced with IPTG. The fusion protein was obtained following purification and confirmed by SDS-PAGE, and then was used to immune the rabbit. Finally, the anti-TopBP1 serum was obtained and checked by Westeru-blot recognizing the specific phosphorylation band under the induction of CPT. The successful preparation of the polyclonal antibody against TopBP1 will provide an important tool for further study of its role in the pathway of DNA damage and repair response.

关 键 词：DNA拓扑异构酶Ⅱβ结合蛋白1 原核表达 多克隆抗体 DNA损伤与修复 

分 类 号：Q7[生物学—分子生物学] R3[医药卫生—基础医学]