RPA32蛋白原核表达、多克隆抗体制备及初步鉴定  

Prokaryotic expression,polyclonal antibody preparation,and identification of human RPA32

作　　者：左斌[1] 王欢[1,2,3] 王海斌[2] 曾鸣[2] 任来峰[1] 李莎[1] 郭莲娣[2] 李明远[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,成都610041 [2]四川大学第二医院发育与干细胞研究所 [3]遵义医学院微生物学教研室,563000

出　　处：《免疫学杂志》2012年第2期120-123,共4页Immunological Journal

基　　金：国家自然科学基金(81071362,30970950);贵州省教育厅自然科学青年基金(C-588c);中央高校基本科研业务费(2010SCV21004)

摘　　要：目的原核表达人RPA32蛋白并制备其多克隆抗体,为后续RPA32的功能研究奠定基础。方法以人cDNA文库为模板,构建原核表达质粒PET41a-RPA32,转化受体菌E.coli BL21,经IPTG诱导表达,采用GST亲和纯化方法获得GST-RPA32融合蛋白,将该融合蛋白免疫家兔制备RPA32多抗血清,最后采用Western blot检测其在多种肝细胞株中的特异性。结果在受体菌E.coli BL21中成功诱导表达GST-RPA32融合蛋白,免疫家兔后获得高效价RPA32抗血清,Western blot检测证实该血清能够识别多种肝细胞株中的RPA32及CPT作用后出现的RPA32磷酸化条带。结论成功制备兔抗人RPA32多克隆抗体,为RPA32在DNA损伤中的功能研究奠定了基础。This study aims to express and purify the protein of RPA32（replication protein A 32） in prokaryocytes and prepare anti-RPA32 polyclonal antibody.First,RPA32 gene was amplified from human cDNA library,and then cloned into recombinant expression vector PET41a.GST-RPA32 fusion protein was expressed in E.coli BL21 after being induced with isopropylthio-soprgalactoside（IPTG）.After purification and confirmed by SDS-PAGE,the fusion protein was used to immune the rabbit.Finally,the anti-RPA32 serum was obtained and analyzed by Western-blot in several liver cell lines.The successfully prepared anti-RPA32 polyclonal antibody provides a basis for further exploring the role of RPA32 in the DNA damage response.

关 键 词：RPA32 原核表达 多克隆抗体 复制蛋白A 

分 类 号：R392.1[医药卫生—免疫学]