人载脂蛋白A5 基因的克隆, 表达, 纯化及多克隆抗体制备.pdf

应用与环境生物学报 2009,15 ( 1 ): 095~099 2009-02-25
Chin J Appl Environ Biol=ISSN 1006-687X DOI: .
人载脂蛋白A5基因的克隆、表达、纯化
及多克隆抗体制备
任东升1, 2 官家发1* 李德华3 梁波3 吴洽庆1, 3*
(1中国科学院成都生物研究所成都 610041)
(2中国科学院研究生院北京 100049)
(3成都地奥制药集团有限公司成都 610041)
摘要人载脂蛋白A5 (ApoA5)是血浆三酰甘油水平的重要决定因素之一,与心血管疾病密切相关,因此获得高纯度
的载脂蛋白A5及其抗体具有重要的意义. 本文利用PCR技术,从人肝癌细胞系7721的cDNA中扩增出ApoA5基因,并克
隆到pThioHisD载体中导入大肠杆菌Escherichia coli BL21 (DE3)中进行表达. 经优化条件,发酵得到高效表达的融合蛋
白. 利用融合蛋白上的一段组氨酸序列,用镍离子亲和柱进行纯化,得到纯度大于95%的融合蛋白. 使用纯化蛋白免
疫新西兰大耳兔,得到多克隆抗体,Western印迹结果显示此多克隆抗体能与ApoA5特异性结合. 图6 参28
关键词载脂蛋白A5;原核表达;发酵;纯化;抗体.
CLC Q78 : Q592
Cloning, Expression, Purification and Polyclonal Antibody Preparation of
Human Apolipoprotein A5
REN Dongsheng1, 2, GUAN Jiafa1*, LI Dehua3, LIANG Bo3 & WU Qiaqing1, 3*
(1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)
(2Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
(3Chengdu Di'ao Pharmaceutical Group Co., Ltd., Chengdu 610041, China )
Abstract Apolipoprotein A5 (ApoA5) gene has been shown to play an important role in determining plasma triglyceride
concentrations in humans, and is a major independent risk factor of cardiovascular disease. It is important to obtain the purified
ApoA5 and antiserum. In this paper, full-length ApoA5 gene was amplified from human hepatoma cell line 7721 cD