|本期目录/Table of Contents|

 REN Dongsheng,GUAN Jiafa,LI Dehua,et al.Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5[J].Chinese Journal of Applied & Environmental Biology,2009,15(01):95-99.[doi:10.3724/SP.J.1145.2009.00095]





Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5
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
REN Dongsheng GUAN Jiafa LI Dehua LIANG Bo WU Qiaqing
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
apolipoprotein A5 prokaryotic expression fermentation purification antibody
Q78 : Q592
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
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 cDNA, and cloned into expression plasmid pThioHisD named as pTh-ApoA5. The pTh-ApoA5 was introduced into Escherichia coli BL21 (DE3). After optimizing its fermentation condition, recombinant ApoA5 was highly expressed. The recombinant ApoA5 protein that contained a 6-His sequence was separated by Ni2+ affinity column and the purity of recombinant ApoA5 was more than 95%. The purified recombinant ApoA5 protein was employed for immunizing rabbits and the antiserum was obtained. Western blot showed that the antibody was specific. Fig 6, Ref 28


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更新日期/Last Update: 2009-03-05