人载脂蛋白A5基因的克隆、表达、纯化及多克隆抗体制备

Cloning, Expression, Purification and Polyclonal Antibody Preparation of Human Apolipoprotein A5

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.

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 eDNA, 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 Ni^2＋ 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, Ref28