Detecting expression of spindle protein gene INMAP in hepatoma cells by polyclonal antibody
ZHU Yan1,2, KANG Jingyan1,4, WANG Yu1, SUN Le3, ZHANG Haijiang3, LIANG Qianjin1,2
1. Beijing Key Laboratory of Gene Resource and Molecular Development, School of Life Sciences, Beijing Normal University, Beijing 100875, China;
2. Key Laboratory for Cell Proliferation and Regulation Biology, Ministry of Education; School of Life Sciences, Beijing Normal University, Beijing 100875, China;
3. AbMax Biotechnology Co., Ltd., Beijing 101111, China;
4. No. 4 High School of Lanzhou City, Lanzhou 730050, China;
5. Beijing Health Guard Biotechnology Inc., Beijing 100176, China
Abstract：To investigate the function of INMAP of spindle protein and its role in malignant cell proliferation, an INMAP polyclonal antibody is prepared. The prokaryotic expression of His-INMAP fusion protein is induced at 16℃ by adding 0.1 mmol/L isopropyl-β-D-thiogalactoside (IPTG), which is identified by SDS-PAGE analysis and western blotting assay. The inclusion body is treated with 4.0 mol/L urea to obtain soluble His-INMAP fusion protein antibody, fusion protein is purified using Ni Sepharose High Performance, and then the protein concentration and purity are detected. The purified protein antibody is injected into 4 Balb/c mice, then blood samples are collected from their hearts, and the anti- serum is isolated. The specificity of polyclonal anti- INMAP antibodies in unpurified and purified prokaryotically expressed products are analysed. In addition, the expression difference between normal liver cell L-02 and 5 hepatoma cell (PLC, HepG2, SUN449, SMMC-7721 and BEL-7402) is determined by western blotting assay. The results show that His-INMAP fusion protein mainly exists in insoluble inclusion bodies. Soluble protein is obtained with 4.0 mol/L urea treatment to solubilise inclusion bodies. Highly protein purity (94.1%) is harvested after purification. The polyclonal anti-INMAP antibody can bind antigen specifically. Moreover, INMAP is found existing polymorphically in hepatoma cells and its gene expression is down-regulated significantly in all tested hepatoma cells except PLC cell. Obviously, in this study the anti-INMAP polyclonal antibody is of high specificity, which lays a foundation of further study of INMAP functions.
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2015, Vol. 33 
