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.

INMAP  /  hepatoma (liver cancer) cell  /  polyclonal antibody  /  inclusion body