为探究纺锤体蛋白INMAP 的功能及其在细胞恶性增殖中发挥的作用,制备INMAP 多克隆抗体.利用0.1 mmol/L IPTG于16℃诱导His-INMAP 融合蛋白进行原核表达,SDS-PAGE 及免疫印迹检测蛋白表达.4.0 mol/L 尿素处理包涵体获得可溶性融合蛋白,镍亲和柱分离纯化融合蛋白抗原并检测蛋白浓度及纯度.以所获抗原免疫4 只Balb/c 小鼠,收集心脏抗血清.以纯化前、后的原核表达产物作为抗原,检测INMAP 多克隆抗体的特异性.通过免疫印迹分析INMAP 在正常肝细胞系L-02 及5个肝癌细胞系(PLC、HepG2、SUN449、SMMC-7721 和BEL-7402)中的表达差异.结果显示,His-INMAP 融合蛋白主要以包涵体形式存在,经4.0 mol/L 尿素处理可获得可溶性目的蛋白,且纯化后的抗原纯度较高(94.1%).INMAP 多克隆抗体能与INMAP 抗原特异结合.INMAP 在6 种肝细胞中具有多态性,除PLC 外,在其他肝癌细胞中其基因表达水平显著下调.本研究制备的INMAP 多克隆抗体特异性高,为INMAP 基因功能的进一步研究奠定了基础.
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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