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科技导报  2015 

利用多克隆抗体检测纺锤体蛋白基因INMAP在肝癌细胞中的表达

DOI: 10.3981/j.issn.1000-7857.2015.01.002, PP. 17-21

Keywords: INMAP,肝癌细胞,多克隆抗体,包涵体

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Abstract:

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0.1mmol/LIPTG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.4.0mol/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4.0mol/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.

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