利用多克隆抗体检测纺锤体蛋白基因INAIAP在肝癌细胞中的表达  

Detecting expression of spindle protein gene INMAP in hepatoma cells by polyclonal antibody

作　　者：朱艳[1,2] 康璟妍 王钰[1] 孙乐 张海江 梁前进[1,2] 

机构地区：[1]北京师范大学生命科学学院,抗性基因资源与分子发育北京市重点实验室,北京100875 [2]北京师范大学生命科学学院,细胞增殖与调控生物学教育部重点实验室,北京100875 [3]兰州市第四中学,兰州730050 [4]京天成生物技术(北京)有限公司,北京101111 [5]北京康乐卫士生物技术股份有限公司,北京100176

出　　处：《科技导报》2015年第1期17-21,共5页Science & Technology Review

基　　金：《科技导报》博士生创新研究资助计划项目(kjdb2012003);北京市自然科学基金项目(5122017);北京师范大学抗性基因资源与分子发育北京市重点实验室开放基金项目(201204,201307);北京师范大学细胞增殖与调控生物学教育部重点实验室开放基金项目(201001)

摘　　要：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、Hep G2、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 with4.0 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,Hep G2,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.

关 键 词：INMAP 肝癌细胞 多克隆抗体 包涵体 

分 类 号：R735.7[医药卫生—肿瘤] Q78[医药卫生—临床医学]