人宫颈癌致癌基因的原核表达与多克隆抗体制备  

Prokaryotic Expression of Human Cerivical Cancer Oncogene, Preparation of Polyclonal Antibodies against HCCR Protein

作　　者：孙慎侠[1] 张革[2] 伦永志[1] 王芳[1] 

机构地区：[1]大连大学医学院,辽宁大连116622 [2]中山大学药学院,广东广州510080

出　　处：《中国医疗前沿》2008年第15期3-5,共3页China Healthcare Innovation

摘　　要：目的构建人宫颈癌致癌基因HCCR-1167-360片段的重组质粒,在原核细胞中表达并鉴定。方法从培养的人肝癌细胞株HepG2中提取总RNA,经RT-PCR扩增出HCCR-1167-360的cDNA片段,将其克隆到pGEM-T-Easy克隆载体中,将测序正确的HCCR-1167-360基因连接到pET30a(+)表达载体中,IPTG诱导在BL21中进行表达,用SDS-PAGE检测表达产物。采用初步纯化和割胶纯化方法,获得较纯的重组蛋白,采用弗氏佐剂方法免疫兔子,收获多克隆抗体,用Wastern blot鉴定重组蛋白的免疫原性。结果经测序证明获得了HCCR-1167-360基因片段。SDS-PAGE分析,HCCR-1167-360在BL21中获得表达,其相对分子质量为26kD左右,主要以包涵体形式存在。免疫兔子获得了HCCR-1167-360多克隆抗体,经Wastern blot检测结果表明获得了特异性的多克隆抗体。结论获得具有抗原性的HCCR-1167-360原核表达蛋白。Objective To construct the recombinant plasmides of human cerivical cancer oncogene (HCCR) and express fusion proteins in E.coli BL21. Develop the polyclonal antibody against HCCR-1167-360. Methods HCCR-1167-360 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-30a+, then expresed in E.coli BL21 which was induced by IPTG. Recombinant protein was purified by SDS-PAGE ,then its antigenicity was analyzed by Western blot analysis. Polyclonal antibodies was developed by immunizing hea...

关 键 词：HCCR RT-PCR 基因表达 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]