LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备  

Prokaryotic Expression of LINE-1 Encoded Protein L1-ORF1 and Preparation of Anti-L1-ORF1 Antibody

作　　者：胡明明[1,2] 朱运峰[1] 王越[2] 王宇[2] 张亮[2] 高旭东[1] 董金凯 于继云[2] 

机构地区：[1]军事医学科学院附属医院,北京100071 [2]军事医学科学院基础医学研究所,北京100850 [3]中国人民锵放军总医院,北京100853

出　　处：《中国生物工程杂志》2010年第10期7-11,共5页China Biotechnology

基　　金：国家科技重大专项基金(2008ZX10002-003);国家自然科学基金(30772002)资助项目

摘　　要：目的:制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。Aim：to detect L1-ORF1,an ubiquitous protein in tumor tissue,its polyclonal antibody with rabbits were prepared.Methods：L1-ORF1 gene was prepared by PCR amplification and cloned into pGEX-6P-1 with GST fusion.After expressing in E.coli,GST-L1-ORF1 fusion protein was purified by Glutathione Sepharose 4B and subjected to immunity with rabbits.The titer of anti-serum was measured by ELISA and the specificity was examined by Western blot with the cell lysates derived from HepG2 tumor cells.Result：The anti-L1-ORF1p antibody exhibits a high sensitivity and specificity,which was evaluated by L1-ORF1p transfected normal cells and tumor cells.Conclusion：Anti L1-ORF1p polyclonal antibody with high sensitivity and specificity were successfully acquired.

关 键 词：L1-ORF1p 原核表达 蛋白纯化 多克隆抗体 间接免疫荧光 

分 类 号：R730.21[医药卫生—肿瘤]