人CD96基因胞外区的原核表达及抗血清制备  

Human CD96 gene cloning,expression and identification

作　　者：曾建明[1] 刘飞[2] 谭坪海[3] 王丽娜[1] 李沫[1] 陈中华[1] 李松[1] 龙一飞[1] 李有强[1] 陈茶[1] 

机构地区：[1]广东省中医院检验科,广东广州510120 [2]南方医科大学南方医院检验科,广东广州510515 [3]广东医学院检验学院,广东东莞523808

出　　处：《南方医科大学学报》2011年第7期1232-1235,共4页Journal of Southern Medical University

基　　金：广东省医学科学技术研究基金(B2008066)

摘　　要：目的原核表达并纯化人CD96胞外区蛋白,免疫家兔制备抗体。方法构建pET32-CD96重组质粒,转化大肠埃希菌BL21(DE3),以IPTG诱导表达,Ni2+-NTA树脂纯化蛋白并免疫家兔,以间接ELISA检测抗体效价,Westernblot鉴定抗体特异性。结果质粒pET32-CD96经测序鉴定,通过SDS电泳显示能在BL21(DE3)中高效表达,相对分子质量约37000;Westernblot实验结果显示制备的多克隆抗体可与HL-60细胞提取蛋白及原核表达的人CD96蛋白胞外区特异性结合。结论成功构建并表达人CD96胞外区蛋白,免疫家兔获得高滴度抗血清,为后续研究奠定了基础。Objective To construct and express human CD96 gene outer membrane domain（hCD96om） in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.Methods hCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a（＋） to construct the recombinant plasmid pET32-CD96.The expression of hCD96om was induced by IPTG in BL21（DE3） cells,and the expression product was identified by Western blotting.The anti-hCD96 polyclona1 antibody was prepared by immunization of rabbits with the fusion protein.The specificity of anti-hCD96 antibody was determined by Western blotting.Results hCD96om protein was expressed in E.coli BL21（DE3） cells in the form of inclusion body,with a relative molecular mass around 37 kD.Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 000 hCD96om fusion protein.Conclusion The CD96 gene of human has been successfully cloned and expressed in BL21（DE3） cells,and its rabbit polyclonal antibody has been obtained.

关 键 词：人CD96基因 原核表达 白血病干细胞 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]