抗11β-羟类固醇脱氢酶1的单克隆抗体的制备与鉴定  

Generation and characterization of monoclonal antibody against HSD11B1

作　　者：王婉[1] 杨金菊[2] 刘莉[1] 陈志成[1] 高媛[2] 高建恩[1] 安立国[3] 孙启鸿[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所免疫学研究室,北京100850 [2]北京蛋白质组研究中心抗体工程研究室,北京102206 [3]山东师范大学生命科学学院,山东济南250014

出　　处：《细胞与分子免疫学杂志》2008年第1期38-40,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(20060102A3006);国家重点基础研究发展计划(973)资助项目(2006CB91083)

摘　　要：目的:制备抗人11β-羟类固醇脱氢酶1(homo sapi-ens hydroxysteroid 11-beta dehydrogenase 1,HSD11B1)的单克隆抗体(mAb)并初步鉴定其特性。方法:将正常成人肝组织匀浆离心并分离线粒体,用线粒体总蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb,并通过间接ELISA法、Westernblot、免疫组化的方法对mAb进行特性鉴定,通过免疫沉淀、Uni-ZAP XR肝脏cDNA表达文库筛选鉴定抗原。结果:通过间接ELISA筛选获得1株可稳定分泌抗人HSD11B1 mAb的杂交瘤细胞系。其分泌的mAb的Ig亚类(型)为IgG1(κ),Western blot结果显示该mAb可以特异地识别相对分子质量(Mr)为35 000的蛋白;应用mAb CBF245对人肝脏cDNA表达文库(Uni-ZAP XR)进行筛选,阳性噬菌斑测序结果显示5个阳性克隆插入序列均为HSD11B1。结论:mAb BAD062特异地识别抗原HSD11B1,该mAb可用于ELISA检测、Westernblot、免疫组化和免疫沉淀实验,为HSD11B1的研究提供了有力的工具。AIM： To generate and identify monoclonal antibodies （mAb） against homo sapiens hydroxysteroid 11- beta dehydrogenase 1 （ HSD11 B1 ）. METHODS： Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to generate mAb by hybridoma technique. The mAb were characterized by ELISA, Western blot and immunohistochemistry. The antibody specificity was identified by immunoprecipitation （IP）, and confirmed by Uni-ZAP expression library screening. RESULTS： One hybridoma CBF245 secreting specific mAb against HSD11 B1 was established. The Ig subclass of this mAb was IgG1, and it could be used in ELISA, Western blot, immunohistochemistry. Our data showed that the antigen recognized by CBF245 mAb was localized in the hepatocyte cytoplasm, with molecular weight of Mr 35 000 in the mitochondria of human liver tissue. The CBF245 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. CONCLUSION： A hybridoma cell line stably secreting specific mAb against HSD11 B1 is established and characterized. This mAb reacted with HSD11B1 in ELISA, Western blot, immunohistochemistry assay, IP, and would be very useful for the HSD11 B1 studies.

关 键 词：HSD11B1 单克隆抗体 线粒体 

分 类 号：R392.11[医药卫生—免疫学]