抗丙酮酸脱氢酶复合体E2亚基单克隆抗体的制备与鉴定  

Preparation and characterization of monoclonal antibody against PDC-E2

作　　者：刘莉[1] 鞠艳芳[1] 杨金菊[2] 柳晓兰[1] 高媛[2] 王婉[3] 刘蓉[1] 曲海霞[4] 陈志成[1] 刘莹[2] 高建恩[1] 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,济南250014 [4]第二军医大学东方肝胆医院,上海200438

出　　处：《免疫学杂志》2008年第1期90-92,共3页Immunological Journal

基　　金：国家科技攻关计划资助(2004BA711A20、2002BA711A11);国家重点基础研究发展规划(973)资助(2001CB510208)

摘　　要：目的制备抗人丙酮酸脱氢酶复合体E2亚基(pyruvate dehydrogenase complex E2,PDC-E2)的单克隆抗体(mAb)并进行初步鉴定。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、Western blot及免疫组化的方法对mAb进行特性鉴定,通过免疫沉淀联合质谱、Uni-ZAP XR肝脏cDNA表达文库筛选鉴定抗原。结果获得1株可稳定分泌抗人PDC-E2mAb的杂交瘤细胞系。mAb的Ig亚类(型)为IgG1(κ),该mAb可用于ELISA检测、Western blot、免疫组化和免疫沉淀实验。结论成功制备了抗人PDC-E2的mAb,为PDC-E2的研究提供了有力的工具。Objective To prepare monoclonal antibodies （mAbs） against pyruvate dehydrogenase complex E2 （PDC-E2）. Methods Normal human liver tissues were homogenized and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice for preparation of rnA~ by hybridoma technique. The mAbs were analyzed by ELISA, Western blot, and immunohistochemistry. The antibody specificities were identified by immunoprecipitation （IP） and confirmed by Uni- ZAP expression library screening. Results One hybridoma AEBO18 secreting specific mAb against PDC-E2 was established. The Ig subclass of this mAb was IgG1. The mAB could be used in ELISA, Western blot, immunohistochemistry, and IP. The protein recognized by AEBO18 was about Mr 70000 and localized in bile duct and hepatocytes of human liver tissue. Conclusion A hybridoma cell line stably secreting specific mAb against PDC-E2 is established. The specific mAb against PDC-E2 would be very useful for the studies of PDC-E2 functions and distribution.

关 键 词：PDC-E2 单克隆抗体 线粒体 

分 类 号：R392[医药卫生—免疫学]