应用单克隆抗体鉴定人类肝脏线粒体蛋白质组中的抗原优势蛋白  

Antigenic Dominant Proteins in Human Liver Mitochondrial Proteome Identified by Monoclonal Antibodies

作　　者：鞠艳芳[1] 杨金菊[2] 刘蓉[2] 柳晓兰[2] 杜雪梅[2] 刘莉[2] 陈志成[2] 迟俊[2] 刘淑娥[2] 高媛[2] 高建恩[2] 贺福初[2] 孙启鸿[2] 

机构地区：[1]中国人民解放军总医院肿瘤内科,北京100853 [2]军事医学科学院放射与辐射医学研究所北京蛋白质组研究中心,蛋白质组学国家重点实验室,北京102206

出　　处：《中国科学：生命科学》2010年第9期834-842,共9页Scientia Sinica(Vitae)

基　　金：国家重点基础研究发展计划(批准号:2006CB910803);国家高技术研究发展计划(批准号:2006AA02A311)资助项目

摘　　要：对线粒体蛋白质组的鉴定和分析有助于理解线粒体的功能和相关疾病的发病机制,包括能量代谢、凋亡、自由基产生、产热作用、钙离子信号通路等.本实验旨在鉴定人类肝脏线粒体蛋白质组中的抗原优势蛋白.用线粒体蛋白质作为免疫原,经过细胞融合、筛选和克隆,制备了240多个单克隆抗体杂交瘤细胞系.单克隆抗体识别的线粒体蛋白抗原通过人类肝脏cDNA表达文库筛选方法鉴定,相应的线粒体蛋白质的亚细胞定位通过免疫组化证实.发现了肝脏线粒体中6个抗原优势蛋白,分别被至少两种特异性的单克隆抗体所识别.这6个蛋白分别是乙酰辅酶A酰基转移酶(线粒体3-酮酯酰辅酶A硫解酶)2、醛脱氢酶1家族A1、氨甲酰磷酸合成酶1、二氢硫辛酰胺S乙酰转移酶(丙酮酸脱氢酶复合物的E2组分)、烯酰辅酶A水合酶1和羟基类固醇(11β)脱氢酶1.这些单克隆抗体有望应用于人类肝脏蛋白质组计划的相关研究,如去除优势蛋白、蛋白与蛋白之间相互作用的研究和验证等.The identification and analysis of the mitochondrial proteome would be valuable for understanding the functions of mitochondrion and the mechanisms for mitochondrion-related diseases, such as energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. To identify antigenic dominant proteins in human liver mitochondrial proteins, we generated more than 240 hybridoma cell lines using native mitochondrial proteins as the immunogens after cell fusion, screening, and cloning. The antibody-recognized mitochondrial proteins were identified by screening of human liver cDNA expression libraries. We found that 6 protein antigens were the antigenic dominant proteins in liver mitochondria with at least 2 mAbs specific for each of these protein antigens. The antigenic dominant proteins in liver mitochondria included acetyl-Coenzyme A acyltransferase 2 （mitochondrial 3-oxoacyl-Coenzyme A thiolase）, aldehyde dehydrogenase 1 family member A1, carbamyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase （E2 component of pyruvate dehydrogenase complex）, enoy! coenzyme A hydratase 1, and hydroxysteroid （11-beta） dehydrogenase 1. The subcellular localization of these proteins in mitochondria was further confirmed by immunohistocytochemistry. We believe these well-characterized antibodies would be very useful in various applications for Human Liver Proteome Project （HLPP）, such as the depletion of dominant proteins and the validation of protein-protein interactions.

关 键 词：线粒体 单克隆抗体 cDNA文库筛选 抗原优势蛋白 肝脏 

分 类 号：R392[医药卫生—免疫学]