Clusterin抗原的表达、抗体制备及其初步鉴定  

Preparation and characterization of antibodies against clusterin

作　　者：张迎春[1,2] 刘莹[1] 杨金菊[1] 柳晓兰[1] 迟俊[1] 高建恩[1] 唐晓波[2] 朱大岭[2] 孙启鸿[1] 

机构地区：[1]蛋白质组学国家重点实验室,北京蛋白质组研究中心抗体工程研究室,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]哈尔滨医科大学药学院,黑龙江哈尔滨150081

出　　处：《细胞与分子免疫学杂志》2008年第1期45-48,共4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(2006AA02A311);国家重点基础研究发展计划(973)资助项目(2006CB910803)

摘　　要：目的:制备Clusterin(CLU)多克隆和单克隆抗体(mAb),并进行特性鉴定。方法:以成人肝cDNA表达文库为模板,构建重组表达质粒pGEX-4T-1-CLU和PET-32a-CLU。GST-CLU融合蛋白在大肠杆菌中表达,被作为免疫原制备兔多抗和鼠mAb。采用ELISA法和Western blot鉴定抗CLU抗血清在重组蛋白和天然蛋白中的特异性。采用West-ern blot,间接免疫荧光,免疫组化鉴定mAb的特异性。结果:GST-CLU融合蛋白在相对分子质量(Mr)约54 000处呈现明显表达条带。Western blot鉴定表明,制备的抗CLU兔多克隆抗体可特异地识别成人肝总蛋白中Mr约52 000和58 000的CLU蛋白。获得9株可稳定分泌抗CLU的杂交瘤细胞株可识别重组人CLU蛋白,其中有2株可特异性结合HepG2细胞质中的蛋白,4株可特异性结合成人肝脏组织肝细胞质中的蛋白。结论:成功地制备出兔抗人CLU抗血清和9株抗CLU的mAb,为进一步研究CLU在肿瘤中的功能奠定了实验基础。AIM： To prepare rabbit polyclonal antibodies （pAb） and mouse monoclonal antibodies （mAbs） against human clusterin （CLU） and characterize these antibodies＇ properties. METHODS. CLU fragment was amplified from human liver cDNA library, and recombinant expression vectors pGEX-4T-1-CLU and PET-32a-CLU were constructed, GST-CLU fusion protein was expressed in E, coli and then used as the immunogen. Properties of antiserum against human CLU were identified by ELISA, Western blot, and the mAbs against human CLU was characterized by Westem blot, indirect immunofluorescent staining and immunohistochemistry staining, RESULTS： The GST-CLU fusion protein was highly expressed with a molecular weight of 54000. Westem blot analysis proved that the rabbit pAb could specifically recognize 52 000 and 58 000 proteins in human liver total protein. All of the nine established mAbs recognized recombinant human CLU protein, two of which specifically bound to proteins in the cytoplasm of HepG2 cells and four of which specifically bound to proteins in the cytoplasm of adult liver tissue. CONCLUSION： pAb and mAbs against human CLU were successfully prepared, which will provide efficient tools for functional studies of CLU expressed in human tumors.

关 键 词：CLUSTERIN 原核表达 多克隆抗体 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]