人核迁移蛋白单克隆抗体的制备及其在人巨核细胞中的表达  

Preparation of the monoclonal antibody against human hNudC and its expression in human megakaryocytes

作　　者：陈彦球[1] 葛怡琛[2] 杨燕[3] 

机构地区：[1]中山大学第一附属医院,广东广州510008 [2]广东省职业病防治院毒理科,广东广州510275 [3]中山大学公共卫生学院,广东广州510008

出　　处：《细胞与分子免疫学杂志》2008年第9期894-897,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：广东省自然科学基金博士启动资助项目(06300645);教育部博士点基金新教师资助项目(20070558275);中山大学2006学生业余科研项目(1163198);中山大学2007实验室开放基金项目(KF200707)

摘　　要：目的:制备人核迁移蛋白(hNudC)的单克隆抗体(mAb)并鉴定其特性。方法:从人胎肝组织中克隆得到hNudC基因,构建重组表达质粒pET-28b/hNudC,表达带有6-His标记的重组hNudC,用纯化的重组hNudC蛋白免疫BALB/c小鼠制备mAb,用ELISA筛选抗体阳性的细胞克隆,用免疫荧光染色法及Western blot试验鉴定mAb的特异性。结果:获得2株杂交瘤细胞系2C16和2D8,其分泌的mAb的Ig亚类(型)分别为IgG1和IgM(κ),杂交瘤细胞培养上清的ELISA效价分别为1∶64和1∶32;腹水mAb的效价分别为1∶1×105和1∶5×104。mAb与重组hNudC蛋白有较强的特异性反应,免疫荧光染色和Western blot试验证明,制备的mAb可以识别人巨核系白血病细胞株Dami、Meg-01和人脐带血来源的CD41+巨核细胞中表达的hNudC蛋白。结论:成功地制备了特异性抗hNudC mAb,为研究hNudC蛋白的功能、天然分布及异常表达奠定了基础。AIM： To prepare and characterize the monoclonal antibody （mAb） against human nuclear distribution C （hNudC）. METHODS： The coding region of hNudC was amplified from human embryo liver tissue and the recombinant prokaryotic expression vector pET28b was constructed. hNudC protein was expressed as a fusion protein with an Nterminal 6-His tag. The purified recombinant hNudC protein was used to immunize BALB/c mice for preparing mAb. The hybridoma ceils procuced from mAB were screened by ELISA and the specificity of mAb was analyzed by munohistochemical staining and Western blot. RESULTS： Two hybridoma cells （2C16 and 2D8） secrting mAb against hNudC were developed. The isotypes of the two mAbs were IgG1 and IgM, respectively. ELISA detection showed that the titer of mAbs, 2C16 and 2D8 was 1：64, 1：32 in cultured supernatant and 1：1 × 10^5, 1：5 × 10^4 in ascites, respectively. They could specifically bind to recombinant hNudC. The results of immunohistochemical staining and Western blot indicated that mAb could specifically recognize hNudC in human Dami, Meg-01 coil lines and human marrow CD41^＋ megakaryocytes generated from umbilical core blood. CONCLUSION： Monoclonal antibodies against hNudC with high titers and specificity have been successfully prepared, which will be useful for assessing the function, native distribution and aberrant expression of hNudC protein.

关 键 词：人核迁移蛋白 单克隆抗体 巨核细胞 

分 类 号：R392.11[医药卫生—免疫学]