鼠抗人CMTM7单克隆抗体的制备和鉴定  

Preparation and characterization of monoclonal antibodies against CMTM7

作　　者：许兰俊[1] 张婷[2] 邵陆宁[1] 宋泉声[1] 韩文玲[1] 陈英玉[1] 

机构地区：[1]北京大学基础医学院免疫学系,北京100083 [2]北京大学人类疾病基因研究中心,北京100083

出　　处：《北京大学学报（医学版）》2008年第3期236-240,共5页Journal of Peking University:Health Sciences

基　　金：国家高技术研究发展计划专项经费(2006AA02A305)资助~~

摘　　要：目的：制备鼠抗人CMTM7（CKLF-like MARVEL transmembrane domain containing 7）蛋白的单克隆抗体，探讨CMTM7蛋白的分子结构和生物学功能。方法：通过生物信息学对CMTM7的蛋白序列进行分析，选取了3段序列合成多肽并与钥孔戚血蓝素（keyhole limpet hemocyanin，KLH）偶联，混合后免疫Balb／c小鼠。取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2／0进行常规融合，间接ELISA方法进行筛选和有限稀释克隆化，获得鼠抗CMTM7蛋白单克隆抗体的杂交瘤细胞株，用Westernblot、免疫荧光以及免疫细胞化学等方法对其特性进行鉴定。结果：成功地建立了2株稳定分泌抗CMTM7蛋白的单克隆抗体杂交瘤细胞株，分别命名为MC9和2C9，其免疫球蛋白亚类均为IgG1。MC9单抗识别的抗原表位位于CMTM7氨基酸残基163～175区域（CMTM7163-175），可用于Western blot、免疫荧光和免疫细胞化学分析（immunocytochemistry，ICC）。2C9单抗识别的抗原表位位于CMTM7氨基酸残基19～44区域（CMTM719-44），该单抗只能用于Westernblot分析。初步的功能研究提示，CMTM7蛋白在植物血凝素（phytohemagglutinin，PHA）活化早期的外周血淋巴细胞中表达上调。结论：获得了特异性好、能够识别不同抗原表位以及不同用途的鼠抗人CMTM7蛋白的单克隆抗体，为研究CMTM7蛋白的分子结构以及功能特性奠定了基础。Objective：To obtain monoclonal antibodies membrane domain containing 7 ） for further study of the against CMTM7 （CKLF-like MARVEL transstructure and biological function of CMTM7. Methods： Three polypeptides were synthesized based on the bioinformatics analysis of the CMTM7 and coupled with keyhole limpet hemocyanin （KLH）. Balb/c mice were immunized with these mixed CMTM7 polypeptides. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Resulting hybridomas producing anti-CMTM7 antibodies were screened by enzyme- linked immunosorbent assay （ELISA）. The specificities of these monoclonal antibodies were determined by Western blot, immunofluorescecence and immunocytochemistry （ICC）. Results： Two hybridoma cell lines （MC9 and 2C9） stable in secreting anti-CMTM7 monoclonal antibodies （MAbs） were generated. Both of them produced immunoglobulin G1 （IgG1） against CMTM7. 2C9 and MC9 recognized a region between amino acid residues 19 -44 and 163 - 175 of CMTM7, respectively. MC9 antibody could be used for Western blot, immunofluorescecence and immunocytochmistry assay. However, 2C9 antibody could be only used for Western blot. Data obtained from immunofluorescence and ICC indicated that CMTM7 protein expression was upregulated in the early stage of lymphocyte activation treated with phytohemagglutinin （PHA）. ConcLusion： Monoclonal antibodies of high specificity against CMTM7 have been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of CMTM7.

关 键 词：趋化因子类 CMTM7 抗体 单克隆 肽类 杂交瘤 

分 类 号：R392.1[医药卫生—免疫学]