人TEM7膜外段单克隆抗体的制备及其在不同来源肿瘤组织中的定位研究  

Preparation of monoclonal antibody against human tumor endothelial marker-7 extracellular fragment and study of the localization of its protein in carcinoma of different origins

作　　者：张琳[1] 郑莉[1] 任加强[1] 陈琦[1] 朱虹光[1] 

机构地区：[1]复旦大学上海医学院病理学系,上海200032

出　　处：《中华微生物学和免疫学杂志》2004年第10期780-784,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的　利用制备的抗人肿瘤血管内皮标志物 7(TEM7)单克隆抗体通过免疫组织化学的方法 ,检测TEM7蛋白在不同来源的肿瘤组织中的定位 ,为以TEM7为靶标的肿瘤血管导向治疗提供必要的理论基础。方法　利用PCR的方法 ,将TEM7膜外段重组到原核表达载体pET 32b中 ,在大肠杆菌中表达 ,蛋白经离子吸附层析纯化后免疫雌性BALB/c小鼠 ,传统的杂交瘤融合技术制备鼠抗人TEM7膜外段单克隆抗体。以酶联免疫吸附实验 (ELISA)、Westernblot筛选及鉴定分泌特异性抗人TEM7膜外段单克隆抗体的杂交瘤细胞株。最后采用免疫组织化学ABC的方法 ,分别对不同来源的70例肿瘤组织及癌旁正常组织进行了TEM7蛋白定位的研究。结果　制备了抗人TEM7单克隆抗体 ,经过Westernblot鉴定具有高度的特异性。对肿瘤及癌旁正常组织免疫组织化学ABC染色的结果可见TEM7蛋白定位于 :大肠癌、胃癌、乳腺癌、肺癌、肝细胞癌、肾细胞癌和前列腺癌的血管内皮细胞 ;大肠癌、胃癌、乳腺癌、肺癌和肝细胞癌的肿瘤细胞 ;大肠癌、胃癌和乳腺癌的血管平滑肌细胞 ;胃癌的胃壁平滑肌细胞 ;肝细胞癌的胆管上皮细胞。结论　成功制备了特异性抗人TEM7膜外段的单克隆抗体 ;TEM7在肿瘤组织中并非特异性地表达于肿瘤血管内皮细胞 ,还可见于肿瘤组织的癌细胞。Objective To prepare monoclonal antibody specifically against human tumor endothelial marker-7(TEM7), then to examine the localization of TEM7 protein in solid tumors and related normal tissues of different origins by immunohistochemical staining in order to provide a valuable theoretical basis for TEM7-based vascular targeting cancer therapy. Methods TEM7 extracellular cDNA fragment was cloned with PCR to pET-32b vector and expressed in E. coli BL21(DE3). The purified TEM7 fusion protein was used to immunize BALB/c mice and monoclonal antibody against TEM7 extracellular fragment was prepared. The hybridoma cells were primarily screened by enzyme-linked immunoabsorbent assay (ELISA), and identified by Western blot. With the previous prepared monoclonal antibody, tumor tissues and related normal tissues were examined immunohistochemically(ABC method) for the localization of TEM7 protein. Results A specific monoclonal antibody against human TEM7 extracellular domain was characterized by Western blot and human TEM7 protein was detected in different tumor tissue. Conclusion The TEM7 isn′t only present in tumor endothelia, but also in cancer cells, even in some normal tissues. Our results suggested that to examine the protein markers may be a more effective and reliable way in the research of vascular targeting cancer therapies.[

关 键 词：单克隆抗体 肿瘤组织 肿瘤血管 胃癌 大肠癌 肝细胞癌 乳腺癌 蛋白定位 雌性 原核表达载体 

分 类 号：R73-3[医药卫生—肿瘤]