ANKIB1单克隆抗体的制备及应用  

Development and application of monoclonal antibody against ANKIB1

作　　者：杨子善[1] 谢云飞[1] 解博红[1,2] 任太芳[1,3] 王永淑[1,3] 丰慧根[1] 刘世饶[1] 马振玲[1] 

机构地区：[1]新乡医学院生命科学技术学院,河南省"遗传性疾病与分子靶向药物"高校重点实验室培育基地,新乡453003 [2]新乡医学院基础医学院免疫学教研室,新乡453003 [3]新乡医学院第三附属医院妇产科,新乡453003

出　　处：《中国免疫学杂志》2013年第7期745-749,共5页Chinese Journal of Immunology

基　　金：新乡医学院博士科研启动基金资助

摘　　要：目的:制备抗人ANKIB1的小鼠单克隆抗体。方法:PCR扩增人ANKIB1(1-480)的编码序列,构建pGEX-5X-ANKIB1(1-480)表达载体。在大肠杆菌中诱导表达GST-ANKIB1(1-480),利用谷胱甘肽琼脂糖凝胶纯化重组蛋白。使用纯化蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,筛选杂交瘤细胞株,通过ELISA、Western blot和免疫沉淀实验鉴定抗体,并检测ANKIB1在哺乳动物细胞中的表达。结果:获得了一株杂交瘤细胞株(7A9),所分泌的抗体对人、大鼠和小鼠ANKIB1均具有很高的灵敏性和特异性。ANKIB1在哺乳动物细胞中高表达。结论:成功制备了抗ANKIB1的单克隆抗体,ANKIB1在人、大鼠和小鼠脑以及人胚肾细胞293ET中均有较高水平的表达。Objective：To develop and characterize monoclonal antibody against human ANKIB1.Methods： The coding DNA sequence of human ANKIB1 aa 1-480 was amplified by PCR and inserted into the prokaryotic expression vector pGEX-5X-3.The recombinant plasmid was transformed into E.coli BL21（DE3）.After induction with IPTG,the fusion protein GST-ANKIB1（1-480） was purified by Glutathione-sepharose 4B affinity chromatography and used to vaccinate BALB/c mouse.Splenocytes cells were fused with SP2/0 myeloma cells,and hybridoma cells were screened.Monoclonal antibodies（McAbs） secreted from the hybridomas were characterized through ELISA,Western blot and immunoprecipitation.Expression of ANKIB1 in mammalian cells was analysed by Western blot.Results： A hybridoma（7A9） secreting antibodies against human ANKIB1 was developed.The McAb was demonstrated to have high sensitivity and specifity to ANKIB1 from human,rat and mouse.ANKIB1 was demonstrated of high expression in mammalian cells.Conclusion： Monoclonal antibody against ANKIB1 was successfully prepared.ANKIB1 is expressed at high level in human,rat and mouse brain and human embryonic kidney（HEK）293ET cells.

关 键 词：ANKIB1 单克隆抗体 表达 RBR结构域 

分 类 号：R392.33[医药卫生—免疫学]