抗人FXYD6特异性多肽单克隆功能性抗体制备及生物学作用鉴定  

Preparation and biological identification of monoclonal and functional antibody against human specific polypeptide FXYD6

作　　者：陈雄飞[1] 袁俊建[1] 张执全[1] 郭忠健[2] 郭尧 刘汝海[1] 李凤山[1] CHEN Xiongfei;YUAN Junjian;ZHANG Zhiquan;GUO Zhongjian;GUO Yao;LIU Ruhai;LI Fengshan(Department of General Surgery Cangzhou Municipal Central Hospital,Cangzhou,Hebei 061001,China;Department of Pathology Cangzhou Municipal Central Hospital,Cangzhou,Hebei 061001,China;Central Laboratory,Cangzhou Municipal Central Hospital,Cangzhou,Hebei 061001,China)

机构地区：[1]河北省沧州市中心医院普外一科,061001 [2]河北省沧州市中心医院病理科,061001 [3]河北省沧州市中心医院中心实验室,061001

出　　处：《重庆医学》2018年第10期1309-1313,共5页Chongqing medicine

摘　　要：目的研制抗人转运调节因子(FXYD)6功能区单克隆抗体库,筛选分泌胞内、胞外区单克隆抗体的杂交瘤细胞株并对鉴定胞外区单克隆抗体生物学作用。方法原核表达并纯化去除跨膜区域FXYD6功能区重组蛋白,FXYD6重组蛋白免疫BALB/c小鼠,将免疫后的小鼠脾细胞与SP2/0骨髓瘤细胞融合,多次筛选及克隆化,建立稳定分泌抗人FXYD6胞外区或胞内区的单克隆抗体杂交瘤。分别应用间接酶联免疫吸附法(ELISA)、Western blot和免疫组织化学法鉴定抗体特异性及亚型,细胞流式技术筛选可识别天然构象的胞外区单克隆抗体,用腹水诱导法对胞外区单抗进行制备、纯化并检测亲和常数,高表达FXYD6的HepG2细胞系检测胞外区单抗的功能。结果成功获得分泌抗人FXYD6胞外区或胞内区单克隆抗体的杂交瘤细胞库,制备出具有功能阻断性的胞外区单抗。结论制备的抗人FXYD6胞外区单抗可抑制HepG2细胞的增殖。Objective To study and prepare the monoclonal antibody library against human FXYD6 functional region,to screen the hybridoma cell lines secreting the monoclonal antibodies against intracellular or extracellular region of human FXYD6,and to identify the biological function of monoclonal antibody against extracellular domain.Methods FXYD6 functional region recombinant protein which did not contain the transmembrane region was prokaryotically expressed,purified,and FXYD6 recombinant protein was used to immunize BALB/c mice.Then splenocytes after immunization were fused with myeloma cells SP2/0.After several rounds of screening and cloning,the hybridomas which secreted the antibodies against the extracellular domain or the intracellular domain of human FXYD6 were established.The antibody specificity and subtype were identified with indirect ELISA,western blot and immunohistochemistry.The monoclonal antibodies against the extracellular domain which recognized the native conformation were screened with flow cytometry.The antibody against extracellular region was prepared with the ascites revulsion method and purified.The affinity constants were measured with indirect ELISA.The function of extracellular monoclonal antibody was detected by HepG2 cell line with high expression of FXYD6.Results The hybridoma cell library which secreted the monoclonal antibody against extracellular domain or the intracellular domain of human FXYD6 was successfully obtained,and extracellular region monoclonal antibodies with the functional blocking were prepared.Conclusion The prepared anti-human FXYD6 extracellular monoclonal antibodies could inhibit HepG2 cell proliferation.

关 键 词：转运调节因子6 单克隆抗体 制备 生物学鉴定 

分 类 号：R73-3[医药卫生—肿瘤]