一株中和人IL-17免疫活性的单克隆抗体的制备  

Preparation of a neutralizing monoclonal antibody to human IL-17

作　　者：孙阳[1] 谷学佳[1] 叶贤龙[1] 徐黎明[1] 陈睿[1] 任桂萍[1] 王秋颖[1] 丁良军[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,哈尔滨150030

出　　处：《免疫学杂志》2011年第7期606-609,614,共5页Immunological Journal

基　　金：国家自然科学基金(31001084);哈尔滨科技创新人才研究专项资金(2010RFQXN109);哈尔滨市科技创新人才发展计划(2006RFXXS002);东北农业大学创新团队发展计划(CX2001)

摘　　要：目的制备抗人IL-17单克隆抗体,并鉴定其中和活性。方法用hIL-17作为免疫和检测抗原,用间接ELISA法筛选分泌抗人IL-17抗体的杂交瘤细胞株,并对抗体进行中和活性鉴定。结果获得1株稳定分泌抗人IL-17单克隆抗体的杂交瘤细胞株,所分泌的单抗类型重链为IgG2b,轻链为κ;该株杂交瘤细胞腹水效价为1∶8.192×105;传30代及液氮中保存6个月,抗体效价稳定;Western Blot检测证明该单抗与人IL-17蛋白特异地结合;单抗亲和常数为8.192×10-9 mol/L;ELISA及Real-time-PCR检测证明该单抗能够有效地阻断人IL-17刺激Hela细胞产生IL-6的作用。结论所制备的抗人IL-17单克隆抗体具有高度的特异性、稳定性及中和活性,为针对IL-17为靶点的抗体药物的开发奠定了基础。We aim to prepare a monoclonal antibody against human IL-17,and to examine its neutralizing activity.hIL-17 was used as an immunizing and detective antigen;hybridoma cell lines secreting mAb against human IL-17 was screened by indirect ELISA;and the secreted monoclonal antibodies were characterized.A stable hybridoma cell line secreting mAb against human IL-17 was obtained,and the heavy and light chains of the secreted mAb were IgG2b and κ respectively.The titers of the ascites was 1∶8.192×105,which was remained stable after subculture of hybridoma cells for 30 passages or storage at liquid nitrogen for 6 months.Western blot test showed that the mAb could specifically bind to human IL-17,with an affinity constant（K） of 3.84×109 L/mol;ELISA and realtime PCR detection showed that the mAb could effectively block human IL-17 activity of stimulating HeLa cell to express IL-6.The prepared mAb against human IL-17 shows high specificity,stability and neutralizing activity,and lays a foundation for antibody drug development aimed at IL-17 as a target antigen.

关 键 词：人白细胞介素-17 单克隆抗体 中和活性 IL-6 

分 类 号：R392[医药卫生—免疫学]