抗人DcR3单克隆抗体研制及其鉴定.docVIP

抗人DcR3单克隆抗体研制及其鉴定 　 作者：吴春风, 马忠森, 王秀丽, 杨俊玲, 杨洋, 张越, 图们乌力吉, 乔俊华 【摘要】 　　目的: 制备抗人DcR3单克隆抗体（mAb）, 并鉴定其特异性。方法: 纯化的HisDcR3融合蛋白免疫小鼠, 应用淋巴细胞杂交瘤技术制备抗人DcR3 mAb并进行纯化, 纯化后的抗体经Western blot和ELISA方法鉴定其特异性、 Ig亚型和效价。结果: 获得5株稳定分泌抗DcR3 mAb的杂交瘤细胞, 均属IgG1亚型, 其腹水抗体效价为1×10-5～1×10-7, Western blot显示5株细胞分泌的mAb均可识别DcR3蛋白, 其中1株（1B1）可与SW480细胞成分反应。结论: 成功建立稳定分泌抗人DcR3 mAb的杂交瘤细胞株, 其分泌的抗体特异性强、 效价高, 为研究DcR3在组织中的表达、 分布及研制ELISA试剂盒奠定基础。 【关键词】 DcR3 单克隆抗体 制备 　　[Abstract] AIM: To prepare monoclonal antibodies(mAbs) against human DcR3 and identify their characterization. METHODS: BALB/c mice were immunized with purified HisDcR3 protein, and mAbs against DcR3 which prepared by hybridoma technique were purified and identified by their specificity, subtype, titers via ELISA and Western blot. RESULTS: Five hybridoma cell lines secreting mAbs against human DcR3 were obtained, which were determined as IgG1 subtype and ascites titers of five mAbs against DcR3 reached 1×10-5-1×10-7. Five mAbs were proved to recognize HisDcR3 protein specifically, one of which (1B1) could recognize SW480 cell. CONCLUSION: mAbs against DcR3 with high titers and specificity have been prepared and purified successfully, which laid a foundation for the study of DcR3 expression, distribution in tissu and development of ELISA kit. 　　[Keywords]DcR3; monoclonal antibody; preparation 　　诱骗受体3(decoy receptor 3, DcR3, 又称TR6)为近年新发现的一种可溶性肿瘤坏死因子受体(TNFR)超家族成员, 可竞争性结合肿瘤坏死因子(TNF)成员LIGHT、 FasL及TL1A[1, 2], 但并不介导细胞凋亡, 在细胞的生长、 分化、 死亡以及免疫调节等方面发挥重要作用, 与肿瘤、 自身免疫疾病、 移植排斥等密切相关。应用DcR3抗体研究DcR3在上述病变组织或细胞中的表达现已成为研究热点。目前, 国外已有商品化的抗人DcR3 mAb, 但价格昂贵, 国内尚无这方面的研究报道。我们运用淋巴细胞杂交瘤技术制备了抗人DcR3 mAb, 并进行了鉴定, 为研究DcR3在组织中的表达、 分布及研制ELISA试剂盒奠定基础。 　　1 材料和方法 　　1.1 材料 纯化的HisDcR3融合蛋白为本室自制[3]; RPMI1640购自Hyclone公司; PEG4000购自Merck公司; HAT、 HT购自Sigma公司; HRP标记的羊抗鼠及羊抗兔IgG抗体购自Santa Cruze公司; 抗体亚类测定试剂盒购自Serotech公司; Protein A亲和层析胶购自Pierce公司; PVDF膜购自Amresco公司; ECL化学发光试剂盒购自Amersham公司。Sp2/0细胞株取自东北师范大学细胞遗传所; 高表达DcR3的人结肠癌SW480细胞株由本室保存; BALB/c小鼠(6～8周龄, 雌性, 清洁级)购自吉林省生物制品所