人单核细胞趋化因子受体5(CCR5)N-端膜外第一襻特异抗体F(ab′)_2的制备及鉴定  

The preparation of first extracellular membrane loop1 of human monocyte chemotactic protein receptor 5 N- terminal's specific antibody F(ab′) 2

作　　者：张莉[1] 郭葆玉[2] 苗红[1] 张冉[1] 

机构地区：[1]第二军医大学药学院生化药学教研室,上海200433 [2]上海国和路325号生化药学教研室,上海200433

出　　处：《中国免疫学杂志》2003年第4期236-239,共4页Chinese Journal of Immunology

摘　　要：目的 :研究人 β趋化因子受体 5 (huCCR5 )NH2 端膜外第一襻特异抗体F(ab′) 2 的制备及鉴定方法。方法 :计算机分析定位超基因家族中同源顺序最低的结构域NH2 端膜外第一襻 ,PCR扩增出该基因片段后克隆到原核表达载体pGEX 1N中 ,经测序鉴定正确后 ,在E coli中表达 ,纯化后免疫新西兰白兔。经A蛋白 SepharoseCL 4B亲和层析纯化后 ,用胃蛋白酶消化IgG ,经S 2 0 0凝胶过滤柱分离制备F(ab′) 2 。结果 :经还原和非还原聚丙烯酰胺凝胶电泳和ELISA阻断实验分析证明得到抗huCCR5NH2 端的特异性抗体。结论 :这一简捷快速的特定功能结构域抗体F(ab′) 2 的制备方法 ,对研究该基因在体内的表达及生物学功能提供了重要的实验材料 ,同时也对超基因家族中亚家族特异抗体研制提供了一种研究思路和方法。Objective:To prepare the first extracellular domain of human β chemokine receptor 5(huCCR5)NH 2 terminal's specific antibody F(ab′) 2 and its detection method Methods:Use computer analyzed and located the least homologous domain of the extracellular first loops The gene fragment was amplified by PCR and cloned into the pGEX IN vector A recombinant GST fusion protein was constructed After comfirming the correctness of the inserted sequence,the transformation and expression of this fusion protein were performed in E coli The expression products of the fusion protein were purified and 2 New Zealand rabbits were immunized An anti huCCR5 NH 2 terminal antibody F(ab′) 2 was prepared by protein A Sepharose CL 4B affinity chromatography ,pepsin digestion and S 200 column seperation.Results:Reduced,unreduced SDS PAGE and ELISA block examination analysis demonstrated that this F(ab′) 2 had high specificity to combine with huCCR5 Conclusion:In this paper,not only introduce a simple and quick method to get a specific antibody F(ab′) 2 of certain functional domain but also a good idea and technique to study other high similar superfamily members

关 键 词：人单核细胞趋化因子受体5 融合蛋白 抗体 制备 

分 类 号：R392.12[医药卫生—免疫学]