人趋化因子受体5N端膜外第一襻的克隆与表达及其特异抗体的制备  

Cloning and expression of human first extracellular membrane loop chemokine receptor 5 N-terminal and preparation of its specific antibody

作　　者：张莉[1] 郭葆玉[1] 苗红[1] 邱磊[1] 张冉[1] 道书艳[1] 卞广兴[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

出　　处：《第二军医大学学报》2002年第11期1201-1203,共3页Academic Journal of Second Military Medical University

基　　金：国家重点基础研究发展规划项目(G199800H14).

摘　　要：目的:获得人β趋化因子受体5(huCCR5)N端膜外第一襻基因片段的序列,并构建表达huCCR5 N端膜外第一襻的重组体,实现其有效表达及其特异抗体的鉴定。方法:计算机分析比较趋化因子超家族部分成员,定位超家族中同源性最低的N端膜外第一襻结构域。PCR扩增出该结构域114个核苷酸序列基因片段,构建融合表达载体pGEX-IN/NR5,经测序确定基因片段正确后,在大肠杆菌中表达。表达产物经纯化后免疫新西兰兔。ELISA鉴定CCR5 N端肽段特异性抗体。结果:序列测定结果与文献报道一致,经计算机作图分析ELISA实验数据证明得到抗huCCR5 N端肽段的特异性抗体。结论:本方法采用pGEX-lN在大肠杆菌DE3中有效表达CCR5 N端膜外第一襻基因片段,并实现了对其特异性抗体的鉴定,为进一步的研究奠定基础。Objective:To obtain the first extracellular domain of β chemokine receptor 5(CCR5) N-terminal gene fragment with high level expression in E.coli and to detect its specific antibody. Methods:Some β chemokine superfamily members were analyzed with computer and the least homologous domain of the extracellular loops were located. The first extracellular domain (114 nucleotides) was defined,sequenced and amplified by PCR. The expression vector pGEX-IN/NR5 of a recombi-nant GST fusion protein was constructed. After confirming the correctness of the inserted sequence,the transformation and expression of this fusion protein were performed in E. coli. The expression products were purified and used to immunize 2 New Zealand rabbits. Anti-CCR5 N-terminal antibody was detected by competed method of ELISA. Results:The sequence of the PCR product was consistent with that reported, ELISA showed the antibody was specific to huCCR5 N-terminal. Conclusion: huCCR5 N-terminal gene fragment is successfully cloned and its specific antibody is detected.

关 键 词：人趋化因子受体5 基因克隆 融合表达 抗体 制备 

分 类 号：R392－33[医药卫生—免疫学]