降钙素基因相关肽受体单克隆抗体的制备与初步鉴定  

Preparation and Characterization of Monoclonal Antibodies Specific for Calcitonin Gene-Related Peptide Receptor

作　　者：王佳星 许永亮 付楚溪 张丹雨 薛冲 张惟材 王升厚[1] 熊向华 WANG Jia-Xing;XU Yong-Liang;FU Chu-Xi;ZHANG Dan-Yu;XUE Chong;ZHANG Wei-Cai;WANG Sheng-Hou;XIONG Xiang-Hua(Shenyang Normal University,Shenyang 110034;Beijing Institute of Biotechnology,Beijing 100071;Liaoning University Academy of Science,Shenyang 110036,China)

机构地区：[1]沈阳师范大学生命科学学院,辽宁沈阳110034 [2]军事科学院军事医学研究院生物工程研究所,北京100071 [3]辽宁大学生命科学学院,辽宁沈阳110036

出　　处：《生物技术通讯》2019年第6期775-779,785,共6页Letters in Biotechnology

摘　　要：目的:制备与鉴定抗降钙素基因相关肽受体(CGRPR)单克隆抗体。方法:构建GST-CGRPR胞外区融合表达载体,诱导表达后经GST亲和层析得到的重组蛋白作为抗原免疫BALB/c小鼠,利用杂交瘤技术筛选阳性细胞株,通过腹水制备鼠源单克隆抗体,采用ELISA、SDS-PAGE、Western印迹和抗体分型试剂盒等方法进行分析鉴定。结果:纯化得到的GST-CGRPR胞外区融合蛋白纯度大于90%,筛选到2株针对CGRPR的单克隆抗体1F9、1F10,轻、重链均分别为κ型、IgG1型,细胞上清效价分别为1∶3200、1∶1600,腹水效价都达到105,抗体纯度均达到90%以上,且都以线性表位结合GST-CGRPR,亲和力常数分别为3.95×10^-8、1.49×10^-8 mol/L。结论:获得2株具有高亲和力针对CGRPR的单克隆抗体,为偏头痛治疗药物研发奠定了基础。Objective:To prepare and identify monoclonal antibodies against calcitonin gene-related peptide receptor(CGRPR).Methods:A GST-CGRPR extracellular domain fusion expression vector was successfully constructed.After induced expression,the recombinant protein was obtained by GST affinity chromatography,which was further used as an antigen to immunize BALB/c mice.Mouse monoclonal antibodies were screened from positive hybridoma lines and prepared by ascites,which were analyzed and identified by ELISA,SDS-PAGE,Western blot,and antibody typing kit.Results:The purity of GST-CGRPR extracellular domain fusion protein was over 90%.Two monoclonal antibodies(1F9,1F10)against CGRPR were successfully screened,and the titers of the corresponding cell culture supertenant were 1∶3200 and 1∶1600,respectively.Our results showed that their light chain belongs toκtype,and heavy chain belongs to IgG1 type.The ascites titer achieved 105,the purified monoclonal antibodies both bind to GST-CGRPR with a linear epitope.Their affinity constants are 3.95×10^-8 and 1.49×10^-8 mol/L,respectively.Conclusion:Two high-affinity monoclonal antibodies against CGRPR were obtained,which laid a foundation for the development of migraine drugs.

关 键 词：降钙素基因相关肽受体 抗原 单克隆抗体 杂交瘤 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]