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抗人Kv1.5胞外环肽段E215抗体的制备与鉴定

Preparation and verification of antibodies against the peptide fragment E215 from the extracellular loop of human Kv1.5
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摘要 目的:为研究新的人Kv1.5(hKv1.5)通道/超快激活延迟整流钾通道(Ikur)阻滞剂,通过免疫法制备抗hKv1.5胞外环肽段E215的特异性抗体,并进行鉴定。方法:雄性新西兰大白兔,随机分为免疫组和假性免疫组。从hKv1.5胞外环中筛选出一段短肽E215作为抗原肽免疫动物。免疫组予抗原免疫,2周1次,假性免疫组予0.9%氯化钠溶液免疫,方法同前。实验63 d处死,留取血清,亲合层析法提取、纯化抗肽段E215抗体。酶联免疫吸附法(ELISA)动态地观察抗体在体内的产生及效价,免疫荧光组织化学及免疫印迹(Western-blot)观察制备的抗体与人心房肌细胞膜及hKv1.5/Ikur通道蛋白的结合。结果:ELISA结果表明,免疫组于实验第28、42、56、63 d血清抗体滴度分别达到1∶6 400、1∶6 400、1∶6 400、1∶12 800;免疫荧光组织化学显示,制备的抗体能结合于人心房肌细胞的细胞膜;Western-blot结果表明,此抗体能特异性结合人心房肌细胞膜上分子量为75000的蛋白(hKv1.5/Ikur通道蛋白)。而假性免疫组未发现相同抗体产生。结论:所制备胞外环肽段E215的抗体能与人心房肌细胞膜上hKv1.5/Ikur通道特异性结合,为下一步实验提供了新的药理学干预手段。 Objective:To prepare and verify antibodies against a peptide fragment E215 located at the extracellular loop of human Kv1.5(hKv1.5) by immunizing rabbits in order to explore a novel hKv1.5 or Ikur channel blocker.Method:New Zealand white rabbits were randomly divided into two groups: the immunization group(n=5) and the sham immunization group.A peptide fragment E215 from the extracellular loop of hKv1.5 channel was predicted and synthesized as an antigenic determinant in the immunization group,the antigen was used to immunize the rabbits every two weeks.0.9% NaCl solution was adopted instead in the sham immunization group.On the 63th day of the experiment the rabbits were sacrificed and the sera were obtained.The antibodies against the peptide fragment E215 were purified by affinity chromatography method.The titer of the antibodies was detected dynamically by ELISA during the experiment.Immunofluorescence technique and Western blotting were applied to determine the binding of the antibody with hKv1.5 channel.Result:In the immunization group,the titer of the antibody against the peptide fragment E215 reached to 1∶6 400,1∶6 400,1∶6 400,1∶12 800 respectively on the 28th,42nd,56th and 63th day.Immunofluorescence showed that the antibody binded on the membranes of human atrial myocytes.Western blotting indicated the antibody specifically recognized 75KD protein from human atrial myocytes membrane(hKv1.5 or Ikur channel protein).However the antibodies could not be detected in the sham immunization group.Conclusion:The antibodies against the peptide fragment E215 can specifically recognize hKv1.5 or Ikur channel,which provides a novel pharmacological tool in further experiments.
作者 戴芝银 刘坤 曾秋棠 刘金平 祝聪聪 杨晓芳 廖玉华 王敏 陈志坚 黄丹 刘鸿涛 DAI Zhiyin ,LIU Kun, ZENG Qiutang, LIU Jinping, ZHU Congcong,YANG Xiaofang ,LIAO Yuhua, WANG Min, CHEN Zhijian,HUANG Dan ,LIU Hongtao(1Department of Cardiology,2Department of Cardial Surgery,Union Hospital,Huazhong University of Science and Technolog,Wuhan,430022,China)
出处 《临床心血管病杂志》 CAS CSCD 北大核心 2010年第5期389-392,共4页 Journal of Clinical Cardiology
基金 国家自然科学基金(No:30700747) 湖北省科技攻关重大项目资助(No:2006AA301A04) 国家973重点基础研究发展规划基金资助项目(No:2007CB512000 No:2007CB512005)
关键词 抗体 人心房肌细胞 KV1.5 胞外环 antibody human atrial myocyte Kvl.5 extracellular loop
作者简介 通信作者:刘坤,Email:liukun@mail.hust.edu.cn 通信作者:曾秋棠
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