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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 study a novel human Kv1.5 (hKv1.5) channel / ultra-fast activating delayed rectifier potassium channel blocker (Ikur) and to prepare specific antibodies against hKv1.5 extracellular cyclic peptide segment E215 by immunoassay, And identification. Methods: Male New Zealand rabbits were randomly divided into immunized group and pseudo-immunized group. A short peptide E215 was screened from hKv1.5 extracellular loop to immunize animals as antigenic peptide. Immune group were immunized with antigen, once every two weeks, and the immunized group was immunized with 0.9% sodium chloride solution. The method was the same as before. The experiment was executed at 63 days, serum was collected, and the anti-peptide E215 antibody was purified by affinity chromatography. The production and titer of antibody in vivo were observed by enzyme-linked immunosorbent assay (ELISA). The immunofluorescence histochemistry and Western blotting were used to observe the production of antibody and human atrial myocyte membrane and hKv1.5 / Ikur channel protein Combined. Results: The results of ELISA showed that the serum antibody titers of the immunized group reached 1: 400, 1: 400, 1: 400 and 1: 1200 respectively at the 28th, 42th, , The prepared antibody could bind to the cell membrane of human atrial myocytes; Western-blot results show that this antibody can specifically bind to a 75,000 molecular weight protein (hKv1.5 / Ikur channel protein) on human atrial myocyte membrane. However, the same antibody was not found in the pseudo-immunity group. Conclusion: The antibody of E215 can specifically bind to hKv1.5 / Ikur channel in human atrial myocyte membrane, providing a new pharmacological intervention for further experiments.