Kir2.1_ Kir2.3嵌合体的构建与表达.docVIP

  Kir2.1/ Kir2.3 嵌合体的构建与表达# 5 10 15 20 25 30  3. 河北省人民医院临床医学研究中心，河北省老年医学重点实验室，河北石家庄 050000）） 摘要：目的：构建 Kir2.1/ Kir2.3 通道嵌合体，为进一步研究 Kir2.3 通道的调控机制打下基 础。方法：利用重叠延伸 PCR 方法构建不同的 Kir2.1/ Kir2.3 嵌合体质粒： N1P3C3， N3P1C1， N3P3C1，N1P1C3，NIP3C1，N3P1C3。将不同的嵌合体质粒分别用 NheI 线性化 后，转录为 cRNA 表达于非洲爪蟾卵母细胞，用双电极电压钳记录电流。结果：不同的 Kir2.1/ Kir2.3 嵌合体质粒构建成功，在非洲爪蟾卵母细胞上可以记录到电流表达。结论： 成功构建 Kir2.1/ Kir2.3 嵌合体并完成了嵌合体通道的异源性表达和电压钳记录。 关键词：分子药理学；Kir2.3 通道；Kir2.1 通道；嵌合体；重叠延伸 PCR 中图分类号：R966; Q73 Construction and expression of Kir2.1/ Kir2.3 chimeras ZHAO Zhiying1, ZHU Hongqian2, ZHANG Zhe3, LIU Li1, ZHANG Guohong1 (1. Department of Pharmacology, Hebei Medical University, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, The Key Laboratory of New Drug Pharmacology and Toxicology, Hebei Province, Shijiazhuang 050017,China; 2. National security corps of Public Security Department of Hebei Province, Shijiazhuang, 050000 China; 3. People’s Hospital of Hebei Province, The Clinical Trial Center, Hebei Provincial Key Laboratory of Geriatrics, Shijiazhuang 050000, China) Abstract: Objective: to construct Kir2.1 / Kir2.3 Chimeras, and this would lay a basis for a future research. Methods: Kir2.1/ Kir2.3 Chimeras were constructed by over-lap extension PCR including N1P3C3, N3P1C1, N3P3C1, N1P1C3, NIP3C1, and N3P1C3. The Chimeras would be expressed in Xenopus oocytes, and the currents of chimeras were recorded by TEVC. Results: the Chimeras between Kir2.1 and Kir2.3 were constructed successfully; the currents could be recorded and analyzed by TEVC. Conclusion: The Kir2.1/ Kir2.3 Chimeras were successfully constructed. Key words: Molecular Pharmacology; Kir2.3; Kir2.1; chimera; over-lap extension PCR 35 0 引言 内向整流钾通道 (inwardly rectifying potassium channel, Kir) 最初是 1949 年 Bernard Katz 在骨骼肌细胞上发现的一种钾通道，在各种组织中广泛分布[1]。Kir 通道至少由 7 个家 族组成，Kir2 亚族是它的重要一员，主要存在于心脏、神经系统、骨骼肌等许多组织, 并表 40 现出强烈的电压依赖性内向整流特性，这种通道的整流作用有利于维持细胞的静息电位并参 与复极化过程[2]。Kir2.1 和 Kir2.3 是 Kir2.0 家族中的重要成员，许多对 Kir2.0 家族的研究都 是以 Kir2.1 和 Ki