作　　者： ; ; ;

机构地区： 广东省人民医院

出　　处： 《医学研究生学报》 2013年第11期1125-1128,共4页

摘　　要： 目的目前认为KCNA5通道蛋白是治疗房颤的一个理想靶点,文中旨在验证FHL2蛋白与KCNA5基因编码的心脏Kv1.5钾通道是否存在相互作用。方法①质粒构建:应用PCR法构建质粒pcDNA3.1/myc-His C-KCNA5和pcDNA3.0-FHL2;②基因转染:应用Lipofectamine 2000将pcDNA3.1/myc-His C-KCNA5质粒和pcDNA3.0-FHL2质粒共转染HEK293细胞;③免疫共沉淀:应用抗FHL2抗体和Protein A/G Plus-Agarose沉淀目的蛋白,应用抗Myc抗体进行Western blot分析;④免疫荧光细胞化学分析:应用抗FHL2一抗和标记有FITC的二抗检测FHL2蛋白,应用抗Myc一抗和标记有Cy3的二抗检测KCNA5-Myc融合蛋白。结果①酶切和测序结果表明质粒pcDNA3.1/myc-His C-KCNA5和pcDNA3.0-FHL2构建正确;②抗FHL2抗体能够将KCNA5从总蛋白中沉淀下来;③KCNA5蛋白和FHL2蛋白共定位于细胞膜上。结论初步证实FHL2蛋白与KCNA5蛋白存在相互作用。 Objective KCNA5 channel protein is thought of as a desirable target for the treatment of fibrillation atrial. The aim of this study was to identify the interaction between protein FHL2 and cardiac Kvl. 5 potassium channel encoded by the KCNA5 gene. Methods We construct peDNA3.1/mye-His C-KCNA5 plasmid and pcDNA3.0-FHL2 plasmid with the PCR method, and then cuhured HEK293 cells and co-transfected them with the two plasmids using Lipofectamine 2000. We precipitated the interacting protein with anti-FHL2 antibodies and Protein A/G Plus-Agarose, conducted Western blot analysis using anti-Myc antibodies, and de- tected the FHL2 protein with anti-FHL2 primary antibodies and FITC-eonjugated secondary antibodies, as well as the KCNA5-Mye fu- sion protein with anti-Myc primary antibodies and Cy3-conjugated secondary antibodies. Results The successful construction of the plasmids pcDNA3.1/myc-His C-KCNA5 and pcDNA3. O-FHL2 was verified by sequencing and restriction enzyme digestion analysis. KCNA5 was precipitated from the total protein by anti-FHL2 antibodies. The KCNA5 and FHL2 proteins were co-localized on the cell membrane. Conclusion Co-immuoprecipitation and immunofluorescence cytochemistry manifested an interaction between the KCNA5 and FHL2 proteins.

关 键 词： 钾通道 免疫共沉淀 免疫荧光细胞化学

领　　域： [文化科学]