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张莉, 秦桂秀, 张新华, 刘克战, 吴博威, 刘清华, 焦咪, 范辰辰, 宋瑞瑞, 鲍春丽, 李瑜. 大鼠心脏kir2.2、kir2.3通道重组质粒构建及真核表达[J]. 中国公共卫生, 2014, 30(3): 367-370. DOI: 10.11847/zgggws2014-30-03-36
引用本文: 张莉, 秦桂秀, 张新华, 刘克战, 吴博威, 刘清华, 焦咪, 范辰辰, 宋瑞瑞, 鲍春丽, 李瑜. 大鼠心脏kir2.2、kir2.3通道重组质粒构建及真核表达[J]. 中国公共卫生, 2014, 30(3): 367-370. DOI: 10.11847/zgggws2014-30-03-36
ZHANG Li, QIN Gui-xiu, ZHANG Xin-hua.et al, . Construction of eukaryotic expression vectors for rat cardiac kir2.2, kir2.3 channels and their expressions in HEK293 cells[J]. Chinese Journal of Public Health, 2014, 30(3): 367-370. DOI: 10.11847/zgggws2014-30-03-36
Citation: ZHANG Li, QIN Gui-xiu, ZHANG Xin-hua.et al, . Construction of eukaryotic expression vectors for rat cardiac kir2.2, kir2.3 channels and their expressions in HEK293 cells[J]. Chinese Journal of Public Health, 2014, 30(3): 367-370. DOI: 10.11847/zgggws2014-30-03-36

大鼠心脏kir2.2、kir2.3通道重组质粒构建及真核表达

Construction of eukaryotic expression vectors for rat cardiac kir2.2, kir2.3 channels and their expressions in HEK293 cells

  • 摘要: 目的 构建大鼠心脏kir2.2、kir2.3通道真核表达质粒,并鉴定其在人胚胎肾细胞(HEK293)中的表达。方法 提取大鼠心脏组织细胞RNA,逆转录扩增kir2.2、kir2.3通道编码基因,克隆至真核表达质粒pEGFP-N1中,构建重组质粒并转染HEK293细胞,应用全细胞膜片钳法定kir2.2、kir2.3通道电流。结果 重组质粒pEGFP-N1-kir2.2及pEGFP-N1-kir2.3 经双酶切和测序证实构建正确,并在HEK293细胞中成功表达,且记录到相应通道电流。结论 成功构建了大鼠心脏kir2.2、kir2.3通道真核表达质粒,并在HEK293细胞中成功表达。

     

    Abstract: Objective To construct eukaryotic expression vectors for rat cardiac kir2.2,kir2.3 channels and to determine their expressions in HEK293 cells.Methods Rat cardiac orthologs of Kir2.2 and Kir2.3 were cloned by reverse transcriptase-PCR and were subcloned into the eukaryotic expression vector pEGFP-N1.The constructed recombinant plasmids pEGFP-N1-kir2.2 and pEGFP-N1-kir2.3 were transfected into human embryonic kidney-293(HEK293)cells.The currents of kir2.2 and kir2.3 channels were identified by whole-cell patch-clamp technique.Results Restriction analysis and sequencing proved that the recombinant plasmids pEGFP-N1-kir2.2 and pEGFP-N1-kir2.3 were constructed correctly. The Kir2.2 and Kir2.3 channels were successfully expressed in HEK293 cells and Kir2.2 current(IKir2.2)and Kir2.3 current(IKir2.3)were recorded.Conclusion The eukaryotic expression vectors for rat cardiac kir2.2 and kir2.3 channels were successfully constructed and expressed in HEK293 cells.

     

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