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pEGFPN1-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。
Objective To construct eukaryotic expression vectors for rat cardiac kir2. 2, kir2. 3 channels and to deter- mine 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-NI-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 a- nalysis and sequencing proved that the recombinant plasmids pEGFP-NI-ldr2. 2 and pEGFP-NI-kir2. 3 were constructed correctly. The Kir2. 2 and Kir2. 3 channels were successfully expressed in HEK293 cells and Kir2. 2 current（ IKi^2.2 ） and Kit2. 3 current（ IKir2.3 ） were recorded. Conclusion The eukaryotic expression vectors for rat cardiac kix2. 2 and kir2. 3 channels were successfully constructed and expressed in HEK293 cells.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2014年第3期367-370,共4页
Chinese Journal of Public Health
基金
国家自然科学基金(31200864)
山西省卫生厅科技攻关项目(2011055)
山西医科大学大学生创新基金
