【摘要】 目的构建FLAG标记的内向整流钾通道(inwardly rectifying K channel,Kir)2.3通道蛋白,为进一步研究通道的生理功能和调节机制奠定基础。方法运用聚合酶链反应(polymerase chain reaction,PCR)技术,将FLAG标记短肽DNA碱基序列插入到Kir2.3通道氨基末端,构建重组质粒DNA,FLAG-Kir2.3-pGEMHE;采用菌落PCR方法挑取阳性克隆,表达于非洲爪蟾卵母细胞,验证加入标记后是否影响Kir2.3通道蛋白的功能;进行免疫细胞化学实验,检测FLAG标记短肽表达情况。结果经测序验证,FLAG-Kir2.3-pGEMHE重组质粒DNA构建成功,没有碱基突变。FLAG标记短肽没有影响Kir2.3通道功能,FLAG-Kir2.3成功表达于非洲爪蟾卵母细胞,双电极电压钳可以记录到电流。免疫细胞化学实验证实FLAG标记短肽表达。结论成功构建重组质粒DNA,FLAG标记短肽已与Kir2.3通道蛋白成功融合并有效表达。更多还原

【Abstract】 Objective Flag tag will be inserted into the upstream of Kir2.3 - pGEMHE DNA sequence,which will lay a basis for future research.Methods The DNA base sequence corresponding to the amino acid sequence of Flag peptide was inserted into the N - terminus of Kir2.3 by polymerase chain reaction(PCR).The recombinant plasmid DNA FLAG - Kir2.3 - pGEMHE was constructed.The correct clones were chosen by the method of colony PCR and sent for sequencing.Flag - Kir2.3 was expressed in the Xenopus oocytes.The function of FLAG tagged Kir2.3 channel was studied.Immunocytochemistry method was applied to confirm that the Flag - Kir2.3 was expressed on the membrane of the Xenopus oocytes.Results After examination by sequencing,the Flag tag was found to be inserted into the N - terminus of Kir2.3-pGEMHE.FLAG - Kir2.3 was expressed in the Xenopus oocytes successfully. Channel currents were recorded by two-microelectrode voltage clamp(TEVC).Immunocytochemistry experiments showed that the Flag tag was fused with Kir2.3 channel protein successfully.Conclusion FLAG - Kir2.3 - pGEMHE was constructed by means of PCR successfully.Moreover,the Flag tag did not affect Kir2.3 channel function.These results laid a basis for future research.更多还原