| 左广锋1 陈绍良1△ 徐艳2 肖杭2.人HCN2 真核表达载体的构建及在HEK293 细胞中的表达[J].,2011,11(6):1068-1071 |
| 人HCN2 真核表达载体的构建及在HEK293 细胞中的表达 |
| Construction of eukaryotic expressing vector of human HCN2 geneand its expression in HEK293 |
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| DOI： |
| 中文关键词: HCN4 基因 真核表达载体 转染 异源性表达 |
| 英文关键词: HCN2 gene Eukaryon expression vector Transfection Heterologous expression |
| 基金项目: |
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| 中文摘要: |
| 目的：构建含有人HCN2 基因的真核表达载体，并观察在人胚胎肾细胞（HEK293）中的表达情况。方法：对人HCN2 基因全
序列进行分析，进行oligo 设计，通过PCR，扩增HCN2 全长cDNA，通过双酶切（XhoI 和BamHI） 装入真核表达载体
pIRES2-EGFP 中，脂质体法转染入HEK293 细胞中，利用真核表达载体中带有绿色荧光蛋白GFP 报告基因,对转染效率进行监
测，采用反转录- 聚合酶链反应检测HCN2 mRNA 表达，全细胞膜片钳技术检测HCN2 通道电流。结果：测序及酶切结果表明
HCN2 基因正确，荧光显微镜下，转染细胞观察到绿色荧光，反转录- 聚合酶链反应检测到HCN2 mRNA 表达，膜片钳检测到
hHCN2 基因编码的通道电流。结论：成功地构建了HCN2 真核表达载体并进行了起搏通道HCN2 基因的异源性表达。 |
| 英文摘要: |
| Objective: To construct the eukaryon expression vector of human HCN2 gene and to investigate its expression in
human embryonic kidney cells (HEK293). Methods: HCN2 gene entire sequence was analysed and designed by oligo. cDNA encoding
human HCN2 gene was amplified by polymerase chain reaction, and digested by the restriction endonucleases Xho1 and BamHI, then
inserted into eukaryotic expressing vector pIRES2-EGFP. pIRES2- HCN2-EGFP was transfected into HEK293 cells by Lipofacta
mine2000. The transfection rate of target gene was determined by the green fluorescent protein (GFP) expression in the eukaryotic expressing
vector and the mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Whole-cell patch clamp was
used to detected whether the hHCN2 gene was transfected into HEK293cells. Results: The HCN2 gene was completely accurate, GFP
was observed in the transfected 293 cells under a fluorescent microscope, HCN2 mRNA expression was confirmed by RT-PCR, Whole
cell patch clamp recorded ionic currents of transfected hHCN2. Conclusion: The pIRES-HCN2-EGFP eukaryon expression vector was
successfully constructed, and pacemaker channel HCN2 gene heterologous expression was acquired. |
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