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 gene and its expression in HEK293

作　　者：左广锋[1] 陈绍良[1] 徐艳[2] 肖杭[2] 

机构地区：[1]南京医科大学附属南京市第一医院心内科,江苏南京210006 [2]南京医科大学江苏省医药动物实验基地,江苏南京210029

出　　处：《现代生物医学进展》2011年第6期1068-1071,共4页Progress in Modern Biomedicine

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。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 ex-pressing 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.

关 键 词：HCN4基因 真核表达载体 转染 异源性表达 

分 类 号：R54[医药卫生—心血管疾病] Q75[医药卫生—内科学]