起搏通道HCN2基因和增强型绿色荧光蛋白双基因载体在人胚胎肾细胞的共转染表达  

Cotransfection and expression of HCN2 and enhanced green fluorescent protein in human embryonic kidney cells

作　　者：刘丹妮[1] 刘铁牧[1] 姚成立[2] 刘兴亚[1] 李泱[3] 

机构地区：[1]宁夏回族自治区人民医院,宁夏回族自治区银川市750021 [2]宁夏银川市第一人民医院,宁夏回族自治区银川市750001 [3]解放军总医院老年心血管病研究所,北京市100853

出　　处：《中国组织工程研究与临床康复》2008年第42期8323-8326,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30570739)~~

摘　　要：背景:获得高效、安全的基因转移载体是目前研究的目标。目的:构建携带起搏通道(HCN2)基因和增强型绿色荧光蛋白(EGFP)双基因真核表达载体,并观察在人胚胎肾细胞(HEK293)表达情况。设计、时间及地点:单一样本观察,实验于2006-07/2007-08在全军重点实验室解放军总医院老年心血管病研究所完成。材料:质粒pIRES-EGFP为Clontech公司产品,PCI-HCN2质粒,含有人全长HCN2基因为解放军总医院老年心血管病研究所惠赠。方法:利用脑炎心肌炎病毒的内部核糖进入位点(IRES),构建HCN2与增强型绿色荧光蛋白基因真核表达载体pIRES-EGFP-HCN2。将脂质体和pIRES-EGFP-HCN2基因混合后转染人胚胎肾细胞,用反转录-聚合酶链反应检测HCN2 mRNA表达。主要观察指标:①重组质粒pIRES2-EGFP-HCN2的构建。②起搏通道基因HCN2 cDNA的克隆。③增强型绿色荧光蛋白表达的荧光检测。结果:构建的pIRES-EGFP-HCN2在转染8h后开始表达,48h达到最高。经酶切鉴定含有增强型绿色荧光蛋白和HCN2基因。结论:实验成功地构建了pIRES-EGFP-HCN2真核共表达载体,具有HCN2、增强型绿色荧光蛋白的双重活性,IRES能够介导外源基因HCN2在人胚胎肾细胞表达。BACKGROUND： To obtain highly effective and safe gene transfer vector is the aim of present study. OBJECTIVE： To construct hyperpolarization activated cyclic nucleotide gated cation channel （HCN2） and enhanced green fluorescent protein （EGFP） fusion gene eukaryotic expression vector, and to investigate its expression in human embryonic kidney cells （HEK293）. DESIGN, TIME AND SETTING： The single sample observation was performed at the Key Laboratory, Institute of Geriatric Cardiology, General Hospital of Chinese PLA from July 2006 to August 2007. MATERIALS： Plasmid plRES-EGFP was purchased from Clontech Company. PCI-HCN2 plasmid containing human full-length HCN2 gene was presented by Institute of Geriatric Cardiology, General Hospital of Chinese PLA. METHODS： Human HCN2 cDNA was inserted into mammalian expressed plasmid plRES-EGFP. The recombinant expression plasmid plRES-EGFP-HCN2 was transfected into human embryonic kidney. The mRNA expression was detected by reverse transcriptase-polymerase chain reaction after being transfected into the HEK293 cells. MAIN OUTCOME MEASURES： Construction of recombinant plasmid pIRES2-EGFP-HCN2; Clone of HCN2 cDNA; Flourescence detection of EGFP expression. RESULTS： Constructed pIRES-EGFP-HCN2 expressed at 8 hours, and reached a peak at 48 hours. Enzyme digestion demonstrated that HCN2/EGFP was expressed in the HEK293. CONCLUSION： The pIRES-EGFP-HCN2 eukaryon expression vector is successfully constructed, with double activity of HCN2 and EGFP. IRES-induced exogenous gene HCN2 can express in human embryonic kidney cells.

关 键 词：离子通道 发光蛋白质类 转染 

分 类 号：R329.251[医药卫生—人体解剖和组织胚胎学]