构建永生化人肝细胞系人端粒酶反转录酶真核表达质粒  

Construction of eukaryotic expression vector of human telomerase reverse transcriptase in immortalized hepatocytes

作　　者：赵擎[1] 孙颖[1] 胡燕[2] 侯俊[2] 游绍莉[3] 赵军[3] 辛绍杰[3] 貌盼勇[2] 

机构地区：[1]解放军总医院军医进修学院,北京市100853 [2]解放军第三○二医院传染病研究所病毒研究室,北京市100039 [3]解放军第三○二医院感染三科,北京市100039

出　　处：《中国临床康复》2006年第45期47-50,I0005,共5页Chinese Journal of Clinical Rehabilitation

基　　金：军队医药卫生"十一五"课题(06MA362);国家自然科学基金面上项目(30671849)~~

摘　　要：目的:构建人端粒酶反转录酶真核表达质粒,为人端粒酶反转录酶稳定转染细胞提供简单、快速的检测方法。方法:实验于2005-06/2006-03在解放军第三○二医院病毒研究室完成。①聚合酶链反应扩增人端粒酶反转录酶基因和绿色荧光蛋白基因。②利用基因重组技术构建真核表达质粒VR1012-GFP-hTERT和pEGFP-C1-hTERT,筛选阳性克隆进行双酶切、聚合酶链反应和序列测定。③转染HepG2细胞,荧光显微镜下观察人端粒酶反转录酶基因和绿色荧光蛋白的表达情况。结果:①聚合酶链反应扩增目的基因:电泳显示人端粒酶反转录酶基因的聚合酶链反应产物在3.5kb处有特异性目的条带,绿色荧光蛋白基因的聚合酶链反应产物在750bp处显示特异性目的条带。②质粒VR1012-GFP-hTERT和pEGFP-C1-hTERT的构建和鉴定结果:双酶切、聚合酶链反应鉴定和测序结果与预期完全相符。③转染HepG2细胞结果:荧光显微镜下观察人端粒酶反转录酶和绿色荧光蛋白的融合蛋白主要分布在细胞核内。结论:①初步验证所构建的真核表达质粒VR1012-GFP-hTERT和pEGFP-C1-hTERT的正确性。两种质粒可使转染细胞同时表达人端粒酶反转录酶和绿色荧光蛋白的融合蛋白,通过观察绿色荧光蛋白的表达对转染效率和结果进行及时、快捷的观察。②重组真核表达质粒VR1012-GFP-hTERT和pEGFP-C1-hTERT的构建成功,为人端粒酶反转录酶转染细胞提供简洁、快速、实时的检测方法,并为建立永生化人肝细胞系奠定基础。AIM： Through constructing eukaryotic expression vectors containing human telomerase reverse transcriptase, to provide an effective and fast method for evaluating transfection of human telomerase reverse transcriptase. METHODS： The experiment was conducted at the Virus Research Room, the 302 Hospital of Chinese PLA from June 2005 to March 2006. ① Human telomerase reverse transcriptase cDNA and green fluorescent protein cDNA were cloned by polymerase chain reaction （PCR）. ②The recombinant vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT were formed with gene recombination technique. The positive clones were checked up with enzymes digestion, PCR and sequencing. ③Transfection HepG2, the location and expression of human telomerase reverse transcriptase and green fluorescent protein were studied under fluorescent microscope. RESULTS： ①PCR amplification goal gene： Electrophoresis showed that the special goal strap appeared at 3.5 kb human telomerase reverse transcriptase DNA fragment and 750 bp green fluorescent protein DNA fragment. ②Construction and identification of plasmid VR1012-GFP-hTERT and pEGFP-C1-hTERT： The consequences of enzymes digestion, PCR and sequencing were match to the anticipation. ③Transfection of HepG2 cell： The fusion protein of human telomerase reverse transcriptase and green fluorescent protein distributed mainly in the nuclei of HepG2 under fluorescent microscope. CONCLUSION： ①The construction of recombinant eukaryotic expression vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT is verified successfully. These two plasmids can express fusion protein of human telomerase reverse transcriptase DNA fragment and green fluorescent protein at the same time. Transfection efficiency and result are observed timely and rapidly by observing the expression of green fluorescent protein.② The successful construction of recombinant eukaryotic expression vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT provides a effective and fast method for evaluating stable transfection of human

关 键 词：端粒 末端转移酶 发光蛋白质类 质粒 

分 类 号：R394.2[医药卫生—医学遗传学]