人HP1αpEGFP-N1真核表达载体的构建及其表达和定位.pdfVIP

泸州医学院学报 2016年 第39卷 第6期 Journal ofLuzhou MedicalCollege Vol.39 No.6 2016 537 论 著 人HP1琢/pEGFP-N1真核表达载体的构建及 其表达和定位* 1 1 1 1,2 吴 婷 袁杨 茂 袁何 涛 袁杨文理 渊西南医科大学基础医学院: 肿瘤医学研究所曰 生物化学教研室,四川泸州 646000冤1 2 摘 要 目的院用增强型绿色荧光蛋白作为标记物袁观察人类异染色质相关蛋白HP1琢在HeLa细胞中的表达及定位遥方法院 用RT-PCR方法扩增获得HP1琢基因编码序列袁将其定向克隆至含增强型绿色荧光蛋白渊enhanced greenfluorescence protein, EGFP 冤的真核表达载体pEGFP-N1 中袁构建HP1琢/pEGFP-N1荧光蛋白融合基因表达载体遥 并由脂质体介导将其转染HeLa 细胞袁 荧光显微镜观察其表达情况及亚细胞定位遥 结果院 双酶切与测序结果表明目的基因序列完全正确曰 荧光观察结果证实 HP1琢主要存在于细胞核内遥 结论院成功构建了目的基因HP1琢的真核表达载体袁并在HeLa细胞中获得有效表达和定位遥 关键词 异染色质相关蛋白曰绿色荧光蛋白曰基因表达定位曰HeLa细胞 中图分类号 R78 文献标志码 A doi:10.3969/j.issn.1000-2669.2016.06.012 ConstructionandexpressionofhumanpEGFP-N1-HP1琢in eukaryotic cells 1 1 1 1,2 WuTing,Yang Mao,HeTao,Yang Wenli 1 2 Institute for Cancer Medicine, Department of Biochemistry and Molecular Biology, School of Preclinical Medicine, SouthwestMedical University,Luzhou, SichuanProvince 646000, China Abstract Objective: To observe the expression and localization of human HP1琢, an eukaryotic expression vector HP1琢/EGFP-N1 was constructedand introduced into HeLa cells. Methods:HP1琢 cDNAwere obtainedby RT-PCR, and cloned into the eukaryotic expression vector pEGFP-N1 to construct HP1琢/EGFP-N1 that was transfected into HeLacellswith Lipofectamine2000.The expression andlocation of HP1琢intransfected cellswas detected by fluorescence microscope. Results: Diagnostic restriction digestion and sequencing analysis verified the identity of the plasmid, HP1琢/EGFP-N1. After transfection, HP1琢 is localized in