用分泌型萤光素酶报告系统比较TTR启动子与CMV启动子的体内外表达特性  

Comparison of TTR and CMV Promoters in vivo and in vitro via a Secreted Luciferase Reporter System

作　　者：罗顺涛[1,2] 田文洪[2] 王刚[2] 董小岩[2,3] 杨莉[1] 吴小兵[2] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,成都610041 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [3]北京五加和分子医学研究所有限公司,北京100176

出　　处：《病毒学报》2009年第6期424-429,共6页Chinese Journal of Virology

基　　金：病毒基因工程国家重点实验室开放课题;国家重点基础研究发展计划(973)2004CB518806资助项目。

摘　　要：GLuc（GaussiaLuciferase）是一种高灵敏度分泌型萤光素酶。本研究利用GLuc的易分泌、高灵敏性和检测方法简单快速的特点,初步比较了TTR启动子（PTTR）与CMV启动子（PCMV）的体内外表达特性。首先构建了两种启动子-报告基因表达质粒pAAV2-neo-TTR-GLuc和pAAV2-neo-CMV-GLuc。用脂质体转染法将它们分别转染至两株肝细胞源的细胞株Huh7与HepG2以及两株非肝细胞源细胞株HEK293与HeLaS3。在转染后不同时间点取细胞培养上清液检测GLuc的表达。此外采用水动力注射法,将这两种质粒分别以注射剂量0.1μg、1μg和10μg/只经尾静脉注射至BALB/c小鼠体内,注射后2h开始在不同时间点上经尾静脉采集全血（2.5μl/次）并测定血液中的GLuc水平。细胞实验结果显示,PCMV介导的GLuc表达都明显高于PTTR;然而,在HEK293和HeLaS3细胞中PCMV比PTTR的表达水平高50-300倍,而在Huh7和HepG2中仅高10倍以内,提示PTTR具有相对的肝细胞特异性。在小鼠实验中,PCMV介导的GLuc表达在各剂量组中也明显高于PTTR,然而它们表达水平变化特征明显不同：PCMV介导的GLuc表达在质粒注射后约10h达到最大值，此后迅速下降；而PTTR介导的GLuc表达在质粒注射后约48h达到峰值，随后缓慢下降。上述实验结果提示，PTTR虽然在表达强度方面不及PCMV但其介导的表达水平维持长时间，下降较缓慢，比较适于目的基因在肝脏内的较长时间表达。GLuc（Gaussia luciferase） is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLue. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then,4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/e mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5μl tail tip blood since 2h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLue driven by CMV promoter was 50-300 times higher than that of GLue driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PcMv＇s. Thus, it is more suitable for maintaining longer expression of target genes in liver.

关 键 词：分泌型萤光素酶 水动力注射 启动子 转甲状腺素蛋白 巨细胞病毒 

分 类 号：Q786[生物学—分子生物学]