腺病毒介导的双荧光素酶报告系统的构建及其应用  

The construction and application of an adenovirus mediated dual-luciferase reporter system

作　　者：李振海[1] 吴红平[2] 徐增辉[2] 吕赛群[2] 施军霞[2] 刘品一 李林芳[2] 金华君[2] 吴孟超[2] 钱其军[1,2] 

机构地区：[1]浙江理工大学生命科学学院新元医学与生物技术研究所,浙江杭州310018 [2]第二军医大学东方肝胆外科医院基因-病毒治疗实验室,上海200438

出　　处：《中国肿瘤生物治疗杂志》2015年第4期420-426,共7页Chinese Journal of Cancer Biotherapy

基　　金："重大新药创制"科技重大专项资助(No.2013ZX09102-060);上海工程技术研究中心专项资助(No.12DZ2251600)~~

摘　　要：目的:构建腺病毒介导的双荧光素酶报告系统,并利用其筛选一种广谱高活性启动子。方法:将待检测启动子控制的萤火虫荧光素酶(Fluc)基因表达框插入腺病毒E1区,CMV启动子控制的海肾荧光素酶(Rluc)基因表达框插入E3区作为内参,构建腺病毒介导的双荧光素酶报告系统。检测不同病毒感染量条件下测得的启动子活性数据差异、组内差异,难转染细胞内的转染情况,分析该系统的操作简便性、稳定可靠性。应用该系统检测常用启动子CAG、CASI及新启动子CCAU在一系列细胞内的活性,从中筛选出一种广谱高活性启动子。结果:成功构建了腺病毒介导的双荧光素酶报告系统;不同MOI值下测得的各启动子相对活性值无显著差异(P>0.05);在所实验的细胞内均能有效且准确地测定各启动子的活性,且复孔间方差小;CCAU在所实验的9株细胞中的表达活性显著(P<0.05)或极显著地(P<0.01)高于CASI以及CAG的表达活性。结论:构建获得的腺病毒介导双荧光素酶报告系统操作简便、稳定可靠、通用性强,可作为启动子活性筛选的一种新方法;利用该系统筛选获得了一种新的广谱高活性启动子CCAU。Objective: To construct anadenovirus mediated dual- luciferase reportersystemthat can be used for screening promoters with high activity in a broad of cells. Methods: The testing promoter controlled expressioncassette of Firefly luciferase and CMV promoter controlled expressioncassette of Renilla luciferase were cloned into the E1 and E3 region of adenoviral vector respectively. The capacity of the system was assessed in the following three aspects: the activities of each promoter detected at different MOI,the variance within the same group,and the availability of the system for examining the promoter activity in cell lines that were hard to be transfected. Finally,the activities of CAG,CASI and a new CCAU promoters were examined by the system in 9 cell lines to find the promoter with high and broad-spectrum activity. Results:The adenovirus mediated dual-luciferase reporters system was successfully constructed. The activities of Rluc and Fluc of individual promoter had no significant difference at different MOI conditions( P > 0. 05),and the variations in each test group were small. The system worked effectively in K562,Jurkat,and primary skin cells,which are hard to be transfected. We found that the CCAU promoter produced higher luciferase activity than the CAG and CASI promoters in 9 cell lines tested( P < 0. 05 and P < 0. 01 respectively). Conclusion: The adenovirus mediated dual- luciferase reporters is an easy- to- use and reliable assay system suitable for assessing promoter activity. The new designed promoter CCAU has broadspectrum strongactivity and can be used in transgenic over- expression.

关 键 词：腺病毒 双荧光素酶报告系统 启动子 CCAU 

分 类 号：Q939.4[生物学—微生物学]