新型双荧光素酶启动子报告分析系统的构建与验证  

Construction and Validation of A Novel Dual-Luciferase PromoterAssay System

作　　者：吕环环 王胜利 谢娜娜[1,2] 党洁 霍正浩[1,2] 马占兵[1,2] LYU Huanhuan;WANG Shengli;XIE Nana;DANG Jie;HUO Zhenghao;MA Zhanbing(School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Fertility Conservation of Ministry of Education,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院,银川750004 [2]生育力保持教育部重点实验室,银川750004

出　　处：《宁夏医科大学学报》2025年第1期23-30,共8页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金面上项目(2023AAC03154);国家自然科学基金地区项目(82060301;81560273)。

摘　　要：目的 构建一种多功能的5’双荧光素酶启动子分析报告系统,该系统同时包含萤火虫荧光素酶和海肾荧光素酶元件,以弥补传统分析启动子活性时需同时转入双质粒的不足。方法 通过基于PCR的长DNA序列准确合成法(PAS)技术,制备含有双酶切位点的海肾荧光素酶基因和SV40启动子片段,采用重组克隆技术将其插入pGL3-basic质粒中,构建pGL-Duo双荧光素酶启动子分析报告质粒。随后,采用同源重组克隆技术将PGK启动子亚克隆到pGL-Duo质粒的多克隆位点(MCS)区。经酶切分析、DNA测序、293T细胞瞬时转染以及荧光素酶活性检测,验证pGL-Duo双荧光素酶报告系统的功能是否正常。结果 通过酶切分析和DNA测序,确认pGL-Duo报告质粒及其含PGK启动子的版本均构建正确。瞬时转染实验及荧光素酶活性检测显示,PGK启动子能有效启动萤火虫荧光素酶基因表达(P<0.01),双荧光素酶启动子报告分析系统功能正常。结论 成功构建并验证了一种新型双荧光素酶启动子报告分析系统,可为启动子转录活性及转录因子调控研究提供高效且便利的方案,适用于大规模启动子筛选、验证以及转录因子表达调控的研究。Objective This study aims to construct a multifunctional 5’dual-luciferase promoter analysis reporting system.The system incorporates both Firefly luciferase and Renilla luciferase components to address the current limitation of requiring the independent transfection of dual plasmids for analyzing promoter activity.Methods Through PAS technology,a dual-luciferase fragment containing luciferase and SV40 promoter was prepared and inserted into the pGL3-basic plasmid using recombinant cloning techniques to construct the pGL-Duo dual-luciferase promoter analysis reporter plasmid.Subsequently,in order to verify whether the pGL-Duo vector could work properly,the PGK promoter was subcloned into the multicloning site(MCS)area of the pGL-Duo plasmid using homologous recombination cloning techniques.The functionality of the pGL-Duo dual-luciferase reporter system was verified through enzyme digestion analysis,DNA sequencing,transient transfection of 293T cells,and luciferase activity detection.Results Confirmation through enzyme digestion analysis and DNA sequencing has verified that the pGL-Duo reporter plasmid and its version containing the PGK promoter have been constructed correctly.Functional validation through transient transfection experiments and monitoring of luciferase activity confirms that the PGK promoter effectively initiates the expression of the Firefly luciferase gene(P<0.01),demonstrating the normal functionality of the dual luciferase reporter analysis system.Conclusion A novel dual-luciferase promoter reporter assay system was successfully constructed and validated,which provides an efficient and convenient solution for studying promoter transcriptional activity and transcription factor regulation.It is suitable for large-scale promoter screening,validation,and research on transcription factor expression regulation.

关 键 词：双荧光素酶 质粒 基因表达 启动子 

分 类 号：R349.64[医药卫生—基础医学]