鸭IFN-β启动子双荧光素酶报告基因系统的构建及活性检测  

Construction and activity detection of dual luciferase reporter gene system for duck IFN-β promoter

作　　者：高全新 刘云霞[2] 程玉强[2] 严亚贤[2] 孙建和[2] GAO Quan-xin;LIU Yun-xia;CHENG Yu-qiang;YAN Ya-xian;SUN Jian-he(Animal Disease Prevention and Control Center of Baoshan District,Shanghai 201900, China;Shanghai Key Laboratory of Veterinary Bioteehnology/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China)

机构地区：[1]上海市宝山区动物疫病预防控制中心,上海201900 [2]上海交通大学农业与生物学院上海市兽医生物技术重点实验室,上海200240

出　　处：《上海农业学报》2018年第3期66-71,共6页Acta Agriculturae Shanghai

基　　金：国家自然科学基金(31672524)"鸡MDA5-STING-IFNβ天然免疫通路中STING介导信号传导和抗病毒分子机制"

摘　　要：通过PCR方法扩增获得6个不同长度的鸭IFN-β启动子DNA片段,并连接至萤火虫荧光素酶报告质粒p GL3.0,构建包含6个鸭IFN-β启动子的报告质粒。分别将构建的报告质粒与内参报告质粒p RL-TK转染至DEF细胞,检测报告质粒在不同刺激下的荧光素酶活性。结果表明:包含鸭IFN-β启动子全长的报告质粒p GL-IFN-β-1593能够有效地被相应刺激物激活;截短研究表明,包含390 bp鸭IFN-β启动子的报告质粒p GL-IFN-β-453的荧光素酶活性与全长基本一致,且一定程度上可降低冗余碱基的不良影响,因此选取该质粒用于鸭IFN-β启动子活性检测。本研究建立的基于荧光素酶双报告基因系统的鸭IFN-β启动子活性检测方法,为后续鸭IFN-β通路研究提供了检测工具。Six duck IFN-β promoter DNA fragments were amplified by PCR,and then sub-cloned into fireflyluciferase reporter gene vector p GL3. 0 to construct different recombinant plasmids. The constructed reporterplasmid and internal reference plasmid p RL-TK were transfected into DEF cells respectively to detect theluciferase activity of the reporter plasmid under different stimulation. The results showed that the reporter plasmidp GL-IFN-β-1593,which contained the full length of the duck IFN-β promoter,could be activated effectively bythe corresponding stimulus. The luciferase activity of the reporter plasmid p GL-IFN-β-453 containing 390 bpduck IFN-β promoter was basically the same as that of the full-length,and the adverse effects of redundant basecould be reduced to some extent. Therefore,the recombinant plasmid p GL-IFN-β-453 was selected for detectionof duck IFN-β promoter activity. The detection method of duck IFN-β promoter activity based on dual luciferasereport gene system was established in this study to provide a detection tool for subsequent IFN-β pathwayresearch.

关 键 词：鸭 IFN-β启动子 双荧光素酶报告基因 

分 类 号：S852.65[农业科学—基础兽医学]