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.
作者
高全新
刘云霞
程玉强
严亚贤
孙建和
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)
出处
《上海农业学报》
CSCD
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"
作者简介
高全新（1971-），男，本科，高级兽医师，研究方向：动物疾病防控。E-mail：gqx2588@sina．com，Tel：13801946930;通信作者，E-mail：sunjhe@sjtu．edu．cn
