IFN-β启动子分泌性荧光素酶稳定表达细胞系的构建及鉴定  

Construction and Characterization of a Cell Line Stably Integrating an IFN-β-Inducible Gluc Reporter Construct

作　　者：王飒 屈阳 孟春春[2] 仇旭升[2] 廖瑛[2] 谭磊[2] 宋翠萍[2] 刘炜玮 孙英杰[2] 丁铲[2] WANG Sa;QU Yang;MENG Chunchun;QIU Xusheng;LIAO Ying;TAN Lei;SONG Cuiping;LIU Weiwei;SUN Yingjie;DING Chan(College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]福建农林大学动物科学学院,福州350002 [2]中国农业科学院上海兽医研究所,上海200241 [3]西北农林科技大学动物医学院,杨凌712100

出　　处：《中国动物传染病学报》2022年第4期31-38,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划项目(2017YFD0500800);国家自然科学基金面上项目(31872453)。

摘　　要：先天性免疫是细胞抵御病毒感染的第一道防线,I型干扰素(IFN)在抗病毒先天性免疫中发挥关键作用。干扰素β(IFN-β)作为I型IFN成员,是IFN通路激活的效应蛋白和指征蛋白,IFN-β启动子活性则是通路是否激活的重要指标。Gaussia luciferase(Gluc)是近年来发现的荧光素酶家族中最小的成员,呈分泌性表达,与常规荧光素酶相比具有诸多优势。为了建立一种新型、简易、稳定的IFN-β启动子活性检测方法,本研究首先将人IFN-β基因启动子以及下游Gluc克隆至慢病毒载体中,将其包装成慢病毒后感染A549细胞;使用杀稻瘟菌素筛选及有限稀释法稀释培养得到单克隆细胞,经PCR初步筛选得到稳定表达IFN-β-Gluc的A549细胞株,新城疫病毒和poly(I:C)处理单克隆细胞,筛选得到能够高水平诱导Gluc的阳性细胞株。进一步通过正向诱导(转染IFN-β信号通路相关蛋白质粒)和反向抑制实验(转染新城疫病毒V蛋白质粒)证实,并从基因及酶活性水平验证其表达IFN-β-Gluc的稳定性。该细胞系提供了检测IFN通路激活的简易方法,为进一步研究病毒诱导IFN-β的转录、调控,动态监测IFN-β启动子活性,高通量筛选等研究奠定了基础。Innate immunity is the fi rst line of cellular defense against virus infection.Type I interferon(IFN)plays a key role in antiviral innate immunity.IFN-β,which belongs to type I IFN,is the effector and indicator of IFN pathway.IFN-βpromoter activity is an important indicator of whether the pathway is activated or not.Gaussia luciferase(Gluc)is the smallest member of luciferase family found in recent years.It is the secreted luciferase with many advantages over conventional luciferase.In order to develop a new,simple and stable method for detection of IFN-βpromoter activity,the human IFN-βpromoter and downstream Gluc were cloned into lentivirus vector and packed into lentivirus to infect A549 cells.The monoclonal cells were obtained by blasticidin screening and limited dilution culture,and then A549 stably expressing pIFN-β-Gluc was initially screened by PCR.Subsequently,monoclonal cells were treated with Newcastle disease virus or poly (I: C) to obtain the positive cell line with a high level of inducible Gluc. This positive cell line was further confi rmed by positive (transfection of IFN-β signal pathway related plasmids) and negative verifi cations (transfection of Newcastle disease virus V plasmids). Finally, the stability of the A549 cell line expressing IFN-β-Gluc was verifi ed for its gene level and enzyme activity. The availability of this A549 cell line provided a simple method to detect the activation of IFN pathway, which laid a foundation for further study on the transcription and regulation of IFN-β induced by virus, dynamic monitoring of IFN-β promoter activity and high-throughput screening.

关 键 词：干扰素Β 启动子 报告基因 分泌性荧光素酶 

分 类 号：S852.4[农业科学—基础兽医学]