一种检测蛋白酶活性的荧光素酶报告基因系统的构建及应用  

Construction and application of a luciferase reporter gene system for detecting protease activity

作　　者：周仕君 宋洁 李帅 刘佳 李江南[1,2] 翁长江 ZHOU Shi-jun;SONG jie;LI Shuai;LIU Jia;LI Jiang-nan;WENG Chang-jiang(Division of Fundamental Immunology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China;Heilongjiang provincial Key Laboratory of Veterinary Biotechnology,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/基础免疫创新团队,黑龙江哈尔滨150069 [2]黑龙江省免疫学重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第5期535-539,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金优秀青年项目(YQ2019C033);国家自然科学基金面上项目(31872448)。

摘　　要：为建立一种检测蛋白酶活性的方法,本研究将IL-1β前体基因和荧光素酶基因融合,克隆至p CAGGS真核载体,构建了基于荧光素酶的报告基因系统(iGLuc),同时构建GLuc真核表达质粒作为对照。以PRRSV nsp4和ASFV S273R蛋白酶为检测对象,利用该报告基因系统对病毒蛋白酶活性进行检测,将iGLuc中蛋白酶酶切位点(FVCDAN)相对应的基因序列替换为Kpn I酶切位点,构建重组质粒Kpn I-iGLuc,然后分别将PRRSV nsp4和ASFV S273R蛋白酶切割位点对应的核酸序列插入至报告基因Kpn I-iGLuc中的Kpn I位点,构建重组质粒nsp4-iGLuc和S273R-i GLuc。分别将报告基因质粒(iGLuc、GLuc、Kpn I-i GLuc、nsp4-i GLuc和S273R-iGLuc)与不同剂量的相应蛋白酶真核表达质粒共转染HEK293T细胞,24 h后检测上清中荧光素酶GLuc的活性。结果显示,转染i GLuc、nsp4-iGLuc或S273R-iGLuc质粒的上清中荧光信号随着相应蛋白酶的剂量增加而升高,呈现剂量依赖性,而缺失蛋白酶切位点的对照质粒无该现象,表明构建的荧光素酶报告基因能够特异性地检测细胞蛋白酶和病毒蛋白酶的活性。本研究首次建立了一种检测蛋白酶活性的方法,并以PRRSV nsp4和ASFV S273R蛋白酶验证了该方法的应用价值,为后续开展高通量化筛选抗PRRSV、ASFV等药物的研究奠定了基础。To detect the activity of a protease,a fusion gene,i GLuc,was constructed by fusing porcine pro-IL-1βwith GLuc And the eukaryotic expression plasmid of GLuc was constructed as a control.To further explore the applicability of the protease reporter viral proteases,the protease cleavage site in pro-IL-1βwas replaced by Kpn I(KpnI-iGLuc),and then the sequences corresponding to the protease cleavage sites of PRRSV nsp4 or ASFV S273R were inserted into the Kpn I site,forming reporter plasmids of nsp4-iGLuc or S273R-iGLuc.The reporter plasmids were co-transfected into HEK293T cells with the protease expression construct in a dose gradient.And the activity of luciferase GLuc in the supernatant was detected at 24 hours post-transfection.The results showed that the luciferase signal in the supernatant transfected with i GLuc,nsp4-iGLuc or S273R-iGLuc was increased in a dose-dependent manner with the increase amount of indicated protease,while the control reporters did not,indicating that the constructed luciferase reporters could detect the activity of cellular protease or viral protease.This study provides a method to detect protease activity,which laid a foundation for subsequent high-throughput quantitative screening of anti-PRRSV and-ASFV drugs.

关 键 词：蛋白酶活性 荧光素酶报告基因 病毒蛋白酶 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]