一种验证内部核糖体进入位点的双荧光素酶报告载体构建  

Construction of dual-luciferase reporter vector for identification of internal ribosome entry site

作　　者：史冰田 宋芹芹[1] 宋娟[1] 夏志强 夏冬[1] 刘宓 王文军 王瑞芳 韩俊[1] Shi Bingtian;Song Qinqin;Song Juan;Xia Zhiqiang;Xia Dong;Liu Mi;Wang Wenjun;Wang Ruifang;Han Jun(State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,Beijing 102206 China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,传染病预防控制国家重点实验室传染病诊断和治疗协同中心,北京102206

出　　处：《中华实验和临床病毒学杂志》2021年第1期106-110,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重大科技专项(2018ZX10102001,2018ZX10711001,2018ZX10734404);传染病预防控制国家重点实验室发展基金(2011SKLID104)。

摘　　要：目的构建一种能够验证内部核糖体进入位点的双荧光素酶报告载体,为验证内部核糖体进入位点(internal ribosome entry site,IRES)活性提供了方法。方法以双荧光素酶报告基因载体psiCHECK-2为基础,将发卡结构插入到海肾荧光素酶(R-Luc)基因与萤火虫荧光素酶(F-Luc)基因之间,构建载体psiCHECK-IRES。为了验证载体是否有效,通过同源重组技术将Encephalomyocarditis virus(EMCV)的IRES序列插入到psiCHECK-IRES载体的发卡结构与F-Luc之间,形成载体psiCHECK-IRES-EMCV。以psiCHECK-2质粒做模板建立F-Luc与R-Luc的扩增标准曲线。将psiCHECK-IRES与psiCHECK-IRES-EMCV质粒转染BHK-21细胞,利用RT-qPCR检测F-Luc与R-Luc的拷贝数;将psiCHECK-IRES与psiCHECK-IRES-EMCV质粒分别转染BHK-21细胞,24 h后利用双荧光素酶报告基因检测系统检测荧光素酶活性。结果经过测序证实带有发卡样结构的双荧光素酶表达载体psiCHECK-IRES的载体序列。RT-qPCR结果显示F-Luc与R-Luc mRNA的拷贝数大致相同,证明未出现异常的单顺反子转录本,避免F-Luc读数出现假阳性;插入EMCV IRES的psiCHECK-IRES-EMCV载体表达的F-Luc/R-Luc比值是空载体的53.35倍,证明EMCV的IRES序列能够在构建的载体上起始F-Luc基因的表达。结论本研究构建了可用于验证病毒或者细胞依赖IRES表达的双荧光素酶报告载体psiCHECK-IRES。Objective To construct the dual-luciferase reporter vector for identification of internal ribosome entry site(IRES).Methods The hairpin structure was inserted between Renilla luciferase(R-Luc)and Firefly luciferase(F-Luc)genes based on psiCHECK-2 to form plasmid psiCHECK-IRES.IRES of Encephalomyocarditis virus(EMCV)was inserted between the hairpin structure and F-Luc genes of psiCHECK-IRES to form vector psiCHECK-IRES-EMCV.After psiCHECK-IRES-EMCV or psiCHECK-IRES was transfected into BHK-21 cells respectively,expressions of F-Luc and R-Luc were detected by RT-qPCR.Then Luciferase activity of transfected cells was detected with the dual-luciferase reporter assay system at 24 h post-transfection.Results The hairpin structure was successfully inserted into psiCHECK-2 to form psiCHECK-IRES by sequencing.RT-qPCR result showed that there were the approximate expressing levels of mRNA between F-Luc and R-Luc.The result indicated that no aberrant monocistronic transcripts,which caused false positive F-Luc readings,were produced.Then IRES of EMCV was introduced into psiCHECK-IRES to form psiCHECK-IRES-EMCV.The F-Luc/R-Luc ratio in psiCHECK-IRES-EMCV-transfected cells was 53.35 times that of psiCHECK-IRES-transfected cells.The result confirmed that IRES of EMCV initiated effectively the translation of F-Luc.Conclusions Dual-luciferase reporter vector psiCHECK-IRES was successfully constructed,which could be used to validate viruses and eukaryotic genes,the translation thereof was IRES-dependent.

关 键 词：内部核糖体进入位点 海肾荧光素酶 萤火虫荧光素酶 

分 类 号：Q78[生物学—分子生物学]