Construction of dual-luciferase reporter vector for identification of internal ribosome entry site
10.3760/cma.j.cn112866-20200310-00057
- VernacularTitle: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
- Author:
Bingtian SHI
1
;
Qinqin SONG
;
Juan SONG
;
Zhiqiang XIA
;
Dong XIA
;
Mi LIU
;
Wenjun WANG
;
Ruifang WANG
;
Jun HAN
Author Information
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
- Keywords:
Internal ribosome entry site;
Renilla luciferase;
Firefly luciferase
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(1):106-110
- CountryChina
- Language:Chinese
-
Abstract:
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.
