Novel multifunctional dual-luciferase reporter gene plasmid

Abstract

The invention discloses a novel multifunctional dual-luciferase reporter gene plasmid. The plasmid contains a luc firefly luciferase gene, a Rluc renilla luciferase gene, a SV40 poly(A) termination signal part and two independent multiple cloning sites, wherein the two independent multiple cloning sites are located on the upstream and the downstream of a luc firefly luciferase gene coder frame respectively. The dual-luciferase reporter gene plasmid containing detection gene and reference gene is successfully constructed, not only can the detection gene be transferred into a subject cell conveniently, but also the reference gene can be taken in correspondingly, the complexity of experiment operation and experiment errors are greatly reduced, the plasmid has the multiple cloning sites at thetwo ends of the firefly luciferase gene, the multiple cloning site located on the upstream of firefly luciferase can introduce a promoter sequence which can be used for genetic transcription adjustment, control and detection, and the multiple cloning site located on the downstream of the firefly luciferase can introduce a 3'UTR sequence which can be used for miRNA target identification, so that switching of different application of the same plasmid is achieved.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a novel multifunctional double luciferase reporter gene plasmid. Background technique [0002] Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or adipose fluorescein in organisms. Luciferase can catalyze luciferin to form oxidized luciferin. During the luciferin oxidation process, bioluminescence is emitted, and the intensity of bioluminescence can be detected and recorded by a chemiluminescence instrument. The luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect luciferase activity. It can detect gene expression extremely sensitively and efficiently, and is an important detection method for detecting gene transcription or translation regulation. [0003] When using luciferase to quantitatively detect gene expression, the second reporter gene is usu...

AI Technical Summary

Problems solved by technology

However, the fixed promoter in pmirGLO can only express the promoter gene in most mammalian cells, which greatly limits the scope of application of this miRNA target identification plasmid, and pmirGLO only introduces polyclonal expression downstream of firefly luciferase site, the plasmid can only be used for miRNA target identification, but not for other dual-luciferase reporter gene detection studies such as promoter activity regulation identification

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