Preparation method and application of dual luciferase reporter expression vector pFireRluc

Abstract

The invention relates to a preparation method and application of a dual luciferase reporter expression vector pFireRluc. According to the invention, the problem that a testing system is affected by cell activity and transfection efficiency of two different vectors in the transfection process can be effectively solved; a Rluc sequence fragment is obtained through amplification by taking a pmirGLO vector as a template and taking R1 / R2 as a primer; SalI restriction enzyme cutting sites are added at two ends of the sequence, a pGL4.10 vector is subjected to SalI single restriction digest, and theRluc sequence fragment is inserted into the pGL4.10 vector, and thus a recombinant pFireRluc dual luciferase reporter vector is constructed; the recombinant pFireRluc dual luciferase reporter vector is effectively used for expression and identification of eukaryotic cells and activity testing of the dual luciferase reporter expression vector pFireRluc. The method disclosed by the invention has theadvantages that novelty and uniqueness are realized, the credibility of experimental data is improved, the influence of cell activity and transfection efficiency on experiment result is reduced or can be eliminated, and further the stability and the reliability of a detection result are improved.

Description

technical field [0001] The invention relates to molecular biology, in particular to a preparation method and application of a dual-luciferase reporter gene expression vector pFireRluc. Background technique [0002] The Luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase (fireflyluciferase). The bioluminescence released during the luciferin oxidation process can be sensitively and efficiently detected by the luciferase assay (luminometer). The detection system is a detection method for detecting the interaction between the transcription factor and the DNA in the promoter region of the target gene. The dual-luciferase reporter gene test system co-expresses firefly and Renilla luciferase, uses firefly luciferase to quantify the expression of the target gene, and uses a second reporter gene (Renilla luciferase) to reduce experimental variability. Traditional co-reporter genes including CAT, β-Gal,...

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Problems solved by technology

[0003] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a dual-luciferase reporter gene expression vector pFireRluc, which can effectively solve the problem of the test system being affected by cell activity and two different vectors. Influence of transfection efficiency during transfection

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