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Dual-luciferase reporter gene plasmid applicable to screening of orphan nuclear receptor activity regulating agent, and construction and application thereof

A dual luciferase and reporter gene technology, applied in the field of construction of dual luciferase reporter gene plasmids, can solve the problems of high labor intensity, complicated operation, poor stability, etc., and achieve strong specificity, high sensitivity, and easy operation Effect

Active Publication Date: 2018-04-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has poor stability, complicated operation, high labor intensity, and cannot adapt to the simultaneous screening of a large number of drugs.

Method used

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  • Dual-luciferase reporter gene plasmid applicable to screening of orphan nuclear receptor activity regulating agent, and construction and application thereof
  • Dual-luciferase reporter gene plasmid applicable to screening of orphan nuclear receptor activity regulating agent, and construction and application thereof
  • Dual-luciferase reporter gene plasmid applicable to screening of orphan nuclear receptor activity regulating agent, and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0022] 1. Preparation of strains, plasmids and reagents.

[0023] Strain: Escherichia coli competent cell DH5a was purchased from Dalian TAKARA Company;

[0024] Cell line: COS-7 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0025] Plasmids: pGL3-Basic, pRL-TK, and pmirGLO were purchased from Promega.

[0026] Reagent sources: 2×PCR Taq Mix, T4DNA Ligase were purchased from Fermentas; endotoxin-free plasmid mini-extraction kit, high-purity plasmid mini-extraction kit, DNA extraction kit were purchased from Kangwei Century Company; restriction endonuclease NheI , BglII, BamHI, HindIII were purchased from NEB Company, the agarose gel recovery kit was purchased from Epigenetics Company; the luciferase detection kit was purchased from Promega Company; lipofectamine TM 3000 was purchased from Invitrogen; DMEM medium, fetal bovine serum, trypsin, PBS buffer, OPTI-MEM medium, and penicillin and streptomycin were purchased from Hangzhou Jinuo...

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Abstract

The invention discloses a dual-luciferase reporter gene plasmid applicable to screening of an orphan nuclear receptor activity regulating agent. The plasmid is a chimeric plasmid (p-TK(DR1)<3>-Fluc-hRluc) of DR1-containing firefly luciferase and the internal reference renilla luciferase. A construction method for the plasmid is to insert a fragment as shown in SEQ ID No. 1 into a hRluc-containingframework plasmid obtained after NheI / BglII double-digestion of the commercial plasmid pmirGLO. High-efficiency screening of the orphan nuclear receptor activity regulating agent can be realized through cotransfection of cells with the plasmid and an orphan nuclear receptor-overexpressed plasmid and activity detection via dual luciferases. The method is simple to operate, and the dual-luciferase reporter gene plasmid has high sensitivity and good specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a dual-luciferase reporter gene plasmid, which can be used for screening orphan nuclear receptor activity regulators. Background technique [0002] Orphan nuclear receptors are nuclear receptors that have no ligand or a ligand that has not yet been discovered. At present, more than 150 kinds of orphan nuclear receptors have been discovered, some of which are involved in metabolic regulation, embryonic development, cell differentiation and gene expression in animals. In recent years, with the discovery of partial orphan nuclear receptor activity modulators, especially the discovery of endogenous agonists, ligand-dependent nuclear receptors have been developed, such as retinoic acid X receptor, farnesic acid X receptor body, peroxisome proliferator-activated receptor gamma, etc. It can be seen that the discovery of orphan nuclear receptor activity modu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/66C12Q1/66
CPCC12N15/63C12N15/65C12N15/66C12Q1/66
Inventor 夏李群李恭会沈丹阳
Owner ZHEJIANG UNIV
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