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Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method

A fluorescent reporter gene and technology platform technology, which is applied in the field of high-throughput drug screening on the expression PXR-MRP2 fluorescent reporter gene technology platform, to achieve the effect of saving initial investment and avoiding blindness

Inactive Publication Date: 2010-12-08
CENT SOUTH UNIV
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AI Technical Summary

Problems solved by technology

[0007] Using the technology platform of transient transfection of HepG2 cells expressing PXR and the Renilla luciferase internal reference reporter gene containing the MRP2 promoter binding sequence with possible active lead drugs, screening the target gene-drug transporter MRP2 by activating PXR Produce induced possible active lead drug ligands, this method has not been reported at home and abroad

Method used

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  • Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method
  • Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method
  • Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: Construct the expression plasmid containing MRP2 gene promoter sequence luciferase reporter gene and PXR, and transiently transfect HepG2 cell technology platform, the steps are as follows:

[0061] 1. Construction of MRP2 gene promoter sequence luciferase reporter gene plasmid

[0062] 1.1 Blood sample collection and DNA extraction

[0063] Collect 5 mL of peripheral venous blood from healthy volunteers, and store in EDTA anticoagulated glass tubes in a -40°C refrigerator for later use.

[0064] 1.2 DNA extraction method

[0065] Genomic DNA was extracted according to the standard phenol-chloroform method and stored in a 4°C refrigerator for later use.

[0066] 1.3 MRP2 promoter PCR

[0067] 1.3.1 Primer design

[0068] 1.3.2 Selection of amplified fragments

[0069] 1.3.3 Amplification system design

[0070] 1.3.4 Selection of amplification conditions

[0071] 1.4 Amplified product recovery

[0072] 1.5 Ligate the PCR product of MRP2 to the pGEM-...

Embodiment 2

[0092] Embodiment 2: High-throughput screening of possible active drugs The method is as follows:

[0093] 1. Culture HepG2 cells;

[0094] 2. HepG2 cells were treated with 2×10 per well 5 Cells were seeded in 24-well culture plates in DMEM medium without fetal bovine serum and antibiotics. Each hole was transferred into 600ng MRP2 reporter gene plasmid or control plasmid, 100ngPXR plasmid and 10ng pRL-TK respectively. Dilute the DNA and Lipofectamine 2000 liposomes with an appropriate amount of culture medium that does not contain antibiotics and fetal bovine serum, mix the two, place them at room temperature for 20 minutes, add them to the wells of the culture plate, and shake the wells gently to make them even. After 6 hours, change to fresh DMEM containing 10% calf serum, add DMSO, positive drugs and various concentrations of candidate possible active drugs, and incubate for 24 hours.

[0095] 3. After 24 hours of incubation, the growth medium was aspirated from the cel...

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Abstract

The invention discloses a pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method, which screens candidate potential activity pilot medicaments by directly starting from a medicament metabolism dynamic target which is used in a terminal medicament clinic research stage in a new medicament research. In the method, a luciferase reporter gene containing a MRP2 gene promoter sequence and expression plasmids of RXR is constructed and human hepatocellular carcinoma (HepG 2) cells are transfected transiently; and the high-flux screening of in-vitro RXR induction potential activity pilot medicaments is carried out by detecting the activity of the luciferase in a self-luminous detector and reflecting the influences of different potential activity pilot medicaments on the activity of a MRP2 promoter according to the ratio of the fluorescence of a glowworm to the fluorescence of a sea pansy. The method can avoid severe untoward effects aroused by activity medicaments obtained by other medicament screening methods due to medicament interaction generated in a final clinical medication process, save a big amount of preliminary investment in the new medicament research and reduce blindness.

Description

technical field [0001] The invention belongs to the field of molecular biology and pharmacology, in particular, relates to a method for expressing pregnane X receptor (pregnane X receptor, PXR) and drug transporter-organic anion transporting polypeptide (multidrug resistance-associated protein) in HepG2 cells , MRP2) and Renilla luciferase internal reference reporter gene technology platform, using this technology platform to screen the possible active drug ligands that can induce the target gene-drug transporter MRP2 by activating PXR, and are used for the method of new drug screening. Background technique [0002] The research and development of new drugs is a high-input, high-risk, but also high-yield industry. In the U.S. Food and Drug Administration, the FDA often requires a scientific research team to spend more than 10 years and hundreds of millions of dollars in expenditures to finally bring a new drug to the market. During the pre-clinical laboratory research stage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/66
Inventor 高利臣曹杉俞竞范岚李智王连生张伟刘昭前周宏灏
Owner CENT SOUTH UNIV
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