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Method for in-vitro screening of PXR activation characteristics

A characteristic and carrier technology, applied in the field of in vitro PXR activation characteristic screening, to achieve the effect of improving safety

Inactive Publication Date: 2012-07-04
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Noracharttiyapot W et al. stably transfected HepG2 cells with the reporter gene vector alone, and obtained a functional monoclonal cell line with a clear induction effect on rifampicin, but it needs to lyse the cells every time the fluorescence is detected

Method used

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  • Method for in-vitro screening of PXR activation characteristics
  • Method for in-vitro screening of PXR activation characteristics
  • Method for in-vitro screening of PXR activation characteristics

Examples

Experimental program
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Effect test

experiment example 1

[0027] 1 Materials and methods

[0028] 1.1 Materials

[0029] HepG2 cell line was purchased from Xiehe Cell Center, MEM medium, non-essential amino acid (NEAA), fetal bovine serum were purchased from Hyclone Company, rifampicin was purchased from Merck Company, T4 ligase, pGLuc-Basic reporter gene plasmid, restriction endonuclease Dicer and luciferase activity assay kits were purchased from NEB Company, transfection reagent Lipofectamine TM LTX and G418 were purchased from Invitrogen Company, plasmid medium extraction kit, mini-extraction kit and gel recovery kit were purchased from Promega Company, Probest Taq enzyme system was purchased from TaKaRa Company, and the constructed pGL3-3A4 dual luciferase reporter gene plasmid, The hPXR expression vector was provided by Zhejiang University.

[0030] 1.2 Method

[0031]1.2.1 Construction of the reporter gene vector: design primers (see Table 1 for the sequence), use the pGL3-3A4 dual luciferase reporter gene plasmid as a tem...

Embodiment 1

[0054] Example 1: Method for Screening PXR Activation Properties in Vitro

[0055] 1), the construction of reporter gene carrier:

[0056] Primers were designed, using the pGL3-3A4 dual-luciferase reporter gene plasmid as a template, and the primer pairs dis-F / dis-R and pro-F / pro-R were used to amplify the remote regulatory sequence of the CYP3A4 promoter (-7833~- 7208) and the proximal promoter sequence (-361 ~ +11), the distal regulatory sequence and the proximal regulatory sequence were subjected to EcoR I-EcoR V and EcoR V-Hind III double enzyme digestion, and the two double The digested products were ligated, and then the primer pair dis-F / pro-R was used to clone the linear tandem fragments of the distal regulatory sequence and the proximal regulatory sequence, which were connected to the EcoR I / HindIII site of the pGLuc-Basic vector, and transformed into Escherichia coli , verified by enzyme digestion and sequencing, the vector was named pGLuc-NEB-3A4-1, that is, No. 1; t...

Embodiment 2

[0062] Embodiment 2: A method for screening the activation characteristics of PXR in vitro, the method comprising the following steps:

[0063] 1), the construction of reporter gene carrier:

[0064] Primers were designed, using the pGL3-3A4 dual-luciferase reporter gene plasmid as a template, and the primer pairs dis-F / dis-R and pro-F / pro-R were used to amplify the remote regulatory sequence of the CYP3A4 promoter (-7833~- 7208) and the proximal promoter sequence (-361 ~ +11), the distal regulatory sequence and the proximal regulatory sequence were subjected to EcoR I-EcoR V and EcoR V-Hind III double enzyme digestion, and the two double The digested products were ligated, and then the primer pair dis-F / pro-R was used to clone the linear tandem fragments of the distal regulatory sequence and the proximal regulatory sequence, which were connected to the EcoR I / Hind III site of the pGLuc-Basic vector, and transformed into large intestine Bacillus, verified by enzyme digestion ...

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Abstract

The invention relates to a method for in-vitro screening inducers, particularly to a method for in-vitro screening of PXR (pregnane X receptor) activation characteristics, which comprises the following steps: constructing a reporter gene vector; culturing cells, and screening G418 working concentration; carrying out transient transfection of cells; screening stably transfected cell clones; inducing and identifying stably transfected cell strain with a tested drug (ligand drug); and screening the PXR activation characteristics. When the PXR is activated by the ligand drug, the PXR can regulate the expression CYP3A gene. PXR gene-deficient mice lose drug inductivity of CYP3A. Contrarily, hPXR (human pregnane X receptor) receptor in activation state expressed in liver of a transgenic mouse can lead to continuous high expression of CYP3A enzyme. The method of the invention establishes critical technology for high-throughput screening based on the passway and carries out screening of PXR activation characteristics from mass compounds in a compound library at the early stage of drug development, and can reduce the risk of adverse drug interactions after the new drug comes into the market, and greatly reduce development cost.

Description

technical field [0001] The invention relates to a method for screening in vitro inducers, in particular to a method for screening in vitro PXR activation characteristics. Background technique [0002] To maintain normal physiological functions, the body must metabolize some exogenous substances into polar compounds and excrete them. This process of clearance and detoxification mainly depends on the regulation of cytochrome P450 (CYP) gene expression in the liver system. At the same time, more and more drugs are used to treat various diseases, and combined drug use has become a common treatment method, and adverse reactions caused by drug-drug interactions have also increased, most of which are related to drug interactions. The mechanism of most drug interactions is related to drug metabolism, especially the metabolism related to cytochrome P450 (cytochrome P450, CYP450). Cytochrome P450 is a super family composed of many isoenzymes. The main ones involved in drug metabolism...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N15/85C12N5/10
Inventor 高月陆倍倍王宇光刘浩生马增春梁乾德肖成荣谭洪玲洪倩
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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