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Recombinant plasmid containing OATP1B1 promoter and reporter gene and method for screening OATP1B1 inducer

A reporter gene and recombinant plasmid technology, applied in the field of plasmids containing fluorescent reporter genes, can solve the problems of rarely used, human hepatocyte model ethics, and tissue source culture conditions can not achieve high throughput, etc., to achieve easy materials, The effect of fast detection speed and high sensitivity

Inactive Publication Date: 2012-03-14
CENT SOUTH UNIV
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  • Claims
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Problems solved by technology

However, the human hepatocyte model is seldom used in actual research due to many limitations such as ethics, tissue sources, culture conditions, and inability to achieve high throughput.

Method used

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  • Recombinant plasmid containing OATP1B1 promoter and reporter gene and method for screening OATP1B1 inducer
  • Recombinant plasmid containing OATP1B1 promoter and reporter gene and method for screening OATP1B1 inducer
  • Recombinant plasmid containing OATP1B1 promoter and reporter gene and method for screening OATP1B1 inducer

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Experimental program
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Effect test

Embodiment 1

[0030] The OATP1B1 promoter fluorescent reporter gene plasmid was constructed, and transiently transfected into HepG2 cells to construct a dual fluorescent reporter gene technology platform.

[0031] 1. Materials

[0032] 1. Plasmid, cell line

[0033] pGL-4.17 fluorescent reporter gene vector, pPEM-T vector, pcDNA3.1-myc / hisB(-) eukaryotic expression vector: American Promega Company.

[0034] HepG2 cell line: Tumor Cell Bank, Chinese Academy of Medical Sciences.

[0035] 2. Main reagents

[0036] Fetal bovine serum (Hangzhou Sijiqing); MEM medium (Gibco, USA); 50ml cell culture flask, 24-well cell culture plate (Corning, USA); RevertAid TM First Strand cDNA Reverse Transcription Kit (Fermentas); Lipofectamine TM 2000 liposome (Invitrogen, USA); DNA extraction kit (promega); Trizol (Invitrogen, USA).

[0037] 3. Main instruments

[0038] PCR instrument (Thermo, USA); electrophoresis equipment (Beijing Liuyi); CO 2 Cell incubator (ThermoForm, Series II, USA); ultra-cle...

Embodiment 2

[0158] The experimental method established above was used to study whether the traditional Chinese medicine monomer compound baicalin could induce the expression of OATP1B1 by activating PXR.

[0159] Steps:

[0160] 1. Culture HepG2 cells;

[0161] When HepG2 cells grow to the logarithmic phase, digest with 2×10 per well 5 Cells were seeded into 24-well plates until the cells grew to 80%.

[0162] 2. Co-transfection

[0163] 2.1 Each well needs to be transferred into 600ng PXR plasmid, 300ng OATP1B1 promoter fluorescent reporter gene plasmid and 50ng sea cucumber luciferase respectively. Dilute the DNA and Lipofectamine2000 liposomes with an appropriate amount of culture medium without antibiotics and fetal bovine serum, mix the two, and place them at room temperature for 20 minutes.

[0164] 2.2 Wash the cells three times with PBS, and then add 1ml of MEM medium without fetal bovine serum to each well.

[0165] 2.3 Add the above mixture (DNA / liposome) into a 24-well pla...

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Abstract

The invention belongs to the field of molecular pharmacology, and relates to a method for screening an inducer capable of regulating expression of a downstream target gene organic anion transporter 1B1 through activation on a pregnane X receptor (PXR). The method for screening OATP1B1 inducer comprises cloning organic anion transporter 1B1 (OATP1B1) gene promoter specific sequences (of -128 to -111 and -9957 to -9940), recombining the cloned organic anion transporter 1B1 (OATP1B1) gene promoter specific sequences and firefly luciferase reporter gene vectors, simultaneously, constructing a human PXR expression plasmid, then co-transfecting the constructed OATP1B1-luciferase reporter gene recombinant plasmid, the human PXR expression plasmid and renilla luciferase into a human liver cancer cell HepG2, and simultaneously, and detecting transcription activity of active reaction OATP1B1 gene promoters of firefly luciferase and renilla luciferase so that the method for targetedly screening an OATP1B1 inducer is established. The method for targetedly screening an OATP1B1 inducer can provide a mass of scientific basises for disease treatment, drug interaction, safe and reasonable utilization of drugs, and preclinical study of novel drugs.

Description

technical field [0001] The invention relates to an inducer screening method for regulating the expression of the downstream target gene organic anion transporter 1B1 by activating the pregnane receptor (PXR). [0002] The invention also relates to plasmids containing fluorescent reporter genes. Background technique [0003] The liver is the main organ for biotransformation and detoxification. There are many types of drug transporters in the liver, which play an important role in the absorption, distribution and excretion of drugs, such as multidrug resistance-associated proteins, breast cancer resistance proteins, organic anion transporters Peptide family, etc. OATP1B1 is the most important member of the organic anion transporting polypeptide family. The coding gene SLCO1B1 of OATP1B1 is not only highly polymorphic, but also has multiple functionally significant mutants. Studies have confirmed that OATP1B1 not only targets the transport of unconjugated bilirubin, bile acid...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12Q1/66C12Q1/02G01N21/64
Inventor 郭成贤吴兰香张伟周宏灏
Owner CENT SOUTH UNIV
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