High-throughput drug screening model using human pregnane X receptor (hPXR)-mediated dual-luciferase reporter gene technique
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A luciferase and reporter gene technology, applied in the fields of molecular biology and pharmacology, can solve problems affecting the expression and adverse effects of metabolic enzymes and transporters
Inactive Publication Date: 2015-05-06
SUN YAT SEN UNIV
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[0004] However, after stimulating PXR, it often produces unexpected adverse eff
Method used
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Embodiment 1
[0026] Routine culture of HEK293T cells
[0027] HEK293T cells were grown in DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin and streptomycin, and adhered to the wall at 37°C, 5% CO2, and saturated humidity. When the cells grow to 80% ~ 90% confluent, they are digested with 0.25% trypsin digestion solution, the digestion time is 1 ~ 2min, and passaged at 1:3.
Embodiment 2
[0029] Plasmid extraction
[0030] 1) Pick and inoculate the Escherichia coli containing the plasmid in 30 mL LB liquid medium for culture, shake at 37 °C, 260 rpm / min, and shake for 12-16 h to obtain a small shake liquid. Collect the bacteria by centrifuging at 3500–5000 g for 30 min at room temperature.
[0031] 2) Discard the medium supernatant. Add 250 μL Solution I / RNaseA resuspension solution and vortex to completely suspend the cells.
[0032] 3) Add 250 μL of Solution II lysate to the resuspension mixture, immediately invert and mix 4-6 times, then place the mixture at room temperature for 2 minutes to increase the yield, until the solution is clear. Avoid vigorous mixing of the lysate and the lysis reaction should not exceed 5 min.
[0033] 4) Add 300 μL Solution III neutralizing solution, and gently invert the centrifuge tube several times until a white flocculent precipitate is formed. high speed centrifugation
[0034] 5) Carefully transfer the supernatant to ...
Embodiment 3
[0042] According to the optimal transfection conditions obtained, HEK293 cells transiently transfected with PXR expression plasmids, reporter gene plasmids, and internal reference plasmids were given corresponding positive drugs, and the dual luciferase activity was detected after drug stimulation for 24 h. The results were as follows: figure 1 As shown, the relative fluorescence intensity increases dose-dependently with the positive drug, and the relative fluorescence intensity of 50 μM Rif can reach about 8.
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Abstract
The invention discloses a high-throughput drug screening model using a human pregnane X receptor (hPXR)-mediated dual-luciferase reporter gene technique. The invention discloses a method for screening a human pregnane X receptor stimulant and an antagonist; by virtue of the method which is a new drug screening method, active drug ligands having regulating effect on a target gene, namely cytochrome CYP450 gene, of the PXR. A human embryonic renal HEK293T cell is subjected to the instantaneous co-transfection of an hPXR expression plasmid, a firefly luciferase reporter gene plasmid and an internal reference renilla luciferase plasmid by virtue of the method. The activities of the firefly luciferase and the renilla luciferase are simultaneously detected to show the regulating effect of the drug on the PXR activity, so as to establish a method for screening the hPXR stimulant and the hPXR antagonist. On one hand, the method can screen out compounds having an hPXR stimulating activity at high throughput from a massive compound library so as to avoid a risk that adverse drugs interact caused by the combined use of new drugs; and on the other hand, the method can screen out compounds having PXR antagonistic activity to inhibit the inducing effect of ligands on drug metabolic enzyme and transporter, so as to reduce the occurrence of adverse DDI and to reverse the drug resistance of anticancer drugs.
Description
technical field [0001] The invention belongs to the field of molecular biology and pharmacology, in particular the invention relates to a method for screening human pregnane X receptor agonists and antagonists, which can be used to screen for the target gene of PXR—cytochrome CYP450 The active drug ligand for the regulation of gene production can be applied to the screening of new drugs. Background technique [0002] PXR belongs to the NR1I subfamily of nuclear receptors. The human PXR gene contains 10 exons and 9 introns, with a total length of about 35kb. In 1998, three research groups independently discovered pregnane-activated receptors, steroid and xenobiotic receptors PXR in humans and mice using homologous cloning and database technology, because they can be activated by high concentrations of progesterone, Hence the name, sometimes also called steroid xenobiotic receptor (SXR). The functional proteins that PXR can regulate involve drug distribution, metabolism, and...
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