CN101906469A - Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method - Google Patents
Pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method Download PDFInfo
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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
The invention belongs to molecular biology and area of pharmacology, specifically, relate to a kind of at HepG2 cell inner expression pregnane X acceptor (pregnane X receptor, PXR) and the drug transport body-organic anion transhipment polypeptide (multidrug resistance-associated protein, MRP2) and renilla luciferase confidential reference items reporter gene technology platform, utilize this technology platform screening to come target gene-drug transport body MRP2 is produced inductive possibility active medicine part, be used for the method for new medicament screen by excited PXR.
Background technology
The research and development of new drug are a high investment, excessive risk, also are the industries of high yield simultaneously.Often need scientific effort team to take the time in more than 10 years at the FDA of U.S. food Drug Administration, the expenditure of multi-billion dollar just can make a kind of new drug finally go on the market.In the preclinical laboratory study stage preliminary pharmacodynamics that may active medicine and general toxicology research and the I in clinical study stage, II, the research of III phase all there are a lot of uncertain factors during this time, make the new drug major part of research and development die on the vine, thereby wasted a large amount of manpowers and material resources cost.Therefore, can high flux screening and the effective examination to the active lead drug of possibility realize finally that the clinical application method of this medicine becomes the focus of pharmacy and medical worker's research.Restrict a new drug and can go up three main factors of municipalization, that is: whether " safe, effective, controlled ".New drug in research and development or the medicine that has gone on the market are even its curative effect is outstanding, quality controllable, if produced all can be under an embargo research and development and selling of serious adverse reaction.Therefore, be characterized as the target high flux screening with Clinical Researches of New Drugs stage pharmacokinetics and may be thought the frontier that the new drug high flux screening is studied by the world of medicine for drug candidate.
Multidrug-associated protein MRP2 is that the medicine that is expressed on the timid periosteum effluxes ATP in conjunction with boxlike superfamily protein transporter, is the target gene of extraneous signal nuclear receptor PXR.It is relevant with the medicine-drug interaction pharmacokinetics feature that can not expect with adverse drug reaction that MRP2 is considered to.Confirm that on evidence ATP is bringing into play keying action in conjunction with the boxlike superfamily protein in the clinical medicine disposal process.Particularly the liver internal specific is being expressed a large amount of MRP2 and can transported a lot of substrate medicines: anticancer class Zorubicin, Etoposide, mitoxantrone, cis-platinum, vincristine(VCR), vinealeucoblastine(VLB), camptothecine, HIV medicine class Indinavir, lamivudine, Saquinavir, Adefovir, cidofovir, viracept see nelfinaivr, statins Pravastatin, endogenous material have bile acide, bilirubin, leukotriene, estradiol, gsh etc.MRP2 under expression or the function causes that bile acide in the blood plasma, bilirubin and blood plasma increase in conjunction with statins, thereby produces cholestasis and Jaundice disease and medicine source property rhabdomyolysis.Simultaneously, PXR induces it to express by combining with MRP2 promoter region specific sequence, and some possible active lead drugs part of PXR exactly.Therefore, find and induce PXR to regulate the possible active lead drug that MRP2 expresses, just can effectively treat cholestasis, Jaundice disease and because the caused medicine of substrate drug accumulation source property untoward reaction disease.Use possible active lead drug to handle the HepG2 cell of expressing PXR and containing the renilla luciferase confidential reference items reporter gene transient transfection of MRP2 promoter region binding sequence, just can on the luminous detector, detect uciferase activity, reflect of the influence of different possible active lead drugs with the ratio of sea pansy fluorescence MRP2 drug transport body promoter activity with Lampyridea fluorescence.The final method high flux screening of utilization fluorescence report gene of realizing has inducing action to regulate the possible active lead drug that MRP2 expresses to PXR.This screening approach finally search out aspect the new active medicine of effective treatment cholestasis, Jaundice disease, medicine source property rhabdomyolysis significant.Also can evade the caused serious toxicity of medicine-drug interaction that the resulting medicine of some other medicines screening methods produces because of final clinical application process, and reduce the adverse drug reaction of drug combination.
The medicine that has research to damage for the scheming of finding the treatment oxidative stress to cause is playing vital p66 in the apoptosis path that causes at oxidative stress
ShcThe gene promoter fragment cloning has obtained p66 to the upstream of luciferase reporter gene
ShcThe luciferase reporter gene expression plasmid of gene promoter control, and, obtain stably transfected cell line A549/p66 with its transfection people's lung cell A549
ShcAnd utilize the reporter gene screening system, and screened hundreds of Chinese medical extracts and monomeric compound, found 2 kinds of Chinese medical extracts and 8 kinds of monomeric compounds.
Research is also arranged in order to find the medicine of the cardiovascular diabetes of treatment, set up that (Farnesoid X receptor FXR) is the FXR agonist high flux screening model of target spot with Farnesoid X receptor.Amplify FXR ligand activation binding domains (ligand bingding domain, LBD) gene order, and made up fusion expression vector pBind-FXR and report carrier pG13-GAL4 from liver cell exactly.By expression plasmid and reporter plasmid cotransfection, transient transfection L102 cell, by measuring the expression of reporter gene luciferase, 880 fungies and actinomycetic secondary metabolite and 120 purification of samples are carried out the agonist screening of FXR, obtained two active compounds of CoQ10 and F01WA2076A.
Also have research to cause the CYPs inhibitor lead compound of serious adverse reaction in order to screen the medicine-drug interaction that is suppressed to produce owing to drug metabolism enzyme-Cytochrome P450 (CYPs), in people's hepatomicrosome, utilization LC-MS (LC/MS/MS) technology is carried out high flux screening in conjunction with the cocktail model to 20 kinds of terpenes and 32 kinds of flavones traditional Chinese medicine monomer, has obtained 4 kinds and has suppressed CYPs enzymic activity traditional Chinese medicine monomer.
Use possible active lead drug to handle this technology platform of renilla luciferase confidential reference items reporter gene transient transfection HepG2 cell of expressing PXR and containing MRP2 promoter region binding sequence, screening can come target gene-drug transport body MRP2 is produced the active lead drug part of inductive possibility by exciting PXR, and this method yet there are no report both at home and abroad.
Summary of the invention
The objective of the invention is to overcome the deficiency of existing medicament high flux screening method from the pharmacokinetics angle, a kind of expression PXR-MRP2 fluorescence report gene engineering platform high-flux medicaments sifting method is provided, utilizes this method can filter out effective treatment cholestasis and Jaundice disease and the new active medicine of medicine source property rhabdomyolysis.
Technical scheme of the present invention: a kind of expression PXR-MRP2 fluorescence report gene engineering platform high-flux medicaments sifting method, this method is directly started with the screening candidate from the pharmacokinetics target of the terminal clinical drug conceptual phase of new drug research may active lead drug, structure contains the expression plasmid of MRP2 gene promoter sequence luciferase reporter gene and PXR, transient transfection HepG2 cell; By on the luminous detector, detecting uciferase activity, reflect of the influence of the active lead drug of different possibilities respectively with the fluorescent worm fluorescence and the ratio of sea pansy fluorescence, carry out the high flux screening that external PXR induces the active lead drug of possibility the MRP2 promoter activity.
The present invention makes up the expression plasmid that contains MRP2 gene promoter sequence luciferase reporter gene and PXR, and the step of transient transfection HepG2 cell technology platform is as follows:
1.MRP2 the structure of gene promoter sequence luciferase reporter gene plasmid
1.1 blood specimen collection and DNA extracting
Gather healthy volunteer's peripheric venous blood 5mL, place the glass test tube of EDTA anti-freezing, it is standby to be stored in-40 ℃ of refrigerators.
1.2 DNA method for extracting
According to standard benzene phenol-chloroform method extracting genomic dna, place 4 ℃ of refrigerators standby.
1.3?MRP2?promoter?PCR
1.3.1 design of primers
1.3.2 amplified fragments is selected
1.3.3 amplification system design
1.3.4 amplification condition is selected
1.4 amplified production reclaims
1.5 the PCR product of MRP2 is connected on the pGEM-T carrier, extract plasmid, enzyme is cut and is checked order.
Confirm to insert plasmid DNA and the PGL4.17 carrier enzymes double zyme cutting that fragment does not have the PCR mispairing 1.6 will check order, reclaim respectively with agarose electrophoresis and insert fragment and carrier, use gel piece to link fast.Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme is cut and is checked order.
1.7 recombinant clone is further cultivated amplification.
2.PXR the structure of cDNA expression plasmid
2.1 RNA extracting and reverse transcription
From the former foster liver cell of being commissioned to train, extract total RNA with Trzol, use RevertAid
TMFirst Strand cDNA synthetic agent box synthesizes cDNA, is that template is carried out pcr amplification with cDNA.
2.2?PXR?PCR
2.2.1 design of primers
2.2.2 the amplification coding section length is selected
2.2.3 amplification system design
2.2.4 amplification condition is selected
2.3 be connected in the PCR product of PXR on the pPEM-T carrier and order-checking
Confirm to insert plasmid DNA and pcDNA3.1/hisB (-) carrier Hind III and the EcoR I double digestion simultaneously that fragment does not have the PCR mispairing 2.4 will check order, reclaim respectively with agarose electrophoresis and insert fragment and carrier, use gel piece to link fast.Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme is cut and is checked order.
2.5 recombinant clone is further cultivated amplification, the extracting plasmid DNA.Plasmid DNA is kept in 70% ethanol and keeps sterile state, and-20 ℃ frozen stand-by.
3.MRP2 the evaluation of reporter gene plasmid and PXR cDNA expression plasmid
3.1 MRP2 and PXR be double digestion respectively
3.2 1% agarose gel electrophoresis
3.3 sequencing analysis
4. use the reporter gene plasmid, pRL-TK (internal reference plasmid) and pcDNA3.1/hisB (-)-PXR transient transfection HepG2 cell
4.1 extract the test kit plasmid DNA purification with amount in the plasmid, the fluorescence report gene plasmid, pRL-TK, PXR and control plasmid illustrate transfection HepG 2 cell according to Lipofectamine2000.All comprise following sample sets in each experiment, carry out cotransfection as internal reference and PXR expression plasmid with each fluorescence report genophore to be measured with another reporter gene carrier pRL-TK, by measuring the activity of its gene product Renilla Luciferas, proofread and correct the transfection efficiency between each hole.
The present invention makes up transient transfection HepG2 cell technology platform, and the possible active lead drug method of high flux screening is as follows:
1. cultivate the HepG2 cell;
2.HepG2 cell is with every hole 2 * 10
5The density of individual cell is with not containing foetal calf serum and antibiotic DMEM culture medium inoculated in 24 well culture plates.Every hole changes 600ng MRP2 reporter gene plasmid or control plasmid respectively over to, 100ngPXR plasmid and 10ng pRL-TK.Behind nutrient solution difference dilution DNA that does not contain microbiotic and foetal calf serum in right amount and Lipofectamine2000 liposome, with the two mixing, room temperature is placed 20min, adds and cultivates in the plate hole, rocks orifice plate gently and makes evenly.Behind the 6h, renew the DMEM that aquatic foods contain 10% calf serum, add candidate's possibility active medicine of DMSO, positive drug and various different concns, hatch 24h.
3. after hatching 24h, with growth medium from cell sucking-off to be checked.
4. with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off.
5. in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB).
6. the activation analysis of reporter gene
6.1 the activity of reporter gene detects
6.1.1 principle: as the fluorescence report gene series plasmid of the pRL-TK plasmid of internal reference and to be measured or the contrast two kinds of different protein of renilla luciferase and Photinus pyralis LUC of encoding respectively.In forming the oxyluciferin process, by the mediation transfer transport chemical energy is converted into luminous energy, thereby luminous.Two reporter genes all do not have endogenous activity in the experiment host cell, can continuously measured in single sample.In measuring process, at first add luciferase detection reagent II and produce the Lampyridea fluorescent signal, signal continues 1min at least, measures the Photinus pyralis LUC reporter gene so earlier.Quantitatively after the Lampyridea fluorescence intensity, in same sample, add Stop ﹠amp again; Glo
Reagent with above-mentioned reaction quencher, and starts the renilla luciferase reaction simultaneously, carries out the second time simultaneously and measures.
6.1.2 the activity of reporter gene detects step
(1) hatch 24h after the transfection, with growth medium from cell sucking-off to be checked.
(2) with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off.
(3) in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB).
(4) detect uciferase activity.On the luminous detection instrument, detecting uciferase activity with two fluorescence report kit genes after the lysis, reflecting the influence of different pharmaceutical respectively the MRP2 promoter activity with the ratio of Lampyridea fluorescence and sea pansy fluorescence.
Adopt the resulting active medicine of the present invention can evade the caused serious adverse reaction of medicine-drug interaction that the resulting active medicine of some other medicines screening methods produces because of final clinical application process, can save huge infusion of financial resources in advance in the new drug research, thereby avoid the blindness that drops in advance; Once more, this screening approach is for finally searching out effective treatment cholestasis and Jaundice disease and the new active medicine aspect of property rhabdomyolysis, medicine source provides the feasibility screening method.
Description of drawings
Fig. 1 makes up the expression plasmid that contains MRP2 gene promoter sequence luciferase reporter gene and PXR, transient transfection HepG2 cell technology platform technology route map for the present invention
The transient transfection HepG2 cell technology platform that Fig. 2 makes up for the present invention, the active medicine method and technology route map that high flux screening is possible
Concrete embodiment
Embodiment 1: make up the expression plasmid that contains MRP2 gene promoter sequence luciferase reporter gene and PXR, and transient transfection HepG2 cell technology platform, step is as follows:
1.MRP2 the structure of gene promoter sequence luciferase reporter gene plasmid
1.1 blood specimen collection and DNA extracting
Gather healthy volunteer's peripheric venous blood 5mL, the glass test tube as for the EDTA anti-freezing, it is standby to be stored in-40 ℃ of refrigerators.
1.2 DNA method for extracting
According to standard benzene phenol-chloroform method extracting genomic dna, place 4 ℃ of refrigerators standby.
1.3?MRP2?promoter?PCR
1.3.1 design of primers
1.3.2 amplified fragments is selected
1.3.3 amplification system design
1.3.4 amplification condition is selected
1.4 amplified production reclaims
1.5 the PCR product of MRP2 is connected on the pGEM-T carrier, extract plasmid, enzyme is cut and is checked order.
Confirm to insert plasmid DNA and the PGL4.17 carrier enzymes double zyme cutting that fragment does not have the PCR mispairing 1.6 will check order, reclaim respectively with agarose electrophoresis and insert fragment and carrier, use gel piece to link fast.Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme cut and identify and order-checking.
1.7 recombinant clone is further cultivated amplification.
2.PXR the structure of cDNA expression plasmid
2.1 RNA extracting and reverse transcription
From the former foster liver cell of being commissioned to train, extract total RNA with Trzol, use RevertAid
TMFirst Strand cDNA synthetic agent box synthesizes cDNA, is that template is carried out pcr amplification with cDNA.
2.2?PXR?PCR
2.2.1 design of primers
2.2.2 the amplification coding section length is selected
2.2.3 amplification system design
2.2.4 amplification condition is selected
2.3 be connected in the PCR product of PXR on the pGEM-T carrier and order-checking
Confirm to insert plasmid DNA and pcDNA3.1/hisB (-) carrier Hind III and the EcoR I double digestion simultaneously that fragment does not have the PCR mispairing 2.4 will check order, reclaim respectively with agarose electrophoresis and insert fragment and carrier, use gel piece to link fast.Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme is cut and is checked order.
2.5 recombinant clone is further cultivated amplification, the extracting plasmid DNA.Plasmid DNA is kept in 70% ethanol and keeps sterile state, and-20 ℃ frozen stand-by.
3..MRP2 the evaluation of reporter gene plasmid and PXR cDNA expression plasmid
3.1 MRP2 and PXR be double digestion respectively
3.2 1% agarose gel electrophoresis
3.3 sequencing analysis
4. use the reporter gene plasmid, pRL-TK (internal reference plasmid) and pcDNA3.1/hisB (-)-PXR transient transfection HepG2 cell
4.1 extract the test kit plasmid DNA purification with amount in the plasmid, the fluorescence report gene plasmid, pRL-TK, PXR and control plasmid illustrate transfection HepG 2 cell according to Lipofectamine2000.All comprise following sample sets in each experiment, carry out cotransfection as internal reference and PXR expression plasmid with each fluorescence report genophore to be measured with another reporter gene carrier pRL-TK, by measuring the activity of its gene product Renilla Luciferas, proofread and correct each spatial transfection efficiency.
Embodiment 2: the possible active medicine method of high flux screening is as follows:
1. cultivate the HepG2 cell;
2.HepG2 cell is with every hole 2 * 10
5The density of individual cell is with not containing foetal calf serum and antibiotic DMEM culture medium inoculated in 24 well culture plates.Every hole changes 600ng MRP2 reporter gene plasmid or control plasmid respectively over to, 100ngPXR plasmid and 10ng pRL-TK.Behind nutrient solution difference dilution DNA that does not contain microbiotic and foetal calf serum in right amount and Lipofectamine2000 liposome, with the two mixing, room temperature is placed 20min, adds and cultivates in the plate hole, rocks orifice plate gently and makes evenly.Behind the 6h, renew the DMEM that aquatic foods contain 10% calf serum, add candidate's possibility active medicine of DMSO, positive drug and various different concns, hatch 24h.
3. after hatching 24h, with growth medium from cell sucking-off to be checked.
4. with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off.
5. in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB).
6. the activation analysis of reporter gene
6.1 the activity of reporter gene detects
6.1.1 principle: as the fluorescence report gene series plasmid of the pRL-TK plasmid of internal reference and to be measured or the contrast two kinds of different protein of renilla luciferase and Photinus pyralis LUC of encoding respectively.In forming the oxyluciferin process, by the mediation transfer transport chemical energy is converted into luminous energy, thereby luminous.Two reporter genes all do not have endogenous activity in the experiment host cell, can continuously measured in single sample.In measuring process, at first add luciferase detection reagent II and produce the Lampyridea fluorescent signal, signal continues 1min at least, measures the Photinus pyralis LUC reporter gene so earlier.Quantitatively after the Lampyridea fluorescence intensity, in same sample, add Stop ﹠amp again; Glo
Reagent with above-mentioned reaction quencher, and starts the renilla luciferase reaction simultaneously, carries out the second time simultaneously and measures.
6.1.2 the activity of reporter gene detects step
(1) hatch 24h after the transfection, with growth medium from cell sucking-off to be checked.
(2) with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off.
(3) in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB).
(4) detect uciferase activity.On the luminous detection instrument, detecting uciferase activity with two fluorescence report kit genes after the lysis, reflecting the influence of different pharmaceutical respectively the MRP2 promoter activity with the ratio of Lampyridea fluorescence and sea pansy fluorescence.
Claims (3)
1. express PXR-MRP2 fluorescence report gene engineering platform high-flux medicaments sifting method for one kind, it is characterized in that: this method is directly started with the screening candidate from the pharmacokinetics target of the terminal clinical drug conceptual phase of new drug research may active lead drug, structure contains the expression plasmid of MRP2 gene promoter sequence luciferase reporter gene and PXR, transient transfection HepG2 cell; By on the luminous detector, detecting uciferase activity, reflect of the influence of the active lead drug of different possibilities respectively with the fluorescent worm fluorescence and the ratio of sea pansy fluorescence, carry out the high flux screening that external PXR induces the active lead drug of possibility the MRP2 promoter activity.
2. expression PXR-MRP2 fluorescence report gene engineering platform high-flux medicaments sifting method according to claim 1, it is characterized in that: described structure contains the expression plasmid of MRP2 gene promoter sequence luciferase reporter gene and PXR, and the step of transient transfection HepG2 cell is as follows:
Step 1: the structure of MRP2 gene promoter sequence luciferase reporter gene plasmid, process is:
(1) blood specimen collection and DNA extracting;
Gather healthy volunteer's peripheric venous blood 5mL, place the glass test tube of EDTA anti-freezing, it is standby to be stored in-40 ℃ of refrigerators;
(2) DNA method for extracting;
According to standard benzene phenol-chloroform method extracting genomic dna, place 4 ℃ of refrigerators standby;
(3)MRP2?promoter?PCR；
(4) amplified production reclaims;
(5) the PCR product of MRP2 is connected on the pGEM-T carrier, extracts plasmid, enzyme is cut and is checked order;
(6) will check order and confirm insert plasmid DNA and the PGL4.17 carrier enzymes double zyme cutting that fragment does not have the PCR mispairing, and reclaim respectively with agarose electrophoresis and insert fragment and carrier, the use gel piece links fast; Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme is cut and is checked order;
(7) recombinant clone is further cultivated amplification;
Step 2: the structure of PXR cDNA expression plasmid, process is:
(1) RNA extracting and reverse transcription;
From the former foster liver cell of being commissioned to train, extract total RNA with Trzol, use RevertAid
TMFirst Strand cDNA synthetic agent box synthesizes cDNA, is that template is carried out pcr amplification with cDNA;
(2)PXR?PCR；
(3) be connected in the PCR product of PXR on the pPEM-T carrier and order-checking;
(4) will check order and confirm insert fragment and do not have the plasmid DNA of PCR mispairing and pcDNA3.1/hisB (-) carrier with Hind III and EcoR I double digestion simultaneously, and reclaim respectively with agarose electrophoresis and insert fragment and carrier, and use gel piece to link fast; Transformed into escherichia coli DH5 α, picking mono-clonal extracting plasmid, enzyme is cut evaluation;
(5) recombinant clone is further cultivated amplification, the extracting plasmid DNA; Plasmid DNA is kept in 70% ethanol and keeps sterile state, and-20 ℃ frozen stand-by;
Step 3: the evaluation of MRP2 reporter gene plasmid and PXR cDNA expression plasmid, process is:
(1) MRP2 and PXR difference double digestion;
(2) 1% agarose gel electrophoresis;
(3) sequencing analysis;
Step 4: use the reporter gene plasmid, pRL-TK (internal reference plasmid) and pcDNA3.1/hisB (-)-PXR transient transfection HepG2 cell, process is: extract the test kit plasmid DNA purification with amount in the plasmid, the fluorescence report gene plasmid, pRL-TK, PXR and control plasmid illustrate transfection HepG 2 cell according to Lipofectamine2000; All comprise following sample sets in each experiment, carry out cotransfection as internal reference and PXR expression plasmid with each fluorescence report genophore to be measured with another reporter gene carrier pRL-TK, by measuring the activity of its gene product Renilla Luciferas, proofread and correct the transfection efficiency between each hole.
3. expression PXR-MRP2 fluorescence report gene engineering platform high-flux medicaments sifting method according to claim 1 and 2 is characterized in that: described external PXR induces the high flux screening step of the active lead drug of possibility as follows:
Step 1: cultivate the HepG2 cell;
Step 2: the HepG2 cell is with every hole 2 * 10
5The density of individual cell is with not containing foetal calf serum and antibiotic DMEM culture medium inoculated in 24 well culture plates; Every hole changes 600ng MRP2 reporter gene plasmid or control plasmid respectively over to, 100ngPXR plasmid and 10ng pRL-TK.Behind nutrient solution difference dilution DNA that does not contain microbiotic and foetal calf serum in right amount and Lipofectamine2000 liposome, with the two mixing, room temperature is placed 20min, adds and cultivates in the plate hole, rocks orifice plate gently and makes evenly; Behind the 6h, renew the DMEM that aquatic foods contain 10% calf serum, add candidate's possibility active medicine of DMSO, positive drug and various different concns, hatch 24h;
Step 3: after hatching 24h, with growth medium from cell sucking-off to be checked;
Step 4: with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off;
Step 5: in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB);
Step 6: the activity of reporter gene detects and analyzes;
The process that reporter gene activity detects is:
(1) hatch 24h after the transfection, with growth medium from cell sucking-off to be checked;
(2) with 1 * PBS rinsing cell, do not contact attached cell, as much as possible rinsing is used the PBS sucking-off;
(3) in the cell cultures hole, add an amount of 1 * passive lysis buffer (PLB);
(4) detect uciferase activity: on the luminous detection instrument, detecting uciferase activity with pair fluorescence report kit genes after the lysis, reflecting the influence of different pharmaceutical respectively to the MRP2 promoter activity with the ratio of Lampyridea fluorescence and sea pansy fluorescence.
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| CN102533925A (en) * | 2011-12-06 | 2012-07-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for in-vitro screening of PXR activation characteristics |
| CN104181221A (en) * | 2013-05-21 | 2014-12-03 | 成都先导药物开发有限公司 | Medicine target capturing method |
| CN108728417A (en) * | 2018-01-12 | 2018-11-02 | 湖南农业大学 | Method and products thereof for screening fodder antibiotics substitute |
| CN109913487A (en) * | 2019-02-14 | 2019-06-21 | 湖北大学 | A method of based on double fluorescent reporter gene system identification biological elements |
| CN111239385A (en) * | 2020-01-20 | 2020-06-05 | 河南科技大学 | Screening method and application of small molecule compound for targeted inhibition of vitamin K epoxide reductase |
| CN114891708A (en) * | 2022-05-26 | 2022-08-12 | 四川大学 | Luciferase reporter gene strain for monitoring up-regulation of gtfD and ftf gene level expression, preparation method and application thereof |
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- 2010-07-06 CN CN2010102216322A patent/CN101906469A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102533925A (en) * | 2011-12-06 | 2012-07-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for in-vitro screening of PXR activation characteristics |
| CN104181221A (en) * | 2013-05-21 | 2014-12-03 | 成都先导药物开发有限公司 | Medicine target capturing method |
| CN104181221B (en) * | 2013-05-21 | 2017-02-01 | 成都先导药物开发有限公司 | Medicine target capturing method |
| CN108728417A (en) * | 2018-01-12 | 2018-11-02 | 湖南农业大学 | Method and products thereof for screening fodder antibiotics substitute |
| CN109913487A (en) * | 2019-02-14 | 2019-06-21 | 湖北大学 | A method of based on double fluorescent reporter gene system identification biological elements |
| CN111239385A (en) * | 2020-01-20 | 2020-06-05 | 河南科技大学 | Screening method and application of small molecule compound for targeted inhibition of vitamin K epoxide reductase |
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