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
2 Cites 8 Cited by

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Problems solved by technology

However, the human hepatocyte model is seldom used in actual research due to many limitations...
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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.

Application Domain

Technology Topic

InducerDrug interaction +15

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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

Examples

  • Experimental program(2)

Example Embodiment

[0029] Example 1
[0030] Construct the OATP1B1 promoter fluorescent reporter gene plasmid and transiently transfect it into HepG2 cells to construct a dual fluorescent reporter gene technology platform.
[0031] 1. Material
[0032] 1. Plasmids, cell lines
[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 of 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 liposomes (Invitrogen, USA); DNA extraction kit (promega); Trizol (Invitrogen, USA).
[0037] 3. Main instruments
[0038] PCR machine (Thermo, USA); electrophoresis equipment (Beijing Liuyi); CO 2 Cell incubator (U.S. ThermoForm, SeriesII); ultra-clean workbench (U.S. thermal); Sirius C single-tube chemiluminescence detector (Berthold); GIS-2009 gel imaging analysis system (Tanon); low-temperature microcentrifuge (Eppendorf).
[0039] 2. Experimental method
[0040] 1. Construction of OATP1B1 promoter fluorescent reporter gene plasmid
[0041] 1.1 Blood sample collection and DNA extraction
[0042] Collect 5 mL of peripheral venous blood from healthy volunteers. As for the EDTA anticoagulated glass test tube, store it in a refrigerator at -40°C for later use. Genomic DNA was extracted according to the standard phenol-chloroform method and placed in a refrigerator at 4°C for later use.
[0043] 1.2OATP1B1PCR
[0044] 1.2.1 Primer design
[0045] Design primers based on the human OATP1B1 promoter sequence and the region where PXR binds to its promoter:
[0046] Binding region 1 (-128to-111): Primer F is the upstream 5'end primer, which contains 19 complementary bases of OATP1B1 gene promoter, a KpnI recognition site, and 2 protective bases;
[0047] Primer R is the upstream 3'primer, which contains 19 complementary bases of the OATP1B1 gene promoter, a Nhel recognition site, and 2 protective bases;
[0048] Primer F: TC GGTACC CCAGTGATGATTAACCACC
[0049] Primer R: CC CGATCG CCAGGTGGTATCTCCAGTC
[0050] Binding region 2 (-9957to-9940): Primer F is the upstream 5'end primer, which contains 19 complementary bases of OATP1B1 gene promoter, a Hind III recognition site, and 2 protective bases;
[0051] Primer R is the upstream 3'primer, which contains 19 complementary bases of OATP1B1 gene promoter, one Xhol recognition site, and 2 protective bases;
[0052] Primer F: TC AAGCTT CAAGCAGGGGCAATCTGTC
[0053] Primer R: CC CTCGAG GAGGGAGTAGGCAATGCTC
[0054] 1.2.2 Amplified fragment: (-128to-111); (-9957to-9940)
[0055] 1.2.3 Amplification system:
[0056] Front primer (10μM) 0.5μl
[0057] Back primer (10μM) 0.5μl
[0058] dNTP(2.5mM) 1.5μl
[0059] rTaq enzyme (5u/μl) 0.20μl
[0060] 10×PCR buffer 2.5μl
[0061] MgCl2 0.5μl
[0062] ddH2O 17.30μl
[0063] cDNA 2μl
[0064]
[0065] Final reaction system 25μl
[0066] 1.2.4 Amplification conditions
[0067] Binding region 1 amplification conditions
[0068] 95℃ 5 minutes
[0069] 95°C for 30 seconds
[0070] 56℃ 30 seconds 40 cycles
[0071] 72℃ 1 minute
[0072] 72℃ 5 minutes
[0073] Binding zone 2 amplification conditions
[0074] 95℃ 5 minutes
[0075] 95°C for 30 seconds
[0076] 60℃ 30 seconds 40 cycles
[0077] 72℃ 1 minute
[0078] 72℃ 5 minutes
[0079] 1.3 Recovery of amplified products
[0080] The amplified product was electrophoresed on a 1.0% agarose gel for 60 minutes, stained with ethidium bromide, and cut off the target strip gel under an ultraviolet light. Use QIAGEN's gel recovery kit to recover the target fragments. The steps are as follows:
[0081] ① After weighing the cut rubber block, place it in a 1.5EP tube, add 3 times the gel weight of QX1buffer, and add 10ul QIAEX II (glass powder); mix upside down and resuspend QIAEX II fully.
[0082] ②50 ℃ water bath for 10 minutes, mix EP tube upside down every 2 minutes, and resuspend the glass powder.
[0083] ③Centrifuge at 13000r/min for 1min and discard the supernatant.
[0084] ④Add 500ul QX1buffer to resuspend the glass powder.
[0085] ⑤ Centrifuge at 13000r/min for 1min, discard the supernatant.
[0086] ⑥Add 500ul buffer PE and resuspend the glass powder.
[0087] ⑦Centrifuge at 13000r/min for 1min, discard the supernatant.
[0088] ⑧ Dry in a 50 oven for 3-5 minutes, until the precipitate turns white.
[0089] ⑨Add 20ul ddH2O and incubate for 5min at room temperature to fully dissolve.
[0090] 1.4 Connect the PCR product of OATP1B1 to pPEM-T vector, extract the plasmid, digest by restriction enzyme and sequence.
[0091] The amplified products were all heated with rTaq enzyme at 70°C for 10 minutes and "A" was added. Carry out ligation reaction with pGEM-T vector, reaction system: 2×ligation buffer 5.0ul, add "A" PCR product 3.5ul, T vector 0.5ul, T4ligase 1.0ul; 16℃ water bath overnight.
[0092] The ligation product is transformed into JM109 E. coli, the steps are as follows:
[0093] ① Add 20ul of the ligation product to 200ul of self-made JM109 competent bacteria, rotate gently several times to mix, and place on ice for 30min.
[0094] ②Heat shock in 42℃ water bath for 80s.
[0095] ③Quickly place on ice and cool for 2-3 minutes.
[0096] ④Add 800ul of LB liquid medium without Amp.
[0097] ⑤ 37℃, 150r/min, shake for 1h.
[0098] ⑥Centrifuge at 13000r/min for 10s and remove 800ul of supernatant.
[0099] ⑦Resuspend the pellet and spread it evenly on the Amp-positive LB solid medium.
[0100] ⑧Invert the culture plate and incubate at 37℃ for 16h.
[0101] Pick the colony for culture, extract the T plasmid linked to the target fragment from E. coli, extract a small amount of plasmid DNA by alkaline lysis method, and conduct restriction digestion identification. Recombinant clones use SPIN column to extract and purify plasmids for sequencing.
[0102] The steps for plasmid extraction with OMEGA D6942-01 kit are as follows:
[0103] Ⅰ Centrifuge to collect 1.5-5ul bacterial liquid precipitate.
[0104] Ⅱ Add 250ul of RNase-containing solution I (bacterial resuspension) to fully resuspend the pellet.
[0105] Ⅲ Add 250ul solution II (bacterial lysis solution), gently invert and mix 4-6 times, incubate at room temperature for 2 minutes to fully lyse bacteria.
[0106] Ⅳ Add 350ul solution III (protein precipitation solution), slightly invert and mix 6-8 times to make the protein fully precipitate.
[0107] Centrifuge at 13000g for 10min at room temperature.
[0108] Ⅵ Transfer the supernatant to HiBind column and centrifuge at 13000r/min for 1min.
[0109] ⅦAdd 500ul Buffer HB and centrifuge at 13000r/min for 1min.
[0110] ⅧAdd 700ul Wash Buffer and centrifuge at 13000r/min for 1min.
[0111] Ⅸ Repeat step Ⅷ.
[0112] Ⅹ13000r/min idling for 2min.
[0113] ⅪReplace a new sterile 1.5EP tube, add 60ul ddH2O in HiBind column, and incubate for 2min at room temperature.
[0114] Centrifuge at 13000r/min for 1min to collect plasmids.
[0115] 1.5 The plasmid DNA and PGL4.10 vector whose inserts were confirmed to be free of PCR mismatch by sequencing were digested with endonuclease, and the insert and vector were recovered by agarose electrophoresis, and the gel block was used for quick linking. Transform JM109 Escherichia coli, pick a single clone to extract the plasmid, and identify by restriction enzyme digestion. The steps are the same as above.
[0116] 1.6 Recombinant clones are further cultivated and expanded.
[0117] Prepare plasmid DNA using PROMEGA Medium Plasmid Extraction Kit. Plasmid DNA is stored in 70% ethanol to maintain a sterile state, and frozen at -20°C for later use. Proceed as follows:
[0118] Ⅰ 50ul bacterial liquid was added to 50ml LB liquid medium and shaken at 220rpm/min for 12-16h.
[0119] ⅡCentrifuge at 5000g for 5min to collect bacteria, discard the supernatant, and leave the remaining liquid on the filter paper.
[0120] Ⅲ Add 3ml of cell resuspension solution and mix thoroughly with a gun.
[0121] IV Add 3ml of cell lysis solution, gently invert 3-5 times, and let stand for 3min.
[0122] ⅤAdd 5ml neutralization solution, gently invert 5-6 times, stand upright for 2-3min, until white precipitate appears.
[0123] Ⅵ Put the clean blue spin column on a new 50ml centrifuge tube.
[0124] ⅦPut the supernatant into a clean spin column and let it stand for 2 min.
[0125] ⅧCentrifuge at 3000g for 5min.
[0126] Ⅸ The filtrate is introduced into the binding column, and the subnatant is collected with a waste 50ml centrifuge tube, centrifuged at 3000g for 3min, and the subnatant is discarded.
[0127] ⅩAdd 20ml of eluent to the binding column, centrifuge at 1500g for 5min, discard the supernatant, and repeat centrifugation at 3000g for 5-10min.
[0128] ⅪAfter idling, the filter paper absorbs ethanol completely.
[0129] ⅫAdd 800ul ddH2O, centrifuge at 3000g for 5min, collect the subnatant in a clean 50ml centrifuge tube, and finally transfer it into a 1.5EP tube and store it at -40℃ for later use.
[0130] 2. Construction of PXR cDNA expression plasmid
[0131] 2.1 RNA extraction and reverse transcription
[0132] Use Trzol to extract total RNA from primary cultured hepatocytes, use RevertAid TM First Strand cDNA reverse transcription kit synthesizes cDNA, and uses cDNA as a template for PCR amplification.
[0133] 2.2PXRPCR
[0134] 2.2.1 Primer
[0135] 2.2.2 Amplified coding region length
[0136] 2.2.3 Amplification system
[0137] 2.2.4 Amplification conditions
[0138] 2.3 Connect the PCR product of PXR to pPEM-T vector and sequence
[0139] 2.4 Double enzyme digestion of plasmid DNA and pcDNA3.1-myc/hisB(-) vector confirmed by sequencing that there is no PCR mismatch in the insert, and the insert and vector were recovered by agarose electrophoresis, and the gel block was used for quick linking. Transform JM109 Escherichia coli, pick out the monoclonal extraction plasmid, and identify it by restriction enzyme digestion. The recombinant clones were further cultivated and amplified, and plasmid DNA was extracted. Plasmid DNA is stored in 70% ethanol to maintain a sterile state, and frozen at -20°C for later use.
[0140] 3. Double fluorescent reporter gene detection
[0141] Proceed as follows:
[0142] 3.1. Culture HepG2 cells;
[0143] When HepG2 cells grow to log phase, digest to 2×10 per well 5 The density of each cell is seeded on a 24-well plate, and the cells are grown to 80%.
[0144] 3.2. Co-transfection
[0145] 3.2.1 Each well needs to be transferred into 600ng PXR plasmid, 300ng OATP1B1 promoter fluorescent reporter gene plasmid and 50ng sea cucumber luciferase. After diluting DNA and Lipofectamine 2000 liposomes with an appropriate amount of culture medium without antibiotics and fetal bovine serum, they were mixed and placed at room temperature for 20 minutes.
[0146] 3.2.2 Wash the cells three times with PBS, and then add 1ml of MEM medium without fetal bovine serum to each well.
[0147] 3.2.3 Add 200ul of the above mixture (DNA/liposome) to a 24-well plate per well, and gently shake the culture plate to mix.
[0148] 3.3. Treatment with positive drug rifampicin
[0149] After 6h, fresh MEM containing 10% fetal bovine serum was changed, and medicine was added at the same time. Set up a control group and a positive drug rifampicin group (10umol), each with 4 replicate holes.
[0150] 3.4. The effect of rifampicin on the activity of reporter genes
[0151] 3.4.1 After 24 hours of incubation, aspirate the medium from the cells to be tested.
[0152] 3.4.2 Gently rinse the cells with 1×PBS and wash 3 times.
[0153] 3.4.3 Add an appropriate amount of 1× passive lysis buffer (PLB) to the cell culture wells.
[0154] 3.4.4 Detection of luciferase activity. a) Add 20ul of cell lysate to a 1.5ml centrifuge tube, then add 100ul of LARII solution, mix and put it into a luminescence detector to detect the first luminescence value (firefly fluorescence value); b) Then add Stop&Glo detection solution to stop the first Once the light is emitted, the second light is started at the same time, and the mixture is put into the luminescence detector to detect the second luminescence value (renilla fluorescence value).
[0155] 4. Results judgment
[0156] The ratio of firefly fluorescence to Renilla fluorescence was used to reflect the influence of different compounds to be tested to activate PXR on OATP1B1 promoter activity. The starting activity is to look at the ratio of two luciferases, the larger the ratio, the stronger the activity, the smaller the ratio, the weaker the activity. Then the evaluation standard is to look at the difference between the ratio of the drug-added group and the ratio of the non-medicine group. If there is a difference, the drug is considered active. For example, the fluorescence ratio of the cell group with rifampicin was 5.013±0.894, and the cell group without rifampicin treatment had the fluorescence value of 2.305±0.630, (p<0.05) (see Figure 4 ).

Example Embodiment

[0157] Example 2
[0158] The experimental method established above was used to study whether baicalin, a monomer compound of traditional Chinese medicine, can induce the expression of OATP1B1 by activating PXR.
[0159] Steps:
[0160] 1. Culture HepG2 cells;
[0161] When HepG2 cells grow to log phase, digest to 2×10 per well 5 The density of each cell is seeded on a 24-well plate, and the cells are grown 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. After diluting DNA and Lipofectamine 2000 liposomes with an appropriate amount of culture medium without antibiotics and fetal bovine serum, they were mixed and placed at room temperature for 20 minutes.
[0164] 2.2 Wash the cells three times with PBS, and then add 1 ml of MEM medium without fetal bovine serum to each well.
[0165] 2.3 Add 200ul of the above mixture (DNA/liposome) per well to a 24-well plate, and gently shake the culture plate to mix.
[0166] 3. Add Chinese medicine monomer compound treatment
[0167] After 6h, fresh MEM containing 10% fetal bovine serum was changed, and medicine was added at the same time. Set up a control group, a positive drug rifampicin group (10umol), a single compound of Chinese medicine to be tested baicalin group (1, 10, 100umol three concentration groups), each group has 4 multiple holes.
[0168] 4. The effect of baicalin, a monomer compound of traditional Chinese medicine, on the activity of reporter genes
[0169] 4.1 After 24 hours of incubation, aspirate the medium containing baicalin, a monomer compound of traditional Chinese medicine, from the cells to be tested.
[0170] 4.2 Gently rinse the cells with 1×PBS and wash 3 times.
[0171] 4.3 Add an appropriate amount of 1× passive lysis buffer (PLB) to the cell culture wells.
[0172] 4.4 Detection of luciferase activity. a) Add 20ul of cell lysate to a 1.5ml centrifuge tube, then add 100ul of LARII solution, mix and put it into a luminescence detector to detect the first luminescence value (firefly fluorescence value); b) Then add Stop&Glo detection solution to stop the first Once the light is emitted, the second light is started at the same time, and the mixture is put into the luminescence detector to detect the second luminescence value (renilla fluorescence value). The fluorescence ratio of firefly fluorescence value and Renilla fluorescence value was used to reflect the effect of traditional Chinese medicine monomer compound baicalin activated PXR on OATP1B1 promoter activity.
[0173] 5. Result judgment
[0174] The ratio of firefly fluorescence to Renilla fluorescence reflects the influence of different possible active compounds to activate PXR on the activity of OATP1B1 promoter. Studies have found that baicalin can enhance the transcriptional activity of OATP1B1 by activating PXR (see Figure 5 ). When baicalin is at three concentrations of 1, 10, and 100umol, the measured fluorescence ratios are: 4.527±0.291, 4.810±0.846, 5.102±0.586, and the P value is less than 0.05 compared with the control group 2.348±0.404.
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