SLCO1B1 overexpression cell strain as well as construction method and application thereof

Abstract

The invention provides an SLCO1B1 overexpression cell strain as well as a construction method and application thereof, and belongs to the technical field of molecular biology. The cell strain constructed by the invention has a good probe substrate transfer function, and due to the characteristic, the cell strain has important application value in drug development and research. The invention not only provides a construction method, but also covers the application of the overexpression cell strains in a plurality of fields, including research on a drug liver uptake mechanism, a drug liver metabolic pathway, drug interaction, a disease mechanism, drug screening or optimization, drug toxicity evaluation and drug curative effect evaluation. The applications show wide potential of the SLCO1B1 overexpression cell strain in drug development and research, and a powerful tool is provided for researchers in related fields.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a SLCO1B1 overexpression cell line and a construction method and application thereof. Background Art

[0002] SLC (solute carrier) uptake transporters are an important class of membrane proteins that play a key role in the absorption, distribution, metabolism, and excretion of drugs by the human body. These transporters actively or passively transport drug molecules from the extracellular environment to the cell or vice versa, thereby affecting the bioavailability and efficacy of the drug. Among them, organic anion transporting polypeptides OATP1B1 and OATP1B3 are important members of the SLC family. They are mainly expressed in the liver and are closely related to the process of drug distribution from blood vessels to hepatocytes. OATP1B1 and OATP1B3 can recognize and transport a variety of endogenous and exogenous compounds, including a variety of drugs, and therefore play a decisive role in drug metabolism and interactions. Among them, OATP1B1 is mostly expressed in the liver and is related to the distribution of drugs from blood vessels to hepatocytes.

[0003] The function and expression of these SLC uptake transporters are affected by multiple factors such as genetic polymorphism, disease status, and drug interactions, which in turn affect individual responses to drugs and drug efficacy. Therefore, the study of these transporters is of great significance for understanding the mechanism of drug action, predicting drug interactions, and guiding personalized medication. By deeply studying the structure, function, and regulatory mechanism of these transporters, it can provide important scientific basis for drug design, drug screening and optimization, and drug efficacy evaluation. Summary of the invention

[0004] The object of the present invention is to provide a SLCO1B1 overexpressing cell line and a construction method and application thereof, which have good probe substrate transport function.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a SLCO1B1 overexpressing cell line, comprising the following steps:

[0007] S1. Constructing a SLCO1B1 recombinant plasmid, wherein the SLCO1B1 recombinant plasmid contains the target gene shown in SEQ ID NO.1;

[0008] S2, lentiviral packaging, using the SLCO1B1 recombinant plasmid, together with virus packaging helper plasmid 1 and virus packaging helper plasmid 2 to form a three-plasmid lentiviral system, and co-transfecting into HEK-293T cells;

[0009] S3. Screen and obtain stably transfected SLCO1B1 overexpressing cell lines.

[0010] Preferably, the virus packaging helper plasmid 1 is a psPAX2 vector containing the pol gene and the rev gene.

[0011] Preferably, the virus packaging helper plasmid 2 is a pMD2G vector containing the vsvg gene.

[0012] Preferably, the SLCO1B1 recombinant plasmid uses pHBLV series vectors as starting vectors.

[0013] Preferably, the SLCO1B1 recombinant plasmid structure includes a CMV promoter, three FLAG tag sequences, an EF1 promoter and a puromycin resistance gene.

[0014] Preferably, the screening comprises screening stably expressing cell lines by puromycin.

[0015] Preferably, the target gene is amplified by a primer set containing the following primers:

[0016] The upstream primer as shown in SEQ ID NO.2;

[0017] The downstream primer is shown as SEQ ID NO.3.

[0018] Preferably, the co-transfection uses liposomes as carriers.

[0019] The present invention also provides a SLCO1B1 overexpressing cell line prepared by the above construction method.

[0020] The present invention also provides the use of the above-mentioned SLCO1B1 overexpressing cell line in studying drug liver uptake mechanism, drug liver metabolic pathway, drug interaction, disease mechanism, drug screening or optimization, drug toxicity evaluation or drug efficacy evaluation.

[0021] Beneficial effects of the present invention:

[0022] The SLCO1B1 overexpression cell line construction method provided by the present invention realizes efficient overexpression of the SLCO1B1 gene in HEK-293T cells through precise molecular biological technology, which is essential for studying and understanding the role of SLCO1B1 in drug liver uptake, metabolism and interaction. By utilizing a combination of CMV promoter, FLAG tag sequence, EF1 promoter and puromycin resistance gene, the method ensures stable expression of the target gene and screening efficiency of the cell line, and enhances the repeatability and reliability of the experiment.

[0023] The psPAX2 vector and pMD2G vector were used as virus packaging auxiliary plasmids, combined with the SLCO1B1 recombinant plasmid to form an efficient three-plasmid lentiviral system, which not only improved the virus packaging efficiency, but also increased the success rate of transfection. The use of liposomes as co-transfection vectors further optimized the transfection process, reduced toxicity to cells, and improved transfection efficiency.

[0024] The present invention also screens stably expressed cell lines by puromycin, and this step ensures that the screened cell lines have stable SLCO1B1 overexpression characteristics, which are suitable for long-term experiments and drug research. In addition, the method also provides detailed steps for amplifying the target gene using a specific primer set, which increases the accuracy and convenience of the experimental operation.

[0025] The cell lines constructed by the present invention have good probe substrate transport function, and this characteristic makes them have important application value in drug development and research. The present invention not only provides a construction method, but also covers the application of these overexpression cell lines in multiple fields, including studying drug liver uptake mechanism, drug liver metabolic pathway, drug interaction, disease mechanism, drug screening or optimization, drug toxicity assessment and drug efficacy assessment. These applications demonstrate the broad potential of SLCO1B1 overexpression cell lines in drug development and research, and provide powerful tools for researchers in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the pHBLV-CMV-MCS-3flag-EF1-puro vector map;

[0027] Figure 2 This is the result of observing the cell status under a microscope. DETAILED DESCRIPTION

[0028] The present invention provides a SLCO1B1 overexpression cell line and a construction method and application thereof.

[0029] In the present invention, the SLCO1B1 overexpressing cell line can be applied in the following aspects:

[0030] Drug metabolism and transport studies:

[0031] The OATP1B1 protein encoded by the SLCO1B1 gene is mainly present in the liver and is responsible for the transport of a variety of endogenous and exogenous compounds, including statins. Overexpression cell lines can be used to study the liver uptake mechanism and metabolic pathways of specific drugs.

[0032] Drug Interaction Studies:

[0033] Using SLCO1B1 overexpressing cell lines, we can study the interactions between different drugs, especially how they compete for the same transporter and affect the pharmacokinetics and efficacy of drugs.

[0034] Study on individual differences in drug responsiveness:

[0035] Since SLCO1B1 gene polymorphism is significantly associated with the lipid-lowering efficacy and clinical side effects of statins, overexpression cell lines can be used to explore how gene polymorphism affects drug responsiveness.

[0036] Drug safety assessment:

[0037] SLCO1B1-overexpressing cell lines can be used to evaluate potential drug-induced hepatotoxicity, especially for those drugs that are primarily metabolized and excreted by the liver.

[0038] Drug screening and optimization:

[0039] During drug development, SLCO1B1-overexpressing cell lines can be used to screen and optimize new drug candidates and predict the in vivo behavior of drugs by evaluating their transport properties in the liver.

[0040] Disease Mechanism Research:

[0041] SLCO1B1 overexpressing cell lines can be used to study diseases associated with SLCO1B1, such as certain inherited liver diseases, and how they affect the disposition of drugs.

[0042] Pharmacology and Toxicology Studies:

[0043] In pharmacology and toxicology studies, SLCO1B1 overexpressing cell lines can be used to evaluate the efficacy and toxicity of drugs, especially in terms of drug hepatic distribution and clearance.

[0044] In summary, the SLCO1B1 overexpressing cell line provided by the present invention has wide applications in multiple fields such as drug development, drug interaction and disease mechanism research.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example

[0047] Construction of h-SLCO1B1 lentiviral vector

[0048] Vector information: pHBLV-CMV-MCS-3flag-EF1-puro vector map as shown Figure 1 shown.

[0049] Target gene sequence information:

[0050]

[0051] Vector digestion

[0052] Add each reagent in the order shown in the table below, gently pipette to mix, and place in a 37°C water bath to react for 1-2 hours; after the enzyme digestion is completed, perform agarose gel electrophoresis to recover the target fragment;

[0053] The vector enzyme digestion system is as follows Table 1:

[0054] Table 1 Vector restriction enzyme digestion system

[0055] Reagents Volume (μL) Vector DNA (1 μg / uL) 1 10*buffer 4 <![CDATA[ddH2O]]> 32 EcoRI 1.5 B H 1.5 total 40

[0056] Acquisition of target fragment

[0057] Primer design:

[0058] LV-h-SLCO1B1-E / BF:

[0059] actagaggatctatttccggtGAATTCGCCACCatggaccaaaatcaac, as shown in SEQ ID NO.2;

[0060] LV-h-SLCO1B1-E / BR:

[0061] GTCACTTAAGCTTGGTACCGAGGATCCacaatgtgtttcactatctgc, as shown in SEQ ID NO.3;

[0062] PCR amplification of target fragment

[0063] Prepare the following system, mix gently, and place in PCR instrument for reaction;

[0064] The fragment PCR amplification system is shown in Table 2:

[0065] Table 2 PCR amplification system

[0066] Component name Volume (μL) 2XPCRBuffer 25 dNTPsmix (10 mM each) 1 F primer (10 μM) 2 R primer (10 μM) 2 Template DNA (200ng / uL) 1 <![CDATA[ddH2O]]> 18 phantaSuper-FidelityDNAPolymerase 1 Total volume 50

[0067] The PCR program is shown in Table 3:

[0068] Table 3 PCR program

[0069]

[0070] Connect the target fragment to the vector

[0071] HB infusion TM One-step cloning and ligation system:

[0072] Prepare the following reaction system in an ice-water bath. If the liquid accidentally sticks to the tube wall, it can be centrifuged briefly to make it sink to the bottom of the tube. After the ligation reaction solution reacts at 50℃ for 30 minutes, place it on ice for 5 minutes and transform immediately.

[0073] The reaction system is shown in Table 4:

[0074] Table 4 Reaction system

[0075] Component name Volume (μL) Target gene fragment X(≥100ng) Linearized vector Y(≥50ng) 2×HBinfusionTMMastermix 10 <![CDATA[ddH2O]]> Z(=10-XY) Total volume 20

[0076] Conversion

[0077] 1) After taking out the DH5α competent cells from the -80℃ refrigerator, put them on ice to thaw immediately. The competent cells should be packaged gently to reduce mechanical damage;

[0078] 2) After the competent cell melts, dispense it into 50 μL volumes per tube, add 5 μL of the ligation product, and place it on ice for 20-30 min;

[0079] 3) Heat shock at 42℃ for 90s, and immediately incubate on ice for 2-3min;

[0080] In a clean bench, add 500 μL of antibiotic-free LB medium and gently invert 3-5 times;

[0081] 5) Incubate at 37°C, 230 rpm with shaking for 45-60 min;

[0082] 6) Apply the bacterial solution to the solid plate of the corresponding resistance, spread it evenly, and then incubate the plate upside down at 37°C incubator for 12-16 hours;

[0083] Bacterial liquid PCR identification

[0084] The bacterial liquid PCR identification system is shown in Table 5:

[0085] Table 5 Bacterial liquid PCR identification system

[0086]

[0087]

[0088] The bacterial liquid PCR identification procedure is shown in Table 6:

[0089] Table 6 Bacterial liquid PCR identification procedures

[0090]

[0091] The sequencing results were consistent with the target sequence, and the target plasmid was successfully constructed.

[0092] The three-plasmid lentiviral system is used for virus packaging. The three-plasmid lentiviral system includes the following plasmids:

[0093] The vector plasmid carrying the target gene, the virus packaging auxiliary plasmid (psPAX2 vector) and the virus packaging auxiliary plasmid (pMD2G vector), the psPAX2 vector contains the gag gene, encoding the main structural protein of the virus; the pol gene, encoding the virus-specific enzyme; the rev gene, encoding the regulatory factor that regulates the expression of the gag and pol genes. The pMD2G vector contains the vsvg gene, which provides the envelope protein required for virus packaging.

[0094] After the three plasmid vectors were extracted to high purity and without endotoxin, the three plasmids were co-transfected into 293T cells using a transfection reagent. The viral supernatant was collected at 48 and 72 hours after transfection. The specific steps are as follows:

[0095] 293T cells were passaged in advance for transfection and placed in an incubator at 37°C and 5% CO2 after the operation;

[0096] Observe the cell density before transfection, and transfection can be performed when the confluence reaches 70-80%;

[0097] The components of the transfection system are shown in Table 7:

[0098] Table 7 Liposome transfection system

[0099] Plasmids and reagents Dosage pSPAX2 10 μg pMD2G 5μg Shuttle Plasmid 10 μg <![CDATA[Lipofiter TM Dosage]]> 75μL

[0100] After the lipid transfer complex is mixed and incubated at room temperature for 15 min, it is slowly added dropwise to the 293T cells and cultured in a cell culture incubator at 37°C and 5% CO2;

[0101] 16 h after transfection, fresh complete medium containing 10% fetal bovine serum (FBS) was replaced;

[0102] Virus collection: Collect the virus supernatant twice at 48h and 72h after transfection (replace with fresh complete culture medium after 48h collection). When collecting the virus at 48h, pour the culture medium in the 100mm dish into a 50mL centrifuge tube. Be careful not to let the wall of the culture dish touch the mouth of the centrifuge tube to prevent bacterial contamination. Then add 10mL of fresh complete culture medium containing 10% fetal bovine serum (FBS) and place it steadily in a constant temperature incubator at 37°C and 5% CO2 to continue culturing. When collecting the virus at 72h, pour the culture medium in the 100mm dish directly into a 50mL centrifuge tube. Also, be careful not to let the wall of the culture dish touch the mouth of the centrifuge tube to prevent bacterial contamination.

[0103] Ultracentrifugation: The virus supernatant in the 50mL centrifuge tube was centrifuged at 4°C, 2000×g for 10 min to remove cell debris; then the supernatant of the virus stock solution was collected and placed in an ultracentrifuge tube, and centrifuged at 4°C, 82700×g for 120 min. The virus precipitate was resuspended in complete culture medium, and finally the ultracentrifuge resuspension was dispensed into sterilized virus tubes to obtain the target virus HBLV-h-SLCO1B1-PURO.

[0104] Construction of SLCO1B1 gene overexpression stable strain using HEK-293 cells:

[0105] Cell plating (day 1)

[0106] After the cells in the 10 cm culture dish were fully grown, they were digested with trypsin, collected by centrifugation, and diluted to a density of 1.5 × 10 5 A cell suspension of 100 cells / ml (adjusted for different cell sizes and viability) was inoculated into a six-well plate, 2 ml per well, so that the cell fusion rate was about 60% on the second day, which was conducive to infection.

[0107] Virus infection (day 2)

[0108] Infection method: To promote the infection efficiency of the virus, first discard the original culture medium during infection, add 2 mL of fresh culture medium containing 5% FBS, and then add the infection-assisting reagent polybrene to a final concentration of 7 μg / ml. Since the optimal MOI values ​​of different cells are different, different MOI gradients are designed during infection. After 48 hours of infection, the gradient with high infection efficiency and good cell status is selected for subsequent experiments.

[0109] Calculation method of virus dosage: (cell number × MOI value / virus stock solution titer) × 10 3 =Amount of virus added (μl) The experimental groups: the virus MOI values ​​were 5, 10, and 20, as shown in Table 8 below.

[0110] Table 8 Virus MOI values

[0111]

[0112] Change medium 24 hours after infection (day 3)

[0113] After 24 h of infection, the virus infection medium was discarded and replaced with fresh complete medium.

[0114] 48h observation after infection (day 4)

[0115] After 48 hours of infection, it was observed that the three groups of HEK-293-SLCO1B1-PURO cells were in relatively ideal conditions. After comparison, the group with MOI=20 was selected for subsequent experiments; the group with MOI=20 was selected for subsequent experiments in the HEK-293-PURO group. After virus infection, when the cell fusion rate reached 80-90%, the cells were transferred to a 10 cm culture dish.

[0116] Stable strain screening (day 5)

[0117] Puromycin concentration exploration: Because the viral vector carries the puromycin resistance gene, cells infected with the virus will acquire puromycin resistance. Based on this characteristic, cells infected with the virus can be screened out by treating the virus-infected cells with puromycin. Before screening, the lowest puromycin concentration that can kill empty cells needs to be explored: cells are plated in six-well plates so that the fusion rate is about 60% on the second day, and fresh culture medium containing different concentrations of puromycin is replaced after 24 hours. The gradient is set to 0, 0.1, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 μg / ml and other gradient puromycin treatment for 48 hours, and the cell death is observed. The lowest concentration that can kill all cells is selected, and the lowest puromycin concentration that can kill HEK-293 empty cells is 1.5 μg / ml.

[0118] Resistance treatment: When the cells to be passaged grow to 60% in the culture dish, treat them with the found puromycin concentration for 48 hours. After 48 hours, if the fusion rate of HEK-293 cells is less than 80%, change to fresh puromycin medium and continue treatment. When the cell density exceeds 80%, passage treatment is required. Subsequently, culture medium containing puromycin is changed every 2-3 days to obtain a stable strain with overexpression. The cell status is observed under a microscope. Figure 2 .

[0119] Experimental example

[0120] The obtained cell lines were functionally verified using positive substrates and inhibitors.

[0121] Test methods

[0122] The experimental system is shown in Table 9 below:

[0123] Table 9 Experimental system

[0124]

[0125] In this experiment, the SLC transporter cell line prepared in the example was recorded as OATP1B1, the probe substrate was β-estradiol 17-(β-D-glucuronic acid) (E217βG), and the positive inhibitor was rifampicin (Rif).

[0126] The grouping and dosing solution composition table is shown in Table 9 below:

[0127] Table 9 Grouping and composition of dosing solution

[0128]

[0129] Experimental Procedure

[0130] Cell recovery

[0131] (1) Add 9 mL of pre-warmed culture medium (10% FBS DMEM) to a 15 mL centrifuge tube.

[0132] (2) Thaw the cells in a 37°C water bath until 90% of the cells are thawed (a small portion of unthawed clumps may still be observed).

[0133] (3) Pipette the cell suspension in the cryopreservation tube into a centrifuge tube containing 9 mL of culture medium.

[0134] (4) Rinse the cryotube with 1 mL of culture medium and transfer the solution to a centrifuge tube.

[0135] (5) Gently invert the centrifuge tube to evenly disperse the cells.

[0136] (6) After centrifugation at 300 g for 5 minutes, discard the supernatant and resuspend the cell pellet in 12.5 mL of fresh culture medium.

[0137] (7) Add 500uL of cell suspension to each well of a 24-well poly-lysine cell plate and gently shake the cell plate to spread the cells evenly.

[0138] (8) Place in an incubator at 37°C and 5% CO2 for incubation.

[0139] (9) After 3-4 hours, replace with fresh pre-warmed culture medium.

[0140] (10) After continuing to culture in a CO2 incubator for 14-27 hours, transporter experiments can be performed.

[0141] Intake test

[0142] Before cell lysis, the operation process needs to be gentle to avoid cells from detaching from the plate, and the solution needs to be gently added along the wall of the well to avoid hitting the cells directly.

[0143] (1) Preheat the buffer at 37°C (for dosing) and precool some buffer at 4°C (for termination of the experiment).

[0144] (2) Preparation of dosing solution:

[0145] Use DMSO to prepare stock solutions of positive substrate, test substances of various concentrations, and positive inhibitors at a concentration of 400 times (400×) the final concentration, and then dilute 400 times with buffer. The required solutions are the following: Solutions containing (or not containing) positive inhibitors and probe substrates: Use buffer to prepare solutions containing substrates and inhibitors (or 0.25% DMSO) and preheat at 37°C.

[0146] (3) Remove the culture medium from the wells and wash the cells once with 500 uL of preheated buffer.

[0147] (4) Remove the washing buffer and add 500uL of preheated buffer and incubate for 10 minutes.

[0148] (5) After 10 minutes, remove the buffer solution and add 250 uL of preheated dosing solution (step 2) to start the uptake experiment.

[0149] (6) After 90 min of incubation, the drug solution was immediately removed to terminate the uptake experiment.

[0150] (7) Wash the cells quickly three times with pre-cooled buffer, 500 μL / well / time.

[0151] (8) Add 250 μL of ultrapure water and freeze-thaw the cells three times in a liquid nitrogen-water bath.

[0152] (9) Detection: LC-MS / MS was used to determine the drug concentration, and the BCA method was used to determine the protein concentration.

[0153] Note: For other types of transiently transfected SLC cells, MATE1 and MATE2-K, the pH values ​​of the dosing solution and pre-chilled buffer were 8.0 and 8.4, respectively.

[0154] Data processing

[0155] (1) Substrate test:

[0156] 1) The uptake rate of the probe substrate (U, pmol / mg / min) was calculated using the following formula:

[0157]

[0158] Among them, C lysate is the amount of probe substrate transported in the cell lysate (pmol); P is the amount of cell protein (mg); T is the incubation time (min).

[0159] 2) The uptake ratio (UR) was calculated using the following formula:

[0160]

[0161] U is the uptake rate of substrate in the SLC transporter cell, U MOCK is the uptake rate of substrate in MOCK cells.

[0162] 3) The inhibition rate (IR) was calculated using the following formula:

[0163]

[0164] Among them, U withinhibitor is the uptake rate of the probe substrate or test substance in the presence of a selective inhibitor, U withoutinhibitor is the uptake rate of the probe substrate or test substance in the absence of a selective inhibitor.

[0165] When the UR value of the positive control group is greater than or equal to 2.0 and the inhibition rate exceeds 50%, the test system is considered qualified and can be used to evaluate the transport effect of SLC transporter cells on the test substance.

[0166] According to the USFDA's "Drug Interaction Guidelines", when the uptake ratio of the test substance through the transporter is less than 2.0, it indicates that the test substance is not a substrate for the transporter; when the uptake ratio is greater than or equal to 2.0 and the inhibition ratio exceeds 50%, it indicates that the test substance is a substrate for the transporter.

[0167] The results are shown in Table 10:

[0168] Table 10 Uptake rate, uptake ratio and inhibition rate of E217βG in MOCK and OATP1B1 cells

[0169]

[0170]

[0171] -1 group is the group without inhibitor; -2 group is the group with inhibitor.

[0172] As can be seen from the table, E217βG, as a positive control substrate of the OATP1B1 transporter, has an uptake ratio of 10.3 without adding an inhibitor, and an inhibition rate of 82%. The results show that the cell line OATP1B1 constructed by the present invention has an uptake ratio greater than 2.0 and an inhibition rate greater than 50%, and has a good probe substrate transport function.

[0173] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a SLCO1B1 overexpressing cell line, characterized in that: The following steps are involved: S1. Constructing a SLCO1B1 recombinant plasmid, wherein the SLCO1B1 recombinant plasmid contains the target gene shown in SEQ ID NO.1; S2, lentiviral packaging, using the SLCO1B1 recombinant plasmid, together with virus packaging helper plasmid 1 and virus packaging helper plasmid 2 to form a three-plasmid lentiviral system, and co-transfecting into HEK-293T cells; S3. Screen and obtain stably transfected SLCO1B1 overexpressing cell lines.

2. The construction method according to claim 1, characterized in that: The virus packaging auxiliary plasmid 1 is a psPAX2 vector containing pol gene and rev gene.

3. The construction method according to claim 1, characterized in that: The virus packaging auxiliary plasmid 2 is a pMD2G vector containing a vsvg gene.

4. The construction method according to claim 1, characterized in that: The SLCO1B1 recombinant plasmid uses the pHBLV series vector as the starting vector.

5. The construction method according to claim 4, characterized in that: The SLCO1B1 recombinant plasmid structure includes a CMV promoter, three FLAG tag sequences, an EF1 promoter and a puromycin resistance gene.

6. The construction method according to claim 5, characterized in that: The screening includes screening stably expressing cell lines by using puromycin.

7. The construction method according to claim 1, characterized in that: The target gene was amplified by a primer set containing the following primers: The upstream primer as shown in SEQ ID NO.2; The downstream primer is shown as SEQ ID NO.

3.

8. The construction method according to claim 1, characterized in that: The co-transfection uses liposome as a carrier.

9. A SLCO1B1 overexpressing cell line prepared by the construction method according to any one of claims 1 to 8.

10. Use of the SLCO1B1 overexpressing cell line according to claim 9 in studying drug liver uptake mechanism, drug liver metabolic pathway, drug interaction, disease mechanism, drug screening or optimization, drug toxicity evaluation or drug efficacy evaluation.

Images