Method for instantaneously and efficiently expressing human SLC transporter OATP1B3

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for instantaneously and efficiently expressing a human SLC transporter OATP1B3, which comprises the following steps: S1, preparing an expression plasmid of the transporter OATP1B3; step S2, recovering the HEK293H cells, and subculturing the HEK293H cells to a logarithmic phase to obtain an HEK293H cell suspension; s3, a transfection compound is added into an HEK293H cell suspension for transfection for 45-50 h, the volume ratio of the transfection compound to the cell suspension is 1: (8-12), and the transfection compound comprises a transfection reagent TA-293 and the expression plasmid in the step S1; s4, after transfection is finished, cells are collected, pyrolysis and purification are conducted at the room temperature, and the product is obtained. According to the method, a cell strain for transient expression of a human SLC transporter OATP1B3 transporter is constructed by using a genetic engineering technology, and a specific transfection reagent is combined, so that the transient expression efficiency of the human SLC transporter OATP1B3 is obviously improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a method for transiently and efficiently expressing human SLC transporter OATP1B3. BACKGROUND

[0002] OATP1B3 belongs to the Organic Anion-Transporting Polypeptide (OATP) branch SLCO subfamily in the solute carrier superfamily (SLC). Members of this subfamily are all transmembrane glycoproteins that mediate the transmembrane uptake of substrates in a sodium-independent manner. OATP1B3 has a wide substrate specificity and can transport a variety of endogenous substances and drugs (bilirubin, bile acids (such as chenodeoxycholic acid glucuronide), steroid conjugates, thyroid hormones T4 / T3, etc.). Statins (atorvastatin, pravastatin, rosuvastatin), antitumor drugs (paclitaxel, methotrexate, SN-38), antibiotics (rifampicin, erythromycin), sartans, bosentan, gliclazide, etc.). OATP1B3 is almost specifically expressed in the sinusoidal membrane of human hepatocytes and is the "key portal" for the uptake of drugs by the liver. The US FDA, the Chinese NMPA, the European EMA, etc. have issued relevant "Guidelines for Drug Interaction Studies". These documents clearly list OATP1B3 as a clinically relevant transporter that needs to be focused on. In the application for new drug clinical trials (IND) and new drug marketing authorization (NDA), sufficient in vitro and / or in vivo data must be provided to demonstrate that the potential risks of the interaction of the new drug with key transporters such as OATP1B3 have been assessed.

[0003] There are many methods for expressing human SLC transporter OATP1B3 in the prior art, but there are many defects: 1. The preparation of stable cell strains is long and costly, and adaptive changes or selection may occur during long-term culture, which indirectly affects the overall physiological state of the cells, thereby introducing unpredictable experimental variables. 2. The engineering cell strains used are diverse (such as HEK293, CHO or HepG2 cells), and different genetic backgrounds of cells may affect the transport properties of drugs. 3. The preparation method is not optimized, and the standardization degree is low, resulting in low expression of the target protein of the transiently prepared cell strain, and the transport activity cannot meet the requirements.

[0004] Therefore, there is an urgent need in the art to develop a preparation method for human OATP1B3 with high expression and high activity and standardization to meet the technical needs of transporter function research and drug interaction evaluation in the drug research and development process. SUMMARY

[0005] To overcome the above technical problems, the application provides a method for transiently and efficiently expressing human SLC transporter OATP1B3, which uses genetic engineering technology to construct a cell strain for transiently expressing human SLC transporter OATP1B3 transporter protein and combines with a specific transfection reagent, thereby significantly improving the transient expression efficiency of human SLC transporter OATP1B3.

[0006] In one aspect, the application provides a method for transiently and efficiently expressing human SLC transporter OATP1B3, which comprises the following steps, Step S1, preparing an expression plasmid of transporter OATP1B3; Step S2, recovering HEK293H cells and subculturing them to the logarithmic growth phase to obtain a HEK293H cell suspension; Step S3, adding a transfection complex to the HEK293H cell suspension for transfection for 45-50 h, the volume ratio of the transfection complex to the cell suspension being 1:8-12, and the transfection complex comprising a transfection reagent TA-293 and the expression plasmid of step S1; Step S4, collecting the cells after transfection, lysing them at room temperature, and purifying them to obtain the product.

[0007] By using the above technical solution, a transfection method using specific HEK293H cells as a transfection cell strain and combining with a specific transfection reagent TA-293 is screened for human SLC transporter OATP1B3 protein, and the transfection efficiency and substrate transfection activity of the transporter OATP1B3 can be significantly improved by using the transfection method.

[0008] Further, the cell density of the HEK293H cell suspension in step S2 is 1.5-2.5 x 10 6 cells / mL, preferably 2 x 10 6 cells / mL.

[0009] Further, the HEK293H cell suspension in step S2 is pretreated with DMSO before transfection in step S3.

[0010] By using the above technical solution, the HEK293H cell suspension is pretreated with DMSO before transfection, which can increase the fluidity and permeability of the cell membrane, so that the exogenous nucleic acid can more easily penetrate the cell membrane into the cell interior, and at the same time, it can also help the nucleic acid complex of the cell to be released from the endosome to the cytoplasm, thereby avoiding degradation, thereby greatly improving the transfection efficiency.

[0011] Further, the mass-volume ratio of the DMSO is 0.4-0.6%, preferably, the mass-volume ratio is 0.5%, and the pre-treatment time is 3-5h, preferably, the treatment time is 4h.

[0012] According to the above technical solution, as the concentration of DMSO increases and the treatment time increases, the toxicity of DMSO to cells also increases. Through strict experimental adjustment and determination, it is found that after the HEK293H cell suspension is pre-treated with 0.5% DMSO for 4h, the relative expression amount of the gene OATP1B3 can be obviously increased, and the activity of the cells can be ensured.

[0013] Further, in the step S3, an endoplasmic reticulum protein homeostasis regulator is added to the cell suspension containing the transfection complex after 5.5-6.5h of transfection.

[0014] In some embodiments, the endoplasmic reticulum protein homeostasis regulator is AA147, sodium phenylbutyrate, sodium tauroursodeoxycholate, tunicamycin, thapsigargin or brefeldin A, preferably AA147, sodium phenylbutyrate or sodium tauroursodeoxycholate, and more preferably AA147.

[0015] According to the above technical solution, by adding an endoplasmic reticulum protein homeostasis regulator, the function of the endoplasmic reticulum can be interfered, a stress state can be artificially created or the endoplasmic reticulum can be helped to cope with stress, the load of unfolded proteins can be reduced, or the folding capacity of cells can be enhanced. In this application, the endoplasmic reticulum protein homeostasis regulator is preferably AA147. This small molecule substance can activate the endoplasmic reticulum-related degradation pathway to activate the processing capacity of the endoplasmic reticulum for unfolded or misfolded proteins, thereby obviously increasing the relative expression amount of the gene OATP1B3, and effectively improving the transfection efficiency of the specific cell HEK293H. At the same time, in this method, the endoplasmic reticulum protein homeostasis regulator AA147 can also be used to treat the cell strain HEK293H in cooperation with DMSO, thereby obviously improving the transfection efficiency of the cell HEK293H for the OATP1B3 protein.

[0016] Further, the molar volume ratio of the endoplasmic reticulum protein homeostasis regulator to the cell suspension is 1:8-10.

[0017] Further, in the step S3, the transfection reagent TA-293 and the expression plasmid are mixed with the culture medium respectively, and the volume ratio of the transfection reagent TA-293 to the expression plasmid is 4-6:1.

[0018] Further, the volume ratio of the transfection reagent TA-293 to the culture medium is 1:8-12, and the volume ratio of the expression plasmid to the culture medium is 1:45-55.

[0019] In the present application, the culture medium used is serum-free DMEM culture medium.

[0020] In another aspect, the present application also provides a human SLC transporter OATP1B3 protein obtained by transient expression using the above method.

[0021] Using the above technical solution, the functional activity of the transporter OATP1B3 protein obtained is improved by nearly 50% compared with the OATP1B3 protein obtained by conventional methods in the art.

[0022] The present application has the following technical effects relative to the prior art.

[0023] 1) The transient expression method of the transporter OATP1B3 provided by the present application selects a specific HEK293H cell as a transfection cell strain and combines a specific transfection reagent TA-293, and the transfection efficiency and substrate transfection activity of the transporter OATP1B3 can be significantly improved by the transfection method.

[0024] 2) Using the method of the present application, high expression, high activity, and small batch difference of the transient transfection cell strain can be obtained in 48 hours, and efficient expression of the transporter OATP1B3 is realized, which meets the urgent needs of drug interaction evaluation in the drug research and development process. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0026] The following examples are used to illustrate the present application, but not to limit the scope of the present application. Based on the specific examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] In the examples, all raw material components are commercially available products well known to those skilled in the art unless otherwise specified; in the examples of the present application, the technical means used are conventional means well known to those skilled in the art unless otherwise specified.

[0028] The main reagents used in the examples are as follows: TA-293 transfection reagent: Zhuhai Kai Rui Company, item number: K20001; Lipofectamine 3000: Invitrogen Company, item number: L3000015; PEI Max: Kyfora bio by Polysciences Company, item number: 24765-1; HEK293H cells: Shanghai Bingsui Biotechnology Co., Ltd. HEK293T cells: National Biomedical Research Cell Bank CHO-K1 cells: National Biomedical Research Cell Bank HeLa cells: National Biomedical Research Cell Bank Endoplasmic reticulum protein homeostasis regulator AA147: MCE Company, Catalog No: HY-124293 Endoplasmic reticulum stress inhibitor sodium phenylbutyrate: MCE Company, Catalog No: HY-15654 Endoplasmic reticulum stress inhibitor sodium tauroursodeoxycholate: MCE Company, Catalog No: HY-19696A.

[0029] Example 1

[0030] 1. Preparation of expression plasmid of transporter OATP1B3

[0031] 1.1. Design PCR primers according to human OATP1B3 coding sequence (NCBI Reference Sequence: NM_019844.3): upstream primer: 5'-TTCCATTTCAGGTGTCGTGAGGATCATGGACCAACATCAACATTTGAAT-3' (SEQ ID NO. 1), downstream primer: 5'-TCTAGAGCCGGCGCGGCCGCGGATCTTAGTTGGCAGCAGCATTGTCTT-3' (SEQ ID NO. 2).

[0032] 1.2. Amplify human OATP1B3 coding sequence by PCR method with human cDNA as template.

[0033] 1.3. After linearizing the vector, OATP1B3 gene is constructed into expression vector pcDNA3.1(+) by homologous recombination method.

[0034] 1.4. Plasmid transformation, positive clones are identified by colony PCR method, and the correctness of OATP1B3 coding sequence is verified by sequencing.

[0035] 2. Cell preparation: HEK293H cells are cultured in a 37℃, 5% CO2 incubator using complete culture medium (DMEM + 10% FBS, F-12K + 10% FBS for CHO-K1), and passaged regularly to ensure that all cells are in the logarithmic growth phase at the time of transfection, with a viability of >95%.

[0036] 3. Transient transfection: TA-293 transfection reagent is used when the cell density reaches 1.5×10 6cells / mL. Transfection complex preparation (for 80 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, mix gently. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, mix gently. Add A tube solution to B tube, mix gently and stand at room temperature for 15 minutes. Add the transfection complex to the cell suspension and put into a shaker (37°C, 5% CO2, 110 rpm) for 45 hours.

[0037] 4. Cell cryopreservation: 45 hours after transfection, collect the cells and centrifuge at 300g for 5 minutes. Resuspend the cells with pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, transfer into liquid nitrogen for storage.

[0038] Example 2

[0039] 1. Preparation of expression plasmid of transporter OATP1B3 (same as Example 1).

[0040] 2. Cell preparation: use complete culture medium (DMEM + 10% FBS, CHO-K1 uses F-12K + 10% FBS) to culture HEK293H cells in a 37°C, 5% CO2 incubator, regularly passaged, and ensure that all cells are in the logarithmic growth phase at the time of transfection, with a viability of >95%.

[0041] 3. Transient transfection: use TA-293 transfection reagent, and transfect when the cell density reaches 2 x 10 6 cells / mL. Transfection complex preparation (for 100 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, mix gently. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, mix gently. Add A tube solution to B tube, mix gently and stand at room temperature for 15 minutes. Add the transfection complex to the cell suspension and put into a shaker (37°C, 5% CO2, 110 rpm) for 48 hours.

[0042] 4. Cell cryopreservation: 48 hours after transfection, collect the cells and centrifuge at 300g for 5 minutes. Resuspend the cells with pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, transfer into liquid nitrogen for storage.

[0043] Example 3

[0044] 1. Preparation of expression plasmid of transporter OATP1B3 (same as Example 1).

[0045] 2. Cell preparation: HEK293H cells were cultured in a 37°C, 5% CO2 incubator using complete medium (DMEM + 10% FBS, CHO-K1 used F-12K + 10% FBS), and passaged regularly to ensure that all cells were in the logarithmic growth phase at the time of transfection, with a viability of >95%.

[0046] 3. Transient transfection: TA-293 transfection reagent was used when the cell density reached 2.5 x 10 6 cells / mL. Transfection complex preparation (take 120 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, gently mix. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, gently mix. Add the solution in tube A to tube B, gently mix and stand at room temperature for 15 minutes. Add the transfection complex to the cell suspension and place it in a shaker (37°C, 5% CO2, 110 rpm) for 50 hours.

[0047] 4. Cell cryopreservation: 50 hours after transfection, the cells were collected and centrifuged at 300g for 5 minutes. Resuspend the cells with pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, transfer to liquid nitrogen for storage.

[0048] Comparative Example 1

[0049] Comparative Example 1 differs from Example 2 only in that HEK293H cells are replaced by HEK293T, and the remaining steps are the same as Example 2.

[0050] Comparative Example 2

[0051] Comparative Example 2 differs from Example 2 only in that HEK293H cells are replaced by CHO-K1, and the remaining steps are the same as Example 2.

[0052] Comparative Example 3

[0053] Comparative Example 3 differs from Example 2 only in that HEK293H cells are replaced by HeLa, and the remaining steps are the same as Example 2.

[0054] Comparative Example 4

[0055] Comparative Example 4 differs from Example 2 only in that the transfection reagent used in step S3 is different, which is Lipofectamine 3000, and the remaining steps are the same as Example 2.

[0056] Transient transfection: Lipofectamine 3000 transfection reagent was used, and the transfection was performed when the cell density reached 2 x 10 6 cells / mL. The transfection complex was prepared according to the instructions, and the plasmid and reagent were diluted with Opti-MEM, respectively, and mixed and stood for 5-10 minutes. The transfection complex was added to the cell suspension and placed in a shaker (37°C, 5% CO2, 110 rpm) for 48 hours.

[0057] Comparative Example 5

[0058] Comparative Example 5 is compared with Example 2, and the only difference is that the transfection reagent used in step S3 is PEI Max, and the other steps are the same as Example 2.

[0059] Transient transfection: Lipofectamine 3000 transfection reagent was used, and the transfection was performed when the cell density reached 2 x 10 6 cells / mL. The mass ratio of plasmid: PEI was calculated to be 1:3, and the plasmid and PEI Max were diluted with serum-free medium, respectively, and mixed and stood for 15 minutes. The transfection complex was added to the cell suspension and placed in a shaker (37°C, 5% CO2, 110 rpm) for 48 hours.

[0060] Comparative Example 6

[0061] Comparative Example 6 is compared with Example 2, and the only difference is that the transfection reagent used in step S3 is calcium phosphate, and the other steps are the same as Example 2.

[0062] Transient transfection: The plasmid-CaCl2 solution and 2x HBS buffer were prepared according to the classic method, mixed to form a precipitate, and then added dropwise to the cell suspension and placed in a shaker (37°C, 5% CO2, 110 rpm) for 48 hours.

[0063] Example 1

[0064] Transient transfection efficiency and functional activity detection were performed for Examples 1-3 and Comparative Examples 1-6.

[0065] 1. Cell recovery and plating: Quickly recover the frozen samples of each cell strain, resuspend and centrifuge with complete culture medium. Resuspend the cells in HBSS buffer, count and adjust the density to 0.5-0.8 x 10 6 cells / mL. Seed the cells into polylysine-coated 24-well plates at 0.5 mL of cell suspension per well (i.e. 0.25-0.4 x 10 6 cells / well), and incubate in a 37°C incubator for 12-16 hours to allow the cells to adhere.

[0066] 2. Transport assay: Aspirate the culture medium in the well, and wash the cells twice with pre-warmed HBSS buffer. Add 450 μL pre-warmed HBSS buffer to each well, and incubate at 37 °C for 10 min. Add 50 μL of HBSS buffer containing the probe substrate estradiol-6,7-3H(N)]-estradiol-7β-D-glucuronide (final concentration 1 μM) to each well, and incubate at 37 °C for 5 min. Aspirate the transport solution immediately, and wash the cells with ice-cold HBSS buffer for 5 times to terminate the reaction.

[0067] 3. Sample processing and detection: Add 100 μL acetonitrile to each well to lyse the cells, and incubate at room temperature for 10 min. Aspirate the lysate, and centrifuge at 10,000 g at 4 °C for 5 min. Take the supernatant. Detect the content of the probe substrate in the supernatant by LC-MS / MS. At the same time, determine the protein concentration of the lysate in each well by BCA method, which is used for standardization of the uptake amount (pmol / min / mg protein).

[0068] 4. Data analysis and comparison: Calculate the transfection efficiency of OATP1B3 gene and the transport activity (protein-standardized substrate uptake) of each cell strain, and the results are shown in Table 1.

[0069] Table 1 Transfection efficiency and functional activity of different cell strains and transfection reagents

[0070]

[0071] As can be seen from Table 1, the transfection efficiency and functional activity of OATP1B3 obtained by using different cell strains or different transfection reagents are different. In terms of the type of transfected cells, the transfection efficiency and substrate transport activity from high to low are HEK293H > HEK293T > CHO-K1 > HeLa, in terms of the transfection reagent, the substrate transport activity from high to low is TA-293 ≈ PEI Max > Lipofectamine 3000 > calcium phosphate method, the cost evaluation of the transfection reagent is TA-293 ≈ PEI Max < calcium phosphate method < Lipofectamine 3000, the repeatability evaluation (batch difference) is TA-293 > Lipofectamine 3000 > PEI Max > calcium phosphate method. Therefore, HEK293H cells are selected as the transfection cell strain, and the transfection reagent TA-293 is combined to perform transient transfection on the transport body OATP1B3.

[0072] Example 4

[0073] 1. Preparation of expression plasmid of transport body OATP1B3 (same as Example 1).

[0074] 2. Cell preparation: HEK293H cells were cultured in complete medium (DMEM + 10% FBS, F-12K + 10% FBS for CHO-K1) at 37°C, 5% CO2 incubator, passaged regularly to ensure all cells were in log phase growth and viability > 95% at the time of transfection.

[0075] 3. The cultured HEK293H cells were pretreated with 0.5% DMSO for 4h.

[0076] 4. Transient transfection: TA-293 transfection reagent was used when the cell density reached 2 x 10 6 cells / mL. Transfection complex preparation (for 120 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, gently mixed. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, gently mixed. The solution in A tube was added to B tube, gently mixed and then incubated at room temperature for 15 minutes. The transfection complex was added to the cell suspension and placed in a shaker (37°C, 5% CO2, 110 rpm) for 48 hours.

[0077] 5. Cell cryopreservation: 50 hours after transfection, the cells were collected and centrifuged at 300g for 5 minutes. The cells were resuspended with pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, the cells were transferred to liquid nitrogen for storage.

[0078] Example 5

[0079] 1. Preparation of the expression plasmid of the transporter OATP1B3 (same as Example 1).

[0080] 2. Cell preparation: HEK293H cells were cultured in complete medium (DMEM + 10% FBS, F-12K + 10% FBS for CHO-K1) at 37°C, 5% CO2 incubator, passaged regularly to ensure all cells were in log phase growth and viability > 95% at the time of transfection.

[0081] 3. Transient transfection: TA-293 transfection reagent was used when the cell density reached 2 x 10 6cells / mL. Transfection complex preparation (for 120 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, gently mix. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, gently mix. Add A tube solution to B tube, gently mix and stand at room temperature for 15 minutes. Add the transfection complex to the cell suspension, and put into a shaker (37°C, 5% CO2, 110 rpm) for 6 hours, then add endoplasmic reticulum protein homeostasis regulator AA147 at a concentration of 10 μM, continue transfection for 48 hours.

[0082] 4. Cell cryopreservation: 50 hours after transfection, collect the cells and centrifuge at 300 g for 5 minutes. Resuspend the cells with pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, transfer to liquid nitrogen for storage.

[0083] Example 6

[0084] 1. Preparation of expression plasmid of transporter OATP1B3 (same as Example 1).

[0085] 2. Cell preparation: HEK293H cells were cultured in a 37°C, 5% CO2 incubator using complete culture medium (DMEM + 10% FBS, CHO-K1 uses F-12K + 10% FBS), and were passaged regularly to ensure that all cells were in the logarithmic growth phase at the time of transfection, with a viability of >95%.

[0086] 3. The cultured HEK293H cells were pretreated with 0.5% DMSO for 4 hours.

[0087] 4. Transient transfection: TA-293 transfection reagent was used, and transfection was performed when the cell density reached 2 x 10 6 cells / mL. Transfection complex preparation (for 120 mL system as an example): A tube: 5 mL serum-free DMEM + 500 μL TA-293, gently mix. B tube: 5 mL serum-free DMEM + 100 μg pCDNA3.1(+)-OATP1B3 plasmid, gently mix. Add A tube solution to B tube, gently mix and stand at room temperature for 15 minutes. Add the transfection complex to the cell suspension, and put into a shaker (37°C, 5% CO2, 110 rpm) for 6 hours, then add endoplasmic reticulum protein homeostasis regulator AA147 at a concentration of 10 μM, continue transfection for 48 hours.

[0088] 5. Cell cryopreservation: 50 hours after transfection, collect cells and centrifuge at 300g for 5 minutes. Resuspend cells in pre-cooled cryopreservation solution (10% DMSO + 90% FBS) at a density of 10 x 10 6 cells / mL. After programmed cooling, transfer into liquid nitrogen for preservation.

[0089] Comparative Example 7

[0090] Comparative Example 7 is compared with Example 4, the only difference is that 0.5% DMSO is replaced by 100 μM menthol pretreatment for 2 hours in step 3, and the rest of the steps are the same as Example 4.

[0091] Comparative Example 8

[0092] Comparative Example 8 is compared with Example 5, the only difference is that 10 μM endoplasmic reticulum protein homeostasis regulator AA147 is replaced by 5 mM endoplasmic reticulum stress inhibitor sodium phenylbutyrate in step 3, and the rest of the steps are the same as Example 5.

[0093] Comparative Example 9

[0094] Comparative Example 9 is compared with Example 5, the only difference is that 10 μM endoplasmic reticulum protein homeostasis regulator AA147 is replaced by 100 μM endoplasmic reticulum stress inhibitor sodium tauroursodeoxycholate in step 3, and the rest of the steps are the same as Example 5.

[0095] Effect Example 2

[0096] 1. Cell cryopreservation and functional activity detection (method same as Effect Example 1), after recovery, detect OATP1B3 protein expression (WB), functional activity (substrate uptake).

[0097] 2. To quantitatively evaluate the membrane localization efficiency of OATP1B3, immunofluorescence staining is used in combination with a semi-quantitative scoring system. Briefly, after fixation, permeation, and blocking of the cells after transfection, anti-OATP1B3 primary antibody, fluorescent secondary antibody, membrane dye (WGA-Alexa Fluor 647), and nuclear dye (DAPI) are used for staining in sequence. Images are obtained by confocal microscopy, and three researchers who do not know the grouping information independently score according to the predetermined 0-5 point standard. A score of 0 represents no signal, and a score of 5 represents strong and precise localization of the protein on the cell membrane. The final score is the average of the scores of the three people.

[0098] The detection results of Examples 4-6 and Comparative Examples 7-9 are shown in Table 2.

[0099] Table 2 Influence of different treatment methods on OATP1B3 expression and functional activity

[0100]

[0101] From the test results of Example 2, Example 4 and Comparative Example 7, it can be seen that the pretreatment of HEK293H cells with DMSO before transient transfection improves the transfection efficiency, substrate transport activity, OATP1B3 relative expression and membrane localization score; while the pretreatment of HEK293H cells with menthol is not as effective as DMSO.

[0102] From the test results of Example 2, Example 5 and Comparative Examples 8-9, it can be seen that the treatment of transfected cells with endoplasmic reticulum protein homeostasis regulator AA147 after transient transfection for a period of time improves the transfection efficiency, substrate transport activity, OATP1B3 relative expression and membrane localization score; while the treatment of transfected cells with endoplasmic reticulum stress inhibitor sodium phenylbutyrate or sodium tauroursodeoxycholate is not as effective as AA147.

[0103] From the test results of Example 2, 4-6, it can be seen that the pretreatment with DMSO and the treatment with endoplasmic reticulum protein homeostasis regulator AA147 are mutually synergistic, whether it is transfection efficiency, substrate transport activity, or OATP1B3 relative expression and membrane localization score, compared with the method of pretreatment with DMSO alone or treatment with endoplasmic reticulum protein homeostasis regulator AA147 alone, all have been significantly improved, especially the substrate transport activity is improved by 40%, far beyond the expectation.

[0104] The specific embodiments are merely an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make modifications to the present embodiments without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for transient and efficient expression of the human SLC transporter OATP1B3, characterized in that, The method includes the following steps: Step S1: Prepare the expression plasmid of transporter OATP1B3; Step S2: Resuscitate HEK293H cells, passage them to the logarithmic growth phase to obtain HEK293H cell suspension, and pretreat the HEK293H cell suspension with DMSO. Step S3: Add the transfection complex to HEK293H cell suspension for transfection for 45-50 hours. 5.5-6.5 hours after the start of transfection, add the endoplasmic reticulum protein homeostasis regulator AA147 to the cell suspension containing the transfection complex. The volume ratio of the transfection complex to the cell suspension is 1:8-12. The transfection complex includes the transfection reagent TA-293 and the expression plasmid described in step S1. Step S4: After transfection, collect the cells, lyse them at room temperature, and purify them to obtain the final product.

2. The method according to claim 1, characterized in that, In step S2, the cell density of the HEK293H cell suspension is 1.5-2.5 × 10⁻⁶ cells / mL. 6 cells / mL.

3. The method according to claim 1, characterized in that, The mass-to-volume ratio of DMSO is 0.4-0.6%, and the pretreatment time is 3-5 hours.

4. The method according to claim 1, characterized in that, The molar volume ratio of the endoplasmic reticulum protein homeostasis regulator AA147 to the cell suspension is 1:8-10.

5. The method according to claim 1, characterized in that, In step S3, the transfection reagent TA-293 and the expression plasmid are mixed with culture medium, and the volume ratio of the transfection reagent TA-293 to the expression plasmid is 4-6:

1.

6. The method according to claim 5, characterized in that, The volume ratio of the transfection reagent TA-293 to the culture medium is 1:8-12, and the volume ratio of the expression plasmid to the culture medium is 1:45-55.

7. A method for transiently expressing the human SLC transporter OATP1B3 protein using the method described in any one of claims 1-6.

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