A method for constructing a KCNB1 ion channel stably transfected cell line and its application in toxicity screening of plastic mobile substances and degradation products

Abstract

The present invention discloses a method for constructing a KCNB1 ion channel stably transfected cell line and its application in toxicity screening of plastic mobile substances and degradation products, and relates to the field of biotechnology. The construction method includes the construction of a plasmid vector, which is to clone the KCNB1 gene sequence by PCR technology using cDNA as a template, and connect it to the plasmid pcSLenti‑EF1‑EGFP‑P2A‑Puro‑CMV‑MCS‑3xFLAG‑WPRE by double enzyme digestion technology to obtain a pcSLenti‑EF1‑EGFP‑P2A‑Puro‑CMV‑KCNB1‑3xFLAG‑WPRE lentiviral vector. The KCNB1 ion channel stably transfected cell line constructed by the present invention is more sensitive to the toxicity of pollutants than conventional cells, and can realize high-throughput excitotoxicity screening of mobile substances and degradation products in biodegradable plastics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a KCNB1 ion channel stably transfected cell line and an application thereof in toxicity screening of plastic migratable substances and degradation products. Background Art

[0002] Ion channels are an important mechanism associated with a variety of toxicities, including skin sensitization, pain, and neurotoxicity. Voltage-gated potassium (K+) channels are a large and diverse family of ion channels that play a fundamental role in regulating neuronal excitability. Abnormal neural excitation may lead to many adverse outcomes, such as Alzheimer's disease.

[0003] Chinese patent CN116144705A discloses a method for efficiently preparing a voltage-dependent calcium channel cell model, which discloses a group of vectors for co-expressing human Cav1.2 calcium channel protein, including vector 1 and vector 2, wherein the human Cav1.2 calcium channel protein includes three subunits: α1, α2 / δ1 and β; the vector 1 contains a TetON promoter, and the expression sequences of α2 / δ1 and β subunits are located downstream of the TetON promoter; at the same time, the vector 1 has an rtTA expression element, which is driven by the CMV promoter for expression, and the vector 1 also contains a resistance gene 1; the vector 2 contains a TetON promoter, and the expression sequences of EGFP and α1 subunits are located downstream of the TetON promoter; the vector 2 also contains a resistance gene 2; the expression sequences of the α2 / δ1 and β subunits are connected by the expression sequence of the T2A peptide; the expression sequences of the EGFP and α1 subunits are connected by the expression sequence of the P2A peptide. The invention also discloses a method for efficiently preparing a voltage-dependent calcium channel cell model based on this; through doxycycline induction, the cell line of the invention has the characteristics of stably expressing and functional human Cav1.2 calcium channels. The invention provides an alternative cell line other than human cardiomyocytes - the Cav1.2 calcium channel-expressing HEK293T cell line, as an effective and economical in vitro cardiac safety evaluation of drugs. However, it only targets the Cav1.2 calcium channel.

[0004] At present, there is no standard animal experimental method for toxicity testing of ion channels, and the species specificity of ion channels makes it impossible for animal-derived experimental systems to solve the problem of species extrapolation. The degradation products and intermediates present in biodegradable plastics may have the effect of disturbing ion channels, but their types are numerous and their content is extremely low. The use of normal cell lines cannot achieve the purpose of low-dose identification and rapid screening due to the low sensitivity of ion channel expression. The lack of human cell lines with a complete system and stable expression of ion channels has limited the establishment of relevant toxic effect testing systems.

[0005] Therefore, the present invention constructs a stable expression cell model of KCNB1 ion channel, conducts excitatory or inhibitory neurotoxicity tests on mobile substances and degradation products in biodegradable plastics, and thus establishes a detection and screening technology based on ion channel-related toxicity. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a KCNB1 ion channel stably transfected cell line.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a method for constructing a KCNB1 ion channel stably transfected cell line, including the construction of a plasmid vector, which is to clone the KCNB1 gene sequence by PCR technology using cDNA as a template, connect it to the plasmid pcSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE by double enzyme digestion technology, and use ligase to connect to obtain the pcSLenti-EF1-EGFP-P2A-Puro-CMV-KCNB1-3xFLAG-WPRE lentiviral vector.

[0009] Specifically, the KCNB1 gene sequence is NCBI Reference Sequence: NM_004975.4.

[0010] Specifically, the KCNB1 gene sequence was cloned by PCR using cDNA as a template to obtain a fragment with an XbaI restriction site at the front end and an EcoRI restriction site at the back end.

[0011] Furthermore, the ligase is T4 DNA ligase.

[0012] Specifically, the construction method comprises the following steps:

[0013] (1) Cultivating cells in culture medium;

[0014] (2) Construction of plasmid vector;

[0015] (3) adding the DNA transfection reagent complex to the cells cultured in step (1) for transfection, centrifuging after transfection, adding a buffer to dissolve the precipitate, and obtaining the virus;

[0016] (4) Digest the cells and then inoculate them, adding viruses for infection;

[0017] (5) The cells are arranged into (1-10)×10 5cells / mL cell suspension, incubate for 12-20 hours, infect with virus, add polybrene, and obtain KCNB1 ion channel stably transfected cell line.

[0018] Specifically, the cells described in step (1) include HEK293 cells, SH-SY5Y cells, and HaCaT cells.

[0019] Furthermore, the cells described in step (1) are HEK293 cells.

[0020] Specifically, the components of the culture medium in step (1) include fetal bovine serum, L-glutamine and double antibody.

[0021] Furthermore, the concentration of the fetal bovine serum is 5-15%, and the concentration of L-glutamine is 1-5 mM;

[0022] Furthermore, the concentration of the fetal bovine serum is 10%, and the concentration of L-glutamine is 2 mM.

[0023] Furthermore, the dual antibiotics include penicillin and streptomycin, the concentration of penicillin is 50-150 U / mL, and the concentration of streptomycin is 50-150 μg / mL.

[0024] Furthermore, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 100 μg / mL.

[0025] Specifically, the culture conditions in step (1) are 37°C, 5% CO 2 .

[0026] Specifically, during the transfection in step (3), the density of the cells during transfection is controlled to be 70-80%.

[0027] Specifically, the DNA transfection reagent complex in step (3) includes a transfection reagent and a plasmid complex.

[0028] Furthermore, the plasmid complex includes a backbone plasmid and a shuttle plasmid;

[0029] Furthermore, the mass ratio of the backbone plasmid to the shuttle plasmid is 1-2:1; preferably, the mass ratio of the backbone plasmid to the shuttle plasmid is 1:1.

[0030] Specifically, the backbone plasmid is pcSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE, and the shuttle plasmid is pCMV-VSVG.

[0031] Specifically, the plasmid complex is obtained by dissolving the viral vector plasmid in the culture medium.

[0032] Furthermore, the transfection reagent is prepared by dissolving the transfection reagent in a culture medium.

[0033] The culture medium is selected from one or more of Opti-MEM culture medium, DMEM culture medium, and RPMI1640 culture medium;

[0034] Furthermore, the culture medium is Opti-MEM culture medium.

[0035] Specifically, the mass volume ratio of the transfection reagent to the plasmid complex in the DNA transfection reagent complex is (30-40) μg:500 μL.

[0036] Furthermore, the mass volume ratio of the transfection reagent to the plasmid complex in the DNA transfection reagent complex is 32 μg:500 μL.

[0037] Specifically, in step (3), the first virus harvest is performed 48 hours after transfection; fresh culture medium is replaced after virus harvest; and the second virus harvest is performed 72 hours after transfection.

[0038] Specifically, the number of centrifugation in step (3) is 2 times, the speed of the first centrifugation is 3500 rpm, the centrifugation time is 10 min, and the centrifugation temperature is room temperature.

[0039] Furthermore, after the first centrifugation, the supernatant was collected and a second centrifugation was performed; the speed of the second centrifugation was 30,000 rpm, the centrifugation time was 2 h, and the centrifugation temperature was 4°C.

[0040] Furthermore, the volume of the buffer added in step (3) is 100 μL.

[0041] Furthermore, the buffer is DPBS buffer.

[0042] Specifically, the digestive enzyme used to digest the cells in step (4) is pancreatic enzyme.

[0043] Specifically, the inoculation amount of cells in step (4) is (1-10)×10 5 Furthermore, the cell inoculation volume was 1×10 5 .

[0044] Specifically, the final concentration of polybrene in step (5) is (4-10) μg / mL;

[0045] Furthermore, the final concentration of polybrene in step (5) is 5 μg / mL.

[0046] Specifically, in step (5), after 72 hours, polybrene is added at a final concentration of (1-3) μg / mL.

[0047] Furthermore, after 72 h, polybrene was added at a final concentration of 2 μg / mL.

[0048] In another aspect, the present invention provides a KCNB1 ion channel stably transfected cell line constructed by the above construction method.

[0049] In yet another aspect, the present invention provides application of the above-mentioned construction method in high-throughput excitotoxicity screening of mobile substances and degradation products in biodegradable plastics.

[0050] Specifically, the migratable substances include heat stabilizers, chain extenders, organic phosphate flame retardants, UV absorbers, antioxidants and their degradation products, phthalates, and perfluorinated compounds. The degradation products include phenyltin trichloride, di-tert-butyl peroxide, and diphenylmethane diisocyanate.

[0051] Furthermore, the heat stabilizer is an organic tin compound.

[0052] Furthermore, the chain extender includes dimethylthiotoluene diamine and isocyanates.

[0053] Furthermore, the anti-ultraviolet absorber includes benzotriazole.

[0054] Furthermore, the antioxidant degradation products include p-tert-butylphenol and di-tert-butyl peroxide.

[0055] Furthermore, the degradation product is phenyltin trichloride among the organotin substances.

[0056] The beneficial effects of the present invention are:

[0057] 1. The KCNB1 ion channel stably transfected cell line constructed by the present invention is more sensitive to the toxicity of pollutants than conventional cells;

[0058] 2. The constructed gene sequence is a membrane receptor fragment, which can better reflect its ion channel activity;

[0059] 3. The constructed vector carries both EGFP tag and Flag tag, which is conducive to the observation and screening of ion channel stably transfected cells;

[0060] 4. The virus can infect HEK293 and other cells, meeting the toxicity screening of cells from different sources;

[0061] 5. It can realize high-throughput excitotoxicity screening of mobile substances and degradation products in biodegradable plastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Fluorescence images of stably transfected cell lines.

[0063] Figure 2 This is the WB test result of membrane protein. DETAILED DESCRIPTION

[0064] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0065] The DMEM medium, fetal bovine serum, and carbon-adsorbed fetal bovine serum used in the cell culture of the present invention were purchased from Gibco (Thermo Fisher scientific, MA, USA). The trypsin used for cell digestion was purchased from Gibco (Thermo Fisher scientific, MA, USA).

[0066] Reagents: Puromycin hydrochloride (58-58-2) was purchased from Bio-Tech. Flag antibody was purchased from Abcam. Membrane protein extraction kit was purchased from Bio-Tech (P0033).

[0067] Example 1 Construction of infectious virus

[0068] 1. Cell Culture

[0069] HEK293 cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, and double-antibody (100 U / mL penicillin and 100 μg / mL streptomycin). The cells were incubated at 37°C with 5% CO 2 Culture in an incubator.

[0070] 2. Plasmid vector construction

[0071] The KCNB1 gene sequence (NCBI Reference Sequence: NM_004975.4) was cloned by PCR using human cDNA template (Heyuan Biotechnology) to obtain a fragment with an XbaI restriction site at the front end and an EcoRI restriction site at the back end.

[0072] The PCR reaction conditions are shown in Table 1:

[0073] Table 1 PCR reaction conditions

[0074]

[0075] The plasmid vector pcSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE and the DNA fragment were digested with XbaI enzyme (purchased from Takara, catalog number 1093S) and EcoRI enzyme (purchased from Takara, catalog number 1040S), reacted at 37°C for 2 h, and inactivated in a metal bath at 65°C for 20 min after the digestion. Use T4 ligase (1 μL T4 ligase, 1 μL buffer, 200 ng DNA fragment, 50 ng vector, make up to 10 μL with water) for overnight ligation, mix 10 μL of overnight ligated DNA with DH5α competent E. coli, and place on ice for 30 min to allow the plasmid vector to contact the competent E. coli cells; after 30 min, place in a 42°C water bath for 90 s to allow the plasmid vector to enter the competent E. coli cells; then place it back on ice for 2 min and add 1 mL LB culture medium solution and culture it in a 37°C shaker for 90 min; after centrifugation, evenly apply the bacterial solution to the LB solid culture medium containing the corresponding antibiotics. Pick a single colony on the culture medium the next day and place it in the LB liquid culture medium containing the corresponding resistance and sequence it.

[0076] The one with correct sequencing is the pcSLenti-EF1-EGFP-P2A-Puro-CMV-KCNB1-3xFLAG-WPRE lentiviral vector.

[0077] 3. Virus encapsulation and collection

[0078] One day before transfection, cells were seeded into 100 mm dishes, and the density of cells during transfection was controlled to be 70-80%. One hour before transfection, the cell culture dish was removed, the original cell culture medium was removed, 10 ml of Opti-MEM culture medium was added, and the cells were returned to the incubator.

[0079] Prepare the complex of transfection reagent and plasmid:

[0080] Dissolve 32 ug of the viral vector plasmid to be transfected (backbone plasmid: shuttle plasmid = 1:1, the backbone plasmid is pcSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE, and the shuttle plasmid is pCMV-VSVG) in Opti-MEM medium to a total volume of 500 μl, mix gently, and let stand for 5 minutes;

[0081] The transfection reagent Polybrene (H9268, Sigma) was dissolved in Opti-MEM medium to a total volume of 500 μl and a final concentration of 5 μg / mL. The mixture was gently mixed and allowed to stand for 5 minutes.

[0082] Add the transfection reagent dilution solution dropwise to the plasmid dilution solution, mix gently while adding, and leave at room temperature for 20 minutes to allow the DNA and transfection reagent to fully combine to form a stable transfection complex.

[0083] Take out the cell culture plate, add the prepared DNA transfection reagent complex (500 μL) to the cell culture plate, and return it to the incubator. After 6 hours, remove the culture medium, wash once with PBS, and add 10 mL of fresh complete culture medium for culture.

[0084] 48 hours after transfection, harvest the culture medium for the first time into a 50 ml centrifuge tube, mark it, and replace the cells with fresh complete culture medium.

[0085] 72 hours after transfection, harvest the virus for the second time, collect the culture medium into a 50 ml centrifuge tube, mark it, and discard the cells.

[0086] After centrifugation at 3500 rpm, 10 min, RT, pour the supernatant into a new 50 ml centrifuge tube immediately, filter the supernatant with 0.45um and transfer it to an ultracentrifuge tube. Divide the supernatant into 12 ultracentrifuge tubes, balancing them in pairs. After centrifugation at 30000 rpm, 2h, 4℃ in a Hitachi high-speed centrifuge, white virus precipitate can be observed on one side of the tube wall, mark it.

[0087] In the safety cabinet, open the centrifuge tube, carefully discard the supernatant, turn the centrifuge tube upside down on the sterilized absorbent paper, and discard the supernatant that has not been removed. Add DPBS to each tube, 100 ul / tube, seal the tube mouth with sealing film, and dissolve the precipitate at 4℃ overnight. For the same virus, collect the virus to the last tube by tube collection method, and then collect it twice with 100 ul DPBS, and collect all into 1.5 ml EP tubes, package the virus as required, and store in a -80℃ refrigerator.

[0088] 1 mL trypsin digested HEK293 cells, 1 × 10 5 Cells were inoculated into each well of a 24-well plate. Viruses were added after the cells adhered to the plate (4-6 hours after plating). The medium was changed after 12-20 hours, and the medium containing the virus was removed. Fluorescence was recorded 72 hours after infection, and the cells were then harvested to extract genomic DNA and perform quantitative PCR experiments to determine the titer.

[0089] 4. Testing of lentiviral titer:

[0090] Real-time fluorescence quantitative PCR (Real-time PCR) was performed on ABI7500. PCR premix reagent (SYBR Master Mixture) was from TAKARA, catalog number: 639676.

[0091] 4.1. Prepare the reaction system according to the following proportions:

[0092] SYBR premix ex taq: 10 μl;

[0093] Inert reference dye: 0.4 μL;

[0094] Upstream primer (25 μM): 0.5 μl, 5′- CGCAAATGGGCGGTAGGCGTG-3′ (SEQ ID NO: 1);

[0095] Downstream primer (25 μM): 0.5 μl, 5′-CATAGCGTAAAAGGAGCAACA-3′ (SEQ ID NO: 2);

[0096] Genomic DNA (extracted from samples): 2.0 μl;

[0097] Water 6.6 μl.

[0098] 4.2. Set the program to two-step Real-Time quantification. Pre-denaturation at 95℃, 15S, then denaturation at 95℃, 5S, annealing and extension at 60℃, 34S, for a total of 40 cycles. Read the absorbance value at each extension stage.

[0099] Cycle 1: (1X):

[0100] Program 1: 95.0℃ for 00:15;

[0101] Cycle 2: (40X):

[0102] Program 1: 95.0℃ for 00:05;

[0103] Program 2: 60.0℃ for 00:34.

[0104] Supports data collection and real-time analysis.

[0105] 4.3. Make a melting curve. After the PCR is completed, denature at 95℃ for 1 min. Then cool to 55℃ to allow the DNA double strands to fully combine. Start from 55℃ to 95℃, increase 0.5℃ each step, keep for 30S, and read the absorbance value at the same time.

[0106] Cycle 3: (1X):

[0107] Program 1: 95.0 ℃ for 01:00.

[0108] Cycle 4: (1X):

[0109] Program 1: 55.0 ℃ for 01:00.

[0110] Cycle 5: (81X):

[0111] Program 1: 55.0 ℃-95.0 ℃ for 00:30.

[0112] After 2 cycles, the set point temperature was increased by 0.5°C.

[0113] TU -1 = (CxNxDx1000) / V;

[0114] C = average number of integrated viral copies per genome;

[0115] N = number of cells at the time of infection (approximately 2 × 10 5 );

[0116] D = dilution factor of viral vector;

[0117] V = volume of diluted virus added (μL).

[0118] The test results are shown in the following table:

[0119] Table 2

[0120]

[0121] The average titer of this coating is 6.01*10^8.

[0122] Example 2 Construction of KCNB1 stable cell line model and high-throughput excitotoxicity screening results for mobile substances and degradation products in biodegradable plastics

[0123] 1. Screening of stable transgenic lines

[0124] HEK293 cells were seeded into 6-well plates at a confluence of 30%, and HEK293 cells were diluted to 1×10 5 cells / mL cell suspension, ready for plating. 2 mL per well, i.e. 2×10 5 cells / well, spread on a 6-well plate. Infect with virus 12-20 hours later.

[0125] Add polybrene: Add 10 μL of 1 mg / mL polybrene to each well, and the final concentration of polybrene in the cell sample is 5 μg / mL.

[0126] Change the medium 12-20 hours after infection: discard the medium and add 2 mL of fresh medium to each well.

[0127] After 72 hours, add puromycin hydrochloride at a final concentration of 2 μg / mL. Replace the culture medium with fresh puromycin hydrochloride at a final concentration of 2 μg / mL every 2-3 days. After drug screening, take fluorescent photos ( Figure 1 ). Stable cell lines were cryopreserved.

[0128] 2. Functional verification of stable transfected cells

[0129] The cells were collected by centrifugation, membrane proteins were extracted using the Biotech Membrane Protein Extraction Kit, and membrane protein expression levels were verified using WB antibodies (Flag, primary antibody dilution concentration of 1:1000, overnight incubation).

[0130] The specific steps are as follows:

[0131] Protein extraction: Thaw an appropriate amount of membrane protein lysis buffer on ice in advance and add protease inhibitors and phosphatase inhibitors to it; let it stand on ice for 30 min to wait for cell lysis; centrifuge at 4°C, 12000 rpm for 15 min, carefully pipette the supernatant into a new EP tube, and discard the precipitate.

[0132] Western Blot:

[0133] 1. Prepare 10% lower separation gel and solidify it by pressing methanol for 30 minutes. Then prepare 4% concentrated gel and insert the comb and wait for solidification (about 30 minutes).

[0134] 2. Dilute the protein solution with SDS 5× loading buffer (protein solution: buffer = 4:1), heat it at 99°C for 5 min using a PCR instrument or metal bath to denature the protein, then cool it on ice and wait for loading;

[0135] 3. The sample volume per well is 20 μg;

[0136] 4. Gel running: 80 V, 30 min, through the concentrated gel; 120 V, 60 min-70 min to run the separation gel, and stop when the protein marker is completely separated;

[0137] 5. Transfer: The transfer solution is pre-cooled in a 4°C refrigerator. Cut the PVDF membrane according to the size of the gel and soak it in methanol for 5 minutes to activate the PVDF membrane. Transfer the membrane in an enamel tray with transfer solution. Place the black bottom plate of the transfer clamp on the bottom layer, and lay a layer of sponge pad and three layers of filter paper in sequence, and use a roller to drive away the bubbles in the interlayer. Spread the gel after electrophoresis on the filter paper, and lay the soaked membrane on the gel seamlessly to ensure that there are no bubbles between the gel and the membrane. Lay three layers of filter paper and sponge pads on the membrane in sequence, and fasten the transfer clamp. Put the clamp into the transfer tank, so that the black side of the clamp faces the black side of the tank, and the red side of the clamp faces the red side of the tank. When transferring, put an ice bag on the other side of the tank to cool it down, and place the transfer tank on ice to keep it low temperature to maintain a constant voltage. The constant voltage is 100 V during transfer, and the transfer time is adjusted according to the molecular weight;

[0138] 6. Blocking: Prepare blocking solution (5% skim milk powder in PBST), take out the transferred PVDF membrane and block it in the blocking solution at room temperature for 2-5 hours;

[0139] 7. Apply primary antibody: dilute the primary antibody with blocking solution (see Table 3) and incubate overnight on a shaker in a 4°C refrigerator;

[0140] 8. Elute the primary antibody: Use PBST elution buffer, elute 5 times on a shaker, each wash for 6 minutes;

[0141] 9. Apply secondary antibody: dilute the secondary antibody (depending on the species of the primary antibody) with PBST to a concentration of 1:5000 and incubate on a shaker at room temperature for 1 h;

[0142] 10. Wash the secondary antibody: Wash with PBST 3 times on a shaker, 5 min each time;

[0143] 11. Add developer for development: Mix solution A and solution B in the fluorescent detection reagent ECL kit in a ratio of 1:1 to prepare 1 mL of developer. Place the rinsed membrane on the developer plate, evenly add developer, and after a period of reaction, use a fully automatic chemiluminescence immunoassay analyzer for development analysis. Note: The protein imaging software needs to be opened in advance and pre-cooled to -30℃ before use.

[0144] The antibody information used is as follows:

[0145] Table 3 Antibody information

[0146]

[0147] The results are as follows Figure 2 shown.

[0148] 3. Comparison of the results of the phenyltin trichloride excitotoxicity screening with the cytotoxicity screening results of HEK293 cells not transfected with KCNB1

[0149] The specific steps are as follows: HEK293 cells stably expressing KCNB1 were cultured in a 96-well plate using a liquid medium containing 2 µg / mL puromycin hydrochloride. 4 HEK293 cells stably expressing KCNB1 were cultured in the same volume of culture medium in each well, and the wells were divided into a zero adjustment group, a negative group, and an experimental group.

[0150] Different concentrations of phenyltin trichloride (contaminant to be screened) were added to the wells of the experimental group, and the specific concentration gradient was as follows: only culture medium was added to the negative group; there were no cells in the zero adjustment group.

[0151] After 24 hours of treatment, the culture medium was discarded, the cells were washed three times with PBS, 10 μL of CCK-8 reagent was added, and the cells were incubated at room temperature (37°C) for 1 hour and 30 minutes. The absorbance was measured at 450 nm, and the cytotoxicity IC50 value was calculated. The results are shown in the following table:

[0152] Calculation method of cytotoxicity IC50 value: Use GraphPrism to perform curve fitting and calculate the corresponding IC50 value.

[0153] Table 4

[0154]

[0155] The above results indicate that HEK293 cells stably expressing KCNB1 are more sensitive in cell-based potassium ion excitotoxicity screening and are more sensitive to the risks that may be caused by low-dose exposure.

[0156] Example 3 Comparison of the results of screening the excitotoxicity of mobile substances and degradation products in biodegradable plastics using cell models with stabilized and unstabilized KCNB1 ion channels

[0157] The results of the phenyltin trichloride excitotoxicity screening were compared with the cytotoxicity screening results of HEK293 cells not transfected with KCNB1.

[0158] The specific steps are as follows: HEK293 cells stably expressing KCNB1 were cultured in a 96-well plate with a liquid culture medium containing 2 µg / mL puromycin hydrochloride. Each well contained 104 HEK293 cells stably expressing KCNB1 and the culture medium volume in each well was the same. The wells were divided into a zeroing group, a negative group, and an experimental group. Different concentrations of phenyltin trichloride (contaminant to be screened) were added to the wells of the experimental group; only culture medium was added to the negative group; there were no cells in the zeroing group. After 24 hours of treatment, the culture medium was discarded, washed three times with PBS, and CCK-8 reagent was added. After incubation at room temperature (37°C) for 1h30min, the absorbance was tested at 450nm, and the cytotoxicity IC50 value was calculated. The results are as follows:

[0159] Table 5

[0160]

[0161] The above results indicate that HEK293 cells stably expressing KCNB1 have higher sensitivity in potassium ion excitotoxicity screening and are more sensitive to the risks that may be caused by low-dose exposure.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a KCNB1 ion channel stably transfected cell line, characterized in that: The invention comprises the construction of a plasmid vector, which comprises cloning a KCNB1 gene sequence by PCR technology using cDNA as a template, connecting the KCNB1 gene sequence to a plasmid pcSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE by double enzyme digestion technology, and connecting the KCNB1 gene sequence by ligase to obtain a pcSLenti-EF1-EGFP-P2A-Puro-CMV-KCNB1-3xFLAG-WPRE lentiviral vector; the KCNB1 gene sequence is NCBI Reference Sequence: NM_004975.

4.

2. The construction method according to claim 1, characterized in that: The ligase is T4 DNA ligase.

3. The construction method according to claim 1, characterized in that: The following steps are involved: (1) Cultivating cells in culture medium; (2) Construction of plasmid vector; (3) adding the DNA transfection reagent complex to the cells cultured in step (1) for transfection, centrifuging after transfection, adding a buffer to dissolve the precipitate, and obtaining the virus; (4) Digest the cells and then inoculate them, adding viruses for infection; (5) The cells are arranged into (1-10)×10 5 cells / mL cell suspension, incubate for 12-20h, infect with virus, add polybrene, and obtain KCNB1 ion channel stable transfection cell line; The final concentration of polybrene in step (5) is 4-10 μg / mL.

4. The construction method according to claim 3, characterized in that: The cells described in step (1) include HEK293 cells, SH-SY5Y cells, and HaCaT cells.

5. The construction method according to claim 4, characterized in that: The cells described in step (1) are HEK293 cells.

6. The construction method according to claim 3, characterized in that: In step (3), the DNA transfection reagent complex comprises a transfection reagent and a plasmid complex; the plasmid complex comprises a backbone plasmid and a shuttle plasmid; and the mass ratio of the backbone plasmid to the shuttle plasmid is 1-2:

1.

7. The construction method according to claim 6, characterized in that: The mass ratio of the backbone plasmid to the shuttle plasmid is 1:

1.

8. The construction method according to claim 6, characterized in that: The plasmid complex is prepared by dissolving the viral vector plasmid in a culture medium, and the culture medium is selected from one or more of Opti-MEM culture medium, MRS culture medium, M17 culture medium, PDA culture medium, LB culture medium, TB culture medium, SOB culture medium, DMEM culture medium, and RPMI1640 culture medium.

9. The construction method according to claim 8, characterized in that: The culture medium is Opti-MEM culture medium.

10. The construction method according to claim 3, characterized in that: The digestive enzyme used to digest the cells in step (4) is pancreatic enzyme.

11. A KCNB1 ion channel stably transfected cell line constructed by the construction method according to any one of claims 1 to 10.

12. Application of the construction method according to any one of claims 1 to 10 in high-throughput excitotoxicity screening of phenyltin trichloride.

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