CN114134197A - Luciferase reporter gene-based cytotoxicity rapid detection method, cell strain construction method and application thereof - Google Patents
Luciferase reporter gene-based cytotoxicity rapid detection method, cell strain construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a luciferase reporter gene-based rapid cytotoxicity detection method, which comprises the following steps: (1) constructing a cell strain based on a luciferase reporter gene; (2) the sample to be tested is added to the culture solution after being prepared into a solution, then the culture solution is added to a culture medium containing cell strains, and after the culture solution is sucked, luciferase activity is measured to detect cytotoxicity. A method for constructing a cell line, comprising: s1: constructing a fthl29-pGL6-TA plasmid; s2: transfecting cells with the fthl29-pGL6-TA plasmid; s3: and (4) screening a stable cell line. The cell strain is applied to the aspects of characterizing pollutant toxicity and actual water sample comprehensive toxicity. The reporter gene cell strain constructed by the iron death related gene promoter can rapidly indicate early cytotoxicity, has high specificity and sensitivity, and is convenient for high-throughput detection; and the method is suitable for detecting the toxic effect of most toxic pollutants, characterizing the cytotoxicity related to iron death, and can also be used for the toxicity characterization of complex practical water samples.
Description
Technical Field
The invention belongs to the technical field of pollutant cytotoxicity detection, and particularly relates to a luciferase reporter gene-based rapid cytotoxicity detection method, a cell strain construction method and application thereof.
Background
According to the incomplete statistics, the method has the advantages that,the number of chemicals registered in the world is 2.5 million, and about 13 to 14 ten thousand chemicals are sold, produced and used, about 4000 more chemicals are frequently used, and the number increases at a rate of 2 ten thousand every year. Chemicals such as chemical drugs and practical, food additives, agricultural chemicals, building decoration materials, cosmetics, etc. are frequently present in the production and life of people, and the effects of these chemicals on human health and living things are receiving close attention. When various chemical substances generated in daily life of people directly or indirectly enter a water body and exceed the water body load to cause water pollution, aquatic organisms are influenced or human health is indirectly influenced. However, conventional water quality indicators such as COD, BOD indicating water pollution5Total nitrogen and total phosphorus, etc. cannot indicate the toxic effect of the water body; traditional toxicity detection methods such as a luminescent bacteria test, an Ames test, a micronucleus test, a comet assay and the like and an in vivo toxicity test can detect the water toxicity, but the method is time-consuming and labor-consuming. The toxicity detection technology based on the luciferase reporter gene is favored by the advantages of simplicity, rapidness, high sensitivity, good repeatability and the like.
Many environmental pollutants cause serious toxic effects by inducing cell death. Traditional cell death methods include apoptosis, necrosis, and autophagy. Iron death is a newly discovered form of iron-dependent, non-apoptotic programmed cell death in recent years. Iron metabolism disorders and lipid peroxidation are two major links of iron death, and the processes are accompanied by disorders of energy metabolism, inflammation, up-regulation of oxidative stress and functional impairment of important organelles in cells. Recent studies have shown that iron death is one of the common toxic effects of pollutants. Therefore, a reporter gene cell strain capable of indicating early iron death related cytotoxicity effect is constructed, and pollutants and early cytotoxicity of actual water can be detected more sensitively.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting cytotoxicity based on a luciferase reporter gene, a method for constructing a cell strain and application thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly detecting cytotoxicity based on a luciferase reporter gene comprises the following steps in sequence:
(1) constructing a cell strain based on a luciferase reporter gene;
(2) the sample to be tested is added to the culture solution after being prepared into a solution, then the culture solution is added to a culture medium containing cell strains, and after the culture solution is sucked, luciferase activity is measured to detect cytotoxicity.
A construction method of a cell strain based on a luciferase reporter gene comprises the following steps in sequence:
s1: constructing fthl29-pGL6-TA plasmid:
s1.1: promoter synthesis: obtaining a fthl29 gene promoter fragment;
s1.2: connection transformation: firstly, carrying out enzyme digestion on a pGL6-TA vector by KpnI and XhoI, then carrying out enzyme ligation on the pGL6-TA vector subjected to enzyme digestion and a fthl29 gene promoter fragment, and transforming the pGL6-TA vector into an escherichia coli competent cell after the enzyme ligation;
s1.3: screening and verifying: coating the transformed escherichia coli on a flat plate containing antibiotics, carrying out inverted culture for 12-16h, selecting bacteria for PCR identification, extracting plasmids from positive colonies, carrying out enzyme digestion identification on recombinant plasmids, sequencing positive clones, and extracting plasmids after sequencing comparison is successful to obtain a constructed recombinant expression plasmid fthl29-pGL 6-TA;
s2: fthl29-pGL6-TA plasmid transfected cells:
s2.1: reviving and culturing HepG2 cells: dissolving the frozen HepG2 cells in a water bath at 40 ℃, transferring the dissolved HepG2 cells into a water bath at 37 ℃ for resuscitation, centrifuging the cells at 800r/min for 5min after resuscitation, sucking a supernatant, adding an optimized DMEM culture solution, and culturing the cells in an incubator;
s2.2: cell transfection: plating HepG2 cells, culturing for 12h, adding the cells into a transfection system, culturing for 48h at 37 ℃, discarding transfection liquid, adding culture liquid, and when the cell growth approaches confluence, adding the culture liquid according to the ratio of 1: 4, carrying out density passage, continuously culturing, and carrying out positive monoclonal screening when the cell confluency is 30% to obtain transfected cells;
s3: screening for stable cell lines:
s3.1: g418 screening: washing the transfected cells with PBS, digesting with trypsin, inoculating to a culture dish, adding a culture medium containing G418, replacing the screening culture medium once in 2-3 days, transferring the cells to a culture bottle for continuous culture when the confluency of the cells is above 90%, adding the culture medium containing G418, growing the transfected cells to above 70%, picking the monoclonal cells for continuous culture, adding the culture medium containing G418, and reducing the concentration of G418 by half;
s3.2: picking a single clone and carrying out amplification culture: and screening positive clones by adopting a limiting dilution method to obtain cell strains.
Preferably, in step S1.1, the nucleotide sequence 3kb upstream of the transcription start site of zebrafish fthl29 gene is obtained from NCBI as a promoter fragment.
Preferably in any of the above protocols, in step S1.2, the E.coli competent cells are TOP10 competent cells.
Preferably in any of the above embodiments, in step S1.3, the antibiotic is ampicillin.
In any of the above schemes, preferably, in step S2.1, the optimized DMEM culture solution is a DMEM culture solution containing 10% fetal bovine serum and 1% streptomycin double antibiotics, and the culture condition in the incubator is 5% CO2And 37 ℃.
Preferably in any of the above protocols, in step S2.2, the transfection system comprises 2.5. mu.g fthl29-pGL6-TA plasmid, 1.25. mu.g pSV-. beta. -lacosasidase internal control plasmid, 5. mu. L P3000TMReagents, 3.75 μ L Lipofectamine3000 reagent and 250 μ L DMEM medium.
Preferably in any of the above embodiments, in step S3.2, the step of limiting dilution screening for positive clones is: preparing cell suspension, counting cells, diluting the cells to 1/10 mu L by using a culture solution, adding a culture medium containing G418 to a 96-well plate at the concentration of 250 mu G/mL, adding 10 mu L/well of the cell suspension, picking positive clones under a microscope, transferring the positive clones to a new well for culture, repeatedly screening, and carrying out PCR identification when the cultured cells basically do not die any more to obtain a cell strain.
Before step S3.1, the optimal screening concentration of G418 needs to be determined, wherein the main determination step is to inoculate HepG2 cells in a 24-well plate, remove the original culture medium when the confluence of the cells is about 30%, add culture solutions containing different concentrations of G418, set 7 concentrations: 200. 300, 400, 500, 600, 700, 800. mu.g/mL. Changing the culture solution every three days, observing the cell death condition, and finding that the minimum G418 concentration capable of killing all cells within 10-14 days is 500 mug/mL, so that the optimal screening concentration of G418 is determined to be 500 mug/mL.
The application of the cell strain based on the luciferase reporter gene in the aspect of characterizing the toxicity of the pollutants has high specificity and sensitivity, is convenient for high-throughput detection, and can quickly indicate the early toxicity of the pollutants related to cell death.
The luciferase reporter gene-based cell strain is applied to characterization of comprehensive toxicity of an actual water sample, and the toxicity detection method has the advantages that the types of pollutants in the actual water body are various, such as heavy metals, medicines, personal care products, pesticides, persistent organic pollutants and the like, part of pollutants exist in waste water at low concentration, and the response sensitivity is low. The luciferase reporter gene cell strain provided by the invention can comprehensively characterize the toxicity of a complex practical water body, sensitively indicates the iron death-related cytotoxic effect of a complex pollutant in water through the luciferase activity detection of the cell, and has the advantages of high specificity and low detection limit.
The invention has the technical effects and advantages that: 1. the reporter gene cell strain constructed by the iron death related gene promoter can rapidly indicate early cytotoxicity, has high specificity and sensitivity, and is convenient for high-throughput detection;
2. the cell strain constructed by the invention is not only suitable for detecting the toxic effect of most toxic pollutants to characterize iron death-related cytotoxicity, but also can be used for characterizing the toxicity of a complex actual water sample;
3. the detection method using luciferase as the reporter gene is simple, and can rapidly indicate early toxicity of pollutants and water samples related to cell death.
Drawings
FIG. 1 is the fthl29-pGL6-TA reporter plasmid constructed in example 1;
FIG. 2 shows the restriction enzyme identification of fthl29-pGL6-TA recombinant plasmid in example 1;
FIG. 3 is a graph showing the cell activity of example 2 after various concentrations of sodium arsenite have been infected;
FIG. 4 is a graph showing the relative luciferase activity of example 2 after various concentrations of sodium arsenite have been infected;
FIG. 5 shows the cell viability of the water samples of example 3 after exposure to different concentrations of the virus;
FIG. 6 is the relative luciferase activity after contamination of different concentrations of water sample in example 3.
Wherein, the left lane in the figure 2 is fthl29-pGL6-TA recombinant plasmid, the middle lane is fthl29-pGL6-TA plasmid restriction enzyme product, and the right lane is DNA Marker; control in FIG. 4 shows the relative luciferase activity of untransfected HepG2 cells that were not infected with sodium arsenite, and the remaining groups show the relative luciferase activity of HepG2 cells after successful transfection that were infected with the corresponding concentration of sodium arsenite; control in FIG. 6 indicates the relative luciferase activity of untransfected HepG2 cells without contamination, 0 indicates the relative luciferase activity of non-contaminated HepG2 cells after successful transfection, and the remaining groups indicate the relative luciferase activity of water samples at corresponding concentrations of infected HepG2 cells after successful transfection.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1:
a method for rapidly detecting cytotoxicity based on a luciferase reporter gene comprises the following steps in sequence:
(1) constructing a cell strain based on a luciferase reporter gene;
(2) the sample to be tested is added to the culture solution after being prepared into a solution, then the culture solution is added to a culture medium containing cell strains, and after the culture solution is sucked, luciferase activity is measured to detect cytotoxicity.
Example 2:
a construction method of a cell strain based on a luciferase reporter gene comprises the following steps in sequence:
s1: constructing fthl29-pGL6-TA plasmid:
s1.1: promoter synthesis: acquiring a base sequence of 3kb upstream of a transcription initiation site of a zebra fish fthl29 gene from NCBI, taking the base sequence as a promoter fragment, and synthesizing a fthl29 gene promoter fragment by a chemical synthesis method;
s1.2: connection transformation: firstly, carrying out enzyme digestion on a pGL6-TA vector by KpnI and XhoI, then carrying out enzyme ligation on the pGL6-TA vector subjected to enzyme digestion and a fthl29 gene promoter fragment, and transforming the pGL 3578-TA vector into a TOP10 competent cell after enzyme ligation;
s1.3: screening and verifying: coating the transformed escherichia coli on a flat plate containing antibiotics, wherein the antibiotics are ampicillin, carrying out inverted culture for 12-16h, selecting bacteria for PCR identification, extracting plasmids from positive colonies, carrying out enzyme digestion identification on recombinant plasmids, sequencing positive clones, and extracting plasmids after sequencing comparison is successful to obtain a constructed recombinant expression plasmid fthl29-pGL 6-TA;
s2: fthl29-pGL6-TA plasmid transfected cells:
s2.1: reviving and culturing HepG2 cells: dissolving frozen HepG2 cells in a water bath at 40 ℃, transferring the dissolved HepG2 cells into a water bath at 37 ℃ for resuscitation, centrifuging the cells for 5min at 800r/min after resuscitation, sucking the supernatant, adding an optimized DMEM culture solution, placing the optimized DMEM culture solution into an incubator to culture, and setting the culture condition in the incubator to be 5% CO2And 37 ℃;
s2.2: cell transfection: plating HepG2 cells, culturing for 12h, adding into transfection system containing 2.5. mu.g fthl29-pGL6-TA plasmid, 1.25. mu.g pSV-beta-galactosidase internal control plasmid, 5. mu. L P3000, culturing at 37 deg.C for 48hTMReagent, 3.75 μ L Lipofectamine3000 reagent and 250 μ L DMEM medium, discarding the transfection solution, adding the culture solution, waiting for the cellsIncrease near confluence as per 1: 4, carrying out density passage, continuously culturing, and carrying out positive monoclonal screening when the cell confluency is 30% to obtain transfected cells;
s3: screening for stable cell lines:
s3.1: g418 screening: washing the transfected cells with PBS, digesting with trypsin, inoculating to a culture dish, adding a culture medium containing G418, replacing the screening culture medium once in 2-3 days, transferring the cells to a culture bottle for continuous culture when the confluency of the cells is above 90%, adding the culture medium containing G418, growing the transfected cells to above 70%, picking the monoclonal cells for continuous culture, adding the culture medium containing G418, and reducing the concentration of G418 by half;
s3.2: picking a single clone and carrying out amplification culture: and screening positive clones by adopting a limiting dilution method to obtain cell strains.
Preferably, in step S1.1,.
Preferably in any of the above embodiments, in step S3.2, the step of limiting dilution screening for positive clones is: preparing cell suspension, counting cells, diluting the cells to 1/10 mu L by using a culture solution, adding a culture medium containing G418 to a 96-well plate at the concentration of 250 mu G/mL, adding 10 mu L/well of the cell suspension, picking positive clones under a microscope, transferring the positive clones to a new well for culture, repeatedly screening, and carrying out PCR identification when the cultured cells basically do not die any more to obtain a cell strain.
Before step S3.1, the optimal screening concentration of G418 needs to be determined, wherein the main determination step is to inoculate HepG2 cells in a 24-well plate, remove the original culture medium when the confluence of the cells is about 30%, add culture solutions containing different concentrations of G418, set 7 concentrations: 200. 300, 400, 500, 600, 700, 800. mu.g/mL. Changing the culture solution every three days, observing the cell death condition, and finding that the minimum G418 concentration capable of killing all cells within 10-14 days is 500 mug/mL, so that the optimal screening concentration of G418 is determined to be 500 mug/mL.
Example 3: the application of a luciferase reporter gene-based cell strain in characterization of pollutant toxicity is as follows:
sodium arsenite was selected as a contaminant, HepG2 cell activity was first detected using CCK-8 kit, exposure concentrations were set at 0, 0.25, 0.5, 1, 2.5, 5 μ M, and the maximum concentration at which cell activity reached 95% was taken as the maximum exposure concentration of sodium arsenite, and the experimental results are shown in fig. 3, and 0.5 μ M was finally determined as the maximum exposure concentration of sodium arsenite.
Set 6 exposure concentrations: 0. 0.01, 0.05, 0.1, 0.25, 0.5 μ M, and control groups of untransfected HepG2 cells and not contaminated with sodium arsenite were set, each set in 6 replicates. Before exposure, the stably transformed cells are inoculated in a 96-well plate for 12h, the original culture medium is removed, 150 mu L of culture solution containing sodium arsenite with each concentration is added, after exposure for 48h, the culture solution is completely aspirated, PBS is used for washing the cells, and luciferase activity is detected by using a firefly luciferase reporter gene detection kit, wherein the detection method comprises the following steps: adding 100 mu L of cell lysate into each well to lyse cells, fully lysing, centrifuging at 10000g for 3-5 min, taking 100 mu L of supernatant, adding 100 mu L of luciferase detection reagent, mixing uniformly, measuring on an enzyme-linked immunosorbent assay, setting the measuring interval to be 2 seconds, and setting the measuring time to be 10 seconds. The more toxic the sodium arsenite, the higher the luciferase activity and the stronger the bioluminescence. The results of the experiment are shown in FIG. 4.
As can be seen from FIG. 4, significant differences (p <0.05) exist between the sodium arsenite contaminated group and the control group with different concentrations, the luciferase activity is enhanced by the exposure of the sodium arsenite, a certain dose effect relationship exists between the luciferase activity and the sodium arsenite, and the luciferase activity is increased to 2.86 times by the 0.5 μ M sodium arsenite contamination. The results show that the reporter gene cell strain constructed by the invention can detect the iron death-related cytotoxicity of pollutants and can detect pollutants with low concentration.
Example 5: an application of a luciferase reporter gene-based cell strain in characterization of comprehensive toxicity of an actual water sample is as follows:
collecting effluent of a sewage treatment plant, firstly detecting HepG2 cell activity by using a CCK-8 kit, re-dissolving a water sample in DMSO after solid-phase extraction, and diluting the water sample to different concentrations by using a culture solution: the maximum exposure concentration of the water sample was determined as 0, 1, 25, 50, 75, 100, 125 times the raw water concentration, expressed as the relative concentration multiple REF, and the maximum exposure concentration of the water sample was determined as the maximum concentration at which the cell activity reached 95%, as shown in fig. 5, and finally REF was determined as 75.
Setting the exposure concentration of the water sample: REF was 0, 1, 25, 50, 75 and control groups of untransfected HepG2 cells and non-infected water samples were set up in 6 replicates per group. Before exposure, the stably transformed cells are inoculated in a 96-well plate for 12h, the original culture medium is removed, 150ul of water samples with various concentrations are added, after exposure for 48h, the culture solution is completely aspirated, PBS is used for washing the cells, and luciferase activity is detected by using a firefly luciferase reporter gene detection kit. The detection method comprises the following steps: adding 100 mu L of cell lysate into each well to lyse cells, fully lysing, centrifuging at 10000g for 3-5 min, taking 100 mu L of supernatant, adding 100 mu L of luciferase detection reagent, mixing uniformly, measuring on an enzyme-linked immunosorbent assay, setting the measuring interval to be 2 seconds, and setting the measuring time to be 10 seconds. The stronger the toxicity of the water sample, the higher the luciferase activity and the stronger the bioluminescence. The results of the experiment are shown in FIG. 6.
As can be seen from FIG. 6, the significant difference (p <0.05) exists between the water sample toxicant-infected groups with different concentrations and the control group, the luciferase activity is enhanced by the water sample exposure, a certain dose effect relationship exists between the luciferase activity and the water sample concentration, and the luciferase activity is increased to 2.6 times by the water sample toxicant-infected with the relative concentration multiple of 75. The result shows that the reporter gene cell strain constructed by the invention can detect the iron death related cytotoxicity of the actual water body and can detect the water sample with the raw water concentration.
As can be illustrated by the graphs in FIGS. 1 to 6, the reporter gene cell strain constructed by the invention can detect iron death-related cytotoxicity of actual water, and has lower pollutant concentration and detection limit of actual water concentration.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A luciferase reporter gene-based rapid cytotoxicity detection method is characterized in that: the method comprises the following steps in sequence:
(1) constructing a cell strain based on a luciferase reporter gene;
(2) the sample to be tested is added to the culture solution after being prepared into a solution, then the culture solution is added to a culture medium containing cell strains, and after the culture solution is sucked, luciferase activity is measured to detect cytotoxicity.
2. The method for constructing a cell line based on a luciferase reporter gene according to claim 1, wherein the method comprises the following steps: the method comprises the following steps in sequence:
s1: constructing fthl29-pGL6-TA plasmid:
s1.1: promoter synthesis: obtaining a fthl29 gene promoter fragment;
s1.2: connection transformation: firstly, carrying out enzyme digestion on a pGL6-TA vector by KpnI and XhoI, then carrying out enzyme ligation on the pGL6-TA vector subjected to enzyme digestion and a fthl29 gene promoter fragment, and transforming the pGL6-TA vector into an escherichia coli competent cell after the enzyme ligation;
s1.3: screening and verifying: coating the transformed escherichia coli on a flat plate containing antibiotics, carrying out inverted culture for 12-16h, selecting bacteria for PCR identification, extracting plasmids from positive colonies, carrying out enzyme digestion identification on recombinant plasmids, sequencing positive clones, and extracting plasmids after sequencing comparison is successful to obtain a constructed recombinant expression plasmid fthl29-pGL 6-TA;
s2: fthl29-pGL6-TA plasmid transfected cells:
s2.1: reviving and culturing HepG2 cells: dissolving the frozen HepG2 cells in a water bath at 40 ℃, transferring the dissolved HepG2 cells into a water bath at 37 ℃ for resuscitation, centrifuging the cells at 800r/min for 5min after resuscitation, sucking a supernatant, adding an optimized DMEM culture solution, and culturing the cells in an incubator;
s2.2: cell transfection: plating HepG2 cells, culturing for 12h, adding the cells into a transfection system, culturing for 48h at 37 ℃, discarding transfection liquid, adding culture liquid, and when the cell growth approaches confluence, adding the culture liquid according to the ratio of 1: 4, carrying out density passage, continuously culturing, and carrying out positive monoclonal screening when the cell confluency is 30% to obtain transfected cells;
s3: screening for stable cell lines:
s3.1: g418 screening: washing the transfected cells with PBS, digesting with trypsin, inoculating to a culture dish, adding a culture medium containing G418, replacing the screening culture medium once in 2-3 days, transferring the cells to a culture bottle for continuous culture when the confluency of the cells is above 90%, adding the culture medium containing G418, growing the transfected cells to above 70%, picking the monoclonal cells for continuous culture, adding the culture medium containing G418, and reducing the concentration of G418 by half;
s3.2: picking a single clone and carrying out amplification culture: and screening positive clones by adopting a limiting dilution method to obtain cell strains.
3. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S1.1, a base sequence of 3kb upstream of the transcription initiation site of the zebrafish fthl29 gene is obtained from NCBI as a promoter fragment.
4. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S1.2, the E.coli competent cells were TOP10 competent cells.
5. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S1.3, the antibiotic is ampicillin.
6. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S2.1, the optimized DMEM culture solution is a DMEM culture solution containing 10% fetal calf serum and 1% streptomycin double antibiotics, and the culture condition in the incubator is 5% CO2And 37 ℃.
7. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S2.2, the transfection system comprises 2.5. mu.g fthl29-pGL6-TA plasmid, 1.25. mu.g pSV-. beta. -galactosidase internal control plasmid, 5. mu. L P3000TMReagents, 3.75 μ L Lipofectamine3000 reagent and 250 μ L DMEM medium.
8. The method for constructing a cell line based on a luciferase reporter gene as claimed in claim 2, wherein the method comprises the following steps: in step S3.2, the limiting dilution method for screening positive clones comprises the steps of: preparing cell suspension, counting cells, diluting the cells to 1/10 mu L by using a culture solution, adding a culture medium containing G418 to a 96-well plate at the concentration of 250 mu G/mL, adding 10 mu L/well of the cell suspension, picking positive clones under a microscope, transferring the positive clones to a new well for culture, repeatedly screening, and carrying out PCR identification when the cultured cells basically do not die any more to obtain a cell strain.
9. Use of the luciferase reporter gene-based cell line of claim 1 or 2 for characterizing contaminant toxicity.
10. Use of the luciferase reporter gene-based cell line as claimed in claim 1 or 2 for characterization of the comprehensive toxicity of a real water sample.
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