CN110951692B - Transgenic cell assay for glucocorticoid endocrine disruption combined effect - Google Patents
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Abstract
The invention discloses a transgenic cell strain with glucocorticoid endocrine disrupting combined effect, which is characterized in that a Hela cell is selected to transfect a glucocorticoid receptor with a green fluorescent protein label, and the Hela cell strain stably transfected with GFP-GR is obtained by antibiotic screening. Also discloses a method for determining the transgenic cell strain, which comprises the following steps: step 1: constructing a transgenic cell strain with glucocorticoid endocrine disrupting combined effect according to action mechanisms of glucocorticoid and a glucocorticoid receptor; step 2: by administering the transgenic cell line, the rate of the cell line moving from the cytoplasmic region to the nuclear region was quantitatively determined. The cell test carrier can be infinitely amplified and used in the method, and no extra loss cost is caused; through a high content screening technology, synchronous detection of a plurality of samples can be realized, and the method has the characteristics of low cost, extremely high flux and higher sensitivity. The method can be used for detecting glucocorticoid EDCs of dairy products, sewage, urine of cultured animals and the like on a living body layer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a transgenic cell assay method for glucocorticoid endocrine disruption combined effect.
Background
Endocrine Disrupting Chemicals (EDCs), also known as Environmental hormones (Environmental hormones), are substances present in food and the environment that can interfere with the endocrine system of humans or animals and cause abnormal effects. The EDCs can interfere the synthesis, secretion, transportation and metabolism of natural hormones in animals, and have influence on a plurality of endocrine systems such as reproduction, mammary gland development, neuroendocrine, thyroid metabolism, cancer, obesity and cardiovascular system, even if the substances are trace, the endocrine disorders of the animals can be caused by long-term exposure. Endocrine effects are related to estrogen, androgen, glucocorticoid and mineralocorticoid effects, and the like. Glucocorticoid is a kind of adrenocortical hormone, and its function is exerted by a Glucocorticoid Receptor (GR) and a Mineralocorticoid Receptor (MR), respectively. Glucocorticoid deficiency is accompanied by a series of complex symptoms, which can be life threatening in severe cases, and under natural metabolic conditions, the body carries out glucocorticoid release and regulation through day-night circulation. However, an excess of glucocorticoids can cause immunosuppression and various side effects, such as metabolic disorders, obesity, hypertension, atherosclerosis, etc., accelerated sexual maturation in children, decreased male fertility, etc.
Glucocorticoid is a common anti-inflammatory and antiallergic drug in the breeding industry, can be used for treating livestock inflammation, immune diseases and the like, and is widely used because the drug has a certain growth promoting effect. The common medicines include dexamethasone, betamethasone, fluorometholone, prednisolone, etc. A large amount of glucocorticoid EDCs enter an environment and agricultural water source system and are remained in foods such as meat, dairy products and products thereof through biological chain enrichment and industrial chain migration, but no effective method for degrading the substances exists at present, and the health of a human body and the environmental biodiversity are finally influenced. According to the report, the Hong Chang of Beijing university adopts LC-ESI-MS/MS to detect (LOD:0.2-0.5 mu g/kg) 5 steroid hormones in rivers and sewage in Beijing area of China, and the monitoring results are respectively as follows: glucocorticoid (river 52ng/L, sewage 390ng/L), estrogen (river 9.8ng/L, sewage 25ng/L), androgen (river 480ng/L, sewage 1887ng/L), progestogen (river 50ng/L, sewage 75ng/L), but there is still no effective curtailment method to prevent and control such risks. Therefore, effective monitoring of glucocorticoid EDCs residues in food and environment is very important and is very slow.
At present, the national standard and the like relate to 9 sets of glucocorticoid endocrine disruptor detection methods, wherein 8 sets of glucocorticoid endocrine disruptor detection methods are based on a liquid chromatography-tandem mass spectrometry method, and the method comprises a liquid chromatography-mass spectrometry/mass spectrometry method for detecting multiple hormone residues in animal-derived food GB/T21981 and 2008, a liquid chromatography/tandem mass spectrometry method and a thin layer chromatography method for determining forty-one glucocorticoid in cosmetics GB/T24800.2-2009 and the like; an enzyme linked immunosorbent assay (ELISA)1 set comprises an enzyme linked immunosorbent assay (SN/T4141-2015 method for detecting the residual quantity of glucocorticoid in livestock and animal products). The detection limit of the gas chromatography-mass spectrometry method is 0.2 mu g/kg (calculated by dexamethasone), and the ELISA detection limit of the enzyme-linked immunosorbent assay can reach 0.125-4 mu g/kg (calculated by dexamethasone). In addition, there are studies on in vitro bioassay methods based on cell proliferation, and in vivo bioassay methods based on animal experiments such as organ weight gain, growth and development, but the above methods all have some bottlenecks and disadvantages, mainly expressed in: (1) glucocorticoid EDCs are ubiquitous in the environment and food, hospital discharge, plasticizer and packaging material use, agricultural input residue, environmental heavy metal background and the like, a large number of unknown or known glucocorticoid EDCs prototypes and metabolites thereof can be missed or cannot be detected, the existing methods such as mass spectrometry and ELISA and the like can meet the imbalance among high flux, sensitivity, time and cost, and the investigation and monitoring of the problems are not carried out in a large number at present, mainly because the cost is high and the detection time is long, the development of a novel detection method which has higher flux, lower cost, good sensitivity and reasonable detection time is of great importance; (2) the glucocorticoid-inducing effector substances are widely available, various known and unknown chemicals and structural analogues thereof exist, the development of a method which is more broad-spectrum and non-targeting is realized, and the urgency of realizing the overall screening of glucocorticoid EDCs is particularly prominent.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention aims to provide a transgenic cell strain with glucocorticoid endocrine disruption combined effect, which is obtained by transfecting Hela cells by using green fluorescent protein as a traceable label and using a glucocorticoid receptor translocated from cytoplasm to nucleus as a target protein.
The invention also provides a method for measuring the transgenic cell strain with glucocorticoid endocrine disruption combined effect, the cell test carrier can be infinitely amplified and used without extra loss, and a plurality of samples can be synchronously detected by a high content screening technology based on high resolution picture imaging and model analysis means, so that the cost is very low and the flux is extremely high.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a transgenic cell strain with glucocorticoid endocrine disrupting combined effect, which is characterized in that a Hela cell is selected to transfect a glucocorticoid receptor with a green fluorescent protein label, and the Hela cell strain stably transfected with GFP-GR is obtained by antibiotic screening.
The invention also provides a method for measuring the transgenic cell strain with glucocorticoid endocrine disruption combined effect, which comprises the following steps:
step 1: constructing a transgenic cell strain with glucocorticoid endocrine disrupting combined effect according to action mechanisms of glucocorticoid and a glucocorticoid receptor;
step 2: by administering the transgenic cell line, the rate of the cell line moving from the cytoplasmic region to the nuclear region was quantitatively determined.
Preferably, the construction of the transgenic cell line with glucocorticoid endocrine disruption combined effect in the step 1 specifically comprises the following steps:
s1: plasmids pTet-GFP-GR, pTet-tTAK and pSV2-neo were transformed into E.coli competent cells by heat shock method, respectively, plated on LB plate containing ampicillin, and cultured, and then monoclonal strains were picked up, respectively; culturing in LB liquid culture medium containing ampicillin, extracting plasmid and sequencing;
s2: respectively linearizing and purifying three plasmids with correct sequencing;
s3: the purified plasmids were mixed with pTet-GFP-GR, pTet-tTAK and pSV2-neo, and Hela cells were transfected by the liposome-mediated method, and transgenic cells stably expressing the green fluorescent protein signal were selected using antibiotics G418 and tet.
Preferably, pSV2-neo is single-digested with restriction enzymes XmnI for pTet-GFP-GR, NotI for pTet-tTAK, and EcoRI, respectively, and the results of the cleavage are identified by 1% agarose gel electrophoresis.
Preferably, the plasmid concentration after linearization is approximately 166-393 ng/. mu.L.
Preferably, pTet-GFP-GR: pTet-tTAK: pSV2-neo, according to the mass ratio of 10: 10: 1 and mixing.
Preferably, the liposome-mediated transfection method further comprises the following steps before the Hela cells are transfected: the Hela cells were subjected to concentration screening using G418 antibiotic and 5% CO at 37 deg.C2Culturing for 14d under conditions to obtain a minimum concentration that results in total cell death;
the minimum concentration of G418 that causes total cell death is 800. mu.g/mL.
Preferably, the HeLa cells are in the form of 105And (4) inoculating each plate to a 60cm plate, and growing until the confluence rate is about 80%, so that the plate can be used for transfection experiments.
The invention also provides application of the transgenic cell strain, which is applied to the glucocorticoid endocrine disrupting combined effect induced by chemicals in samples in the fields of environmental safety, food safety or clinical medicine.
Preferably, the sample is from any one of monitoring sewage, human and cultured animal body fluids, milk powder and products thereof.
The invention discloses the following technical effects:
the invention discloses a transgenic cell strain with glucocorticoid endocrine disruption combined effect, which is obtained by transfecting Hela cells by using Green Fluorescent Protein (GFP) as a traceable label and Glucocorticoid Receptor (GR) shifted from cytoplasm to nucleus as a target protein. The Glucocorticoid Receptor (GR) is a proton-nuclear transfer receptor, and in the absence of the corresponding hormone, GR exists in the cytoplasm and binds to multiple heat shock proteins and immunophilins, forming a complex multiprotein complex; when glucocorticoids enter cells, GR binds to the corresponding hormone ligand and is dissociated from polyprotein complexes, moves from the cytoplasm to the nuclear region, interacts with GR Regulatory Elements (GREs) in the nuclear region, and finally causes glucocorticoid receptor-specific transcriptional regulation. After the glucocorticoid EDCs with different concentrations act, the mass-nuclear transfer ratios are different, and the values are in direct proportion to the glucocorticoid EDCs, and the values can be used as a cell toxicology evaluation means, and compared with blank control, negative control and positive (dexamethasone) control, the qualitative and quantitative results of a background and a sample to be detected can be obtained.
The invention also discloses a determination method of the transgenic cell strain, the gene cell line simultaneously transfects three plasmids, namely pTet-GFP-GR, pTet-tTAK and pSV2neo, by a liposome transfection method; wherein both pTet-GFP-GR and pTet-tTAK are under the control of the tet promoter, and together they complete the tetracycline-off system. That is, when tetracycline tet is added exogenously, tet is combined with tTK protein contained in cells, so that the tet promoter cannot be turned on, and at the moment, tTK and GFP-GR (the gene sequence is shown as SEQ ID NO: 1) are not expressed; when tet is removed, tTK protein can bind to the tet promoter, simultaneously turn on expression of tTK and GFP-GR, and the massive expression of tTK further promotes the tet promoter to be turned on continuously; pSV2neo was used to provide the neo resistance gene, facilitating selection of stable cell lines by G418 antibiotics. The result of the screened stable cell strain can be obtained by the ratio of the fluorescent signal moving from the cytoplasm region to the nucleus region after administration, and the determination and calculation of the result are realized by a high-content screening instrument.
In the method for determining the transgenic cells, the cell test vector can be infinitely amplified for use, no extra loss cost is caused, and the cost is greatly reduced; and by high content screening technology, based on high resolution picture imaging and model analysis means, synchronous detection of 20-25 samples (3 parallel) can be simultaneously realized on a 96-well plate, the cost is very low, the flux is extremely high, the sensitivity is extremely high, and the sensitivity can reach 10-9mol/L-10-8mol/L (calculated by dexamethasone) or 0.39-3.9 mug/L (calculated by dexamethasone). The method can be applied to food and environment such as milk product, sewage, urine of cultured animals, etcIn the detection of the DCs, the method is very convenient and has the characteristics of low cost, high flux, high sensitivity and the like.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a mass spectrum of pTet-Splice of the present invention;
FIG. 2 is a mass spectrum of pTet-tTAK of the present invention;
FIG. 3 is a mass spectrum of pSV2-neo according to the present invention;
FIG. 4 shows the single cleavage of pTet-GFP-GR, pTet-tTAK, pSV2-neo of the present invention; wherein, lanes 1-8 are sequentially arranged from left to right, and lanes 1 and 2 are markers respectively; lanes 3 and 4 are the results before and after digestion with pTet-GFP-GR, respectively; lanes 5 and 6 are the results before and after pTet-tTAK cleavage, respectively; lanes 7 and 8 are the results before and after digestion with pSV2-neo, respectively;
FIG. 5 shows the clone Hela-GFP-GR selected by the present invention; A. b is two clones screened out of the Hela-GFP-GR, the number of A is 2-C11, B is 3-D5, and the bright field, the GFP-GR and the GFP-GR after cell fixation are respectively photographed;
FIG. 6 shows that the Hela-GFP-GR cells of the invention are screened for stable cell lines after dexamethasone (Dexa) standard is added; a is Control, namely Dexa standard substance is not added; b is 10-7A mol/L Dexa standard;
FIG. 7 shows the plasmid-nucleus transfer of Hela-GFP-GR stable strain cells detected by Dexa according to the present invention;
FIG. 8 shows the results of the cell region sorting according to the present invention;
FIG. 9 shows the effect of different Dexa concentrations on the migration of nuclei of Hela-GFP-GR with different time periods; a nucleoplasmic ratio of 1 is the blank control (DMSO) baseline expression level;
FIG. 10 shows the sequencing results of pTet-GFP-GR of the present invention; wherein the first is the sequencing result and the second is the sequence information.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Some of the test materials and instrument sources used in the present invention, if not specifically listed, are commercially available through conventional commercial channels.
pTet-splice、pTet-tTAK(thermofisher)、pSV2-neo(ATCC)。
GFP-GR complete gene synthesis and recombination into pTet-GFP-GR plasmid, accomplished by Nanjing Kinshire.
Trans5 α competent cell (Beijing Quanjin Biotechnology Co., Ltd.).
Tryptone, yeast extract (OXOID), NaCl (Skyo science, Inc.), Amp (Beijing Quanjin Biotechnology, Inc.).
Endotoxin-free plasmid Mini-drawer kit (Gene star), plasmid purification kit (Tiangen Biochemical technology Co., Ltd. (Beijing)).
Hela cell line (ATCC). MEM medium, phenol red-free MEM medium, fetal bovine serum, pancreatin, PBS solution (Gibco), HEPES solution (Beijing Solebao), activated carbon-treated fetal bovine serum CD-FBS (Hyclone).
Lipo3000, opti-MEM (Thermofoisher); g418 (beijing solibao), tetracycline tet (sigma); dexamethasone standard (CAS: 50-05-2; Tianjin Alta).
25cm2Consumables (corning, NEST) such as culture flasks, 60cm culture plates, 12-well plates, 6-well plates, 96-well plates (black, transparent bottom), 50ml and 15ml centrifuge tubes.
High content screening system (PerkinElmer, Operetta CLS)TM) Desk centrifuge, fluorescence inverted microscope (Olympus, IX73/DP 80).
Example 1
Screening method of transgenic cell strain with glucocorticoid endocrine disruption combined effect
1) Plasmid extraction and sequencing
The plasmids pTet-GFP-GR [ comprising pTet-splice (shown in FIG. 1) and GFP-GR recombinant as a whole gene ], pTet-tTAK (shown in FIG. 2) and pSV2-neo (shown in FIG. 3) were transformed into E.coli Trans 5. alpha. competent cells by heat shock method, plated on LB (Amp) plates, and cultured overnight at 37 ℃ to pick up a monoclonal strain. The obtained monoclonal strain was inoculated into 30mL of LB (Amp) liquid medium and cultured overnight, and plasmid extraction was performed using an endotoxin-free plasmid miniprep kit.
The plasmid concentration was 147-508 ng/. mu.L (OD)260/280At 1.82-1.85), 20 μ L of each sample was taken and sent for sequencing, the sequencing results were all correct, as shown in FIG. 10, and the rest were kept for use.
2) Plasmid linearization and purification
pSV2-neo was subjected to single digestion with restriction enzymes XmnI for pTet-GFP-GR, NotI for pTet-tTAK, and EcoRI, respectively, and the digestion results were identified by 1% agarose gel electrophoresis.
The linearized plasmid successfully digested by enzyme is subjected to desalting and enzyme removing by using a plasmid purification kit, the concentration of the linearized plasmid is about 166-393 ng/. mu.L, and the digestion result is identified by agarose gel electrophoresis as shown in FIG. 4.
3) Liposome-mediated transfection of Hela cells
Before the lipofection experiment, Hela cells are treated according to the formula 105And (4) inoculating each plate to a 60cm plate, and growing until the confluence rate is about 80%, so that the plate can be used for transfection experiments.
Before the plasmid is transfected into the cells, the G418 antibiotic is also used for carrying out action concentration screening on the Hela cells to be transfected so as to obtain the optimal antibiotic screening concentration. The method specifically comprises the following steps: hela cell press 105Each well was placed in a 6-well plate, and G418 concentration was set to 0. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 600. mu.g/mL, 800. mu.g/mL and 1000. mu.g/mL, respectively, and the plate was placed at 37 ℃ under 5% CO2Cultured for 14d under conditions to obtain a minimum concentration that results in total cell death. At least 3 replicates were set up for each experiment and the final resulting G418 screen concentration was 800. mu.g/mL.
Three plasmids were used for transfection as pTet-GFP-GR: pTet-tTAK: the mass ratio of pSV2-neo is 10: 10: 1, mixing in proportion; negative control: equal volume of sterile water was used instead. The plasmid, the transfection reagent and the opti-MEM are respectively mixed according to lipo3000 transfection reagent instructions and recommended transfection plasmid dosage, and are finally mixed and then are stood for 15min, Hela cell plates are dripped into the mixture and are placed in an incubator for culture, the solution can be changed according to the cell condition after 24h, and the mixture is changed into a fresh MEM complete culture medium with 800 mu G/mL antibiotic G418 and 5 mu G/mL tet after 48h [ abbreviated as MEM complete culture medium (+ T + G) ].
And (4) continuously culturing for 7-10 days after the antibiotics are added until all cells in the negative control group die, and the cell clones in the experimental group are not paved on the whole plate in a connected manner, so that the subsequent screening can be carried out.
4) Stable strain screening transgenic cell strain
a: cells for primary screening of transfection were digested, centrifuged and collected, diluted to an inoculation concentration of 1 cell/well using MEM complete medium (+ T + G), plated into 96-well plates, and cultured continuously until obvious monoclonal or mixed clonal cells were present in the visible well.
The clones were individually expanded and cultured, 50% were further cultured, and 50% were used for fluorescence signal detection. Before detection, tet was removed and cultured for 1d, and clones with green fluorescent protein signals were selected by fluorescence inverted microscope detection (as shown in FIG. 5).
b: clones with green fluorescent protein signal were selected and expanded, and the clones were further subjected to drug selection, 1d after tet was removed, negative control MEM complete medium, and 10 additions were made-7mol/L dexamethasone (Dexa) in MEM complete medium, taken photograph and observed 24h after administration. The selected clone is continuously maintained by MEM complete medium (+ T + low concentration G), after passage for 2-3 times, tet is removed for fluorescence signal detection, and the fluorescence signal can be maintained without loss after G418 is removed, namely the selected stable transgenic cell (shown in figure 6).
Example 2
Method for measuring transgenic cell strain with glucocorticoid endocrine disruption combined effect (dexamethasone is taken as an example)
1) And testing the administration time and the administration concentration of the selected Hela-GFP-GR stable strain cells.
In order to eliminate the influence factors in the culture medium and serum as much as possible, phenol-free red MEM medium and activated carbon treatment were usedIt was subjected to CD-FBS. Removing tet from Hela-GFP-GR, culturing for 1d, digesting, counting, and performing according to 104Inoculating into black 96-well plate, culturing overnight, adding Dexa 10-6mol/L、10-7mol/L、10-9mol/L、10-11mol/L and 10-13mol/L, with blanks (DMSO), at least 3 per concentration in parallel, cell fixation and Hochest33342 (Ex/Em 350/361nm) staining were performed 0.5h, 1h and 4h after dosing, respectively.
The results are shown in FIG. 7: the results of high content images showed that Dexa induced GFP-GR cytoplasmic nucleus transfer, and that the control group added DMSO without Dexa, and no GFP-GR cytoplasmic nucleus transfer occurred. And add Dexa groups when adding 10-7Cytoplasmic nuclear transfer occurred after 0.5h after mol/L Dexa.
2) Data processing and result calculation
After the image is obtained by the high content screening system, the cell nucleus and the whole cell region need to be selected. First, cell nuclei were sorted by hochests 33342, and then, the entire cell region was sorted by GFP signal. The difference between the two regions then gives the cytoplasmic region we need. Finally, the intensity of GFP signal in the nucleus and cytoplasm regions was calculated by using the high content system, and the result is shown in FIG. 8.
As shown in FIG. 9, the nuclear to cytoplasmic ratio increased with the increase in the Dexa concentration, and the optimum Dexa action concentration was 10-7mol/L; since at 1h, 10-6The expression is reduced at the mol/L level, so the optimal time is about 0.5h or more. As Dexa time of action increased, 10-7The core-to-mass ratio of Dexa with mol/L and higher concentration is reduced rather than that of the experimental group with short action time; while the low concentration of Dexa nucleoplasm increased slightly. The condition may be related to the conditions that the cells start to proliferate and divide under the action of a long time, and the cells form connected sheets and overlapped cells, and the like, and the factors influence the division of the whole area of the cells by software and finally influence the fluorescence intensity calculation.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> institute of agricultural quality standards and testing technology of Chinese academy of agricultural sciences
<120> transgenic cell assay for glucocorticoid endocrine disruption combined effect
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atggactttt ataaaagcct gaggggagga gctacagtca aggtttctgc atcttcgccc 900
tcagtggctg ctgcttctca ggcagattcc aagcagcaga ggattctcct tgatttctcg 960
aaaggctcca caagcaatgt gcagcagcga cagcagcagc agcagcagca gcagcagcag 1020
cagcagcagc agcagcagca gcagcagcca ggcttatcca aagccgtttc actgtccatg 1080
gggctgtata tgggagagac agaaacaaaa gtgatgggga atgacttggg ctacccacag 1140
cagggccaac ttggcctttc ctctggggaa acagactttc ggcttctgga agaaagcatt 1200
gcaaacctca ataggtcgac cagcgttcca gagaacccca agagttcaac gtctgcaact 1260
gggtgtgcta ccccgacaga gaaggagttt cccaaaactc actcggatgc atcttcagaa 1320
cagcaaaatc gaaaaagcca gaccggcacc aacggaggca gtgtgaaatt gtatcccaca 1380
gaccaaagca cctttgacct cttgaaggat ttggagtttt ccgctgggtc cccaagtaaa 1440
gacacaaacg agagtccctg gagatcagat ctgttgatag atgaaaactt gctttctcct 1500
ttggcgggag aagatgatcc attccttctc gaagggaaca cgaatgagga ttgtaagcct 1560
cttattttac cggacactaa acctaaaatt aaggatactg gagatacaat cttatcaagt 1620
cccagcagtg tggcactacc ccaagtgaaa acagaaaaag atgatttcat tgaactttgc 1680
acccccgggg taattaagca agagaaactg ggcccagttt attgtcaggc aagcttttct 1740
gggacaaata taattggtaa taaaatgtct gccatttctg ttcatggtgt gagtacctct 1800
ggaggacaga tgtaccacta tgacatgaat acagcatccc tttctcagca gcaggatcag 1860
aagcctgttt ttaatgtcat tccaccaatt cctgttggtt ctgaaaactg gaataggtgc 1920
caaggctccg gagaggacag cctgacttcc ttgggggctc tgaacttccc aggccggtca 1980
gtgttttcta atgggtactc aagccctgga atgagaccag atgtaagctc tcctccatcc 2040
agctcgtcag cagccacggg accacctccc aagctctgcc tggtgtgctc cgatgaagct 2100
tcaggatgtc attacggggt gctgacatgt ggaagctgca aagtattctt taaaagagca 2160
gtggaaggac agcacaatta cctttgtgct ggaagaaacg attgcatcat tgataaaatt 2220
cgaaggaaaa actgcccagc atgccgctat cggaaatgtc ttcaggctgg aatgaacctt 2280
gaagctcgaa aaacaaagaa aaaaatcaaa gggattcagc aagccactgc aggagtctca 2340
caagacactt cggaaaatcc taacaaaaca atagttcctg cagcattacc acagctcacc 2400
cctaccttgg tgtcactgct ggaggtgatt gaacccgagg tgttgtatgc aggatatgat 2460
agctctgttc cagattcagc atggagaatt atgaccacac tcaacatgtt aggtgggcgt 2520
caagtgattg cagcagtgaa atgggcaaag gcgatactag gcttgagaaa cttacacctc 2580
gatgaccaaa tgaccctgct acagtactca tggatgtttc tcatggcatt tgccttgggt 2640
tggagatcat acagacaatc aagcggaaac ctgctctgct ttgctcctga tctgattatt 2700
aatgagcaga gaatgtctct acccggcatg tatgaccaat gtaaacacat gctgtttgtc 2760
tcctctgaat tacaaagatt gcaggtatcc tatgaagagt atctctgtat gaaaacctta 2820
ctgcttctct cctcagttcc taaggaaggt ctgaagagcc aagagttatt tgatgagatt 2880
cgaatgactt atatcaaaga gctaggaaaa gccatcgtca aaagggaagg gaactccagt 2940
cagaactggc aacggtttta ccaactgaca aagcttctgg actccatgca tgaggtggtt 3000
gagaatctcc ttacctactg cttccagaca tttttggata agaccatgag tattgaattc 3060
ccagagatgt tagctgaaat catcactaat cagataccaa aatattcaaa tggaaatatc 3120
aaaaagcttc tgtttcatca aaaatga 3147
Claims (8)
1. A transgenic cell strain with glucocorticoid endocrine disruption combined effect is characterized in that Hela cells are selected and transfected with glucocorticoid receptors with green fluorescent protein labels, and the Hela cell strain which is stably transfected with GFP-GR is obtained by screening antibiotics, wherein three plasmids are used simultaneously: pTet-GFP-GR containing tTK gene, pTet-tTAK containing GFP-GR and pSV2neo containing neo resistance gene are co-transfected into Hela cells to obtain;
wherein both pTet-GFP-GR and pTet-tTAK plasmids are under the control of the tet promoter; pSV2neo is used for providing neo resistance genes, so that stable cell strains can be screened by G418 antibiotics; wherein the plasmid pTet-GFP-GR is formed by the whole gene recombination of pTet-splice and GFP-GR.
2. The transgenic cell line for glucocorticoid endocrine disruption in combination with effects of claim 1, wherein the transfection of Hela cells comprises the steps of:
s1: plasmids pTet-GFP-GR, pTet-tTAK and pSV2-neo were transformed into E.coli competent cells by heat shock method, respectively, plated on LB plate containing ampicillin, and cultured, and then monoclonal strains were picked up, respectively; culturing in LB liquid culture medium containing ampicillin, extracting plasmid and sequencing;
s2: respectively linearizing and purifying three plasmids with correct sequencing;
s3: the purified plasmids were mixed with pTet-GFP-GR, pTet-tTAK and pSV2-neo, and Hela cells were transfected by the liposome-mediated method, and transgenic cells stably expressing the green fluorescent protein signal were selected using antibiotics G418 and tet.
3. The transgenic cell line for glucocorticoid endocrine disruption in combination with effects of claim 2, wherein in step S3, the ratio of pTet-GFP-GR: pTet-tTAK: pSV2-neo, according to the mass ratio of 10: 10: 1 and mixing.
4. The transgenic cell line with combined glucocorticoid endocrine disrupting effect according to claim 2, wherein the step S3 further comprises the following steps before transfection of Hela cells by liposome-mediated method: the Hela cells were subjected to concentration screening using G418 antibiotic and 5% CO at 37 deg.C2Culturing for 14d under conditions to obtain a minimum concentration that results in total cell death;
the minimum concentration of G418 that causes total cell death is 800. mu.g/mL.
5. The transgenic cell line for glucocorticoid endocrine disruption in combination with the effect of claim 2, wherein in step S3, Hela cells are treated as 105And (4) inoculating each plate to a 60cm plate, and growing until the confluence rate is about 80%, so that the plate can be used for transfection experiments.
6. Use of a transgenic cell line according to any one of claims 1 to 5 for detecting glucocorticoid EDCs residual.
7. The use of claim 6, wherein the test sample is from any one of monitoring sewage, milk powder or products thereof.
8. The use according to claim 6 or 7, wherein, when the detection is required, the transgenic cell line is cultured by replacing the serum culture medium treated without tet, a sample is added to the culture medium to culture the transgenic cell line, and the determination is made by measuring the ratio of the nucleus pulposus migration reflected by the fluorescence value of GFP as a result.
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| CN1197483A (en) * | 1995-08-03 | 1998-10-28 | 曾尼卡有限公司 | Inducible herbicide resistance |
| CN101052726A (en) * | 2003-05-09 | 2007-10-10 | 森托科尔公司 | IL-23p40 specific immunoglobulin derived proteins, compositions, methods and uses |
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