CN111500545A

CN111500545A - Method for determining glucocorticoid mixture based on transgenic engineering cell strain

Info

Publication number: CN111500545A
Application number: CN202010362024.7A
Authority: CN
Inventors: 李耘; 刘畅; 钱永忠; 邱静; 任亚林; 李金娟
Original assignee: Institute of Agricultural Quality Standards and Testing Technology for Agro Products of CAAS
Current assignee: Institute of Agricultural Quality Standards and Testing Technology for Agro Products of CAAS
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-07

Abstract

The invention discloses a transgenic engineering cell strain and a method for determining glucocorticoid mixture based on the cell strain. Based on Hela cell strain stably expressing GFP-GR, by measuring the migration rate of GFP-GR binding hormone ligand from a cytoplasmic region to a nuclear region, the positive detection rate of glucocorticoid mixtures in different samples can be quickly detected with high throughput and low cost, and the sensitivity can reach 10 by using dexamethasone^‑9mol/L, can be used for rapidly screening the glucocorticoid mixture content in the environment and food, determining the hazard degree and guiding the risk prevention and control.

Description

Method for determining glucocorticoid mixture based on transgenic engineering cell strain

Technical Field

The invention relates to the technical field of biology, in particular to a method for determining a glucocorticoid mixture based on a GFP-GR-puro Hela cell transgenic engineering cell strain.

Background

The lentivirus (L entivirus) vector is a gene therapy vector developed on the basis of human immunodeficiency virus I (HIV-1). The virus vectors commonly used in genetic engineering include retrovirus, adenovirus and lentivirus, wherein the retrovirus vector can only infect cells in a division stage, the capacity of the vector is limited, a target gene of the adenovirus after infecting the cells can not be integrated on a chromosome of the host cell generally and can only be expressed by transient infection, and the lentivirus is different from the two viruses, not only has the capacity of infecting the cells in the division stage and in a non-division stage, but also has the characteristics that the vector can contain large exogenous gene segments and can express the target gene for a long time.

Glucocorticoids, which are one type of adrenocortical hormone, can exert their functions via the Glucocorticoid Receptor (GR) and the Mineralocorticoid Receptor (MR), respectively. Under natural conditions, the body carries out glucocorticoid release and regulation through day and night circulation, and excessive or long-term exposure of low-dose glucocorticoid medicaments can cause immunosuppression of the body and generation of various side effects, such as metabolic disorder, hypertension, obesity, premature sexual maturity, reduced fertility and the like.

The glucocorticoid medicine can be used for treating adrenal cortex insufficiency, inflammation, allergy and various skin diseases; the traditional Chinese medicine composition is also widely used in the breeding industry, and common medicines comprise dexamethasone, prednisolone, hydrocortisone, betamethasone and the like. With the wide use of the drugs, the glucocorticoid drugs with known molecular structures may have structural changes after the migratory metabolism of livestock and poultry and the migration of ecological environment through the human sources and livestock and poultry feeding and discharge, thereby generating unknown pollutants with similar structures and glucocorticoid endocrine disrupting effects. These known and unknown contaminants are enriched and migrated in the biological and ecological chain in a more complex and diverse manner, which will ultimately affect human health and the diversity of environmental organisms. A large number of known and unknown glucocorticoid mixtures and their metabolites may be missed or undetectable, and existing mass spectrometry and enzyme-linked immunosorbent assays are unbalanced in terms of high throughput, sensitivity, time and cost.

Currently, a great deal of investigation and risk monitoring aiming at glucocorticoid mixtures are not performed, mainly because of high cost and long detection time. Therefore, it is important to develop a novel monitoring and detection method with higher throughput, lower cost, broader spectrum/non-targeting property and detection time saving.

Disclosure of Invention

The invention aims to provide a detection method for detecting glucocorticoid mixtures based on a genetically engineered cell strain, which comprises the steps of cloning a fusion protein of GFP and GR into a lentiviral vector plasmid by using a genetic engineering means as a target gene to construct the genetically engineered cell strain, and detecting the migration ratio of GFP-GR binding hormone ligand from a cytoplasmic region to a nuclear region based on the cell strain, so that the content of the glucocorticoid mixtures in different samples can be quickly detected with high flux and low cost.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a transgenic engineering cell strain, which integrates green fluorescent protein GFP and glucocorticoid receptor GR fusion protein genes into Hela cell chromosome in a lentivirus infection mode, and finally obtains the Hela cell strain stably expressing GFP-GR by puromycin puro screening.

The invention also provides a method for determining glucocorticoid mixture based on the transgenic engineering cell strain, which comprises the following steps:

step 1: packaging GFP-GR gene by using lentivirus, infecting Hela cell, and obtaining GFP-GR-puro Hela cell transgenic engineering cell strain;

step 2: after a glucocorticoid mixture is added into the constructed GFP-GR-puro Hela transgenic engineering cell strain, the glucocorticoid content is quantitatively and semi-quantitatively determined (specifically, the positive detection rate is determined) by measuring the migration rate of a fluorescence signal from cytoplasm to a nucleus region, and the sensitivity can reach 10 by using dexamethasone^-9mol/L。

Preferably, the construction of the GFP-GR-puro Hela cell transgenic engineering cell strain in the step 1 specifically comprises the following steps:

step a, connecting the synthesized GFP-GR target gene to a viral vector FV026 to construct L enti-GFP-GR vector;

step b, mixing L enti-GFP-GR vector, psPAX2 vector and pMD2G vector according to the mass ratio of 2:1:1, transfecting 293T cells according to a liposome-mediated method, and packaging viruses;

step c: and c, infecting Hela cells with the qualified virus suspension detected in the step b, and screening by using a DMEM complete culture medium containing puro to obtain the GFP-GR-puro Hela cell transgenic engineering cell strain.

Preferably, the 293T cell in the step b is digested before being used for transfection, and is re-inoculated into a cell culture dish, and the cell culture dish is grown until the confluence rate is 80% -90% and then can be used for transfection experiments.

Preferably, the titer of the virus suspension that is qualified for the assay is 1E +7TU/m L.

The invention provides an application of the transgenic engineering cell strain,

the method is applied to the fields of medicine health, food safety and environmental protection, and is used for quantitatively detecting the content of the glucocorticoid mixture in body fluid, food and environmental water sources.

Preferably, the method is applied to quantitatively detect the content of the glucocorticoid mixture in different samples of human body, livestock and poultry body fluid, dairy products and sewage.

The invention discloses the following technical effects:

the slow virus vector used in the invention mainly comprises three plasmids of vector plasmid FV026 helper plasmid psPAX2(pHelper1) and helper plasmid pMD2G (pHelper 2). Taking a fusion protein gene of green fluorescent protein and a glucocorticoid receptor as a target gene, and cloning the target gene into a lentiviral vector plasmid by a genetic engineering means; then co-transfecting the carrier plasmid and the two auxiliary plasmids to 293T cells to complete virus packaging; purifying the prepared virus supernatant, infecting 293T cells again, and detecting the virus titer; and finally infecting host Hela cells by using the detected virus supernatant, and obtaining the Hela cells stably transfected with GFP-GR by puromycin screening.

The screened Hela cells stably transfected with GFP-GR can be used for detecting glucocorticoid endocrine disrupting effects, and when no glucocorticoid mixture exists in a cell culture environment, GFP-GR exists in cytoplasm in the form of protein complexes combined with multiple heat shock proteins and immunoaffinity proteins; when a mixture of glucocorticoids is present, GFP-GR binds to the corresponding hormone ligand and is released from the protein complex and migrates to the nuclear region. In the process, a GFP-GR green fluorescence signal moves from cytoplasm to a nucleus area, and the ratio of the cytoplasmic nucleus migration is measured, wherein the ratio can be used as a basis for positive judgment and semi-quantitative detection of the glucocorticoid content level of a sample. The method realizes high-throughput, low-cost and rapid quantitative detection of the glucocorticoid mixture in the sample.

Drawings

FIG. 1 is a schematic diagram of the monitoring principle of GFP-GR-puro Hela transgenic engineering cell strain; a is a schematic diagram of principle of slow virus packaging and host cell infection; b is a schematic diagram of the cytoplasmic nucleus migration caused by adding glucocorticoid medicaments to the transgenic cells; c is a high content screening system;

FIG. 2 shows L enti-GFP-GR electrophoretogram, lanes M, 6, 7 and 8 marker and L enti-GFP-GR vector (FC-2338), respectively;

FIG. 3 shows three plasmids co-transfected 293T cells, which collect the bright field and GFP images of the transfected cells for 24h and 48h, respectively;

FIG. 4 shows the titer of virus-infected 293T cells, and bright field and GFP images of 293T cells infected with 100. mu. L and 10. mu. L virus supernatants, respectively, were taken;

FIG. 5 is bright field and GFP images of GFP-GR-puro Hela cells after screening; brightfield is the bright field and GFP is the fluorescent signal; 100x and 200x represent magnification 100 times and 200 times, respectively;

FIG. 6 shows the fluorescent signal substance nuclear transfer of GFP-GR-puro Hela cells, DMSO (0.1%) in the Control group, and 10 in the 17 β -E2 group^-7mol/L estradiol, Dexa group addition 10^-7The method comprises the following steps of (1) adding dexamethasone/L, incubating for 1h after administration, fixing cells, dyeing by Hochest33324, collecting images by high content, 20 × watermirror and confocal mode, wherein DPC is a digitalphase contrast channel, and Merge is the superposition effect of GFP and Hochest33324 channels;

FIG. 7 shows the result of cell area sorting, which uses Hochest33342 and DPC channels to sort the cell nucleus and the whole cell area respectively, and obtains the cytoplasm area through the difference between the two areas;

FIG. 8 shows the effect of DMSO and 8 drugs such as 17 β -Estradiol, Dexamethasone, Betamethasone, Beclomethisone, Prednisone, Meprednison, Methylprednisone, etc. on the cytoplasmic nuclear migration of GFP-GR-puro Hela at 1 h;

FIG. 9 shows the molecular structure of four drugs;

FIG. 10 shows 1 × 10^-13mol/L、1×10^-11mol/L、1×10^-9mol/L、1×10^-7mol/L and 1 × 10^-6The effect of Dexamethasone at mol/L5 concentration levels on the nuclear migration of GFP-GR-puro Hela cytoplasm for 0.5h, 1h and 3h respectively;

FIG. 11 is a map of FV026 vector (size 8.1kb), helper plasmid psPAX2, helper plasmid pMD 2G.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

Method for determining glucocorticoid mixture based on GFP-GR-puro Hela cell transgenic engineering cell strain

1. Synthesis of the Gene of interest

GFP-GR complete Gene (SEQ ID NO: 1) (pTet-GFP-GR) was synthesized by Nanjing Kinsley.

2. Plasmid extraction and sequencing

pTet-GFP-GR was transformed into E.coli Trans5 α competent cells by heat shock, plated on L B (Amp) plates, cultured at 37 deg.C, monoclonal strains were picked, cultured overnight in 100m L L B (Amp) liquid medium, and plasmid extraction was performed using the Gene star endotoxin free plasmid extraction kit.

Plasmid concentration 857.6 ng/. mu. L₂₆₀And taking 20u L samples, and performing sequencing and identification.

3. Vector construction

Sequencing the correct plasmid, amplifying a GFP-GR target fragment by using PCR, purifying a PCR product, carrying out enzyme digestion linearization treatment on a lentiviral vector plasmid FV026, and then connecting the purified GFP-GR fragment with a lentiviral vector to construct L enti-GFP-GR vector (marked as FC-2338, shown in figure 2).

4. Viral packaging and titer detection

1) Before a lipofection experiment, 293T cells are digested and re-inoculated into a cell culture dish, and the three co-transfected plasmids are mixed according to the mass ratio of L enti-GFP-GR, psPAX2 and pMD2G of 2:1:1, the plasmids, the transfection reagent and opti-MEM are respectively mixed according to the operation instruction of the transfection reagent, and are finally mixed and then are kept stand for 15min, and then are added into the 293T cell culture dish, and after the three co-transfected plasmids are cultured for 6-8h, the three co-transfected plasmids are replaced by fresh DMEM complete culture medium (10% FBS);

2) after 24h of transfection, the transfection efficiency was judged by fluorescence microscopy (as shown in FIG. 3). When the proportion of the fluorescent cells is more than 70 percent, the transfection is successful, and cell culture solution can be collected and stored at 4 ℃. Replacing a fresh DMEM complete culture medium in the cell culture dish;

3) after transfection for 48 hours, observing the expression condition of the fluorescent protein again, and collecting culture solution for the second time;

4) filtering the harvested culture solution containing the virus particles through a 0.22 mu m filter membrane, centrifuging for 4h at 80000g, re-suspending the precipitate with a virus store buffer, and filtering and sterilizing through the filter membrane;

5) 293T cells were treated as 1 × 10 before virus titer detection⁴And inoculating the cells into a 96-well plate in a DMEM (2% FBS) culture medium, taking purified and sterilized virus supernatant, performing gradient dilution on the plate, namely, the virus supernatant is 100 mu L, 10 mu L, 1 mu L and 0.1 mu L, observing GFP expression after infection for 48 hours, and judging the final titer to be 1E +7TU/m L according to the fluorescence ratio (as shown in figure 4).

5. Infection of host Hela cells

1) Before infection experiment, Hela cells are treated according to 3 × 10⁴One/well was seeded in 96-well plates and the virus was diluted with L V-enhance to a titer of 1 × 10⁶TU/m L and 1 × 10⁵TU/m L, and dripping into the hole for culture, observing the cell state after 8h, optionally replacing with DMEM complete culture medium, infecting for 3-4d, observing the GFP fluorescence expression to judge the infection efficiency, and selecting the optimal infection titer;

2) hela cells are inoculated in a 6-well plate, a virus infection experiment can be carried out when the confluence rate is 70-80%, sterile water is used for replacing virus supernatant in a blank control, a fresh culture medium is replaced by a DMEM complete culture medium (containing 2 mu g/m L puro) after infection is carried out for 24-48h, the cells are continuously cultured until the cells in the blank control die, and the cells in an infected group survive.

6. Selection of stable cell line

1) Culturing with DMEM complete medium (containing puro) until uninfected negative cells in the infected group die, removing dead cells, and replacing with fresh medium;

2) after the cell culture is stable, the cell culture is replaced by a complete culture medium without puro for continuous culture;

3) when the cell confluence rate reached about 90%, the cells were digested, cultured in an expanded manner, continuously cultured for three generations stably, and then frozen, and named GFP-GR-puro Hela (as shown in FIG. 5).

Example 2

Detection and validation of glucocorticoid mixtures

1. Experimental methods

The stable strain cell GFP-GR-puro Hela selected in the example 1 is subjected to 17 β -Estradiol (17 β -Estradiol) (negative), dexamethasone (Dexa) (positive) and other glucocorticoid medicaments for 0.5h, and then the cytoplasmic nuclear migration response is detected.

To exclude as much influence as possible from the medium and serum, the cell culture medium was changed to phenol red-free MEM (+ 10% charcoal-treated serum, 10% CD-FBS) complete medium 1d in advance. Subsequently, GFP-GR-puro Hela digestions were counted as 10⁴After culturing overnight, respectively adding blank control (DMSO), negative control estradiol (17 β -E2), positive drug dexamethasone (Dexa) and other glucocorticoid drugs (prednisone and methylprednisolone, etc.) with the concentration of 10^-7mol/L, 5 per concentration in parallel, 37 deg.C CO₂After being placed in the incubator for 1h, the cells were fixed and stained with Hochestt 33342(Ex/Em 350/361nm) (as shown in FIG. 6).

2. Data processing

The high content screening system performs area sorting and GFP signal intensity derivation: the obtained image is used for sorting cell nucleuses by a Hochest33342 channel, and then sorting the whole cell area by a DPC signal channel; subsequently, the cytoplasmic region was obtained by the phase difference of the two regions (as shown in FIG. 7); finally, the average GFP signal intensity of the nucleus and cytoplasm regions of each well is calculated and given by using a high content system;

3. calculation of results

Calculation of the ratio of proton-nuclear migration for the different drug groups: the ratio of nuclear migration of the drug groups of Dexa et al (as shown in figure 8) was adjusted to 1 for the blank control (DMSO) nuclear migration ratio. All drugs involved in the experiment had a concentration of 10^-7mol/L, wherein, the ratio of proton-core migration of two glucocorticoids prednisone (prednisone) and methylprednisolone (meprednisone) is higher than that of the negative control, but lower than that of the other drugs, because the active sites of the two drugs are aldehyde groups (as shown in the red circle part of FIG. 9), and belong to the inactive state(ii) a The other drugs have hydroxyl at the site and belong to an activated state.

Example 3

1. Experimental methods

The selected confluent cells of example 1, GFP-GR-puro Hela, were similarly subjected to blank control (DMSO) and 1 × 10^-13mol/L、1×10^-11mol/L、1×10^-9mol/L、1×10^--7mol/L and 1 × 10^-6Dexa administration experiments under mol/L5 concentration levels, wherein the action time is 0.5h, 1h and 3h respectively, the influence on the GFP-GR-puro Hela cytoplasm nuclear migration condition is analyzed, and the optimal action time and method sensitivity are detected and screened;

to exclude as much as possible the influencing factors in the medium and serum, the cell culture medium was also changed to phenol red-free MEM (+ 10% charcoal-treated serum, 10% CD-FBS) complete medium 1d earlier. Subsequently, GFP-GR-puro Hela digestions were counted as 10⁴One/well access black 96-well plates (3603, corning). After overnight culture, 5 concentration levels of Dexa were added, each concentration being set at 5 replicates; 37 ℃ and 5% CO₂After 0.5h, 1h and 3h of action in the incubator respectively, the cells are taken out for cell fixation and Hochestt 33342(Ex/Em 350/361nm) staining.

The high-content data acquisition method and process are completely the same as those in example 2.

2. Calculation of results

1×10^-13mol/L、1×10^-11mol/L、1×10^-9mol/L、1×10^-7mol/L and 1 × 10^-6Dexa et al at mol/L5 concentration levels (L og treatment) obtained ratios of cytoplasmic nuclear transfer minus the DMSO blank level (as shown in FIG. 10). The results show a concentration at 1 × 10^-13mol/L-1×10^-9Dexa expression was almost at DMSO baseline levels between mol/L, but at>1×10^-9After the mol/L level, a significant increase in the ratio of proton-nuclear transport was observed, and the sensitivity of the method was seen to be approximately equal>1×10^- ⁹mol/L (calculated as Dexa), and under the condition of respectively acting for 0.5h and 1h, the reduction of the nucleus pulposus migration rate is not obvious, and the cell expression is stable, so the optimal time range of the drug acting on GFP-GR-puro Hela is 0.5h-1 h.

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

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Claims

1. A transgenic engineering cell strain is characterized in that a Green Fluorescent Protein (GFP) and Glucocorticoid Receptor (GR) fusion protein gene is integrated into a Hela cell chromosome in a lentivirus infection mode, and the Hela cell strain capable of stably expressing GFP-GR is finally obtained through puromycin puro screening.

2. A method for determining glucocorticoid mixture based on the genetically engineered cell line of claim 1, comprising the steps of:

step 2: after the glucocorticoid mixture is added into the GFP-GR-puro Hela transgenic engineering cell strain, the content of the glucocorticoid mixture (calculated by dexamethasone) can be determined semi-quantitatively/quantitatively by measuring the migration rate of a fluorescence signal from cytoplasm to a nucleus region.

3. The method for assaying glucocorticoid-like mixture according to claim 2, wherein the step 1 of constructing the GFP-GR-puro Hela cell transgenic engineered cell line specifically comprises the following steps:

4. The method for assaying glucocorticoid mixtures according to claim 3, wherein the 293T cells are digested before being used for transfection in step b, and re-inoculated into a cell culture dish until the cells grow to reach 80% -90% confluence for transfection experiments.

5. The method for assaying glucocorticoid-like mixture according to claim 3, wherein the titer of the qualified virus suspension is 1E +7TU/m L.

6. The use of the genetically engineered cell line of claim 1 in the fields of medical health, food safety and environmental protection for quantitative determination of the glucocorticoid compound content in body fluids, food and environmental water sources.

7. The use of the genetically engineered cell line of claim 6 for quantitative determination of the glucocorticoid content of different samples of human and animal body fluids, milk and sewage.