CN112126680B - Kit and method for screening trace endocrine disruptors based on fluorescent quantitative PCR - Google Patents

Abstract

The invention discloses a kit and method for screening trace amounts of endocrine disruptors based on fluorescence quantitative PCR, comprising a biological magnetic bead-NR-LBD protein solution, an NCoA-DNA tag chimera solution, a pair of specific primers, a binding reaction solution and fluorescence quantitative PCR reaction system. In the present invention, the activated biological magnetic beads-NR-LBD protein solution, the NCoA-DNA label chimera solution and the appropriate volume of the substance to be tested are incubated together with the binding reaction solution, and then the detection system is magnetically separated and cleaned, and 1× fluorescent The quantitative PCR reaction system was resuspended, and then specific primers were added to carry out fluorescence quantitative PCR amplification, and the nuclear receptor activity of the substance to be tested was quantitatively characterized according to the Ct value. The kit and the detection method have the advantages of strong specificity, good repeatability, low detection limit, ultra-high sensitivity, high test throughput, rapidity and simplicity.

Description

Kit and method for screening trace endocrine disruptors based on fluorescence quantitative PCR

technical field

The invention relates to the technical field of detection of environmental compounds, in particular to a kit and method for screening trace endocrine disruptors based on fluorescence quantitative PCR.

Background technique

Ecological environment safety and food safety are two important factors related to human survival. With a good ecological environment and sufficient and safe food supply, human beings can survive and develop more healthily. However, while promoting the rapid economic development, human industry and agriculture also bring a heavy burden to environmental safety and food safety. More and more compounds enter the human living environment through various ways, and they exist stably in the environment and have strong bioaccumulation. Endocrine Disrupting Chemicals (EDCs), also known as environmental hormones, refer to chemical substances existing in the environment that interfere with various aspects of the endocrine system of humans or animals and cause abnormal effects. They have abnormal effects on organisms through various indirect ways such as ingestion, exposure, accumulation, etc. Even extremely low concentrations may cause reproductive disorders, developmental abnormalities, behavioral abnormalities, immune dysfunction, and larval death. Endocrine disruptors have a wide variety of types, long-lasting toxicity and long incubation period. They can pass through biological enrichment and food chain transmission, and eventually cause harm to higher animals and humans.

There are many kinds of environmental endocrine disrupting substances with complex and changeable structures. Many synthetic chemicals have endocrine disrupting activity. In recent years, typical EDCs have been widely detected in environmental samples, and their detection concentrations have reached the PPt-PPb level. Although enough attention has been paid to the endocrine disrupting effects of these trace compounds, their endocrine disrupting activities are often difficult to detect due to their wide distribution in the environment and usually low levels, as well as complex sample matrices. There is a very complex correlation between the levels of endocrine disruptors in the environment and the health of humans and other organisms, and the detection of endocrine disruptors is necessary to ensure the safety of humans and animals in the environment. The use of biological detection methods can rapidly measure or screen endocrine disruptors, and determine their endocrine disrupting activities. The in vitro test has the advantages of uncomplicated, economical and rapid pretreatment, so it is often used for the preliminary evaluation of the endocrine disrupting activity of environmental compounds. Most of the traditional receptor binding/transcription activation assays require the cultivation of experimental materials (cells, yeast, etc.), which require high laboratory conditions, complex screening procedures, high cost, and poor repeatability due to the individual sensitivity of experimental materials. The detection accuracy is low and the screening ability is limited. In addition, immunoassays are based on the specific reaction of antigen-antibody to detect EDCs, which has high specificity and sensitivity, but the preparation of antibodies is difficult. On-site monitoring. The research on the content, migration and transformation of these trace compounds in environmental media and the exposure of organisms to environmental endocrine disruptors is inseparable from their quantitative analysis. Therefore, it is particularly important to develop new rapid, sensitive, and cost-effective toxicological analysis methods. urgent.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a kit and method for screening trace endocrine disruptors based on fluorescence quantitative PCR, so as to achieve high efficiency, high throughput and high sensitivity of the endocrine disrupting activity of environmental trace pollutants Quick check.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is as follows:

One aspect of the present invention is to provide a kit for screening trace amounts of endocrine disruptors based on fluorescence quantitative PCR, comprising a biological magnetic bead-NR-LBD protein solution, an NCoA-DNA tag chimera solution, a pair of specific primers, a combination of Reaction solution and fluorescence quantitative PCR reaction system.

Further, the biological magnetic beads-NR-LBD protein is prepared by the following steps:

The first is to use the cDNA of human or mouse cells or tissues as a template and refer to the coding sequence of the human or mouse nuclear receptor NR published by NCBI, which is closely related to human or mouse health, and its ligand binding domain NR-LBD. Amplification of the coding sequence;

Then the NR-LBD protein was obtained by prokaryotic expression and purification. It was further incubated with the pretreated biomagnetic beads to obtain the biomagnetic beads-NR-LBD protein.

Further, the NCoA-DNA tag chimera is prepared by the following steps:

The first is to amplify the coding sequence of nuclear receptor coactivator NCoA by referring to the coding sequence of human or mouse nuclear receptor coactivator NCoA published by NCBI using the cDNA of human or mouse cells or tissues as a template;

Then the NCoA protein was obtained by prokaryotic expression and purification. The NCoA protein and DNA tag sequences were further assembled into NCoA-DNA tag chimeras by DNA-protein cross-linking strategy.

Further, the nuclear receptor NR closely related to human or mouse health includes estrogen receptor ER, progesterone receptor PR, androgen receptor AR, thyroid hormone receptor TR, glucocorticoid receptor GR , mineralocorticoid receptor MR, peroxisome proliferator-activated receptor PPAR, constitutive androstane receptor CAR, pregnane X receptor PXR, vitamin D receptor VDR, retinoic acid receptor RAR, retinoic acid Related orphan nuclear receptor ROR, liver X receptor LXR, farnesoid X receptor FXR and estrogen related receptor ERR.

Further, the ER includes ERα, ERβ; the TR includes TRα, TRβ; the GR includes GRα, GRβ; the PPAR includes PPARα, PPARβ/δ and PPARγ; the RAR includes RARα, RARβ, RARγ; The ROR includes RORα, RORβ, and RORγ; the LXR includes LXRα and LXRβ; the ERR includes ERRα, ERRβ, and ERRγ.

Further, the above-mentioned human or mouse nuclear receptor coactivator NCoA includes sterol receptor coactivator (Steriod Receptor Coactivator, SRC), activated signal co-integrator 2 (Activating SignalCointegrator 2, ASC-2), peroxidation Biosome proliferator-activated receptor gamma coactivator 1 (PeroxisomeProliferator Activated Receptor gamma Coactivator 1, PGC 1).

Further, the SRC includes SRC-1/NcoA1, SRC-2/TIF-2/GRIP1/NcoA, SRC3/NCOA3/AIB, NCOA4, NCOA5, NCOA6/NRC/ASC-2/TRBP/PRIP/RAP250, CBP/p300, CARM1/PRMT1, etc.; the PGC 1 includes PGC-1α, PGC-1β, PRC and the like.

Further, the DNA tag sequence is any nucleotide sequence with a length of 50-300 bp derived from a naturally occurring gene or an artificially synthesized gene.

Further, the specific primer is composed of a forward primer and a reverse primer, and is an oligonucleotide sequence with a length of 15-30 bp designed with the DNA tag sequence as the target sequence.

Further, the components of the binding reaction solution include DTT, MgCl ₂ , KCl, Triton X-100, BSA and Tris buffer. Preferably, the components of the binding reaction solution are 1 mM DTT, 5 mM MgCl ₂ , 50 mM KCl, 0.02% Triton X-100, 0.1 mg/mL BSA, 10 mM Tris-HCl, and the pH is 7.5.

Further, the components of the fluorescent quantitative PCR reaction system include 2×SYBR Green I qPCR Mix, 50×Rox and ddH ₂ O, which are all diluted with ddH ₂ O to 1× working solution when used in the fluorescent quantitative PCR reaction.

Another aspect of the present invention is to provide a method for screening trace endocrine disruptors based on fluorescence quantitative PCR, comprising the following steps:

(1) Activation of biomagnetic beads-NR-LBD protein: take an appropriate amount of biomagnetic beads-NR-LBD protein solution, wash and activate with pre-cooled binding reaction solution, magnetically separate and discard the supernatant, repeat the washing once, and use pre-cooling The binding reaction solution was resuspended to obtain the activated biological magnetic beads-NR-LBD protein solution;

(2) Preparation and incubation of detection system: Mix the activated biomagnetic beads-NR-LBD protein solution, NCoA-DNA tag chimera solution and the analyte with the pre-cooled binding reaction solution in a reaction tube to prepare a Detection system, low temperature incubation;

(3) Cleaning of the detection system: after the incubation, the detection system was washed with the pre-cooled binding reaction solution, the supernatant was discarded by magnetic separation, the washing was repeated once, and the pre-cooled 1× fluorescence quantitative PCR reaction system was used to resuspend;

(4) Addition of specific primers and fluorescence quantitative PCR amplification reaction: Add specific primers to the reaction tube for fluorescence quantitative PCR amplification reaction, set melting curve to analyze the specificity of the amplified product, and reflect the Ct value in the system The amount of DNA fragments bound was based on the blank control with distilled water as the base, and the 2- ^ΔΔCt method was used to calculate the relative receptor activity of the analyte.

Further, preparation of the kit: preparation of biomagnetic beads-NR-LBD protein and NCoA-DNA tag chimera:

Among them, the biological magnetic beads-NR-LBD protein is firstly obtained by using the cDNA of human or mouse cells or tissues as a template, referring to the human or mouse nuclear receptor NR published by NCBI which is closely related to human or mouse health. The coding sequence is amplified, and the coding sequence of the ligand binding domain NR-LBD is amplified, and then the NR-LBD protein is obtained by expressing and purifying in prokaryotic cells. It was further incubated with the pretreated biomagnetic beads to obtain the biomagnetic beads-NR-LBD protein. The NCoA-DNA tag chimera firstly uses human or mouse cell or tissue cDNA as a template and refers to the coding sequence of human or mouse nuclear receptor coactivator NCoA published by NCBI to co-activate nuclear receptors. The coding sequence of factor NCoA is amplified, and then expressed and purified by prokaryotic cells to obtain NCoA protein. The NCoA protein and DNA tag sequences were further assembled into NCoA-DNA tag chimeras by using the strategy of DNA-protein cross-linking.

Further, the nuclear receptor NR closely related to human or mouse health includes estrogen receptor ER, progesterone receptor PR, androgen receptor AR, thyroid hormone receptor TR, glucocorticoid receptor GR, Mineralocorticoid receptor MR, peroxisome proliferator-activated receptor PPAR, constitutive androstane receptor CAR, pregnane X receptor PXR, vitamin D receptor VDR, retinoic acid receptor RAR, retinoic acid related Orphan nuclear receptor ROR, liver X receptor LXR, farnesoid X receptor FXR and estrogen-related receptor ERR.

Further, the ER includes ERα, ERβ; the TR includes TRα, TRβ; the GR includes GRα, GRβ; the PPAR includes PPARα, PPARβ/δ and PPARγ; the RAR includes RARα, RARβ, RARγ; The ROR includes RORα, RORβ, and RORγ; the LXR includes LXRα and LXRβ; the ERR includes ERRα, ERRβ, and ERRγ.

Further, the above-mentioned human or mouse nuclear receptor coactivator NCoA includes sterol receptor coactivator (Steriod Receptor Coactivator, SRC), activated signal co-integrator 2 (Activating SignalCointegrator 2, ASC-2), peroxide Peroxisome Proliferator Activated Receptor γ Coactivator 1 (PGC 1).

Further, the SRC includes SRC-1/NcoA1, SRC-2/TIF-2/GRIP1/NcoA, SRC3/NCOA3/AIB, NCOA4, NCOA5, NCOA6/NRC/ASC-2/TRBP/PRIP/RAP250, CBP/p300, CARM1/PRMT1, etc.; the PGC 1 includes PGC-1α, PGC-1β, PRC and the like.

Further, the precooling method in steps (1), (2) and (3) is to place on ice; the precooling and low temperature temperature are 0-8°C, preferably 4°C.

Further, the concentration range of the biological magnetic beads-NR-LBD protein in the detection system (calculated as NR-LBD protein) in step (2) is 0.01-0.05 μg/μL, preferably 0.03 μg/μL; the NCoA - The concentration range of the DNA tag chimera in the detection system (calculated as NCoA protein) is 0.01-0.05 μg/μL, preferably 0.03 μg/μL.

Further, the reaction incubation time in step (2) is 20-60 min, preferably 30 min.

Further, the components of the binding reaction solution in steps (1), (2) and (3) include DTT, MgCl ₂ , KCl, TritonX-100, BSA and Tris buffer. Preferably, the components of the binding reaction solution are 1 mM DTT, 5 mM MgCl ₂ , 50 mM KCl, 0.02% Triton X-100, 0.1 mg/mL BSA, 10 mM Tris-HCl, and the pH is 7.5.

Further, the components of the 1× fluorescence quantitative PCR reaction system in step (3) include 1× SYBR Green I qPCR Mix and 1× Rox diluted with ddH ₂ O.

The invention rapidly detects the endocrine disrupting activity of chemicals based on fluorescence quantitative PCR technology, and the main reaction components are biological magnetic beads-NR-LBD protein solution, NCoA-DNA tag chimera solution and a pair of specific primers. The principle of the present invention is as follows: the tested substance (compound with endocrine disrupting activity) in the sample to be tested is combined with the NR-LBD receptor protein on the solid phase carrier (biological magnetic bead), and the conformation of the receptor protein is changed, so that the It can further combine with NCoA on the NCoA-DNA tag chimera to form a polymer. Then, wash the plate to remove the unbound material and resuspend it with 1× fluorescent quantitative PCR reaction system. At this time, specific primers are added to amplify the signal by fluorescent quantitative PCR amplification, and the double-stranded DNA fragment in the SYBR Green I dye-binding polymer is emitted. Fluorescence. Therefore, the fluorescence intensity obtained by DNA amplification during the amplification cycle is monitored, and the strength of the binding between different analytes and receptor proteins can be indirectly characterized according to the Ct values at different analyte concentrations.

Compared with the prior art, the beneficial effects of the present invention are:

(1) Strong specificity, good repeatability, low detection limit, and ultra-high sensitivity:

The NR-LBD receptor protein provided by the present invention is immobilized on a solid-phase carrier (biomagnetic bead), and has more stable structure and properties, and can resist certain environmental factors. Then combined with real-time fluorescent quantitative PCR technology as a quantitative method, the polymerized DNA fragments in the reaction system are amplified exponentially by PCR, thereby amplifying the detection signal millions of times, the detection limit can reach 1 × 10 ^-13 M, and the sensitivity Much higher than the common immunodetection methods such as ELISA and immunofluorescence.

(2) Small reaction volume, high test throughput, and easy automation:

The reaction tubes used in the detection method provided by the present invention are real-time quantitative PCR consumables such as 96-well plate, 384-well plate or chip, etc., and the endocrine disrupting activity of multiple samples or the activities of multiple nuclear receptors of the same sample can be simultaneously performed on the same reaction plate. The detection is easy to realize automation and high-throughput detection, and can be used for the screening of a large number of samples. Taking the 384-well plate test as an example, the reaction volume per well is only 10-25 μL, which can realize the high-throughput determination of the specific receptor agonist or antagonist activity of 20 chemicals at one time. The specific calculation is as follows: 20 chemical substances (6 concentration gradients × 3 parallels/substance) = 360 wells + 1 positive control substance (5 concentration gradients × 3 parallels/substance) = 21 wells + 3 wells (blank control) + 3 wells (negative control). Therefore, using the detection scheme provided by the present invention, the throughput can be increased by 5 times compared with the prior art, and the test can be increased from 4 kinds of substances to 20 kinds of substances at one time.

(3) Quick and easy:

The detection method provided by the present invention does not require living cells as experimental materials, is simple to operate, easy to grasp and use, and has a short analysis period. Taking the 384-well plate test as an example, the entire detection time is only 3.5 hours. The specific calculation is as follows: 384-well loading of activated premixed reagents and test substances (1.5 hours) + low temperature incubation reaction (0.5 hours) + on-board amplification reading (1.5 hours) = 3.5 hours. Therefore, using the detection scheme provided by the present invention, the speed can be increased by more than 3 times compared with the prior art, and one test can be completed in 3.5 hours from the current 12 hours.

Description of drawings

Figure 1 is a schematic diagram of a method for screening trace endocrine disruptors based on real-time PCR.

Figure 2 is a graph showing the mERα receptor activity curves of different chemicals. (A) is the mERα receptor activity of bisphenols; (B) is the mERα receptor activity of salicylates; (C) and (D) are the mERα receptor activities of alkylphenols.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to FIG.

1. Preparation of biological magnetic beads-mouse mERα-LBD protein

Total RNA from mouse liver tissue was extracted with Trizol reagent, and reversed into cDNA as template. Referring to the coding sequence of mouse estrogen-related receptor mERα (NCBI GenBank: NP_001289460.1) published by NCBI, primers were designed to amplify the sequence. The encoding gene of mERα-LBD (314..551aa) was ligated into the prokaryotic expression vector pGEX-4T-1 by double digestion method, transformed into E. coli DH5α competent cells, screened for positive clones and sequenced to obtain the recombinant expression vector pGEX- 4T-mERα-LBD. The recombinant plasmid was extracted and transformed into E. coli BL21 competent cells to obtain mERα-LBD recombinant expression bacteria. The recombinant bacteria were amplified and cultured at 37°C, and IPTG was added to induce expression. Then, the recombinant protein was purified by sonication and affinity chromatography to obtain mERα-LBD protein. The expression and purification of the target protein were detected by SDS-PAGE electrophoresis, and the concentration of the purified protein was determined to analyze the enzymatic properties of the recombinant protein. It was further incubated with the pretreated biomagnetic beads to obtain the biomagnetic beads-mERα-LBD protein.

2. Preparation of mNCoA2-DNA tag chimeras

Total RNA from mouse liver tissue was extracted with Trizol reagent, and reversed into cDNA as template. Referring to the coding sequence of mouse nuclear receptor coactivator mNCoA2 (NCBI GenBank: NP_001289631.1) published by NCBI, primers were designed to amplify the sequence. Using the method of double digestion, mNCoA2 was connected to the prokaryotic expression vector pET-28a(+), transformed into E. coli DH5α competent cells, and screened for positive clones. The recombinant plasmid was extracted and transformed into E. coli BL21 competent cells to obtain mNCoA2 recombinant expression bacteria. The recombinant bacteria were amplified and cultured at 37°C, and IPTG was added to induce expression. Then, the recombinant protein was purified by sonication and affinity chromatography to obtain mNCoA2 protein. The expression and purification of the target protein were detected by SDS-PAGE electrophoresis, the concentration of purified protein was determined, and the enzymatic properties of the recombinant protein were analyzed. A DNA sequence between the restriction sites Nco I and Xho I on the plasmid pET-28a(+) was selected as the tag sequence during PCR amplification, and the mNCoA2 protein was further assembled with the DNA tag sequence through the biotin-avidin system into mNCoA2-DNA tag chimeras.

3. Rapid determination of mERα receptor activity of chemicals at different concentrations

Using tamoxifen, an inverse agonist of mERα, as a standard sample, the estrogen-related receptor activity of chemicals was detected. Compounds tested include: Bisphenols: Bisphenol F (BPF), Bisphenol S (BPS), Bisphenol Fluorene (BHPF), Bisphenol AP (BPAP), Bisphenol BP (BPBP); Salicylates: Propyl salicylate, butyl salicylate, isobutyl salicylate, and ethylene glycol salicylate; alkylphenols: 4-methylphenol, 4-ethylphenol, 4-isopropylphenol , 4-sec-butylphenol, 4-tert-butylphenol, 4-pentylphenol, 4-tert-amylphenol, 4-tert-octylphenol, nonylphenol and 4-benzylphenol.

Take ≥1.2 mg of biomagnetic beads-mERα-LBD protein solution with pre-cooled binding reaction solution (1 mM DTT, 5 mM MgCl ₂ , 50 mM KCl, 0.02% Triton X-100, 0.1 mg/mL BSA, 10 mM Tris-HCl, pH 7.5) Wash and activate, magnetic separation, discard the supernatant, repeat the washing once, and resuspend with 500 μL of pre-cooled binding reaction solution to obtain an activated biological magnetic beads-mERα-LBD protein solution. The 384-well plate was pre-cooled on ice, and 10 μL of activated biomagnetic beads-mERα-LBD protein, 10 μL of mNCoA2-DNA tag chimera, and 1 μL of different concentrations of the test substance were added to each reaction well. Combine the reaction solution and mix to form a detection system, and incubate at low temperature for 30 min. After the incubation, wash the detection system with the pre-cooled binding reaction solution, discard the supernatant by magnetic separation, repeat the washing once, and use 8 μL of pre-cooled 1× fluorescence quantitative PCR reaction system (1×SYBRGreen I qPCR Mix and 1×Rox) Resuspended. Then 1 μL of specific forward primer (whose sequence is 5′ATGGGCAGCAGCCATCAT 3′) and 1 μL of specific reverse primer (whose sequence is 5′ATCCGCGACCCATTTGCT3′) were then added to each reaction well. Each well was sealed with a sealing film, and placed in an ABI 7500 fluorescence quantitative PCR instrument for fluorescence quantitative PCR amplification reaction. The amplification program was as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 40 cycles; and final extension at 72 °C for 5 min. After the reaction, the amplification curve and melting curve of real-time PCR were confirmed, and the mERα receptor activity of different test substances was calculated and compared. The results are shown in Figure 2. The mERα receptor activity of BPF and BPS in bisphenols is higher than that of the other three bisphenols. BPAP has estrogenic activity only at low concentrations, and is a weak estrogen receptor active substance, while BHPF and BPBP have almost no estrogen receptor activity; among salicylates, isobutyl salicylate has the highest mERα receptor activity, followed by butyl salicylate with more significant estrogen activity, and propyl salicylate and ethylene glycol salicylate are weak in activity; among the alkylphenols, 4-ethylphenol, 4-isopropylphenol, 4-tert-butylphenol, 4-tert-amylphenol, 4-tert-octyl Phenol, 4-pentylphenol, 4-benzylphenol, 4-sec-butylphenol, nonylphenol and 4-methylphenol all have high estrogen receptor activity and are estrogen receptor agonists.

4. Rapid determination of different nuclear receptor activities of 4-octylphenol

The activities of different nuclear receptors of 4-octylphenol were tested by 384-well plate. The tested nuclear receptor mNR activities included ERα, ERβ, TRα, TRβ, PPARα, PPARβ/δ, PPARγ, VDR, RARα, RARγ, LXRα, LXRβ , FXR, ERRα, ERRβ, ERRγ, the receptor activity test also used the natural ligand of each receptor as a control. According to the method shown in Example 1, the above-mentioned receptor mNR-LBD proteins were prepared and immobilized with biological magnetic beads. Take ≥1.2mg biomagnetic beads-mNR-LBD protein solution with pre-cooled binding reaction solution (1mM DTT, 5mM MgCl ₂ , 50mM KCl, 0.02% Triton X-100, 0.1mg/mL BSA, 10mM Tris-HCl, pH 7.5) Wash and activate, magnetic separation, discard the supernatant, repeat the washing once, and resuspend with 500 μL of pre-cooled binding reaction solution to obtain the activated biological magnetic beads-mNR-LBD protein solution. The 384-well plate was pre-cooled on ice, and 10 μL of activated biomagnetic beads-mNR-LBD protein, 10 μL of mNCoA2-DNA tag chimera and 1 μL of 4-octylphenol solution were added to each reaction well. Combine the reaction solution and mix to form a detection system, and incubate at low temperature for 30 min. After the incubation, the detection system was washed with the pre-cooled binding reaction solution, the supernatant was discarded by magnetic separation, and the washing was repeated once. )Resuspended. Then 1 μL of specific forward primer (whose sequence is 5′ATGGGCAGCAGCCATCAT 3′) and 1 μL of specific reverse primer (whose sequence is 5′ATCCGCGACCCATTTGCT 3′) were then added to each reaction well. Each well was sealed with a sealing film, and placed in an ABI 7500 fluorescence quantitative PCR instrument for fluorescence quantitative PCR amplification reaction. The amplification program was as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 40 cycles; and final extension at 72 °C for 5 min. After the reaction, the amplification curve and melting curve of fluorescence quantitative PCR were confirmed, and the different mNR receptor activities of 4-octylphenol were calculated and compared. The results showed that 4-octylphenol has strong ERα, ERβ and RARγ activities, weak RARα, LXRα, FXR and ERRα activities, but no activity on the remaining receptors.

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Those skilled in the art can modify or equivalently replace the technical solutions of the present invention. The protection scope of the present invention is subject to the claims.

Claims (9)

1. a test kit for screening trace endocrine disruptors based on fluorescence quantitative PCR, is characterized in that, comprises biological magnetic beads-NR-LBD protein solution, NCoA-DNA label chimera solution, a pair of specific primers, binding reaction liquid and fluorescence quantitative PCR reaction system; wherein, first, using the cDNA of human or mouse cells or tissues as a template, referring to the coding sequence of the human or mouse nuclear receptor coactivator NCoA published by NCBI, the nuclear receptor coactivator The coding sequence of NCoA is amplified, and the NCoA protein is obtained by expression and purification in prokaryotic cells. Then, the NCoA protein and the DNA tag sequence are assembled by the DNA-protein cross-linking strategy to obtain the NCoA-DNA tag chimera. The DNA-protein cross-links The strategy is to assemble mNCoA2 protein and DNA tag sequence into mSRC2-DNA tag chimera through biotin-avidin system; the components of real-time quantitative PCR reaction system include 2 × SYBR Green I qPCR Mix, 50 × Rox and ddH ₂ O. 2. test kit as claimed in claim 1 is characterized in that, described biological magnetic bead-NR-LBD protein is prepared by the following steps: with the cDNA of human or mouse cell or tissue as template, with reference to NCBI published and The coding sequence of the human or mouse nuclear receptor NR, which is closely related to human or mouse health, amplifies the coding sequence of the NR ligand-binding domain NR-LBD, and expresses and purifies the NR-LBD protein in prokaryotic cells; the NR -LBD protein was incubated with pretreated biomagnetic beads to obtain biomagnetic beads-NR-LBD protein. 3. test kit as claimed in claim 2 is characterized in that, described nuclear receptor NR closely related to human or mouse health comprises estrogen receptor ER, progesterone receptor PR, androgen receptor AR, Thyroid hormone receptor TR, glucocorticoid receptor GR, mineralocorticoid receptor MR, peroxisome proliferator-activated receptor PPAR, constitutive androstane receptor CAR, pregnane X receptor PXR, vitamin D Receptors VDR, retinoic acid receptor RAR, retinoic acid related orphan nuclear receptor ROR, liver X receptor LXR, farnesoid X receptor FXR and estrogen related receptor ERR. 4. the test kit for screening trace endocrine disruptors based on fluorescence quantitative PCR as claimed in claim 1, is characterized in that, described human or mouse nuclear receptor coactivator NCoA comprises sterol receptor coactivator, activated Signal co-integrator 2, peroxisome proliferator-activated receptor gamma co-activator 1. 5. The kit of claim 1, wherein the DNA tag sequence is any nucleotide sequence with a length of 50-300 bp derived from a naturally occurring gene or an artificially synthesized gene. 6. test kit as claimed in claim 5 is characterized in that, described specific primer is made up of forward primer and reverse primer, is designed with described DNA tag sequence as target sequence, and the length is 15-30 bp Oligonucleotide sequence. 7. The kit of claim 1, wherein the components of the binding reaction solution comprise DTT, MgCl ₂ , KCl, Triton X-100, BSA and Tris buffer. 8. a method for screening trace endocrine disruptors based on fluorescence quantitative PCR according to claim 1, is characterized in that, comprises the following steps: (1) Activation of biological magnetic beads-NR-LBD protein: Take the biological magnetic beads-NR-LBD protein solution, wash and activate it with pre-cooled binding reaction solution, remove the supernatant by magnetic separation, repeat the washing once, and use pre-cooled The activated biological magnetic beads-NR-LBD protein solution was obtained by resuspending the combined reaction solution; (2) Preparation and incubation of detection system: Mix the activated biomagnetic beads-NR-LBD protein solution, NCoA-DNA tag chimera solution and the analyte with the pre-cooled binding reaction solution in the reaction tube to prepare Detection system, low temperature incubation; (3) Cleaning of the detection system: after the incubation, wash the detection system with the pre-cooled binding reaction solution, discard the supernatant by magnetic separation, repeat the washing once, and resuspend with the pre-cooled 1 × fluorescence quantitative PCR reaction system; (4) Addition of specific primers and fluorescence quantitative PCR amplification reaction: Add specific primers to the reaction tube for fluorescent quantitative PCR amplification reaction, set the melting curve, and reflect the amount of DNA fragments bound in the system by the Ct value. Taking the blank control added with distilled water as the background, the

The relative receptor activity of the analyte was calculated by the method. 9 . The method according to claim 8 , wherein the method for pre-cooling in steps (1), (2) and (3) is to place on ice, and the temperature of pre-cooling is 0-8° C.; step 9 . The concentration range of the biomagnetic beads-NR-LBD protein in the detection system in (2) is 0.01-0.05 µg/µL, and the concentration range of the NCoA-DNA tag chimera in the detection system is 0.01-0.05 µg/µL, The incubation time at low temperature is 20-60 min.

Images