CN109827934B - Kit and method for rapidly detecting endocrine disrupting activity of chemicals - Google Patents
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Abstract
The invention discloses a kit for rapidly detecting endocrine disrupting activity of chemicals, which comprises a recombinant human nuclear receptor ligand binding domain hNR-LBD protein solution, a recombinant human nuclear receptor coactivator-EcoR I fusion protein solution, a specific Taqman probe, an A-DTT buffer solution and a B buffer solution. The invention also discloses a method for rapidly detecting the endocrine disrupting activity of chemicals, which realizes rapid detection of the endocrine disrupting activity of chemicals on a microporous plate by applying the kit. The invention realizes the endocrine disrupting activity of chemicals such as alkylphenol, p-alkoxy phenol, paraben, bisphenol, biphenyl, diphenyl ether, organophosphorus and halogen compounds with high flux, multiple targets and rapid detection by a Taqman fluorescent probe method, and the upper limit of detection reaches 10‑ 11M。
Description
Technical Field
The invention relates to the technical field of detection of environmental compounds, in particular to a kit and a method for rapidly detecting endocrine disrupting activity of chemicals based on a fluorescent probe.
Background
There are hundreds of thousands of newly synthesized chemicals worldwide each year, and there are currently over 1.2 million registered compounds, as counted by the Chemical Abstracts Service (CAS), and the number is still increasing. Endocrine Disrupting Chemicals (EDCs) refer to environmental compounds that interfere with the synthesis, secretion, transport, metabolism, binding and removal processes of endogenous hormones to affect the homeostasis, reproductive and developmental processes of an organism. Research shows that many chemical substances in the environment have endocrine disrupting effects, are wide in source and extremely different in structural properties, and have persistent organic pollutants (such as polychlorinated biphenyl, polybrominated diphenyl ether, organochlorine pesticides and the like), natural phytoestrogens (such as flavonoids and the like), microbial toxins, pesticides and the like. In recent years, the incidence of intestinal cancer, cervical cancer and ovarian cancer in humans has increased, and diseases such as infertility, thyroid dysfunction, congenital defects, behavioral disorders, nervous system depression and genital malformations have increased, and may be related to endocrine disturbance. Therefore, a great deal of manpower and material resources are invested in many countries, regions and international organizations (such as the U.S. environmental protection agency, the environmental protection agency of japan, the environmental planning agency of the united nations, the world health organization, etc.) to identify the EDCs, and research the harmful effects and action mechanisms of the EDCs so as to better manage chemicals. However, to date, a large number of compounds with potential endocrine disrupting activity have not been effectively identified.
From the technical perspective, there are technical bottlenecks such as flux is little, time is long, complex operation, lack multicomponent check out test set equipment at present in the categorised detection of chemical health hazard, and in endocrine interference effect detects, it is urgent to wait to improve the detection flux, shortens to detect for a long time, promotes the degree of automation that conventional detected, solves the difficult problem of external metabolism activation. Therefore, the existing detection of the endocrine disrupting activity of chemicals has the technical problems of low flux, long test period, few toxic targets in one-time test, low content of toxic effect spectrums and the like.
For Estrogen receptors (ER; including ER alpha, ER beta), Estrogen Related receptors (ERR; including ERR alpha, ERR beta, ERR gamma), Androgen receptors (Androgen receptors, AR), Glucocorticoid receptors (Glucocorticoid receptors, GR; including GR alpha, GR beta, GR gamma), Retinoic Acid receptors (Retinoic Acid receptors, RAR; including RAR alpha, RAR beta, RAR gamma), Retinoic Acid Related Orphan nuclear receptors (ROTinoid receptors, ROR; including ROR alpha, ROR beta, ROR gamma), Peroxisome proliferator-activated receptors (Peroxisome proliferator receptors, PPAR; including PPAR alpha, gamma/PPAR beta, and Vitamin D), Thyroid receptors (LXR; LXR Receptor alpha, RAR beta; LXR beta, LR gamma; LXR; R; LXR; R; LXR; R; LXR, TR beta) and the like which are closely related to human health, and establishing a test method for analyzing the activity of the receptor agonist or antagonist has important significance for evaluating the potential health risk of the chemicals.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a kit and a method for rapidly detecting the endocrine disrupting activity of chemicals based on a fluorescent probe, so that the endocrine disrupting activity of chemicals can be rapidly screened and confirmed with high flux and multiple targets.
The invention utilizes a fluorescent probe to quickly detect the endocrine disrupting activity of chemicals, and comprises hNR-LBD (Ligand-binding Domain, LBD) receptor protein of a recombinant human nuclear receptor Ligand binding Domain, a recombinant human nuclear receptor coactivator-EcoR I fusion protein and 1 Taqman probe in a reaction system for quantitatively detecting the endocrine disrupting activity of a compound by a Taqman probe method. The 5 'end of the Taqman probe is marked with a Reporter group Reporter, such as FAM, VIC and the like, and the 3' end is marked with a fluorescence quenching group Quencher, such as TAMRA and the like. When the probe is complete, the fluorescent energy emitted by the reporter group is absorbed by the quencher group, and no signal can be detected by the instrument. When hNR-LBD receptor protein-ligand is combined, the receptor conformation can be changed, and then the receptor can be combined with the human nuclear receptor coactivator-EcoR I fusion protein; the EcoR I enzyme cuts off the Taqman probe through a G AATTC site on the Taqman probe, the reporter group is far away from the quenching group, the energy of the reporter group cannot be absorbed, namely, a fluorescence signal is generated, and the intensity of the signal represents the strength of the combination of different environmental compounds and the receptor.
The invention provides a kit for rapidly detecting the endocrine disrupting activity of chemicals, which comprises a recombinant human nuclear receptor ligand binding domain hNR-LBD protein solution, a recombinant human nuclear receptor coactivator-EcoR I fusion protein solution, a specific Taqman probe, an A-DTT buffer solution and a B buffer solution.
Furthermore, the recombinant human nuclear receptor ligand binding domain hNR-LBD protein is obtained by amplifying the coding sequence of the nuclear receptor ligand binding domain closely related to human health by using cDNA of a cancer cell strain related to human endocrine disturbance as a template, and further performing prokaryotic cell expression and purification.
Furthermore, the recombinant human nuclear receptor coactivator-EcoR I fusion protein is obtained by taking cDNA of cancer cells related to human endocrine disturbance as a template, amplifying the human nuclear receptor coactivator, and further carrying out coexpression and purification on prokaryotic cells and Escherichia coli restriction endonuclease EcoR I.
Furthermore, the cancer cells related to human endocrine disturbance are human tumor cells, including human colorectal adenocarcinoma cells Caco-2, human breast cancer cells MCF7, human cervical carcinoma cells HeLa and human liver cancer cells Hep G2, and the cancer cells can be obtained from national experimental cell resource sharing platform (http:// www.cellresource.cn/index. aspx).
Further, the nuclear receptors closely related to human health include human Estrogen Receptor (ER), estrogen-related receptor (ERR), Androgen Receptor (AR), Glucocorticoid Receptor (GR), Retinoic Acid Receptor (RAR), retinoic acid-Related Orphan Receptor (ROR), peroxisome proliferator-activated receptor (PPAR), Vitamin D Receptor (VDR), Liver X Receptor (LXR), and thyroid hormone receptor (TR) published by NCBI.
Still further, the ER includes ER α, ER β; the ERR comprises ERR alpha, ERR beta and ERR gamma; the GR comprises GR alpha, GR beta and GR gamma; the RAR comprises RAR alpha, RAR beta and RAR gamma; the ROR comprises ROR alpha, ROR beta and ROR gamma; the PPARs include PPAR α, PPAR β and PPAR γ; the LXRs comprise LXR alpha and LXR beta; the TR comprises TR alpha and TR beta.
Further, the human nuclear Receptor coactivators include Sterol Receptor Coactivators (SRC), Activated Signal Cointegrator 2(ASC-2), Peroxisome Proliferator-Activated Receptor gamma coactivators 1(Peroxisome Proliferator-Activated Receptor gamma Coactivator 1, PGC 1), and the like.
Still further, the SRC include 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 comprises PGC-1 alpha, PGC-1 beta, PRC and the like.
Furthermore, the 5 'end of the specific fluorescence labeled Taqman probe is labeled with a reporter group reporter (R), the 3' end is labeled with a fluorescence quenching group Quencher (Q), the sequence comprises an EcoR I enzyme cutting site, and the nucleic acid sequence is as follows: 5'R-acagtG AATTCtacgCCCCAAaaCgtaGAATTCactga-Q3'; still further, the reporter group R may be selected from FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), or VIC, and the quencher group Q may be selected from TAMRA (6-carboxytetramethylrhodamine), DABCYL (4- (4-oxaneaminophenylazo) benzoic acid), BHQ.
Further, when the kit is used for detecting the endocrine disrupting activity of chemicals, a microplate can be preferably used for realizing high-throughput determination; still further, the microwell plate includes 96-well plates and 384-well plates.
Further, the A-DTT buffer solution is used for washing the hole of the micropore plate before the human nuclear receptor coactivator-EcoR I fusion protein is added; and the buffer solution B is used for washing the hole of the micropore plate before the specific Taqman probe is added.
Another aspect of the present invention provides a method for rapidly detecting endocrine disrupting activity of a chemical, comprising:
(1) preparation of the above kit: prokaryotic cell expression, purification and protein solution preparation containing recombinant human nuclear receptor ligand binding domain hNR-LBD protein and recombinant human nuclear receptor coactivator-EcoR I fusion protein;
wherein, the recombinant human nuclear receptor ligand binding domain hNR-LBD protein is obtained by taking cDNA of cancer cells related to human endocrine disturbance as a template, amplifying coding sequences of nuclear receptor ligand binding domains closely related to human health, and further performing prokaryotic cell expression and purification; the human recombinant nuclear receptor coactivator-EcoR I fusion protein is obtained by taking cDNA of cancer cells related to human endocrine interference as a template, amplifying the human nuclear receptor coactivator, and further co-expressing and purifying prokaryotic cells and E.coli restriction enzyme EcoR I;
(2) hNR-adsorption of LBD protein: adding a pre-cooled hNR-LBD solution into each hole of the pre-cooled microporous plate, sealing each hole by using a sealing plate membrane, standing at a low temperature overnight, removing the sealing plate membrane, pouring out liquid in each hole, and washing each hole by using a pre-cooled A-DTT buffer solution;
(3) addition of recombinant human nuclear receptor coactivator-EcoR I fusion protein and test sample: pouring out the A-DTT buffer solution in the holes, respectively adding a precooled recombinant human nuclear receptor coactivator-EcoR I solution and a substance sample to be detected which is diluted in a DMSO gradient into each hole, standing at a low temperature, pouring out the liquid in the holes, and washing each hole by using a precooled B buffer solution;
(4) addition of specific Taqman probes and determination of fluorescent signals: adding specific Taqman probes into each hole of the microplate, standing at 37 ℃ for 5min, and detecting the fluorescence intensity by using a fluorescence spectrophotometer.
Further, the cancer cells related to endocrine disturbance in the step (1) comprise human tumor cells which can be obtained from national laboratory cell resource sharing platform (http:// www.cellresource.cn/index. aspx), preferably, the cancer cells related to endocrine disturbance are human colorectal adenocarcinoma cells Caco-2, human breast cancer cells MCF7, human cervical carcinoma cells HeLa, human liver cancer cells Hep G2 and the like, and the extraction of the total RNA of the human tumor cells can adopt Trizol reagent. Further, the nuclear receptors closely related to human health in step (1) include human Estrogen Receptor (ER), Estrogen Related Receptor (ERR), Androgen Receptor (AR), Glucocorticoid Receptor (GR), Retinoic Acid Receptor (RAR), retinoic acid related orphan nuclear receptor (ROR), peroxisome proliferator-activated receptor (PPAR), Vitamin D Receptor (VDR), Liver X Receptor (LXR), and thyroid hormone receptor (TR) published by NCBI.
Still further, the ER includes ER α, ER β; the ERR comprises ERR alpha, ERR beta and ERR gamma; the GR comprises GR alpha, GR beta and GR gamma; the RAR comprises RAR alpha, RAR beta and RAR gamma; the ROR comprises ROR alpha, ROR beta and ROR gamma; the PPARs include PPAR α, PPAR β and PPAR γ; the LXRs comprise LXR alpha and LXR beta; the TR comprises TR alpha and TR beta.
Further, the recombinant human nuclear receptor coactivators in the step (1) comprise Sterol Receptor Coactivators (SRC) published by NCBI, activated signaling coactivator 2(ASC-2), and peroxisome proliferator-activated receptor gamma coactivator 1(PGC 1).
Still further, the SRC include SRC-1/NcoA1, SRC-2/TIF-2/GRIP1/NcoA, SRC3/NCOA3/AIB, NCOA4, NCOA5, NCOA6/NRC/ASC-2/TRBP/PRIP/RAP250, CBP/p300, CARM1/PRMT 1; the PGC 1 comprises PGC-1 alpha, PGC-1 beta and PRC.
Specifically, prokaryotic cell expression and purification of the recombinant human nuclear receptor ligand binding domain hNR-LBD protein in the step (1): the sequence of the ligand binding domain of the polypeptide is obtained by designing and amplifying primers by referring to the human nuclear receptor sequence published by NCBI. Connecting the coding sequence of the amplified nuclear receptor ligand binding domain to a pGEX-4T-1 vector by adopting a double enzyme digestion method, transforming E.coli DH5 alpha competent cells, screening positive clone sequencing, and obtaining a recombinant expression vector pGEX-4T-hNR-LBD. Extracting recombinant plasmid, transforming E.coli BL21 competent cell to obtain hNR-LBD recombinant expression strain. The recombinant strain is subjected to amplification culture at 37 ℃, and IPTG is added for induction expression. Then, the recombinant protein was purified by ultrasonication and Sepharose 4B to obtain hNR-LBD protein. The expression and purification of the target protein were examined by SDS-PAGE electrophoresis, and the concentration of the purified protein was determined to analyze the enzymatic properties of recombinant hNR-LBD.
Specifically, prokaryotic cell expression and purification of the recombinant human nuclear receptor coactivator-EcoR I fusion protein in the step (1): the sequence of the human nuclear receptor coactivator disclosed by NCBI is obtained by designing and amplifying primers. Coli genome is extracted and amplified to obtain the coding sequence of E.coli EcoR I (GenBank: BAE78340.1) by using it as template. Connecting the human nuclear receptor coactivator and the EcoR I to a pET-28a vector by using a double-enzyme cutting method, transforming E.coli DH5 alpha competent cells, screening positive clone sequencing, and obtaining the human nuclear receptor coactivator and the EcoR I coexpression vector. Extracting recombinant plasmid, transforming E.coli strain ER2566(NEB #1312) competent cells to obtain the human nuclear receptor coactivator-EcoR I coexpression strain. The recombinant strain is subjected to amplification culture at 37 ℃, and IPTG is added for induction expression. Then purifying the recombinant protein by ultrasonic disruption and Ni-NAT affinity chromatography to obtain the human nuclear receptor coactivator-EcoR I fusion protein. The expression and purification conditions of the target protein are detected through SDS-PAGE electrophoresis, the concentration of the purified protein is determined, and the enzymatic properties of the recombinant human nuclear receptor coactivator-EcoR I fusion protein are analyzed.
Further, the microplate in the step (2) includes a 96-well plate and a 384-well plate. Preferably, the microplate in step (2) is a 384-well plate; the precooling method can be optionally placed on ice; the low temperature is 2-8 ℃; the washing times of the A-DTT buffer solution are at least 3.
Further, the substance to be detected in the step (3) includes alkylphenols, p-alkoxyphenols, parabens, bisphenols, biphenyls, diphenyl ethers, organophosphorus compounds and halogen compounds, and the concentration of the substance to be detected is 10-4M-10-11M。
Further, the microplate in the step (3) includes a 96-well plate and a 384-well plate. Preferably, the microplate in step (3) is a 384-well plate; the low-temperature standing temperature is 2-8 ℃, and the time is 60 min; the washing times of the buffer B are at least 3 times.
Preferably, the fluorescence intensity results obtained by the step (4) may be calculated as EC50 or IC50 using GraphPad Prism, and compared with a standard dose-response curve made by a standard substance to determine the receptor activity of the test substance.
Compared with the prior art, the invention has the beneficial effects that:
(1) high flux: taking a 384-well plate assay as an example, a high throughput assay of 20 chemical-specific receptor agonist or antagonist activities can be achieved at one time. The specific measurement is as follows: 20 chemicals (6 concentration x 3 replicates) + 360 wells +1 positive control (5 concentration x 3 replicates) + 21 wells +3 wells (negative control) +3 wells (blank control). In addition, if each sample concentration test is changed to 2 parallels, more than 30 chemical species can be tested. Thus, with the detection scheme provided by the present invention, the flux can be increased by 5 times compared to the prior art, from 4 substances to 20 substances tested at a time.
(2) Multiple targets: taking a 384-well plate test as an example, high-throughput determination of the activity of 6 nuclear receptor agonists or antagonists of the same chemical substance can be achieved at one time. The specific measurement is as follows: receptor agonist activity assay: 1 test substance x 6 concentration x 3 replicates/substance +1 positive control x 4 concentration x 2 replicates +3 wells (negative control) +3 wells (blank) 32 wells; receptor antagonist activity assay: 1 test substance x 6 concentration x 3 replicates/substance +1 positive control x 4 concentration x 2 replicates +3 wells (negative control) +3 wells (blank) 32 wells; a total of 64 wells is required for receptor agonist and antagonist activity assays for such a chemical; the 6 receptor synchronization tests only require: 6X 64-384 wells. Therefore, by using the detection scheme provided by the invention, the target can be improved by more than 6 times, and the target of 6 receptors can be completed by increasing the number of the targets from one test to one test aiming at only one receptor.
(3) And (3) fast: the recombinant protein obtained by the invention can be frozen and stored for a long time, and the analysis time can be shortened to 3 hours by taking a 384-well plate test as an example. The specific measurement is as follows: premix reagent and test substance were applied to 384 wells (1.5 hr) + warm bath reaction (1 hr) + machine reading (0.5 hr) ═ 3 hr. Therefore, by using the detection scheme provided by the invention, the speed can be improved by more than 4 times compared with the prior art, and the test can be completed in 3 hours instead of the current 12 hours.
Drawings
FIG. 1 shows hERR gamma receptor activity of various chemicals;
FIG. 2A is a graph showing ER α receptor activity for various concentrations of bisphenol A;
FIG. 2B is a graph showing ERR γ receptor activity for various concentrations of bisphenol A;
FIG. 2C shows AR receptor activity at various concentrations of bisphenol A.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1:
prokaryotic expression and purification of human estrogen related receptor gamma-ligand binding domain hERR gamma-LBD recombinant protein
Extracting the total RNA of human colorectal adenocarcinoma cells Caco-2 by using Trizol reagent, and inverting the total RNA into cDNA serving as a template. The sequence of the human estrogen related receptor ERR gamma (NCBI GenBank: NP-001230435.1) published by NCBI is obtained by designing and amplifying primers. A double-enzyme cutting method is utilized to connect the coding gene of hERR gamma-LBD (44..263aa) to a pGEX-4T-1 vector, transform E.coli DH5 alpha competent cells, screen positive clone sequencing and obtain a recombinant expression vector pGEX-4T-hERR gamma-LBD. Extracting recombinant plasmid, transforming E.coli BL21 competent cell to obtain hERR gamma-LBD recombinant expression strain. The recombinant strain is subjected to amplification culture at 37 ℃, and IPTG is added for induction expression. Then, the recombinant protein is purified by ultrasonic disruption and Sepharose 4B to obtain hERR gamma-LBD protein. Detecting the expression and purification condition of the target protein by SDS-PAGE electrophoresis, determining the concentration of the obtained protein to be 1mg/mL by concentration determination, and subpackaging at-80 ℃ for later use.
Second, prokaryotic expression and purification of human hTIF2-EcoR I recombinant protein
Total RNA of human colorectal adenocarcinoma cells Caco-2 was extracted with Trizol reagent, inverted to cDNA as a template, and its sequence was obtained by referring to human nuclear receptor co-activator hTIF2 published by NCBI (NCBI GenBank: NP-006531.1) and designed for primer amplification. Coli genome is extracted and amplified to obtain the coding sequence of E.coli EcoR I (GenBank: BAE78340.1) by using it as template. A double-enzyme cutting method is utilized to connect human nuclear receptor coactivator hTIF2 and EcoR I to a pET-28a vector, transform E.coli DH5 alpha competent cells, screen positive clone sequencing, and obtain a recombinant expression vector pET-28a-hTIF2-EcoR I. Extracting pET-28a-hTIF2-EcoR I recombinant plasmid, transforming E.coli strain ER2566(NEB #1312) competent cells, and obtaining hTIF2-EcoR I recombinant expression bacteria. The recombinant strain is subjected to amplification culture at 37 ℃, and IPTG is added for induction expression. Then purifying the recombinant protein by ultrasonic disruption and Ni-NAT affinity chromatography to obtain the hTIF2-EcoR I protein. Detecting the expression and purification condition of the target protein by SDS-PAGE electrophoresis, determining the concentration of the obtained protein to be 1mg/mL by concentration determination, and subpackaging at-80 ℃ for later use.
Three, quick determination of hERR gamma receptor activity of different chemicals
And (3) detecting the estrogen receptor activity of the chemicals by using the hERR gamma inverse agonist tamoxifen as a standard sample. The compounds tested included: bisphenol A (BPA), bisphenol E (BPE), 2-tert-butylphenol (2t-BP), 4-sec-butylphenol (s-BP), 4-tert-butylphenol (4t-BP), 4-tert-amylphenol (t-AP), 2-tert-butyl-5-methylphenol (2t-5-MP), 4-Cinnamylphenol (CP), 4-Butoxyphenol (BOP), 4-tert-butoxyphenol (t-BOP), 4-phenoxyphenol (PhoP), 3-chloro-4-methylphenol (CPMC), 4-chloro-3-methylphenol (PCMC), 4-chloro-3-ethylphenol (CLMEP), 4-chloro-3, 5-dimethylphenol (PCMX), 4-tert-butylchlorobenzene (t-Cl), 4-Ethylbromobenzene (EBB), 4-Propylbromobenzene (PBB), 4-tert-butylbromobenzene (t-Br), 4-tert-butyliodobenzene (t-I). The assay uses 384-well plates and the chemicals tested are diluted to 1X 10 in DMSO-4–1×10-11And M. More than or equal to 1.2mg of hERR gamma-LBD protein was dissolved in 40mL of precooled 0.1M NaHCO3(pH 8.4), the 384 well plates were placed on ice for precooling, and 100. mu.L of precooled 1 × hERR γ -LBD protein solution was added to each well. The wells were sealed with a sealing membrane and allowed to stand overnight at 4 ℃. The plate-sealing membrane was removed, the wells were decanted, and the wells were washed 3 times with 180. mu.L of pre-chilled A-DTT buffer (20mM Tris-HCl, 100mM KCl, 0.25mM EDTA, 5% Glycerol, 0.05% Tween 20, 0.5mM DTT, pH 7.4) to adsorb hERR γ -LBD receptor onto 384-well plates. More than or equal to 1.2mg of hTIF2-EcoR I fusion protein is dissolved in 40mL of precooled A-DTT buffer, 100. mu.L of 1 XHTIF 2-EcoR I protein solution and 1. mu.L of DMSO gradient diluted test sample are added to each well, and the mixture is placed for 60min at 4 ℃. The well was decanted and 180. mu.L of precooled B buffer (50mM Tris-HCl, 100mM KCl, 5mM MgCl)20.10% Nonidet P-40, pH 7.2) each well was washed 3 times. Then, 100. mu.L of 5nM Taqman probe (nucleic acid sequence FAM-acagtG AATTCtacgCCCCAAaaCgtaG AATTCactga-TAMRA) was added to each well, and the mixture was left at 37 ℃ for 5min and subjected to fluorescence spectrophotometryThe meter detects the fluorescence intensity. The results obtained were finally used to calculate EC50 using GraphPad Prism, comparing the hERR γ receptor activity of the different test substances. The results are shown in fig. 1, indicating that these compounds all have strong hERR γ activity.
Rapid determination of activity of different nuclear receptors of tetra-bisphenol A
The test uses 384-well plates and the substance tested is bisphenol A (BPA) diluted to 1X 10 with DMSO-4–1×10-10And M. The nuclear receptor activities tested included ER α, ERR γ, AR, LXR α, RAR α, ROR α, with the receptor activity test being performed with its natural ligand as a control. Respectively dissolving hNR-LBD purified protein of more than or equal to 1.2mg in 40mL precooled 0.1M NaHCO3(pH 8.4), the 384-well plate was placed on ice for precooling, and 100. mu.L of precooled 1X hNR-LBD solution was added to each well. The wells were sealed with a sealing membrane and allowed to stand overnight at 4 ℃. The plate-sealing membrane was removed, the wells were decanted, and the wells were washed 3 times with 180. mu.L of pre-chilled A-DTT buffer (20mM Tris-HCl, 100mM KCl, 0.25mM EDTA, 5% Glycerol, 0.05% Tween 20, 0.5mM DTT, pH 7.4) to allow hNR-LBD receptor protein to be adsorbed onto 384-well plates. More than or equal to 1.2mg of hTIF2-EcoR I protein was dissolved in 40mL of pre-cooled A-DTT buffer, 100. mu.L of 1 XHTIF 2-EcoR I protein solution, 1. mu.L of DMSO-diluted test sample or natural ligand sample was added to each well, and left at 4 ℃ for 60 min. The well was decanted and 180. mu.L of precooled B buffer (50mM Tris-HCl, 100mM KCl, 5mM MgCl)20.10% Nonidet P-40, pH 7.2) each well was washed 3 times. Then, 100. mu.L of 5nM Taqman probe was added to each well, and the mixture was left at 37 ℃ for 5min, and the fluorescence intensity was measured with a fluorescence spectrophotometer. The results were finally calculated as EC50 using GraphPad Prism, comparing the receptor activity of different concentrations of bisphenol A. FIGS. 2A and 2B show that bisphenol A has ER α and ERR γ activity, respectively, and is an agonist of ER α and ERR γ; figure 2C shows that bisphenol a has anti-androgenic activity and is an antagonist of AR.
Claims (10)
1. A kit for rapidly detecting the endocrine disrupting activity of chemicals is characterized by comprising a recombinant human nuclear receptor ligand binding domain hNR-LBD protein solution, a recombinant human nuclear receptor coactivator-EcoR I fusion protein solution, a specific fluorescence labeled Taqman probe, an A-DTT buffer solution and a B buffer solution.
2. The kit for rapidly detecting the endocrine disrupting activity of chemicals according to claim 1, wherein the recombinant human nuclear receptor ligand binding domain hNR-LBD protein is obtained by amplifying the coding sequence of the nuclear receptor ligand binding domain closely related to human health by using cDNA of a cancer cell line related to human endocrine disruption as a template, and further performing prokaryotic cell expression and purification.
3. The kit for rapidly detecting the endocrine disrupting activity of chemicals according to claim 1, wherein the recombinant human nuclear receptor coactivator-EcoR I fusion protein is obtained by amplifying the human nuclear receptor coactivator using cDNA of cancer cells associated with human endocrine disruption as a template, and further co-expressing and purifying the human nuclear receptor coactivator with Escherichia coli restriction endonuclease EcoR I through prokaryotic cells.
4. The kit for rapidly detecting the endocrine disrupting activity of chemicals according to claim 2 or 3, wherein the cancer cells related to human endocrine disruption are human-derived tumor cells, including human colorectal adenocarcinoma cells Caco-2, human breast cancer cells MCF7, human cervical cancer cells HeLa, and human liver cancer cells Hep G2.
5. The kit for rapidly detecting the endocrine disrupting activity of a chemical according to claim 2, wherein the nuclear receptors closely related to human health comprise human estrogen receptors, estrogen-related receptors, androgen receptors, glucocorticoid receptors, retinoic acid-related orphan receptors, peroxisome proliferator-activated receptors, vitamin D receptors, liver X receptors, and thyroid hormone receptors.
6. The kit for rapidly detecting the endocrine disrupting activity of chemicals according to claim 3, wherein the human nuclear receptor coactivators comprise sterol receptor coactivators, activated signal coactivators 2, peroxisome proliferator-activated receptor gamma coactivators 1.
7. The kit for rapidly detecting the endocrine disrupting activity of chemicals according to claim 1, wherein the nucleotide sequence of the specifically fluorescently-labeled Taqman probe is 5'R-acagtG AATTCtacgCCCCAAaaCgtaG AATTCactga-Q3', wherein R is a reporter group comprising FAM, TET, JOE, HEX, VIC; q is a quenching group, including TAMRA, DABCYL, BHQ; the nucleotide sequence of the specific fluorescence labeled Taqman probe contains an EcoR I enzyme cutting site.
8. The kit for rapidly detecting the endocrine disrupting activity of a chemical according to claim 1, wherein a microplate is used for high-throughput assay when the kit is used for detecting the endocrine disrupting activity of the chemical; the A-DTT buffer solution is used for washing a micropore plate hole before the recombinant human nuclear receptor coactivator-EcoR I fusion protein is added; and the buffer solution B is used for washing the hole of the micropore plate before the specific Taqman probe is added.
9. A method for rapidly detecting chemical endocrine disrupting activity using the kit of claim 1, comprising:
(1) hNR-adsorption of LBD protein: adding the hNR-LBD solution which is precooled in the kit of claim 1 into each well of the precooled microplate, sealing each well by a sealing plate membrane, standing overnight at low temperature, removing the sealing plate membrane, pouring off liquid in each well, and washing each well by the A-DTT buffer solution which is precooled in the kit;
(2) addition of recombinant human nuclear receptor coactivator-EcoR I fusion protein and test sample: pouring out an A-DTT buffer solution in each hole, respectively adding a pre-cooled recombinant human nuclear receptor coactivator-EcoR I fusion protein solution and a substance sample to be detected diluted in DMSO gradient in the kit into each hole, standing at a low temperature, pouring out liquid in each hole, and washing each hole by using a pre-cooled B buffer solution in the kit;
(3) addition of specific Taqman probes and determination of fluorescent signals: and respectively adding the specific Taqman probe in the kit into each hole of the microporous plate, placing for 5min at 37 ℃, and detecting the fluorescence intensity by using a fluorescence spectrophotometer.
10. The method according to claim 9, wherein the substance to be detected in step (2) comprises alkylphenols, p-alkoxyphenols, parabens, bisphenols, biphenyls, diphenyl ethers, organophosphates, and halogens, and the concentration of the substance to be detected is 10-4M-10-11M。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN112126680B (en) * | 2020-09-27 | 2022-10-14 | 北京大学 | Kit and method for screening trace endocrine disruptors based on fluorescent quantitative PCR |
| CN112420122B (en) * | 2020-11-04 | 2021-06-22 | 南京大学 | Method for identifying allosteric site of action of endocrine disruptor and nuclear receptor |
| CN113092782A (en) * | 2021-03-31 | 2021-07-09 | 北京大学 | Method for screening chemical nuclear receptor activity based on Alpha technology high-throughput and multi-target |
| CN114703227B (en) * | 2022-01-27 | 2023-11-10 | 中国科学院生态环境研究中心 | RAR alpha effect in vitro screening method constructed based on MCF-7 cell line |
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| JP2009124990A (en) * | 2007-11-22 | 2009-06-11 | Tokyoto Igaku Kenkyu Kiko | Method for determining antigen |
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