A recombinant vector and method for determining the activity of dioxins based on mouse aryl hydrocarbon receptor

Abstract

The present invention discloses a recombinant vector and application method for determining the activity of dioxin-like substances based on the mouse aromatic hydrocarbon receptor, and relates to a genetic engineering. This study mainly selects and intercepts the efficient mouse aromatic hydrocarbon receptor AHR expression sequence through molecular cell biology means, amplifies and subclones it into the pBIND expression vector dedicated to mammalian single hybridization; after successful sequencing verification, the pBIND expression vector and the mammalian single hybridization reporter gene vector pG5luc are co-transfected, and then dioxin-like substances are added to carry out compound activity determination, as well as sensitivity indicators related to the mouse AHR receptor. The present invention is based on the systematic optimization of the mammalian single hybridization reporter gene technology, establishes a toxicological test method, solves the problem of efficient cross-species toxicity assessment, and provides an effective tool for studying the ecotoxicity risk of new dioxin-like pollutants by enhancing the detection sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant vector and method for determining the activity of dioxin substances based on mouse aromatic hydrocarbon receptor. Background Art

[0002] Dioxins are persistent organic pollutants (POPs) that are widely present in the environment and are extremely toxic. They are unintentionally produced during waste incineration and industrial production. They are semi-volatile, have long-range migration, cold trapping effects, and bioaccumulation / amplification. Their main site of action in cells is the aryl hydrocarbon receptor (AHR). By activating AHR, they produce a series of toxic effects, which can pose a serious threat to the ecological environment and human health. They are one of the first 12 persistent organic pollutants restricted for use in the Stockholm Convention. The new similar pollutants that are being discovered (for example, halogenated carbazoles PHCZs, polychlorinated diphenyl sulfides PCDPSs, polychlorinated naphthalenes PCNs, etc.) have similar properties to dioxins in terms of molecular structure and biological effects. Among them, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetrachlorodibenzo- p TCDD) is the most toxic monomer among dioxins and has been identified as a Class I human carcinogen by the International Agency for Research on Cancer (IRAC) under the World Health Organization. TCDD is a substance with a strong ability to bind to AHR among such pollutants and is often used as a standard to verify the sensitivity of the dioxin measurement system.

[0003] The molecular initiation event of AHR transcriptional activation is the first event in the toxic effects of dioxins and their analogs, which briefly includes: 1) ligands bind to AHR in the cytoplasm and translocate to the nucleus; 2) AHR binds to the chaperone protein ARNT and is recruited to the dioxin receptor AHR response element DRE; 3) cooperates with the basic transcription machinery to start the transcription of regulated genes (such as CYP1A1, CYP1B1). Based on this mechanism, many quantitative methods for evaluating AHR activity, ligand screening and environmental samples have been developed. Among them, the AHR reporter gene method has also become an important tool for toxicity assessment of new dioxins and similar pollutants. However, due to the different sensitivities of the aromatic hydrocarbon receptor AHR, which mainly mediates dioxin toxicity in different organisms, the risks caused by exposure to dioxins in different organisms are significantly different, and the activation efficiency of different dioxin monomers on AHR in different species is also significantly different, so targeted research is needed.

[0004] Currently, the technical route of the typical reporter gene method for evaluating the activity of AHR in different species is: based on the in vitro transient expression of COS-7 cells with expression vectors that express AHR subtypes of different species, and simultaneously transfer into a reporter gene vector containing the dioxin receptor AHR response element DRE sequence, followed by adding the test compound and detecting the activity / sensitivity of the AHR through the reporter gene method, as well as the compound's ability to activate specific AHR subtypes.

[0005] However, due to the errors in transient transfection efficiency, the difficulty in quality control of the transferred plasmids, the weak sensitivity, and the interference of a small amount of endogenous monkey AHR in COS-7, the existing technology is not sufficient to efficiently carry out AHR activity assessment in multiple species. Therefore, a more sensitive and accurate method is needed to study the sensitivity of AHR subtypes of different species to dioxins and analogs.

[0006] In addition to the above methods, a derived mammalian single-hybrid reporter gene system based on Gal4-UAS is an alternative new method for detecting the activity of transcription factors and nuclear receptors. By fusing the LBD-TAD region (ligand binding region-transcription activation region) of the nuclear receptor in series to the rear of the Gal4 domain in the expression vector, the fusion protein after transfection and expression will be able to bind to the upstream activation sequence Gal4-UAS region of the reporter gene vector, and the expression of the reporter gene will be enhanced through the ligand-dependent transcriptional activation ability of the nuclear receptor's own TAD. The level of nuclear receptor activation and the activation efficacy of the ligand can be reflected by analyzing the strength of the reporter gene activity. Summary of the invention

[0007] Dioxins and their analogs produce a series of toxic effects by activating aromatic hydrocarbon receptors in cells. Since the sensitivity of AHR, the main mediator of dioxin toxicity, is different in different organisms, the risks caused by exposure to dioxins in different organisms are significantly different, and targeted research is needed.

[0008] At present, in the analysis of the sensitivity of AHR subtypes of different species to dioxins and analogs, the method based on transient transfection of COS-7 cells in vitro has the following shortcomings: transient transfection efficiency error, difficult quality control of the transferred plasmids, weak sensitivity, and interference from a small amount of endogenous monkey AHR in COS-7 cells. Therefore, this method is mostly used in the study of the strong AHR ligand TCDD in the study of AHR function identification, and subsequent studies on dioxin analogs with weaker AHR activation ability found that this method performed poorly. However, in most cases, the activity of new dioxin analogs is weak, and this method is difficult to effectively carry out the analysis of AHR activation ability in multiple species due to its own characteristics.

[0009] Therefore, the present invention provides a recombinant vector for determining the activity of dioxin substances based on the mouse aromatic hydrocarbon receptor, and uses a mammalian single hybrid reporter gene system based on Gal4-UAS to determine the activity of rodent-mouse AHR and evaluate the activation ability of dioxin substances, i.e., toxicity. The system allows the yeast Gal4 domain to express fusion with the mouse AHR sequence of different segments that are preferably intercepted, so that the fusion-expressed Gal4-AHR can bind to the artificial binding region Gal4-UAS and trigger the expression of downstream reporter genes under the stimulation of external dioxin substances, avoiding the potential endogenous interference effect caused by the simultaneous binding of endogenous AHR to the dioxin response element DRE in the traditional reporter gene method, and can be used to determine the activity of rodent-mouse AHR in mammals, and can be used to analyze and determine the potential activation ability (i.e., toxicity) of dioxins and dioxin analogs. The present invention provides two highly efficient recombinant vectors and related assay methods for dioxin-like substance activity assay based on mouse AHR by optimizing and intercepting highly efficient AHR receptor sequence regions. Compared with the prior art, the present method eliminates the interference of endogenous AHR; reduces the background value of nonspecific reporter genes; transfers fewer plasmids to reduce transient transfection errors, and systematically optimizes relevant technical parameters to improve the sensitivity and stability of the system. A toxicological test method is established to solve the problem of highly efficient toxicity assessment across species, and to provide an effective tool for studying the ecotoxicity risks of new dioxin-like pollutants.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0011] A recombinant vector for determining the activity of dioxins based on the mouse aryl hydrocarbon receptor, wherein the nucleotide sequence thereof is selected from SEQ ID NO. 1 to SEQ ID NO. 4.

[0012] A recombinant vector for determining the dioxin activity of the mouse aryl hydrocarbon receptor, wherein the primer pair of the nucleotide sequence is selected from one of SEQ ID NO. 5-SEQ ID NO. 8 and SEQ ID NO. 9.

[0013] A detection reagent or kit contains a recombinant vector.

[0014] A recombinant vector / detection reagent or kit is used in the sensitivity determination of mouse AHR receptor and the evaluation of the activation ability / toxicity of dioxin-like substances.

[0015] Beneficial Effects

[0016] The present invention provides a recombinant vector for determining the activity of dioxin-like substances based on the mouse aromatic hydrocarbon receptor by designing and optimizing the mouse AHR gene segment, so as to reduce the non-specific reporter gene background value and eliminate the interference of the endogenous AHR of the detection cell line used. The present invention conducts systematic optimization based on the mammalian single-hybrid reporter gene technology, establishes a dioxin toxicology test method, solves the problem of efficient cross-species toxicity assessment, and provides an effective tool for studying the ecotoxicity risk of new dioxin-like pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the response diagram of the recombinant vector to TCDD;

[0018] Figure 2 This is the dose-effect curve of the recombinant vector. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments.

[0020] Example 1

[0021] Taking the construction of the recombinant plasmid pBIND-mAHR-2 with the best sensitivity as an example (the construction steps of pBIND-mAHR-1, pBIND-mAHR-3, and pBIND-mAHR-4 are the same), the details are as follows:

[0022] Step 1: Optimization of mouse AHR sequence segment and primer design

[0023] 1) Based on the sequence information of mouse AHR, the locations of DNA Binding Domain (DBD) and Ligand Binding Domain (LBD) were predicted, and a pair of primers mAHR-2F and mAHR-R were designed.

[0024] mAHR-2F:5'-CGTCGACTTGACGCGTTGATAGGGAGGTTAAAGTATCTTCATGGACAGAA-3'

[0025] mAHR-R:5'-CTGCGGCCGCTCTAGATGATTCAACTCTGCACCTTGCTTAGGAATGCC-3'

[0026] The sequence was amplified by PCR using mAHR-2F and mAHR-R as primers and the pcDNA3.1(+) expression plasmid solution of mouse AHR as a template. This step used PrimeSTAR high-fidelity PCR enzyme.

[0027] Add ddH2O in a 1.5 ml centrifuge tube.2 O 10μl, mAHR-2F primer 5μl, mAHR-R primer 5μl, pcDNA3.1(+)-mAHR bacterial solution 5μl, PrimeSTAR high-fidelity PCR enzyme 25μl, total volume 50μl.

[0028] PCR reaction parameter settings:

[0029]

[0030] After the reaction, 50 μl of PCR product was identified by 1% agarose gel electrophoresis. A single fragment of about 1700 bp that met the expected size appeared on the electrophoresis pattern. The amplified product was recovered using a gel recovery kit. At the same time, the empty plasmid vector pBIND was digested with EcoRV and recovered by gel. Then, the target sequence and the vector fragment were connected at 50°C for 20 minutes using the ligase of the homologous recombination ligation kit to obtain the pBIND-mAHR-2 ligation product.

[0031] 2) Add 50 μl of competent cells to the centrifuge tube containing 10 μl of ligation product, mix well, and incubate on ice for 30 min.

[0032] 3) Heat shock at 42℃ for 60s, then ice bath for 2min, add 350μl fresh LB medium (without ampicillin), shake at 37℃ at 200rpm for 60min, centrifuge, discard supernatant, add 100μl fresh LB medium (with ampicillin), take 50μl to plate (with Amp 100μg / ml), and culture at 37℃ overnight.

[0033] Pick the pBIND-mAHR-2 single clone and perform colony PCR verification. Specifically, add ddH 2 O 4μl, mAHR-2F primer 2μl, pBIND-R primer 2μl, pBIND-mAHR-2 bacterial solution 2μl, PrimeSTAR high-fidelity PCR enzyme 10μl, total volume 20μl.

[0034] The successfully verified pBIND-mAHR-2 clone was sent to Sangon Biotechnology Co., Ltd. for sequencing using universal primers to further verify the accuracy of the obtained plasmid sequence.

[0035] The methods for the other vectors pBIND-mAHR-1, pBIND-mAHR-3, and pBIND-mAHR-4 are the same as above, and the insert sequences of the four recombinant vectors obtained are SEQ ID NO. 1 to SEQ ID NO. 4.

[0036] Example 2 Transfection of recombinant plasmid

[0037] First, the modified recombinant plasmid was subjected to a preliminary screening experiment.

[0038] 1. 36 hours before transfection, HEK-293T cells with good growth (density above 70%) were digested and counted, diluted and inoculated into 96-well plates at 40,000 cells / well, so that the density on the day of transfection was above 90%. Cells were plated in complete medium containing serum and antibiotics for normal growth.

[0039] 2. Before transfection, discard the original cell culture medium, dilute 100ng DNA (40ng PG5luc (reporter vector pG5luc containing Gal4-UAS) + 60ng pBIND-mAHR-2) with Opti-MEM, and mix with lipo8000 transfection reagent to prepare a transfection mixture, and add the transfection mixture to the culture well.

[0040] At 37°C, 5% CO 2 After 10 h of transfection under the conditions of Opti-MEM, the medium was replaced with a normal growth medium containing serum and antibiotics. Then, the drug exposure was performed. The control group used 1% DMSO, and the experimental group used 1×10 -7 MTCDD.

[0041] 3. At 37°C, 5% CO 2 36 hours after cell transfection, the cell extracts were analyzed and the luciferase reporter gene activity was detected using the Reporter Assay System.

[0042] The results of the analysis and testing showed that each recombinant plasmid had a single dose of 1×10 -7 The optimal recombinant vector was obtained by measuring the response value under M TCDD. Figure 1 Compared with other recombinant plasmids, the modified pBIND-mAHR-2 recombinant plasmid showed the highest sensitivity to TCDD, and the pBIND-mAHR-1 recombinant plasmid also showed high sensitivity to TCDD.

[0043] Example 3

[0044] The recombinant vector was exposed to a series of TCDD concentration gradients to test the dose-effect curve of the recombinant vector performance.

[0045] 1. 36 hours before transfection, HEK-293T cells with good growth (density above 70%) were digested and counted, diluted and inoculated into 96-well plates at 40,000 cells / well, so that the density on the day of transfection was above 90%. Cells were plated in complete medium containing serum and antibiotics for normal growth.

[0046] 2. Before transfection, discard the original cell culture medium, dilute 100ng DNA (40ng PG5luc (reporter vector pG5luc containing Gal4-UAS) + 60ng pBIND-mAHR-2) with Opti-MEM, and mix with lipo8000 transfection reagent to prepare a transfection mixture, and add the transfection mixture to the culture well.

[0047] At 37°C, 5% CO 2 After 12 hours of transfection under the conditions of Opti-MEM, the culture medium was replaced with a normal growth medium containing serum and antibiotics. The control group used 1% DMSO, and the experimental groups used 1.69×10 -12 , 5.08×10 -12 , 1.52×10 -11 , 4.57×10 -11 , 1.37×10 -10 , 4.12×10 -10 , 1.23×10 -9 , 3.70×10 -9 , 1.11×10 -8 , 3.33×10 -8 , 1×10 -7 MTCDD.

[0048] 3. At 37°C, 5% CO 2 After TCDD was added to the cells for 24 hours, the cell extracts were analyzed and the luciferase reporter gene activity was detected using the Reporter Assay System.

[0049] The dose-effect curve of each recombinant vector under exposure to a series of TCDD concentration gradients was analyzed and tested. The results are as follows Figure 2 The modified pBIND-mAHR-1, pBIND-mAHR-2, pBIND-mAHR-3, and pBIND-mAHR-4 all responded to the strong AHR activator TCDD, and the responses of pBIND-mAHR-1 and pBIND-mAHR-2 recombinant plasmids were the most obvious. With the increase of TCDD concentration, the response values ​​of cell reporter genes containing pBIND-mAHR-1 and pBIND-mAHR-2 recombinant plasmids showed obvious trend changes, with a positive correlation. Among them, the pBIND-mAHR-2 recombinant plasmid had the highest effect multiple compared with other recombinant plasmids, with the highest multiple (compared with the solvent control DMSO) reaching more than 30 times, and the EC50 was 2.355×10 -8; and pBIND-mAHR-1 also increased by about 20 times, with an EC50 of 4.655×10 -8 Therefore, the present invention has tested and selected pBIND-mAHR-1 and pBIND-mAHR-2 as efficient recombinant vectors, and can be used for the sensitivity determination of mouse AHR receptors and the evaluation of the activation ability / toxicity of dioxins.

[0050] In summary, the present invention designed to intercept different segments of mouse AHR sequences and subclone them into pBIND single-hybrid vectors, and constructed a series of recombinant vectors for dioxin activity assay based on mouse aryl hydrocarbon receptor: pBIND-mAHR-1, pBIND-mAHR-2, pBIND-mAHR-3, and pBIND-mAHR-4. Among them, pBIND-mAHR-1 and pBIND-mAHR-2 were selected as efficient recombinant vectors through testing, and their highest multiples (compared with solvent control DMSO) reached 20 times and 30 times, respectively, and the EC50 was 4.655×10 -8 , 2.355×10 -8 , which is convenient for efficient evaluation of the toxic effects of dioxins and new analogs, and can also reflect the sensitivity of mice to specific compounds. The present invention can stably transfer Gal4-UAS reporter gene plasmids and screen high-performance cell lines, provide two recombinant vectors for the activity determination of dioxin-like substances based on mouse aromatic hydrocarbon receptors, systematically optimize the mammalian single-hybrid reporter gene technology, establish a toxicology test method, solve the problem of efficient cross-species toxicity evaluation, and provide an effective tool for studying the ecotoxicity risk of new dioxin-like pollutants.

Claims

1. A recombinant vector for determining the activity of dioxins based on the mouse aryl hydrocarbon receptor, wherein the insertion sequence of the recombinant vector is SEQ ID NO. 1 or SEQ ID NO.

2.

2. A detection reagent or kit comprising the recombinant vector according to claim 1.

3. Use of the recombinant vector according to claim 1 and the detection reagent or kit according to claim 2 in the sensitivity determination of mouse AHR receptors and the evaluation of the activation ability / toxicity of dioxins.

4. The use according to claim 3, characterized in that: The sensitivity test method is: (1) Before transfection, HEK-293T cells with good growth conditions were digested and counted, diluted and seeded into 96-well plates at a concentration of 40,000 cells / well so that the density on the day of transfection was above 90%; the cells were plated in normal growth complete medium containing serum and antibiotics; (2) Before transfection, the original cell culture medium was discarded, 100 ng DNA was diluted with Opti-MEM, and mixed with lipo8000 transfection reagent to prepare a transfection mixture, and the transfection mixture was added to the culture well; the DNA included 40 ng PG5luc and 60 ng of the recombinant vector described in claim 1, and PG5luc contained a Gal4-UAS reporter vector; Transfection was carried out for 10 h at 37°C and 5% CO2, and Opti-MEM was replaced with a normal growth complete medium containing serum and antibiotics. Drug exposure was then performed, with 1% DMSO used in the control group and 1×10-7M TCDD used in the experimental group. (3) Culture the cells at 37°C, 5% CO2 for 26 h. Analyze the cell extracts 36 h after cell transfection and detect the luciferase reporter gene activity using the Reporter Assay System.

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