CN114703227B

CN114703227B - RAR alpha effect in vitro screening method constructed based on MCF-7 cell line

Info

Publication number: CN114703227B
Application number: CN202210133063.9A
Authority: CN
Inventors: 周群芳; 徐汉卿; 杨晓溪; 刘倩; 苏佳惠; 江桂斌
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2023-11-10
Anticipated expiration: 2042-01-27
Also published as: CN114703227A

Abstract

The application relates to an RAR alpha effector in-vitro screening method constructed based on an MCF-7 cell line, which comprises the following steps: step1, construction of RARα expression vector; step2, cell transfection; step3, compound exposure and reporter gene detection. The application uses the isolated human cells as host cells, and can reflect the potential endocrine disrupting effect of exogenous chemicals on human health to the greatest extent. Furthermore, the method of the application has higher sensitivity and EC of the agonist AM580 ₅₀ A value of 0.87nM and a minimum observable effect concentration (LOEC) of 0.1nM; IC for antagonist Ro41-5253 ₅₀ The value was 2.67. Mu.M and the LOEC value was 0.625. Mu.M.

Description

RAR alpha effect in vitro screening method constructed based on MCF-7 cell line

Technical Field

The application belongs to the technical field of in-vitro screening and detection of environmental Endocrine Disruptors (EDCs), and particularly relates to a retinoic acid receptor alpha (RARalpha) transcriptional activation test method.

Background

Endocrine disruptors (Endocrine disrupting compounds, EDCs) refer to a class of compounds that are capable of interfering with hormonal balance in an organism. Since most EDCs have structural similarity to endogenous hormonal compounds, they exhibit corresponding functions, which in turn unbalance the natural hormone levels in the body. After the organism ingests the compounds, the normal functions of the endocrine system can be interfered, and the endocrine system is promoted or inhibited, so that the occurrence and development of diseases such as nerves, reproduction, immunity and the like are caused. With the continuous production and release of more and more novel chemicals, more than 10 tens of thousands of chemicals have been frequently detected in our living environment, involving industries such as industry, agriculture, food, and commodity. While more than 1,000 EDCs are reported at present, which are only the iceberg corners of known chemicals, more unknown environmental pollutants still need to be further screened and identified for biological effects.

In view of the potential harm of EDCs to human health, screening detection of endocrine disrupting effects of compounds and their mechanism of action are particularly important for the health risk assessment of compounds. Compared with the in-vitro screening method, the in-vitro screening method has more advantages, including high response sensitivity, high detection speed, good economic benefit, suitability for high-throughput detection and the like. Because the way by which EDCs interfere with the endocrine system of the body is mediated primarily through nuclear receptors, in ex vivo screening, key nuclear receptors are also used as independent targets for screening of the corresponding ligands. The existing more effective in vitro screening methods which directly affect the function of nuclear receptors mainly comprise a transcriptional activation test and a competitive binding test based on Estrogen Receptors (ER), a competitive binding test based on Yu Xiong hormone receptors (AR), and the like. In addition, there are some EDCs in which hormone synthesis is effected to interfere with the endocrine system, and in vitro screening methods for such compounds mainly include steroid synthesis assays, aromatase synthesis assays, and the like.

Several bioassays have been developed to screen potential ligands for Retinoic Acid Receptors (RARs) to find more contaminants with endocrine disrupting effects and to assess health risks associated with environmental pollution. The in vitro screening method of the EDCs based on RARs mainly comprises a yeast two-hybrid test and a Luc reporter gene test.

Recombinant yeast models constructed by using reporter genes, human RARs genes and coactivator TIF2 are one popular method of yeast two-hybrid detection. This method has been successfully applied to various studies including screening for RARs agonistic activity of various chemicals, and identifying pollution of EDCs with retinoic acid receptor alpha (Retinoic acid receptor alpha, rarα) agonism or antagonism in wastewater and urban rivers. The plasmid containing human retinoic acid receptor gamma (RARgamma) gene and the plasmid containing coactivator TIF2 are transferred into yeast cells to obtain recombinant yeast, and compound screening is carried out by utilizing the yeast cells, so that various compounds such as organochlorine pesticide, alkylphenol and the like are found to have certain RARgamma activating effect. In addition to the use of yeast cells to construct screening systems, human and mammalian ex vivo cells are also commonly used to construct screening systems. For example, recombinant cell lines containing reporter genes and different subtype RARs genes constructed with human Hela cells are used for screening organochlorine pesticides to distinguish compounds for agonism or antagonism of RARα, RARβ and RARγ.

The development of in vitro screening methods for RARs is still in the beginning and more recent methods are being developed to evaluate the endocrine disrupting effects of compounds modulating retinoic acid receptor production. Among them, RAR alpha subtype is important in physiological and pathological processes such as reproduction, homeostasis, synaptic plasticity, tumorigenesis, etc. of vertebrates, making it more likely to be a target of EDCs. Thus, there is a need to establish a more complete EDCs in vitro screening system for rarα subtypes.

The inventors found in the study that: the existing RARα -mediated in-vitro screening system adopts a yeast two-hybrid system, and although the method provides a certain support for the RARα -targeted EDCs screening, certain limitations still exist: 1. it is known that lower yeast cells lack more cofactors necessary for transcription, whereas the actual cellular environment has repressors, so that the transfer of only one TIF2 coactivator does not truly reflect the transcriptional activation or inhibition of rarα by the compound, possibly resulting in inconsistent results with human health effects; 2. the yeast cells are passaged less frequently than mammalian cells, and are not able to maintain cell viability and are not suitable for automated high throughput screening. Thus, there remains a need to construct rarα -mediated screening systems on the basis of more complex mammalian cells to more accurately assess the potential endocrine disrupting effects of contaminants on humans.

Disclosure of Invention

In order to solve the problems, the application provides an in-vitro screening method for RARα effect, which is constructed by taking a human breast cancer cell line MCF-7 as a host and transiently transfecting a Luc reporter gene vector and a human RARα expression vector, and aims to more truly reflect the agonism/antagonism of a compound on the generation of RARα, thereby playing a role in accurately evaluating the safe use of chemicals. The application is realized by the following technical scheme:

an RARα effector in vitro screening method constructed based on an MCF-7 cell line comprises the following steps:

step1, construction of RARα expression vector

1) The transcript sequence NM_000964.4 of the human RARα receptor is synthesized by genes, and both ends of the transcript sequence NM_000964.4 contain cleavage sites of NheI and EcoRI;

2) The commercial vector pEF1α -IRES-TagRFP and RARα synthetic fragment are digested by NheI and EcoRI, respectively, so as to obtain a new vector skeleton fragment and RARα fragment with sticky ends;

3) Recombining the two fragments by using T4 DNA ligase to form an RARalpha expression vector pEF1alpha-RARalpha-RFP, and converting the RAF1alpha-RARalpha-RFP into an E.coli DH5 alpha strain for plasmid amplification;

4) Carrying out large-scale extraction of endotoxin removal on the RARα expression vector pEF1α -RARα -RFP to ensure that the final concentration of the plasmid is more than 500 ng/. Mu.L;

step2, cell transfection

1) Resuscitating MCF-7 cells at 37deg.C with 5% CO ₂ Subculturing a cell culture box, wherein the culture medium is a complete culture medium;

2) A seed plate; after stable cell passage, MCF-7 cells were seeded into the tape Kong Baiban at a cell density of 4X 10 ⁴ Cells/well and placed at 37℃in 5% CO ₂ Allowing the cells to grow on the wall of the cell incubator overnight;

3) Transfection; when the cell confluency is 80%, starting to perform cell transfection, wherein the transfection method is a cationic lipid exchange method;

step3, compound exposure and reporter gene detection

1) Exposing the compound; after the transfection process is finished, exposing the cells with compounds to be tested with different concentrations and no obvious cytotoxicity for 24 hours, wherein the exposure culture medium is phenol red-free DMEM supplemented with 1% CS-FBS and 1% diabody;

2) Firefly luciferase (Luc) activity assay; the Luc activity of the compounds after 24h exposure was determined using a detection kit by the monoluciferase assay (mono-Luc assay).

Further, in Step2, the complete medium means: 10% Fetal Bovine Serum (FBS) and 1% diabody were added to DMEM high-glucose basal medium.

Further, in Step2, the plates were seeded with MCF-7 cells in 96 Kong Baiban, transparent at the bottom, at a cell density of 4X 10 ⁴ Cells/well cells were cultured in transfection medium in the presence of phenol red free DMEM supplemented with 10% char-threaded FBS (CS-FBS) and 1% diabody.

Further, in Step 1) of Step3, in order to detect the agonistic activity of the compound, the transfected cells were directly exposed to test compounds of different concentrations, wherein the DMSO content in the test compounds was less than or equal to 0.1%, and 0.1% DMSO was used as a control group.

Further, in Step3, step 1), 25nM of AM580 was added to the compounds at different concentrations in order to examine antagonistic activity of the compounds, wherein the total amount of DMSO contained in the test compound and AM580 was 0.1% or less, and the AM580 group containing only 25nM was used as a control group.

Further, the diabody refers to: penicillin at a concentration of 100U/mL and streptomycin at a concentration of 100. Mu.g/mL.

Further, if the test compound exhibits rarα agonistic or antagonistic activity, the test compound is judged to be a potential endocrine disruptor.

Further, if a significant increase in firefly luciferase (Luc) relative activity (fold change) occurs, the test compound is judged to have rarα agonistic activity.

Further, if the firefly luciferase (Luc) relative activity (%) is less than 50%, the test compound is judged to have rarα antagonistic activity.

Further, EC of agonist AM580 ₅₀ A value of 0.87nM and a minimum observable effect concentration (LOEC) of 0.1nM; IC for antagonist Ro41-5253 ₅₀ The value was 2.67. Mu.M and the minimum observable effect concentration (LOEC) value was 0.625. Mu.M.

Compared with the prior art, the application has the following advantages:

1. the isolated human cells are used as host cells, so that potential endocrine disrupting effects of exogenous chemicals on human health can be reflected to the greatest extent.

2. The positive agonist AM580 and the antagonist Ro41-5253 both produced good dose-response relationships, exhibiting a typical "S" profile or an inverted "S" profile.

3. The method has high sensitivity, and EC of agonist AM580 ₅₀ A value of 0.87nM and a minimum observable effect concentration (LOEC) of 0.1nM; IC for antagonist Ro41-5253 ₅₀ The value was 2.67. Mu.M and the LOEC value was 0.625. Mu.M.

4. The response interval of the method is wider, the linear range of the agonist AM580 is 0.02-50nM, and the linear range of the antagonist Ro41-5253 is 0.125-10 mu M.

5. The reproducibility of the method was better, and the Relative Standard Deviation (RSD) of each concentration induced by AM580 and Ro41-5253 was <25%.

6. The identification of the double luciferase test proves that the accuracy and the reliability of the result of the method are good.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic flow chart of an in vitro screening method for RARα effect constructed based on MCF-7 cell line of the present application;

FIG. 2 is a block diagram of a vector used in the RARα -responsive in vitro screening method constructed based on the MCF-7 cell line of the present application;

FIG. 3 shows the results of the identification of positive agonists and antagonists by the screening system of the RARα in vitro screening method constructed based on the MCF-7 cell line of the present application;

FIG. 4 shows the application of the screening system of the RARα in vitro screening method constructed based on MCF-7 cell line of the present application to the actual screening of exogenous compounds.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

As shown in FIGS. 1-4, MCF-7 cells were transfected with the vector of FIG. 2 according to the procedure of FIG. 1, and the results of FIG. 3 are the sensitivity and stability of a screening system for detection of positive agonist AM580 and positive antagonist Ro41-5253 as exogenous compounds, and the results of FIG. 4 are the identification of the effect of the screening system on 7 novel phenolic compounds. The details are as follows.

In the first step, RARα expression vectors as shown in FIG. 2-B were constructed.

1. The transcript sequence NM_000964.4 of the human RARα receptor is synthesized by genes, and both ends of the transcript sequence comprise cleavage sites of NheI and EcoRI. Wherein,

NheI cleavage site: GCTAGC

EcoRI cleavage site: GAATTC

2. The commercial vector pEF1α -IRES-TagRFP and RARα synthetic fragment are digested by NheI and EcoRI double enzyme digestion respectively, so as to obtain a new vector skeleton fragment with cohesive end and RARα fragment.

3. The two fragments were recombined with T4 DNA ligase to form the RARα expression vector pEF1α -RARα -RFP and transformed into E.coli (E.coli) DH 5. Alpha. Strain for plasmid amplification.

4. The RARα expression vector is subjected to massive endotoxin removal extraction, so that the final concentration of the plasmid is more than 500 ng/. Mu.L, and the transfection requirement is met.

In the second step, the cells are transfected.

Mcf-7 cells were resuscitated and subcultured. The medium used was complete medium, i.e.DMEM high glucose basal medium, to which 10% Fetal Bovine Serum (FBS) and 1% diabody (final concentration of 100U/mL penicillin and 100. Mu.g/mL streptomycin) were added. The cells were exposed to 5% CO at 37 ℃ ₂ The cell culture chamber is used for culturing.

2. And (5) a seed plate. After stable cell passage (typically 2-5 passages), MCF-7 cells were seeded in 96 Kong Baiban with a clear bottom and a cell density of 4X 10 ⁴ CellsCells were cultured in transfection medium at this time, i.e. phenol red free DMEM supplemented with 10% char-threaded FBS (CS-FBS) and 1% diabody. The cells were exposed to 5% CO at 37 ℃ ₂ Cells were grown in the incubator overnight to allow adherent growth.

3. And (5) transfection. When the cell confluency was 80%, cell transfection was started using a cationic lipid exchange method and a transfection reagent of Lipofectamine 3000.a. 2 centrifuge tubes (1.5 mL) were prepared, and 5. Mu.L/well of reduced serum medium Opti-MEM I and 0.2. Mu.L/well of Lipofectamine3000 transfection reagent were added to tube 1; mu.L/well of Opti-MEM I medium, 50 ng/well of the reporter vector pRARE-TA-Luc (FIG. 2-A, SEQ ID NO: 4), 50 ng/well of the RARα expression vector pEF1α -RARα -RFP (FIG. 2-B, SEQ ID NO: 3) and 0.2. Mu.L/well of the P3000 reagent were added sequentially to tube 2. Wherein Lipofectamine3000 and P3000 are both reagents in the transfection kit; the reporter vector pRARE-TA-Luc is a commercial vector (FIG. 2-A) and the final concentration of plasmid was determined by endotoxin removal prior to use>500 ng/. Mu.L. b. The solution in tube 2 was added to tube 1, mixed well and incubated for 15min at room temperature. c. The incubated transfection complexes were added to the cell culture medium at 96 Kong Baiban at a rate of 10. Mu.L/well. d. At 37℃with 5% CO ₂ The cells were cultured in an incubator for 4 hours to complete the plasmid transfection process. The transfection procedure above was referred to the Lipofectamine3000 transfection kit (Thermo Fisher Scientific, USA) instructions.

And thirdly, exposing the compound and detecting a reporter gene.

1. Exposing the compound. After the transfection process has ended, the cells are exposed with test compounds at the respective concentrations without significant cytotoxicity (cell viability > 80%). Stock solutions of compounds formulated with dimethyl sulfoxide (DMSO) were diluted to a range of concentrations with exposure medium. a. If the compound is to detect the agonistic activity of the compound, the transfected cells are directly exposed to the diluted compound at each concentration (DMSO content is less than or equal to 0.1%) and 0.1% DMSO is used as a control group; b. in order to measure the antagonistic activity of the compounds, 25nM of AM580 should be contained in each concentration of the compounds, and the total amount of the test compound and the solvent DMSO contained in AM580 is 0.1% or less, and the AM580 group containing only 25nM is used as the control group. The compound exposure time was 24h, with at least 3 well replicates per test compound concentration. Wherein, the exposure culture medium is supplemented with 1% CS-FBS and 1% diabody for phenol red-free DMEM; positive compounds are a selective rara agonist AM580, an rara natural endogenous agonist all-trans retinoic acid (atRA) and a selective rara antagonist Ro41-5253, respectively; 7 novel phenolic compounds to be identified include tetrabromobisphenol A (TBBPA), triclosan (TCS), dibromophenol (2, 4-DBP), tribromophenol (2, 4, 6-TBP), 3-tert-butyl-4-p-hydroxyanisole (3-BHA), bisphenol A (BPA) and 4-hexylphenol (4-HP).

2. Firefly luciferase (Luc) activity assay. The Luc activity of the compounds after 24h exposure was determined using a Luciferase Assay System assay kit as a monoluciferase assay (mono-Luc assay). a. Diluting 5 XLysis Buffer into 1 Xlysate with sterile water, and balancing to room temperature; b. washing the cells 2 times with Phosphate Buffered Saline (PBS); c. adding 20 mu L/hole of diluted lysate to cells, incubating for 20min at room temperature, and sealing a transparent bottom of 96 Kong Baiban with a white back cover film; d. under light-shielding conditions, 80. Mu.L/well of Luciferase Assay Reagent substrate reagent was added to the cell lysate; e. the chemiluminescent intensity generated by each well was read in an microplate reader, and the read time period was set to 1 s/well. The detection process is described in reference to Luciferase Assay System detection kit (Promega, USA).

3. And (5) analyzing results. The in vitro screening methods for the effectors of rarα agonism or antagonistic activity were evaluated using the positive selective agonist AM580 and the selective antagonists Ro41-5253, respectively. As shown in fig. 3-a and B, under the mono-Luc assay, the positive agonist AM580 and the antagonist Ro41-5253 both produced good dose-response relationships, exhibiting a typical "S" profile or an inverted "S" profile; the method has high sensitivity, and EC of AM580 ₅₀ IC for value sum Ro41-5253 ₅₀ The values were 0.87nM and 2.67. Mu.M, respectively, and the minimum observable effect concentrations (LOEC) of AM580 and Ro41-5253 were 0.1nM and 0.625. Mu.M, respectively; the method has a wider response interval, and the linear ranges of AM580 and Ro41-5253 are respectively 0.02-50nM and 0.125-10 mu M; the method has the advantages ofGood reproducibility, relative Standard Deviation (RSD) of concentrations induced by AM580 and Ro41-5253<25％。

For 7 novel phenolic compounds to be identified, the occurrence of significant increase in Luc relative activity (fold change) was taken as a judgment that the compound had rarα agonistic activity, and Luc relative activity (%) was less than 50% as a judgment that the compound had rarα antagonistic activity. As shown in fig. 4-a, none of the 7 compounds exhibited agonistic activity, whereas the cognate rarα natural endogenous substrate, atRA, exhibited good agonistic activity. As shown in FIG. 4-B, 7 compounds showed some antagonism, but only the Luc relative activity (%) of both TBBPA and TCS compounds was less than 50%, so both compounds had RARα antagonism. Whether a compound exhibits rarα agonistic or antagonistic activity, it is predicted that the compound is a potential endocrine disruptor.

Fourth, a dual luciferase reporter assay.

In the development of the method, to test the accuracy and reliability of the agonism/antagonism effect of positive compounds on rarα, we used a dual-Luc assay to test and compare with the results of the single-luciferase assay. We introduced the Renilla luciferase (RLuc) expression vector pGL4.74 (FIG. 2-C) as an internal reference for correction of Luc activity. In accordance with the cell transfection procedure described in the second step, the procedure was unchanged in tube 2 except that the Luc reporter vector and the RARα receptor vector were added, and 10 ng/well of the RLuc reporter vector pGL4.74 (FIG. 2-C) was added. In the detection of the reporter gene, the activity of Luc and RLuc in the system was detected by using Dual-Glo luciferase assay system double-luciferase assay kit (Promega, USA) according to the requirements of the specification, and the ratio of the chemiluminescent intensity of Luc to RLuc in each well was used as the corrected Luc activity of the well sample. As shown in FIG. 3, the results of the single and double luciferase assays are highly similar from the results, confirming the reliability of the mono-Luc assay system in response to agonist and antagonist induction, and the Luc single reporter gene was selected for screening exogenous compounds with RARα agonism/antagonism from the viewpoint of simplification of the transfection system and the cost of the detection reagent. This step is used to verify the correctness of the method of the application.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.

Sequence listing

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aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 2460

gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 2520

agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 2580

ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg 2640

aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 2700

cgatgataat atggccacaa ccatggtgtc taagggcgaa gagctgatta aggagaacat 2760

gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga 2820

gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg 2880

ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcagaacctt 2940

catcaaccac acccagggca tccccgattt ctttaagcag tccttccctg agggcttcac 3000

atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag 3060

cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa 3120

cggccctgtg atgcagaaga aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc 3180

cgctgacggc ggcctggaag gcagaaccga catggccctg aagctcgtgg gcgggggcca 3240

cctgatctgc aacttcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat 3300

gcccggcgtc tactatgtgg accacagact ggaaagaatc aaggaggccg acaaagagac 3360

ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg 3420

gcacaaactt aatggcatgg acgagctgta caagtgagcg gccgcgactc tagatcataa 3480

tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 3540

tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 3600

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 3660

attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt 3720

aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 3780

gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 3840

gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 3900

aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 3960

gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 4020

tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 4080

gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 4140

aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 4200

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4260

taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga 4320

atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 4380

gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 4440

gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 4500

ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 4560

tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 4620

gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg 4680

tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 4740

ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 4800

ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 4860

gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 4920

gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 4980

gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 5040

gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 5100

catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 5160

gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg 5220

cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 5280

gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 5340

caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 5400

cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 5460

cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 5520

ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 5580

gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 5640

tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa 5700

cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc 5760

ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt 5820

ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag 5880

gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat 5940

atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6000

tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6060

accccgtaga aaagatcaa 6079

<210> 3

<211> 7436

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 60

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 120

aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 180

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 240

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 300

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 360

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 420

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 480

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 540

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 600

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 660

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt 720

aatgagtaat tcatacaaaa ggactcgccc ctgccttggg gaatcccagg gaccgtcgtt 780

aaactcccac taacgtagaa cccagagatc gctgcgttcc cgccccctca cccgcccgct 840

ctcgtcatca ctgaggtgga gaagagcatg cgtgaggctc cggtgcccgt cagtgggcag 900

agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg 960

cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt 1020

ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc 1080

gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc 1140

tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac 1200

gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct 1260

taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ttgggcgctg gggccgccgc 1320

gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt 1380

taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg 1440

ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt 1500

gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga 1560

cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc 1620

gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg 1680

ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg 1740

gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt 1800

gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt 1860

acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg 1920

gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt 1980

tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt 2040

ccatttcagg tgtcgtgacg ctagcatggc cagcaacagc agctcctgcc cgacacctgg 2100

gggcgggcac ctcaatgggt acccggtgcc tccctacgcc ttcttcttcc cccctatgct 2160

gggtggactc tccccgccag gcgctctgac cactctccag caccagcttc cagttagtgg 2220

atatagcaca ccatccccag ccaccattga gacccagagc agcagttctg aagagatagt 2280

gcccagccct ccctcgccac cccctctacc ccgcatctac aagccttgct ttgtctgtca 2340

ggacaagtcc tcaggctacc actatggggt cagcgcctgt gagggctgca agggcttctt 2400

ccgccgcagc atccagaaga acatggtgta cacgtgtcac cgggacaaga actgcatcat 2460

caacaaggtg acccggaacc gctgccagta ctgccgactg cagaagtgct ttgaagtggg 2520

catgtccaag gagtctgtga gaaacgaccg aaacaagaag aagaaggagg tgcccaagcc 2580

cgagtgctct gagagctaca cgctgacgcc ggaggtgggg gagctcattg agaaggtgcg 2640

caaagcgcac caggaaacct tccctgccct ctgccagctg ggcaaataca ctacgaacaa 2700

cagctcagaa caacgtgtct ctctggacat tgacctctgg gacaagttca gtgaactctc 2760

caccaagtgc atcattaaga ctgtggagtt cgccaagcag ctgcccggct tcaccaccct 2820

caccatcgcc gaccagatca ccctcctcaa ggctgcctgc ctggacatcc tgatcctgcg 2880

gatctgcacg cggtacacgc ccgagcagga caccatgacc ttctcggacg ggctgaccct 2940

gaaccggacc cagatgcaca acgctggctt cggccccctc accgacctgg tctttgcctt 3000

cgccaaccag ctgctgcccc tggagatgga tgatgcggag acggggctgc tcagcgccat 3060

ctgcctcatc tgcggagacc gccaggacct ggagcagccg gaccgggtgg acatgctgca 3120

ggagccgctg ctggaggcgc taaaggtcta cgtgcggaag cggaggccca gccgccccca 3180

catgttcccc aagatgctaa tgaagattac tgacctgcga agcatcagcg ccaagggggc 3240

tgagcgggtg atcacgctga agatggagat cccgggctcc atgccgcctc tcatccagga 3300

aatgttggag aactcagagg gcctggacac tctgagcgga cagccggggg gtggggggcg 3360

ggacgggggt ggcctggccc ccccgccagg cagctgtagc cccagcctca gccccagctc 3420

caacagaagc agcccggcca cccactcccc gtgagaattc tgcagtcgac ggtaccgcgg 3480

gcccgggatc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga 3540

ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa 3600

tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc 3660

tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc 3720

ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg 3780

cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca 3840

accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag 3900

cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct 3960

ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc 4020

cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca 4080

tggtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg tacatggagg 4140

gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg 4200

gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca 4260

tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc cagggcatcc 4320

ccgatttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg 4380

aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct 4440

acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa 4500

cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc ctggaaggca 4560

gaaccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac ttcaagacca 4620

catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac tatgtggacc 4680

acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag cacgaggtgg 4740

ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat ggcatggacg 4800

agctgtacaa gtgagcggcc gcgactctag atcataatca gccataccac atttgtagag 4860

gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat 4920

gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc 4980

atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5040

ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt 5100

aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 5160

taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 5220

actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 5280

cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 5340

aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 5400

ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 5460

ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 5520

aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 5580

ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 5640

aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga 5700

aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 5760

accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 5820

aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 5880

agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 5940

gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 6000

ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 6060

attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 6120

acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 6180

tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg 6240

gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 6300

agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 6360

ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 6420

tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 6480

tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 6540

gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt 6600

gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 6660

catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 6720

tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 6780

cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc 6840

gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 6900

gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 6960

tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg 7020

ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa 7080

agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag 7140

ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct 7200

tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc 7260

ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa 7320

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 7380

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaa 7436

<210> 4

<211> 5663

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ggcctaactg gccggtaccg ctagcctcga taggtcacct ggaggtcacg gaggtcacct 60

ggaggtcacg ggcgcgtaga tctgcagaag cttagacact agagggtata taatggaagc 120

tcgacttcca gcttggcaat ccggtactgt tggtaaagcc accatggaag atgccaaaaa 180

cattaagaag ggcccagcgc cattctaccc actcgaagac gggaccgccg gcgagcagct 240

gcacaaagcc atgaagcgct acgccctggt gcccggcacc atcgccttta ccgacgcaca 300

tatcgaggtg gacattacct acgccgagta cttcgagatg agcgttcggc tggcagaagc 360

tatgaagcgc tatgggctga atacaaacca tcggatcgtg gtgtgcagcg agaatagctt 420

gcagttcttc atgcccgtgt tgggtgccct gttcatcggt gtggctgtgg ccccagctaa 480

cgacatctac aacgagcgcg agctgctgaa cagcatgggc atcagccagc ccaccgtcgt 540

attcgtgagc aagaaagggc tgcaaaagat cctcaacgtg caaaagaagc taccgatcat 600

acaaaagatc atcatcatgg atagcaagac cgactaccag ggcttccaaa gcatgtacac 660

cttcgtgact tcccatttgc cacccggctt caacgagtac gacttcgtgc ccgagagctt 720

cgaccgggac aaaaccatcg ccctgatcat gaacagtagt ggcagtaccg gattgcccaa 780

gggcgtagcc ctaccgcacc gcaccgcttg tgtccgattc agtcatgccc gcgaccccat 840

cttcggcaac cagatcatcc ccgacaccgc tatcctcagc gtggtgccat ttcaccacgg 900

cttcggcatg ttcaccacgc tgggctactt gatctgcggc tttcgggtcg tgctcatgta 960

ccgcttcgag gaggagctat tcttgcgcag cttgcaagac tataagattc aatctgccct 1020

gctggtgccc acactattta gcttcttcgc taagagcact ctcatcgaca agtacgacct 1080

aagcaacttg cacgagatcg ccagcggcgg ggcgccgctc agcaaggagg taggtgaggc 1140

cgtggccaaa cgcttccacc taccaggcat ccgccagggc tacggcctga cagaaacaac 1200

cagcgccatt ctgatcaccc ccgaagggga cgacaagcct ggcgcagtag gcaaggtggt 1260

gcccttcttc gaggctaagg tggtggactt ggacaccggt aagacactgg gtgtgaacca 1320

gcgcggcgag ctgtgcgtcc gtggccccat gatcatgagc ggctacgtta acaaccccga 1380

ggctacaaac gctctcatcg acaaggacgg ctggctgcac agcggcgaca tcgcctactg 1440

ggacgaggac gagcacttct tcatcgtgga ccggctgaag agcctgatca aatacaaggg 1500

ctaccaggta gccccagccg aactggagag catcctgctg caacacccca acatcttcga 1560

cgccggggtc gccggcctgc ccgacgacga tgccggcgag ctgcccgccg cagtcgtcgt 1620

gctggaacac ggtaaaacca tgaccgagaa ggagatcgtg gactatgtgg ccagccaggt 1680

tacaaccgcc aagaagctgc gcggtggtgt tgtgttcgtg gacgaggtgc ctaaaggact 1740

gaccggcaag ttggacgccc gcaagatccg cgagattctc attaaggcca agaagggcgg 1800

caagatcgcc gtgtaataat tctagagtcg gggcggccgg ccgcttcgag cagacatgat 1860

aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 1920

ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 1980

taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 2040

ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt ttgcgtattg 2100

ggcgctcttc cgctgatctg cgcagcacca tggcctgaaa taacctctga aagaggaact 2160

tggttagcta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt 2220

ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 2280

gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 2340

ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 2400

cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 2460

gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 2520

ggcttttgca aaaagctcga ttcttctgac actagcgcca ccatgatcga acaagacggc 2580

ctccatgctg gcagtcccgc agcttgggtc gaacgcttgt tcgggtacga ctgggcccag 2640

cagaccatcg gatgtagcga tgcggccgtg ttccgtctaa gcgctcaagg ccggcccgtg 2700

ctgttcgtga agaccgacct gagcggcgcc ctgaacgagc ttcaagacga ggctgcccgc 2760

ctgagctggc tggccaccac cggcgtaccc tgcgccgctg tgttggatgt tgtgaccgaa 2820

gccggccggg actggctgct gctgggcgag gtccctggcc aggatctgct gagcagccac 2880

cttgcccccg ctgagaaggt ttctatcatg gccgatgcaa tgcggcgcct gcacaccctg 2940

gaccccgcta cctgcccctt cgaccaccag gctaagcatc ggatcgagcg tgctcggacc 3000

cgcatggagg ccggcctggt ggaccaggac gacctggacg aggagcatca gggcctggcc 3060

cccgctgaac tgttcgcccg actgaaagcc cgcatgccgg acggtgagga cctggttgtc 3120

acacacggag atgcctgcct ccctaacatc atggtcgaga atggccgctt ctccggcttc 3180

atcgactgcg gtcgcctagg agttgccgac cgctaccagg acatcgccct ggccacccgc 3240

gacatcgctg aggagcttgg cggcgagtgg gccgaccgct tcttagtctt gtacggcatc 3300

gcagctcccg acagccagcg catcgccttc taccgcttgc tcgacgagtt cttttaatga 3360

tctagaaccg gtcatggccg caataaaata tctttatttt cattacatct gtgtgttggt 3420

tttttgtgtg ttcgaactag atgctgtcga ccgatgccct tgagagcctt caacccagtc 3480

agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt 3540

atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc 3600

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 3660

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 3720

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 3780

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 3840

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 3900

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 3960

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 4020

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 4080

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 4140

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 4200

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 4260

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 4320

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 4380

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 4440

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 4500

aacttggtct gacagcggcc gcaaatgcta aaccactgca gtggttacca gtgcttgatc 4560

agtgaggcac cgatctcagc gatctgccta tttcgttcgt ccatagtggc ctgactcccc 4620

gtcgtgtaga tcactacgat tcgtgagggc ttaccatcag gccccagcgc agcaatgatg 4680

ccgcgagagc cgcgttcacc ggcccccgat ttgtcagcaa tgaaccagcc agcagggagg 4740

gccgagcgaa gaagtggtcc tgctactttg tccgcctcca tccagtctat gagctgctgt 4800

cgtgatgcta gagtaagaag ttcgccagtg agtagtttcc gaagagttgt ggccattgct 4860

actggcatcg tggtatcacg ctcgtcgttc ggtatggctt cgttcaactc tggttcccag 4920

cggtcaagcc gggtcacatg atcacccata ttatgaagaa atgcagtcag ctccttaggg 4980

cctccgatcg ttgtcagaag taagttggcc gcggtgttgt cgctcatggt aatggcagca 5040

ctacacaatt ctcttaccgt catgccatcc gtaagatgct tttccgtgac cggcgagtac 5100

tcaaccaagt cgttttgtga gtagtgtata cggcgaccaa gctgctcttg cccggcgtct 5160

atacgggaca acaccgcgcc acatagcagt actttgaaag tgctcatcat cgggaatcgt 5220

tcttcggggc ggaaagactc aaggatcttg ccgctattga gatccagttc gatatagccc 5280

actcttgcac ccagttgatc ttcagcatct tttactttca ccagcgtttc ggggtgtgca 5340

aaaacaggca agcaaaatgc cgcaaagaag ggaatgagtg cgacacgaaa atgttggatg 5400

ctcatactcg tcctttttca atattattga agcatttatc agggttacta gtacgtctct 5460

caaggataag taagtaatat taaggtacgg gaggtattgg acaggccgca ataaaatatc 5520

tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc 5580

tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag 5640

tgcaggtgcc agaacatttc tct 5663

<210> 5

<211> 4237

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ggcctaactg gccggtacct gagtctaaat gagtcttcgg acctcgcggg ggccgcttaa 60

gcggtggtta gggtttgtct gacgcggggg gagggggaag gaacgaaaca ctctcattcg 120

gaggcggctc ggggtttggt cttggtggcc acgggcacgc agaagagcgc cgcgatcctc 180

ttaagcaccc ccccgccctc cgtggaggcg ggggtttggt cggcgggtgg taactggcgg 240

gccgctgact cgggcgggtc gcgcgcccca gagtgtgacc ttttcggtct gctcgcagac 300

ccccgggcgg cgccgccgcg gcggcgacgg gctcgctggg tcctaggctc catggggacc 360

gtatacgtgg acaggctctg gagcatccgc acgactgcgg tgatattacc ggagaccttc 420

tgcgggacga gccgggtcac gcggctgacg cggagcgtcc gttgggcgac aaacaccagg 480

acggggcaca ggtacactat cttgtcaccc ggaggcgcga gggactgcag gagcttcagg 540

gagtggcgca gctgcttcat ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg 600

gaagaaatat atttgcatgt ctttagttct atgatgacac aaaccccgcc cagcgtcttg 660

tcattggcga attcgaacac gcagatgcag tcggggcggc gcggtcccag gtccacttcg 720

catattaagg tgacgcgtgt ggcctcgaac accgagcgac cctgcagcga cccgcttaaa 780

agcttggcaa tccggtactg ttggtaaagc caccatggct tccaaggtgt acgaccccga 840

gcaacgcaaa cgcatgatca ctgggcctca gtggtgggct cgctgcaagc aaatgaacgt 900

gctggactcc ttcatcaact actatgattc cgagaagcac gccgagaacg ccgtgatttt 960

tctgcatggt aacgctgcct ccagctacct gtggaggcac gtcgtgcctc acatcgagcc 1020

cgtggctaga tgcatcatcc ctgatctgat cggaatgggt aagtccggca agagcgggaa 1080

tggctcatat cgcctcctgg atcactacaa gtacctcacc gcttggttcg agctgctgaa 1140

ccttccaaag aaaatcatct ttgtgggcca cgactggggg gcttgtctgg cctttcacta 1200

ctcctacgag caccaagaca agatcaaggc catcgtccat gctgagagtg tcgtggacgt 1260

gatcgagtcc tgggacgagt ggcctgacat cgaggaggat atcgccctga tcaagagcga 1320

agagggcgag aaaatggtgc ttgagaataa cttcttcgtc gagaccatgc tcccaagcaa 1380

gatcatgcgg aaactggagc ctgaggagtt cgctgcctac ctggagccat tcaaggagaa 1440

gggcgaggtt agacggccta ccctctcctg gcctcgcgag atccctctcg ttaagggagg 1500

caagcccgac gtcgtccaga ttgtccgcaa ctacaacgcc taccttcggg ccagcgacga 1560

tctgcctaag atgttcatcg agtccgaccc tgggttcttt tccaacgcta ttgtcgaggg 1620

agctaagaag ttccctaaca ccgagttcgt gaaggtgaag ggcctccact tcagccagga 1680

ggacgctcca gatgaaatgg gtaagtacat caagagcttc gtggagcgcg tgctgaagaa 1740

cgagcagtaa ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat 1800

tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 1860

ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 1920

caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 1980

gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag 2040

ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta 2100

tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc 2160

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2220

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 2280

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 2340

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 2400

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 2460

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 2520

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 2580

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 2640

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 2700

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 2760

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 2820

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 2880

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 2940

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 3000

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 3060

agtatatatg agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt 3120

accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag 3180

tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca 3240

gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc 3300

agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt 3360

ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag 3420

ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca 3480

actctggttc ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag 3540

tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca 3600

tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg 3660

tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct 3720

cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca 3780

tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca 3840

gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg 3900

tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac 3960

gaaaatgttg gatgctcata ctcgtccttt ttcaatatta ttgaagcatt tatcagggtt 4020

actagtacgt ctctcaagga taagtaagta atattaaggt acgggaggta ttggacaggc 4080

cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata 4140

gtactaacat acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct 4200

gtccccagtg caagtgcagg tgccagaaca tttctct 4237

Claims

1. An in vitro RARα effect screening method constructed based on an MCF-7 cell line, comprising the steps of:

step1, construction of RARα expression vector

3) Recombining the two fragments by using T4 DNA ligase to form an RARalpha expression vector pEF1alpha-RARalpha-RFP, and converting the RARalpha expression vector pEF1alpha-RARalpha-RFP into an E.coli DH5 alpha strain for plasmid amplification, wherein the sequence of the pEF1alpha-RARalpha-RFP is shown as SEQ ID NO. 3;

step2, cell transfection

1) Resuscitating MCF-7 cells at 37deg.C, 5% CO ₂ Subculturing a cell culture box, wherein the culture medium is a complete culture medium;

3) Transfection; when the cell confluency was 80%, cell transfection was started by the cationic lipid exchange method:

a. 2 centrifuge tubes (1.5 mL) were prepared, and 5. Mu.L/well of reduced serum medium Opti-MEM I and 0.2. Mu.L/well of Lipofectamine3000 transfection reagent were added to tube 1; 5 mu L/well of Opti-MEM I culture medium, 50 ng/well of report vector pRARE-TA-Luc, 50 ng/well of RARα expression vector pEF1α -RARα -RFP and 0.2 mu L/well of P3000 reagent are sequentially added into a tube 2, wherein the sequence of the report vector pRARE-TA-Luc is shown as SEQ ID NO. 4;

b. adding the solution in the tube 2 into the tube 1, fully and uniformly mixing, and incubating for 15min at room temperature;

c. adding the incubated transfection complex to a cell culture medium at 96 Kong Baiban at an amount of 10. Mu.L/well;

d. at 37℃with 5% CO ₂ Continuously culturing for 4 hours in a cell incubator to finish the plasmid transfection process;

step3, compound exposure and reporter gene detection

1) Exposing the compound; after the transfection process is finished, exposing the cells with different concentrations of the compound to be tested without obvious cytotoxicity for 24 hours, wherein the exposure culture medium is phenol red-free DMEM supplemented with 1% CS-FBS and 1% diabody;

2) Firefly luciferase (Luc) activity assay; the Luc activity of the compound after 24 hours exposure is measured by a detection kit, wherein the detection method is a single luciferase assay (mono-Luc assay);

step4, dual luciferase reporter assay

In the cell transfection process described in Step2, when preparing the transfection complex, except for adding the Luc report vector and the RARα receptor vector, and then adding the RLuc report vector pGL4.74 of 10 ng/hole, the rest processes are unchanged;

when the reporter gene is detected, the Dual-Glo luciferase assay system double-luciferase detection kit is used for respectively detecting the activity of Luc and RLuc in the system, and the ratio of the chemiluminescent intensity of Luc to that of RLuc in each hole is used as the corrected Luc activity of the hole sample.

2. The method for in vitro screening of rarα effect constructed based on MCF-7 cell line according to claim 1, wherein in Step2, the complete medium is: 10% Fetal Bovine Serum (FBS) and 1% diabody were added to DMEM high-glucose basal medium.

3. The method of claim 1, wherein in Step2, the plates are prepared by inoculating MCF-7 cells into 96 Kong Baiban with a bottom transparent, and the cell density is 4X 10 ⁴ Cells/well cells were cultured in transfection medium in the presence of phenol red free DMEM supplemented with 10% char-threaded FBS (CS-FBS) and 1% diabody.

4. The method according to claim 1, wherein in Step 1) of Step3, transfected cells are directly exposed to test compounds of different concentrations for detecting agonistic activity of the compounds, wherein the test compounds have a DMSO content of 0.1% or less and 0.1% DMSO is used as a control.

5. The method according to claim 1, wherein in Step 1) of Step3, 25nM of AM580 is added to different concentrations of the compound for detecting antagonistic activity, wherein the total amount of DMSO contained in the test compound and AM580 is less than or equal to 0.1%, and only 25nM of AM580 is used as a control group.

6. The method for in vitro screening of rarα effect constructed based on MCF-7 cell line according to claim 2, wherein the diabody is: penicillin at a concentration of 100U/mL and streptomycin at a concentration of 100. Mu.g/mL.

7. The method of claim 1, wherein the test compound is identified as a potential endocrine disruptor if the test compound exhibits rarα agonistic or antagonistic activity.

8. The method of claim 7, wherein the test compound is determined to have RARα agonistic activity if there is a significant increase in firefly luciferase (Luc) relative activity (fold change).

9. The method according to claim 7, wherein the test compound is judged to have RARα antagonistic activity if the relative activity (%) of firefly luciferase (Luc) is less than 50%.

10. An in vitro method for screening for the RARα effect based on the construction of an MCF-7 cell line according to any one of claims 1 to 9, wherein the EC of agonist AM580 ₅₀ A value of 0.87nM and a minimum observable effect concentration (LOEC) of 0.1nM; IC for antagonist Ro41-5253 ₅₀ The value was 2.67. Mu.M and the minimum observable effect concentration (LOEC) value was 0.625. Mu.M.