JP5390221B2 - Polynucleotide for detecting test substance, vector containing said polynucleotide, and detection method using them - Google Patents

Description

  The present invention relates to a polynucleotide which is a dioxin response element, a vector containing the polynucleotide, and a method for detecting a test substance using the polynucleotide. In particular, the present invention is specific to, for example, a protein complex formed when a test substance is an endocrine disrupting substance and such a trace chemical binds to a receptor that is a substrate-dependent transcription factor. And a method for detecting a trace chemical substance using the detection nucleic acid.

  In recent years, the effects of chemical substances present in the environment, such as resin-related chemical substances, plastic plasticizers, and agricultural chemicals, on the living body have attracted attention, and there are concerns about diseases caused by those chemical substances. Of these, many have been found to disrupt endocrine related to the reproductive function of animals, and these are called endocrine disruptors. Dioxins, one of endocrine disrupting substances, are unintentionally produced in the manufacturing process of various chemical substances, and then released into the environment as chemical impurities, which has an impact on the environment and health. I'm worried. Dioxins here are a general term for polychlorinated dibenzo-para-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (coplanar PCB), and there are many structural isomers. Some of these isomers are known to be highly toxic and are considered to be at risk of affecting the human body at current levels of environmental pollution. It is known that most of the toxic expression of these dioxins is mediated by its receptor, aryl hydrocarbon receptor (hereinafter referred to as “AhR”). In the absence of a ligand, AhR exists in the cytoplasm, but by binding to a ligand such as dioxin, AhR undergoes a structural change and moves into the nucleus (Non-patent Document 4). The translocated AhR forms a heterodimer with an aryl hydrocarbon receptor nuclear translocator (hereinafter referred to as “Arnt”). AhR / Arnt heterodimers activate transcription by binding to a dioxin response element called a xenobiotic response element (XRE) present on the transcriptional regulatory region of the target gene (see Figure 1) ).

  In general, high resolution gas chromatography / mass spectrometry defined by official methods is used as a standard method for the analysis of environmental samples and foods. However, this method requires expensive and specialized equipment, highly skilled analytical technicians, dedicated analytical facilities and complex pretreatment. Therefore, it takes a long time to obtain the analysis result, and the analysis cost is high. In addition, the combined influence of a plurality of chemical substances in the same sample on a living body is not considered in chemical analysis.

  Under these circumstances, enzyme immunization that detects the amount of a specific ligand by antigen-antibody reaction is a simple and inexpensive primary screening method to supplement the difficulties of chemical analysis in recent years or to select samples for chemical analysis. Measurement methods (enzyme immunoassay; EIA) and bioassay methods have been attracting attention. In the early stages of the development of bioassays for dioxins, detection of dioxins in samples from the EROD activity of cells using the induction of 7-ethoxyresorufin O-deethylase (EROD) activity by dioxins Was attempted. At present, the chemical-activated luciferase expression (CALUX) method is widely used with an increase in reporter gene expression caused by dioxin exposure as an index. In this method, a vector in which an enhancer sequence containing XRE, which is an AhR recognition sequence derived from the mouse CYP1A1 gene strongly induced by dioxins, and a promoter sequence derived from the mouse mammary tumor virus gene are inserted upstream of the luciferase gene is stably introduced into the cell. This is a method for detecting dioxins in a sample from the introduced luciferase activity.

Although the detection methods listed above are simple and sensitive, the EIA method can detect only limited types of dioxins due to the specificity of the antibody and is susceptible to interfering substances. EROD bioassay has a narrow detection range and is susceptible to substrate inhibition. On the other hand, using the fact that the expression of tyrosine hydroxylase (hereinafter referred to as “TH”), which is involved in dopamine biosynthesis, is increased by the activation of AhR, the activity of this transcriptional regulatory ability is measured to determine the substrate. There is a method for measuring the activity of a small amount of a substance to be detected with respect to the control region of a transcription factor having binding properties (Patent Document 1). This method uses a cell into which an enhancer sequence containing a 67 bp AhR recognition sequence derived from the mouse TH gene and a TH gene-derived promoter sequence inserted upstream of the luciferase gene is strongly induced by dioxins. It is a method to do. The 67 bp enhancer sequence used in this method does not include XRE (see Non-Patent Document 1), which is a known AhR binding sequence.
JP 2007-202555 Boutros PC, Moffat ID, Franc MA, Tijet N, Tuomisto J, PohjanvirtaR, and Okey AB Biochem Biophys Res Commun, 321, 707-715 (2004).

  An object of the present invention is to provide a polynucleotide that is a dioxin response element, a vector containing the polynucleotide, and a method for detecting a compound that induces complex formation between AhR and Arnt using the same.

Means for achieving the above object are as follows:
(A) the polynucleotide represented by SEQ ID NO: 1;
(B) A method for detecting a compound that leads to complex formation between AhR and Arnt,
(1) contacting a test substance, AhR, Arnt, and a detection nucleic acid containing the sequence represented by SEQ ID NO: 1 (wherein N contained in SEQ ID NO: 1 is an arbitrary nucleotide; And the 5 'end NNNN is not CATG, and the 3' end NNNNNNN is not CTAGGGC)
(2) A method comprising detecting binding of the test substance, AhR, Arnt, and a nucleic acid for detection;
(C) A method for detecting a substance to be detected in a sample,
(1) contacting the sample with AhR, Arnt, and a nucleic acid for detection that is a 19-base long double-stranded nucleic acid containing the sequence represented by SEQ ID NO: 1 (herein included in SEQ ID NO: 1) N is an arbitrary nucleotide, and NNNN at the 5 ′ end is not CATG, and NNNNNNN at the 3 ′ end is not CTAGGGC)
(2) detecting binding of the substance to be detected, AhR, Arnt, and the nucleic acid for detection obtained by the contact;
(3) From the detection result of (2), determining whether the detected substance is included in the sample;
A detection method comprising:
(D) an enhancer region that enhances the transcription activity of the downstream gene in response to a test substance in the transcription control region of the TH gene, a promoter operably linked downstream of the enhancer, and a functional downstream of the promoter A vector having a reporter gene linked to, wherein the polynucleotide represented by SEQ ID NO: 1 is an enhancer region;
It is.

  The present invention provides a method for detecting a polynucleotide that is a dioxin response element, a vector containing the polynucleotide, and a compound that induces complex formation between AhR and Arnt using the polynucleotide.

  As a result of intensive studies, the present inventors have identified an AhR binding sequence in the enhancer region of the TH gene that has not been elucidated so far, and a sequence for stably functioning the AhR binding sequence. This sequence is the sequence represented by SEQ ID NO: 1. The sequence represented by SEQ ID NO: 1 is NNNNTCGTGTNNNNNNNGG containing any 4 bases (ie, NNNN) on the 5 'end side and any 7 bases (ie, NNNNNNN) on the 3' end side. In order for the sequence to function stably, it is necessary to include any polynucleotide having 4 bases at the 5 'end and 7 bases at the 3' end, and GG at the 3 'end. Here, “AhR binding sequence works stably” means that the AhR binding sequence works in the same way as it originally works in vivo. That is, this indicates that the allyl hydrocarbon receptor binds to a substance to be originally bound, and the AhR binding sequence exerts the binding ability to the activated AhR, thereby controlling the transcription of the gene. means.

  In the present specification, the base is indicated by a single letter in uppercase or lowercase. That is, the symbol “A” or “a” is adenine, “T” or “t” is thymine, “G” or “g” is guanine, “C” or “c” is cytosine, “N” or “n” Represents adenine, guanine, cytosine or thymine.

  TCGTGT contained in this AhR binding sequence is a 6 bp sequence upstream (that is, 5 ') from the transcription start site of the TH gene. This sequence is determined by gel shift assay using a labeled nucleic acid probe to which mutation has been added. The gel shift assay method is a method for detecting a binding between a nucleic acid and a protein using the fact that a nucleic acid bound to a protein has a lower mobility during electrophoresis than a nucleic acid not bound to a protein. Usually, a labeled nucleic acid composed of a double-stranded oligonucleotide of several tens of bases is reacted with a nucleic acid binding factor such as AhR and Arnt, and then electrophoresed on a non-denaturing gel to detect the mobility of the labeled nucleic acid. A binding nucleic acid probe containing 9 types of unlabeled DNA probes each having a base substitution every 3 bases from the 5 ′ end sequence of the labeled nucleic acid probe and a labeled nucleic acid probe having binding ability to AhR and Arnt protein [ Composition: 100 mM Hepes pH 7.6, 5 mM EDTA, 50 mM DTT, 1% (w / v) Tween 20, 150 mM KCl], left at room temperature for 20 minutes. Detection of the binding between the labeled nucleic acid probe, AhR and Arnt is performed by an electrophoretic measurement method. In addition to the electrophoretic measurement method, the detection can also be performed by using a measurement method using an electro mass sensor element, an electrochemical measurement method, a fluorescence measurement method, a radiation measurement method, or the like.

  Analysis by the gel shift assay identifies an AhR binding sequence in the -228 to -223 nucleotide sequence upstream of the mouse TH gene (numbers represent positions where the transcription start point is +1 and the immediately preceding base is -1). Furthermore, the 3 'terminal GG from the 25-base sequence that is -238 to -213 base sequences upstream of the mouse TH gene, including the 6 bases of -228 to -223 base sequences upstream of the mouse TH gene having AhR binding properties. According to the analysis by gel shift assay using the labeled nucleic acid sequence consisting of the deleted sequence, the labeled nucleic acid sequence consisting of the sequence deleted from the 3 ′ end GG does not bind to the AhR, Arnt complex. It can be confirmed that the 3 'terminal GG sequence is essential for binding to the complex.

  Furthermore, in order to determine the base sequence essential for binding, the 19-base sequence represented by the -223 to -214 base sequence upstream of the mouse TH gene obtained by deleting 6 bases on the 5 'end side, and the above In the analysis by gel shift assay using a labeled nucleic acid sequence in which substitution is made in the sequence of 4 bases on the 5 ′ end side and 7 bases other than GG on the 3 ′ end side among the 19 base sequences shown in FIG. Since the binding between each labeled nucleic acid probe and the AhR / Arnt complex can be confirmed, the 6-base upstream of the mouse TH gene -228 to -223 sequence, and any 4 bases of NNNN at the 5 'end side, It can be confirmed that a 19-base sequence composed of an arbitrary 7 bases of NNNNNNN and a 9-base sequence of GG on the 3 ′ end side is an AhR-binding sequence that functions stably. Here, in the naturally occurring AhR binding sequence, the 5 'terminal NNNN is not CATG, and the 3' terminal NNNNNN is not CTAGGGC.

  The polynucleotide represented by SEQ ID NO: 1 may be synthesized by a method known per se or may be prepared from natural materials.

  The polynucleotide represented by SEQ ID NO: 1 may be used in an assay system that does not use cells or may be incorporated into a vector, or a vector containing the polynucleotide is introduced into a cell and the cell is used. May be used in assay systems.

  When used in such an assay system, the polynucleotide represented by SEQ ID NO: 1 detects the complex formation of AhR and Arnt (that is, the binding of AhR and Arnt) stably and with high specificity. It becomes possible.

  Such detection is performed by using the polynucleotide shown in SEQ ID NO: 1 (wherein N contained in SEQ ID NO: 1 is any nucleotide, and NNNN on the 5 ′ end side is not CATG, but NNNNNNN on the 3 ′ end side) Is not CTAGGGC), the test substance, AhR, and Arnt, and then the binding of the test substance, AhR, Arnt, and the nucleic acid for detection may be detected. By such a method, it is possible to easily detect or detect the complex formation of AhR and Arnt with a stable and simple configuration.

Thus, according to one aspect of the invention,
A method for detecting a compound that leads to complex formation between AhR and Arnt, comprising:
(1) contacting a test substance, AhR, Arnt, and a detection nucleic acid containing the sequence represented by SEQ ID NO: 1 (wherein N contained in SEQ ID NO: 1 is an arbitrary nucleotide; And the 5 'terminal NNNN is not CATG, and the 3' terminal NNNNNN is not CTAGGGC)
(2) A method comprising detecting the binding of the test substance, AhR, Arnt, and a detection nucleic acid is provided.

  This method uses the phenomenon in which AhR, Arnt, AhR ligand, and XRE bind in the cell shown in FIG. 1, and detects the compound that induces the binding of AhR and Arnt, AhR, Arnt, and This is a method for detecting a compound that induces the binding between AhR and Arnt by detecting the binding with the nucleic acid for use in a non-cellular system.

  Please refer to FIG. The principle of the method will be described. In the reaction system 11, the test substance 12, AhR13, Arnt14, and the detection nucleic acid 15 are brought into contact (FIG. 2 (A)). As a result, when the test substance 12 is a compound that induces the bond between AhR and Arnt, a complex is formed as shown in FIG. Therefore, by detecting this complex, it is possible to determine that the test substance is a compound that induces the binding between AhR and Arnt. In addition, when the test substance is a compound that does not induce the bond between AhR and Arnt, or when there is no compound that induces the bond between AhR and Arnt in the reaction system 11, FIG. As shown in FIG. 2, since the complex is not formed, the complex is not detected.

  Using such a principle, it is possible to determine whether or not the test substance is a compound that induces the bond between AhR and Arnt. Further, using the same principle, it is possible to determine whether or not a sample contains a compound that induces a bond between AhR and Arnt. Similarly, it is possible to screen whether a desired compound is a compound that induces the binding between AhR and Arnt.

  Here, the “test substance” may be a known or unknown natural product or compound used in the detection method or determination method according to the present invention, or a mixture containing at least one of them.

  Here, the “substance to be detected” may be an object to be detected in the detection method of the present invention.

  Here, the “sample” is a sample used in the detection method or determination method of the present invention, such as foods, reagents, chemicals, lake water, seawater, river water, sewage and waste, and AhR and Arnt. Any substance may be included as long as it can include a compound that induces the binding of AhR and Arnt.

  Here, the “compound that induces the complex formation of AhR and Arnt” is synonymous with the “compound that induces the bond between AhR and Arnt”, and these can be used interchangeably. Such a compound may be, for example, an AhR ligand-like substance. AhR ligand-like substances may include endocrine disrupting substances such as dioxins. Endocrine disruptors are also commonly referred to as “environmental hormones”.

  The “nucleic acid” used herein may be a synthetic or naturally-occurring DNA, oligonucleotide, or the like. It may also be an artificial nucleic acid such as LNA or peptide nucleic acid. Here, for convenience, a nucleic acid formed by combining a plurality of nucleotides is referred to as “polynucleotide”.

1. Nucleic acid for detection The polynucleotide represented by SEQ ID NO: 1 may be used as a nucleic acid for detection. Such a nucleic acid for detection may be a nucleic acid containing at least a part of the sequence represented by SEQ ID NO: 1 in a double strand. When the nucleic acid for detection is double-stranded, one of the nucleic acids contained in the double-stranded nucleic acid is a 19-base nucleic acid represented by SEQ ID NO: 1, and a nucleic acid that hybridizes to these sequences under stringent conditions If it is. The other nucleic acid contained in the double strand may be a complementary strand of a 19-base nucleic acid represented by SEQ ID NO: 1 and a complementary strand of a nucleic acid that hybridizes to these sequences under stringent conditions. The important site of the nucleic acid for detection is the sequence represented by SEQ ID NO: 1, and at least this portion may form a double strand together with another single strand having complementarity. The other part may form a double strand by a complementary strand, may form a double strand together with a single strand that hybridizes to these sequences under stringent conditions, or may be a single strand. May be.

  Here, “stringent conditions” refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Further, the sequence that can “hybridize under stringent conditions” may be any sequence that has a certain degree of homology. “A certain degree of homology” means, for example, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more. It may be sufficient, and may have 100% homology, that is, complementarity.

  Such a nucleic acid having a certain homology or more may be a nucleic acid having a base sequence in which one or more bases of a nucleic acid having a desired sequence are substituted, deleted, and / or inserted.

  In the present invention, it is also possible to use a nucleic acid for detection of SEQ ID NO: 1 linked multiple times, for example, 2 to 10 nucleic acids.

  Such a nucleic acid for detection may be chemically synthesized, or a naturally occurring nucleic acid may be isolated. In either case, it may be produced by a method known per se.

2. AhR and Arnt
As used herein, the term “AhR” refers to AhR protein unless otherwise noted. Similarly, the term “Arnt” as used herein refers to an Arnt protein unless otherwise noted.

  AhR and Arnt used in the present invention are DNA binding factors. AhR and Arnt may be isolated from any naturally occurring organism or synthesized using genetic engineering.

  AhR and Arnt are preferably derived from a rat, but are not limited thereto. For example, AhR and Arnt derived from mammals such as mice and humans, AhR and Arnt derived from birds such as terns, or AhR and Arnt derived from fishes such as sturgeon and medaka may be used. These may be natural proteins, as long as their functions are not lost, modified proteins whose amino acid sequences have been modified by genetic manipulation, or fusion proteins in which tag sequences or other proteins are fused to the ends of the proteins It may be.

  For example, as an example of a fusion protein that can be used in the present invention, a fusion rat AhR encoded by the nucleotide sequence shown in SEQ ID NO: 7 and having a histidine tag / V5 tag added to the C-terminus, and SEQ ID NO: 8 Examples include fused rat Arnt encoded by the nucleotide sequence described in (1) with a histidine tag / V5 tag added to the C-terminus.

  AhR and Arnt used in the present invention may be prepared from any biological tissue as a cell extract. Alternatively, it may be prepared using a protein expression system using E. coli, yeast, insect or animal cultured cells. Alternatively, it can be prepared by a cell-free expression system (ie, in vitro transcription / translation).

  When preparing AhR and Arnt as a cell extract, AhR and Arnt may be prepared by extracting proteins from cells or nuclei from cells expressing AhR and Arnt by a method known per se. Examples of the cells that can be used in the present invention include cells having the deposit numbers FERM BP-10341 and FERM BP-10342.

  When AhR and Arnt are prepared using a protein expression system using E. coli, yeast, insect or animal cultured cells, the AhR gene or Arnt gene is incorporated into an appropriate expression vector and then introduced into the host cell to force It can be expressed.

  In such a protein expression system, a vector suitable for protein expression is a human cytomegalovirus immeadiate-early enhancer / promoter region incorporated in the case of cultured animal cells, and a T7 promoter sequence / multicloning site downstream thereof. PTargetT vector having SV40 enhancer and SV40 early promoter (Promega), pBK-CMV, CMV-Script, pCMV-Tag and pBK-RSV (Stratagene) and pcDNA4 vector series (Invitrogen) And so on.

  In addition, vectors suitable for expression in microbial hosts such as E. coli and yeast include, for example, pET series vector expression systems (for example, pET3a, pET27b (+) and pET28a (+); Novagen) having an E. coli T7 promoter. In yeast systems, it may be a Pichia expression vector pIC series vector (for example, pPIC9K or PIC3.5K; Invitrogen) having an alcohol oxidase promoter.

  When preparing AhR and Arnt in a cell-free expression system (i.e., in vitro transcription-translation), a method of performing translation only in vitro using RNA as a template and transcription / translation using DNA as a template simultaneously in vitro. There are two ways to do it.

  When RNA is used as a template, total RNA, mRNA, in vitro transcripts and the like can be used. When DNA is used as a template, a plasmid vector containing a transcription promoter and a target gene incorporated downstream of the translation initiation point, or a PCR / RT-PCR product can be used.

  Suitable vectors for such methods include, for example, the pTargetT vector, which incorporates the immeadiate-early enhancer / promoter region of human cytomegalovirus and has a T7 promoter sequence / multicloning site downstream thereof, and SV40 enhancer and SV40 Examples include pSI vectors having early promoters (Promega), pBK-CMV, CMV-Script, pCMV-Tag and pBK-RSV (Stratagene) and pcDNA4 vector series (Invitrogen).

  Selection of the optimal system for cell-free expression systems takes into account the origin of the protein gene to be synthesized (prokaryotic / eukaryotic), the type of template (DNA / RNA), or the intended use of the protein after synthesis. There is a need to do. Generally, problems rarely occur when eukaryotic sequences are translated in eukaryotic systems or prokaryotic sequences are translated in prokaryotic systems. For protein expression using a cell-free expression system, a commercially available protein expression system may be used. For example, TnT (registered trademark) Reticulocyte Lysate (Promega), Wheat Germ Extract Plus (Promega), or the like can be used.

3. Detection and Screening Methods According to the present invention,
A method for determining whether or not a test substance is a compound that induces a bond between AhR and Arnt,
(1) contacting the test substance, AhR, Arnt, and a nucleic acid for detection consisting of 19 consecutive bases containing the sequence represented by SEQ ID NO: 1,
(2) detecting binding of the test substance, AhR, Arnt, and the detection nucleic acid obtained by the contact;
(3) From the detection result of (2), determining whether or not the test substance is a compound that induces a bond between AhR and Arnt;
Is provided.

  In addition, the method of the present invention may be provided as a method for detecting a compound that induces the binding between AhR and Arnt as follows, or as a method for screening a compound that induces the binding between AhR and Arnt.

Such a method is, for example,
A method for detecting a compound that induces binding to AhR and Arnt, comprising:
(A) contacting the detection nucleic acid according to the present invention with AhR and Arnt in the presence of a sample containing the test compound, and (b) detecting the binding between the detection nucleic acid, AhR and Arnt,
A method comprising:
A method for screening a compound that leads to the binding between AhR and Arnt,
(A) contacting the nucleic acid for detection according to the present invention with AhR and Arnt in the presence of a sample containing a test compound;
(B) detecting the binding between the detection nucleic acid and the test compound and AhR and Arnt, and (c) selecting a compound that induces the binding between the detection nucleic acid, AhR and Arnt,
A method comprising:
Furthermore, a method for screening a compound that induces the binding between AhR and Arnt,
(d) the nucleic acid for detection is an enhancer region, characterized by having a promoter operably linked downstream of the enhancer and a reporter gene operably linked downstream of the promoter A step of culturing a cell in which a vector to be introduced into a host cell is present in the presence and absence of a test substance,
(e) measuring the expression level of the reporter gene in the cells cultured in the step (d),
(f) a step of evaluating that the test substance is a compound that induces the binding between AhR and Arnt when the expression level of the reporter gene in a cell cultured in the absence of the test substance is higher than the measured value; And the like.

  In the present invention, the test substance, the nucleic acid for detection, AhR and Arnt can be brought into contact under the following conditions. For example, in a solvent such as a binding buffer [composition: 100 mM Hepes pH7.6, 5 mM EDTA, 50 mM DTT, 1% (w / v) Tween 20, 150 mM KCl], 25 ° C. to 30 ° C., preferably May be contacted at 30 ° C. for 60 minutes or longer, most preferably 120 minutes or longer.

  In this contact, AhR, Arnt, and a test substance (or a sample or a test substance) may be first contacted, and then a nucleic acid for detection may be contacted. In addition, after contacting AhR and a test substance (or sample or target substance), Arnt may be contacted and then contacted with a nucleic acid for detection.

  Detection of the binding between the detection nucleic acid, AhR and Arnt may be performed using an electrophoresis measurement method, a measurement method using a mass sensor element, an electrochemical measurement method, a fluorescence measurement method, a radiation measurement method, or the like.

  These methods are known methods of appropriate vendors (edited by Hiroto Okada, “New Cell Engineering Experimental Protocol”, Shujunsha, 1993, edited by Takaaki Tamura, “Biomanual Series 5 Transcription Factor Research Method”, Yodosha 1993, Inouye, C. et al., DNA Cell Biol., 13, 731-742 (1994)), for example, methods using affinity columns, Southwestern methods, footprinting methods, EMSA (electrophoretic mobility shift assay) method, one-hybrid method, etc. may be used. Or by pull-down assay using DNA-immobilized beads, etc., measurement using quartz crystal microbalance, binding measurement using surface plasmon resonance such as BIACORE, or interaction analysis between DNA proteins by fluorescence polarization measurement You can also

  For example, as an example of the electrophoretic measurement method, there is an EMSA method (generally called a gel shift method). This measurement method is a method for detecting a binding between a nucleic acid and a protein using the fact that a nucleic acid bound to a protein has a lower mobility during electrophoresis than a nucleic acid not bound to a protein. Usually, a labeled nucleic acid composed of a double-stranded oligonucleotide of several tens of bases is reacted with a nucleic acid binding factor such as AhR and Arnt, and then electrophoresed on a non-denaturing gel to detect the mobility of the labeled nucleic acid.

  The EMSA in the present invention reacts with AhR and Arnt prepared by any of the above-described methods with a labeled nucleic acid to bind AhR and Arnt to the labeled nucleic acid. This reaction solution is electrophoresed on a non-denaturing polyacrylamide gel. As a result, the mobility of the nucleic acid to which the protein is bound becomes smaller than the mobility of the unbound nucleic acid. Therefore, it is possible to determine whether or not a complex of AhR, Arnt, and a detection nucleic acid has been formed based on the difference in mobility.

Specifically, for example, oligonucleotides that are completely complementary to each other are synthesized and made into single strands at 95 ° C. for 5 minutes in the heat block, and then the heat block is turned off and left until it returns to room temperature (ie, , Double-stranded single-stranded DNA). The labeling of the nucleic acid for detection is not particularly limited. For example, in addition to radioactive substances represented by ³² P, fluorescent substances represented by fluorescein and Alexa dye can be used.

The reaction is carried out, for example, by mixing AhR and ARNT synthesized by in vitro transcription-translation with a test compound for 15 minutes to 2 hours at 15 to 37 ° C. After that, in the presence of 10 mM Tris-Cl (pH 7.5), 1 mM DTT, 1 mM EDTA, 10% Glycerol, 1 mM MgCl ₂ , 0.15 M KCl, a labeled detection nucleic acid is added, and 15-37 ° C. After 15 minutes of reaction, electrophoresis is performed on a 4-10% non-denaturing polyacrylamide gel.

  If necessary, when reacting the label binding nucleic acid, AhR and Arnt, unlabeled nucleic acid (10 to 500 times the amount of the label binding nucleic acid) may be added as a competitor. Thereby, the specificity of the binding between the binding nucleic acid, AhR and Arnt can be confirmed. As a competitor, it is common to use a nucleic acid containing the same sequence as the label binding nucleic acid.

  After the electrophoresis is completed, the label detection nucleic acid may be detected by any means known per se according to the characteristics of the labeling substance used.

  For example, when a labeled nucleic acid is labeled with a fluorescent substance, the pattern is analyzed by detecting the labeled detection nucleic acid with a fluorescence detection device such as a fluorescent scanner, and in the case of a radioactive substance with a radiation measuring device such as an X-ray film or image analyzer. I do. Thus, if a labeled detection nucleic acid with reduced mobility is detected, it can be confirmed that the detection nucleic acid, AhR and Arnt are bound in the reaction solution.

  As a control, if a reaction solution to which the test compound is not added is prepared, the mobility of the label detection nucleic acid in this reaction solution is compared with the mobility of the label detection nucleic acid in the mixed solution to which the test compound is added. It is possible to evaluate the influence of the test compound on the binding between the nucleic acid for detection, AhR and Arnt. In addition, in a reaction solution containing unlabeled nucleic acid as a competitor, if the signal of the labeled detection nucleic acid whose mobility has decreased decreases or disappears, the binding between the detection nucleic acid, AhR and Arnt is confirmed to be specific. can do.

  The type of protein that binds to the labeled detection nucleic acid can be identified by, for example, a supershift assay. For example, if an antibody against a known DNA binding factor is added to the reaction solution and a supershift is observed, it can be seen that the DNA binding factor protein to which the added antibody binds forms a complex with the probe. By this method, it can be confirmed that AhR and Arnt bind to the detection nucleic acid to form a complex.

  In the detection in the present invention, in addition to the electrophoretic measurement method, a mass change measurement method, an electrochemical method, a fluorescence measurement method, and a radiation measurement method may be used.

  Detection by a mass change measurement method using a mass sensor element can be performed by, for example, a quartz crystal microbalance method or an SPR method.

  A crystal oscillator has a structure in which a metal thin film is attached to both sides of a slice of a crystal crystal cut into a very thin plate. When an alternating electric field is applied to each metal thin film, a certain frequency (resonance frequency) Shows the property of vibrating. When a nanogram substance is adsorbed on a metal thin film, the resonance frequency decreases in proportion to the mass of the substance, so it can be used as a microbalance. This methodology is known as QCM (Quartz Crystal Microbalance). Called.

  A biotin-labeled double-stranded oligonucleotide of usually 15 to 30 bases is immobilized as a nucleic acid for detection on the surface of a QCM sensor chip on which an irreversible bond, for example, avidin is immobilized. By immersing a QCM sensor chip on which detection nucleic acid is immobilized in a reaction tank containing Tris buffer, and adding AhR, Arnt, and a test compound to the reaction tank, the nucleic acid for detection of the protein complex is added. Can be evaluated in real time as a decrease in frequency. In the SPR method, a refractive index change and / or a layer thickness change near the surface of a transparent substrate coated with a thin metal layer is detected as a change in reflected light intensity. When the immobilized substance, that is, the detection nucleic acid and the substance in the liquid, that is, AhR and Arnt interact, a change in refractive index near the surface and / or a change in the thickness of the layer occurs, so that the detection nucleic acid or AhR or The interaction can be analyzed without labeling Arnt.

  In detection by an electrochemical measurement method, the binding between the detection nucleic acid, AhR and Arnt is detected using an electrically active compound or an enzyme that catalyzes a chemical reaction that produces an electrically active compound. May be. When labeling the nucleic acid for detection with this measurement method, the labeled detection nucleic acid and the test compound are reacted with AhR or Arnt supported on a conductive carrier, and the non-specific binding product is washed. Then, the electrochemical response may be measured. When labeling AhR or Arnt, the labeled AhR or Arnt is reacted with the detection nucleic acid supported on a conductive carrier, and after washing away non-specific binding substances, What is necessary is just to measure a chemical response. The labeling to the detection nucleic acid or AhR or Arnt may be performed by directly binding a labeled compound or a labeling enzyme, or indirectly using a biotin-streptavidin bond or an antigen-antibody bond. May be combined. In the case of AhR and Arnt, labeling may be performed by forming a fusion protein with a labeling enzyme.

  In detection characterized by fluorescence measurement, a fluorescent substance typified by fluorescein or Alexa dye, or a fluorescent protein typified by GFP, or an enzyme that catalyzes a reaction that produces a fluorescent substance, and the detection nucleic acid or AhR or Arnt may be labeled. The label may directly bind a desired fluorescent substance, fluorescent protein, or enzyme to the detection nucleic acid or AhR or Arnt, and indirectly using a biotin-streptavidin bond or an antigen-antibody bond. May be labeled. In the case of AhR or Arnt, labeling may be performed by using a fusion protein with a fluorescent protein. When labeling the detection nucleic acid, react the labeled detection nucleic acid with AhR or Arnt supported on the carrier, wash away non-specific binding substances, and then measure the fluorescence intensity. Good. When labeling AhR or Arnt, measure the fluorescence intensity after reacting the labeled AhR or Arnt with the detection nucleic acid supported on the carrier, washing away the non-specific binder, and removing it. That's fine.

In the detection method characterized by radiation measurement, the nucleic acid for detection or AhR or Arnt may be labeled with a radioactive substance typified by ³² P or ³⁵ S. When labeling the detection nucleic acid, react the labeled detection nucleic acid with AhR or Arnt supported on the carrier, wash away non-specific binding substances, and then measure the radiation intensity. Good. When labeling AhR or Arnt, measure the radiation intensity after reacting the labeled AhR or Arnt with the detection nucleic acid supported on the carrier and washing away non-specific binding substances. do it.

4). Detection Device In the present invention, a multi-analyte detection device in which the detection nucleic acid or AhR or Arnt is immobilized in an array on a carrier can be prepared and used for detection and evaluation.

  Such devices can be made such that detection by mass sensor elements, electrochemical measurements, fluorescence measurements, radiation measurements and evaluation can be conveniently performed. Such a device may be a detection probe-immobilized chip in which the detection nucleic acid is immobilized as a probe on a carrier, or a DNA binding factor protein-immobilized chip in which AhR or Arnt is immobilized on a carrier. Good.

  The carrier for the device may be any conventionally used substrate such as a glass substrate and a silicon substrate, a bead and a container. The fixing means can be fixed using means known per se to those skilled in the art, such as means using a spotter or the like, means using a general semiconductor technology, and the like.

  In the case of a probe-immobilized chip or DNA-binding factor protein-immobilized chip detected by an electrochemical method, the detection nucleic acid or AhR or Arnt is covalently bonded to a desired substrate, for example, an electrode substrate, an ion The immobilization may be performed by bonding, physical adsorption, chemical adsorption, or the like.

  According to a further aspect of the present invention, there is provided a probe-immobilized chip comprising a substrate and the nucleic acid for detection according to the present invention immobilized on the substrate, and a substrate and AhR immobilized on the substrate. A DNA binding factor protein-immobilized chip and a DNA binding factor protein-immobilized chip comprising a substrate and Arnt immobilized on the substrate are also provided as the present invention. Further, these chips may be provided in combination with any of the above-described labeling means or a labeling means known per se, depending on the detection means.

  According to a further aspect of the present invention, a probe-immobilized chip comprising a substrate and the above-described detection nucleic acid according to the present invention immobilized on the substrate, and a detection kit comprising AhR and Arnt are also provided. The In addition, a detection kit comprising a DNA binding factor protein immobilization chip comprising a substrate and AhR immobilized on the substrate, the detection nucleic acid according to the present invention, and Arnt described above may be provided. Furthermore, a DNA binding factor protein immobilization chip comprising a substrate and Arnt immobilized on the substrate, a detection kit comprising the detection nucleic acid and AhR according to the present invention described above may be provided. Such a detection kit is also within the scope of the present invention. These kits may be provided in combination with any of the above-described labeling means or a labeling means known per se, depending on the detection means.

  According to a further aspect of the present invention, the nucleic acid for detection for detecting the above-mentioned test compound, or AhR or Arnt itself comprises the DNA detection probe for detecting the test compound and / or Alternatively, the present invention may be provided as a DNA binding factor protein for detecting a test compound. Such detection nucleic acids and / or DNA binding factor proteins and their use for detecting test compounds are also within the scope of the present invention.

  According to the present invention, a compound that induces the binding between AhR and Arnt can be easily detected with a simple configuration without using an antibody antigen reaction or cultured cells. Further, according to the present invention, the compound that induces the binding between AhR and Arnt can be detected specifically, quickly and simply. Therefore, it is possible to easily and inexpensively detect a compound that induces a bond between AhR and Arnt present in a trace amount in the environment. In addition, compounds that lead to the binding of AhR and Arnt are likely to work as endocrine disrupting substances when taken up in the body, so endocrine disrupting substances present in the environment and ingested substances can be detected easily and with high sensitivity. It is also possible to screen easily and with high sensitivity whether or not any test substance is an endocrine disrupting substance.

  Conventionally, in order to detect trace chemical substances, methods using antibodies and cultured cells are used. According to the present invention, it is possible to detect a trace chemical substance by a simple method using a material that can be prepared easily and inexpensively.

5. Vector Hereinafter, the vector according to the present invention will be described.

  The vector according to the present invention has an enhancer region consisting of a 6 bp sequence having an AhR binding property in the transcriptional regulatory region of a tyrosine hydroxylase (TH) gene, an arbitrary base sequence of 11 bp, and a 19 bp base sequence containing two Gs, A vector having a promoter and a reporter gene operably linked downstream of the region;

(1) Enhancer region The transcription control region of the TH gene is a 19 bp sequence described in SEQ ID NO: 1. In the present invention, the “TH gene transcription control region” can be the 5 ′ upstream region of the TH gene of any organism, and can be, for example, the 5 ′ upstream region of the TH gene of a human, mouse, or rat. Further, the sequence described in SEQ ID NO: 1 contains a 6 base sequence in the transcription control region of TH gene derived from mouse, but the transcription control region of TH gene corresponding to SEQ ID NO: 1 derived from other species Can be used.

  The enhancer region may be contained as a tandem repeat sequence in the vector of the present invention.

(2) Promoter In addition to the enhancer region comprising the sequence set forth in SEQ ID NO: 1, the vector according to the present invention comprises a promoter and reporter gene operably linked downstream of the region. “Functionally connected” means that the connected regions perform the functions of the regions, that is, are operatively connected. For example, the expression “operably linked” of a promoter or reporter gene means that the promoter or reporter gene is linked so as to exhibit promoter activity and enhance expression of the reporter gene in the vector of the present invention. In addition, the reporter gene is “operably linked” means that the reporter gene is expressed in the vector of the present invention by the action of a “region where the transcriptional activity of the gene is enhanced in response to a test substance” or a promoter. It is connected so that. Any promoter can be used as long as it is functional in the host cell. Preferably, it is a promoter having activity in mammalian cells. For example, the core promoter of the TH gene of SEQ ID NO: 2 can be used. Alternatively, for example, the simian virus (SV40) early promoter (SEQ ID NO: 3) or SV40 late promoter (SEQ ID NO: 4), human herpesvirus 1 thymidine kinase (TK) promoter (SEQ ID NO: 5), cytomegalovirus (CMV) promoter (SEQ ID NO: 6). A person skilled in the art is also very easy to select a suitable promoter suitable for the host organism.

(3) Reporter gene As the reporter gene of the present invention, any reporter gene known in the art can be used. The reporter gene is preferably one whose activity of the product can be easily measured and having a low measurement background.For example, as such a gene, a luciferase gene capable of detecting the gene product by luminescence, a green fluorescent protein gene capable of detecting by fluorescence, Examples thereof include a β-galactosidase gene that can be detected by color development and a chloramphenicol acetyltransferase gene that can be detected by radioactivity.

(4) Other elements The vector of the present invention may contain various elements in addition to the above regions. For example, an origin of replication and a drug resistance gene that function in an appropriate microorganism can be incorporated. Moreover, in order to incorporate a vector on the chromosome of a cell and to hold it stably, a drug system gene for mammals can be incorporated into the vector. Examples of such drug resistance genes for mammals include a zeocin resistance gene, a hygromycin resistance gene, and a kanamycin resistance gene. It can also have an appropriate restriction enzyme site such as a multicloning site.

  The vector according to the present invention may be in any form such as circular plasmid DNA, viral vector DNA, linear DNA fragment.

(5) Method for producing vector The vector can be produced using any method known to those skilled in the art. For example, the vector can be produced as follows.

(A) Preparation of enhancer sequence from within the transcription control region of TH gene The enhancer sequence optimal for the present invention is preferably chemically synthesized.

(B) Preparation of vector Next, the enhancer sequence is incorporated into the vector. The vector is prepared by linking the enhancer sequence prepared in (a) so that the reporter gene functions. It is preferable that the prepared vector is sequenced at least in the base region of the enhancer region to confirm that no mutation has been introduced into the gene.

A commercially available vector can be used as the vector used in the present invention. For example, PGV-
By using a B2 vector or a PGV-P2 vector (TOYOB-NET), the vector of the present invention can be produced simply by incorporating the enhancer sequence produced in (a) into the vector.

6). Cells Furthermore, the present invention provides cells into which the above vector has been introduced. The transformed cell into which the vector is introduced can be used to detect a substance that induces the binding between AhR and Arnt.

  The host cell for introducing the vector of the present invention can be any cell, but is preferably a mammalian cell. The mammalian cell may be of any type as long as the introduced enhancer region functions. For example, the mammalian cell may be a primary cultured cell or an immortalized cultured cell. That is, the kind of mammalian cell should just be a cell which has AhR. Alternatively, a gene encoding AhR may be introduced into mammalian cells.

  Preferably, the host cell is a mammalian cell expressing AhR. The mammalian cell is, for example, a human cell, a mouse cell or a rat cell. The “mammalian cell expressing AhR” may be a mammalian cell that naturally expresses AhR, or a gene transfer prepared by introducing a gene encoding AhR into a mammalian cell. It may be a cell.

  When a gene encoding AhR (AhR gene) is introduced into a mammalian cell, the introduced AhR gene can be an AhR gene derived from any mammal, for example, an AhR gene derived from a human, an AhR derived from a mouse It can be a gene or an AhR gene derived from a rat. The AhR gene derived from SD rat is shown in SEQ ID NO: 7, and the AhR gene derived from C57 / BL6 mice is shown in SEQ ID NO: 9. The AhR gene may contain several base substitutions, deletions, and additions as long as the AhR encoded by the gene functions as a ligand-binding transcription factor.

  In particular, the host cell is desirably a cell derived from a nerve, particularly a cell derived from the central nervous system. The nerve-derived cells are, for example, human nerve-derived cells, mouse nerve-derived cells, or rat nerve-derived cells. Specifically, neuroblastoma, in particular Neuro2a which is a mouse neuroblastoma can be used. Particularly preferably, a cell in which a gene encoding AhR is introduced into a mouse neuroblastoma Neuro2a can be used as a host cell. As an example, cells in which a rat AhR-encoding gene was introduced into mouse neuroblastoma Neuro2a were deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, an international depositary agency under the Budapest Treaty. Accession numbers FERMBP-10341 and FERMBP-10342 are assigned.

  Moreover, the introduction of the vector of the present invention into the host cell may be either transient or stable. For example, in the case of transient introduction of a vector, mammalian cells are seeded in a culture vessel and 5% in a medium such as MEM Dulbecco Ham F 1 2 equimixed medium (DF1: 1) medium containing 10% fetal calf serum. Incubate for several hours to overnight at 37 ° C under% CO2. The vector is introduced into the cells thus cultured. As a method for introducing the vector into a cell, any method known to those skilled in the art, such as the lipofectamine method, the electroporation method, the DEAE-dextran method, and the calcium phosphate method, can be used. For example, Lipofectamine 2000 (manufactured by Invitrogen) can be used, and it is preferable to determine in advance the amount of vector to be introduced, the amount of Lipofectamine 2000, the number of cells, and the like according to a commercially available manual. Further, the vector to be introduced into the cell may be introduced after digestion with an appropriate restriction enzyme to make it linear.

  On the other hand, the stable introduction of the vector of the present invention can be carried out using any method known in the art. For example, when preparing the vector, a vector containing a drug resistance gene is prepared, and the vector containing the drug resistance gene is introduced into the cell in the same manner as in the transient case. Next, the cells into which the vector has been introduced are cultured in a medium containing an appropriate concentration of drug for an appropriate period. As a result, only cells containing the drug resistance gene, that is, cells into which the vector has been stably introduced will survive.

7). Detection Method Using Cells Next, a method for detecting a compound that induces binding between AhR and ARNT using cells into which the vector of the present invention has been introduced will be described.

  First, cells into which the above-described vector of the present invention has been introduced are cultured in the presence and absence of the test substance (that is, the test substance is unexposed and exposed). As for the culture time in the presence of the test substance, it is desirable to preliminarily determine an appropriate time by preliminary experiments according to the mammalian cells to be used. An appropriate culture time is such that the expression product can be detected after the expression of the reporter gene is induced by the presence of the test substance. It will be apparent to those skilled in the art that such a culture time varies depending on various conditions such as the cells used, the culture medium, the culture temperature, and the test substance concentration. And it is thought that those skilled in the art can easily determine an appropriate culture time according to the above conditions, usually about 2 hours to 3 days, for example, 1 hour, 10 hours, 24 hours, It is considered that the expression product of the reporter gene can be detected by culturing for 30 hours or more.

  Next, in the method according to the present invention, the expression level of the expression product of the reporter gene in each cell is measured. The expression product can be detected by various detection methods depending on the reporter gene used. For example, according to the kind of expression product, the process of extracting protein is included. The protein extraction method may be a known optimum extraction method depending on the type of reporter gene used in the vector. Next, the expression product amount of the reporter gene in the extracted protein is measured by a method according to the type of the reporter gene. The expression level of the reporter gene is compared in the presence and absence of the test substance.

  As a result, the activity of the test substance on the “enhancer sequence that enhances the transcriptional activity of the gene in response to the test substance” based on the difference in the expression level of the reporter gene in the presence and absence of the test substance Can be judged. That is, when the measured value of the expression level of the reporter gene in the cells cultured in the presence of the test substance is higher than the measured value of the expression level of the reporter gene in the cells cultured in the absence of the test substance, It is evaluated that the test substance has an activity leading to the binding between AhR and ARNT.

[Example]
Hereinafter, examples according to the present invention will be described in detail, but the present invention is not limited to these examples.

[Example 1]
Determination of AhR and Arnt complex binding sequences by gel shift analysis using labeled detection nucleic acids represented by SEQ ID NOs: 10 to 18 (1) Preparation of AhR and ARNT
AhR gene (SEQ ID NO: 7) and Arnt gene (SEQ ID NO: 8) were respectively introduced into plasmid DNA pcDNA4 having a T7 promoter, and two plasmids, namely pcDNA4-rAhR (FIG. 3) and pcDNA4-rArnt (FIG. 4), were introduced. Produced. Thereafter, AhR and Arnt proteins were prepared by in vitro transcription-translation method using TNT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega) based on the template plasmid DNA.

(2) Formation of a complex containing AhR, Arnt and TCDD 1 μM 2,3,7,8-tetrachlorobenzo-p-dioxin (5 μl each of in vitro translation products AhR and Arnt mixed in a solution 0.1 μl of 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) (Kanto Chemical) was added and reacted at 30 ° C. for 90 minutes to form a complex of AhR, Arnt and TCDD. A control experiment was performed by adding 0.1 μl of DMSO to a solution in which 5 μl of AhR and Arnt were mixed, and reacting at 30 ° C. for 90 minutes.

(3) Preparation of Alexa Fluor (Registered Trademark) 532 Label Probe Oligonucleotide consisting of the sequence shown in SEQ ID NOs: 10 to 18 and its complementary strand include Alexa Fluor (Registered Trademark) 532 label at each 5 'end Was synthesized as follows. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes. Thereafter, the heat block was turned off and allowed to stand until it returned to room temperature. As a result, the single-stranded nucleic acid was made double-stranded. Thus, a labeled detection nucleic acid was prepared as an Alexa Fluor (registered trademark) 532 label probe.

(4) Reaction of complex containing AhR, ARNT, and TCDD and Alexa Fluor (registered trademark) 532 label probe In the mixed solution prepared in (2), binding buffer [composition: 100 mM Hepes, pH 7.6, 5 mM EDTA, 50 mM (NH ₄ ) 2 SO ₄ , 5 mM DTT, 1% (w / v) Tween 20, 150 mM KCl], 100 ng / ul Poly [(dI-C)] solution, 1.4 M KCl solution were added Later, 75 fmol Alexa Fluor (registered trademark) 532 label probe was added and reacted at room temperature for 15 minutes. The reaction solution was electrophoresed on a 6% polyacrylamide gel under conditions of 75 V for 1 hour and 30 minutes. The polyacrylamide gel was then subjected to band detection using a 532 nm laser with an 800 V excitation light source using a gel imaging analyzer Typhoon (GE Healthcare Bioscience). Thus, formation of a complex containing the probe, AhR, ARNT, and TCDD was confirmed, and a sequence consisting of 6 bases necessary for binding of the complex of AhR, Arnt, and TCDD was found (FIG. 5).

[Example 2]
Determination of AhR, Arnt and TCDD complex binding sequence by gel shift assay analysis using the nucleic acid shown by SEQ ID NO: 19 (1) Preparation of AhR and Arnt
AhR gene and Arnt gene were introduced into plasmid DNA pcDNA4 having T7 promoter to prepare two plasmids, namely pcDNA4-rAhR (FIG. 3) and pcDNA4-rArnt (FIG. 4). Based on the template plasmid DNA, AhR and Arnt proteins were prepared by in vitro transcription-translation method using TNT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega).

(2) Formation of a complex containing AhR, Arnt and TCDD 1 μM 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) in a solution containing 5 μl each of in vitro translation products AhR and Arnt ) (Kanto Chemical Co., Inc.) 0.1 μl was added and reacted at 30 ° C. for 90 minutes to form a complex of AhR, Arnt and TCDD. A control experiment was performed by adding 0.1 μl of DMSO to a solution obtained by mixing 5 μl each of AhR and Arnt and reacting at 30 ° C. for 90 minutes.

(3) Preparation of Alexa Fluor (Registered Trademark) 532 Label Probe An oligonucleotide consisting of the sequence shown in SEQ ID NO: 19 and its complementary strand are added so that each 5 'end contains Alexa Fluor (Registered Trademark) 532 label. Synthesized. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes. Thereafter, the heat block was turned off and allowed to stand until it returned to room temperature. As a result, the single-stranded nucleic acid was made double-stranded. Thus, a labeled detection nucleic acid was prepared as an Alexa Fluor (registered trademark) 532 label probe.

(4) Reaction of complex containing AhR, Arnt and TCDD with Alexa Fluor (registered trademark) 532 label probe In the above mixed solution, a binding buffer (100 mM Hepes, pH 7.6, 5 mM EDTA, After adding 50 mM (NH ₄ ) 2SO ₄ , 5 mM DTT, Tween 20, 1% (w / v), 150 mM KCl], 100 ng / ul Poly [(dI-C)] solution, 1.4 M KCl solution 75 fmol Alexa Fluor (registered trademark) 532 label probe was added and allowed to react at room temperature for 15 minutes. The reaction solution was electrophoresed on a 6% polyacrylamide gel at 75 V for 1 hour and 30 minutes. Subsequently, the polyacrylamide gel was detected using a gel imaging analyzer Typhoon 8600 (GE Healthcare Bioscience Co., Ltd.) and an 800 V excitation light source with a 532 nm laser. As a result, formation of a complex between the labeled nucleic acid probe consisting of SEQ ID NO: 19 and AhR, Arnt and TCDD was not confirmed. From this, it was confirmed that the GG sequence at the 3 ′ end is necessary for binding to the AhR, Arnt and TCDD complex. (Figure 6)
[Example 3]
Detection of specific binding activity with a labeled nucleic acid probe consisting of the sequences shown in SEQ ID NOs: 21 to 24 consisting of SEQ ID NO: 20 and a sequence obtained by adding a single base mutation to SEQ ID NO: 20 (gel shift method: EMSA)
(1) Preparation of AhR and Arnt
AhR gene (SEQ ID NO: 7) and Arnt gene (SEQ ID NO: 8) were respectively introduced into plasmid DNA pcDNA4 having a T7 promoter, and two plasmids, namely pcDNA4-rAhR (FIG. 3) and pcDNA4-rArnt (FIG. 4), were introduced. Produced. Thereafter, AhR and Arnt proteins were prepared by in vitro transcription-translation method using TNT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega) based on the template plasmid DNA.

(2) Formation of a complex containing AhR, Arnt and TCDD 1 μM 2,3,7,8-tetrachlorobenzo-p-dioxin (5 μl each of in vitro translation products AhR and Arnt mixed in a solution 0.1 μl of 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) (Kanto Chemical) was added and reacted at 30 ° C. for 90 minutes to form a complex of AhR, Arnt and TCDD. A control experiment was performed by adding 0.1 μl of DMSO to a solution in which 5 μl of AhR and Arnt were mixed, and reacting at 30 ° C. for 90 minutes.

(3) Preparation of Alexa Fluor (Registered Trademark) 532 Label Probe Oligonucleotide consisting of the sequence shown in SEQ ID NOs: 20 to 24 and its complementary strand include Alexa Fluor (Registered Trademark) 532 label at each 5 'end Was synthesized as follows. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes. Thereafter, the heat block was turned off and allowed to stand until it returned to room temperature. As a result, the single-stranded nucleic acid was made double-stranded. Thus, a labeled detection nucleic acid was prepared as an Alexa Fluor (registered trademark) 532 label probe.

(4) Reaction of complex containing AhR, Arnt and TCDD and Alexa Fluor (registered trademark) 532 label probe In the mixed solution prepared in (2), binding buffer [composition: 100 mM Hepes, pH 7.6, 5 mM EDTA, 50 mM (NH ₄ ) 2 SO ₄ , 5 mM DTT, 1% (w / v) Tween 20, 150 mM KCl], 100 ng / ul Poly [(dI-C)] solution, 1.4 M KCl solution were added Later, 90 fmol Alexa Fluor (registered trademark) 532 label probe was added and allowed to react at room temperature for 15 minutes. The reaction solution was electrophoresed on a 6% polyacrylamide gel under conditions of 75 V for 1 hour and 30 minutes. The polyacrylamide gel was then subjected to band detection using a 532 nm laser with an 800 V excitation light source using a gel imaging analyzer Typhoon (GE Healthcare Bioscience). This confirmed the formation of a complex containing the probe, AhR, Arnt, and TCDD (FIG. 7). From this result, it was shown that the sequence involved in the binding of the complex was NNNNTCGTGTNNNNNNNGG containing the GG sequence at the 3 ′ end in addition to TCGTGT. N included in the sequence shown here is an arbitrary nucleotide, and NNNN on the 5 ′ end side is not CATG, and NNNNNNN on the 3 ′ end side is not CTAGGGC.

[Example 4]
Detection of specific binding activity by the nucleic acid described in SEQ ID NO: 1 (QCM method: quartz crystal microbalance method)
(1) Immobilization of probes on the QCM sensor chip After the gold surface of the sensor chip was washed with 1% SDS solution, a piranha solution (sulfuric acid: hydrogen peroxide = 3: 1) was dropped onto the gold surface and allowed to stand for 5 minutes. Thereafter, washing with distilled water was performed. By repeating this operation twice, the gold surface on the sensor chip was activated. 100 μl of 5 mM DTDP (3,3′-Dithiodipropionicacid (manufactured by Sigma Aldrich)) solution was added dropwise and allowed to stand at room temperature for 30 minutes for reaction. After the reaction, the sensor chip surface was washed with distilled water. The 5 mM DTDP solution to be used was prepared by preparing a 100 mM stock solution using ethanol as a solvent and then diluting the stock solution with distilled water to prepare a 5 mM DTDP solution. 100 mg / ml NHS DW solution (N-Hydroxysuccinimide (WAKO)) and 100 mg / ml EDC DW solution (1- (3-Dimethylaminopropyl) -3ethylcarbodiimide, hydrochloride (Sigma)) were prepared. An NHS / EDC mixed solution obtained by mixing equal amounts of the solution was dropped on the surface of the sensor chip by 100 μl, and allowed to stand at room temperature for 30 minutes for reaction. After the reaction, the sensor chip surface was washed with distilled water.

  100 μl of 10 μl / ml Neutravidin (manufactured by PIERCE) was dropped, and the reaction was allowed to stand at room temperature for 2 hours to immobilize Neutravidin. After immobilization of Neutravidin, wash with distilled water, and then add blocking solution (TBS buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl) 500 μl with Block Ace stock solution 20 μl) on a 100 μl sensor chip. The sensor chip was blocked by dropping and allowing to stand for 1 hour.

  An oligonucleotide consisting of the sequence shown in SEQ ID NO: 1 and its complementary strand were synthesized so as to contain a biotin label at each 5 'end. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes. Thereafter, the heat block was turned off and allowed to stand until it returned to room temperature. As a result, the single-stranded nucleic acid was made double-stranded. Thereby, a labeled detection nucleic acid was prepared as a biotin-labeled probe.

  Immobilization of the double-stranded biotin label probe on the sensor chip was performed using QCM. After adding 1.6 ml of a reaction solution (TBS buffer) to a reaction tank of Affix-Q (Initium Co., Ltd.), a Neutravidin-immobilized sensor chip was inserted into the apparatus, and the sensor chip was set in the reaction solution. It was left still in the reaction solution until the vibration was stabilized (about 15 minutes). After confirming the stabilization of vibration, 8 μl of double-stranded biotin-labeled probe (10 μM) was added, and measurement was performed until the vibration was stabilized. Assuming that 30 pg of DNA was immobilized at 1 Hz, the amount of nucleic acid immobilized was calculated from the fluctuation value of the frequency after stabilization.

(2) Preparation of AhR and Arnt
PcDNA4-rAhR (FIG. 3) and pcDNA4-rArnt (FIG. 4) in which AhR gene and ARNT gene were introduced into plasmid DNA pcDNA4 having T7 promoter were prepared. Based on the template plasmid DNA, AhR and Arnt were prepared by in vitro transcription-translation method using TNT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega).

(3) Formation of a complex containing AhR, Arnt and TCDD 1 μM 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) in a solution containing 5 μl each of in vitro translation products AhR and Arnt ) (Kanto Chemical Co., Inc.) 0.1 μl was added and reacted at 30 ° C. for 90 minutes to form a complex of AhR, Arnt and TCDD. A control experiment was performed by adding 0.1 μl of DMSO to a solution in which 5 μl of AhR and Arnt were mixed, and reacting at 30 ° C. for 90 minutes.

(4) Detection of AhR-Arnt-TCDD complex by QCM A sensor chip on which a biotinylated and double-stranded nucleic acid represented by SEQ ID NO: 1 and a biotin label probe consisting of its complementary strand are immobilized is set in the QCM device. , The binding capacity of the sensor chip when the same amount of the mixed solution of AhR, Arnt and DMSO, and the solution formed by adding AhR-Arnt and TCDD to form a complex was dropped into the QCM reaction vessel. The presence or absence of the AhR-Arnt-TCDD complex was detected by comparing the change in frequency decrease with the control experiment.

(5) Device FIG. 8 shows a sensor chip on which the above-described biotin label probe is immobilized. In the sensor chip 81, a biotin label probe 74 is immobilized on an immobilization region 73 of a substrate 82 as a carrier.

  The device according to the present invention may be a device in which AhR or Arnt is used as a probe instead of the biotin-labeled probe in the immobilization region 83.

The schematic diagram which shows the transcriptional activation mechanism by AhR ligand in a cell. The figure which shows the outline | summary of the method concerning 1 aspect of this invention. 1 is a schematic diagram showing a vector pcDNA4-rAhR according to one embodiment of the present invention. 1 is a schematic diagram showing a vector pcDNA4-rArnt according to one embodiment of the present invention. Electrophoresis showing the results of a gel shift assay for confirming the binding of a complex of AhR, ARNT, and TCDD to each Fluor® 532 label probe having the nucleic acid sequence described in SEQ ID NOs: 10 to 18 Figure. FIG. 20 is an electrophoretogram showing the results of a gel shift assay for confirming the binding of a complex of AhR, Arnt, and TCDD and a Fluor (registered trademark) 532 label probe having the nucleic acid sequence shown in SEQ ID NO: 19; The electrophoretic diagram which shows the result of the gel shift assay which confirmed the presence or absence of the coupling | bonding of the complex of AhR, Arnt, and TCDD, and the Fluor (trademark) 532 label probe which has a nucleic acid sequence of sequence number 20-22. The figure which shows the concept of 1 aspect of this invention.

Claims (5)

In that sequence, N is any nucleotide, 5 'in the distal NNNN is CATG without and 3' of the distal NNNNNNN is not a CTAGGGC, polynucleotide shown in SEQ ID NO: 1. A method for detecting a compound that leads to the formation of a complex between an allyl hydrocarbon receptor (hereinafter referred to as AhR) and an allyl hydrocarbon receptor nuclear translocator (hereinafter referred to as Arnt),
(1) contacting a test substance, AhR, Arnt, and a nucleic acid for detection containing the sequence represented by SEQ ID NO: 1 ,
In here, N included in SEQ ID NO: 1 is any nucleotide, and 5 'of the terminal-side NNNN instead CATG, 3' not NNNNNNN end side in CTAGGGC,
(2) A detection method comprising detecting binding of the test substance, AhR, Arnt, and a detection nucleic acid.   The detection method according to claim 2, wherein the nucleic acid for detection is a 19-base long double-stranded chain comprising the sequence represented by SEQ ID NO: 1.   The detection of the binding in the above (2) compares the result obtained by the contact in the above (1) with the result obtained by the contact between the nucleic acid for detection, AhR and Arnt in the absence of the test substance. The detection method of Claim 2 or 3 performed by this. A method for detecting a substance to be detected in a sample,
(1) contacting the sample with AhR, Arnt, and a 19-base long nucleic acid for detection containing the sequence represented by SEQ ID NO: 1 ,
In here, N included in SEQ ID NO: 1 is any nucleotide, 5 'NNNN distal rather than CATG, and 3' NNNNNNN distal is not a CTAGGGC,
(2) detecting binding of the substance to be detected, AhR, Arnt, and the nucleic acid for detection;
(3) From the detection result of (2), determining whether the detected substance is included in the sample;
A detection method comprising:

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