JP2010075064A - Nucleic acid for detecting trace chemical substance and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for specifically, quickly and simply detecting a substance such as a compound for introducing a binding between AhR and ARNT, which simply detects the substance by a simple constitution without using an antibody-antigen reaction and a cultured cell. <P>SOLUTION: The method for detecting a compound introducing a binding between AhR and ARNT includes (1) bringing a test substance into contact with AhR and ARNT and a detecting nucleic acid comprising continuous 25-100 bases containing at least a part of sequence number 1 in double strand in a solution and (2) detecting a binding between the test substance, AhR and ARNT and the detecting nucleic acid obtained by the contact. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a detection nucleic acid for detecting a trace chemical substance and a method for detecting a trace chemical substance using the detection nucleic acid. In particular, the present invention provides for detection that specifically binds to a protein complex formed when, for example, a trace chemical is an endocrine disruptor and such trace chemical binds to a substrate-dependent transcription factor. The present invention relates to a nucleic acid for detection and a method for detecting a trace chemical substance using the detection nucleic acid.

  Along with the development of human life and industrial activities, the effects of chemical substances released into the environment have become a serious social problem. In particular, due to the increased awareness of environmental pollution such as environmental water and soil, there is an increasing need to accurately grasp the pollution status of so-called endocrine disrupting substances such as dioxins and PCBs.

  Allyl hydrocarbon receptors (also referred to as “AhR” here), known as receptors for dioxins and PCBs, are receptors to which polycyclic aromatic hydrocarbons (herein also referred to as “PAH”) bind, and the bHLH family. (Non-Patent Document 1). So far, many chemical substances having AhR binding have been reported to have endocrine disrupting properties and induce immunotoxicity and reproductive toxicity (Non-patent Document 2). Therefore, there is a demand for rapid and simple detection of AhR ligands.

  Please refer to FIG. Once AhR ligand-like substance 1 such as dioxin enters cell 5, it reversibly binds to AhR2 and activates AhR2. The activated AhR2 is transported to the nucleus 6 of the cell 5 while being bound to the AhR ligand-like substance 1. In nucleus 6, activated AhR2 forms AhR-ARNT complex with AhR nuclear translocation factor 3 (herein also referred to as “ARNT”), and this complex recognizes and binds to a specific sequence of DNA. . This DNA sequence is called casting response element 4 (Xenobiotics Responsive Elements, hereinafter referred to as “XRE”). When the activated AhR-ARNT complex binds to XRE4, the gene under the control of the AhR-ARNT complex downstream of XRE4 is transcribed. The transcribed and generated mRNA is transported out of the nucleus 6, enters the cytoplasm, is translated in the cytoplasm, and a new protein is synthesized. This protein is considered to be a toxic expression of dioxins (Non-patent Document 3) (FIG. 1).

  In this kind of detection method for AhR ligand-like substances, a known ligand that competitively binds to the detected substance and binds to the AhR receptor is used to compete with the detected substance and the free state is not bound to the AhR receptor. A known method is known in which the amount of a known ligand is detected by an antigen-antibody reaction to detect the presence of a substance to be detected (Patent Document 1).

  Some detection methods for this type of AhR ligand-like substance use living cells to detect the presence of a substance to be detected that exhibits activity by a reporter gene assay. For example, using the fact that the expression of tyrosine hydroxylase involved in dopamine biosynthesis is increased by AhR activation (Non-patent Document 4), by measuring the activity of this transcriptional regulatory ability, substrate binding 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 a characteristic (Patent Document 2).

  The measurement method involves introducing a vector having a reporter gene linked downstream of the recognition sequence of AhR into the cell, and measuring the expression level of the reporter gene when the target substance is brought into contact with the cell and cultured. Thus, there is a method for measuring the activity of a substance to be detected with respect to the transcription control of a transcription factor having a ligand binding property.

The above method is a method for detecting a conventional trace chemical substance as a substance to be detected. In such a method, a skillful technique is required for the production of an antibody, and the production takes a long time and the production cost is also expensive. In addition, in the method using cultured cells, skilled techniques for handling cultured cells are required, and expensive equipment for cell culture is also required.
JP 2000-283984 A Japanese Unexamined Patent Publication No. 2007-202555 Mark E. Hahn, Comparative Biochemistry and Physiology Part C 121 (1998): 23-53. Frank JG et al., Drug Metabolism and Disposition (1998) 26: 1194-1198. Mimura J et al., Genes Dev (1999) 13; 20-25. Akahoshi E et al., Environ Health. (2006) 7; 5; 24

  The present invention can detect a substance that induces the binding between AhR and ARNT, for example, a compound with a simple configuration without using an antibody-antigen reaction or cultured cells, and can detect it specifically, quickly and easily. It aims to provide a method.

To achieve the above object, the present invention provides:
A method for detecting a compound that induces binding between AhR and ARNT, comprising:
(1) contacting a test substance, AhR, ARNT, and a nucleic acid for detection consisting of continuous 25 to 100 bases containing at least a portion of the sequence represented by SEQ ID NO: 1 in a double strand in a solution; ,
(2) detecting the binding of the test substance obtained by the contact, AhR, ARNT, and a nucleic acid for detection;
It is the method which comprises.

  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, and a method for specific, quick and simple detection is provided. The

According to one aspect of the invention,
A method for detecting a compound that induces binding between AhR and ARNT, comprising:
(1) contacting a test substance, AhR, ARNT, and a nucleic acid for detection consisting of continuous 25 to 100 bases containing at least a portion of the sequence represented by SEQ ID NO: 1 in a double strand;
(2) detecting the binding of the test substance obtained by the contact, AhR, ARNT, and a nucleic acid for detection;
Is provided.

  This method utilizes the phenomenon of binding of AhR, ARNT, AhR ligand, and XRE 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 of 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.

According to a further aspect of the invention,
A method for determining whether 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 continuous 25 to 100 bases containing a portion of the sequence represented by SEQ ID NO: 1 in double strands;
(2) detecting the 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.

  Please refer to FIG. The principle of the present invention 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. 2A). 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. 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 the compound that induces the bond between AhR and ARNT does not exist in the reaction system 11, FIG. As shown, 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. By using the same principle, it is possible to determine whether or not a sample contains a compound that induces the 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. For example, food, reagent, medicine, lake water, seawater, river water, sewage, waste, etc. Any substance that can be included, or suspected to include a compound that induces the binding of AhR and ARNT, may be included.

  Here, the “compound that induces the binding between AhR and ARNT” 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”.

  Such a method according to the present invention is based on the results of diligent research, in which the present inventors bind a compound that induces the binding of AhR and ARNT to a complex of AhR and ARNT and detect the nucleic acid that enables the detection. It was achieved by finding.

  That is, the present inventors are useful for detecting a compound in which a nucleic acid having a DNA sequence of 25 bases located upstream of the transcription start site of mouse TH (Tyrosine Hydroxylase) gene induces the binding between AhR and ARNT. And was isolated as a nucleic acid for detection.

  Therefore, the nucleic acid for detection of the present invention may include a nucleic acid represented by a continuous 25-base DNA sequence existing in the upstream region of the transcription start site of mouse TH (Tyrosine Hydroxylase) gene. Specifically, the DNA sequence of 25 bases is a mouse TH gene upstream -274 to -249 base sequence (numbers represent positions where the transcription start point is +1 and the immediately preceding base is -1). This 25-base DNA sequence is shown in the sequence listing as SEQ ID NO: 1. In addition, the nucleic acid for detection of the present invention needs to be double-stranded at this 25 bases, that is, at least a site corresponding to the upstream -274 to -249 base sequence of the mouse TH gene.

  The “nucleic acid” used herein may be a nucleotide such as a synthetic or naturally-derived DNA or oligonucleotide. Artificial nucleic acids such as LNA and peptide nucleic acids may also be used.

  The present invention will be further described in detail below.

1. Nucleic acid for detection The nucleic acid for detection according to the present invention may be a nucleic acid containing at least a portion of the sequence represented by SEQ ID NO: 1 in a double strand.

  One of the nucleic acids contained in the double strand is a 25-base nucleic acid represented by SEQ ID NO: 1, a 25-100 base nucleic acid comprising the nucleic acid represented by SEQ ID NO: 1, a 61-base nucleic acid represented by SEQ ID NO: 2, And any nucleic acid that hybridizes to these sequences under stringent conditions. The other nucleic acid contained in the double strand is a complementary strand of a 25 base nucleic acid represented by SEQ ID NO: 1, a complementary strand of a 25 to 100 base nucleic acid containing a nucleic acid represented by SEQ ID NO: 1, and SEQ ID NO: 2. The complementary strand of the 61-base nucleic acid shown and the complementary strand of the nucleic acid that hybridizes to these sequences under stringent conditions may be used. The important site of the nucleic acid for detection is the sequence shown in SEQ ID NO: 1, and at least this part needs to form a double strand with another single strand having complementarity. Other portions may form a double strand by complementary strands, may form a double strand together with a single strand that hybridizes to these sequences under stringent conditions, or may be a single strand There may be.

The nucleic acid comprising the sequence represented by SEQ ID NO: 2 is a nucleic acid comprising a nucleic acid comprising the sequence represented by SEQ ID NO: 1 at the first position to the 25th position.

  Furthermore, the nucleic acid for detection according to the present invention may be a nucleic acid comprising a sequence capable of hybridizing under stringent conditions to the sequence represented by SEQ ID NO: 1 or 2. Further, it may be a 25 to 100 base nucleic acid containing a sequence capable of hybridizing under stringent conditions to the sequence represented by SEQ ID NO: 1 or 2. Even in this case, when the nucleic acid for detection is used, the 25-base portion corresponding to SEQ ID NO: 1 needs to form a double strand with its complementary strand.

  The nucleic acid that hybridizes under stringent conditions may be any nucleic acid having a certain degree of homology with the single strand to be hybridized.

  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. “Homology above a certain level” 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. 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, a nucleic acid having 25 to 100 bases comprising SEQ ID NO: 1 linked several times, for example, 2 to 10 nucleic acids can be used as the detection nucleic acid.

  Such a nucleic acid for detection may be chemically synthesized, a naturally occurring nucleic acid may be isolated, and in any case, it may be easily 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 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, and may be synthesized using genetic engineering.

  AhR and ARNT are preferably derived from rats, 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: 3 and having a histidine tag / V5 tag added to the C-terminus, and SEQ ID NO: 4 Examples include fused rat ARNT encoded by the nucleotide sequence described in 1 and having a histidine tag / V5 tag added to the C-terminus.

本発明において使用されるAhRおよびARNTは、細胞抽出液として何れかの生物組織から調製されてもよい。また、大腸菌、酵母、昆虫または動物培養細胞を用いる蛋白質発現系を用いて調製されてもよい。或いは、無細胞発現系（即ち、インビトロトランスクリプション/トランスレーション）によって調製することも可能である。

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 AhR and ARNT are prepared as a cell extract, AhR and ARNT may be prepared by extracting proteins from cells expressing AhR and ARNT by whole known methods or nuclei 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 produced 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 in the downstream region. PTargetT vector having SV40 enhancer and pSI vector having SV40 early promoter (Promega), pBK-CMV, CMV-Script, pCMV-Tag and pBK-RSV (Stratagene) and pcDNA4 vector series (Invitrogen) And so on.

  Examples of 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, and yeast. The system may be a Pichia expression vector pIC series vector (for example, pPIC9K or PIC3.5K; Invitrogen) having an alcohol oxidase promoter.

  When AhR and ARNT are prepared 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.

  As a vector suitable for such a method, for example, a pTargetT vector having an immeadiate-early enhancer / promoter region of human cytomegalovirus and a T7 promoter sequence / multicloning site downstream thereof, SV40 enhancer and SV40 Examples include pSI vectors having an early promoter (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 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 continuous 25 to 100 bases containing a portion of the sequence represented by SEQ ID NO: 1 in a double strand in a solution; ,
(2) detecting the 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.

  Further, 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 a solution in the presence of a sample containing the test compound; and (b) detecting the binding between the detection nucleic acid, AhR and ARNT. To do
A method comprising:
A method of screening for a compound that induces binding between AhR and ARNT,
(A) contacting the nucleic acid for detection according to the present invention with AhR and ARNT in a solution in the presence of a sample containing the test compound;
(B) detecting the binding of the detection nucleic acid, the test compound, AhR, and ARNT;
(C) selecting a compound that induces binding between the nucleic acid for detection, AhR and ARNT;
A method comprising:
Etc.

  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 pH 7.6, 5 mM EDTA, 50 mM DTT, 1% (w / v) Tween 20, 150 mM KCl), 25 ° C. to 30 ° C., preferably The contact may be performed 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 with 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 nucleic acid for detection, 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 AhR and ARNT prepared by any of the methods described above 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 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 a single strand at 95 ° C. for 5 minutes in a heat block, and then the heat block is turned off and left to return 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 typified by ³² P, fluorescent substances typified by fluorescein and Alexa dye can be used.

The reaction is performed, 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. Then, labeled detection nucleic acid was added 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 at 15-37 ° C. After 15 minutes of reaction, run on a 4-10% non-denaturing polyacrylamide gel.

  If necessary, when reacting the nucleic acid for label binding, AhR and ARNT, unlabeled nucleic acid (10 to 500 times the amount of nucleic acid for label binding) 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 AC electric field is applied to each metal thin film, the crystal oscillator has a certain frequency (resonance frequency). Shows vibration properties. When a substance of about nanogram is adsorbed on a metal thin film, the resonance frequency decreases in proportion to the mass of the substance, so that it can be used as a microbalance. Such a methodology is called QCM (Quartz Crystal Microbalance).

  A biotin-labeled double-stranded oligonucleotide of usually 15 to 30 bases is immobilized as a detection nucleic acid on the surface of a QCM sensor chip on which an irreversible bond, for example, avidin is immobilized. By immersing a QCM sensor chip with detection nucleic acid immobilized in a reaction vessel containing Tris buffer and adding AhR and ARNT to the reaction vessel, the binding property of the protein to the detection nucleic acid can be determined in real time. It can be evaluated 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. If the immobilized substance, that is, the detection nucleic acid and the substance in the liquid, that is, AhR and ARNT interact with each other, a refractive index change and / or a layer thickness change occur near the surface, so that the detection nucleic acid or AhR Alternatively, the interaction can be analyzed without labeling ARNT.

  In detection by electrochemical measurement, the binding between the detection nucleic acid, AhR and ARNT is detected using an enzyme that catalyzes a chemical reaction that produces an electrically active compound or an electrically active compound. May be. When the detection nucleic acid is labeled by the measurement method, the labeled detection nucleic acid is reacted with AhR or ARNT supported on a conductive carrier, and non-specific binding substances are washed away. Later, the electrochemical response may be measured. In the case of labeling AhR or ARNT, the labeled AhR or ARNT is reacted with the detection nucleic acid supported on a conductive carrier, and the non-specific binding substance is washed and removed. What is necessary is just to measure a chemical response. The labeling to the nucleic acid for detection 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, react the labeled AhR or ARNT with the nucleic acid for detection carried on the carrier, wash away non-specific binding substances, and measure the fluorescence intensity. 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 measure the radiation intensity. That's fine In the case of labeling AhR or ARNT, after reacting labeled AhR or ARNT with the nucleic acid for detection supported on the carrier and washing away non-specific binders, the radiation intensity Can be measured.

4). Detection Device In the present invention, it is also possible to produce a multi-analyte detection device in which the detection nucleic acid or AhR or ARNT is immobilized in an array on a carrier and use it for detection and evaluation.

  Such devices are made so that measurement by mass sensor elements, electrochemical measurements, fluorescence measurements, and radiation measurements can be conveniently performed and evaluated. Such a device may be a detection probe-immobilized chip in which the detection nucleic acid is immobilized on a carrier as a probe, 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 a 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 You may fix | immobilize by a coupling | bonding, physical adsorption, or chemical adsorption.

  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 immobilization chip, and a DNA binding factor protein immobilization 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, there is also provided a detection kit comprising a probe-immobilized chip comprising A substrate and the detection nucleic acid according to the present invention immobilized on the substrate, AhR and ARNT. The Further, a detection kit comprising a DNA binding factor protein-immobilized chip comprising a substrate and AhR immobilized on the substrate, a detection nucleic acid according to the present invention, and an ARNT 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 detection nucleic acid 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, it 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, a compound that induces the binding between AhR and ARNT present in a trace amount in the environment can be detected easily, inexpensively, and with high accuracy. In addition, compounds that lead to the binding of AhR and ARNT are likely to work as endocrine disrupting substances when taken into the body, so endocrine disrupting substances present in the environment and ingestions 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, a trace chemical substance can be easily detected by a simple method using a material that can be prepared at low cost.

[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
Detection of specific binding activity with the nucleic acid for detection shown by SEQ ID NO: 1 (gel shift method: EMSA)
(1) Preparation of AhR and ARNT
AhR gene (SEQ ID NO: 3) and ARNT gene (SEQ ID NO: 4) were introduced into plasmid DNA pcDNA4 having a T7 promoter, respectively, and two plasmids, namely pcDNA4-AhR (FIG. 3) and pcDNA4-ARNT (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 of 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: 1 and its complementary strand are included at their 5 'ends so that Alexa Fluor (Registered Trademark) 532 label is included. Synthesized. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes to form a single strand, and then 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 Binding buffer solution (composition: 100 mM Hepes, pH 7.6, 5 mM) in the mixed solution prepared in (2) 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, 15 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 at 75 V for 1 hour and 30 minutes. Thereafter, the polyacrylamide gel was 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 Co., Ltd.). This confirmed the formation of a complex containing the probe, AhR, ARNT, and TCDD (FIG. 5).

Example 2
Detection of specific binding activity by the nucleic acid represented by SEQ ID NO: 2 (gel shift method: EMSA)
(1) Preparation of AhR and ARNT
AhR gene and ARNT gene were introduced into plasmid DNA pcDNA4 having a T7 promoter to prepare two plasmids, namely pcDNA4-AhR (FIG. 3) and pcDNA4-ARNT (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 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 An oligonucleotide consisting of the sequence shown in SEQ ID NO: 2 and its complementary strand are included at their 5 'ends so that Alexa Fluor (Registered Trademark) 532 label is included. Synthesized. This synthesized product was treated in a heat block at 95 ° C. for 5 minutes to form a single strand, and then 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 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 15 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 30 minutes. Subsequently, the polyacrylamide gel was detected with a 532 nm laser with an 800 V excitation light source using a gel imaging analyzer Typhoon 8600 (GE Healthcare Bioscience). Thereby, the complex formation of the probe, AhR, ARNT, and TCDD was confirmed (FIG. 6).

Example 3
Detection of specific binding activity with 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 is washed with 1% SDS solution, a piranha solution (sulfuric acid: hydrogen peroxide = 3: 1) is dropped on the gold surface and left for 5 minutes. And washed with distilled water. By repeating this operation twice, the gold surface on the sensor chip was activated. After adding 100 μl of 5 mM DTDP (3,3′-Dithiodipropionicacid (Sigma Aldrich)) solution dropwise, the reaction was allowed to stand at room temperature for 30 minutes. After the reaction, the sensor chip surface was washed with distilled water. The 5 mM DTDP solution to be used was prepared after preparing a 100 mM stock solution using ethanol as a solvent, and 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 made into a single strand at 95 ° C. for 5 minutes in a heat block, and then 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-AhR (FIG. 3) and pcDNA4-ARNT (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 obtained by mixing 5 μl each of AhR and ARNT 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-labeled probe consisting of its complementary strand are immobilized is set in the QCM device. The sensor chip has a binding property to the sensor chip when a mixed solution of AhR, ARNT and DMSO, and a solution formed by adding AhR-ARNT and TCDD to form a complex are dropped into the QCM reaction tank. The presence or absence of AhR-ARNT-TCDD complex was detected by comparing the change in frequency decrease with the control experiment.

(5) Device FIG. 7 shows a sensor chip on which the above-described biotin label probe is immobilized. The sensor chip 71 has a biotin label probe 74 immobilized on an immobilization region 73 of a substrate 72 as a carrier.

  The device according to the present invention may be a device immobilized on the immobilization region 73 using AhR or ARNT as a probe instead of the biotin label probe.

Schematic diagram showing the mechanism of transcriptional activation by AhR ligands in cells The figure which shows the concept of 1 aspect of this invention. 1 is a schematic diagram showing a vector pcDNA4-AhR according to one embodiment of the present invention. 1 is a schematic diagram showing a vector pcDNA4-ARNT according to one embodiment of the present invention. The electrophoretic diagram which shows the result of the gel shift assay which confirmed 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 1. The electrophoretic diagram which shows the result of the gel shift assay which confirmed 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 2. The figure which shows the concept of 1 aspect of this invention.

Explanation of symbols

  1. 1. AhR ligand-like substance AhR 3. AhR nuclear translocation factor Casting response array 5. Cells 6. Nuclear 11. Reaction system 12. Test substance 13. AhR 14. ARNT 15. Nucleic acid for detection 71. Sensor chip 72. Substrate 73. Immobilization region 74. Biotin label probe

Claims (28)

A method for detecting a compound that induces binding between AhR and ARNT, comprising:
(1) contacting a test substance, AhR, ARNT, and a nucleic acid for detection consisting of continuous 25 to 100 bases containing at least a portion of the sequence represented by SEQ ID NO: 1 in a double strand in a solution; ,
(2) detecting the binding of the test substance obtained by the contact, AhR, ARNT, and a nucleic acid for detection;
A method comprising:   The detection of the binding in (2) above is the result obtained by contacting the test substance, AhR, ARNT and the detection nucleic acid, and the detection nucleic acid, AhR and ARNT in the absence of the test substance. The method according to claim 1, wherein the method is carried out by comparing the result obtained by contact with each other.   2. The method according to claim 1, wherein the nucleic acid for detection is a single-stranded nucleic acid comprising a sequence represented by SEQ ID NO: 1 and a double-stranded nucleic acid comprising a complementary strand thereof.   2. The method according to claim 1, wherein the nucleic acid for detection is a single-stranded nucleic acid comprising a sequence represented by SEQ ID NO: 2 and a double-stranded nucleic acid comprising a complementary strand thereof.   2. The method according to claim 1, wherein the detection nucleic acid comprises a single-stranded nucleic acid comprising the sequence represented by SEQ ID NO: 1, and a nucleic acid that hybridizes with the single-stranded nucleic acid under stringent conditions. A method comprising a double-stranded nucleic acid.   2. The method according to claim 1, wherein the detection nucleic acid comprises a single-stranded nucleic acid comprising the sequence represented by SEQ ID NO: 2 and a nucleic acid that hybridizes with the single-stranded nucleic acid under stringent conditions. A method comprising a double-stranded nucleic acid.   The method according to any one of claims 1 to 6, wherein the step of detecting the binding of the test substance, the detection nucleic acid, AhR, and ARNT is based on an electrophoretic measurement method. Method.   The method according to claim 7, wherein the electrophoretic measurement method is an EMSA method.   The detection method according to any one of claims 1 to 6, wherein the step of detecting the binding of the test substance, the detection nucleic acid, AhR and ARNT is a mass change measurement method using a mass sensor element. A method characterized by being performed by:   The detection method according to claim 9, wherein the mass change detection method is a quartz crystal microbalance method.   The detection method according to claim 9, wherein the mass change detection method is a surface plasmon resonance (SPR) method.   The method according to any one of claims 1 to 6, wherein the step of detecting the binding of the test substance, the nucleic acid for detection, AhR, and ARNT is performed by an electrochemical measurement method. And how to.   13. The method according to claim 12, wherein the detection nucleic acid, AhR, ARNT, and the test substance are in contact with the detection nucleic acid carried by a carrier made of a conductor or a carrier having a conductor. Contacting AhR or ARNT labeled with an enzyme protein that catalyzes a chemical reaction that produces an electrochemically active compound or an electrochemically active compound with unlabeled ARNT or AhR and the test substance A method characterized by being performed.   The method according to claim 12, wherein the contact between the nucleic acid for detection, AhR, ARNT, and the test substance is carried by a carrier made of a conductor or a carrier having a conductor, and AhR or ARNT, Contacting the analyte with the nucleic acid for detection labeled with an enzyme protein that catalyzes an electrochemically active compound or a chemical reaction that produces an electrochemically active compound, released ARNT or AhR, and the analyte A method characterized by being performed by:   The method according to any one of claims 1 to 6, wherein the step of detecting the binding of the test substance, the detection nucleic acid, AhR, and ARNT is performed by a fluorescence measurement method. how to.   The method according to claim 15, wherein the detection nucleic acid, AhR, ARNT, and the test substance are contacted with the detection nucleic acid supported by a carrier, and AhR or ARNT labeled with a fluorescent substance. The method is performed by contacting with unlabeled ARNT or AhR.   16. The detection method according to claim 15, wherein the nucleic acid for detection, AhR, ARNT, and the test substance are contacted by AhR or ARNT supported by a carrier, unsupported ARNT or AhR, and a fluorescent substance. The method is carried out by bringing the nucleic acid for detection labeled with the substance into contact with the test substance.   The method according to any one of claims 1 to 6, wherein the step of detecting the binding of the nucleic acid for detection, AhR, ARNT, and the test substance is performed by a radiation measurement method. how to.   19. The method according to claim 18, wherein the detection nucleic acid, AhR, ARNT, and the test substance are contacted with the detection nucleic acid supported by a carrier and the radioisotope-labeled AhR or ARNT. And an unlabeled ARNT or AhR and the test substance are brought into contact with each other.   19. The detection method according to claim 18, wherein the nucleic acid for detection, AhR, ARNT, and the test substance are contacted with AhR or ARNT supported by a carrier, unsupported ARNT or AhR, and radioisotope. A method comprising: bringing the detection nucleic acid labeled with a body into contact with the test substance.   21. The method according to any one of claims 1 to 20, wherein the detection nucleic acid is DNA or oligonucleotide. A method for detecting a substance to be detected contained in a sample,
(1) contacting the sample with AhR, ARNT, and a nucleic acid for detection consisting of continuous 25 to 100 bases comprising at least a portion of the sequence represented by SEQ ID NO: 1 in a double strand;
(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 test compound is included in the sample;
A method comprising: A method for determining whether 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 continuous 25 to 100 bases containing a portion of the sequence represented by SEQ ID NO: 1 in a double strand;
(2) detecting the 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;
A method comprising:   The method according to any one of claims 22 and 23, wherein the substance to be detected or the substance to be detected is an AhR ligand-like substance.   The method according to any one of claims 22 and 23, wherein the substance to be detected or the substance to be detected is an endocrine disrupting substance.   A multi-analyte detection device for use in the method according to any one of claims 7 to 20, comprising a carrier and nucleic acids for detection immobilized in an array on the carrier, AhR or ARNT A device comprising:   A detection kit for use in the method according to any one of claims 7 to 20, comprising the detection nucleic acid, AhR and ARNT, wherein the detection nucleic acid, AhR or ARNT A detection kit, wherein any one of the above is immobilized on a carrier.   A single-stranded nucleic acid comprising a sequence represented by SEQ ID NO: 1 and a double-stranded nucleic acid comprising a complementary strand thereof, which is used for detecting a compound that induces the binding between AhR and ARNT.

Images