JP2002045188A - Dioxin receptor gene and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new dioxin receptor gene utilized for a measurement for dioxin-like substances. SOLUTION: This dioxin receptor gene comprises the base sequence of the following either (a) or (b): (a) a base sequence encoding a specific amino acid sequence derived from a Chinese hamster or CHO(Chinese hamster ovary) cell; or (b) a base sequence encoding a dioxin receptor containing an amino acid sequence which shows the amino acid homogeneity of more than 95% to the above amino acid sequence. A vector containing the above dioxin receptor gene, a virus particle containing the vector and a transformant produced by transducing the above dioxin receptor gene into the host cell consisting of mammal cells or insect cells are obtained. Further, a method for producing dioxin receptor by the culture of the transformant and a receptor-binding assay containing a process for contacting the above dioxin receptor with a test substance and keeping them warm are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a dioxin receptor gene and its use.

[0002]

BACKGROUND OF THE INVENTION Generally, polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) are collectively referred to as dioxins, to which coplanar polychlorinated biphenyl (PCB) has been added. Three kinds of compounds are collectively referred to as dioxin-like substances. Each of these dioxin-like substances has a number of structural isomers and homologs depending on the number and position of substituted chlorine, but some of them cause reproductive abnormalities, immune abnormalities, carcinogenesis, etc. even in extremely small amounts in vivo. For this reason, attempts have been made to measure toxic dioxin-like substances as part of the safety evaluation of living environments and foods. A dioxin-like substance is intracellularly used as a dioxin receptor (also called an allyl hydrocarbon receptor (AhR)) (Mol. Pharmacol., 39, 13-19 (1991); Proc. Natl.
Acad. Sci. USA., 89, 8185-8189 (1992)) to elicit its toxicity (Ann. Rev. Pharmacol. Toxico).
l., 22,517-554 (1982)). When a dioxin-like substance such as 2,3,7,8-TCDD binds to a dioxin receptor, the receptor is activated and translocated into the nucleus, and then Arnt (Scie
nce, 252, 954-958 (1991)), and forms a heterodimer with a dioxin responsive element on the chromosome [Dioxin responsive element (DRE);
Sometimes called an ic responsive element (XRE). J.
Biol. Chem., 263, 17221-17224 (1988)], thereby activating transcription of a gene downstream of the sequence.
Therefore, a reporter gene serving as an indicator of the dioxin receptor activation ability is bound downstream of the dioxin response element, and this is introduced into a cell that expresses a dioxin receptor. A method for measuring the dioxin receptor activation ability of a test substance by using the gene expression level as an index has been proposed (Fund.Appl.Toxicol., 30, 194-203 (199).
6)). By measuring the dioxin receptor activating ability of various test substances using such a method, it becomes possible to detect a chemical substance capable of causing dioxin-like toxicity. There is a strong need for the isolation of a dioxin receptor gene that can be used for E. coli.

[0003]

Means for Solving the Problems Under such circumstances, the present inventors have conducted intensive studies, and as a result, have succeeded in isolating a novel dioxin receptor gene, and have achieved the present invention.
That is, the present invention provides: 1) a dioxin receptor gene (hereinafter, referred to as the gene of the present invention) having any one of the following base sequences (a) and (b): (a) an amino acid sequence represented by SEQ ID NO: 1 Nucleotide sequence encoding (b) a base sequence encoding a dioxin receptor having an amino acid sequence showing 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1; 2) base number 10 in the base sequence represented by SEQ ID NO: 2
3) a dioxin receptor gene having the nucleotide sequence of SEQ ID NO: 2835, 3) a vector containing the gene of the present invention (hereinafter referred to as the vector of the present invention), 4) incorporating the gene of the present invention into a vector capable of replicating in a host cell. 5) a transformant obtained by introducing the gene of the present invention into a host cell (hereinafter referred to as the transformant of the present invention); 6) introducing the gene of the present invention into the host cell 7) a method for producing a dioxin receptor, which comprises culturing the transformant of the present invention to produce a dioxin receptor; and 8) a method for producing a dioxin receptor from the amino acid sequence represented by SEQ ID NO: 1. 9) 95% of the amino acid sequence represented by SEQ ID NO: 1
A dioxin receptor having an amino acid sequence having the above amino acid identity; 10) a transformant which expresses the gene of the present invention, and into which a reporter gene linked downstream of a transcription control region containing a dioxin response element has been introduced; Contact with things,
A method for evaluating the ability of a test substance to regulate dioxin receptor activity, comprising the step of measuring the expression level of the reporter gene in the transformant; 11) the dioxin receptor according to any one of 8) or 9) above; A receptor binding assay comprising a step of contacting the sample with a test substance and keeping the mixture warm.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The gene of the present invention includes a dioxin receptor gene having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, and a dioxin having an amino acid sequence exhibiting 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1. A dioxin receptor gene having a base sequence encoding a receptor, a dioxin receptor gene having a base sequence represented by base numbers 10 to 2835 of the base sequence represented by SEQ ID NO: 2, and the like are included. Here, “dioxin receptor having an amino acid sequence showing 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1” includes:
A dioxin receptor having an amino acid sequence showing 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1 and having a receptor function substantially equivalent to that of the dioxin receptor consisting of the amino acid sequence represented by SEQ ID NO: 1 Can be given. The receptor function can be evaluated based on, for example, a reporter assay and a receptor assay described below. Examples of the gene of the present invention include a dioxin receptor gene having a base sequence encoding a dioxin receptor having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. You can also. Here, "several" means 2 to about 50, preferably 2 to about 20, and more preferably about 2 to about 10. Such amino acid deletions, substitutions, and additions include naturally occurring polymorphic mutations such as differences in amino acid sequences based on differences in animal strains, individuals, organs, tissues, and the like. The gene of the present invention may be a natural gene, or may be a gene created by introducing a mutation into a natural gene, for example, by site-directed mutagenesis or mutagenesis. .

[0005] The gene of the present invention can be obtained, for example, from the tissue of a Chinese hamster (Cricetulus griseus) or from a CHO cell, which is an established cell line, in J. Sambrook, EFFrisc.
h, T. Maniatis; Molecular Cloning 2nd Edition (M
olecular Cloning 2nd edition), Cold Spring Harbor Laborat
ory) can be obtained according to the genetic engineering method described in 1989 and the like. Specifically, first, RNA derived from a tissue such as a Chinese hamster is prepared. For example, CHO cells, a cell line derived from a tissue such as the liver or skin of a Chinese hamster or the ovary of a Chinese hamster, are crushed in a solution containing a strong protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and further crushed. The protein is denatured by adding phenol, chloroform, etc. to the product. After removing the denatured protein by centrifugation or the like, total RNA is extracted from the collected supernatant fraction by a method such as a guanidine hydrochloride / phenol method, an SDS-phenol method, or a guanidine thiocyanate / CsCl method. Commercially available kits based on these methods include, for example, ISOGEN (manufactured by Nippon Gene).
There is. Using the obtained total RNA as a template, an oligo dT primer is annealed to the poly A sequence of the RNA, and a single-stranded cDNA is synthesized by the action of reverse transcriptase.
Next, using the single-stranded cDNA as a template,
A double-stranded cDNA is synthesized using Escherichia coli DNA polymerase I with an RNA fragment obtained by forming a nick and a gap in the RNA strand using aseH as a primer. Further, both ends of the double-stranded cDNA are T4 DN
Smoothing with A polymerase. Obtained double-stranded cD
NA is purified and recovered by ordinary methods such as phenol and chloroform extraction and ethanol precipitation. Commercially available kits based on these methods include, for example, cDNA
Synthetic System Plus (Amersham Pharmacia) and
TimeSaver cDNA synthesis kit (Amersham Pharmacia) and the like. Next, the obtained double-stranded cDNA
Can be inserted into a vector such as plasmid pUC118 or phage λgt10 using ligase to obtain cDNA.
Create a library. From such a cDNA library, for example, a hybridization method using a DNA having a partial base sequence of the base sequence of SEQ ID NO: 2 as a probe, or an oligonucleotide having a partial base sequence of the base sequence of SEQ ID NO: 2 Polymerase chain reaction (hereinafter referred to as P
Recorded as CR. ), The gene of the present invention can be obtained. Examples of hybridization conditions include stringent conditions. Specifically, for example, 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 4
00, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5
% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA, or in a DIG EASY Hyb solution (Boehringer Mannheim) containing 100 μg / ml denatured salmon sperm DNA, and incubated at 65 ° C., then 1 × Incubation was performed twice at room temperature for 15 minutes in the presence of SSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.5% SDS, and further 0.1 × SSC (0.015 M NaCl, 0.0015 M sodium citrate) and 0.5% SDS In the presence, conditions for keeping the temperature at 68 ° C. for 30 minutes can be given. As a primer used for PCR, for example, a base sequence having a length of about 20 bp to about 40 bp and a ratio of G or C bases of about 40% to about 60% is the same as the base sequence represented by SEQ ID NO: 2. It is preferable to select from the 'untranslated region and the 3' untranslated region, design an oligonucleotide based on the base sequence, and synthesize the oligonucleotide. Specifically, for example, the forward primer includes an oligonucleotide having the base sequence of SEQ ID NO: 3, and the reverse primer includes an oligonucleotide having the base sequence of SEQ ID NO: 4. The gene of the present invention thus obtained is, for example, J. Sambrook, EFFrisch, T. Mani
by atis; Molecular Cloning 2nd Edition (Molecular
Cloning 2nd edition), published by Cold Spring Harbor Laboratory, 1989, etc., and can be cloned into a vector according to the genetic engineering method described in 1989 and the like. Specifically, for example, a TA cloning vector (Invitrogen) or pBlu
The gene of the present invention can be cloned using a commercially available plasmid vector such as escriptII (Stratagene). The gene of the present invention can be prepared, for example, by the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984) based on the nucleotide sequence shown in SEQ ID NO: 2.
It can also be prepared by chemically synthesizing a nucleic acid according to an ordinary method such as that described above. The nucleotide sequence of the obtained gene of the present invention is determined by the Maxam Gilbert method (for example, Maxam, AM & W. Gi.
lcad, Proc. Natl. Acad. Sci. USA, 74, 560, 1977) and the Sanger method (for example, Sanger, F. & ARCouls).
on, J. Mol. Biol., 94, 441, 1975; Sanger, F, & Nickle
n and ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 54
63, 1977, etc.).

[0006] The vector of the present invention contains, for example, a vector that can be used in a host cell to be transformed with the gene of the present invention obtained by the above-described method, for example, a genetic information that can be replicated in the host cell, and Which can be isolated and purified from host cells and can be inserted into a vector having a detectable marker (hereinafter referred to as a basic vector) by using a conventional genetic engineering technique. It can be built by doing. As a basic vector that can be used for constructing the vector of the present invention, specifically, when a microorganism, Escherichia coli, is used as a host cell, for example, plasmid pUC119 (manufactured by Takara Shuzo Co., Ltd.)
And phagemid pBluescript II (Stratagene)
Etc. can be given. When budding yeast is used as a host cell, plasmid pACT2 (manufactured by Clontech) and the like can be mentioned. When mammalian cells are used as host cells, plasmids such as pRC / RSV, pRC / CMV (manufactured by Invitrogen), bovine papilloma virus plasmid pBPV (manufactured by Amersham Pharmacia), and EB virus plasmid pCEP4 (Inv
and a vector containing an autonomous origin of replication derived from a virus such as V. itrogen, and a virus such as vaccinia virus. When an insect animal cell (hereinafter referred to as an insect cell) is used as a host cell, an insect virus such as a baculovirus can be used. Vector containing an autonomous origin of replication, for example, the plasmid pACT2 for yeast described above
When the vector of the present invention is constructed using bovine papilloma virus plasmid pBPV, EB virus plasmid pCEP4, or the like, the vector is retained in the cell as an episome when introduced into a host cell. In order to incorporate the gene of the present invention into a virus such as baculovirus or vaccinia virus, a transfer vector containing a nucleotide sequence homologous to the genome of the virus to be used can be used. Specific examples of such transfer vectors include pVL1392, commercially available from Pharmingen,
pVL1393 (Smith, GE, Summers MD et al .: Mol. Cell. Bio
l., 3, 2156-2165 (1983)), pSFB5 (Funahashi, S. et al .: J.
Virol., 65, 5584-5588 (1991)). When the gene of the present invention is inserted into the transfer vector as described above, and the transfer vector and the genome of the virus are simultaneously introduced into a host cell, homologous recombination occurs between the transfer vector and the genome of the virus, and the gene of the present invention is The genome of the integrated virus can be obtained. As the virus genome, genomes of viruses such as Baculovirus, Adenovirus, and Vacciniavirus can be used. More specifically,
For example, when incorporating the gene of the present invention into a baculovirus, the transfer vector pVL1393, after inserting the gene of the present invention into a multiple cloning site such as pVL1392, the DNA of the transfer vector into which the gene of the present invention has been inserted
And Baculovirus genome DNA (Baculogold; manufactured by Pharmingen) are introduced into insect cell strain Sf21 (available from ATCC) by a calcium phosphate method or the like, and the obtained cells are cultured.
When the Baculogold DNA is used, only the virus particles containing the genome into which the gene of the present invention has been inserted are released into the culture solution. By collecting the virus particles from the culture solution by centrifuging the culture solution and subjecting it to deproteinization treatment with phenol or the like, the genome of the virus containing the gene of the present invention can be obtained. Furthermore, a virus containing the gene of the present invention is obtained by introducing the genome of the obtained virus into a host cell having virus particle-forming ability such as insect cell strain Sf21 by a calcium phosphate method or the like, and culturing the obtained cell. Particles can be increased. On the other hand, the gene of the present invention can be directly integrated into a relatively small viral genome such as a mouse leukemia retrovirus without using a transfer vector. For example, the viral vector DC (X) (Eli Gilboa et al., BioT
echniques, 4, 504-512 (1986)) and the like, the gene of the present invention may be incorporated into the cloning site on the vector. The thus-obtained viral vector into which the gene of the present invention has been integrated is used, for example, in Ampli-GPE (J. Virol., 66, 3755).
(1992)), viral particles containing the genome of the virus into which the gene of the present invention has been inserted can be obtained.

[0007] A promoter operable in a host cell is operably linked to the upstream of the gene of the present invention and inserted into a basic vector as described above to express the gene of the present invention in the host cell. Possible vectors of the invention can be constructed. Here, "to operably bind" means that the promoter is linked to the gene of the present invention such that the gene of the present invention is expressed under the control of the promoter in a host cell into which the gene of the present invention is introduced. Means that. As the promoter operable in the host cell, for example, when the host cell is Escherichia coli, the promoter of the lactose operon of Escherichia coli (lac
P), tryptophan operon promoter (trpP),
Arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T
7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR) and the like. Also,
When the host cell is an animal cell or fission yeast, for example, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV
40) The early or late promoter, mouse papilloma virus (MMTV) promoter and the like. When the host cell is budding yeast, examples include the ADH1 promoter. When a basic vector having a promoter operable in a host cell in advance is used, the present invention is placed downstream of the promoter so that the promoter of the basic vector and the gene of the present invention are operably linked. What is necessary is just to insert a gene. For example, the aforementioned plasmids pRC / RSV, pRC / CMV, etc. are provided with a cloning site downstream of a promoter operable in animal cells, and by inserting the gene of the present invention into the cloning site and introducing it into animal cells. The gene of the present invention can be expressed. Since these plasmids contain the SV40 autonomous replication origin (ori) in advance, cultured cells transformed with the ori (-) SV40 genome,
For example, when the plasmid is introduced into a COS cell or the like, the copy number of the plasmid is greatly increased in the cell, and as a result, the gene of the present invention incorporated in the plasmid can be expressed in a large amount. The yeast plasmid pACT2 described above is A
Having the DH1 promoter, if the gene of the present invention is inserted downstream of the ADH1 promoter of the plasmid or its derivative, the gene of the present invention, for example, CG1945 (manufactured by Clontech)
Thus, the vector of the present invention that can be expressed in large amounts in budding yeasts can be constructed.

The transformant of the present invention can be obtained by introducing the constructed vector of the present invention into a host cell. As a method for introducing the vector of the present invention into a host cell, an ordinary method for introduction corresponding to the host cell to be transformed can be applied. For example, when Escherichia coli, which is a microorganism, is used as a host cell, conventional methods such as the calcium chloride method and the electroporation method described in "Molecular Cloning" (J. Sambrook et al., Cold Spring Harbor, 1989) and the like are used. The vector of the present invention can be introduced into a host cell by using the above method. When a mammalian cell or an insect cell is used as a host cell, for example, the vector of the present invention is introduced into the cell by a general gene transfer method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method. can do. When yeast is used as the host cell, for example, yeast based on the lithium method
It can be introduced using a transformation kit (Clontech). When a virus is used as a vector, the virus genome can be introduced into a host cell by a general gene transfer method as described above, and a virus particle containing the virus genome incorporating the gene of the present invention can be used. The genome of the virus can also be introduced into a host cell by infecting the host cell.

In order to select the transformant of the present invention, for example, a marker gene is introduced into a host cell simultaneously with the vector of the present invention, and the host cell into which the vector of the present invention has been introduced by a method according to the properties of the introduced marker gene. May be cultured. For example, when the marker gene is a gene that imparts drug resistance to a selected drug that exhibits lethal activity to the host cell, the host cell into which the vector of the present invention has been introduced is cultured using a medium containing the drug. Good. As the combination of the drug resistance imparting gene and the selected drug, for example,
Examples include a combination of a neomycin resistance imparting gene and neomycin, a combination of a hygromycin resistance imparting gene and hygromycin, and a combination of a blasticidin S resistance imparting gene and blasticidin S. When the marker gene is a gene that complements the auxotrophy of a host cell, cells into which the vector of the present invention has been introduced may be cultured using a minimal medium not containing the nutrient. Furthermore, when the vector of the present invention capable of expressing the gene of the present invention in a host cell is introduced, a detection method based on dioxin binding activity can also be used. In order to obtain the transformant of the present invention in which the gene of the present invention is introduced into the chromosome of a host cell, for example, the vector of the present invention and a vector having a marker gene are linearized by digestion with a restriction enzyme or the like. rear,
These may be introduced into a host cell by the above-described method, and the cell is usually cultured for several weeks, and a target transformant may be selected using the expression of the introduced marker gene as an index. Also,
For example, the vector of the present invention having a gene imparting resistance to the selective drug as a marker gene as described above is introduced into a host cell by the method described above, and the cell is subcultured for several weeks or more in a medium to which the selective drug is added. By purifying and culturing selected drug-resistant clones that survived in colonies,
The transformant of the present invention in which the gene of the present invention has been introduced into the chromosome of a host cell can be selected. In order to confirm that the introduced gene of the present invention has been integrated into the chromosome of the host cell, genomic DNA of the cell is prepared in accordance with ordinary genetic engineering methods, and from the prepared genomic DNA,
DNA having a partial nucleotide sequence of the introduced gene of the present invention
The presence of the gene of the present invention may be detected by using a method such as PCR or Southern hybridization using as a primer or probe. Since the transformant can be cryopreserved and awakened when necessary, it can be used for preparation of the transformant each time an experiment is performed, as compared with a transient gene-transferred strain. Can be omitted, and a test can be performed using a transformant whose properties and handling conditions have been confirmed in advance.

[0010] The dioxin receptor encoded by the gene of the present invention can be produced by culturing the transformant of the present invention obtained as described above. For example, when the transformant of the present invention is a microorganism, the transformant is usually cultured using various media containing a carbon source or a nitrogen source, an organic or inorganic salt, or the like as appropriate for culture in general microorganisms. Is done. Culture is performed according to the usual method for general microorganisms, and solid culture and liquid culture (test tube shake culture, reciprocal shake culture, Jar Fermenter culture, tank culture, etc.) are possible. is there. The culturing temperature can be appropriately changed within a range in which the microorganism grows. For example, culturing is generally performed at a culturing temperature of about 15 ° C to about 40 ° C and a medium pH of about 6.0 to about 8.0. The culture time varies depending on various culture conditions, but is usually about 1 to about 5 days. When an inducible expression vector such as a temperature shift type or an IPTG induction type is used, the induction time is 1
Within days is desirable, usually several hours. When the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium usually used for culturing general cultured cells. When a selective drug is used for selection of the transformant, it is preferable to culture in the presence of the selective drug. For mammalian cells,
For example, using a DMEM medium (manufactured by Nissui) supplemented with FBS so that the final concentration becomes 10%, culturing while changing to a new culture solution every few days at 37 ° C. and in the presence of 5% CO _2. do it. When the cells have grown to confluence, trypsin PBS solution of, for example, about 0.25 (w / v)% is added to disperse the cells, and the obtained cell suspension is diluted several times to obtain new cells. Seed the petri dish and continue the passage.
Similarly, in the case of insect animal cells, culture is performed at a culture temperature of 25 ° C. to 35 ° C. using an insect cell medium such as Grace's medium containing, for example, 10 (v / v)% FBS and 2 (w / v)% yeastlate. do it. At this time, in the case of cells that are easily detached from the petri dish such as Sf21 cells, the cells can be dispersed by pipetting without using a trypsin solution and passage can be performed. In the case of a transformant containing a virus as a vector such as Baculovirus, the culturing time is preferably up to 72 hours before the cells are killed due to the appearance of cytoplasmic effect.
The dioxin receptor produced by culturing the transformant of the present invention can be recovered by appropriately combining ordinary isolation and purification methods. For example, after completion of the culture, cells of the transformant are collected by centrifugation or the like, and if necessary, the collected cells are collected in a normal buffer, for example, 20 mM.
After suspending in a buffer consisting of HEPES pH 7, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, crush it with a polytron, sonication, dounce homogenizer, etc., and ultracentrifuge the crushed liquid at tens of thousands xg for tens of minutes to about 1 hour. By separating and collecting the supernatant fraction after centrifugation, a fraction containing a dioxin receptor can be obtained. Further, the fraction is subjected to ion exchange,
By subjecting it to various types of chromatography such as hydrophobicity, gel filtration, and affinity, a more purified dioxin receptor can be recovered. At this time, the fraction containing the dioxin receptor of the present invention can also be identified by a DNA binding assay using an oligonucleotide having a length of about 15 bp to about 200 bp containing a dioxin response element as a probe. The dioxin receptor of the present invention thus produced can be used, for example, in a receptor binding assay for evaluating the binding ability and amount of a test substance to the dioxin receptor.

The gene of the present invention can also be used, for example, in a reporter assay for evaluating the ability of a test substance to regulate dioxin receptor activity. Examples of the ability to regulate dioxin receptor activity include agonist activity and antagonist activity for dioxin receptors. As the “reporter gene linked downstream of the transcription control region containing a dioxin response element” used in the reporter assay using the gene of the present invention, specifically, for example, a dioxin response element such as a transcription control region of the human CYP1A1 gene And a chimeric gene in which a reporter gene is linked downstream of a transcription control region containing, or a chimeric gene in which a base sequence required for transcription initiation and a reporter gene are linked downstream of a dioxin response element. It can be used to monitor the ability of the dioxin receptor to regulate transcription within the cell. As a reporter gene used for producing such a chimeric gene, a luciferase gene, a secretory alkaline phosphatase gene, a β-galactosidase gene, a chloramphenicol acetyltransferase gene, a growth hormone gene, etc. Genes encoding reporter proteins with relatively high sex are preferred. For example, a reporter gene linked downstream of a transcription control region containing a dioxin response element is introduced into a host cell expressing the gene of the present invention, or a reporter gene linked downstream of a transcription control region containing a dioxin response element. And the gene of the present invention are introduced into, for example, a dioxin receptor non-endogenous host cell, specifically, for example, a HeLa cell or the like to prepare a transformant. Here, the gene of the present invention may be introduced, for example, in a form operably linked to a promoter operable in a host cell and incorporated into a basic vector. A reporter gene linked downstream of a transcription control region containing a dioxin response element may also be used by incorporating it into a vector. Also, for example, a vector incorporating a reporter gene linked downstream of a transcription control region containing a dioxin response element, and a vector carrying the gene of the present invention operably linked to a promoter operable in a host cell. Can be introduced into a host cell together with a vector having a marker gene, and selected by using the expression of the marker gene as an index, so that a reporter gene linked downstream of a transcription control region containing a dioxin response element and a host cell can function. A transformant obtained by introducing the gene of the present invention operably linked to a promoter into the chromosome of a host cell may be obtained and used in a reporter assay. Since the transformant can be cryopreserved and can be awakened and used as needed, once it is obtained, these genes are introduced into host cells every time the assay is performed, and new transformants are used. Since it is not necessary to obtain a transformant and the performance of the transformant can be kept constant, it is convenient, for example, when performing a large-scale screening by an automated robot. While culturing the transformant prepared as described above, for example, for one to several days, a test substance is added to a medium and brought into contact with the transformant, and the expression level of the reporter gene in the transformant Is measured. When the dioxin receptor produced by the transformant is activated by the binding of a dioxin-like substance in the test substance, the transcription of the reporter gene is promoted, and the protein encoded by the reporter gene is intracellularly transformed. Or is secreted into the medium. By measuring the amount of this protein,
The reporter gene expression level per cell of the transformant is measured. Specifically, for example, when a luciferase gene is used as a reporter gene, light is emitted when luciferin, which is a substrate of luciferase, is added to the crude cell extract, and the amount of luminescence is proportional to the amount of luciferase. Therefore, by measuring the amount of luminescence with a measuring device such as a luminometer, the amount of luciferase and, consequently, the expression of luciferase gene can be known. Similarly, the expression level of the reporter gene under the condition that the test substance is not brought into contact with the transformant is measured, and the expression level and the expression level of the reporter gene under the condition that the test substance is brought into contact with the transformant are measured. By comparing with the above, it is possible to evaluate the agonist activity of the dioxin-like substance in the test substance for the dioxin receptor, that is, the ability to activate the receptor. Further, for example, the expression amount of the reporter gene under the condition of contacting the transformant with dioxin such as TCDD, and the reporter gene under the condition of simultaneously contacting the dioxin and the test substance with the transformant. The expression level is measured and compared.
Compared with the expression level of the reporter gene under the condition of contacting dioxin with the transformant, if the expression level of the reporter gene under the condition of simultaneously contacting dioxin and the test article with the transformant is low, This test substance can be evaluated as having an antagonist activity to the dioxin receptor, that is, having an anti-activating ability of the receptor.

The receptor binding assay using the dioxin receptor of the present invention is a test method capable of measuring and quantifying the binding ability of a chemical substance acting on the dioxin receptor, as well as analyzing binding specificity and binding strength. For example, a test substance is allowed to coexist with a labeled receptor ligand (hereinafter, referred to as a labeled ligand) bound to the dioxin receptor of the present invention recovered from the transformant of the present invention as described above. From the competition between the test substance and the labeled ligand, the labeled ligand is released from the receptor in accordance with the affinity of both for the receptor, and the amount of the labeled ligand bound to the receptor decreases. Decrease.
Therefore, by monitoring the amount of free labeled ligand or the amount of bound labeled ligand, the ability of the test substance to bind to the receptor can be indirectly determined. Labeled ligands include, for example, tritium-labeled 2,3,7,8-
TCDD or the like can be used. Binding type of labeled ligand /
The free form can be separated by a hydroxyapatite method or a glycerol density gradient ultracentrifugation method. The reaction system is roughly divided into three groups. One system is a group in which only the solvent is added to the site where the labeled ligand is bound to the dioxin receptor, and corresponds to a system where the concentration of the test substance is zero. The labeling amount indicates the total binding amount of the labeled ligand to the dioxin receptor. In another system, where the labeled ligand is bound to the dioxin receptor, for example, the concentration of unlabeled 2,3,7,8-TCDD is sufficient to saturate the receptor enough to prevent the labeled ligand from binding. (For example, 10 μM), and the amount of the labeled labeled ligand obtained from this system is determined as the amount of nonspecific binding of the labeled ligand to the dioxin receptor. Therefore, the specific binding amount of the labeled ligand to the dioxin receptor is a value obtained by subtracting the non-specific binding amount from the total binding amount. The third system is a system in which a test substance is added to a site where a labeled ligand is bound to a dioxin receptor, for example, to a final concentration of 10 μM (this concentration is arbitrarily changed depending on the purpose). When the test substance has a binding ability to the dioxin receptor, the amount of the labeled labeled ligand obtained from this system is determined by the amount of the labeled ligand to the dioxin receptor when the concentration of the test substance determined as described above is zero. Is smaller than the specific binding amount of By performing a receptor binding assay in this manner, the ability of the test substance to bind to the dioxin receptor of the present invention can be examined, and whether or not a substance having an affinity for the dioxin receptor in the test substance can be examined. it can.
Further, in order to evaluate the binding ability of the test substance to the dioxin receptor of the present invention in more detail, for example,
The assay is performed in the same manner by changing the concentration of the test substance in the second system, and the amount of the bound labeled ligand is measured.
Based on the measured values, the amount of the bound and free ligands in each assay is calculated, for example, by performing Scatchard analysis, whereby the binding affinity between the test substance and the dioxin receptor of the present invention, the binding specificity, The coupling capacity and the like can be evaluated. The reporter assay and receptor binding assay of the present invention can be used for safety evaluation of chemical substances, detection of dioxin-like substances in the environment, and the like.

[0013]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Acquisition of the Gene of the Present Invention) First, Molecular Cloning 2nd editi by J. Sambrook, EFFrisch, T. Maniatis;
on), Cold Spring Harbor Laboratory (Co)
ld Spring Harbor Laboratory), 1989 7.1
According to the description in section 9, total RNA was extracted and separated from one 10 cm plate (purchased from a Chinese hamster ovary-derived cell line CHO-K1, ATCC) by guanidine thiocyanate-CsCl density gradient ultracentrifugation. Next, mRNA was separated from the total amount of total RNA using an Oligotex-dT30 supermRNA purification kit (Takara Shuzo). 1 μg of this mRNA was subjected to a reverse transcription reaction using 1 μg of oligo dT primer (Amersham Pharmacia) and SuperscriptII (Gibco) to prepare a library comprising single-stranded cDNA. Next, in order to obtain the gene of the present invention by the PCR method, an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 5 (forward primer) and an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 6 (reverse primer) Synthesizer (Applied
Biosystems model 394).
Using these oligonucleotides as primers and the above single-stranded cDNA library as a template,
First, the first PCR was performed using PCRsystem9700 (manufactured by Applied Biosystems). The amount of primer in the reaction solution was 10 pmol each, the amount of template DNA was 10 ng, and the total amount was 50 μl using LA-Taq polymerase (manufactured by Takara Shuzo) and a buffer attached to the enzyme. This was carried out for 35 minutes, with the temperature being kept at 72 ° C. for 3 minutes as one cycle. 1 μl was collected from the obtained reaction solution, and the second PCR was performed using this as a template. A primer, LA-Taq polymerase and a buffer attached to the enzyme were added to each at the same concentration as above to prepare a reaction solution having a total volume of 50 μl.
The cycle was kept warm. When the whole amount of the obtained reaction solution was subjected to agarose gel electrophoresis using low-melting point agarose (Agarose L; manufactured by Nippon Gene), 3.1 kbp
A band indicating the mobility was detected. DNA was recovered from the gel of the band portion, and subjected to direct sequencing using a dye terminator sequencing kit FS (manufactured by Applied Biosystems) with an auto sequencer model 377 manufactured by ABI. As a result, the DN
A is a base sequence represented by SEQ ID NO: 2
It was found to have the nucleotide sequence represented by No. 2835.

Example 3 (Construction of the Vector of the Present Invention) An expression plasmid pRC / RSV (Invi
2 μg of trogen) was digested with 10 U of restriction enzymes Not I and Xba I at 37 ° C. for 1.5 hours, blunt-ended using a blunting kit (Takara Shuzo), and then alkaline phosphatase. (BAP) 5U to 65
The reaction was carried out at a temperature of 1 hour. This was subjected to gel electrophoresis using low-melting point agarose (Agarose L; manufactured by Nippon Gene Co., Ltd.), and DNA was recovered from a band portion having a length of about 6 kbp and used as vector DNA. On the other hand, the DNA containing the gene of the present invention obtained in Example 2 was reacted with T4 kinase to phosphorylate the terminal, and then blunt-ended using a blunting kit (Takara Shuzo). The obtained DNA was subjected to low-melting point agarose electrophoresis and then recovered from the gel. About 1 μg of the DNA was mixed with 100 ng of the vector DNA prepared as described above, 5 μl of T4 ligase was added, and the mixture was added at 16 ° C. for 3 hours. Insulated. The reaction solution was used for E. coli DH5α
It was introduced according to the method described in the instructions attached to a competent cell strain (manufactured by TOYOBO), and the base sequence of plasmid DNA was prepared from an ampicillin-resistant colony by an alkaline method. Next, the nucleotide sequences of the plasmid DNAs obtained from these several clones were compared with an oligonucleotide (forward primer designed based on the nucleotide sequence of the dioxin receptor gene coding region) consisting of the nucleotide sequence shown in SEQ ID NO: 7 and the sequence thereof. Number 8
(Reverse direction primer designed based on the nucleotide sequence of the coding region of the dioxin receptor gene), and a plasmid incorporating the gene of the present invention was selected. Furthermore, the nucleotide sequence of the plasmid thus selected was determined using an oligonucleotide consisting of the nucleotide sequence of any of SEQ ID NOs: 9 to 16 as a primer, and was 100% identical with the sequence of SEQ ID NO: 2. One plasmid was selected. The baculovirus transfer vector pVL1392 or pVL1393 (Phar139
mingen) (2 μg each) with restriction enzymes NotI and XbaI
Each was digested with 10 U at 37 ° C. for 1.5 hours, blunt-ended with a blunting kit (Takara Shuzo), and 5 U of alkaline phosphatase (BAP) was added at 65 ° C. for 1 hour.
Let react for hours. This is a low melting point agarose (agarose
L; manufactured by Nippon Gene Co., Ltd.), DNA was recovered from the band, and these were vector DNA
And On the other hand, the DNA containing the gene of the present invention obtained in Example 1 or 2 is reacted with T4 kinase to phosphorylate the terminal, and then blunt-ended with a blunting kit (Takara Shuzo). The obtained DNA was subjected to low melting point agarose electrophoresis and then recovered from the gel.
Is mixed with 100 ng of the vector DNA prepared as described above, 5 U of T4 ligase is added, and the mixture is incubated at 16 ° C. for 3 hours. The DNA in this reaction mixture is introduced into E. coli DH5α competent cells (manufactured by TOYOBO) according to the method described in the attached manual, and plasmid DNA is prepared from an ampicillin-resistant colony by an alkali method.

Example 4 (Preparation of Virus Vector and Virus Particle Containing the Gene of the Present Invention) 1 × 10 ⁶ Sf21 cells (obtained from ATCC) were used for 75 c
Grace's medium (hereinafter, containing FBS) containing 10% FBS and 2% Yeastlate in an m ² T-type flask (Falcon)
Described as Grace medium. ) At 27 ° C overnight. On the other hand, the DNA of the transfer vector in which the gene of the present invention prepared as in Example 3 was incorporated into pVL1392
10 μg of the linearly prepared viral genomic DNA Ba
Grace's medium with 20 ng of culo gold (Pharmingen)
10 μl of Lipofectin (manufactured by GIBCO) diluted twice with sterile water is added to 100 μl, and left at room temperature for 30 minutes. The culture supernatant of the Sf21 cells cultured overnight was removed, and the cells were washed with a small amount of Grace's medium.
ml was added to the cells, and the lipofectin-DN was added thereto.
Add the entire mixture of A and incubate at 27 ° C for 3 hours. Next, after washing the cells with FBS-containing Grace medium, FBS-containing Grace
Add 20 ml of medium to the cells and incubate at 27 ° C. for 5 days. On the 5th day, the culture supernatant is collected, collected in a 50 ml centrifuge tube, and centrifuged at 5000 × g for 15 minutes to precipitate cell debris, and the centrifuged supernatant is collected. The entire amount of the supernatant is ultracentrifuged at 100,000 × g for 24 hours to obtain a virus particle containing the gene of the present invention as a pellet. This pellet is suspended in 100 μl of TE, an equivalent amount of TE-saturated phenol is added, and the mixture is gently mixed at room temperature for 24 hours.
After centrifugation at 10,000 × g for 10 minutes, the aqueous layer is collected, an equivalent amount of chloroform is added thereto, mixed gently for 10 minutes, and centrifuged again at 10,000 × g for 10 minutes. The aqueous layer is recovered, and NaCl in an amount to give a final concentration of 0.2 M and 2.5-fold amount of ethanol are added to the aqueous layer, and the DNA of the virus vector containing the gene of the present invention is recovered as a precipitate.

Example 5 (Construction of a transformant in which a virus vector containing the gene of the present invention is introduced into Sf21 cells and production of a dioxin receptor of the present invention) Sf21 cells (obtained from ATCC) were obtained from a 75 cm ² T-type. A total of 10 1 × 10 ⁶ cells are seeded in a flask (manufactured by Falcon) and cultured at 27 ° C. in a Grace medium containing FBS. A culture supernatant containing virus particles prepared as in Example 4 is added to the cells at a rate of 10 μl / flask, and the cells are cultured as they are for 4 days. The culture supernatant is collected, and 1 ml of the culture supernatant is added to each Sf21 cell cultured in 10 75 cm ² T-type flasks (manufactured by Falcon) in the same manner as described above, and cultured for 60 hours. After 60 hours, the cells were suspended by pipetting and recovered from the flask,
The resulting cell suspension is centrifuged at 5,000 xg for 15 minutes to form a pellet. Transfer the cell pellet to 20 mM HEPES p
After suspending in H7, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF buffer, the obtained cell suspension is homogenized up and down 30 times with a dounce type glass homogenizer to disrupt the cells. The crushed liquid is ultracentrifuged at 30,000 × g for 1 hour, and the supernatant fraction is collected to obtain a fraction containing the dioxin receptor of the present invention.

Example 6 (Preparation of transformant for reporter assay in which the gene of the present invention was introduced into chromosome) Using Isogen reagent (manufactured by Nippon Gene), a human-derived transformant was prepared by the method described in the protocol attached to the reagent. Genomic DNA was purified from HepG2 cells. PCR is performed using the obtained genomic DNA as a template and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 7 (forward primer) and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 8 (reverse primer). 75 from TATAbox upstream of human CYP1A1 gene
The base sequence of the region from 0 bp upstream to 1370 bp upstream (J. Bi
ochem., 110.232-236 (1991)) and a DNA containing a dioxin response element was amplified. The amplified DNA was recovered, and its end was blunted using a Blunting kit (manufactured by Takara Shuzo Co., Ltd.) (this DNA is referred to as XRE DNA hereinafter).
Base sequence and leader sequence near TATA box of mouse metallothionein I gene (Genbank Accession No. J00605)
An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10
NA was reacted with T4 polynucleotide kinase to phosphorylate both ends thereof (the DNA is hereinafter referred to as TATA DNA). On the other hand, a plasmid pGL3 (promega) containing the firefly luciferase gene was digested with restriction enzymes Bgl II and Hind III, and
Erial alkaline phosphatase (BAP) was added, and the mixture was kept at 65 ° C for 1 hour. Next, the heat retaining liquid was mixed with low melting point agarose (Ag
aroseL; manufactured by Nippon Gene Co., Ltd.) and Bgl II-Hi containing pGL3-derived luciferase gene.
DNA showing a migration degree corresponding to the length of the ndIII fragment was recovered. About 100 ng of the recovered DNA and the TATA DNA described above
By mixing with 1 μg and binding with T4 ligase, plasmid pGL3-TATA was prepared. Next, after pGL3-TATA was digested with a restriction enzyme SmaI, BAP was added and the mixture was incubated at 65 ° C. for 1 hour. The incubation solution was subjected to low-melting point agarose gel electrophoresis, and DNA was recovered from the gel in the band portion. The D
After about 100 ng of NA and about 1 μg of the above XRE DNA were mixed and reacted with T4 ligase, the DNA in the reaction solution was added to D
It was introduced into H5α competent cells (manufactured by TOYOBO). The cells were cultured in a medium containing ampicillin, and DNAs of respective plasmids were prepared from several colonies of Escherichia coli showing ampicillin resistance.These DNAs were digested with restriction enzymes Kpn I and Xho I, and the digested solution was digested. Analyzed by agarose gel electrophoresis. About 60 corresponding to XRE DNA
A plasmid having a structure in which one bp of DNA was introduced into the SmaI site of pGL3-TATA by one copy was selected and named plasmid pGL3-TATA-1A1. Then the plasmid
pUCSV-BSD (purchased from Funakoshi) was digested with BamHI to prepare a DNA encoding a blasticidin S deaminase gene expression cassette. The DNA and the DNA obtained by digesting the plasmid pGL3-TATA-1A1 with BamHI and treating with BAP were mixed and reacted with T4 ligase, and the DNA in the reaction solution was subjected to E. coli DH5α competent cells. (Manufactured by TOYOBO). Plasmid DNA was prepared from the obtained ampicillin-resistant Escherichia coli clone, each was digested with a restriction enzyme BamHI, and the digested solution was analyzed by agarose gel electrophoresis. Blast Saidin S
Deaminase gene expression cassette is plasmid pGL3-TA
A plasmid having a structure inserted into the Bam HI cleavage site of TA-1A1 was selected and named plasmid pGL3-TATA-1A1-BSD. Next, the DNA of the plasmid pGL3-TATA-1A1-BSD was digested with the restriction enzyme SalI, and the gene of the present invention was pRC / RSV
Was digested with PvuI. On the other hand, HeLa cells were transformed with DME containing 10% FBS.
M medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. and 5% CO ₂
In the presence, culture is performed using a petri dish (Falcon) having a diameter of about 10 cm. After culturing about 5 × 10 ⁵ cells for one day, the above two kinds of restriction enzymes S were subjected to lipofection using lipofectin (manufactured by GIBCO).
The DNA of the plasmid digested with alI or PvuI is introduced simultaneously. The conditions for the lipofection method were as follows, according to the description of the manual attached to the lipofectin.
Time, total amount of linearized plasmid DNA 7 μg (3.5 μg each) / dish, amount of lipofectin 20 μg
1 / Dish. After lipofection, the cells are cultured in a DMEM medium containing 10% FBS for 3 days. Next, the cells are peeled off from the petri dish by trypsin treatment, seeded on 10 new petri dishes by 1/10 amount, and cultured as it is until the next day. Next, G418 (SIGMA) was added to a final concentration of 400.
blasticidin S
Is added to the medium to a final concentration of 8 μg / ml, and the culture is continued. One week later, G418 and blasticidin S are replaced with a new medium containing the same concentration as above, and the culture is continued. One week later, the same operation is performed again. One week later, the petri dish was observed with an inverted microscope, and 30 colonies each having a diameter of several mm were transferred to each well of a 96-well view plate (manufactured by Berthold) in which the medium had been previously dispensed, and further cultured. to continue. Cells are harvested by trypsinization before harvesting confluence, divided into three equal parts, and seeded on three new 96-well view plates. Subculture and culturing were continued for one of them, and 3-methylcholanthrene was added to one of the remaining two to a final concentration of 50 nM, and the other was cultured for two days without any addition. . Next, each culture supernatant was removed, the cells were rinsed twice with 200 μl / well of PBS (−), and then PGC50 (manufactured by Nippon Gene Co., Ltd.) diluted 5 times.
Is added at a time, and left at room temperature for 30 minutes to lyse the cells. This plate is used as a luminometer LB96p with an automatic enzyme substrate injector (manufactured by Berthold).
And 50 μl of the substrate solution PGL100
The luciferase activity is measured while automatically dispensing (Nippon Gene). In the system to which 3-methylcholanthrene is added, a transformant showing luciferase activity that is at least twice as high as that in the system to which 3-methylcholanthrene is not added is selected.

Example 7 (Reporter assay using a transformant in which the gene of the present invention has been introduced into a chromosome) A transformant of HeLa cells prepared as described in Example 6 was used in a 96-well view plate (Berthold). by about 1.5 × 10 ⁴ cells / well in purchases) from seeding, 10% charcoal dextran-treated FBS, E-MEM medium containing blasticidin S for G418 and 8 [mu] g / ml of 400 [mu] g / ml (hereinafter, FBS and antibiotics Substance-containing E-MEM medium).
Culture is performed at 37 ° C. under 1% CO _{2 for} 1 day. 2,3,7,8-
FBS with TCDD (obtained from Daiichi Kagaku) dissolved in DMSO (manufactured by Wako Pure Chemical Industries) and added to a final concentration of 0.15 pM to 150 nM
And an antibiotic-containing E-MEM medium, and the aforementioned 2,3,7,8
-Prepare an E-MEM medium containing FBS and an antibiotic containing the same amount of DMSO instead of the DMSO solution of TCDD, and replace them with the culture supernatant of the cells. Note that the experiment is performed with six repetitive experiments and the average value is obtained.
The cells are cultured in a CO ₂ incubator, and after 24 hours, the culture supernatant is removed, and the wells are rinsed twice with 100 μl / well of PBS (−) so as not to peel off the cells adhered to the wells, and 5 times. The cell lysing agent PGC50 (manufactured by Nippon Gene Co., Ltd.) diluted at 50 μl / well is added thereto, and left at room temperature for 30 minutes with occasional gentle shaking to lyse the cells. The view plate containing the cell lysate prepared in this way is luminometer equipped with an automatic substrate injector
Set in LB96p (Berthold), 50μl / well
The amount of luminescence is immediately measured for 1 second while adding the enzyme substrate solution PGL100 (manufactured by Nippon Gene Co., Ltd.). As a result, a concentration-dependent increase in the transcriptional activity of 2,3,7,8-TCDD, which is known as a ligand for the dioxin receptor, is detected. By a luciferase reporter assay using such a transformant in which the gene of the present invention has been introduced into a chromosome, a test substance containing a substance having the ability to activate dioxin receptors can be found.

Example 8 (Receptor Binding Assay) The binding reaction buffer had a final composition of 20 mM HEPES-KOH pH
7.9, 10mM sodium molybdate, 1mM DTT, 0.5mM ED
Prepare TA and 0.5 mM PMSF (all manufactured by Wako Pure Chemical Industries). The reaction solution was adjusted to a total volume of 100 μl, to which a cell extract containing the dioxin receptor of the present invention was added in an amount of 10 μg of protein, and tritium-labeled 2,3,7,8-TCDD was added.
Add so that the concentration is about 1 pM to about 10 nM. Unlabeled 2,3,7,8-TCDD for non-specific binding
Is further added to a final concentration of 10 μM. The binding reaction is performed as follows. After keeping the above reaction solution on ice for 15 hours, a charcoal dextran solution [composition: 10 mM Tr
is-HCl, 0.2% pickling activated carbon (Nakarai Tesque's Norit
A), 100 μl of 0.005% Pharmacia Dextran T70] is added and left on ice for 10 minutes. This reaction solution is
Activated carbon was precipitated by centrifugation for 0 minutes, and the supernatant was
An aliquot is taken and its radioactivity is measured with a liquid scintillation counter. Based on this measurement, the label 2,2
3,7,8-TCDD amount, that is, label 2,
Determine the amount of 3,7,8-TCDD (the amount of bound labeled ligand). Sign
The amount of the labeled labeled ligand in the test group to which only 2,3,7,8-TCDD was added corresponds to the total amount of the labeled 2,3,7,8-TCDD bound to the receptor. On the other hand, the amount of bound labeled ligand in the test group to which unlabeled 2,3,7,8-TCDD was added in addition to labeled 2,3,7,8-TCDD was labeled 2,3,7,8-TCDD. It corresponds to the amount of non-specific binding of TCDD to the receptor. Labels of various concentrations 2,3,7,8-
For each reaction mixture to which TCDD has been added, the amount of non-specific binding is subtracted from the total amount of binding to determine the amount of specific binding of the labeled ligand to the receptor in the reaction mixture. Next, the specific binding labeled ligand concentration is plotted on the Y axis (specific binding labeled ligand concentration / free labeled ligand concentration) and the specific binding labeled ligand concentration is plotted on the X axis, and Scatchard analysis is performed to obtain 2,3,2,3 of the dioxin receptor of the present invention. Find the Kd value for 7,8-TCDD. To measure the affinity of the test substance for the dioxin receptor, 1 nM
A test substance is added to a binding assay binding reaction solution containing about tritium-labeled 2,3,7,8-TCDD to a final concentration of about 1%. In a system to which no test substance is added, the same amount of solvent as the test substance is added to the system. When the amount of the labeled 2,3,7,8-TCDD bound to the receptor is decreased by the addition of the test substance, it is determined that the test substance contains a substance that binds to the dioxin receptor.

[0021]

According to the present invention, it is possible to provide a novel dioxin receptor gene or the like which can be used for a method for measuring a dioxin-like substance.

[Sequence Listing Free Text] SEQ ID NO: 3 oligonucleotide primer designed for PCR SEQ ID NO: 4 oligonucleotide primer designed for PCR SEQ ID NO: 5 oligonucleotide primer designed for PCR SEQ ID NO: 5 6 oligonucleotide primer designed for PCR SEQ ID NO: 7 oligonucleotide primer designed for PCR SEQ ID NO: 8 oligonucleotide primer designed for PCR SEQ ID NO: 9 designed to synthesize promoter DNA Oligonucleotide SEQ ID NO: 10 Oligonucleotide designed to synthesize promoter DNA

[0023]

[Sequence List] <110> Sumitomo Chemical Company Limited <120> Dioxin receptor gene <130> P151878 <160> 10 <210> 1 <211> 941 <212> PRT <213> Cricetulus griseus; CHO-K1 cell line <400 > 1 Met Ser Ser Gly Ala Asn Ile Thr Tyr Ala Ser Arg Lys Arg Arg Lys 1 5 10 15 Pro Val Gln Lys Thr Val Lys Pro Val Pro Ala Glu Gly Val Lys Ser 20 25 30 Asn Pro Ser Lys Arg His Arg Asp Arg Leu Asn Thr Glu Leu Asp Arg 35 40 45 Leu Ala Ser Leu Leu Pro Phe Pro Gln Asp Val Ile Asn Lys Leu Asp 50 55 60 Lys Leu Ser Val Leu Arg Leu Ser Val Ser Tyr Leu Arg Ala Lys Ser 65 70 75 80 Phe Phe Asp Val Ala Leu Lys Ser Ser Pro Ala Asp Arg Asn Gly Gly 85 90 95 Gln Asp Gln Cys Arg Ala Gln Phe Ser Asp Gly Leu Asp Leu Gln Glu 100 105 110 Gly Glu Phe Leu Leu Gln Ala Leu Asn Gly Phe Val Leu Val Val Thr 115 120 125 Ala Asp Ala Leu Val Phe Tyr Ala Ser Ser Thr Ile Gln Asp Tyr Leu 130 135 140 Gly Phe Gln Gln Ser Asp Val Ile His Gln Ser Val Tyr Glu Leu Ile 145 150 155 160 His Thr Glu Asp Arg Ala Glu Phe Gln Arg Gln Leu His Trp Ala Leu 16 5 170 175 Asn Pro Ala Gln Cys Thr Asp Ser Ala Gln Ala Gly Asp Glu Ala Pro 180 185 190 Gly Leu Trp Gln Pro Pro Ala Val His Phe Asn Pro Glu Gln Leu Pro 195 200 205 Pro Glu Ser Ala Ser Phe Leu Glu Arg Cys Phe Ile Cys Arg Leu Arg 210 215 220 Cys Leu Leu Asp Asn Ser Ser Gly Phe Leu Ala Met Asn Phe Gln Gly 225 230 235 240 Arg Leu Lys Tyr Leu His Gly Gln Asn Lys Lys Gly Lys Asp Gly Thr 245 250 255 Leu Leu Pro Pro Gln Leu Ala Leu Phe Ala Ile Ala Thr Pro Leu Gln 260 265 270 Pro Pro Ser Ile Leu Glu Ile Arg Thr Lys Asn Phe Ile Phe Arg Thr 275 280 285 Lys His Lys Leu Asp Phe Thr Pro Ile Gly Cys Asp Ala Lys Gly Gln 290 295 300 Leu Val Leu Gly Tyr Thr Glu Val Glu Leu Cys Thr Arg Gly Ser Gly 305 310 315 320 Tyr Gln Phe Ile His Ala Ala Asp Met Leu Tyr Cys Ala Glu Ser His 325 330 335 Val Arg Met Ile Lys Thr Gly Glu Ser Gly Met Thr Val Phe Arg Leu 340 345 350 Leu Ala Lys His Ser Arg Trp Arg Trp Val Gln Ser Asn Ala Arg Leu 355 360 365 Ile Tyr Arg Asn Gly Arg Pro Asp Tyr Ile Ile Ala Thr Gln Arg Pro 370 Three 75 380 Leu Thr Asp Glu Glu Gly Arg Glu His Leu Gln Lys Arg Ser Met Thr 385 390 395 400 Leu Pro Phe Met Phe Ala Thr Gly Glu Ala Val Leu Tyr Glu Ile Ser 405 410 415 Ser Pro Phe Pro Pro Ile Met Asp Pro Leu Pro Val Arg Thr Lys Ser 420 425 430 Ser Thr Ser Ala Lys Asp Trp Val Pro Gln Ser Thr Pro Ser Lys Asp 435 440 445 Ser Leu His Pro Ser Ser Leu Met Asn Cys Ile Ile Gln Gln Asp Glu 450 455 460 Ser Ile Tyr Leu Cys Pro Pro Ser Ser Ala Ala Pro Leu Asp Ser His 465 470 475 480 Phe Leu Thr Ser Ser Val Ser Glu Gly Ser Gly Trp Gln Asp Ser Ile 485 490 495 Ala Pro Ile Gly Ser Asp Ala Met Leu Lys His Gln Gln Ile Gly His 500 505 510 Ala Gln Asp Met Asn Pro Ala Leu Ser Gly Gly Pro Pro Gly Leu Phe 515 520 525 Pro Asp Asn Lys Asn Asn Asp Leu Tyr Ser Ile Met Arg Asn Leu Gly 530 535 540 Ile Asp Phe Asp Asp Ile Lys Arg Met Gln Ser Glu Glu Phe Phe Arg 545 550 555 560 Thr Glu Leu Ser Asp Asp Val Asp Phe Arg Asp Ile Asp Ile Thr Asp 565 570 575 Glu Ile Leu Thr Tyr Val Gln Asp Ser Leu Asn Lys Ser Thr Leu Leu 580 Five 85 590 Asn Ser Ala Ser Gln Gln Gln Pro Val Thr Gln His Leu Ser Cys Met 595 600 605 Leu Gln Glu Arg Leu His Leu Glu Gln Gln Gln Gln Gln Gln Gln Gln Gln 610 615 620 Gln Gln Gln Leu Gln Gln His Gln Thr Gln Ala Ala Glu Pro Gln His 625 630 635 640 Glu Leu Glu Gln Met Glu Ser Gln Gln Gln Leu His His Gln Met Glu 645 650 655 Pro Gln Gln Gln Leu His His Gln Met Glu Pro Gln Gln Gln Leu His 660 665 670 His Gln Met Glu Pro Gln Gln Gln Leu Cys His Gln Met Glu Pro Gln 675 680 685 Gln Gln Leu Cys His Gln Met Glu Leu Gln Gln Gln Leu Cys His Gln 690 695 700 Met Glu Pro Gln Gln Gln Leu Cys His Gln Met Glu Leu Pro Gln Gln 705 710 715 720 Gln Leu Cys His Gln Met Glu Leu Pro Gln Gln Glu Leu Gly Gln Lys 725 730 735 Met Lys His Ala Gln Val Asn Gly Met Phe Thr Ser Trp Asn Pro Thr 740 745 750 Pro Leu Val Pro Phe Ser Phe Pro Gln Gln Glu Leu Lys His Tyr Asn 755 760 765 Val Phe Ser Asp Leu Glu Gly Ala Val Glu Glu Phe Pro Tyr Lys Ser 770 775 780 Glu Val Asp Ser Val Pro Tyr Thr Gln Ser Phe Ala Pro Cys Asn Gln 785 790795 800 Ser Val Leu Pro Gln His Ser Lys Cys Ala Gln Leu Asp Leu Pro Gly 805 810 815 Lys Gly Phe Glu Pro Pro Leu His Pro Asn Thr Ser Asn Leu Gly Asp 820 825 830 Phe Val Thr Cys Leu Gln Val Pro Glu Asn Gln Arg His Gly Ile Asn 835 840 845 Pro Gln Ser Thr Met Val Thr Pro Gln Thr Tyr Tyr Ala Gly Ala Met 850 855 860 Ser Met Tyr Gln Cys Gln Pro Gly Pro Gln His Ile Pro Val Asp Gln 865 870 875 880 Val Gln Tyr Asn Pro Ala Ile Pro Gly Ser Gln Ala Phe Leu Asn Lys 885 890 895 Phe Gln Asn Gln Gly Val Leu Asn Glu Thr Tyr Ser Ser Glu Leu Asn 900 905 910 Ser Val Gly His Arg Gln Asn Thr Ala His Leu His His Pro Ala Glu 915 920 925 Gly Arg Pro Phe Pro Asp Ile Thr Pro Gly Gly Phe Leu 930 935 940 <210> 2 <211> 3110 <212> DNA <213> Cricetulus griseus; CHO-K1 cell line <220> <221> CDS <222> (10) ... (2835) <400> 2 gcgggcacc atg agc agc ggc gcc aac atc acc tat gcc agc cgc aag cgg 51 Met Ser Ser Gly Ala Asn Ile Thr Tyr Ala Ser Arg Lys Arg 1 5 10 cgc aag ccg gtg cag aaa aca gta aag cca gtc cct gct gaa gga gtc 99 Ar g Lys Pro Val Gln Lys Thr Val Lys Pro Val Pro Ala Glu Gly Val 15 20 25 30 aag tca aat cct tct aag cga cat aga gac cgg ctg aac aca gag ttg 147 Lys Ser Asn Pro Ser Lys Arg His Arg Asp Arg Leu Asn Thr Glu Leu 35 40 45 gac cgc ctg gcc agc ctg ctg ccc ttc cca caa gat gtt att aac aag 195 Asp Arg Leu Ala Ser Leu Leu Pro Phe Pro Gln Asp Val Ile Asn Lys 50 55 60 ctg gac aaa ctc tca gtt ctt agg ctc agt gtc agc tac ctg agg gcc 243 Leu Asp Lys Leu Ser Val Leu Arg Leu Ser Val Ser Tyr Leu Arg Ala 65 70 75 aag agc ttc ttc gat gtt gca tta aaa tcc tcc cct gca gac agg aat 291 Lys Ser Phe Phe Asp Val Ala Leu Lys Ser Ser Pro Ala Asp Arg Asn 80 85 90 gga ggc cag gat cag tgt aga gca caa ttc agc gat gga ctg gac ttg 339 Gly Gly Gln Asp Gln Cys Arg Ala Gln Phe Ser Asp Gly Leu Asp Leu 95 100 105 110 caa gaa gga gag ttc tta tta cag gca ctg aac ggc ttt gta ctg gtt 387 Gln Glu Gly Glu Phe Leu Leu Gln Ala Leu Asn Gly Phe Val Leu Val 115 120 125 gtc aca gca gat gcc ttg gtc ttc tat gct atc caa gat 435 Val Thr Ala Asp Ala Leu Val Phe Tyr Ala Ser Ser Thr Ile Gln Asp 130 135 140 tac ctg ggc ttt cag caa tct gat gtc atc cat caa agt gtc tat gag 483 Tyr Leu Gly Phe Gln Gln Ser Asp Val Ile His Gln Ser Val Tyr Glu 145 150 155 ctt atc cat acc gaa gac cga gct gag ttc cag cgc cag ctt cac tgg 531 Leu Ile His Thr Glu Asp Arg Ala Glu Phe Gln Arg Gln Leu His Trp 160 165 170 gct cta aac cca gca cag tgt aca gat tct gca caa gca ggg gat gaa 579 Ala Leu Asn Pro Ala Gln Cys Thr Asp Ser Ala Gln Ala Gly Asp Glu 175 180 185 190 gct cct ggc ctc tgg cag cca cca gca gtc cac ttc aac cca gag cag 627 Ala Pro Gly Leu Trp Pro Pro Ala Val His Phe Asn Pro Glu Gln 195 200 205 ctt ccg cca gag agt gcc tct ttc ctg gag cgg tgc ttc ata tgc cgg 675 Leu Pro Pro Glu Ser Ala Ser Phe Leu Glu Arg Cys Phe Ile Cys Arg 210 215 220 ctc cgg tgt ctg ctg gat aat tcc tct ggt ttt ctg gcg atg aat ttc 723 Leu Arg Cys Leu Leu Asp Asn Ser Ser Gly Phe Leu Ala Met Asn Phe 225 230 235 caa ggg agg ctg aag tat ctc cat gga cag aaag gat 771 Gln Gly Arg Leu Lys Tyr Leu His Gly Gln Asn Lys Lys Gly Lys Asp 240 245 250 gga aca cta ctg ccc cca cag ttg gct ttg ttt gca ata gcg acc cca 819 Gly Thr Leu Leu Pro Pro Gln Leu Ala Leu Phe Ala Ile Ala Thr Pro 255 260 265 270 ctt cag cca ccg tcc ata ctg gaa att cga acc aaa aac ttc atc ttc 867 Leu Gln Pro Pro Ser Ile Leu Glu Ile Arg Thr Lys Asn Phe Ile Phe 275 280 285 agg acc aaa cac aaa cta gac ttc aca cct att ggt tgt gat gcc aaa 915 Arg Thr Lys His Lys Leu Asp Phe Thr Pro Ile Gly Cys Asp Ala Lys 290 295 300 ggg cag ctt gtt ctg ggc tac aca gaa gta gag ctt tgc acc cga ggc 963 Gly Gln Val Leu Gly Tyr Thr Glu Val Glu Leu Cys Thr Arg Gly 305 310 315 tct ggg tac cag ttt atc cat gcc gct gac atg ctg tac tgc gca gag 1011 Ser Gly Tyr Gln Phe Ile His Ala Ala Asp Met Leu Tyr Cys Ala Glu 320 325 330 tcc cac gtc cgc atg att aag acg gga gaa agt ggt atg aca gtt ttc 1059 Ser His Val Arg Met Ile Lys Thr Gly Glu Ser Gly Met Thr Val Phe 335 340 345 350 agg ctt ctg gca aaa cac agt cga tgg agg tgg gtc cag tcc aat gca 1107 Arg Leu Leu Ala Lys His Ser Arg Trp Arg Trp Val Gln Ser Asn Ala 355 360 365 cgc ttg att tat aga aat gga aga cca gat tac atc atc gca act cag 1155 Arg Leu Ile Tyr Arg Asn Gly Arg Pro Asp Tyr Ile Ile Ala Thr Gln 370 375 380 agg cca cta acg gat gaa gaa gga aga gag cat ttg cag aag cga agc 1203 Arg Pro Leu Thr Asp Glu Glu Gly Arg Glu His Leu Gln Lys Arg Ser 385 390 395 atg acg ctg ccg ttc atg ttt gct act gga gag gct gta ttg tat gag 1251 Met Thr Leu Pro Phe Met Phe Ala Thr Gly Glu Ala Val Leu Tyr Glu 400 405 410 atc tcc agc cct ttc ccg ccc ata atg gat ccc ttg cca acct 1299 Ile Ser Ser Pro Phe Pro Pro Ile Met Asp Pro Leu Pro Val Arg Thr 415 420 425 430 aaa agc agc acc agt gca aag gac tgg gtt ccg cag tca aca cca agt 1347 Lys Ser Ser Thr Ser Ala Lys Asp Trp Val Pro Gln Ser Thr Pro Ser 435 440 445 aag gat tct ctc cac ccc agc tcc ctt atg aac tgc ata atc caa cag 1395 Lys Asp Ser Leu His Pro Ser Ser Leu Met Asn Cys Ile Ile Gln Gln 450 455 460 gat gag tcc atc tat c tc tgt cct cct tca agc gct gca cca cta gac 1443 Asp Glu Ser Ile Tyr Leu Cys Pro Pro Ser Ser Ala Ala Pro Leu Asp 465 470 475 agc cat ttt ctc acc agc tct gtg agt gaa ggc agt ggt tgg caa gac 1491 Ser His Phe Leu Thr Ser Ser Val Ser Glu Gly Ser Gly Trp Gln Asp 480 485 490 agc att gca cca ata gga agt gat gct atg ttg aag cat caa caa att 1539 Ser Ile Ala Pro Ile Gly Ser Asp Ala Met Leu Lys His Gln Gln Ile 495 500 505 510 gga cat gct cag gac atg aac cct gca ctc tct gga ggc ccc ccg ggg 1587 Gly His Ala Gln Asp Met Asn Pro Ala Leu Ser Gly Gly Pro Pro Gly 515 520 525 ctc ttt cca gat aat aaa aat gattt tac agc atc atg aga aac 1635 Leu Phe Pro Asp Asn Lys Asn Asn Asp Leu Tyr Ser Ile Met Arg Asn 530 535 540 cta ggg att gat ttt gat gat atc aaa cgc atg cag agt gag gag ttc 1683 Leu Asp Ashe Phe Ile Lys Arg Met Gln Ser Glu Glu Phe 545 550 555 ttc agg act gag ttg tcg gac gac gtt gac ttc aga gac atc gac ata 1731 Phe Arg Thr Glu Leu Ser Asp Asp Val Asp Phe Arg Asp Ile Asp Ile 560 565 570 a ca gat gaa atc ctg aca tat gtg caa gat tct cta aac aag tca acc 1779 Thr Asp Glu Ile Leu Thr Tyr Val Gln Asp Ser Leu Asn Lys Ser Thr 575 580 585 585 590 ttg ctg aat tct gct agc cag caa cag cct gtg act ca cac cta agc 1827 Leu Leu Asn Ser Ala Ser Gln Gln Gln Pro Val Thr Gln His Leu Ser 595 600 605 tgc atg ctg cag gaa cgc ctg cat cta gaa cag cag cag cag cag cag 1875 Cys Met Leu Gln Glu Arg Leu His Leu Glu Gln Gln Gln Gln Gln Gln 610 615 620 cag cag cag cag cag cta caa cag cac caa act cag gca gca gag ccc 1923 Gln Gln Gln Gln Gln Gln Leu Gln Gln His Gln Thr Gln Ala Ala Glu Pro 625 630 635 635 cag cat gag ctg gag cag atg gag tcc cag caa cag cta cat cat cag 1971 Gln His Glu Leu Glu Gln Met Glu Ser Gln Gln Gln Leu His His Gln 640 645 650 atg gag ccc cag caa cag cta cat cat cag atg gag ccc cag caa cag 2019 Met Glu Pro Gln Gln Gln Leu His His Gln Met Glu Pro Gln Gln Gln 655 660 665 670 cta cat cat cag atg gag ccc cag caa cag ctc tgt cat cag atg gag 2067 Leu His His Gln Met Glu Pro Gln Gln Gln Leu Cys His Gl n Met Glu 675 680 685 ccc cag caa cag ctc tgt cat cag atg gag ctc cag caa cag ctc tgt 2115 Pro Gln Gln Gln Leu Cys His Gln Met Glu Leu Gln Gln Gln Leu Cys 690 695 700 cat cag atg gag ccc cag cag ctc tgt cat cag atg gag ctc ccc 2163 His Gln Met Glu Pro Gln Gln Gln Leu Cys His Gln Met Glu Leu Pro 705 710 715 cag caa cag ctc tgt cat cag atg gag ctc ccc cag caa gag ctg ggc 2211 Gln Gln Gln Le His Gln Met Glu Leu Pro Gln Gln Glu Leu Gly 720 725 730 cag aaa atg aag cat gcg caa gtc aat ggc atg ttt aca agt tgg aac 2259 Gln Lys Met Lys His Ala Gln Val Asn Gly Met Phe Thr Ser Trp Asn 735 740 745 750 ccc acc cct ctc gtg cct ttc agc ttt cct cag cag gaa cta aag cat 2307 Pro Thr Pro Leu Val Pro Phe Ser Phe Pro Gln Gln Glu Leu Lys His 755 760 765 tat aat gtc ttt tca gac tta gag ggg gct gtt gaga gag ttc ccc tac 2355 Tyr Asn Val Phe Ser Asp Leu Glu Gly Ala Val Glu Glu Phe Pro Tyr 770 775 780 aaa tct gag gtg gac agt gtg cct tac aca cag agc ttt gct ccc tgt 2403 Lys Ser Glu Val Asp Ser Val Pro Ty r Thr Gln Ser Phe Ala Pro Cys 785 790 795 aat cag tct gtg cta ccc cag cat tcc aag tgt gca cag ttg gac ttg 2451 Asn Gln Ser Val Leu Pro Gln His Ser Lys Cys Ala Gln Leu Asp Leu 800 805 810 cct gga aag ggt ttt gaa cca ccc ctg cac ccc aat act tct aac tta 2499 Pro Gly Lys Gly Phe Glu Pro Pro Leu His Pro Asn Thr Ser Asn Leu 815 820 825 830 gga gat ttt gtc act tgt tta caa gtt ccc gaa aac caa aga cat gga 2547 Gly Asp Phe Val Thr Cys Leu Gln Val Pro Glu Asn Gln Arg His Gly 835 840 845 ata aat cca cag tca acc atg gtc act cct cag aca tac tat gct ggg 2595 Ile Asn Pro Gln Ser Thr Met Val Thr Pro Gln Thr Tyr Tyr Ala Gly 850 855 860 gcc atg tcc atg tac cag tgc cag cca gga cct cag cac atc cct gtg 2643 Ala Met Ser Met Tyr Gln Cys Gln Pro Gly Pro Gln His Ile Pro Val 865 870 875 gac cag gtg caa tac aac cct gca att cca ggc tcc cag gca ttt cta 2691 Asp Gln Val Gln Tyr Asn Pro Ala Ile Pro Gly Ser Gln Ala Phe Leu 880 885 890 aac aag ttc cag aac cag gga gtg tta aat gaa acg tat tcg tcc gaa 2739 Asn Lys Phe Asn Gln Gly Val Leu Asn Glu Thr Tyr Ser Ser Glu 895 900 905 910 tta aac agt gtt ggc cac agg cag aac act gct cat ctt cat cat cca 2787 Leu Asn Ser Val Gly His Arg Gln Asn Thr Ala His Leu His His Pro 915 920 925 gca gaa ggg agg ccg ttt cct gat atc aca ccc ggt gga ttc ctg 2832 Ala Glu Gly Arg Pro Phe Pro Asp Ile Thr Pro Gly Gly Gly Ply Le 930 935 940 tagctttagg aacaagatac tggaactgtc cgtgttgcc tcattgtgttcctcagtt atgcctcagtt aatgggaagc tctgctggag cactgggaga cgccacatgc attttcatag ctttagctat 3012 gcactcagcg tgcacagtgc tcactgtgga gagctttggg attctagaaa gctgagctct 3072 gctcagtgac cagccagaaa gacaacttca <pattern> DNA <110> <a> <110> 3 gcgggcacca tgagcagcgg cgccaacatc accta 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 4 acacggacag ttccagtatc ttgttcctaa agcta 35 <210> 5 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 cggccgccca gccaccatga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> Designed oligonucleotide primer for PCR <400> 6 cgggaatcct gaagttgtct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 7 ttgagctagg cacgcaaata 20 < 210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 8 gctttgattg gcagagcaca 20 <210> 9 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide to synthesize promoter DNA <400> 9 gatctcgact ataaagaggg caggctgtcc tctaagcgtc accacgactt ca 52 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide to synthesize promoter DNA <400> 10 agcttgaagt cgtggtgacg cttagaggac agcctgccct ctttatagtc ga 52

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/02 G01N 33/68 4H045 G01N 33/566 C12R 1:93) 33/68 (C12P 21/02 C // (C12N 5/10 C12R 1:93) C12R 1:93) C12N 15/00 ZNAA (C12P 21/02 5/00 B C12R 1:93) C12R 1:93) F term (reference) 2G045 AA34 AA35 DA12 DA13 DA36 FB01 FB02 FB08 4B024 AA11 AA17 BA63 CA04 DA02 EA02 GA11 GA13 GA18 HA01 4B063 QA01 QA18 QQ08 QQ13 QQ22 QR69 QR80 QS05 QS28 QS36 QS38 QX02 4B064 AG20 CA10 CA19 CC24 DA13 DA164A0AA BAA ABAA4A45 DA50 EA50 FA74 GA01 GA15

Claims (19)

[Claims] 1. A dioxin receptor gene having the following base sequence (a) or (b): (a) a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1; (b) 95% of the amino acid sequence represented by SEQ ID NO: 1
A base sequence encoding a dioxin receptor having an amino acid sequence showing the above amino acid identity. 2. A dioxin receptor gene having a nucleotide sequence represented by nucleotide numbers from 10 to 2835 in the nucleotide sequence represented by SEQ ID NO: 2. 3. A vector containing the dioxin receptor gene according to claim 1 or 2. 4. The vector according to claim 3, wherein a promoter is operably linked to the dioxin receptor gene. 5. The vector according to claim 3, wherein the vector is a virus. 6. A virus particle containing the vector according to claim 5. 7. A method for producing a vector, comprising incorporating the dioxin receptor gene according to claim 1 into a vector capable of replicating in a host cell. 8. A transformant obtained by introducing the dioxin receptor gene according to claim 1 into a host cell. 9. A transformant obtained by introducing the vector according to any one of claims 3 to 5 into a host cell. 10. The transformant according to claim 8, wherein the dioxin receptor gene is introduced into a chromosome of a host cell. 11. The method according to claim 8, wherein the host cell is an animal cell.
0. The transformant according to any one of 0. 12. The transformant according to claim 8, wherein the host cell is a mammalian cell. 13. The transformant according to claim 8, wherein the host cell is an insect animal cell. 14. A method for producing a transformant, comprising introducing the dioxin receptor gene according to claim 1 into a host cell. 15. A method for producing a dioxin receptor, comprising culturing the transformant according to claim 8 to produce a dioxin receptor. 16. A dioxin receptor comprising the amino acid sequence represented by SEQ ID NO: 1. 17. A dioxin receptor having an amino acid sequence showing 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1. 18. A transformant which expresses the dioxin receptor gene according to claim 1 or 2 and which has a reporter gene introduced downstream of a transcription control region containing a dioxin response element, and a test subject. A method for evaluating the ability of a test substance to regulate dioxin receptor activity, comprising the step of contacting the substance with a test substance and measuring the expression level of the reporter gene in the transformant. 19. A receptor binding assay comprising a step of bringing the dioxin receptor according to claim 16 or 17 into contact with a test substance and keeping it warm.