JP2001078782A - Dioxin receptor gene and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new dioxin receptor gene comprising a DNA having a base sequence encoding a specific amino acid sequence and capable of using for assay of a dioxin-like substance causing reproduction abnormality, immunopathy, production of cancer or the like. SOLUTION: This gene is a new dioxin receptor gene comprising either a DNA comprising a base sequence encoding an amino acid sequence shown by the formula or a DNA capable of hybridizing with the DNA comprising the base sequence encoding the amino acid sequence shown by the formula under a stringent condition and encoding a dioxin receptor having an amino acid sequence having more than 95% amino acid identity based on the amino acid sequence shown by the formula, and is useful for a measuring method of a dioxin-like substance. The gene is obtained by extracting a RNA from the liver of a hamster, producing a cDNA library using the RNA through a routine procedure and screening the library using a probe comprising a partial base sequence of the gene by the hybridization method.

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 coplana 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) comprising the DNA of any one of the following (a) or (b): (a) an amino acid represented by SEQ ID NO: 1 DNA consisting of a nucleotide sequence encoding a sequence. (b) hybridizes with a DNA consisting of the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 under stringent conditions, and has 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1 Encoding a dioxin receptor having the amino acid sequence
NA. 2) base number 17 in the base sequence represented by SEQ ID NO: 2
3) a dioxin receptor gene having the nucleotide sequence of SEQ ID NO: 2776, 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 obtained by introducing a gene of the present invention and a reporter gene linked downstream of a transcription control region containing a dioxin response element into a host cell, A method for evaluating the ability of a test substance to regulate dioxin receptor activity, which comprises the step of: bringing the transformant into contact with the test substance and measuring the expression level of the reporter gene in the transformant; It is intended to provide a receptor binding assay, which comprises a step of bringing the dioxin receptor into contact with a test substance and keeping the temperature.

[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 consisting of a DNA consisting of a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and a DNA consisting of a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 under stringent conditions. A dioxin receptor gene consisting of DNA encoding a dioxin receptor having an amino acid sequence having 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1, and a base represented by SEQ ID NO: 2 Among the sequences, a dioxin receptor gene having a base sequence represented by base numbers 17 to 2776 is included. Here, "the DNA hybridizes with a DNA consisting of the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 under stringent conditions, and has 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1". DNA Encoding Dioxin Receptor Having Amino Acid Sequence Showing Sexuality "
Has an amino acid sequence showing 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1, and has a receptor function substantially equivalent to that of the dioxin receptor consisting of the amino acid sequence represented by SEQ ID NO: 1. And DNA encoding a dioxin receptor. The receptor function can be evaluated based on, for example, a reporter assay and a receptor assay described below. As stringent conditions, for example, 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 40)
0, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5%
(w / v) Incubated at 65 ° C. in the presence of 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 then incubated at 1 × SSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.5% SDS are kept twice at room temperature for 15 minutes, and 0.1 × SSC (0.015 M NaCl, 0.0015 M sodium citrate) and 0.5% SDS are also present. The conditions for keeping the temperature at 68 ° C. for 30 minutes can be listed below. Examples of the gene of the present invention include a gene 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. Here, "several" means 2 to about 40, preferably 2 to about 20,
More preferably, the number is 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 encoding such a dioxin receptor may be a natural gene, or may be created by introducing a mutation into the natural gene, for example, by site-directed mutagenesis or mutation treatment. Gene may be used.

[0005] The gene of the present invention can be obtained from tissues such as Syrian hamsters (Mesocricetus auratus) by using
By Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd editio
n) Cold Spring Harbor Laboratory (Col)
d Spring Harbor Laboratory), 1989 and the like. Specifically, first, RNA derived from tissues such as Syrian hamsters is prepared. For example, tissues such as liver and skin such as Syrian hamsters are pulverized in a solution containing a strong protein denaturing agent such as guanidine hydrochloride or guanidine thiocyanate, and phenol, chloroform or the like is further added to the pulverized substance to denature proteins. Let it. 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. A commercially available kit based on these methods includes, for example, ISOGEN (manufactured by Nippon Gene). 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 cDN
A is synthesized. Next, using the single-stranded cDNA as a template,
In addition, a double-stranded cDNA is synthesized by using Escherichia coli DNA polymerase I with an RNA fragment obtained by making a nick and a gap in the RNA chain using Escherichia coli RNase H as a primer. Further, both ends of the double-stranded cDNA are blunted with T4 DNA polymerase. The obtained double-stranded cDNA was extracted with phenol and chloroform,
Purification and recovery are performed by ordinary methods such as ethanol precipitation.
In addition, commercially available kits based on these methods include:
Examples include cDNA synthesis system plus (Amersham Pharmacia) and TimeSaver cDNA synthesis kit (Amersham Pharmacia). Next, the obtained double-stranded cDNA was used, for example, for plasmid pUC118 or phage λ.
A cDNA library is prepared by inserting ligase into a vector such as gt10. Such a cD
From the NA library, for example, a hybridization method using a DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 2 as a probe, or an oligonucleotide having a partial base sequence of the base sequence represented by SEQ ID NO: 2 as a primer The gene of the present invention can be obtained by the polymerase chain reaction (hereinafter referred to as PCR) used. 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 described, for example, by J. Sambrook, EFFrisch, T. Maniatis;
Molecular Cloning 2nd edition (Molecular Cloning
2nd edition), published by Cold Spring Harbor Laboratory, 19
It can be cloned into a vector according to the genetic engineering method described in 1989 and the like. Specifically, for example, TA
Cloning vector (Invitrogen) or pBluescriptI
The gene of the present invention can be cloned using a commercially available plasmid vector such as I (Stratagene).
The gene of the present invention can be prepared, for example, by the phosphite / triester method (Hunk
apiller, M. et al., Nature, 310, 105, 1984) and the like, and can be prepared by chemically synthesizing nucleic acids. The nucleotide sequence of the obtained gene of the present invention is determined by the Maxam Gilbert method (for example, Maxam, AM & W. Gilbert,
Natl. Acad. Sci. USA, 74, 560, 1977) and the Sanger method (for example, Sanger, F. & ARCoulson, J.
Mol. Biol., 94, 441, 1975; Sanger, F, & Nicklen and
ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 19
77 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. First, a reporter gene linked downstream of a transcription control region containing a dioxin response element and the gene of the present invention are, for example, dioxin receptor non-endogenous host cells, specifically, for example, HeLa
Transformants are prepared by introducing the cells into cells or the like. 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,
It may 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 expression level of the reporter gene 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, hamsters (Syrian, male,
Total RNA was extracted and separated from liver. Next, from this total RNA, Oligotex-dT30
mRN using super mRNA purification kit (Takara Shuzo)
A was separated. The operation at this time was in accordance with the description attached to the kit. Using 1 μg of this mRNA, TimeSaver cDNA
Using a synthesis kit (manufactured by Amersham Pharmacia) and random primers contained in the kit,
A library consisting of a double-stranded cDNA to which an EcoRI adapter was bound was prepared. The operation at this time was in accordance with the description attached to the kit. Next, the whole amount of the double-stranded cDNA library thus prepared was mixed with 1 μg of λgt10 (manufactured by Stratagene) digested with EcoRI and treated with alkaline phosphatase, and reacted with T4 ligase. After the reaction, the DNA in the reaction solution was subjected to Gigapack II
Using Igold packaging extract (manufactured by Stratagene), the reagent was packaged into λ phage particles as described in the attached instruction manual. About 1 × 10 ⁶ phage particles obtained in this way, Kazuhiro Makabe, Bio Experimental Illustrated Volume 4, published by Shujunsha, p.12
Screening by plaque hybridization was performed according to the method described in 5-163. As a probe, a human dioxin receptor cDNA (GenBank Acc.
ession No. L19872) corresponding to 52
A 6 bp PvuII-PvuII fragment labeled with ³² P using a RediprimeII kit (Amersham Pharmacia) was used. The hybridization conditions were such that the composition of the hybridization solution was 6XSSPE (0.9M NaCl,
_{0.052M NaH 2 PO 4, 7.5mM EDTA} ), 0.5% SDS, 5xDenhart, 0.
1mg / ml salmon sperm DNA, incubation temperature 55 ° C, incubation time 16 hours, DNA amount of probe used 0.5μ
g, the radioactivity concentration of the hybridization solution is 150 m
It was 10 × 10 ⁸ cpm per liter. The conditions for washing the filter after hybridization are as follows: the composition of the washing solution is 1xSSC
(0.15 M NaCl, 15 mM sodium citrate), 0.5% SDS, the temperature was 62 ° C., and the temperature was kept twice for 40 minutes. The obtained filter was exposed to a film for 2 nights, and autoradiography was performed. As a result, multiple positive signals were detected. Next, using the phage DNA collected from the plaque that gave the positive signal as a template, λgt
PCR was performed with PCRsystem9700 (manufactured by Applied Biosystems) using Long PCR primers (manufactured by Clontech) for 10 insert screening. The amount of each primer in the reaction solution was 10 pmol, and the temperature was kept at 95 ° C. for 1 minute and then at 68 ° C. for 3 minutes as one cycle using LA-Taq polymerase (Takara Shuzo) and a buffer attached to the enzyme. Cycled. Thus, the DNA inserted into each phage was amplified. These inserted DNAs were subjected to direct sequencing using a dye terminator sequencing kit FS (manufactured by ABI) using an auto sequencer model 377 manufactured by ABI. As a result, it was found that the inserted DNA having a length of slightly more than 3 kbp on the electrophoresis gel had the nucleotide sequence represented by SEQ ID NO: 2.

Example 2 (Acquisition of the Gene of the Present Invention) J. Sambrook, EFFrisch, and T. Maniatis from hamster (Syrian male, purchased from Japan SLC) liver
Author; Molecular Cloning 2nd Edition (Molecular Clo
ning 2nd edition), published by ColdSpring Harbor Laboratory, 1
989 Total RNA 1 isolated by guanidine thiocyanate-CsCl density gradient ultracentrifugation according to the description in 7.19.
The μg 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. In order to obtain the gene of the present invention by PCR, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6 were converted to a DNA synthesizer (Applied Biosystems Model 394). Was synthesized using Using these oligonucleotides as primers and the above single-stranded cDNA library as a template,
Rsystem9700 (Applied Biosystems) P
Performed CR. The amount of primer in the reaction solution is 10 pmo each
l, the amount of template DNA was 10 ng, and LA-Taq polymerase (Takara Shuzo) and a buffer attached to the enzyme were used.
This was repeated 35 times with one cycle of incubation at 5 ° C. for 1 minute and then at 68 ° C. for 3 minutes. The entire amount of the reaction solution was used as a low melting point agarose (agarose L; manufactured by Nippon Gene).
When subjected to agarose gel electrophoresis using
A band indicating a mobility of bp was detected. DNA was recovered from the gel in this band, and the auto sequencer ABI was used.
The sample was subjected to direct sequencing using a dye terminator sequence kit FS (manufactured by Applied Biosystems) using model 377 manufactured by the company. As a result, the D
NA is the base number 17 of the base sequence represented by SEQ ID NO: 2.
It was found to have a base sequence represented by # 2776.

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 the 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 is introduced into chromosome) Using Isogen reagent (manufactured by Nippon Gene Co., Ltd.), 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: 17 (forward primer) and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 (reverse primer). A DNA having a base sequence (J. Biochem., 110.232-236 (1991)) of a region 750 bp to 1370 bp upstream of TATAbox upstream of the human CYP1A1 gene and containing a dioxin response element was amplified. Amplified DNA
Was recovered, and its ends were blunt-ended using Bluntingkit (manufactured by Takara Shuzo) (the DNA is hereinafter referred to as XRE DNA). Nucleotide sequence near the TATA box and leader sequence of mouse metallothionein I gene (Genbank Accession No.J
Two oligonucleotides each having a nucleotide sequence derived from SEQ ID NO: 19; and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 20 which is annealed to form a double-stranded DNA. This was reacted with T4 polynucleotide kinase to phosphorylate both ends (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 Bacterial alkaline phosphatase (BAP) was added thereto.
It was kept at 5 ° C for 1 hour. Next, the warm solution was subjected to electrophoresis using low-melting point agarose (AgaroseL; manufactured by Nippon Gene Co., Ltd.), and Bg containing pGL3-derived luciferase gene was analyzed.
l DN showing an electrophoretic mobility corresponding to the length of II-Hind III fragment
A was recovered. About 100 ng of the recovered DNA and the TAT
The plasmid pGL3-TATA was prepared by mixing 1 μg of A DNA and binding with T4 ligase. Next, pGL3
-TATA is digested with the restriction enzyme Sma I, and
Incubated at ℃ 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. About 100 ng of the DNA and about 1 μg of the above XRE DNA
And reacted with T4 ligase, and then
DNA is used for DH5α competent cells (TOYOBO)
Introduced. 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. A plasmid having a structure in which about 600 bp of DNA corresponding to XRE DNA was introduced into SGL site of pGL3-TATA by one copy was selected.
This was named plasmid pGL3-TATA-1A1. Then
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 obtained from the obtained ampicillin-resistant E. coli clone.
Were digested with the restriction enzyme BamHI, and the digested solution was analyzed by agarose gel electrophoresis. The blasticidin S deaminase gene expression cassette is plasmid pG
A plasmid having a structure inserted at the BamHI cleavage site of L3-TATA-1A1 was selected, and plasmid pGL3-TATA-1A1-BSD was selected.
I named it. Next, the plasmid pGL3-TATA-1A1-BSD
NA is digested with the restriction enzyme SalI, and the gene of the present invention is pR
The DNA of the vector of the present invention integrated in C / RSV was digested with PvuI. On the other hand, HeLa cells were transformed with D containing 10% FBS.
5% C at 37 ° C using MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
The cells were cultured in the presence of O ₂ using a petri dish having a diameter of about 10 cm (Falcon). After culturing about 5 × 10 ⁵ cells for one day, lipofectin (manufactured by GIBCO) was added to the cells.
The DNAs of the above two plasmids digested with the restriction enzymes SalI or PvuI were simultaneously introduced by the lipofection method using. The conditions of the lipofection method were as described in the manual attached to Lipofectin, for a treatment time of 5 hours, and a total amount of linearized plasmid DNA of 7 μg.
(3.5 μg each) / Petri dish, Lipofectin amount is 2
0 μl / dish was used. After lipofection, 10% FBS
And cultured for 3 days in a DMEM medium containing Next, the cells were detached from the Petri dish by trypsin treatment, seeded on 10 new Petri dishes by 1/10 amount, and cultured as it was until the next day. Next, G418 (SIGMA) was added to a final concentration of 4
The blasticidin S was further added to the medium so as to have a final concentration of 8 μg / ml, and the culture was continued. One week later, G418 and blasticidin S were replaced with a fresh medium containing the same concentration as above, and the culture was continued. One week later, the same operation was 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. Continued. Cells were harvested by detachment by trypsinization before becoming confluent, 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 5 μl diluted PGC50 (manufactured by Nippon Gene) was added in 20 μl portions. Was dissolved. The plates were set on a luminometer LB96p (Berthold) with an automatic enzyme substrate injector, and luciferase activity was measured while automatically dispensing 50 μl of the substrate solution PGL100 (Nippon Gene). A transformant showing 2- or more-fold higher luciferase activity was selected in the system to which 3-methylcholanthrene was added than in the system to which 3-methylcholanthrene was not added.

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 was performed at 37 ° C. under 1% CO _{2 for} 1 day. 2,3,7,
8-TCDD (obtained from Daiichi Kagaku) dissolved in DMSO (manufactured by Wako Pure Chemical Industries, Ltd.) and added to a final concentration of 0.15 pM to 150 nM F
E-MEM medium containing BS and antibiotics, and 2,3,
Instead of the DMSO solution of 7,8-TCDD, an E-MEM medium containing FBS and an antibiotic containing the same amount of DMSO was prepared, and these were replaced with the culture supernatants of the cells, respectively. In addition,
The experiment was performed with six repetitive experiments, and the average value was 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.) was added in an amount of 50 μl / well 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 manner is set on a luminometer LB96p (manufactured by Berthold) with an automatic substrate injector, and 50 μl / we.
Enzyme substrate liquid PGL100 (manufactured by Nippon Gene)
The amount of luminescence was measured immediately for 1 second while adding. As a result, a concentration-dependent increase in transcriptional activity of 2,3,7,8-TCDD, which is known as a ligand for the dioxin receptor, was detected (FIG. 1). 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.

[0022]

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 List 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: 6 SEQ ID NO: 7 An oligonucleotide primer designed to determine the base sequence SEQ ID NO: 8 An oligonucleotide primer designed to determine the base sequence SEQ ID NO: 9 To determine the base sequence Designed oligonucleotide primer SEQ ID NO: 10 Oligonucleotide primer designed to determine base sequence SEQ ID NO: 11 Oligonucleotide primer designed to determine base sequence SEQ ID NO: 12 Base sequence SEQ ID NO: 13 Oligonucleotide primer designed to determine base sequence SEQ ID NO: 13 Oligonucleotide primer designed to determine base sequence SEQ ID NO: 14 Oligonucleotide primer designed to determine base sequence SEQ ID NO: 15 SEQ ID NO: 16 oligonucleotide primer designed for determining base sequence SEQ ID NO: 17 oligonucleotide primer designed for PCR SEQ ID NO: 18 oligonucleotide designed for PCR Primer SEQ ID NO: 19 Oligonucleotide designed for synthesizing promoter DNA SEQ ID NO: 20 Oligonucleotide designed for synthesizing promoter DNA

[0023]

[Sequence List] <110> Sumitomo Chemical Company Limited <120> Dioxin receptor gene <130> P151495 <150> JP 11/196035 <151> 1999-07-09 <160> 20 <210> 1 <211> 920 <212 > PRT <213> Mesocricetus auratus <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 Ile 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 Glu Gln Cys Arg Ala Phe Arg Asp Pro Leu Asp Leu Gln Glu Gly 100 105 110 Glu Phe Leu Leu Gln Ala Leu Asn Gly Phe Val Leu Val Val Thr Ala 115 120 125 Asp Ala Leu Val Phe Tyr Ala Ser Ser Thr Ile Gln Asp Tyr Leu Gly 130 135 140 Phe Gln Gln Ser Asp Val Ile His Gln Ser Val Tyr Glu Leu Ile His 145 150 155 160 Thr Glu Asp Arg Ala Glu Phe Gln Arg Gln Leu H is Trp Ala Leu Asn 165 170 175 Pro Ala Gln Cys Thr Asp Ser Ala Gln Gly Gly Asp Glu Ser His Gly 180 185 190 Leu Ser Gln Pro Pro Ala Val Tyr Phe Asn Pro Asp Gln Leu Pro Pro 195 200 205 Glu Ser Ala Ser Phe Leu Glu Arg Cys Phe Ile Cys Arg Leu Arg Cys 210 215 220 Leu Leu Asp Asn Ser Ser Gly Phe Leu Ala Met Asn Phe Gln Gly Arg 225 230 235 240 Leu Lys Tyr Leu His Gly Gln Asn Lys Lys Gly Lys Asp Gly Thr Leu 245 250 255 Leu Pro Pro Gln Leu Ala Leu Phe Ala Ile Ala Thr Pro Leu Gln Pro 260 265 270 Pro Ser Ile Leu Glu Ile Arg Thr Lys Asn Phe Ile Phe Arg Thr Lys 275 280 285 His Lys Leu Asp Phe Thr Pro Ile Gly Cys Asp Ala Lys Gly Gln Leu 290 295 300 Val Leu Gly Tyr Thr Glu Ala Glu Leu Cys Thr Arg Gly Ser Gly Tyr 305 310 315 320 Gln Phe Ile His Ala Ala Asp Met Leu Tyr Cys Ala Glu Phe His Val 325 330 335 Arg Met Ile Lys Thr Gly Glu Ser Gly Met Thr Val Phe Arg Leu Leu 340 345 350 Ala Lys His Ser Arg Trp Arg Trp Val Gln Ser Asn Ala Arg Leu Ile 355 360 365 Tyr Arg Asn Gly Arg Pro Asp Tyr Ile Ile Ala ThrGln Arg Pro Leu 370 375 380 Thr Asp Glu Glu Gly Arg Glu His Leu Gln Lys Arg Ser Met Thr Leu 385 390 395 400 400 Pro Phe Met Phe Ala Thr Gly Glu Alu Val Leu Tyr Glu Ile Ser Ser 405 410 415 Pro Phe Pro Pro Ile Met Asp Pro Leu Pro Ile Arg Thr Lys Ser Gly 420 425 430 430 Thr Gly Ala Lys Asp Trp Val Pro Gln Ser Thr Pro Ser Asn Asp Ser 435 440 445 Leu His Pro Ser Ser Leu Met Asn Cys Met Ile Gln Gln Asp Glu Ser 450 455 460 Ile Tyr Leu Cys Pro Pro Ser Ser Ala Ala Pro Leu Asp Ser His Phe 465 470 475 480 Leu Thr His Gly Ser Glu Gly Asp Gly Trp Gln Asp Ser Ile Ala Ser 485 490 495 Ile Gly Ser Glu Ala Glu Leu Lys His Glu Gln Ile Gly His Gly Gln 500 505 510 Asp Met Asn Pro Ala Val Ser Gly Gly Pro Pro Gly Leu Phe Pro Asp 515 520 525 Asn Arg Asn Ser Asp Leu Tyr Ser Ile Met Lys Asn Leu Gly Ile Asp 530 535 540 Phe Asp Asp Ile Lys Arg Met Gln Ser Glu Glu Phe Phe Arg Thr Glu 545 550 555 560 Leu Ala Gly Glu Val Asp Phe Arg Asp Ile Asp Ile Thr Asp Glu Ile 565 570 575 Leu Thr Tyr Val Gln Asp Ser Leu Asn Arg Ser ThrLeu Leu Ser Ser 580 585 590 Ala Ser Gln Gln Gln Pro Val Thr Gln His Leu Ser Cys Met Leu Gln 595 600 605 Glu Arg Leu His Leu Gly Gln Arg Gln Leu Gln Gln His Glu Thr Gln 610 615 620 Ala Ala Glu Pro Gln Gln Gln Leu Gly His Gln Thr Ala Pro Gln Gln 625 630 635 640 Glu Leu Cys His Gln Thr Ala Pro Gln Gln Gln Met Cys Leu Gln Met 645 650 655 Ala Pro Gln Gln Glu Leu Cys His Gln Met Glu Pro Gln Gln Gln Leu 660 665 670 Cys Leu Gln Met Ala Pro Gln Gln Gln Leu Cys His Gln Thr Ala Pro 675 680 685 Gln Gln Gln Leu Cys Leu Gln Met Ala Pro Gln Gln Glu Leu Cys His 690 695 700 Gln Thr Ala Pro Gln Pro Glu Leu Gly Gln Lys Met Asn His Ala Gln 705 710 715 715 720 Val Asn Gly Met Phe Ala Ser Trp Asn Pro Thr Pro Leu Val Pro Phe 725 730 735 Ser Cys Pro Gln Gln Glu Leu Lys His Tyr Asp Val Phe Ser Asp Leu 740 745 750 Gln Gly Ala Ile Glu Glu Phe Pro Tyr Lys Ser Glu Met Asp Ser Met 755 760 765 Pro Tyr Thr Gln Ser Phe Ala Pro Cys Asn Gln Ser Val Leu Pro Gln 770 775 780 780 Arg Ser Lys Cys Ala Gln Leu Asp Leu Pro Gly Lys Gly Phe Glu Pro 785 790 795 800 Ser Leu His Pro Asn Thr Ser Asn Val Gly Asp Phe Val Thr Cys Leu 805 810 815 Gln Val Pro Glu Asn Gln Arg His Glu Val His Pro Gln Ser Ala Met 820 825 830 Val Ala Pro Gln Thr Tyr Tyr Ala Gly Ala Met Ser Met Tyr Gln Cys 835 840 845 Gln Pro Gly Pro Gln His Val Pro Val Glu Gln Met Gln Tyr Ser Pro 850 855 860 Ala Val Pro Asp Ser Gln Ala Phe Leu Asn Lys Phe Gln Asn Gln Gly 865 870 875 880 Val Leu Asn Glu Thr Tyr Ser Ser Glu Leu Asn Ser Val Gly His Arg 885 890 895 Gln Thr Thr Ala His Leu His His Pro Ala Glu Gly Arg Pro Phe Pro 900 905 910 Asp Ile Thr Pro Ser Gly Phe Leu 915 920 <210> 2 <211> 3144 <212> DNA <213> Mesocricetus auratus <220> <221> CDS <222> (17) ... (2779) <400> 2 gcagggcgcg ggcacc atg agc agc ggc gcc aac atc acc tac gcc agc cgc 52 Met Ser Ser Gly Ala Asn Ile Thr Tyr Ala Ser Arg 1 5 10 aag cgg cgc aag ccg gtg cag aaa aca gtg aag cca gtc ccc gct gaa 100 Lys Arg Arg Lys Pro Val Gln Lys Thr Val Lys Pro Val Pro Ala Glu 15 20 25 gga atc aag tca aat cct tct aa g cga cat aga gac agg cta aac aca 148 Gly Ile Lys Ser Asn Pro Ser Lys Arg His Arg Asp Arg Leu Asn Thr 30 35 40 gag ttg gac cgc ctg gct agt ctg ctg ccc ttc cca caa gat gtt att 196 Glu Leu Asp Arg Leu Ala Ser Leu Leu Pro Phe Pro Gln Asp Val Ile 45 50 55 60 aac aag ctg gac aaa ctc tca gtt ctt agg ctc agt gtc agc tac ctg 244 Asn Lys Leu Asp Lys Leu Ser Val Leu Arg Leu Ser Val Ser Tyr Leu 65 70 75 agg gcc aag agc ttc ttc gat gtt gcg tta aaa tcc tcc ccc gct gac 292 Arg Ala Lys Ser Phe Phe Asp Val Ala Leu Lys Ser Ser Pro Ala Asp 80 85 90 agg aat gga ggc cag gag cag tgt aga gcg ttc aga gat cca ctg gac 340 Arg Asn Gly Gly Gln Glu Gln Cys Arg Ala Phe Arg Asp Pro Leu Asp 95 100 105 ctg caa gaa gga gag ttc tta ttg cag gca ctg aac ggc ttc gta ctg 388 Leu Gln Glu Glu Glu Glu Glu Glu Glu Ala Leu Asn Gly Phe Val Leu 110 115 120 gtt gtc aca gca gat gcc ttg gtc ttc tat gcc tcc tct act atc caa 436 Val Val Thr Ala Asp Ala Leu Val Phe Tyr Ala Ser Ser Thr Ile Gln 125 130 135 140 gat tac ctg ggc ttt cag caa tct gat gtc atc cat cag agt gtc tat 484 Asp Tyr Leu Gly Phe Gln Gln Ser Asp Val Ile His Gln Ser Val Tyr 145 150 155 gag ctc atc cat acc gaa gac cga gct gag ttc cag cgc cag ctt cac 532 Glu Leu Ile His Thr Glu Asp Arg Ala Glu Phe Gln Arg Gln Leu His 160 165 170 tgg gct cta aac ccc gca cag tgt aca gac tct gca caa gga ggg gat 580 Trp Ala Leu Asn Pro Ala Gln Cys Thr Asp Ser Ala Gln Gly Gly Asp 175 180 185 gaa tct cat ggc ctc tca cag cct ccg gca gtc tac ttc aac cca gac 628 Glu Ser His Gly Leu Ser Gln Pro Pro Ala Val Tyr Phe Asn Pro Asp 190 195 200 cag ctt cct ccg gag agc gcc tcc ttc ctg gag ttc atc tgc 676 Gln Leu Pro Pro Glu Ser Ala Ser Phe Leu Glu Arg Cys Phe Ile Cys 205 210 215 220 cgg ctc cgg tgt ctg ctg gat aat tcc tct ggt ttt ctg gca atg aat 724 Arg Leu Arg Cys Leu Leu Asp Sern Ser Gly Phe Leu Ala Met Asn 225 230 235 ttc caa ggg cgg cta aag tat ctc cat gga cag aac aag aag ggg aag 772 Phe Gln Gly Arg Leu Lys Tyr Leu His Gly Gln Asn Lys Lys Gly Lys 240 245 250 gat gga aca ctactg ccc ccg cag ctg gct ctg ttt gca ata gcc acc 820 Asp Gly Thr Leu Leu Pro Pro Gln Leu Ala Leu Phe Ala Ile Ala Thr 255 260 265 cca ctt cag ccc ccg tcc ata ctg gaa atc cga acc aaa aac ttc atc 868 Leu Gln Pro Pro Ser Ile Leu Glu Ile Arg Thr Lys Asn Phe Ile 270 275 280 ttc agg acc aaa cac aaa cta gac ttc aca cct att ggt tgc gat gcc 916 Phe Arg Thr Lys His Lys Leu Asp Phe Thr Pro Ile Gly Cys Asp Ala 285 290 295 300 aaa ggg cag ctt gtc ctg ggc tac acg gaa gcg gag ctg tgc acc cga 964 Lys Gly Gln Leu Val Leu Gly Tyr Thr Glu Ala Glu Leu Cys Thr Arg 305 310 315 ggc tcg ggg tac cgccc gcc gac atg ctc tac tgc gcg 1012 Gly Ser Gly Tyr Gln Phe Ile His Ala Ala Asp Met Leu Tyr Cys Ala 320 325 330 gag ttc cac gtc cgc atg att aag act ggg gaa agc ggc atg aca gtt 1060 Glu Phe Val Ile Lys Thr Gly Glu Ser Gly Met Thr Val 335 340 345 tt ttc cgg ctt ctg gca aag cac agt cga tgg agg tgg gtc cag tcc aac 1108 Phe Arg Leu Leu Ala Lys His Ser Arg Trp Arg Trp Val Gln Ser Asn 350 355 360 gct cgc ttg atc tat aga aac ggg agg cca gat tac atc atc gca act 1156 Ala Arg Leu Ile Tyr Arg Asn Gly Arg Pro Asp Tyr Ile Ile Ale Thr 365 370 375 380 cag agg cca cta acg gat gaa gaa gga aga gag cag aag cgg 1204 Gln Arg Pro Leu Thr Asp Glu Glu Gly Arg Glu His Leu Gln Lys Arg 385 390 395 agt atg acg ctg ccc ttc atg ttc gct aca gga gag gct gta ttg tac 1252 Ser Met Thr Leu Pro Phe Mhe Phe Ala Thr Gly Glu Ala Val Leu Tyr 400 405 410 gag atc tcc agc cct ttc cct ccc ata atg gat ccc ttg cca atc cgt 1300 Glu Ile Ser Ser Pro Phe Pro Pro Ile Met Asp Pro Leu Pro Ile Arg 415 420 425 acc aaa agc ggc acc ggt gca aag gac tgg gtt ccg cag tca aca cca 1348 Thr Lys Ser Gly Thr Gly Ala Lys Asp Trp Val Pro Gln Ser Thr Pro 430 435 440 agc aat gat tcc ctc cac ccc agc tcg ctt atg aat tgc atg atc caa 1396 Ser Asn Asp Ser Leu His Pro Ser Ser Leu Met Asn Cys Met Ile Gln 445 450 455 460 cag gat gag tcc atc tat ctc tgt cct cct tcg agt gct gcg ccg cta 1444 Gln Asp Glu Ser Ile Tyr Leu Cys Pro Pro Ser Ser Ala A la Pro Leu 465 470 475 gac agc cat ttt ctc acc cac ggg agt gaa ggc gat ggt tgg cag gac 1492 Asp Ser His Phe Leu Thr His Gly Ser Glu Gly Asp Gly Trp Gln Asp 480 485 490 agt att gca tca ata gga agt gaa gct gag ttg aaa cat gaa caa atc 1540 Ser Ile Ala Ser Ile Gly Ser Glu Ala Glu Leu Lys His Glu Gln Ile 495 500 505 gga cat ggt cag gac atg aac cct gca gtc tct gga ggc ccc ccg ggg 1588 Gly His Gly Gln Asp Met Asn Pro Ala Val Ser Gly Gly Pro Gly 510 515 520 ctc ttt cca gat aat aga aat agt gac ttg tac agc atc atg aaa aac 1636 Leu Phe Pro Asp Asn Arg Asn Ser Asp Leu Tyr Ser Ile Met Lys Asn 525 530 535 540 cta ggg atc gat ttc gat gac atc aaa cgc atg cag agt gag gag ttc 1684 Leu Gly Ile Asp Phe Asp Asp Ile Lys Arg Met Gln Ser Glu Glu Phe 545 550 555 ttc agg act gag ctg gct gac gtt gaga atc gac ata 1732 Phe Arg Thr Glu Leu Ala Gly Glu Val Asp Phe Arg Asp Ile Asp Ile 560 565 570 aca gat gaa atc ctc act tac gtg caa gac tct cta aac agg tcg acc 1780 Thr Asp Glu Ile Leu Thr Tyr Val Gl n Asp Ser Leu Asn Arg Ser Thr 575 580 585 ttg ctg agt tct gcc agc cag cag cag cct gtg act cag cac ctg agc 1828 Leu Leu Ser Ser Ala Ser Gln Gln Gln Pro Val Thr Gln His Leu Ser 590 595 600 tgc atg ctg cag gaa cgc ctg cat ctg ggg cag cgg cag ctg cag cag 1876 Cys Met Leu Gln Glu Arg Leu His Leu Gly Gln Arg Gln Leu Gln Gln 605 610 615 615 620 cac gaa act cag gca gcg gag ccc cag cag cag gag 1924 His Glu Thr Gln Ala Ala Glu Pro Gln Gln Gln Leu Gly His Gln Thr 625 630 635 gcg ccc cag caa gag ctg tgt cac cag acg gcg ccc cag caa cag atg 1972 Ala Pro Gln Gln Glu Leu Cys His Gln Thr Ala Pro Gln Gln Gln Met 640 645 650 tgt ctt cag atg gcg ccc cag caa gag ctg tgt cat cag atg gaa ccc 2020 Cys Leu Gln Met Ala Pro Gln Gln Glu Leu Cys His Gln Met Glu Pro 655 660 665 caa cag cag ctg tgt ctt cag gcg ccc cag cag cag ctg tgt cac 2068 Gln Gln Gln Leu Cys Leu Gln Met Ala Pro Gln Gln Gln Leu Cys His 670 675 680 cag acg gca ccc cag caa cag ctg tgt ctt cag atg gcg ccc cag caa 2116 Gln Thr Ala o Gln Gln Gln Leu Cys Leu Gln Met Ala Pro Gln Gln 685 690 695 700 gag ctg tgt cac cag acg gcg ccc cag ccg gag ctg ggt cag aaa atg 2164 Glu Leu Cys His Gln Thr Ala Pro Gln Pro Glu Leu Gly Gln Lys Met 705 710 715 aat cat gcg caa gtc aat ggc atg ttt gca agt tgg aac ccc acc cct 2212 Asn His Ala Gln Val Asn Gly Met Phe Ala Ser Trp Asn Pro Thr Pro 720 725 730 ctc gtg cct ttc agc tgt cct cag cag gaa aag cat tat gac gtc 2260 Leu Val Pro Phe Ser Cys Pro Gln Gln Glu Leu Lys His Tyr Asp Val 735 740 745 ttt tca gac ttg cag ggg gcg att gag gag ttc ccc tac aaa tct gag 2308 Phe Ser Asp Leu Gln Gly Ala Ile Glu Glu Phe Pro Tyr Lys Ser Glu 750 755 760 atg gac agt atg cct tac aca cag agc ttt gct ccc tgt aat cag tct 2356 Met Asp Ser Met Pro Tyr Thr Gln Ser Phe Ala Pro Cys Asn Gln Ser 765 770 775 780 780 gtg cta ccc cag cgt tcc aag tgt gca cag ctg gac ttg cct gga aag 2404 Val Leu Pro Gln Arg Ser Lys Cys Ala Gln Leu Asp Leu Pro Gly Lys 785 790 795 ggt ttc gaa cca tcc ctg cac ccc aac act tct aat gta ggat ttt 2452 Gly Phe Glu Pro Ser Leu His Pro Asn Thr Ser Asn Val Gly Asp Phe 800 805 810 gtc act tgt tta caa gtt ccc gaa aac caa aga cac gag gtg cat cca 2500 Val Thr Cys Leu Gln Val Pro Glu Asn Gln Arg His Glu Val His Pro 815 820 825 cag tcg gcc atg gtc gct cct cag aca tac tat gct ggg gcc atg tcc 2548 Gln Ser Ala Met Val Ala Pro Gln Thr Tyr Tyr Ala Gly Ala Met Ser 830 835 840 atg tac cag tgc cag cca gga cct cag cac gtc cct gta gag cag atg 2596 Met Tyr Gln Cys Gln Pro Gly Pro Gln His Val Pro Val Glu Gln Met 845 850 855 860 cag tac agc cct gca gtc cca gac tcc cag gca ttt cta aac aag ttt 2644 Gln Tyr Ser Pro Ala Val Pro Asp Ser Gln Ala Phe Leu Asn Lys Phe 865 870 875 cag aat cag gga gtg tta aat gaa acc tat tcg tct gaa tta aac agc 2692 Gln Asn Gln Gly Val Leu Asn Glu Thr Tyr Ser Ser Glu Leu Asn Ser 880 885 890 gtt ggt cac agg cag acc act gct cat ctt cat cac cct gca gaa ggg 2740 Val Gly His Arg Gln Thr Thr Ala His Leu His His Pro Ala Glu Gly 895 900 905 agg ccg ttt cct gat atc aca ccc agc gga t tc ctg tag ctttgggaac aa 2791 Arg Pro Phe Pro Asp Ile Thr Pro Ser Gly Phe Leu 910 915 920 gataccggga ctgtctgtcc atgttggcca tcagcactct ttttccaagc cagattttaa 2851 tctttgtgtt gggaagagga gtttaaaaac tgttatcggt tacctataaa atgggaagct 2911 ctgcagcagc atagggagag accacaagcc tttaagtagc tttagcacct cagcgtgcac 2971 atgcccacaa cggagagctt tggggttctg gagagctgag cgtctccact cagtgaccag 3031 ccagaaagac aacttcagga ttcccgatgg tgtaccaaat acagttagaa gactaattga 3091 ttttaacctc tctgtgtctc agatgttaga ataaatggtt tggtgctttt atc 3144 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotideccagccc400g <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 4 gacgctcagc tctccagaac cccaaagctc tccgt 35 <210> 5 <211> 36 <212> DNA < 213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 agggcgcggg caccatgagc agcggcgcca acatca 36 <210> 6 <211> 35 <212> D NA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 6 gacgctcagc tctccagaac cccaaagctc tccgt 35 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 7 ctcggggtac cagttcatcc a 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 8 tggatgaact ggtaccccga g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 9 ctcggggtac cagttcatcc a 21 <210> 10 <211> 21 <212 > DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 10 gtatgacgct gcccttcatg t 21 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223 > Designed oligonucleotide primer to determine nucleotide sequence <400> 11 tagacagcca ttttctcacc cac 23 <210> 12 <211> 20 <212> DNA <213> Arti ficial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 12 aggactgagc tggctggcga 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 13 cagcaagagc tgtgtcatca gat 23 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 14 agcaggaact gaagcattat 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 15 gtccatgtac cagtgccag 19 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to determine nucleotide sequence <400> 16 tgtccatgtt ggccatcagc a 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 17 ttgagctagg cacgcaaata 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> De signed oligonucleotide primer for PCR <400> 18 gctttgattg gcagagcaca 20 <210> 19 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide to synthesize promoter DNA <400> 19 gatctcgact ataaagaggg caggctgtcc tctaagcgtc accacgactt ca 52 <210> 20 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide to synthesize promoter DNA <400> 20 agcttgaagt cgtggtgacg cttagaggac agcctgccct ctttatagtc ga 52

[Brief description of the drawings]

FIG. 1 shows the results of measuring the dioxin receptor activation ability of 2,3,7,8-TCDD by a reporter assay using the gene of the present invention. Columns are 2,3,
A section to which only DMSO used as the solvent for 7,8-TCDD was added (DMS
O), a section to which TCDD was added to a final concentration of 0.15 pM, a section to which TCDD was added to a final concentration of 1.5 pM, a section to which TCDD was added to a final concentration of 15 pM, and a section to which a final concentration of 150 pM was added.
, TCDD added to a final concentration of 1.5 nM, TCDD added to a final concentration of 15 nM, final concentration 15
The section to which TCDD was added to be 0 nM is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 5/10 C12N 7/00 4H045 7/00 C12P 21/02 C C12P 21/02 C12Q 1/02 C12Q 1 / 02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/566 33/566 C12N 5/00 A F term (reference) 2G045 AA40 BB20 CB21 DA12 DA13 DA14 DA36 FB02 FB04 4B024 AA11 BA63 CA04 DA02 DA05 DA11 EA02 GA11 4B063 QA01 QQ15 QQ16 QQ18 QQ19 QQ20 QR02 QR80 QX02 4B064 AG20 CA02 CA05 CA10 CA19 CC24 DA13 4B065 AA01X AA57X AA83X AA86X AA87X AA90Y AA95X AB01 AC14 BA0 CA40 ACA4 ACA4 ACA4ACA4

Claims (19)

[Claims] 1. A dioxin receptor gene consisting of the following DNA (a) or (b): (a) DNA comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. (b) hybridizes with a DNA consisting of the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 under stringent conditions, and has 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1 Encoding a dioxin receptor having the amino acid sequence
NA. 2. A dioxin receptor gene having a nucleotide sequence represented by nucleotide numbers 17 to 2776 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 obtained by introducing a reporter gene downstream of a transcription control region containing a dioxin response element and the dioxin receptor gene according to claim 1 or 2 into a host cell. 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.