JP2005087077A - Recombinant cell, method for analyzing polycyclic aromatic compound and kit for analyzing polycyclic aromatic compound using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique with which a polycyclic aromatic compound is analyzed in high sensitivity. <P>SOLUTION: The cell is a recombinant cell that contains a guinea pig-derived AhR (arylhydrocarbon receptor) gene and a guinea pig-derived Arnt (AhR nuclear translocator) and expresses the pig-derived AhR and the pig-derived Arnt. Preferably the cell contains a reporter system for indirectly analyzing a complex composed of a polycyclic aromatic compound, the AhR and the Arnt. The cell is useful in a method for analyzing a polycyclic aromatic compound and in a kit for analyzing a polycyclic aromatic compound. The polycyclic aromatic compound, for example, includes dioxins, PCDF (polychlorinated dibenzofuran), PCB (polychlorinated biphenyl), β-naphthoflavone, 3-methylcholanthrene, benzopyrene, benzimidazole, dibenzanthracene, pentachlorophenol, etc. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a genetically modified cell, a method for analyzing a polycyclic aromatic compound using the same, and a kit for analyzing a polycyclic aromatic compound.

  For detection and analysis of polycyclic aromatic compounds such as dioxins, an expensive and large-scale analyzer is required, and the operation is complicated. In instrumental analysis, it is possible to separate and detect various polycyclic aromatic compounds contained in a test substance, but it is possible to judge the total biological toxicity in the environment from the measurement results. It can be difficult.

  AhR (aryl hydrocarbon receptor) is known as a factor that mediates the induction of drug-metabolizing enzymes when polycyclic aromatic compounds such as dioxins are administered to animals. Arnt (AhR nuclear translocator) is known as a factor that forms a heterodimer with AhR and is involved in the regulation of gene expression of ligand-responsive genes for polycyclic aromatic compounds. . A complex of a polycyclic aromatic compound, AhR and Arnt has a specific base sequence (foreign substance responsive element; XER) upstream of a gene of a drug metabolizing enzyme (for example, P450, glutathione S-transferase, etc.). ) And functions as a transcriptional regulator.

At present, there are methods for introducing a reporter assay of chemical substances, etc. using transformed cells obtained by introducing AhR genes derived from rats and hamsters into insect cells and human cultured cells together with reporter genes, A method of performing a receptor binding assay using AhR obtained by culturing transformed cells has been proposed (see, for example, Patent Documents 1 and 2). Moreover, the result of having investigated the ligand responsiveness with respect to several polycyclic aromatic compounds using the yeast ( Saccharomyces cerevisiae ) which introduce | transduced AhR gene derived from a human is reported (refer nonpatent literature 1).
JP 2000-354494 A Japanese Patent Laid-Open No. 2001-78782 Miller, III “A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures” (1998) ) Toxicology and Applied Pharmacology, 160, 297-303

  However, in order to analyze polycyclic aromatic compounds contained in trace amounts in the environment, development of a more sensitive analytical method is expected. Therefore, an object of the present invention is to provide a technique that enables analysis of a highly sensitive polycyclic aromatic compound.

  To achieve the above object, the cell of the present invention comprises a guinea pig-derived AhR (aryl hydrocarbon receptor) gene and a guinea pig-derived Arnt (AhR nuclear translocation factor) gene, and the guinea-pig-derived AhR and guinea pig-derived cells. It is a recombinant cell characterized by expressing Arnt.

  Here, the fact that guinea pigs are highly sensitive to dioxins among vertebrates was already known. However, the present invention is not immediately conceived from this fact. Rather, it can be said that it is difficult for those skilled in the art to arrive at the present invention. That is, various mechanisms are considered that guinea pigs are highly sensitive to dioxins. For example, (1) guinea pig cells have high cell membrane permeability and easily absorb dioxins, (2) high activity of proteins (Hsp90, Ara9, AhR, Arnt, etc.) involved in dioxin initial responses in guinea pigs, or (3) At least one of the P450 protein molecular species induced by dioxin can metabolize dioxin (cannot be decomposed by humans and other animals), and its metabolic intermediate products are toxic. Thus, among various possible mechanisms, the present inventors have found that the complex formation of AhR, Arnt, and dioxin and the transcription-inducing activity of the complex are highly sensitive to dioxins at the individual level of guinea pigs. As a result of further research based on this hypothesis, it was proved that the mechanism of this hypothesis was correct and led to the present invention. It is common knowledge not only in biology but also in the scientific field that even if one hypothesis is made out of various possible mechanisms, it is unlikely that it can be verified.

  Since the cell of the present invention expresses guinea pig-derived AhR and guinea pig-derived Arnt, the polycyclic aromatic compound can be analyzed with high sensitivity by using the cell of the present invention.

  AhR and Arnt expressed by the recombinant cell of the present invention are guinea pig-derived proteins.

As the guinea pig-derived AhR, for example, the following protein (a) or (b) is preferable. In addition, the amino acid sequence of the following SEQ ID NO: 1 is known, and for example, reference can be made to the registration number AY028947 (GenBank / EMBL / DDBJ). The protein of the following (b) has a homology with the protein of the following (a) of, for example, 98% or more, preferably 98.5% or more, more preferably 99% or more, More preferably, it is 99.5% or more. In the protein (b) below, the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 16, preferably 1 to 12, and more preferably 1 to 8, more preferably 1-4.
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein comprising an amino acid sequence in which at least one amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted, added, inserted or deleted, and functions as a guinea pig-derived AhR.

On the other hand, as the Arnt derived from the guinea pig, for example, the following protein (c) or (d) is preferable. The protein of the following (d) has a homology with the protein of the following (c) of, for example, 98.5% or more, preferably 99.0% or more, more preferably 99.2%. Or more, more preferably 99.5% or more. In the protein (d) below, the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 12, preferably 1 to 8, more preferably 1 to 1. 6, more preferably 1-4.
(C) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(D) A protein comprising an amino acid sequence in which at least one amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is substituted, added, inserted or deleted, and functions as an Arnt derived from guinea pigs.

  In order to express the AhR and Arnt in the cell, for example, the AhR gene and Arnt gene derived from guinea pigs may be introduced into the cell.

The guinea pig-derived AhrR gene preferably includes, for example, the following polynucleotide (a) or (b). In addition, the base sequence of the following SEQ ID NO: 3 is known, and for example, reference can be made to the registration number AY028947 (GenBank / EMBL / DDBJ). The polynucleotide (b) below has a homology with the polynucleotide (a) below of, for example, 90% or more, preferably 93% or more, more preferably 95% or more, More preferably, it is 98% or more. In the polynucleotide (b) below, the number of bases substituted, added, inserted or deleted is, for example, 1 to 250, preferably 1 to 200, more preferably 1 to 140. More preferably, it is 1-60.
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 3.
(B) A polynucleotide comprising a nucleotide sequence in which at least one base of the nucleotide sequence set forth in SEQ ID NO: 3 has been substituted, added, inserted or deleted, and which encodes a protein that functions as a guinea pig-derived AhR.

On the other hand, the Arnt gene derived from the guinea pig preferably contains, for example, the following polynucleotide (c) or (d). The polynucleotide (d) below has a homology with the polynucleotide (c) below of, for example, 95% or more, preferably 97% or more, more preferably 98% or more, More preferably, it is 99% or more. In the polynucleotide (d) below, the number of substituted, added, inserted or deleted bases is, for example, 1 to 120, preferably 1 to 70, more preferably 1 to 40. More preferably, it is 1-20.
(C) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(D) A polynucleotide comprising a nucleotide sequence in which at least one base of the nucleotide sequence set forth in SEQ ID NO: 4 has been substituted, added, inserted or deleted, and which encodes a protein that functions as an Arnt derived from guinea pigs.

  The AhR and Arnt genes may be cloned using guinea pig total RNA or the like based on the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, and may be chemically synthesized using the phosphoramidite method. Alternatively, DNA synthesis may be performed. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit. In addition, since both the guinea pig-derived AhR gene of SEQ ID NO: 3 and the guinea pig-derived Arnt gene of SEQ ID NO: 4 have been cloned into the vector of the accession number FERM P-19496 of the present invention, it is easy to use this vector DNA. The AhR and Arnt genes can be cloned.

As a host cell of the genetically modified cell of the present invention, for example, a cell to which gene recombinant technology can be generally applied is preferable, and a cell that can be easily handled such as storage and proliferation is more preferable. Examples of such host cells include animal cells, plant cells, insect cells, yeast, bacteria, etc. Among these, yeast is preferable. Yeast is easier to handle and has a higher growth rate than other cultured cells, and when this cell is used for analysis of polycyclic aromatic compounds, the analysis time can be shortened. Furthermore, handling equipment, culture media, and the like are inexpensive, and the cost of analysis can be reduced. As the yeast, for example, budding yeast, fission yeast, filamentous yeast, and the like can be used, but budding yeast ( Saccharomyces cerevisiae ) is preferable from the viewpoint that the genetic recombination technique is most established.

  The method for introducing the guinea pig-derived AhR and Arnt genes into the host cell is not particularly limited, and can be performed by a conventionally known gene introduction method suitable for the host cell. Examples of the gene introduction method include a lithium acetate method, a calcium phosphate method, a method using a liposome, electroporation, a method using a viral vector, a micropipette injection method, and the like. The introduction of the guinea pig-derived AhR and Arnt genes into the genetically modified cells of the present invention may be a transient type introduction or integration into the host chromosome if it can be used for the analysis of polycyclic aromatic compounds. It is also possible to introduce a type or an artificial chromosome or plasmid type capable of autonomous replication / distribution. The latter gene introduction method is preferable from the viewpoint of ease of handling and storage and rapidness, accuracy, and simplicity of analysis of polycyclic aromatic compounds.

  The guinea pig-derived AhR and Arnt genes to be introduced into the host cell are preferably operably linked to necessary regulatory sequences so that they are constitutively or arbitrarily expressed in the host cell. The regulatory sequence is a base sequence necessary for expression of the gene operably linked in a host cell. For example, examples of regulatory sequences suitable for eukaryotic cells include promoters, polyadenylation signals, enhancers. Etc. Said operatively connected means that the components are juxtaposed so that they can perform their functions.

  Since the genetically modified cell of the present invention expresses AhR and Arnt derived from guinea pigs, when the genetically modified cell of the present invention comes into contact with a polycyclic aromatic compound that is a ligand of AhR, the polycyclic aromatic compound is intracellular. The ternary complex of compound, AhR and Arnt is formed. This complex can bind to a specific recognition sequence on DNA (foreign substance responsive element; also referred to as xenobiotic responsive element, XRE), and can induce gene expression downstream of the sequence.

  Therefore, for example, a method of directly analyzing the complex using an immunological measurement method or the like, or a gene expression product induced by the complex or an enzyme activity thereof, etc. According to the reporter system that can be analyzed analytically, polycyclic aromatic compounds can be analyzed using the genetically modified cells of the present invention. Here, the analysis of the polycyclic aromatic compound is to perform at least one of detection, qualitative analysis, and quantitative analysis. Since such an analysis method is a method that utilizes the function of AhR that is deeply involved in the biosensitivity of polycyclic aromatic compounds such as dioxins, the analysis results are different from instrumental analysis in that toxic equivalents (TEQ) ) And can be an index for knowing the influence on the living body.

  In the analysis of the polycyclic aromatic compound, it is preferable to use the reporter system. In the reporter system, it is preferable to use a gene whose expression is controlled by the complex as a reporter gene. By using a reporter gene, it is possible to simplify the analysis processing operation and shorten the time required for analysis. As the reporter gene, when the host cell has the complex recognition sequence (XRE) and a gene whose expression is controlled by the binding, the gene may be used, or the complex recognition is separately performed in the host cell. A polynucleotide in which the sequence (XRE), the regulatory sequence and the desired reporter gene are operatively linked may be introduced. Here, operatively connected means that the components are juxtaposed so that they can perform their functions. The method for introducing the polynucleotide is not particularly limited, and can be performed in the same manner as the introduction of the AhR and Arnt genes described above. Expression control of the reporter gene by binding of the complex and the complex recognition sequence may be expression suppression or expression promotion.

  The complex recognition sequence (XRE) is preferably a complex recognition sequence (XRE) having high affinity with a guinea pig-derived complex expressed in the recombinant cell of the present invention, more preferably , A guinea pig-derived complex recognition sequence (XRE). Examples of the XRE consensus sequence include 5′-GCCGTG-3 ′ and the like, and the XRE sequence is preferably a repeat sequence of 1 to 6 times for controlling the expression of the reporter gene, More preferably, it is 2 to 5 times, and still more preferably 2 to 3 times.

  The reporter gene is not particularly limited as long as at least one of its gene product, RNA, protein, and enzyme activity can be easily quantified and has a low measurement background in the host cell. Reporter genes can be used. Specific examples of reporter genes used for measuring enzyme activity include β-galactosidase gene, CAT (chloramphenicol acetyltransferase) gene, luciferase gene and the like.

An example of the recombinant cell of the present invention that expresses guinea pig-derived AhR and Arnt and is equipped with the aforementioned reporter system is yeast ( Saccharomyces cerevisiae ) having accession number FERMP P-19497. In this yeast having the accession number FERM P-19497, an Ah receptor gene derived from guinea pig and an Arnt gene derived from guinea pig are introduced so that they can be expressed in the chromosome, and the complex of the above three of polycyclic aromatic compound, AhR and Arnt Holds a plasmid expressing the β-galactosidase gene.

  The analysis of the polycyclic aromatic compound using the genetically modified cell of the present invention is carried out by bringing a test substance into contact with the genetically modified cell of the present invention, and the polycyclic aromatic compound, AhR derived from guinea pig and Arnt derived from guinea pig This can be done by forming a tripartite complex and analyzing the complex directly or indirectly. Although it does not restrict | limit especially as said test object, For example, water, soil, incinerated ash etc. which are afraid of the contamination by dioxins are mention | raise | lifted. Examples of the method for bringing the subject into contact with the genetically modified cell of the present invention include, for example, a method for mixing the genetically modified cell of the present invention or a culture solution containing the cell with the subject, Examples thereof include a method in which the mixed state is maintained for a predetermined time under the culture condition of the cells so that the polycyclic aromatic compound sufficiently acts on the cells.

  Examples of the polycyclic aromatic compound include dioxins, polychlorinated dibenzofuran (PCDF), polychlorinated biphenyl (PCB), β-naphthoflavone, 3-methylcholanthrene, benzopyrene, derivatives thereof, benzimidazole, dibenzoanthracene, penta Examples include chlorophenol, dicohol, octachlorostyrene and the like. In addition, when there exists a possibility that a polycyclic aromatic compound which is easy to adsorb | suck to polystyrene like the said methylcholanthrene may be contained in a test object, it is preferable to use glass containers and instruments for analysis processing operation. .

  Below, an example of the embodiment of the polycyclic aromatic compound analysis using the gene recombinant cell of this invention is shown. In this example, the cell is a yeast transformed with the guinea pig-derived AhR and Arnt genes, a reporter gene encoding an intracellular enzyme is further introduced, and the reporter gene is expressed by the complex. This is the case for induced yeast.

  In the analysis, it is preferable to first carry out pre-culture so as to proliferate the transformed yeast to ensure a sufficient amount of cells. For example, when the preculture is used as a transformed yeast in a state continuously cultured in a medium composed of amino acids, inorganic salts, vitamins, saccharides and the like as shown in Table 1 below, 50 μl of the solution is taken, added to 5000 μl of the medium shown in Table 1 below, and cultured by shaking at 30 ° C. for about 12 to 24 hours. On the other hand, when pre-culturing using lyophilized cells as the transformed yeast, for example, a nutrient medium such as a YPD medium is added, and 50 μl of the culture solution cultured at 30 ° C. for 24 hours is collected. Similarly, in addition to 5000 μl of the medium shown in Table 1 below, culture with shaking at 30 ° C. for about 12 to 24 hours.

(Table 1)
Ingredients Amount
Dropout powder (Note 1) 1.3g
Nitrogen source (Note 2) 1.7 g
(NH ₄ ) ₂ SO ₄ 5 g
Carbon source (glucose or galactose) 20g
(Note 1)
Dropout powder composition adenine 2.5g L-methionine 1.2g
L-Arginine 1.2g L-Phenylalanine 3g
L-aspartic acid 6 g L-serine 22.5 g
L-glutamic acid 6 g L-threonine 12 g
L-Histidine 1.2g L-Tyrosine 1.8g
L-leucine 3.6 g L-valine 9 g
L-Lysine 1.8g Uracil 1.2g
(Note 2)
Yeast Nitrogen W / O Amino Acid & Ammonium Sulfate (Difco)

Once the bacteria are sufficiently grown by pre-culture, the test substance is placed in a medium suitable for the characteristics of the promoter of the reporter gene (for example, when the reporter gene is β-galactosidase, galactose is used as the carbon source). The preculture is mixed and cultured at an appropriate ratio, and the polycyclic aromatic compound in the test substance is allowed to sufficiently act on the cells. The culture temperature is, for example, 28-30 ° C., and the culture time is, for example, 18 hours. When the test substance is a solid, for example, the test substance is dissolved or a polycyclic aromatic compound is extracted from the test substance using a solvent that is not toxic to bacterial cells such as dimethyl sulfoxide (DMSO). After that, it is preferable to mix with the medium.

  Next, the transformed yeast that has been sufficiently brought into contact with the test substance is lysed by a known technique. For this lysis, for example, those obtained by adding a surfactant (sarcosyl) to Z buffer as shown in Table 2 below can be used.

(Table 2)
Ingredient Compounding amount (final concentration)
Na ₂ HPO ₄ 60 mM
NaH ₂ SO ₄ 40 mM
MgCl ₂ 1 mM
KCl 10 mM
Dithiothreitol 2mM
Sarcosil 0.20%

Then, an appropriate amount of the bacterial solution is collected, and the enzyme activity, which is a reporter gene product activated by the binding of the complex of the polycyclic aromatic compound, AhR and Arnt, is measured by an appropriate means. When the reporter gene expresses, for example, β-galactosidase, a color developing reagent such as ONPG (orthonitrophenylgalactopyranoside) is allowed to act, and the absorbance thereof may be measured. In this measurement, it is preferable to prepare a calibration curve by using a polycyclic aromatic compound as a standard substance, and it is preferable to create the calibration curve simultaneously with the measurement of the test object. Furthermore, in the measurement, it is preferable to appropriately dilute the test substance so as to be within the range of dose-effect relationship. With such a polycyclic aromatic compound analysis method, it is only necessary to measure the absorbance, and the analysis can be performed simply and rapidly.

  The kit for analyzing a polycyclic aromatic compound of the present invention is a kit for simply carrying out the method for analyzing a polycyclic aromatic compound using the genetically modified cell of the present invention, and comprises the genetic recombination of the present invention. A kit containing cells. In addition to such a kit, for example, a composition or medium for a medium, a vector containing various reporter genes from which a reporter gene can be selected, a reagent for analyzing the reporter gene product, and a standard substance are used. The known amount of polycyclic aromatic compound may be included. In addition, the kit for analysis may include a vector in which the guinea pig-derived AhR and Arnt genes for producing the recombinant cell of the present invention are cloned in place of the recombinant cell of the present invention. .

  In the analysis kit, the genetically modified cell of the present invention is preferably yeast. If it is yeast, it can be freeze-dried, and the culture composition can also be powdered. Therefore, handling of the analytical kit is simplified and the cost can be reduced.

  Hereinafter, although an example and a comparative example explain the present invention still in detail, the present invention is not limited to these.

(Cloning of guinea pig Arnt gene)
About 30 to 35 mg of guinea pig (STD: Hartley) lung tissue was collected, RNA was extracted using a kit (trade name: Rneasy Mini Kit, manufactured by QIAGEN), and this RNA and kit (trade name: RNA PCR kit ( RT-PCR was performed under the following conditions using AMV) Ver: 2.1, manufactured by TaKaRa.
Forward primer: SEQ ID NO: 5, reverse primer: SEQ ID NO: 6
RT condition 55 ℃ 30min, 99 ℃ 5min, 5 ℃ 5min
PCR condition 94 ℃ 2min 1 cycle
94 ° C. 30 sec, 49 ° C. 30 sec, 72 ° C. 3 min 50 cycles Gel electrophoresis was performed after the RT-PCR, and an Arnt cDNA fragment of about 2.4 kb was confirmed. Based on the sample obtained by the RT-PCR, Then, PCR under the following conditions was performed to obtain an Arnt cDNA fragment.
PCR condition 94 ℃ 2min 1 cycle
94 ° C 30sec, 49 ° C 30sec, 68 ° C 3min 30 cycles
The end of the guinea pig Arnt cDNA fragment thus obtained was phosphorylated using T4Polynucleotide Kinase (manufactured by TOYOBO). This fragment was inserted into the SmaI site of plasmid pBluscript (manufactured by STRATAGENE), and the guinea pig Arnt gene was cloned (completion of pBluscript-ArntcDNA). The sequence of the Arnt gene on this pBluscript-ArntcDNA was sequenced by a DNA sequencer (SEQ ID NO: 4). For cloning, Escherichia coli DH5α strain was used.

(Cloning of guinea pig AhR and Arnt genes into plasmids with bidirectional promoters)
An AhR fragment and an Arnt fragment were inserted into the BsiWI / MfeI site and the XbaI / SbfI site located downstream of both promoters of the plasmid YEplac Gal1,10 (GenBank, pMC262) having the bidirectional promoter regions Gal1,10, respectively, and AhR And a plasmid YEplac having an Arnt gene and a bidirectional promoter interposed between them. For cloning, Escherichia coli DH5α strain was used.

The AhR fragment was subjected to RT-PCR using the RNA extracted from the lungs of the guinea pig and a kit (trade name: Rever Tra Dash, manufactured by TOYOBO) under the following conditions, and the amplified product was restricted with MfeI and BsiWI. It was prepared by enzyme treatment.
Forward primer: SEQ ID NO: 7, reverse primer: SEQ ID NO: 8
RT condition 42 ℃ 20min, 99 ℃ 5min, 4 ℃ 5min
PCR conditions 98 ° C. 10 sec, 60 ° C. 2 sec, 72 ° C. 3 min 30 cycles On the other hand, the Arnt fragment was subjected to PCR under the following conditions using the pBluscript-ArntcDNA and KOD plus polymerase, and the amplified product was restricted by XbaI and SbfI. It was prepared by enzyme treatment.
Forward primer: SEQ ID NO: 9, reverse primer: SEQ ID NO: 10
PCR condition 94 ℃ 2min 1 cycle
94 ° C 15sec, 52 ° C 30sec, 68 ° C 3min 30 cycles
68 ℃ 5min 1 cycle

(Cloning into a chromosome-integrated shuttle vector)
The AhR cDNA-Gal1.10 promoter-Arnt cDNA region of the plasmid YEplac having the AhR and Arnt genes and a bidirectional promoter interposed between them was amplified by PCR under the following conditions, and the amplified product was treated with Sse8387I. It was inserted into the SmaI / Sse8387I site of the chromosomal integration shuttle vector pAUR101 (TaKaRa) to obtain pAUR101gAhR / Arnt (Accession No. FERM P-19496). For cloning, Escherichia coli DH5α strain was used.
Forward primer: SEQ ID NO: 11, reverse primer: SEQ ID NO: 12
PCR condition 94 ℃ 2min 1 cycle
94 ° C 15sec, 52 ° C 30sec, 68 ° C 3min 30 cycles
68 ℃ 5min 1 cycle

(Production of transformed yeast)
The pAUR101gAhR / Arnt (Accession No. FERM P-19496) is treated with the restriction enzyme BsiWI to be linearized, and introduced into the yeast as a host by the lithium acetate method as described below, and further integrated into the chromosome. went. As a host strain, yeast ( Saccharomyces cerevisiae ) W303a strain was used.

First, the host yeast is cultured in YPD medium at 30 ° C. until OD ₆₆₀ becomes 1-2, and either Solution A (0.1 M Lithium acetate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA) After washing at a final concentration), it was resuspended in Solution A so that OD ₆₆₀ = 150, and 100 μl was dispensed into 1.5 ml microtubes and incubated at 30 ° C. for 1 hour. Thereafter, 5 μg of linear pAUR101gAhR / Arnt and 150 μg (total 20 μl) of carrier DNA (trade name: SALMON TESTES DNA for hybridization, manufactured by SIGMA) were added, and polyethylene glycol 4000 was dissolved in Solution B (100 ml of Solution A). 850 μl) was added and gently mixed.

  Next, the mixture was incubated at 30 ° C. for 30 minutes and heat-treated at 42 ° C. for 15 minutes, then left at room temperature for 10 minutes and then collected by centrifugation at 5000 rpm for 1 minute. In YPD medium and cultured at 30 ° C. overnight. Then, the yeast collected and washed after culturing was suspended in 0.9% NaCl solution and applied to a YPD selection plate containing Aureobasidin A (0.5 g / ml) 100 μl at a time and cultured. Aureobasidin A resistant strain was selected as a transformed yeast in which the AhR cDNA-Gal1.10 promoter-Arnt cDNA region was integrated on the chromosome.

(Introduction of reporter plasmid)
A human-derived XRE sequence (SEQ ID NO: 13) is inserted into the BamHI / XmaI site of plasmid pRW95-3 (Wolf et al .; Biotechniques 20, 568-573 (1996)) containing a gene encoding β-galactosidase, A reporter plasmid was prepared. This plasmid was introduced into the transformed yeast by the lithium acetate method, and a transformed yeast into which the AhR cDNA-Gal1.10 promoter-Arnt cDNA region was integrated on the chromosome and the reporter plasmid was introduced was prepared. An example of the transformed yeast thus prepared is OPU-G11 strain (Accession No. FERM P-19497).

(Comparative Example 1)
Instead of the guinea pig-derived AhR gene and Arnt gene, a human-derived AhR gene (registration number XM004988 (GenBank / EMBL / DDBJ)) and a human-derived Arnt gene (registration number NM001668 (GenBank / EMBL / DDBJ)) were used. Except for the above, a transformed yeast having human-derived AhR and Arnt genes was prepared in the same manner as in Example 1.

(Response to 2, 3, 7, 8-TCDD)
The OPU-G11 strain of Example 1 and the transformed yeast colonies of Comparative Example 1 were each inoculated into a YPD medium and cultured at 30 ° C. until the absorbance at 600 nm reached 2. Thereafter, 50 μl of the culture solution was added to 5000 μl of the medium shown in Table 1, and cultured with shaking at 30 ° C. for 12 hours to perform preculture.

  To a 96-well plate, 1 μl each of 2,3,7,8-TCDD dissolved in DMSO so as to have a predetermined concentration was added. Further, in Table 1, 200 μl using galactose as a carbon source in the medium, and Then, 5 μl of the pre-cultured bacterial cells prepared so that the absorbance at 600 nm was 1 was added and cultured at 30 ° C. for 18 hours.

  After the culture, 10 μl of the culture solution is transferred to another new plate, 140 μl of Z buffer having the composition shown in Table 2 above and 50 μl of 4 mg / l ONPG are added, and cultured at 37 ° C. for 1 hour. did. After culturing, the absorbance at 415 nm was measured, and β-galactosidase activity was calculated based on the following formula.

(Equation 1)
β-galactosidase activity (U) =
[Absorbance at 415 nm / absorbance at 540 nm × culture solution (ml) × reaction time (minutes)] × 1000

FIG. 1 shows that OPU-G11 (guinea pig-derived AhR, Arnt gene-transformed yeast) into which the reporter plasmid of Example 1 was introduced and human-derived AhR, Arnt gene-transformed yeast of Comparative Example 1 were each 5 nM in the same conditions. It is the graph which showed the result of having measured the said beta-galactosidase activity at the time of making it contact with 2,3,7,8-TCDD. As shown in FIG. 1, the use of the guinea pig-derived AhR, Arnt gene can analyze dioxins with higher sensitivity than the use of the human-derived AhR, Arnt gene.

  As described above, the genetically modified cells of the present invention express AhR and Arnt derived from guinea pigs, and can analyze polycyclic aromatic compounds easily and with high sensitivity. For example, environmental purification, waste treatment, safety Useful in fields such as evaluation.

FIG. 1 is a diagram comparing the responses of Example 1 cells and Comparative Example 1 cells to 2,3,7,8-TCDD.

SEQ ID NO: 5 RT-PCR forward primer SEQ ID NO: 6 RT-PCR reverse primer SEQ ID NO: 7 RT-PCR forward primer SEQ ID NO: 8 RT-PCR reverse primer SEQ ID NO: 9 PCR forward primer SEQ ID NO: 10 PCR reverse primer SEQ ID NO: 11 Forward primer for PCR SEQ ID NO: 12 Reverse primer for PCR

Claims (17)

  A genetically modified cell characterized by introducing a guinea pig-derived AhR (aryl hydrocarbon receptor) gene and a guinea pig-derived Arnt (AhR nuclear translocation factor) gene, and expressing guinea pig-derived AhR and guinea pig-derived Arnt . The genetically modified cell according to claim 1, wherein the AhR is the following protein (a) or (b), and the Arnt is the following protein (c) or (d).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein comprising an amino acid sequence in which at least one amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted, added, inserted or deleted, and functions as a guinea pig-derived AhR.
(C) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(D) A protein comprising an amino acid sequence in which at least one amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is substituted, added, inserted or deleted, and functions as an Arnt derived from guinea pigs.   The genetically modified cell according to claim 1 or 2, wherein the cell has a reporter system that can indirectly analyze the complex of the three members of a polycyclic aromatic compound, AhR derived from guinea pig and Arnt derived from guinea pig. .   A base sequence in which the reporter system is operatively linked to a base sequence (foreign substance response sequence; XRE) that the complex specifically recognizes and binds, a reporter gene, and a regulatory sequence for expression of the gene. The genetically modified cell of Claim 3 containing this.   The genetically modified cell according to any one of claims 1 to 4, wherein the cell is derived from one cell selected from the group consisting of animal cells, plant cells, insect cells, and microorganisms.   The gene recombinant cell according to any one of claims 1 to 5, wherein the cell is derived from yeast. The recombinant cell according to any one of claims 1 to 6, which is a transformed yeast ( Saccharomyces cerevisiae ) having a deposit number of FERM P-19497.   A method for analyzing a polycyclic aromatic compound, wherein the test substance is brought into contact with the yeast according to claim 1 to 7, and the complex of the three rings of the polycyclic aromatic compound, AhR derived from guinea pig and Arnt derived from guinea pig And analyzing the complex directly or indirectly.   The analysis method according to claim 8, wherein the analysis is at least one of detection, qualitative analysis, and quantitative analysis of a polycyclic aromatic compound.   The polycyclic aromatic compound is dioxins, polychlorinated dibenzofuran (PCDF), polychlorinated biphenyl (PCB), β-naphthoflavone, 3-methylcholanthrene, benzopyrene, its derivatives, benzimidazole, dibenzoanthracene, pentachloro. The method according to claim 8 or 9, which is at least one compound selected from the group consisting of phenol, dicohol and octachlorostyrene. A vector for cell introduction comprising a guinea pig-derived AhR gene comprising a polynucleotide of the following (a) or (b) and a guinea pig-derived Arnt gene comprising the polynucleotide of (c) or (d), which are arranged to be expressed.
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 3.
(B) A polynucleotide comprising a nucleotide sequence in which at least one base of the nucleotide sequence set forth in SEQ ID NO: 3 has been substituted, added, inserted or deleted, and which encodes a protein that functions as a guinea pig-derived AhR.
(C) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(D) A polynucleotide comprising a nucleotide sequence in which at least one base of the nucleotide sequence set forth in SEQ ID NO: 4 has been substituted, added, inserted or deleted, and which encodes a protein that functions as an Arnt derived from guinea pigs.   The vector according to claim 11, which is a chromosomally integrated yeast shuttle vector having an accession number of FERM P-19496.   A kit for the method for analyzing a polycyclic aromatic compound according to any one of claims 8 to 10, wherein the kit comprises the genetically modified cell according to any one of claims 1 to 7. For kit.   In addition, a reagent for directly or indirectly analyzing the ternary complex of polycyclic aromatic compounds, AhR from guinea pigs and Arnt from guinea pigs or a reporter gene product responsive to the complex is included. The kit according to claim 13.   The kit according to claim 13 or 14, further comprising a quantified polycyclic aromatic compound as a standard substance. The following (a) or (b) guinea pig-derived protein.
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which at least one amino acid residue of the amino acid sequence of SEQ ID NO: 2 has been substituted, added, inserted or deleted, and functions as an Arnt derived from guinea pigs. The following guinea pig-derived polynucleotide (a) or (b):
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(B) A polynucleotide comprising a nucleotide sequence in which at least one base of the nucleotide sequence set forth in SEQ ID NO: 4 has been substituted, added, inserted or deleted, and which encodes a protein that functions as a guinea pig-derived Arnt.

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