JP2012070730A - Transformed yeast, and analytical method and analytical kit each for animal intranuclear receptor ligand using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transformed yeast enabling an intranuclear receptor ligand to be analyzed in high detection sensitivity.SOLUTION: In the transformed yeast, an intranuclear receptor gene and reporter gene have been expressibly transferred, and the reporter gene can be expressed through recognizing a complex composed of the intranuclear receptor ligand and an intranuclear receptor. Besides, at least one of the function of cell wall protein and that of efflux-pump protein is deleted. When this transformed yeast is used, the intranuclear receptor ligand can be analyzed in high sensitivity. The intranuclear receptor gene is preferably e.g. a gene encoding a human intranuclear receptor.

Description

  The present invention relates to a transformed yeast, a method for analyzing a nuclear receptor ligand using the same, and an analysis kit.

  As a method for analyzing chemical substances in the environment, in recent years, a technique has been developed in which a host cell incorporating a nuclear receptor gene is used to analyze the chemical substance depending on the level of expression of a reporter gene. According to this method, for example, biological effects can also be evaluated. For example, animal cells and yeast are used as the host cells.

  For example, a human cell line is used as the animal cell. Analysis using human-derived cells has been reported, for example, for testosterone (T), which is a ligand that binds to the androgen receptor (AR). In this method, for example, a human-derived cell line incorporating an androgen receptor (AR) gene is used (Non-patent Document 1).

  On the other hand, in the method using yeast, for example, analysis of estrogen-like activity has been reported for environmental hormones (endocrine disrupting substances) that have attracted attention in the feminization phenomenon. This method includes, for example, a bluegill-derived estrogen receptor gene (Patent Document 1), a giant lizard-derived estrogen receptor gene, (Patent Document 2), a crocodile-derived estrogen receptor gene (Patent Document 3), and a fathead minnow-derived estrogen receptor. Methods have been developed in which estrogen receptor genes of various animals such as somatic genes (Patent Literature 4) are incorporated into yeast and the expression of reporter genes is measured (Patent Literature 1, Patent Literature 2, Patent Literature 3, Patent Literature) 4). In addition to this, for example, a technique for measuring estrogenic activity of an alkylphenol compound using a transformed yeast into which a human-derived estrogen receptor gene is incorporated has been developed (Non-patent Document 2). In addition, for example, in order to measure aryl hydrocarbons such as dioxin, a technique using a transformed yeast into which a guinea pig-derived aryl hydrocarbon receptor gene is incorporated has been developed (Patent Document 5).

  In the method using the yeast, a nuclear receptor gene is expressed in the yeast, and a ligand (for example, a chemical substance) to be measured is bound to the expressed nuclear receptor to form a complex. This complex then expresses a reporter gene in yeast. For example, a β-galactosidase gene is used as the reporter gene, and the expression of the reporter gene can be measured by measuring the enzyme activity of β-galactosidase.

  The analysis method using yeast, for example, can be analyzed in a short time and has advantages such as easy operation and low cost as compared with an analysis method using animal cells. However, the analysis method using yeast has a problem that the analysis sensitivity is lower than the analysis method using animal cells.

JP 2001-197890 A JP 2003-274 A JP 2002-360273 A Japanese Patent Laid-Open No. 2001-352992 JP 2005-87077 A

Toxicology 220 (2006) 90-103 The Journal Of Biological Chemistry Vol. 272, no. 6, Feb. 7 pp. 3280-3288

  Accordingly, an object of the present invention is to provide a transformed yeast capable of analyzing nuclear receptor ligands with high sensitivity, and a method and kit for analyzing nuclear receptor ligands using the transformed yeast. More specifically, for example, transformed yeast capable of analyzing the nuclear receptor ligand with high sensitivity regardless of the type of nuclear receptor expressed in the yeast, and analysis of the nuclear receptor ligand using the same Methods and analysis kits are provided.

  In order to achieve the above object, the transformed yeast of the present invention recognizes a complex of a nuclear receptor ligand and a nuclear receptor in which a nuclear receptor gene and a reporter gene have been introduced. Thus, a transformed yeast capable of expressing the reporter gene is characterized in that at least one of a function of a cell wall protein and a function of an efflux pump protein is deleted.

The analysis method of the present invention is a method for analyzing a nuclear receptor ligand contained in a specimen,
A culture step of culturing the transformed yeast of the present invention in a medium containing a specimen;
And a measuring step of measuring an expression product of the reporter gene produced in the medium after the culturing step.

The analysis kit of the present invention is a kit for analyzing a nuclear receptor ligand contained in a specimen,
(A) a medium;
(B) the transformed yeast of the present invention;
(C) A coloring reagent is provided.

  According to the transformed yeast of the present invention, although the mechanism is unclear, the analysis sensitivity of the ligand for the nuclear receptor can be improved by deleting at least one of the function of the cell wall protein and the function of the efflux pump protein. Therefore, according to the transformed yeast of the present invention, the ligand for the nuclear receptor can be analyzed with high sensitivity. In particular, the transformed yeast of the present invention can improve ligand analysis sensitivity regardless of the type of nuclear receptor gene introduced into the transformed yeast. For example, according to a conventionally known yeast such as a wild type yeast, the ligand may not be detected depending on the type of nuclear receptor to be introduced even though the ligand is present. On the other hand, the transformed yeast of the present invention can detect a ligand that could not be performed by, for example, the wild type yeast or the like, regardless of the type of the nuclear receptor to be introduced. Thus, since the transformed yeast of the present invention can be used regardless of the type of nuclear receptor gene, it can be said that it is a highly versatile tool in analyzing nuclear receptors. Moreover, if the analysis kit of the present invention is used, the analysis of the ligand for the nuclear receptor can be carried out more simply.

1A, 1B, and 1C are graphs showing the measurement results of β-galactosidase activity in Example 2 of the present invention. FIG. 2 is a graph showing the measurement results of β-galactosidase activity in Example 4 of the present invention. FIG. 3 is a graph showing the measurement results of β-galactosidase activity in Example 6 of the present invention. FIG. 4 is a graph showing the measurement results of β-galactosidase activity in Example 8 of the present invention. FIG. 5 is a graph showing the measurement results of β-galactosidase activity in Example 10 of the present invention. FIG. 6 is a graph showing the measurement results of β-galactosidase activity in Example 12 of the present invention. FIG. 7 is a graph showing the measurement results of β-galactosidase activity in Example 14 of the present invention. FIG. 8 is a graph showing the measurement results of β-galactosidase activity in Example 16 of the present invention. FIG. 9 is a graph showing the measurement results of β-galactosidase activity in Example 18 of the present invention. FIG. 10 is a graph showing the measurement results of β-galactosidase activity in Example 20 of the present invention. FIG. 11 is a graph showing the measurement results of β-galactosidase activity in Example 22 of the present invention. FIG. 12 is a graph showing the measurement results of β-galactosidase activity in Example 24 of the present invention.

As described above, in the transformed yeast of the present invention, a nuclear receptor gene and a reporter gene are introduced so that they can be expressed, and a complex of a nuclear receptor ligand and a nuclear receptor is recognized. A transformed yeast capable of expressing a reporter gene,
Furthermore, at least one of the function of the cell wall protein and the function of the efflux pump protein is deficient.

  The transformed yeast of the present invention preferably lacks the function of the cell wall protein by deleting the coding gene of the cell wall protein.

  In the transformed yeast of the present invention, the cell wall protein-encoding gene is preferably a mannan-binding protein encoding gene.

  In the transformed yeast of the present invention, the mannan-binding protein coding gene is selected from the group consisting of CWP1 gene, CWP2 gene, CCW14 gene, CIS3 gene, KRE11 gene, PIR1 gene, PIR3 gene, TIP1 gene, TIR1 gene, and TIR2 gene. It is preferable that at least one selected.

  The transformed yeast of the present invention preferably lacks the function of the efflux pump protein by deleting the coding gene of the efflux pump protein.

  In the transformed yeast of the present invention, the gene encoding the efflux pump protein is preferably an ABC transporter gene.

  In the transformed yeast of the present invention, the ABC transporter gene is PDR5 gene, STE6 gene, PDR12 gene, PDR15 gene, SNQ2 gene, YOR1 gene, YDR061W gene, PDR18 gene, YOL075C gene, YBT1 gene, AUS1 gene, PDR10 gene , Preferably at least one selected from the group consisting of PDR11 genes.

  The transformed yeast of the present invention preferably lacks the cell wall protein coding gene and the efflux pump protein coding gene.

  In the transformed yeast of the present invention, the nuclear receptor is preferably an animal nuclear receptor, and more preferably a human nuclear receptor.

  In the transformed yeast of the present invention, the nuclear receptor is preferably a homodimer, heterodimer, or monomeric receptor. The heterodimeric receptor is preferably a receptor including a retinoid X receptor (RXR), more preferably a heterodimer of RXR and PXR.

  In the transformed yeast of the present invention, the reporter gene is preferably a β-galactosidase gene.

The transformed yeast of the present invention has the nuclear receptor gene on the chromosome,
Having the reporter gene in a plasmid;
The plasmid preferably includes a nuclear receptor responsive element operably incorporated with the reporter gene.

The transformed yeast of the present invention is preferably budding yeast ( Saccharomyces cerevisiae ).

  The transformed yeast of the present invention is preferably used for analysis of a nuclear receptor ligand contained in a specimen.

As described above, the analysis method of the present invention is a method for analyzing a nuclear receptor ligand contained in a specimen,
A culture step of culturing the transformed yeast of the present invention in a medium containing a specimen;
And a measuring step of measuring an expression product of the reporter gene produced in the medium after the culturing step.

In the analysis method of the present invention, the reporter gene is a β-galactosidase gene,
The medium preferably contains at least one of galactose and glucose as a carbon source.

In the analysis method of the present invention, the medium contains galactose and glucose,
The weight ratio of glucose to the total of galactose and glucose is preferably in the range of more than 0 and 60% by weight or less.

  In the analysis method of the present invention, the transformed yeast is preferably a yeast that has been stored in the presence of glycerol in advance.

  In the analysis method of the present invention, the transformed yeast is preferably a yeast that has been previously cultured in a medium containing glucose alone as a carbon source.

As described above, the analysis kit of the present invention is an analysis kit for analyzing a nuclear receptor ligand contained in a specimen,
(A) a medium;
(B) the transformed yeast of the present invention;
(C) A coloring reagent is provided.

  In the analysis kit of the present invention, the medium is preferably a medium containing at least one of glucose and galactose.

  Next, the present invention will be described in detail.

  As described above, the transformed yeast of the present invention is, for example, a ligand in a specimen that binds to a nuclear receptor expressed from the nuclear receptor gene regardless of the type of the nuclear receptor gene introduced. Can be detected. For this reason, in the present invention, the nuclear receptor and the ligand for the nuclear receptor are not particularly limited.

  What is necessary is just to select the said nuclear receptor gene suitably according to the ligand of analysis object, for example. The structure of the nuclear receptor expressed from the nuclear receptor gene is not particularly limited, and examples thereof include monomers and dimers. The dimer may be, for example, a homodimer composed of the same molecule or a heterodimer composed of different molecules.

  The molecule constituting the nuclear receptor is not particularly limited, and examples thereof include the following.

(1) TRα: Thyroid Hormone Receptor alpha
(2) TRβ: Thyroid Horne Receptor beta
(3) RARα: Retinic Acid Receptor alpha
(4) RARβ: Retinic Acid Receptor beta
(5) RARγ: Retinoic Acid Receptor gamma
(6) PPARα: Peroxisome proliferator activated receptor alpha (PPAR-alpha)
(7) PPARδ: Peroxisome proliferator activated receptor delta (PPAR-delta, PPAR-beta, Nuclear harmony receptor 1, NUC1)
(8) PPARγ1: Peroxisome proliferator activated receptor gamma 1 (PPAR-gamma1)
(9) PPARγ2: Peroxisome proliferator activated receptor gamma 2 (PPAR-gamma2)
(10) Rev-erbα: Organic nuclear receptor NR1D1 (V-erbA related protein EAR-1, Rev-erbA-alpha)
(11) Rev-erbβ: Organic nuclear receptor NR1D2 (Rev-erb-beta, EAR-1R, Orphan nuclear home receptor BD73)
(12) RORα: Nuclear receptor ROR-alpha (Nuclear receptor RZR-alpha)
(13) RORβ: Nuclear receptor ROR-beta (Nuclear receptor RZR-beta)
(14) RORγ: Nuclear receptor ROR-gamma (Nuclear receptor RZR-gamma)
(15) LXRβ: Oxysterols receptor LXR-beta (Liver X receptor beta, Nuclear orphan receptor LXR-beta, Ubiquitously-expressed nucleoreceptor)
(16) LXRα: Oxysterols receptor LXR-alpha (Liver X receptor alpha, Nuclear orphan receptor LXR-alpha)
(17) FXR: Bile acid receptor (Farnesoid X-activated receptor, Farnesol receptor HRR-1, Retinoid X receptor-interacting protein 14, RXR-interacting protein 14)
(18) VDR: Vitamin D3 receptor (VDR, 1,25-dihydroxyvitamin D3 receptor)
(19) PXR-1: Orphan nuclear receptor PXR-1 (Pregnane X receptor-1, Orphan nuclear receptor PAR-1, Steroid and xenobiotic receptor-1, SXR-1)
(20) PXR-2: Organic nuclear receptor PXR-2 (Pregnane X receptor-2, Orphan nuclear receptor PAR-2, Steroid and xenobiotic receptor-2, SXR-2)
(21) CAR: Organic nuclear receptor NR1I3 (Constitutive and passive receptor, CAR, Orphan nuclear receptor MB67)
(22) HNF4α: hepatocyte nuclear factor 4-alpha (HNF-4-alpha, transcription factor 14)
(23) HNF4γ: Hepatocyte nuclear factor 4-gamma (HNF-4-gamma)
(24) RXRα: Retinoid X receptor RXR-alpha
(25) RXRβ: Retinoid X receptor RXR-beta
(26) RXRγ: Retinoid X receptor RXR-gamma
(27) TR2: Nuclear Harmone Receptor TR2 (Orphan nuclear receptor TR2)
(28) TR4: Organic nuclear receptor TR4 (Organic nuclear receptor TAK1)
(29) TLX: Orphan nuclear receptor NR2E1 (Nucleer receptor TLX, Tailless homolog, Tll, hTll)
(30) PNR: Photoreceptor-specific nuclear receptor (Retina-specific nuclear receptor)
(31) COUP-TF I: COUP transcription factor 1 (COUP-TF1, COUP-TFα, V-erbA related protein EAR-3)
(32) COUP-TF II: COUP transcription factor 2 (COUP-TF2, COUP-TFβ, Apolipoprotein AI regulatory protein-1, ARP-1)
(33) EAR-2: Organic nuclear receptor EAR-2 (V-erbA related protein EAR-2, COUP-TFγ)
(34) ERα: Estrogen receptor (ER, Estradiol receptor, ER-alpha)
(35) ERβ: Estrogen receptor beta (ER-beta)
(36) ERRα: Steroid homone receptor ERR1 (Estrogen-related receptor, alpha, ERR-alpha, Estrogen receptor-like 1)
(37) ERRβ: Steroid horne receptor ERR2 (Estrogen-related receptor, beta, ERR-beta, Estrogen receptor-like 2, ERR beta-2)
(38) ERRγ: Estrogen-relevant receptor gamma (Estrogen receptor related protein 3, ERR gamma-2)
(39) GRα: Glucocorticoid receptor alpha
(40) GRβ: Glucocorticoid Receptor beta
(41) MR: Mineralcorticoid receptor (MR)
(42) PRα: Progesterone receptor alpha
(43) PRβ: Progesterone receptor beta
(44) AR: Android receptor (Dihydrotestosterone receptor)
(45) NGFI-Bα: Organic nuclear receptor HMR (Early response protein NAK1, TR3 orphan receptor)
(46) NGFI-Bβ: Organic nuclear receptor NURR1 (Immediate-early response protein NOT, Transiently inducible nuclear receptor)
(47) NGFI-Bγ: Nuclear harmony receptor NOR-1 (Neuron-derived orphan receptor 1, Mitogen induced nuclear orphan receptor)
(48) SF-1: Steroidogenic factor 1 (STF-1, SF-1, Steroid harmone receptor AD4BP, Fushi tarazu factor homolog 1, FTZ-FIα)
(49) LRH-1: Organic nucleic receptor NR5A2 (Alpha-1-fetoprotein transcription factor, Hepatic transcription factor, B1 binding factor, B1 binding factor, B1 binding factor, B1 binding factor, B1 binding factor, B1 binding factor, B1 binding factor
(50) GCNF: Germ cell nuclear factor (Organic nuclear receptor NR6A1, Retinoid receptor-related testis specific receptor, RTR)
(51) DAX1: Organic nuclear receptor DAX-1
(52) SHP: Small heterodimer partner (Organic nuclear receptor SHP)
(53) AhR: aryl hydrocarbon receptor (AHR, Dioxin nuclear receptor)

  The nuclear receptor of the homodimer is, for example, TR4, HNF4α, HNF4γ, RXRα, RXRβ, RXRγ, TR2, PNR, COUP-TF I, COUP-TF II, EAR2, ERα, ERβ, GRα, GRβ, MR, PRα, PRβ, AR and the like can be mentioned.

  Examples of the heterodimeric nuclear receptor include a nuclear receptor including RXR and a nuclear receptor including Arnt. The nuclear receptor containing RXR is, for example, TRα, TRβ, RARα, RARβ, RARγ, PPARα, PPARδ, PPARγ, LXRβ, LXRα, FXR, VDR, PXR-1 as the other molecule constituting the dimer. , PXR-2, and CAR. Of these, heterodimers of RXR and PXR-1 (RXR / PXR-1) and heterodimers of RXR and PXR-2 (RXR / PXR-2) are particularly preferred. The nuclear receptor containing Arnt includes, for example, AhR as another molecule constituting the dimer.

  Examples of the monomeric nuclear receptor include Rev-erbα, Rev-erbβ, RORα, RORβ, RORγ, TLX, ERRα, ERRβ, ERRγ, SF-1, LRH, GCNF, DAX1, and SHP.

  Examples of the nuclear receptor that can take any of the monomer, homodimer and heterodimer include NGF1-Bα, NGF1-Bβ, and NGF1-Bγ.

  In the present invention, a ligand for the nuclear receptor can be detected by appropriately setting the type of the nuclear receptor gene. In the present invention, the ligand to be analyzed may be, for example, a known ligand that binds to the nuclear receptor or an unknown ligand that binds to the nuclear receptor.

  In the present invention, the nuclear receptor may be appropriately selected according to, for example, the ligand to be analyzed.

<Transformed yeast>
As described above, in the transformed yeast of the present invention, a nuclear receptor gene and a reporter gene are introduced so that they can be expressed, and a complex of a nuclear receptor ligand and a nuclear receptor is recognized. A transformed yeast capable of expressing a reporter gene, further characterized in that at least one of a cell wall protein function and an efflux pump protein function is deleted.

Examples of the transformed yeast of the present invention include budding yeast, fission yeast, filamentous yeast, etc., and budding yeast ( Saccharomyces cerevisiae ) is preferred. The form of the transformed yeast of the present invention is not particularly limited, and examples thereof include a culture solution, freeze-dried cells, and stored cells (glycerol stock) in the presence of glycerol.

  The transformed yeast of the present invention only needs to lack at least one of the function of the cell wall protein and the function of the efflux pump protein. In the transformed yeast of the present invention, for example, only one of the function of the cell wall protein and the function of the efflux pump protein may be deleted, but both the function of the cell wall protein and the function of the efflux pump protein are It is preferably missing.

  The cell wall protein is not particularly limited, and examples thereof include mannan binding protein. Examples of the mannan-binding protein include CWP1 protein, CWP2 protein, CCW14 protein, CIS3 protein, KRE11 protein, PIR1 protein, PIR3 protein, TIP1 protein, TIR1 protein, TIR2 protein and the like. In the transformed yeast of the present invention, the type of the cell wall protein lacking the function may be, for example, one type or two or more types. In the latter case, the combination of the cell wall proteins lacking in function is not particularly limited, and examples thereof include a combination of CWP1 protein and CWP2 protein.

  The efflux pump protein is not particularly limited, and examples thereof include an ABC (ATP-Binding Cassette) transporter protein, an MFS (major facility superfamily) transporter protein, and the like. Examples of the ABC transporter protein include PDR5 protein, STE6 protein, PDR12 protein, PDR15 protein, SNQ2 protein, YOR1 protein, YDR061W protein, PDR18 protein, YOL075C protein, YBT1 protein, AUS1 protein, PDR10 protein, PDR11 protein and the like. It is done. In the transformed yeast of the present invention, the type of the efflux pump protein lacking the function may be, for example, one type or two or more types. In the latter case, the combination of the evacuation pump lacking the function is not particularly limited, and examples thereof include a combination of PDR5 protein and PDR10 protein.

As described above, the transformed yeast of the present invention preferably lacks both the function of the cell wall protein and the function of the efflux pump protein. The combination of the cell wall protein deficient in function and the efflux pump protein is not particularly limited. Examples of the combination include a combination of the mannan-binding protein and the ABC transporter protein. Specifically, for example,
A combination of CWP1 protein and PDR5 protein,
A combination of CWP1 protein and PDR10 protein,
A combination of CWP2 protein and PDR5 protein,
A combination of CWP1 protein, CWP2 protein and PDR5 protein,
Examples include a combination of CWP1 protein, PDR5 protein, and PDR10 protein.

  In the transformed yeast of the present invention, the function of the cell wall protein and the function of the efflux pump protein may be lost by any mechanism. The function of the protein is, for example, deletion of the coding gene of the protein in the transformed yeast, deletion due to point mutation or sequence insertion in the coding gene of the protein, suppression of transcription from the coding gene of the protein, transcript Can be deleted by suppression of translation from, and among these, deletion of the coding gene is preferred.

  In the transformed yeast of the present invention, for example, the entire region or the partial region of the coding gene for the cell wall protein and / or the coding gene for the efflux pump protein may be deleted. In the latter case, for example, an open reading frame (ORF) in the coding gene may be deleted, or a region involved in the function of the protein may be deleted. In the transformed yeast of the present invention, it is preferable that at least one of the coding gene for the cell wall protein and the coding gene for the efflux pump protein lacks an ORF, and both the coding genes each lack an ORF. Is more preferable.

  The coding gene for the cell wall protein is not particularly limited, and examples thereof include the coding gene for the aforementioned protein, that is, the coding gene for a protein such as a mannan-binding protein. Examples of the mannan protein coding gene include CWP1 gene, CWP2 gene, CCW14 gene, CIS3 gene, KRE11 gene, PIR1 gene, PIR3 gene, TIP1 gene, TIR1 gene, and TIR2 gene. In the transformed yeast of the present invention, for example, one type of the gene encoding the cell wall protein may be deleted, or two or more types may be deleted. In the latter case, for example, the CWP1 gene and the CWP2 gene may be deleted.

  The coding gene of the efflux pump protein is not particularly limited, and examples thereof include coding genes of the aforementioned proteins, that is, protein coding genes such as ABC transporter protein and MFS (major facility superfamily) transporter protein. The ABC transporter protein coding gene is, for example, PDR5 gene, STE6 gene, PDR12 gene, PDR15 gene, SNQ2 gene, YOR1 gene, YDR061W gene, PDR18 gene, YOL075C gene, YBT1 gene, AUS1 gene, PDR10 gene, PDR11 gene Etc. In the transformed yeast of the present invention, for example, one type of the gene encoding the efflux pump protein may be deleted, or two or more types may be deleted. In the latter case, for example, the PDR5 gene and the PDR10 gene may be deleted.

In the transformed yeast of the present invention, for example, it is preferable to delete both the coding gene for the cell wall protein and the coding gene for the efflux pump protein. The combination of the defective coding gene for the cell wall protein and the coding gene for the efflux pump protein is not particularly limited. Examples of the combination include a combination of the mannan-binding protein coding gene and the ABC transporter protein coding gene. In particular,
A combination of the CWP1 gene and the PDR5 gene,
A combination of the CWP1 gene and the PDR10 gene,
Combination of CWP2 gene and PDR5 gene,
A combination of CWP1 gene, CWP2 gene and PDR5 gene,
A combination of a CWP1 gene, a PDR5 gene, and a PDR10 gene can be mentioned, and a combination of a CWP1 gene and a PDR5 gene is preferable.

  Deletion of the coding gene for the cell wall protein and / or the coding gene for the efflux pump protein is not particularly limited. In the present invention, even when various nuclear receptor genes are introduced by deleting at least one of the coding gene of the cell wall protein and the efflux pump gene, it can be used for analysis of the ligand.

  For example, if the deletion of the coding gene of the cell wall protein and the efflux pump gene is set as follows according to the type of the nuclear receptor, better analysis accuracy can be realized.

  When the nuclear receptor is a GR homodimer and a ligand for GR is analyzed, for example, PDR5 gene deletion, PDR10 gene deletion, CWP1 gene and PDR5 gene deletion, CWP1 gene and PDR10 Deletion of gene, deletion of PDR5 gene and PDR10 gene, and deletion of CWP1 gene, PDR5 gene and PDR10 gene are preferable, more preferable deletion of PDR5 gene, deletion of CWP1 gene and PDR5 gene, PDR5 A deletion of the gene and the PDR10 gene, and a deletion of the CWP1 gene, the PDR5 gene and the PDR10 gene.

  When the nuclear receptor is a heterodimer of AhR and Arnt and a ligand for AhR is analyzed, for example, deletion of CWP1 gene, deletion of CWP2 gene, deletion of PDR5 gene, deletion of PDR10 gene Loss, deletion of CWP1 gene and CWP2 gene, deletion of CWP1 gene and PDR5 gene, deletion of CWP1 gene and PDR10 gene, deletion of PDR5 gene and PDR10 gene, and deletion of CWP1 gene, PDR5 gene and PDR10 gene Loss of PDR10 gene, deletion of CWP1 gene and PDR5 gene, deletion of CWP1 gene and PDR10 gene, deletion of PDR5 gene and PDR10 gene, and CWP1 gene, PDR5 gene and PDR 0 gene is a deletion of, particularly preferably, PDR5 genes and PDR10 gene deletion, as well as, CWP1 gene is a deletion of the PDR5 gene and PDR10 gene.

  When the nuclear receptor is a homodimer of ERα and a ligand for ERα is analyzed, for example, deletion of CWP1 gene, deletion of CWP2 gene, deletion of PDR5 gene, deletion of CWP1 gene and CWP2 gene Deletion, deletion of CWP1 gene and PDR5 gene, deletion of CWP2 gene and PDR5 gene, and deletion of CWP1 gene, CWP2 gene and PDR5 gene, more preferably deletion of CWP1 gene and deletion of CWP2 gene. Loss, deletion of CWP1 gene and CWP2 gene, and deletion of CWP1 gene and PDR5 gene.

  When the nuclear receptor is a homodimer of ERβ and a ligand for ERβ is analyzed, for example, deletion of CWP1 gene, deletion of CWP2 gene, deletion of PDR5 gene, deletion of CWP1 gene and CWP2 gene Deletion, deletion of CWP1 gene and PDR5 gene, deletion of CWP2 gene and PDR5 gene, and deletion of CWP1 gene, CWP2 gene and PDR5 gene, more preferably deletion of CWP1 gene and CWP2 gene. .

  When the nuclear receptor is a TRα homodimer and a ligand for TRα is analyzed, for example, deletion of the CWP1 gene and the CWP2 gene is preferable.

  When the nuclear receptor is a TRβ homodimer and a ligand for TRβ is analyzed, for example, deletion of PDR5 gene, deletion of CWP1 gene and CWP2 gene, and CWP1 gene, CWP2 gene and PDR5 gene Of PDR5 gene, more preferably deletion of PDR5 gene.

  When the nuclear receptor is a heterodimer of RXR and PXR-1, and a ligand for the heterodimer is analyzed, for example, deletion of PDR5 gene, deletion of CWP1 gene and PDR5 gene, CWP2 Deletion of gene and PDR5 gene, and deletion of CWP1, CWP2 and PDR5 genes are preferable, and deletion of PDR5 gene is more preferable.

  When the nuclear receptor is a PR homodimer and a ligand for PR is analyzed, for example, deletion of PDR5 gene, deletion of CWP2 gene, deletion of CWP1 gene and PDR5 gene, CWP1 gene and CWP2 Deletion of gene, deletion of CWP2 gene and PDR5 gene, deletion of CWP1 gene and PDR10 gene, deletion of PDR5 gene and PDR10 gene, deletion of CWP1 gene, CWP2 gene and PDR5 gene, and CWP1 gene, PDR5 Deletion of the gene and the PDR10 gene is preferable, and deletion of the PDR5 gene and deletion of the CWP1 gene and the CWP2 gene are more preferable.

  When the nuclear receptor is a homodimer of AR and a ligand for AR is analyzed, for example, deletion of PDR5 gene, deletion of CWP1 gene and PDR5 gene, deletion of CWP2 gene and PDR5 gene, CWP1 Deletion of the gene and CWP2 gene, deletion of the PDR5 gene and PDR10 gene, deletion of the CWP1 gene, PDR5 gene and PDR10 gene, and deletion of the CWP1 gene, CWP2 gene and PDR5 gene are preferred, more preferably CWP1 A deletion of the gene and the PDR5 gene, and a deletion of the PDR5 and PDR10 genes.

  When the nuclear receptor is a homodimer of MR and a ligand for MR is analyzed, for example, deletion of the PDR5 gene is preferable.

  In the transformed yeast of the present invention, for example, the nuclear receptor to be expressed is preferably an animal nuclear receptor, and specifically, is preferably a human nuclear receptor. For this reason, in the transformed yeast of the present invention, an animal nuclear receptor gene is preferably introduced as the nuclear receptor gene, and more preferably a human nuclear receptor gene is introduced.

  According to the transformed yeast of the present invention, a ligand for any nuclear receptor can be analyzed. Therefore, in the transformed yeast of the present invention, the nuclear receptor gene is not particularly limited and can be appropriately determined according to the type of the ligand to be analyzed.

  In the transformed yeast of the present invention, the nuclear receptor to be expressed can be exemplified by the above-mentioned nuclear receptors, and specific examples thereof include androgen receptor (AR), glucocorticoid receptor (GR), aryl Hydrocarbon receptor (AhR), retinoic acid X receptor α (RXRα), estrogen receptor α (ERα), estrogen receptor β (ERβ), thyroid hormone receptor α (TRα), thyroid hormone receptor β (TRβ ), Progesterone receptor (PR), mineralocorticoid receptor (MR), pregnane X receptor (PXR (PXR-1 or PXR-2)) and the like. These nuclear receptors are preferably animal nuclear receptors, more preferably human nuclear receptors.

  In the transformed yeast of the present invention, a transcription coupling factor gene may be further introduced. In the transformed yeast of the present invention, for example, a transcription coupling factor (coactivator) expressed from the transcription coupling factor gene directly binds to the nuclear receptor and activates transcription of the reporter gene. The transcription coupling factor gene can be appropriately determined according to, for example, the type of nuclear receptor gene.

  In the transformed yeast of the present invention, for example, the transcription coupling factor to be expressed is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. For this reason, in the transformed yeast of the present invention, for example, an animal transcription coupling factor gene is preferably introduced as the transcription coupling factor gene, and more preferably a human transcription coupling factor gene is introduced.

  The origin of the transcription coupling factor to be expressed is not particularly limited. For example, it is preferably the same strain as the origin of the nuclear receptor to be expressed, more preferably the same animal. Specifically, when the nuclear receptor is a human nuclear receptor, the transcription coupling factor is preferably a human transcription coupling factor. Therefore, for example, when the animal nuclear receptor gene is introduced as the nuclear receptor gene in the transformed yeast of the present invention, the animal transcription coupling factor gene is preferably introduced, and human When a nuclear receptor gene is introduced, it is preferable that a human transcription coupling factor gene is introduced. Thus, analysis sensitivity can be further improved by making the nuclear receptor gene and the transcription coupling factor gene have the same origin. Although this mechanism is unknown, it is presumed that, for example, the efficiency of transcription of the reporter gene is improved by a complex containing the nuclear receptor and the transcription coupling factor having the same origin. The present invention is not limited to this mechanism.

Examples of the combination of the transcription coupling factor and a nuclear receptor to which it can bind are shown below. The present invention is not limited to these combinations.

  In the transformed yeast of the present invention, the reporter gene is not particularly limited, and examples thereof include known genes. Specific examples include β-galactosidase gene, luciferase gene, fluorescent protein gene, β-glucuronidase gene and the like.

  In the transformed yeast of the present invention, the introduction site of the nuclear receptor gene is not particularly limited. The nuclear receptor gene is preferably introduced, for example, on the yeast chromosome.

  When the transformed yeast of the present invention has the introduced transcription coupling factor gene, the introduction site of the transcription coupling factor gene is not particularly limited. The transformation site of the present invention preferably has the plasmid, and the plasmid has the transcription coupling factor gene.

  In the transformed yeast of the present invention, the introduction site of the reporter gene is not particularly limited. For example, the transformed yeast of the present invention preferably further includes a plasmid, and the plasmid preferably has the reporter gene.

  In the transformed yeast of the present invention, for example, it is preferable that a nuclear receptor response element is further introduced. The nuclear receptor responsive element is not particularly limited, and can be appropriately determined according to the type of the nuclear receptor.

  The origin of the nuclear receptor responsive element is not particularly limited, and for example, it is preferably the same strain as the origin of the nuclear receptor to be expressed, more preferably the same animal. Specifically, when the nuclear receptor is a human nuclear receptor, the nuclear receptor response element is preferably a human nuclear receptor response element. Therefore, in the transformed yeast of the present invention, for example, when the animal nuclear receptor gene is introduced as the nuclear receptor gene, the animal nuclear receptor responsive element is preferably introduced. When a human nuclear receptor gene is introduced, it is preferable that a human nuclear receptor responsive element is introduced.

  In the transformed yeast of the present invention, the introduction site of the nuclear receptor response element is not particularly limited. For example, the transformed yeast of the present invention preferably further comprises a plasmid, and the plasmid preferably has the nuclear receptor response element. The nuclear receptor response element is preferably inserted into the plasmid having the reporter gene, for example. In the plasmid, the nuclear receptor response element and the reporter gene are preferably operably linked and inserted. In the plasmid, the nuclear receptor response element is preferably inserted upstream of the reporter gene. The nuclear receptor response element is preferably inserted into the plasmid as a repeat sequence, for example.

  The reporter gene and the transcription coupling factor gene are preferably inserted into separate plasmids, for example. Hereinafter, the plasmid having a reporter gene and optionally the nuclear receptor responsive element is referred to as a first plasmid, and the plasmid having the transcription coupling factor gene is referred to as a second plasmid. For example, the first plasmid is preferably a low copy plasmid, and the second plasmid is preferably a high copy plasmid, for example.

  Hereinafter, as an example of the transformed yeast of the present invention, the nuclear receptor is the aforementioned AR, GR, RXRα, ERβ, TRα, AhR, ERα, TRβ, PXR-1, PR, or MR. The transformed yeast used for ligand analysis will be described.

  First, the transformed yeast into which the nuclear receptor is AR and the AR gene is introduced will be described.

  Examples of the ligand for AR include androgen or an androgen-like chemical substance. When the ligand is present, AR forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is AR, examples of the response element include a glucocorticoid response element (GRE).

  The AR is not particularly limited, and is preferably an animal AR, more preferably a human AR, as described above. In order to express the human AR in the transformed yeast of the present invention, for example, it is preferable that a human AR gene is introduced on the yeast chromosome.

The human AR gene preferably includes, for example, the following polynucleotide (a) or (b).
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(B) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 1, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as a human AR.

  The polynucleotide (b) has a homology with the polynucleotide (a) 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), the number of bases substituted, added, inserted or deleted is, for example, the number corresponding to the homology.

  The AR gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 1. The nucleic acid may be DNA or RNA such as total RNA, for example. The AR gene may be chemically synthesized using, for example, a phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is AR, the transcription coupling factor for AR is not particularly limited, and examples thereof include SRC1, CBP, P300, and TIF2. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, GRE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is GR and the GR gene has been introduced will be described.

  Examples of the ligand for GR include glucocorticoid or glucocorticoid-like chemical substance. In the presence of the ligand, GR forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is GR, examples of the response element include a glucocortinoid response element (GRE).

  The GR is not particularly limited, and as described above, animal GR is preferable, and human GR is more preferable. In order to express the human GR in the transformed yeast of the present invention, for example, it is preferable to introduce a human GR gene onto the yeast chromosome.

The human GR gene preferably contains, for example, the following polynucleotide (c) or (d).
(C) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2.
(D) A polynucleotide comprising a base sequence represented by SEQ ID NO: 2, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as a human GR.

  The polynucleotide (d) has a homology with the polynucleotide (c) 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 (d), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The GR gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 2. The nucleic acid may be DNA or RNA such as total RNA, for example. The GR gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is GR, the transcription coupling factor for the GR is not particularly limited, and examples thereof include SRC1, CBP, P300, and TIF2. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, GRE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is RXRα and the RXRα gene is introduced will be described.

  Examples of the ligand for RXRα include retinoic acid or a retinoic acid-like chemical substance. When the ligand is present, RXRα forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is RXRα, examples of the response element include a drug response element (DR1 element).

  The RXRα is not particularly limited, and as described above, animal RXRα is preferable, and human RXRα is more preferable. In order to express the human RXRα in the transformed yeast of the present invention, for example, it is preferable to introduce the human RXRα gene onto the yeast chromosome.

The human RXRα gene preferably includes, for example, the following polynucleotide (e) or (f):
(E) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 3.
(F) A polynucleotide encoding a protein that functions as human RXRα, which is a polynucleotide having a base sequence in which at least one base is substituted, added, inserted, or deleted in the base sequence set forth in SEQ ID NO: 3.

  The polynucleotide (f) has a homology with the polynucleotide (e) 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 (f), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The RXRα gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 3. The nucleic acid may be DNA or RNA such as total RNA, for example. The RXRα gene may be chemically synthesized using, for example, a phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is RXRα, the transcription coupling factor for RXRα is not particularly limited, and examples thereof include SRC1, CBP, P300, and ARA70. Of these, SRC1 is preferred. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor response element, for example, the DR1 element is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is ERβ and the ERβ gene has been introduced will be described.

  Examples of the ligand for ERβ include estrogen or an estrogen-like chemical substance. When the ligand is present, ERβ forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is ERβ, examples of the response element include an estrogen response element (ERE).

  The ERβ is not particularly limited, and is preferably animal ERβ, more preferably human ERβ, as described above. In order to express the human ERβ in the transformed yeast of the present invention, for example, it is preferable to introduce the human ERβ gene onto the yeast chromosome.

The human ERβ gene preferably includes, for example, the following polynucleotide (g) or (h).
(G) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(H) A polynucleotide encoding a protein that functions as human ERβ, the polynucleotide comprising a nucleotide sequence in which at least one base is substituted, added, inserted or deleted in the nucleotide sequence set forth in SEQ ID NO: 4.

  The polynucleotide (h) has a homology with the polynucleotide (g) 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 (h), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The ERβ gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 4. The nucleic acid may be DNA or RNA such as total RNA, for example. The ERβ gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is ERβ, the transcription coupling factor for the ERβ is not particularly limited, and examples thereof include SRC1, CBP, P300, ASC2, and ERAP140. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, ERE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is TRα and the TRα gene has been introduced will be described.

  Examples of the ligand for TRα include thyroid hormone or a thyroid hormone-like chemical substance. When the ligand is present, TRα forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is TRα, examples of the response element include a drug response element (IR0 element).

  The TRα is not particularly limited, and human TRα is preferable as described above. In the transformed yeast of the present invention, in order to express the human TRα, it is preferable to introduce the human TRα gene onto the yeast chromosome.

The human TRα gene preferably includes, for example, the following polynucleotide (i) or (j).
(I) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 5.
(J) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 5, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human TRα.

  The polynucleotide (j) can be obtained, for example, by cloning using a human nucleic acid. The nucleic acid may be DNA or RNA such as total RNA, for example. The TRα gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is TRα, the transcription coupling factor for TRα is not particularly limited, and examples thereof include SRC1, CBP, P300, AIB, ARA70, and ASC2. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor response element, for example, the IR0 element is preferably inserted into the first plasmid as a repeat sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is AhR and the AhR gene has been introduced will be described.

  The ligand for AhR is, for example, β-NF (naphthoflavone), dioxin, dioxin-like chemical substance, polychlorinated dibenzofuran (PCDF), polychlorinated biphenyl (PCB), 3-methylcholanthrene, benzopyrene, benzimidazole, dibenzoanthracene. , Pentachlorophenol, dicohol, octachlorostyrene, and derivatives thereof. When the ligand is present, AhR forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is AhR, examples of the response element include a foreign body response element (XRE).

  The AhR is not particularly limited, and human AhR is preferable as described above. In the transformed yeast of the present invention, in order to express the human AhR, it is preferable to introduce the human AhR gene onto the yeast chromosome. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor.

The human AhR gene preferably includes, for example, the following polynucleotide (k) or (l).
(K) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 6.
(L) A polynucleotide encoding a protein that functions as a human AhR, which is a polynucleotide having a base sequence in which at least one base is substituted, added, inserted or deleted in the base sequence set forth in SEQ ID NO: 6.

  The polynucleotide (1) can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 6. The nucleic acid may be DNA or RNA such as total RNA, for example. The AhR gene may be chemically synthesized using, for example, a phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is AhR, the transformed yeast may further have an AhR nuclear translocation factor (Arnt) gene, for example. In the transformed yeast, the introduction site of the Arnt gene is not particularly limited. The Arnt gene is preferably introduced, for example, on the yeast chromosome.

  The Arnt is not particularly limited, and human Arnt is preferable as described above. In the transformed yeast of the present invention, in order to express the human Arnt, it is preferable to introduce the human Arnt gene onto the yeast chromosome.

The human Arnt gene preferably includes, for example, the following polynucleotide (m) or (n).
(M) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 7.
(N) A polynucleotide comprising a base sequence represented by SEQ ID NO: 7, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human Arnt.

  The polynucleotide (n) can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 7. The nucleic acid may be DNA or RNA such as total RNA, for example. The Arnt gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, XRE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is ERα and the ERα gene has been introduced will be described.

  Examples of the ligand for ERα include estrogen or an estrogen-like chemical substance. When the ligand is present, ERα forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is ERα, examples of the response element include an estrogen response element (ERE).

  The ERα is not particularly limited, and is preferably animal ERα, more preferably human ERα, as described above. In order to express the human ERα in the transformed yeast of the present invention, for example, it is preferable to introduce the human ERα gene onto the yeast chromosome.

The human ERα gene preferably includes, for example, the following polynucleotide (q) or (r).
(Q) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 37.
(R) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 37, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human ERα.

  The polynucleotide (r) has a homology with the polynucleotide (q) 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 (r), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The ERα gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 37. The nucleic acid may be DNA or RNA such as total RNA, for example. The ERα gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is ERa, the transcription coupling factor for ERa is not particularly limited, and examples thereof include SRC1, CBP, P300, ASC2, and ERAP140. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  Next, the transformed yeast into which the nuclear receptor is TRβ and the TRβ gene has been introduced will be described.

  Examples of the ligand for TRβ include thyroid hormone or a thyroid hormone-like chemical substance. When the ligand is present, TRβ forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is TRβ, examples of the response element include a drug response element (IR0 element).

  The TRβ is not particularly limited, and human TRβ is preferable as described above. In the transformed yeast of the present invention, in order to express the human TRβ, it is preferable to introduce the human TRβ gene onto the yeast chromosome.

The human TRβ gene preferably includes, for example, the following polynucleotide (s) or (t).
(S) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 38.
(T) A polynucleotide comprising a base sequence represented by SEQ ID NO: 38, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human TRβ.

  The polynucleotide (t) can be obtained, for example, by cloning using a human nucleic acid. The nucleic acid may be DNA or RNA such as total RNA, for example. The TRβ gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is TRβ, the transcription coupling factor for TRβ is not particularly limited, and examples thereof include SRC1, CBP, P300, AIB, ARA70, ASC2, and ERAP140. Among these, for example, SRC1 is preferable. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor response element, for example, the IR0 element is preferably inserted into the first plasmid as a repeat sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is PXR-1 and the PXR-1 gene has been introduced will be described.

  Examples of the ligand for PXR-1 include rifampicin or rifampicin-like chemical substance. When the ligand is present, PXR-1 forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is PXR-1, examples of the response element include a drug response element (DR-3).

  The PXR-1 is not particularly limited, and as described above, animal PXR-1 is preferable, and human PXR-1 is more preferable. In order to express the human PXR-1 in the transformed yeast of the present invention, for example, it is preferable to introduce the human PXR-1 gene onto the yeast chromosome.

The human PXR-1 gene preferably includes, for example, the following polynucleotide (u) or (v).
(U) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 39.
(V) a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 39, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human PXR-1 .

  The polynucleotide (v) has a homology with the polynucleotide (u) 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 (v), the number of bases substituted, added, inserted or deleted is, for example, a number corresponding to the homology.

  The PXR-1 gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 39. The nucleic acid may be DNA or RNA such as total RNA, for example. The PXR-1 gene may be chemically synthesized using, for example, the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is PXR-1, the transcription coupling factor for PXR-1 is not particularly limited, and examples thereof include CBP and P300. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor response element such as DR-3 is preferably inserted into the first plasmid as a repeat sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is PR and the PR gene is introduced will be described.

  Examples of the ligand for PR include progesterone and progesterone-like chemical substances. In the presence of the ligand, PR forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is PR, examples of the response element include a glucocorticoid response element (GRE).

  The PR is not particularly limited, and human PR is preferable as described above. In the transformed yeast of the present invention, in order to express the human PR, it is preferable to introduce the human PR gene onto the yeast chromosome.

The human PR gene preferably includes, for example, the following polynucleotide (w) or (x).
(W) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 40.
(X) a polynucleotide comprising a nucleotide sequence of the base sequence set forth in SEQ ID NO: 40 wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as a human PR.

  The polynucleotide (x) has a homology with the polynucleotide (w) 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 (x), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The PR gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 40. The nucleic acid may be DNA or RNA such as total RNA, for example. The PR gene may be chemically synthesized using, for example, a phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is PR, the transcription coupling factor for PR is not particularly limited, and examples thereof include CBP and P300. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, GRE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

  Next, the transformed yeast into which the nuclear receptor is MR and the MR gene is introduced will be described.

  Examples of the ligand for MR include aldosterone and aldosterone-like chemical substances. In the presence of the ligand, MR forms a complex with the ligand and the transcription coupling factor in the transformed yeast. The complex recognizes and binds to the nuclear receptor response element. The reporter gene existing downstream of the response element is expressed by the binding of the complex. At this time, transcription of the reporter gene is activated by the transcription coupling factor. When the nuclear receptor is MR, examples of the response element include a glucocorticoid response element (GRE).

  The MR is not particularly limited, and human MR is preferable as described above. In the transformed yeast of the present invention, in order to express the human MR, it is preferable to introduce a human MR gene onto the yeast chromosome.

The human MR gene preferably includes, for example, the following polynucleotide (y) or (z).
(Y) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 41.
(Z) A polynucleotide comprising a base sequence represented by SEQ ID NO: 41, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human MR.

  The polynucleotide (z) has a homology with the polynucleotide (y) 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 (z), the number of substituted, added, inserted or deleted bases is, for example, a number corresponding to the homology.

  The MR gene can be obtained, for example, by cloning using a human nucleic acid based on the nucleotide sequence of SEQ ID NO: 41. The nucleic acid may be DNA or RNA such as total RNA, for example. The MR gene may be chemically synthesized using, for example, a phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit.

  When the nuclear receptor is MR, the transcription coupling factor for the MR is not particularly limited, and examples thereof include CBP and P300. As described above, the transcription coupling factor is preferably an animal transcription coupling factor, more preferably a human transcription coupling factor. The transcription coupling factor gene is preferably inserted into the second plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The second plasmid is preferably a high copy type.

  The nuclear receptor responsive element is preferably inserted into the first plasmid, for example, as described above, and this plasmid is preferably introduced into transformed yeast. The nuclear receptor response element and the reporter gene are preferably operably linked and inserted into the first plasmid as described above. The nuclear receptor responsive element, for example, GRE is preferably inserted into the first plasmid as a repetitive sequence. The first plasmid is preferably a low copy type.

As an example of the transcription coupling factor gene, the human SRC1 gene is shown below. The human SRC1 gene preferably includes, for example, the following polynucleotide (o) or (p).
(O) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 8.
(P) A polynucleotide comprising a base sequence represented by SEQ ID NO: 8, wherein at least one base is substituted, added, inserted or deleted, and which encodes a protein that functions as human SRC1.

  The polynucleotide (p) has a homology with the polynucleotide (o) 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 (p), the number of substituted, added, inserted or deleted bases is, for example, the number corresponding to the homology.

  The method for producing the transformed yeast of the present invention is not particularly limited. The transformed yeast of the present invention can be produced, for example, by deleting the coding gene from the yeast serving as a host and introducing the various genes into the yeast.

The yeast used as the host is not particularly limited, and examples thereof include budding yeast, fission yeast, filamentous yeast, etc., and budding yeast ( Saccharomyces cerevisiae ) is preferable.

  The method for deleting at least one of the coding gene for the cell wall protein and the coding gene for the efflux pump protein from the yeast is not particularly limited, and a known method can be adopted. The coding gene can be deleted using, for example, a cre-loxP system.

  The method for introducing the nuclear receptor gene into the yeast is not particularly limited, and a known gene introduction method can be employed. Examples of the gene introduction method include a lithium acetate method, a calcium phosphate method, a method using a liposome, an electroporation method, a method using a virus vector, and a micropipette injection method. In the present invention, the introduction of the nuclear receptor gene may be performed, for example, as long as it can be used for analysis of a ligand for the nuclear receptor. Therefore, the introduction may be, for example, a transient type introduction, an integration type introduction into the host yeast chromosome, or an artificial chromosome type or plasmid type introduction capable of autonomous replication and distribution. Among them, for example, from the viewpoint of easy handling and storage, and rapid, accurate and simple analysis of the ligand, it is preferable to introduce the gene into the yeast chromosome as described above.

  The nuclear receptor gene to be introduced into the yeast is preferably operably linked to necessary regulatory sequences so that it can be constitutively or arbitrarily expressed in yeast, for example. The regulatory sequence is, for example, a base sequence necessary for expression of the operably linked gene in a host cell, that is, yeast. The regulatory sequences suitable for eukaryotic cells are not particularly limited, and examples thereof include promoters, polyadenylation signals, enhancers and the like. In the present invention, “operably connected” means, for example, that the components are juxtaposed so that they can perform their functions.

  In the present invention, the reporter gene for detecting the nuclear receptor and ligand complex is preferably, for example, a β-galactosidase gene. For example, the β-galactosidase gene is operatively linked upstream of the response element that is recognized and bound by the complex. The response element is also referred to as a transcription regulatory element, for example. The response element is appropriately determined depending on, for example, the type of nuclear receptor gene. When the nuclear receptor is AR, GR, PR and MR, the response element is, for example, GRE, and when RXRα is, the response element is, for example, a DR1 element, and in the case of ERβ and ERα, The response element is, for example, ERE; in the case of TRα and TRβ, the response element is, for example, an IR0 element; in the case of AhR, the response element is, for example, XRE, and in the case of PXR-1 The response element is DR-3. The response element and the reporter gene (for example, β-galactosidase gene) are preferably incorporated into a first plasmid and introduced into yeast as described above. The reporter gene is not limited to, for example, a β-galactosidase gene, and other luciferase genes, fluorescent protein genes, β-glucuronidase genes, and the like can be used.

  The transcription coupling factor gene is preferably introduced into yeast after being incorporated into a plasmid (second plasmid) different from the plasmid into which the reporter gene is incorporated (first plasmid), for example.

  Next, an example of a method for analyzing a nuclear receptor ligand contained in a specimen using the transformed yeast of the present invention is shown. In this example, as the transformed yeast, both the mannan-binding protein coding gene and the efflux protein coding gene are deleted, and the nuclear receptor gene, the transcription coupling factor gene, the nuclear receptor The use of a body response element and a product into which a β-galactosidase gene has been introduced as the reporter gene is exemplified. The present invention is not limited to these examples.

  First, the transformed yeast is prepared. For the transformed yeast, for example, a glycerol stock is preferably prepared. The medium used for the preparation of the glycerol stock is not particularly limited, and examples thereof include a medium containing a nitrogen source, a carbon source, amino acids, nucleic acids, vitamins and the like. An example of the composition of the medium used for the preparation of the glycerol stock is shown in Table 2 below.

  The glycerol stock can be prepared, for example, by culturing the transformed yeast as follows. In the case of using the transformed yeast in a state continuously cultured in the medium as the transformed yeast, for example, 50 μL of the culture solution after the continuous culture is taken and put in 5000 μL of the medium shown in Table 2 above. In addition, shake culture at 30 ° C. for about 12 to 24 hours. When freeze-dried cells are used as the transformed yeast, for example, the freeze-dried cells are added with a nutrient medium such as a YPD medium and cultured at 30 ° C. for 24 hours, and 50 μL of the culture solution is collected. Then, in addition to 5000 μL of the medium shown in Table 2, it is cultured with shaking at 30 ° C. for about 12 to 24 hours.

  After sufficiently growing the cells by the culture, a 10-fold concentrated glycerol stock solution is prepared from the obtained culture solution. The composition of the glycerol stock solution includes, for example, 80% culture solution and 20% glycerol. The glycerol stock solution can be stored at, for example, -80 ° C.

  Next, the transformed yeast is subjected to main culture in the presence of a test substance. At this time, it is preferable to use the glycerol stock solution prepared as described above.

  The medium used for the main culture is not particularly limited, and for example, the medium shown in Table 2 above, which contains galactose and glucose as the carbon source. The ratio of galactose and glucose is not particularly limited, and the weight ratio of glucose to the total weight of galactose and glucose is, for example, more than 0 and 60% by weight or less, preferably more than 0 and 40% by weight or less, more preferably It is in the range of 10 to 30% by weight. Thus, when galactose and glucose are added to the medium, the growth of the transformed yeast can be further promoted in the main culture. For this reason, for example, the test substance can be analyzed with high sensitivity and high reliability in a shorter main culture.

  As described above, the main culture is performed by adding a test substance such as a chemical substance to the medium. The method for adding the test substance to the medium is not particularly limited, and for example, it may be added directly, or after mixing, dissolving or suspending in a solvent. When the test substance is a solid, for example, it is preferable that the test substance is mixed with a solvent and then mixed with the medium. The solvent is not particularly limited, and for example, a solvent that has low toxicity or no toxicity to the transformed yeast is preferable, and examples thereof include dimethyl sulfoxide (DMSO).

  The conditions for the main culture are not particularly limited, and the temperature is, for example, 28 to 30 ° C., and the time is, for example, 10 to 48 hours, preferably 15 to 36 hours, more preferably 18 to 24 hours. It is. The main culture may be, for example, shake culture or static culture.

  Then, the transformed yeast after the main culture is lysed. The lysis method is not particularly limited, and can be performed by, for example, a known method. The lysis can be performed using, for example, a buffer containing a surfactant as the lysis solution. The surfactant is not particularly limited, and examples thereof include sarkosyl (N-Lauroylsarcosine sodium salt). The buffer solution is not particularly limited, and examples thereof include Z buffer. An example of the composition of the lysate is shown below.

  Next, the activity of the produced β-galactosidase is measured for the lysate after lysis. The β-galactosidase activity may be measured by, for example, reacting with a coloring reagent and measuring the degree of color development as, for example, absorbance. The coloring reagent is not particularly limited, and known coloring such as ONPG (orthonitrophenyl galactopyranoside, o-nitrophenyl-β-D-galactopyranoside), CPRG (chlorophenol red galactopyranoside), etc. Substrate can be used.

  In the activity measurement, it is preferable to prepare a calibration curve in advance using a standard substance. The calibration curve is preferably created, for example, under the same conditions as the measurement of the test substance, and is preferably created based on the result of measurement performed simultaneously with the measurement of the test substance. In the activity measurement, for example, it is preferable to appropriately dilute the test substance so that it falls within the range of dose-response relationship. According to such an analysis method, for example, it is only necessary to measure the absorbance, and the test substance can be analyzed simply and quickly.

<Analysis method>
The analysis method of the present invention is a method for analyzing a nuclear receptor ligand contained in a specimen, the culture step of culturing the transformed yeast of the present invention in a medium containing the specimen,
After the culturing step, the method further comprises a measurement step of measuring an expression product of the reporter gene produced in the medium.

  The analysis method of the present invention is characterized by using the transformed yeast of the present invention, and other steps and conditions are not limited at all. The analysis method of the present invention can be carried out, for example, by the method described in the explanation of the transformed yeast of the present invention.

  In the present invention, the analysis may be, for example, any of detection, qualification, and quantification of the ligand.

<Analysis kit>
The analysis kit of the present invention is a kit for simply carrying out the analysis method of the present invention. As described above, the analysis kit of the present invention is a kit containing a medium, the transformed yeast of the present invention, and a coloring reagent. Examples of the coloring reagent include a coloring substrate such as ONPG and CPRG as described above. Furthermore, the analysis kit of the present invention may contain a standard substance for preparing a calibration curve. The transformed yeast may be lyophilized to form a powder. The analysis kit of the present invention may further contain instructions for use.

<Screening method and screening kit>
The analysis method and analysis kit of the present invention can also be referred to as, for example, a screening method and a screening kit. According to the present invention, for example, it is possible to easily screen to which nuclear receptor an unknown substance contained in a specimen binds. In the screening method of the present invention, for example, a plurality of types of the transformed yeast of the present invention for each nuclear receptor are preferably used at the same time.

  Next, examples of the present invention will be described. The present invention is not limited to the following examples.

[Example 1]
In this example, a double-deletion yeast in which the CWP1 gene and the PDR5 gene were deleted was produced by the following procedure, and a transformed yeast into which the human GR gene was incorporated was produced.

(1) Preparation of gene-deficient yeast Plasmid pUG6 has a G418 resistance gene sandwiched between loxP sequences, and the G418 resistance gene is a plasmid controlled by a fungal TEF promoter and a terminator (Nucleic Acids Res. ( 1996) 24, 2519-2524). Using the plasmid pUG6 as a template, the loxP-KanMX-loxP region was amplified by PCR using the following primers SH005, SH006, SH013 and SH014. Specifically, loxP-KanMX-loxP (hereinafter referred to as CWP1 nearby sequence-added loxP-KanMX-loxP) to which a neighboring sequence of the CWP1 gene was added and loxP-KanMX-loxP (hereinafter referred to as CWP1 neighboring sequence-added loxP-KanMX-loxP). , PDR5 nearby sequence added loxP-KanMX-loxP). Each of the primers was a sequence including a sequence adjacent to the CWP1 gene or PDR5 gene. In the following sequences, the uppercase region is the complementary sequence of the neighboring sequence of the CWP1 gene or PDR5 gene, and the lowercase region is the complementary sequence of loxP-KanMX-loxP.

(Primer for addition of loxP-KanMX-loxP in the vicinity of CWP1)
SH005 (SEQ ID NO: 9)
5'-CTGAAGTAAAATATCCCGTAACAAACTAATAAATACTACGAcagctgaagcttcgtacgc-3 '
SH006 (SEQ ID NO: 10)
5'-TATTGAAGGAAATAAAACATGCAGGTTTTGTTCTCGTACcataggccactagtggatctg-3 '
(Primer for loxP-KanMX-loxP with added sequences near PDR5)
SH013 (SEQ ID NO: 11)
5'-TAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAAcagctgaagcttcgtacgc-3 '
SH014 (SEQ ID NO: 12)
5'-AAAGTCCATCTTGGTAAGTTTCTTTTCTTAACCAAATTCcataggccactagtggatctg-3 '

(PCR condition 1)
The mold was thermally transformed at 94 ° C. for 2 minutes, and then subjected to 35 cycles of 94 ° C. for 30 seconds, 50 ° C. for 10 seconds and 68 ° C. for 2 minutes.

Then, after PCR, amplified CWP1-near sequence-added loxP-KanMX-loxP or PDR5-near-sequence added loxP-KanMX-loxP was introduced into yeast ( Saccharomyces cerevisiae ) strain W303a by the lithium acetate method. At this time, a G418-containing YPD medium in which G418 (Geneticin, manufactured by GIBCO BRL) was added to the YPD medium so as to be 200 μg / mL was used as the selection medium for the transformed yeast.

  Here, it was confirmed by introduction of the sequence whether the transformed yeast lacks the CWP1 gene or the PDR5 gene. The deletion of each gene was confirmed by PCR using the DNA of the transformed yeast as a template and the following primers. When amplification was confirmed by PCR using the following primers, it was determined that the CWP1 gene or the PDR5 gene was deleted.

(Primer for verification of 5'CWP1 deletion)
SH007 (SEQ ID NO: 13)
5'-gtgctcgaccttctatcccgt-3 '
SH018 (SEQ ID NO: 14)
5'-ctgcgcacgtcaagactg-3 '
(Primer for verification of 3'CWP1 deletion)
SH017 (SEQ ID NO: 15)
5'-cctcgacatcatctgccc-3 '
SH008 (SEQ ID NO: 16)
5'-cttgtattctcctatcagcctgg-3 '
(Primer for verification of 5'PDR5 deletion)
SH015 (SEQ ID NO: 17)
5'-ggactgaaacttaagactgccc-3 '
SH018 (SEQ ID NO: 14)
5'-ctgcgcacgtcaagactg-3 '
(Primer for verification of 3'PDR5 deletion)
SH017 (SEQ ID NO: 15)
5'-cctcgacatcatctgccc-3 '
SH016 (SEQ ID NO: 18)
5'-gcaaaagatccgattatacttacc-3 '

(PCR condition 2)
The mold was thermally transformed at 94 ° C. for 2 minutes, and then subjected to 35 cycles of 94 ° C. for 30 seconds, 50 ° C. for 10 seconds and 68 ° C. for 1 minute.

  Subsequently, Nucleic Acids Res. (1996) According to 24, 2519-2524, plasmid pSH47 was prepared. The plasmid pSH47 has a site-specific recombinase Cre gene and a uracil selection marker URA3 gene, and these genes are controlled by a Gal1-10 promoter and a CYC1 terminator. The plasmid pSH47 was introduced into the transformed yeast by the lithium acetate method. At this time, a glucose medium without addition of uracil was used as a selection medium for the transformed yeast. The uracil-free glucose medium contained glucose as a carbon source in the medium composition shown in Table 2, and a dropout powder having the composition shown in Table 6 was added as the dropout powder.

  The obtained non-uracil-requiring transformed yeast was pre-cultured overnight at 30 ° C. using a glucose liquid medium without uracil. The transformed yeast that had been pre-cultured was collected by centrifugation, and then washed twice with sterile water. The medium was replaced with uracil-free galactose liquid medium, and the washed transformed yeast was cultured at 30 ° C. for 4 hours. After culturing, the appropriately diluted bacterial solution was seeded on a YPD agar medium. And after colony formation, the colony which became G418 sensitivity was selected by the replica method. The uracil-free galactose medium contained galactose as a carbon source in the medium composition shown in Table 2, and the dropout powder having the composition shown in Table 6 was added as the dropout powder.

  Here, it was confirmed that the G418 resistance gene was excised by the Cre gene, and finally the ORF of the CWP1 gene or the PDR5 gene was replaced with the loxP sequence, respectively. This confirmation is carried out by PCR using the DNA of the G418-sensitive colony as a template and using the primers SH007 (SEQ ID NO: 13), SH008 (SEQ ID NO: 16), SH015 (SEQ ID NO: 17) and SH016 (SEQ ID NO: 18). went. The PCR was performed under the PCR condition 2 using the PCR reaction solution 2. When amplification was confirmed by PCR using the primer, it was determined that the CWP1 gene or the PDR5 gene was deleted.

  As described above, a gene deletion yeast strain lacking the CWP1 gene and the G418 resistance gene and a gene deletion yeast strain lacking the PDR5 gene and the G418 resistance gene were obtained. Finally, the single deletion strain in which plasmid pSH47 was dropped and became uracil-requiring again was used as a gene deletion yeast strain in the following experiments.

  Furthermore, from the gene deletion yeast strain lacking the CWP1 gene and G418 resistance gene or the gene deletion yeast strain lacking the PDR5 gene and G418 resistance gene, a double deletion strain is obtained by the same operation as described above. A gene-deficient yeast was prepared that lacks both the PDR5 gene and the CWP1 gene. That is, the PDR5 gene deletion strain was further subjected to the CWP1 gene deletion operation in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and the CWP1 gene were deleted. Further, for the CWP1 gene deletion strain, the PDR5 gene deletion operation was further performed in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and CWP1 gene were deleted. Finally, the double deletion strain in which plasmid pSH47 was dropped and became uracil-requiring again was used as a gene deletion yeast strain in the following experiments.

(2) Preparation of reporter plasmids Wolf et al. PRW95-3 was prepared according to Biotechniques 20, 568-573 (1996). The plasmid pRW95-3 is a plasmid having a β-galactosidase gene.

  The GRE sequence was used as a sequence that responds to GR. The following oligo DNA (SEQ ID NO: 19) containing the GRE sequence and an oligo DNA complementary to it (SEQ ID NO: 20) were prepared. These oligo DNAs were annealed. Annealing was performed at 94 ° C. for 1 minute in one cycle. After annealing, 8 consecutive GRE sequences annealed were purified and inserted into the Bgl II site of pRW95-3. A plasmid into which 8 GRE sequences were inserted in succession was designated as a GR reporter plasmid.

(GRE sequence)
GREcsfBg (SEQ ID NO: 19)
5'-agatctttgagaacaaactgttcttaaat-3 '
GREcsrBc (SEQ ID NO: 20)
3'-aaactcttgtttgacaagaatttactag-5 '

(3) Preparation of Animal Nuclear Receptor Expression Plasmid The ORF of the GR gene cDNA was amplified by PCR using the following primers using human testis cDNA (Clontech) as a template. PCR was performed under the following PCR condition 3 using the following PCR reaction solution 3.

(Primer for GR)
GRfRbBm (SEQ ID NO: 21)
5'-tcgaggatccaacaaaatggactccaaagaatcattaact-3 '
GRrXh (SEQ ID NO: 22)
5'-attcttctcgaggcagtcacttttgatgaaac-3 '

(PCR condition 3)
A cycle of 94 ° C. for 40 seconds, 58 ° C. for 20 seconds, and 68 ° C. for 3 minutes was performed for 40 cycles.

  Next, the cDNA of the amplified GR gene was cleaved with restriction enzymes BamHI and XhoI. The cleaved cDNA was inserted into the restriction enzyme BamHI and SalI cleaved plasmid pUdp6. The plasmid pUdp6 has a CYC terminator downstream of the bidirectional promoter regions gal1, gal10 and gal10, an ADH terminator downstream of gal1, and a uracil selection marker URA3 gene. The cleaved GR gene cDNA was inserted downstream of the GAL10 promoter in the plasmid pUdp6. Thereby, a GR expression plasmid pUdp6GR having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Preparation of animal transcription coupling factor expression plasmid ORF of cDNA of SRC1 gene was amplified by PCR using the following primers, using human testis cDNA (manufactured by Clontech) as a template. PCR was performed under the following PCR condition 4 using the following PCR reaction solution 4.

(Primer for SRC1)
hSRC1bgF (SEQ ID NO: 23)
5'-tggaactcaagatttgaccatatc-3 '
hSRC1xhR (SEQ ID NO: 24)
5'-gagcattcctctagtctgtagtc-3 '

(PCR condition 4)
A cycle of 94 ° C. for 40 seconds, 56 ° C. for 20 seconds, and 68 ° C. for 5 minutes was performed for 40 cycles.

  Next, the amplified cDNA of the SRC1 gene was purified. A restriction enzyme site was added to the cDNA by PCR using the purified cDNA as a template and the following primers. PCR was performed using the PCR reaction solution 4 under the following PCR condition 5.

(PCR condition 5)
A cycle of 94 ° C. for 40 seconds, 56 ° C. for 20 seconds, and 68 ° C. for 5 minutes was performed for 20 cycles.

(Additional primer)
SRC-1eFbg2 (SEQ ID NO: 25)
5'-caaagaagatctcccaggtgtgaag-3 '
SRC-1eRxh2 (SEQ ID NO: 26)
5'-agggccctcgagactctagtctgtag-3 '

  The amplified cDNA was cleaved with restriction enzymes BglII and XhoI and then inserted downstream of the gal1 promoter region of the plasmid pESC-Leu. The obtained plasmid was designated as animal transcription factor expression plasmid pESC-Leu-hSRC1.

(5) Preparation of transformed yeast The GR expression plasmid pUdp6GR was treated with the restriction enzyme EcoRV to obtain a linear form. As shown below, the linear GR expression plasmid pUdp6GR was introduced into yeast as a host by the lithium acetate method, and further integrated on a chromosome. As the host, the gene-deleted yeast strain lacking the PDR5 gene prepared in the above (1) and the gene-deleted yeast strain lacking the PDR5 gene and the CWP1 gene were used.

  First, the gene-deleted yeast strain lacking the PDR5 gene, the gene-deleted yeast strain lacking the PDR5 gene and the CWP1 gene were cultured at 30 ° C. in a YPD medium until the OD660 reached 1-2. . The cultured cells were washed with Solution A and then resuspended in Solution A so that the OD660 was 150. The composition of the solution A was 0.1 mol / L Lithium acetate, 10 mmol / L Tris-HCl (pH 7.5), 1 mmol / L EDTA (all were final concentrations). The cell suspension was dispensed at 100 μL each into a 1.5 mL microtube and incubated at 30 ° C. for 1 hour. After incubation, 5 μg of the linear plasmid pUdp6GR and 150 μg of carrier DNA (trade name SALMON TESTES DNA for hybridization, manufactured by SIGMA) are added to the tube, and then 850 μL of Solution B is added and mixed gently. did. As the solution B, 100 mL of solution A mixed with polyethylene glycol 4000 was used.

  Next, the mixed solution was incubated at 30 ° C. for 30 minutes, heat-treated at 42 ° C. for 15 minutes, and then allowed to stand at room temperature for 10 minutes. The yeast was collected by centrifugation at 5000 rpm for 1 minute. The yeast was suspended in 5 to 10 mL of YPD medium and cultured overnight at 30 ° C. After culturing, the yeast was collected, washed, suspended in 0.9% NaCl solution, and 100 μL of the suspension was applied to a YPD selection plate containing Aureobasidin A (0.5 g / mL) and cultured. The Aureobasidin A resistant strain was selected as a transformed yeast in which the gal1 promoter and the cDNA region of the GR gene were integrated on the chromosome.

  Subsequently, the animal transcription coupling factor expression plasmid pESC-Leu-hSRC1 was introduced into the transformed yeast. At this time, the selective culture medium for transformed yeast was a preculture medium with tryptophan added in which 100 mg of tryptophan was added to the preculture medium 1 shown below. In the following composition, the dropout powder and the nitrogen source were the same as those in Table 2.

  Next, the GR reporter plasmid was introduced into the transformed yeast by the lithium acetate method. At this time, the preculture medium without addition of tryptophan was used as the selection medium for transformed yeast. In this way, the CWP1 gene or the CWP1 gene and the PDR5 gene are deleted, the reporter gene is retained by the GR reporter plasmid, the SRC1 gene is retained as the plasmid pESC-Leu-hSRC1, and the human GR gene is retained in the genome. A transformed yeast strain having Hereinafter, the transformed yeast strain lacking the PDR5 gene is referred to as the PDR5Δ strain, the CWP1 gene, and the transformed yeast strain lacking the PDR gene are referred to as the PDR5Δ-CWP1Δ strain.

[Example 2]
In this example, the ligand for GR was analyzed using the transformed yeast prepared in Example 1. As a comparative example, yeast ( Saccharomyces cerevisiae ) W 303a strain was used.

(1) Preculture of transformed yeast The transformed yeast was precultured at 30 ° C for 18 hours using the following preculture medium 2. As a result, the turbidity was about Abs 595 nm = 1.0, and the growth of the transformed yeast was confirmed. In the following composition, the dropout powder and the nitrogen source were the same as those in Table 2.

(2) Main culture of transformed yeast Using a 96-well plate, 1 μL of ligand solution, 5 μL of pre-cultured cell fluid (OD = 1 to 1.5) and 100 μL of main culture medium were mixed per well. Static culture was performed at 30 ° C. for 18 hours. The ligand solution was prepared by dissolving dexamethasone, corticosterone, or hydrocortisone in DMSO to a predetermined concentration. The drop culture powder of the following main culture medium did not contain tryptophan and leucine. In the following composition, the dropout powder and the nitrogen source were the same as those in Table 2.

(3) Measurement of β-galactosidase activity After the main culture, 5 μL of the culture solution was collected from each well and transferred to each well of a new 96-well plate. After adding 100 μL of the following measurement reagent to each well, the reaction was performed at 37 ° C. for 30 minutes. After the reaction, the β-galactosidase activity and the amount of cells of the reaction solution were measured using a microplate reader. In order to calculate the β-galactosidase activity from the amount of o-nitrophenol produced, the absorbance of the reaction solution at 405 nm was measured. Moreover, the said microbial cell quantity measured the light absorbency of the said reaction liquid in 595 nm. About each reaction liquid, ratio (Abs.405nm / Abs.595nm) of the light absorbency of 405nm and the light absorbency of 595nm was computed. The analysis result was expressed as an induction rate for each reaction solution, where the ratio of the reaction solution without the ligand, that is, the reaction solution added with 1 μL of DMSO instead of the ligand solution was “1”.

  These results are shown in Tables 13 and 14 below and FIG. In FIG. 1, (A) is a graph showing results for dexamethasone, (B) is a graph showing results for corticosterone, and (C) is a graph showing results for hydrocortisone. In each graph, the horizontal axis represents each ligand concentration (log mol / L), specifically, 0, 1.00E-07, 5 × 1E-07, 1.00E-06, 3.3 ×. It is a plot in 1.00E-06, 6.6x1.00E-06, 1.00E-05, and a vertical axis | shaft is the said induction rate. In each graph, the black triangle plot is the result of the PDR5Δ-CWP1Δ strain, the black square plot is the result of the PDR5Δ strain, and the black rhombus plot is the result of the wild strain.

  As shown in Tables 13 and 14 and FIG. 1, the wild-type strain had a detectable dexamethasone concentration of 1 μmol / L or more. In contrast, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain were confirmed to induce β-galactosidase at a dexamethasone concentration of 300 nmol / L. From this result, it can be said that the transformed yeast of the present invention can obtain sufficiently excellent sensitivity even at a low ligand concentration. Moreover, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed a higher induction rate than the wild strain for a given dexamethasone concentration. As a specific example, as shown in Table 14, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed an induction rate of 1.5 times or 2.2 times that of the wild strain at a predetermined concentration, respectively. In particular, the PDR5Δ-CWP1Δ strain lacking both the PDR5 gene and the CWP1 gene was able to further improve the induction rate by 1.5 times compared to the PDR5Δ strain. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 13, the EC50 was 8.3 μmol / L for the wild type, whereas the PDR5Δ strain and the PDR5Δ-CWP1Δ strain were 500 nmol / L, which was reduced to 1/16. It was done.

  As shown in Table 13 and Table 14 and FIG. 1, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed a higher induction rate than the wild strain with respect to the corticosterone concentration. As a specific example, as shown in Table 14, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed an induction rate 1.3 times or 1.5 times that of the wild strain at a predetermined corticosterone concentration, respectively. In particular, the PDR5Δ-CWP1Δ strain lacking both the PDR5 gene and the CWP1 gene was able to further improve the induction rate by 1.2 times compared to the PDR5Δ strain. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 13, the EC50 was 1 μmol / L for the wild strain, whereas the PDR5Δ strain and the PDR5Δ-CWP1Δ strain were 500 nmol / L, which was reduced to ½. .

  As shown in Tables 13 and 14 and FIG. 1, the wild-type strain had a detectable hydrocortisone concentration of 1 μmol / L or more. In contrast, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain were confirmed to induce β-galactosidase at a hydrocortisone concentration of 600 nmol / L. From this result, it can be said that the transformed yeast of the present invention can obtain sufficiently excellent sensitivity even at a low ligand concentration. Moreover, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed a higher induction rate than the wild strain for a given hydrocortisone concentration. As a specific example, as shown in Table 14, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed an induction rate 2.3 times or 2.5 times that of the wild strain at a predetermined concentration, respectively. In particular, the PDR5Δ-CWP1Δ strain lacking both the PDR5 gene and the CWP1 gene was able to improve the induction rate 1.1 times more than the PDR5Δ strain. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 13, the EC50 was 10 μmol / L for the wild strain, whereas the PDR5Δ strain and the PDR5Δ-CWP1Δ strain were 1.5 μmol / L, which was reduced to 1/7. It was done.

  Thus, by deleting the PDR5 gene and / or the CWP1 gene, the transformed yeast was improved in detection sensitivity and / or detection response to the ligand of GR. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 3
In this example, a double-deletion yeast in which any one of the CWP1 gene, the CWP2 gene and the PDR5 gene was deleted was prepared, and a transformed yeast into which a human AhR gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deleted yeast strains deleted in the CWP1 gene, gene-deleted yeast strains deleted in the PDR5 gene, and PDR5 gene and CWP1 gene prepared in Example 1 A gene-deficient yeast strain that lacks.

  Moreover, the gene deletion yeast strain which deleted the CWP2 gene and the gene deletion yeast strain which deleted the CWP1 gene and the CWP2 gene were produced as follows. Unless otherwise indicated, it was carried out in the same manner as (1) preparation of gene-deleted yeast in Example 1 above.

  The loxP-KanMX-loxP region was amplified by PCR using the plasmid pUG6 of Example 1 as a template and the following primers SH009 and SH010. Specifically, loxP-KanMX-loxP (hereinafter referred to as CWP2 neighboring sequence-added loxP-KanMX-loxP) to which the neighboring sequence of the CWP2 gene was added was amplified. Each of the primers was a sequence containing a sequence adjacent to the CWP2 gene. In the following sequences, the uppercase region is the complementary sequence of the neighboring sequence of CWP2, and the lowercase region is the complementary sequence of loxP-KanMX-loxP.

(Primer for loxP-KanMX-loxP added with CWP2 neighboring sequences)
SH009 (SEQ ID NO: 27)
5'-CAAGAATAAATAACTTCATCACATTCGCTACACACTAACAAcagctgaagcttcgtacgc-3 '
SH010 (SEQ ID NO: 28)
5'-TACCTAGTAAAACCGAAAATTTTGAAAAAAGCCATATAGcataggccactagtggatctg-3 '

After PCR, the amplified CWP2-near-sequence added loxP-KanMX-loxP was introduced into the yeast ( Saccharomyces cerevisiae ) strain W303a by the lithium acetate method. At this time, a G418-containing YPD medium in which G418 (Geneticin, manufactured by GIBCO BRL) was added to the YPD medium so as to be 200 μg / mL was used as the selection medium for the transformed yeast.

  Here, it was confirmed by introduction of the sequence whether or not the transformed yeast had deleted the CWP2 gene. The deletion of the gene was confirmed by PCR using the DNA of the transformed yeast as a template and the following primers. When amplification was confirmed by PCR using the following primers, it was determined that the CWP2 gene was deleted.

(Primer for verification of 5′CWP2 deletion)
SH011 (SEQ ID NO: 29)
5'-gctccgaagggcaattccaca-3 '
SH018 (SEQ ID NO: 14)
5'-ctgcgcacgtcaagactg-3 '
(Primer for verification of 3′CWP2 deletion)
SH017 (SEQ ID NO: 15)
5'-cctcgacatcatctgccc-3 '
SH012 (SEQ ID NO: 30)
5'-cccaccgtagcacaccaatatc-3 '

  Here, it was confirmed that the G418 resistance gene was excised by the Cre gene and finally the ORF of the CWP2 gene was replaced with the loxP sequence. This confirmation was performed by PCR using the DNA of the G418-sensitive colony as a template and the primers SH011 (SEQ ID NO: 29) and SH012 (SEQ ID NO: 30). The PCR was performed under the PCR condition 2 using the PCR reaction solution 2. When amplification was confirmed by PCR using the primer, it was determined that the CWP2 gene was deleted.

  As described above, a gene-deleted yeast strain lacking the CWP2 gene and G418 resistance gene was obtained. Finally, the single deletion strain in which plasmid pSH47 was dropped and became uracil-requiring again was used as a gene deletion yeast strain in the following experiments.

  Furthermore, from the gene deletion yeast strain lacking the CWP1 gene and G418 resistance gene or the gene deletion yeast strain lacking the CWP2 gene and G418 resistance gene, a double deletion strain is obtained by the same operation as described above. Then, a gene-deleted yeast lacking both the CWP1 gene and the CWP2 gene was prepared. That is, the CWP1 gene deletion strain was further subjected to the CWP2 gene deletion operation in the same manner as described above to obtain a gene deletion strain in which the CWP1 gene and the CWP2 gene were deleted. Further, the CWP2 gene deletion strain was further subjected to the CWP1 gene deletion operation in the same manner as described above to obtain a gene deletion strain in which the CWP1 gene and the CWP2 gene were deleted. Finally, the double deletion strain in which plasmid pSH47 was dropped and became uracil-requiring again was used as a gene deletion yeast strain in the following experiments.

(2) Preparation of reporter plasmids Wolf et al. PRW95-3 was prepared according to Biotechniques 20, 568-573 (1996). The plasmid pRW95-3 is a plasmid having a β-galactosidase gene.

The XRE sequence was used as the sequence that responds to AhR. The following oligo DNA (SEQ ID NO: 31) containing the XRE sequence and an oligo DNA complementary to it (SEQ ID NO: 32) were prepared. These oligo DNAs were annealed. Annealing was performed at 94 ° C. for 1 minute in one cycle. After annealing, 5 consecutive XRE sequences annealed were purified and inserted into the BamHI / XmaI site of pRW95-3 to create a reporter plasmid. A plasmid in which five XRE sequences were inserted in succession was designated as an AhR reporter plasmid.
(XRE sequence)
XREfBm (SEQ ID NO: 31)
5'-atccttgcgtgaca-3 '
XREr (SEQ ID NO: 32)
5'-ggattgtcacgcaa-3 '

(3) Preparation of animal nuclear receptor expression plasmid ORF of cDNA of AhR gene or Arnt gene was amplified by PCR using the following primers, using human testis cDNA (manufactured by Clontech) as a template. PCR was performed using the PCR reaction solution 3 under the PCR condition 3.

(Primer for AhR)
AhRfBsiWI (SEQ ID NO: 33)
5'-tcgacgtacgatgaacagcagcagcgccaacatc-3 '
AhRrBsiWI (SEQ ID NO: 34)
5'-attccgtacgttacaggaatccactggatgtcaa-3 '
(Primer for Arnt)
ArntfXbaI (SEQ ID NO: 35)
5'-tcgatctagaatggcggcgactactgccaacccc-3 '
ArntrSse8387I (SEQ ID NO: 36)
5'-attccctgcaggctattctgaaaaggggggaaacat-3 '

  Next, the amplified cDNA of AhR gene was cleaved with the restriction enzyme BsiWI. The cleaved cDNA was inserted into the plasmid YEpLacGal1 and 10 cleaved with the restriction enzyme BsiWI. On the other hand, the amplified cDNA of Arnt gene was cleaved with restriction enzymes XbaI and Sse8387I. Then, Arnt gene cDNA was further inserted into the plasmid YEpLacGal 1 and 10 into which the AhR gene cDNA was inserted. Specifically, the cDNA of the cleaved Arnt gene was inserted into the plasmids YEpLacGal1 and 10 cleaved with restriction enzymes XbaI and Sse8387I. The plasmids YEpLacGal1 and 10 are plasmids having bidirectional promoter regions gal1 and gal10. The cleaved AhR gene cDNA was inserted downstream of the Gal1 promoter in the plasmid YEpLacGal1 and 10, and the cleaved Arnt gene cDNA was inserted downstream of the Gal10 promoter in the plasmid YEpLacGal1 and 10. Thus, a plasmid YEpLacAhR / Arnt having the nuclear receptor gene and having a promoter upstream thereof was obtained. E. coli strain HB101 was used for cloning.

  From the plasmid YEpLacAhR / Arnt, a DNA fragment containing the gal1 promoter region, the gal10 promoter region and the cDNA was excised with restriction enzymes SacI and SalI and inserted between the SacI site and the SalI site of the plasmid pAUR101. The obtained plasmid was designated as AhR expression plasmid pAUR101AhR / Arnt.

(4) Preparation of transformed yeast As in Example 1, except that the gene-deleted yeast, the AhR reporter plasmid, and the AhR expression plasmid were used, and the animal transcription coupling factor expression plasmid was not used. A transformed yeast was prepared. Hereinafter, the transformed yeast strain lacking the CWP1 gene, the CWP1Δ strain, the transformed yeast strain lacking the CWP2 gene, the CWP2Δ strain, the transformed yeast strain lacking the PDR5 gene, the PDR5Δ strain, The transformed yeast strain lacking the CWP1 gene and the CWP2 gene is referred to as the CWP1Δ-CWP2Δ strain, and the transformed yeast strain lacking the CWP1 gene and the PDR5 gene is referred to as the PDR5Δ-CWP1Δ strain.

Example 4
In this example, ligands for AhR were analyzed using the transformed yeast prepared in Example 3. As a comparative example, yeast ( Saccharomyces cerevisiae ) W 303a strain was used.

  In this example, the induction rate was determined in the same manner as in Example 2 except that a ligand solution prepared by dissolving β-NF in DMSO so as to have a predetermined concentration was used.

  These results are shown in Table 15 and Table 16 below, and FIG. FIG. 2 is a graph showing the results for β-NF. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, at 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μmol / L. It is a plot, and a vertical axis | shaft is the said induction rate. In the graph, the black triangle plot is the result of the PDR5Δ-CWP1Δ strain, the black inverted triangle plot is the result of the CWP1Δ-CWP2Δ strain, the black circle plot is the result of the CWP1Δ strain, and the black square The plot is the result of the PDR5Δ strain, the white circle plot is the result of the CWP2Δ strain, and the black diamond-shaped plot is the result of the wild strain.

  As shown in Tables 15 and 16 and FIG. 2, the wild-type strain had a detectable β-NF concentration of 100 nmol / L or more. In contrast, for all mutants, induction of β-galactosidase was confirmed at a β-NF concentration of 10 nmol / L. From this result, according to the transformed yeast of the present invention, sufficiently excellent sensitivity can be obtained even at a low ligand concentration. Moreover, all the mutant strains showed a higher induction rate than the wild strain at the detectable ligand concentration. As a specific example, as shown in Table 16, the PDR5Δ strain and the PDR5Δ-CWP1Δ strain showed a maximum induction rate of 5.3 times or 3.5 times that of the wild type strain, respectively. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 15, EC50 was 500 nmol / L for the wild strain, whereas 90 nmol / L for CWP1Δ, 150 nmol / L for CWP2Δ, and 150 nmol / L for PDR5Δ strain, The CWP1Δ-CWP2Δ was 15 nmol / L, and the PDR5Δ-CWP1Δ strain was 60 nmol / L, which was reduced from 1/3 to 1/30.

  Thus, by deleting at least one of the PDR5 gene, the CWP1 gene and the CWP2 gene, the transformed yeast was improved in detection sensitivity and / or detection responsiveness to the AhR ligand. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

  Transformed yeast was prepared by the following method and subjected to ligand analysis.

(A) Preparation of gene deletion yeast Each 1 gene deletion yeast, each 2 gene deletion yeast, and each 3 gene deletion yeast were prepared by the following method.

(1) Production of 1-gene deletion yeast (1-1) CWP1 gene Gene-deleted yeast lacking the CWP1 gene was produced in the same manner as in Example 1.

(1-2) PDR5 gene In the same manner as in Example 1, a gene-deleted yeast lacking the PDR5 gene was prepared.

(1-3) CWP2 gene In the same manner as in Example 3, a gene-deleted yeast lacking the CWP2 gene was prepared.

(1-4) PDR10 gene A gene-deleted yeast lacking the PDR10 gene was produced as follows. Unless otherwise indicated, it was carried out in the same manner as in the preparation of (1) gene-deleted yeast in Example 1 above.

  The loxP-KanMX-loxP region was amplified by PCR using the plasmid pUG6 of Example 1 as a template and the following primers SH049 and SH050. Specifically, loxP-KanMX-loxP (hereinafter referred to as PDR10 neighboring sequence-added loxP-KanMX-loxP) to which the neighboring sequence of the PDR10 gene was added was amplified. Each of the primers was a sequence including a sequence near the PDR10 gene. In the following sequences, the uppercase region is a complementary sequence of the neighboring sequence of the PDR10 gene, and the lowercase region is a complementary sequence of loxP-KanMX-loxP.

(Primer for PDR10 nearby sequence added loxP-KanMX-loxP)
SH049 (SEQ ID NO: 42)
5'-AAAAATCAAACAATAATTTCTGGTCTTACATAGTTGTTAGGcagctgaagcttcgtacgc-3 '
SH050 (SEQ ID NO: 43)
5'-ATTTCTTTAATTTTTTGCTTTTCTTTGGAACCCGCACCAcataggccactagtggatctg-3 '

Then, after PCR, the amplified PDR10 neighboring sequence-added loxP-KanMX-loxP was introduced into the yeast ( Saccharomyces cerevisiae ) strain W303a by the lithium acetate method as follows. At this time, a G418-containing YPD medium in which G418 (Geneticin, manufactured by GIBCO BRL) was added to the YPD medium so as to be 200 μg / mL was used as the selection medium for the transformed yeast.

  Here, it was confirmed by introduction of the sequence whether the transformed yeast had deleted the PDR10 gene. The deletion of the gene was confirmed by PCR using the DNA of the transformed yeast as a template and the following primers. When amplification was confirmed by PCR using the following primers, it was determined that the PDR10 gene was deleted.

(Primer for verification of 5 'PDR10 deletion)
SH051 (SEQ ID NO: 44)
5'-cgcgctataatgccaaaatccg-3 '
SH018 (SEQ ID NO: 14)
5'-ctgcgcacgtcaagactg-3 '
(Primer for verification of 3 ′ PDR10 deletion)
SH017 (SEQ ID NO: 15)
5'-cctcgacatcatctgccc-3 '
SH052 (SEQ ID NO: 45)
5'-catcagacgtatattcagcagc-3 '

  Subsequently, Nucleic Acids Res. (1996) According to 24, 2519-2524, plasmid pSH47 was prepared. The plasmid pSH47 has a site-specific recombinase Cre gene and a uracil selection marker URA3 gene, and these genes are controlled by a Gal1-10 promoter and a CYC1 terminator. The plasmid pSH47 was introduced into the transformed yeast by the lithium acetate method. At this time, a glucose medium without addition of uracil was used as a selection medium for the transformed yeast. The uracil-free glucose medium contained glucose as a carbon source in the medium composition shown in Table 2, and the dropout powder having the composition shown in Table 6 was added as the dropout powder.

  The obtained non-uracil-requiring transformed yeast was pre-cultured overnight at 30 ° C. using a glucose liquid medium without uracil. The transformed yeast that had been pre-cultured was collected by centrifugation, and then washed twice with sterile water. The medium was replaced with uracil-free galactose liquid medium, and the washed transformed yeast was cultured at 30 ° C. for 4 hours. After culturing, the appropriately diluted bacterial solution was seeded on a YPD agar medium. And after colony formation, the colony which became G418 sensitivity was selected by the replica method. The uracil-free galactose medium contained galactose as a carbon source in the medium composition shown in Table 2, and the dropout powder having the composition shown in Table 6 was added as the dropout powder.

  Here, it was confirmed that the G418 resistance gene was excised by the Cre gene and finally the ORF of the PDR10 gene was replaced with the loxP sequence. This confirmation was performed by PCR using the DNA of the G418-sensitive colony as a template and the primers SH051 (SEQ ID NO: 44) and SH052 (SEQ ID NO: 45). The PCR was performed under the PCR condition 2 using the PCR reaction solution 2. When amplification was confirmed by PCR using the primer, it was determined that the PDR10 gene was deleted.

  As described above, a gene-deleted yeast strain lacking the PDR10 gene and the G418 resistance gene was obtained as a single deletion strain. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(2) Preparation of 2-gene deletion yeast (2-1) PDR5 gene and CWP1 gene In the same manner as in Example 1, a gene deletion yeast lacking the PDR5 gene and CWP1 gene was prepared.

(2-2) CWP1 gene and CWP2 gene In the same manner as in Example 3, a gene-deleted yeast lacking the CWP1 gene and the CWP2 gene was prepared.

(2-3) PDR5 gene and CWP2 gene As described below, a gene-deleted yeast strain lacking the PDR5 gene and the CWP2 gene was prepared. Unless otherwise indicated, it was carried out in the same manner as the production of (1) gene-deleted yeast in Example 1 and Example 3 (hereinafter the same).

  From the gene deletion yeast strain lacking the CWP2 gene and the G418 resistance gene or the gene deletion yeast strain lacking the PDR5 gene and the G418 resistance gene, a double deletion strain is obtained by the same operation as described above. A gene-deficient yeast lacking both the PDR5 gene and the CWP2 gene was prepared. That is, for the PDR5 gene deletion strain, the CWP2 gene deletion operation was further performed in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and the CWP2 gene were deleted. Further, for the CWP2 gene deletion strain, a PDR5 gene deletion operation was further performed in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and the CWP2 gene were deleted. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(2-4) CWP1 gene and PDR10 gene From the gene deletion yeast strain lacking the CWP1 gene and G418 resistance gene, or the gene deletion yeast strain lacking the PDR10 gene and G418 resistance gene, the same as described above By operation, a gene-deficient yeast lacking both the CWP1 gene and the PDR10 gene was produced as a double deletion strain. That is, for the CWP1 gene-deleted strain, a PDR10 gene deletion operation was further performed in the same manner as described above to obtain a gene-deleted strain in which the CWP1 gene and the PDR10 gene were deleted. Further, for the PDR10 gene deletion strain, a CWP1 gene deletion operation was further performed in the same manner as described above to obtain a gene deletion strain in which the CWP1 gene and the PDR10 gene were deleted. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(2-5) PDR5 gene and PDR10 gene From the gene deletion yeast strain lacking the PDR5 gene and G418 resistance gene, or the gene deletion yeast strain lacking the PDR10 gene and G418 resistance gene, the same as described above By operation, a gene-deleted yeast lacking both the PDR5 gene and the PDR10 gene was produced as a double deletion strain. That is, the PDR5 gene deletion strain was further subjected to the PDR10 gene deletion operation in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and the PDR10 gene were deleted. Further, the PDR10 gene deletion strain was further subjected to the PDR5 gene deletion operation in the same manner as described above to obtain a gene deletion strain in which the PDR5 gene and the PDR10 gene were deleted. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(3) Production of 3-gene deletion yeast (3-1) CWP1 gene, CWP2 gene and PDR5 gene Gene deletion yeast strain lacking the CWP1 gene, CWP2 gene and G418 resistance gene, or the CWP1 gene and PDR5 gene And a gene deletion yeast strain lacking the G418 resistance gene, or a gene deletion yeast strain lacking the CWP2 gene, the PDR5 gene and the G418 resistance gene, by the same operation as described above, A gene-deleted yeast in which all of the CWP1 gene, CWP2 gene and PDR5 gene were deleted was prepared. That is, for the CWP1 gene and CWP2 gene deletion strains, a PDR5 gene deletion operation is further performed in the same manner as described above, and a gene deletion strain in which the CWP1 gene, the CWP2 gene and the PDR5 gene are deleted. Obtained. In addition, for the CWP1 gene and PDR5 gene deletion strain, a CWP2 gene deletion operation is further performed in the same manner as described above to obtain a gene deletion strain in which the CWP1 gene, CWP2 gene and PDR5 gene are deleted. It was. In addition, for the CWP2 gene and PDR5 gene deletion strain, a CWP1 gene deletion operation is further performed in the same manner as described above to obtain a gene deletion strain in which the CWP1 gene, CWP2 gene and PDR5 gene are deleted. It was. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(3-2) CWP1 gene, PDR5 gene and PDR10 gene Gene deletion yeast strain lacking the CWP1 gene, PDR5 gene and G418 resistance gene, gene deletion lacking the CWP1 gene, PDR10 gene and G418 resistance gene From a yeast strain or a gene-deficient yeast strain that lacks the PDR5 gene, PDR10 gene, and G418 resistance gene, the CWP1 gene, PDR5 gene, and PDR10 gene are all deleted as a triple-deficient strain by the same procedure as described above. A gene-deficient yeast to be lost was produced. That is, for the CWP1 gene and PDR5 gene deletion strain, a PDR10 gene deletion operation is further performed in the same manner as described above, and a gene deletion strain in which the CWP1 gene, PDR5 gene and PDR10 gene are deleted. Obtained. For the CWP1 gene and PDR10 gene-deficient strain, a PDR5 gene deletion operation is further performed in the same manner as described above to obtain a gene-deleted strain in which the CWP1 gene, PDR5 gene and PDR10 gene are deleted. It was. For the PDR5 gene and PDR10 gene-deficient strain, a CWP1 gene deletion operation is further performed in the same manner as described above to obtain a gene-deleted strain in which the CWP1 gene, PDR5 gene and PDR10 gene are deleted. It was. Finally, the strain in which plasmid pSH47 was dropped and became uracil-requiring again was designated as a gene-deleted yeast strain.

(B) Preparation of reporter plasmids Wolf et al. PRW95-3 was prepared according to Biotechniques 20, 568-573 (1996). The plasmid pRW95-3 is a plasmid having a β-galactosidase gene.

  An oligo DNA containing a sequence that responds to a nuclear receptor (response sequence) and a complementary oligo DNA were prepared. Annealing was performed at 94 ° C. for 1 minute in one cycle. After annealing, a plurality of the response elements annealed successively were purified and inserted into a predetermined restriction enzyme site of pRW95-3. A plasmid in which a plurality of the response elements were successively inserted was used as a nuclear receptor reporter plasmid. The oligo DNA for each response element and the oligo DNA complementary thereto are as follows. The oligonucleotide DNA containing the “GRE sequence” as a response sequence and the oligo DNA complementary thereto were the same as those used in Example 1. The oligonucleotide DNA containing the “XRE sequence” which is a response sequence and the oligo DNA complementary thereto were the same as those used in Example 3.

(ERE sequence)
ERE-Bmg2 (SEQ ID NO: 46)
5'-gatcccaggtcaacatgacctagaa-3 '
ERE-Bgm2 (SEQ ID NO: 47)
5'-gatcttctaggtcatgttgacctgg-3 '
(IR-0 sequence)
IR0XbaF (SEQ ID NO: 48)
5'-ctagatctcaggtcatgacctttcatcca-3 '
IR0SpeR (SEQ ID NO: 49)
5'-ctagtggatgaaaggtcatgacctgagat-3 '
(DR-3 sequence)
DR-3 Xb (SEQ ID NO: 50)
5'-ctagaaacagttcataaagttcatctg-3 '
DR-3 Nh (SEQ ID NO: 51)
5'-ctagcagatgaactttatgaactgttt-3 '

(C) Preparation of Animal Nuclear Receptor Expression Plasmid The ORF of the nuclear receptor gene cDNA was amplified by PCR using human cDNA as a template and the following primers. PCR was performed using the PCR reaction solution 3 under the PCR condition 3. For the AhR gene, the ORF of the AhR gene or Arnt gene cDNA was amplified, and for the PXR-1 gene, the ORF of the PXR-1 gene or RXRα gene cDNA was amplified. The primer for the GR gene was the same as that used in Example 1. The same primers as those used in Example 3 were used for the AhR gene or Arnt gene.

(Primer for ERα)
ERaRbSPf (SEQ ID NO: 52)
5'-ccacactagtctaaaaacatgatgaccatgaccctccacac-3 '
ERaSLr (SEQ ID NO: 53)
5'-ggaagtcgactcagaccgtggcagggaaaccctc-3 '
(Primer for ERβ)
ERβfXb (SEQ ID NO: 54)
5'-gcctcttctagaaaggtgttttctcagc-3 '
ERβrHd (SEQ ID NO: 55)
5'-acgcttaagcttgtgacctctgtgggcc-3 '
(Primer for TRα)
TRaRbSpF (SEQ ID NO: 56)
5'-gggcactagtaacaaaatggaacagaagccaagca-3 '
TRaHdR (SEQ ID NO: 57)
5'-ggccaagcttgctttagacttcctgatcctcaaag-3 '
(Primer for TRβ)
TRb1frbSp (SEQ ID NO: 58)
5'-caggactagtaaactatgactcccaacagtatgac-3 '
TRb1rHd (SEQ ID NO: 59)
5'-ttcgtgaagcttcagtctaatcctcgaacacttcc-3 '
(Primer for PXR-1)
PXRfRbSp (SEQ ID NO: 60)
5'-ttccaactagtaaaacatggaggtgagacccaaag-3 '
PXRrHd (SEQ ID NO: 61)
5'-ccttcaagcttctcagctacctgtgatgccgaac-3 '
(Primer for RXRα)
RXRaRbKpnF (SEQ ID NO: 62)
5'-aaaggggtacctcaaaaatggacaccaaacatttcctgcc-3 '
RXRaEcr (SEQ ID NO: 63)
5'-cttaagaattctaagtcatttggtgcggc-3 '
(Primer for PR)
hPRfBg (SEQ ID NO: 64)
5'-gggcagatctacaaaatgactgagctgaaggcaaa-3 '
hPRrXh (SEQ ID NO: 65)
5'-caagacctcgagatcctgaccaaac-3 '
(Primer for AR)
ARfRbBm (SEQ ID NO: 66)
5'-tcgaggatccaacaaaatggaagtgcagttagggctggg-3 '
ARrXh (SEQ ID NO: 67)
5'-agggggctcgagctggggtggggaaatagg-3 '
(Primer for MR)
hMRfBg (SEQ ID NO: 68)
5'-ccgagatctaacacaatggagaccaaaggctacca-3 '
hMRrHi (SEQ ID NO: 69)
5'-gaattcaaagggacacaacaaggtgttcgaattcc-3 '

  Next, the amplified cDNA of the nuclear receptor gene was cleaved with a desired restriction enzyme. The cleaved cDNA was inserted into the following plasmid cleaved with a desired restriction enzyme. Thereby, a nuclear receptor expression plasmid having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(Plasmid pUdp5)
The plasmid pUdp5 has a CYC terminator and a uracil selection marker URA3 gene downstream of the bidirectional promoter regions gal1, gal10 and gal10. For the preparation method of the plasmid pUdp5 and the like, reference can be made, for example, to the descriptions of [0080] to [0084] of JP2009-232697A (Japanese Patent Application No. 2008-79803).
(Plasmid pUdp6)
The plasmid pUdp6 has a CYC terminator downstream of the bidirectional promoter regions gal1, gal10 and gal10, an ADH terminator downstream of gal1, and a uracil selection marker URA3 gene. For the preparation method of the plasmid pUdp6 and the like, for example, the description of “Shizaki et al. Toxicology in Vitro 24, 638-644 (2010) can be referred to.
(Plasmid pUdp8)
The plasmid pUdp8 is a plasmid having a URS mRNA destabilizing sequence, a CYC terminator and a uracil selection marker URA3 gene downstream of the bidirectional promoter regions gal1, gal10 and gal10. For the preparation method of the plasmid pUdp8 and the like, reference can be made, for example, to the descriptions of [0080] to [0087] of JP2009-232697A (Japanese Patent Application No. 2008-79803).
(Plasmid pUra3)
The plasmid pUra3 is a plasmid having bidirectional promoter regions gal1, gal10 and a uracil selection marker URA3 gene. For the preparation method of the plasmid pUdp3 and the like, reference can be made to, for example, the description of [0080] of JP2009-232697A (Japanese Patent Application No. 2008-79803).

(D) Preparation of animal transcription coupling factor expression plasmid An animal transcription coupling factor expression plasmid pESC-Leu-hSRC1 was prepared in the same manner as in Example 1.

(E) Production of transformed yeast The nuclear receptor expression plasmid was treated with the restriction enzyme EcoRV to obtain a linear form. In the case of an ERβ expression plasmid, it was treated with restriction enzyme NcoI to form a linear form. Using the gene-deleted yeast, the reporter plasmid, and the linear nuclear receptor expression plasmid, a transformed yeast was prepared in the same manner as in Example 1. For AhR, the animal transcription coupling factor expression plasmid was not introduced.

The transformed yeast strain lacking each gene is shown as follows.
(1 gene deletion strain)
CWP1 gene deleted: CWP1Δ strain CWP2 gene deleted: CWP2Δ strain PDR5 gene deleted: PDR5Δ strain PDR10 gene deleted: PDR10Δ strain (2 gene deleted strain)
PDR5 gene and CWP1 gene deleted: PDR5Δ-CWP1Δ strain CWP1 gene and CWP2 gene deleted: CWP1Δ-CWP2Δ strain CWP2 gene and PDR5 gene deleted: CWP2Δ-PDR5Δ strain CWP1 gene and PDR10 gene deleted: CWP1Δ- PDR10Δ strain Deletion of PDR5 gene and PDR10 gene: PDR5Δ-PDR10Δ strain (3-gene deletion strain)
Deletion of CWP1, CWP2 and PDR5 genes:
CWP1Δ-CWP2Δ-PDR5Δ strain The CWP1 gene, PDR5 gene and PDR10 gene are deleted:
CWP1Δ-PDR5Δ-PDR10Δ strain

(F) Ligand analysis Using the produced transformed yeast, the ligand for the nuclear receptor was analyzed. As a comparative example, a yeast ( Saccharomyces cerevisiae ) W303a strain (wild strain) was used.

  Specifically, a ligand solution prepared by dissolving the ligand in DMSO so as to have a predetermined concentration was used. Further, as a result of the analysis, instead of the induction rate calculated in Example 2 and Example 4, the value of the reaction solution without the addition of the ligand, that is, the reaction solution to which 1 μL of DMSO was added instead of the ligand solution was expressed as the ligand. The amount of increase in induction obtained by subtracting the value of the added reaction solution was determined. The rest was the same as in Example 2 and Example 4. The amount of increase in induction may be referred to as reporter activity value, and the increase in induction may be referred to as reporter activity (hereinafter the same).

Example 5
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene and the PDR5 gene was deleted was prepared, and a transformed yeast into which the human ERα gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deleted yeast strain that lacks the PDR5 gene, the CWP1 gene, or the CWP2 gene, the PDR5 gene and the CWP1 gene, the CWP1 gene and the CWP2 gene, or the CWP2 A gene-deleted yeast strain lacking the gene and the PDR5 gene and a gene-deleted yeast lacking the CWP1, CWP2 and PDR5 genes were used.

(2) Preparation of reporter plasmid A three-dimensionally annealed ERE sequence that is a sequence that responds to ERα was purified and inserted into the BglII site of pRW95-3. A plasmid into which three ERE sequences were inserted was designated as an ERα reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified ERα gene cDNA was cleaved with restriction enzymes SpeI and SalI. The cleaved cDNA was inserted into the restriction enzyme SpeI and SalI cleaved plasmid pUdp5. In the plasmid pUdp5, the cleaved cDNA was inserted downstream of the Gal10 promoter. Thereby, an ERα expression plasmid pUdp5ERα having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Preparation of transformed yeast Using the gene-deleted yeast, ERα reporter plasmid, linear ERα expression plasmid, and animal transcription factor expression plasmid, the following seven types of transformed yeast were prepared.
1 gene deletion strain: CWP1Δ strain, CWP2Δ strain, PDR5Δ strain 2 gene deletion strain: CWP1Δ-CWP2Δ strain, PDR5Δ-CWP1Δ strain, CWP2Δ-PDR5Δ strain 3 gene deletion strain: CWP1Δ-CWP2Δ-PDR5Δ strain

Example 6
In this example, the transformed yeast produced in Example 5 was used, and 17β-estradiol was used as the ligand, and the increase in induction was determined.

  These results are shown in the following Table 17, Table 18, and FIG. FIG. 3 is a graph showing the results for 17β-estradiol. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, It is a plot in 10 micromol / L, and a vertical axis | shaft is the induction increase amount (reporter activity value) induced | guided | derived with the ligand. In the graph, the black diamond plot (♦) is the result for the wild strain, the black square plot (■) is the result for the CWP1Δ strain, the black triangle plot (▲) is the result for the CWP2Δ strain, Plots (×) are the results of the PDR5Δ strain, asterisk plots (*) are the results of the PDR5Δ-CWP1Δ strain, black circles plots (●) are the results of the CWP2Δ-PDR5Δ strain, and vertical plots ( |) Is the result of the CWP1Δ-CWP2Δ strain, and the white triangle plot (Δ) is the result of the CWP1Δ-CWP2Δ-PDR5Δ strain.

  As shown in Table 17, Table 18, and FIG. 3, the wild strain had a detectable 17β-estradiol concentration of 100 pmol / L or more. In contrast, for all mutant strains, induction of β-galactosidase was confirmed at a 17β-estradiol concentration of 10 pmol / L. From this result, according to the transformed yeast of the present invention, by deleting at least one of the PDR5 gene, the CWP1 gene and the CWP2 gene, a sufficiently excellent sensitivity can be obtained even when the ligand for ERα is at a low concentration. Obtainable. In addition, all mutants showed higher reporter activity than the wild type at detectable ligand concentrations. As specific examples, as shown in Table 18, the CWP1Δ strain, the CWP2Δ strain, the CWP1Δ-CWP2Δ strain, and the PDR5Δ-CWP1Δ strain are 40.6 times, 37.9 times, 41.7 times, and 36, respectively, of the wild type strain. It showed a particularly excellent reporter activity of 1 time. Other strains also showed 7-15 fold reporter activity. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 17, the EC50 was 396 pmol / L in the wild strain, whereas 33.6 pmol / L in the CWP1Δ strain, 25.6 pmol / L in the CWP2Δ strain, and the PDR5Δ strain. However, 69.8 pmol / L, CWP1Δ-CWP2Δ strain was 37.9 pmol / L, and PDR5Δ-CWP1Δ strain was 33.6 pmol / L, which was reduced from 1/6 to 1/15.

  As described above, by deleting either the mannan-binding protein encoding gene or the efflux pump protein encoding gene, the transformed yeast has improved detection sensitivity and / or detection response to the ligand of ERα. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 7
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene and the PDR5 gene was deleted was prepared, and a transformed yeast into which the human ERβ gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deleted yeast strain that lacks the PDR5 gene, the CWP1 gene, or the CWP2 gene, the PDR5 gene and the CWP1 gene, the CWP1 gene and the CWP2 gene, or the CWP2 A gene-deleted yeast strain lacking the gene and the PDR5 gene and a gene-deleted yeast lacking the CWP1, CWP2 and PDR5 genes were used.

(2) Preparation of reporter plasmid A plasmid in which three ERE sequences were inserted was prepared in the same manner as in Example 5. This plasmid was designated as ERβ reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified cDNA of the ERβ gene was cleaved with restriction enzymes XbaI and HindIII. The cleaved cDNA was inserted into the plasmid pUdp5 cleaved with restriction enzymes XbaI and HindIII. In the plasmid pUdp5, the cleaved cDNA was inserted downstream of the Gal10 promoter. Thereby, an ERβ expression plasmid pUdp5ERβ having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Preparation of transformed yeast Using the gene-deleted yeast, ERβ reporter plasmid, linear Eβ expression plasmid, and animal transcription factor expression plasmid, the following seven types of transformed yeast were prepared.
1 gene deletion strain: CWP1Δ strain, CWP2Δ strain, PDR5Δ strain 2 gene deletion strain: CWP1Δ-CWP2Δ strain, PDR5Δ-CWP1Δ strain, CWP2Δ-PDR5Δ strain 3 gene deletion strain: CWP1Δ-CWP2Δ-PDR5Δ strain

Example 8
In this example, the transformed yeast prepared in Example 7 was used, and 17β-estradiol was used as the ligand, and the increase in induction was determined.

  These results are shown in Table 19 and Table 20 below and FIG. FIG. 4 is a graph showing the results for 17β-estradiol. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, It is a plot in 10 micromol / L, and a vertical axis | shaft is the induction increase amount (reporter activity value) induced | guided | derived with the ligand. In the graph, the black diamond plot (♦) is the result for the wild strain, the black square plot (■) is the result for the CWP1Δ strain, the black triangle plot (▲) is the result for the CWP2Δ strain, and an asterisk. Plots (*) are the results for the PDR5Δ-CWP1Δ strain, vertical plots (|) are the results for the CWP1Δ-CWP2Δ strain, and white plots (Δ) are the results for the CWP1Δ-CWP2Δ-PDR5Δ strain .

  As shown in Table 19, Table 20, and FIG. 4, the wild strain had a detectable 17β-estradiol concentration of 100 pmol / L or more. In contrast, for all mutant strains other than CWP2Δ, induction of β-galactosidase was confirmed at a 17β-estradiol concentration of 10 pmol / L. From this result, according to the transformed yeast of the present invention, sufficiently excellent sensitivity can be obtained even when the ligand for ERβ is at a low concentration. Moreover, all the mutant strains showed a higher induction rate than the wild strain at the detectable ligand concentration. As a specific example, as shown in Table 20, CWP1Δ strain, PDR5Δ strain, CWP1Δ-CWP2Δ strain and PDR5Δ-CWP1Δ strain are 1.6 times, 1.8 times, 2.7 times, 2 times, .3 reporter activity was shown. Other strains also showed 1.3 times the reporter activity. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast has improved detection sensitivity and / or detection responsiveness to the ligand of ERβ. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 9
In this example, a deletion yeast in which the CWP1 gene and the CWP2 gene were deleted was prepared, and a transformed yeast into which the human TRα gene was incorporated was prepared.

(1) Production of gene-deleted yeast A gene-deleted yeast that lacks the CWP1 gene and the CWP2 gene was used.

(2) Preparation of Reporter Plasmid Five consecutive IR-0 sequences that respond to TRα were annealed and purified, and inserted into the site at the SpeI position of pRW95-3. A plasmid in which five IR-0 sequences were inserted was designated as a TRα reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified TRα gene cDNA was cleaved with restriction enzymes SpeI and HindIII. The cleaved cDNA was inserted into the plasmid pUdp6 cleaved with restriction enzymes XbaI and HindIII. In the plasmid pUdp6, the cleaved cDNA was inserted downstream of the Gal10 promoter. Thus, a TRα expression plasmid pUdp6TRα having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Production of transformed yeast Using the gene-deficient yeast, TRα reporter plasmid, linear TRα expression plasmid, and animal transcription factor expression plasmid, the following one type of transformed yeast was produced.
2-gene deletion strain: CWP1Δ-CWP2Δ strain

Example 10
In this example, the transformed yeast produced in Example 9 was used, and triiodothyronine was used as the ligand, and the increase in induction was determined.

  These results are shown in Table 21 and Table 22 below and FIG. FIG. 5 is a graph showing the results for triiodothyronine. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μmol / L. It is a plot in L, and a vertical axis | shaft is the induction increase (reporter activity value) induced | guided | derived with the ligand. In the graph, the black rhombus plot (♦) is the result for the wild strain, and the vertical line plot (|) is the result for the CWP1Δ-CWP2Δ strain.

  As shown in Table 21, Table 22, and FIG. 5, the wild strain had a detectable triiodothyronine concentration of 10 nmol / L or more. In contrast, in the CWP1Δ-CWP2Δ strain, the reporter activity was detected at a ligand concentration of 1 nmol / L, and at 10 nmol / L, the activity was higher than that of the wild strain. As a specific example, as shown in Table 22, the CWP1Δ-CWP2Δ strain was 13 times the wild strain. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. In addition, as shown in Table 21, the EC50 was 65.5 nmol / L for the wild strain, whereas 13.6 nmol / L for the CWP1Δ-CWP2Δ strain, which was reduced to 1/5. .

  As described above, by deleting the CWP1 and CWP2 genes, the transformed yeast was improved in detection sensitivity and / or detection response to the TRα ligand. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 11
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene and the PDR5 gene was deleted was prepared, and a transformed yeast into which the human TRβ gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deficient yeast strains that lack the PDR5 gene, gene-deficient yeast strains that lack the CWP1 gene and CWP2 gene, and PDR5, CWP1 and CWP2 genes A gene-deficient yeast strain was used.

(2) Preparation of reporter plasmid In the same manner as in Example 9, a plasmid having 5 IR-0 sequences inserted therein was prepared. This plasmid was designated as TRβ reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified cDNA of TRβ gene was cleaved with restriction enzymes SpeI and HindIII. The cleaved cDNA was inserted into the plasmid pUdp6 cleaved with restriction enzymes XbaI and HindIII. In the plasmid pUdp6, the cleaved cDNA was inserted downstream of the Gal10 promoter. Thus, a TRβ expression plasmid pUdp6TRβ having the nuclear receptor gene and a promoter upstream thereof was obtained.

(4) Production of transformed yeast Using the gene-deleted yeast, TRβ reporter plasmid, linear Tβ expression plasmid, and animal transcription factor expression plasmid, the following three types of transformed yeast were produced.
1 gene deletion strain: PDR5Δ strain 2 gene deletion strain: CWP1Δ-CWP2Δ strain 3 gene deletion strain: CWP1Δ-CWP2Δ-PDR5Δ strain

Example 12
In this example, the transformed yeast produced in Example 11 was used, triiodothyronine was used as the ligand, and the amount of increase in induction was determined.

  These results are shown in Tables 23 and 24 below and FIG. FIG. 6 is a graph showing the results for triiodothyronine. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, a plot at 0.001, 0.01, 0.1, 1, 10 μmol / L, and the vertical axis represents This is the increase in induction (reporter activity value) induced by the ligand. In the graph, the black rhombus plot (♦) is the result for the wild strain, the cross plot (x) is the result for the PDR5Δ strain, and the vertical line plot (|) is the result for the CWP1Δ-CWP2Δ strain, The white triangle plot (Δ) is the result of the CWP1Δ-CWP2Δ-PDR5Δ strain.

  As shown in Table 23, Table 24 and FIG. 6, in all strains, the detectable triiodothyronine concentration was 10 nmol / L or more. From this result, according to the transformed yeast of the present invention, by deleting at least one of the PDR5 gene, the CWP1 gene and the CWP2 gene, a sufficiently excellent sensitivity can be obtained even when the ligand for TRβ is low. Obtainable. All the mutants showed higher reporter activity than the wild type at a ligand concentration of 1 μmol / L. As a specific example, as shown in Table 24, the PDR5Δ strain, the CWP1Δ-CWP2Δ strain, and the PDR5Δ-CWP1Δ-CWP2Δ strain showed 3.21, 2.23, and 1.90 times that of the wild strain, respectively. . From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 23 above, EC50 was 1.31 μmol / L for the wild type, whereas 10.2 nmol / L for the PDR5Δ strain and 92.5 nmol / C for the CWP1Δ-CWP2Δ strain. L, PDR5Δ-CWP1Δ-CWP2Δ strain was 182 nmol / L, which was reduced from 1/128 to 1/7.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast has improved detection sensitivity and / or detection responsiveness to the ligand of TRβ. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 13
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene and the PDR5 gene was deleted was prepared, and a transformed yeast into which the human PXR-1 gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deficient yeast strain that lacks the PDR5 gene, gene-deficient yeast strain that lacks the PDR5 gene and the CWP1 gene, or the CWP2 gene and the PDR5 gene, and Gene-deleted yeast lacking the CWP1 gene, CWP2 gene and PDR5 gene was used.

(2) Preparation of Reporter Plasmid Two consecutive DR-3 sequences that respond to PXR-1 were annealed and purified and inserted into the XbaI site of pRW95-3. A plasmid in which two DR-3 sequences were inserted was designated as a PXR reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified RXRα gene cDNA was digested with restriction enzymes KpnI and EcoRI. The cleaved cDNA was inserted into plasmid pUdp8 cut with restriction enzymes KpnI and EcoRI. On the other hand, the amplified PXR-1 gene cDNA was cleaved with restriction enzymes SpeI and HindIII. Then, the PXR-1 gene cDNA was further inserted into the plasmid pUdp8 into which the RXRα gene cDNA was inserted. Specifically, the cleaved cDNA was inserted into the plasmid pUdp8 cleaved with the restriction enzymes SpeI and HindIII. In the plasmid pUdp8, the cleaved RXRα gene cDNA was inserted downstream of the Gal1 promoter, and the cleaved PXR-1 gene cDNA was inserted downstream of the Gal10 promoter. As a result, plasmid pUdp8PXR / RXRα having the nuclear receptor gene and having a promoter upstream thereof was obtained.

  In this example, the animal nuclear receptor expression plasmid was the plasmid pUdp8PXR / RXRαT from which the Helix12 at the C-terminal of RXRα was deleted in the plasmid pUdp8PXR / RXRα. The plasmid pUdp8PXR / RXRαT was prepared as follows.

  First, using QuickChangeSite-Directed Mutagenesis Kit (manufactured by Stratagene), a nonsense mutation was introduced into the 443rd codon of RXRα cDNA contained in the above pUDp8PXR / RXRα to delete the Helix12 region present at the C-terminus of RXRα. The primers (SEQ ID NOs: 70 and 71) and reaction conditions (PCR reaction solution and PCR conditions) used for the mutation are shown below.

RXR443F (SEQ ID NO: 70)
5'-cttcttcaagctcatcgggtagacacccat-3 '
RXR443R (SEQ ID NO: 71)
5'-ggtgtcaatgggtctacccgatgagctt-3 '

(PCR reaction solution: 1 × reaction buffer)
0.25 mmol / L dNTP, 3 μL Quick solution, pfu ultrapolymerase 10 unit (from Stratagene), 0.25 μmol / L each primer, 10 ng plasmid DNA are contained in 50 μL of the PCR reaction solution.

(PCR conditions: cycle)
18 cycles of 94 ° C. for 50 seconds, 60 ° C. for 50 seconds, and 68 ° C. for 7 minutes were performed.

  After the PCR, the template plasmid DNA was digested with the restriction enzyme DpnI and transformed into E. coli. The obtained plasmid DNA was designated as PXR expression plasmid pUdp8PXR / RXRαT.

(4) Production of transformed yeast Using the gene-deleted yeast, PXR reporter plasmid, linear PXR expression plasmid, and animal transcription factor expression plasmid, the following four types of transformed yeast were produced.
1 gene deletion strain: PDR5Δ strain 2 gene deletion strain: PDR5Δ-CWP1Δ strain, CWP2Δ-PDR5Δ strain 3 gene deletion strain: CWP1Δ-CWP2Δ-PDR5Δ strain

Example 14
In this example, the transformed yeast prepared in Example 13 was used, and rifampicin was used as the ligand, and the increase in induction was determined.

  These results are shown in Table 25 and Table 26 below and FIG. FIG. 7 is a graph showing the results for rifampicin. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, plots at 0.1, 1, 10, 100 μmol / L, and the vertical axis represents induction induced by the ligand. Increase amount (reporter activity value). In the graph, the black rhombus plot (♦) is the result for the wild strain, the cross plot (x) is the result for the PDR5Δ strain, the asterisk plot (*) is the result for the CWP1Δ-PDR5Δ strain, Plots (●) are the results for the CWP2Δ-PDR5Δ strain, and the white triangle plots (Δ) are the results for the CWP1Δ-CWP2Δ-PDR5Δ strain.

  As shown in Table 25, Table 26 and FIG. 7, the rifampicin concentration detectable in all strains was 1 μmol / L or more. From this result, according to the transformed yeast of the present invention, sufficiently excellent sensitivity can be obtained even if the ligand for PXR-1 is at a low concentration. Moreover, all the mutant strains showed higher reporter activity than the wild strain at a ligand concentration of 1 μmol / L. As a specific example, as shown in Table 26, the PDR5Δ strain, the PDR5Δ-CWP1Δ strain, the PDR5Δ-CWP2Δ strain, and the PDR5Δ-CWP1Δ-CWP2Δ strain are 13.35 times, 5.21 times, and 2.2. It showed 23 times and 5.02 times. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. In addition, as shown in Table 25, the EC50 was 31.6 μmol / L for the wild strain, whereas 7.86 μmol / L for the PDR5Δ strain and 7.33 μmol / L for the PDR5Δ-CWP1Δ strain. L, PDR5Δ-CWP2Δ strain was 8.10 μmol / L, and PDR5Δ-CWP1Δ-CWP2Δ strain was 10.0 μmol / L, which was reduced from ¼ to ３.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast can detect and / or detect responsiveness to the ligand of PXR-1. Improved. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 15
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene, the PDR5 gene and the PDR10 gene was deleted was prepared, and a transformed yeast into which the human PR gene was incorporated was prepared.

(1) Production of gene-deficient yeast The PDR5 gene, or a gene-deleted yeast strain that lacks the CWP2 gene, the PDR5 gene and the CWP1 gene, the CWP1 gene and the CWP2 gene, the CWP2 gene, and the PDR5 gene , The CWP1 gene and the PDR10 gene, or the gene-deleted yeast strain lacking the PDR5 gene and the PDR10 gene, and the CWP1 gene, the CWP2 gene and the PDR5 gene, or the CWP1 gene, the PDR5 gene And a gene-deficient yeast lacking the PDR10 gene was used.

(2) Preparation of Reporter Plasmid A plasmid into which 8 GRE sequences that are responsive to PR were inserted was used as a PR reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified PR gene cDNA was cleaved with restriction enzymes BglII and XhoI. The cleaved cDNA was inserted into the restriction enzymes BamHI and SalI cleaved plasmid pUdp6. In the plasmid pUdp6, the cleaved PR gene cDNA was inserted downstream of the Gal10 promoter. Thereby, a PR expression plasmid pUdp6PR having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Production of transformed yeast Using the gene-deficient yeast, PR reporter plasmid, linear PR expression plasmid, and animal transcription factor expression plasmid, the following nine types of transformed yeast were produced.
1 gene deletion strain: CWP2Δ strain, PDR5Δ strain 2 gene deletion strain: CWP1Δ-CWP2Δ strain, PDR5Δ-CWP1Δ strain, PDR5Δ-CWP2Δ strain, CWP1Δ-PDR10Δ strain, PDR5Δ-PDR10Δ strain 3 gene deletion strains: CWP1Δ-CWP -PDR5Δ strain, CWP1Δ-PDR5Δ-PDR10Δ strain

Example 16
In this example, the transformed yeast prepared in Example 15 was used, and progesterone was used as the ligand, and the increase in induction was determined.

  These results are shown in Tables 27 and 28 below and FIG. FIG. 8 is a graph showing the results for progesterone. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, It is a plot in 10 micromol / L, and a vertical axis | shaft is the induction increase amount (reporter activity value) induced | guided | derived with the ligand. In the graph, the black diamond plot (♦) is the result for the wild strain, the black triangle plot (▲) is the result for the CWP2Δ strain, the cross (×) is the result for the PDR5Δ strain, The plot (*) is the result of the CWP1Δ-PDR5Δ strain, the black circle plot (●) is the result of the CWP2Δ-PDR5Δ strain, the vertical line plot (|) is the result of the CWP1Δ-CWP2Δ strain, The plot (Δ) is the result of the PDR5Δ-PDR10Δ strain, the white rhombus plot (◇) is the result of the CWP1Δ-PDR10Δ strain, the white circle plot (◯) is the result of the CWP1Δ-CWP2Δ-PDR5Δ strain, The square plot (□) is the result of the CWP1Δ-PDR5Δ-PDR10Δ strain.

  As shown in Table 27, Table 28, and FIG. 8, the reporter activity was higher than that of the wild strain at the ligand concentration detectable in all strains. As specific examples, as shown in Table 28 above, at a progesterone concentration of 1 μmol / L, CWP2Δ strain, PDR5Δ strain, CWP1Δ-PDR5Δ strain, CWP2Δ-PDR5Δ strain, CWP1Δ-CWP2Δ strain, CWP1Δ-CWP2Δ-PDR5Δ strain, PDR5Δ-PD Strains CWP1Δ-PDR10Δ strain, CWP1Δ-PDR5Δ-PDR10Δ strain are 3.94 times, 48.55 times, 29.98 times, 22.96 times, 77.2 times, 19.56 times of the wild type strain, respectively. The reporter activity was 22.02 times, 22.5 times, and 18.67 times. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. As shown in Table 27, the EC50 was 3.16 μmol / L for the wild strain, whereas 1.34 μmol / L for the CWP2Δ strain, 853 nmol / L for the PDR5Δ strain, and CWP1Δ−. PDR5Δ strain is 1.36 μmol / L, CWP2Δ-PDR5Δ strain is 1.13 μmol / L, CWP1Δ-CWP2Δ strain is 114 nmol / L, CWP1Δ-CWP2Δ-PDR5Δ strain is 689 nmol / L, and PDR5Δ-PDR10Δ strain is The 1.39 μmol / L, CWP1Δ-PDR10Δ strain was 123 nmol / L, and the CWP1Δ-PDR5Δ-PDR10Δ strain was 696 nmol / L, which was reduced from 1/3 to 1/28.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast has improved detection sensitivity and / or detection responsiveness to the PR ligand. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 17
In this example, a deletion yeast in which any one of the CWP1 gene, the PDR5 gene, and the PDR10 gene was deleted was prepared, and a transformed yeast into which the human GR gene was incorporated was prepared.

(1) Preparation of gene-deficient yeast Gene-deficient yeast strain that lacks the PDR5 gene or the PDR10 gene, the PDR5 gene and the CWP1 gene, the CWP1 gene and the PDR10 gene, or the PDR5 gene and the PDR10 gene And a gene-deleted yeast strain that lacks the CWP1 gene, the PDR5 gene, and the PDR10 gene.

(2) Preparation of Reporter Plasmid A plasmid into which 8 GRE sequences, which are sequences that respond to GR, were inserted was used as a GR reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified GR gene cDNA was cleaved with restriction enzymes BamHI and XhoI. The cleaved cDNA was inserted into the restriction enzyme BamHI and SalI cleaved plasmid pUdp6. In the plasmid pUdp6, the cleaved GR gene cDNA was inserted downstream of the GAL10 promoter. Thereby, a GR expression plasmid pUdp6GR having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Production of transformed yeast Using the gene-deficient yeast, the GR reporter plasmid, the linear GR expression plasmid, and the animal transcription factor expression plasmid, the following 6 types of transformed yeast were produced.
1 gene deletion strain: PDR5Δ strain, PDR10Δ strain 2 gene deletion strain: PDR5Δ-CWP1Δ strain, CWP1Δ-PDR10Δ strain, PDR5Δ-PDR10Δ strain 3 gene deletion strain: CWP1Δ-PDR5Δ-PDR10Δ strain

Example 18
In this example, the transformed yeast produced in Example 17 was used, and corticosterone was used as the ligand, and the increase in induction was determined.

  These results are shown in Table 29, Table 30, and FIG. FIG. 9 is a graph showing the results for corticosterone. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, plots at 0.1, 1, 10 μmol / L, and the vertical axis represents the induction increase (reporter activity value). ). In the graph, the black triangle plot (▲) is the result for the PDR5Δ strain, the cross plot (×) is the result for the PDR10Δ strain, and the asterisk plot (*) is the result for the PDR5Δ-CWP1Δ strain. The black circle plot (●) is the result for the PDR10Δ-CWP1Δ strain, the vertical line plot (|) is the result for the PDR5Δ-PDR10Δ strain, and the white triangle plot (Δ) is the result for the CWP1Δ-PDR5Δ-PDR10Δ strain. The black diamond plot (♦) is the result for the wild strain.

  As shown in Table 29, Table 30 and FIG. 9, the wild-type strain had a detectable corticosterone concentration of 1 μmol / L or more. In contrast, for all deletion strains, induction of β-galactosidase was confirmed at a corticosterone concentration of 10 to 30 nmol / L. From this result, according to the transformed yeast of the present invention, it is possible to obtain sufficiently excellent sensitivity even at a low ligand concentration by deleting at least one of the PDR5 gene, the PDR10 gene and the CWP1 gene. it can. In addition, all gene deletion strains showed higher reporter activity than the wild strain at detectable ligand concentrations. As a specific example, as shown in Table 30, the PDR5Δ strain, the PDR10Δ strain, the PDR5Δ-CWP1Δ strain, the PDR10Δ-CWP1Δ strain, the PDR5Δ-PDR10Δ strain, and the CWP1Δ-PDR5Δ-PDR10Δ strain at a ligand concentration of 1 μmol / L, The reporter activity was 13.68 times, 3.58 times, 13.62 times, 6.21 times, 19.78 times, and 14.72 times that of the wild type. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. As shown in Table 29, the EC50 was 6.5 μmol / L for the wild strain, whereas 648 nmol / L for the PDR5Δ strain, 1 μmol / L for the PDR10Δ strain, and PDR5Δ-CWP1Δ strain. However, 789 nmol / L, PDR10Δ-CWP1Δ strain is 1 μmol / L, PDR5Δ-PDR10Δ strain is 316 nmol / L, CWP1Δ-PDR5Δ-PDR10Δ strain is 739 nmol / L, which is reduced from 1/7 to 1/20. It was.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast has improved detection sensitivity and / or detection responsiveness to the ligand of GR. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 19
In this example, a deletion yeast in which any one of the CWP1 gene, the PDR5 gene, and the PDR10 gene was deleted was prepared, and a transformed yeast into which the human AhR gene was incorporated was prepared.

(1) Production of gene-deleted yeast Gene-deleted yeast strain lacking the CWP1 gene, the PDR5 gene, or the PDR10 gene, the PDR5 gene and the CWP1 gene, the CWP1 gene and the PDR10 gene, or the PDR5 A gene-deleted yeast strain lacking the gene and the PDR10 gene, and a gene-deleted yeast lacking the CWP1 gene, the PDR5 gene, and the PDR10 gene were used.

(2) Preparation of Reporter Plasmid A plasmid into which five XRE sequences, which are sequences that respond to AhR, were inserted was used as an AhR reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid Amplified cDNA of AhR gene or Arnt gene was inserted into the plasmids YEpLacGal1 and 10 in the same manner as in Example 3. Thus, a plasmid YEpLacAhR / Arnt having the nuclear receptor gene and having a promoter upstream thereof was obtained. This plasmid was used as an AhR expression plasmid.

(4) Production of transformed yeast Using the gene-deficient yeast, the AhR reporter plasmid, and the linear AhR expression plasmid, the following seven types of transformed yeast were produced.
1 gene deletion strain: CWP1Δ strain, PDR5Δ strain, PDR10Δ strain 2 gene deletion strain: CWP1Δ-PDR5Δ strain, CWP1Δ-PDR10Δ strain, PDR5Δ-PDR10Δ strain 3 gene deletion strain: CWP1Δ-PDR5Δ-PDR10Δ strain

Example 20
In this example, the transformed yeast prepared in Example 19 was used, β-naphthoflavone was used as the ligand, and the induction increase was determined.

  These results are shown in Tables 31 and 32 below and FIG. FIG. 10 is a graph showing the results for β-naphthoflavone. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, a plot at 0.0001, 0.001, 0.01, 0.1, 1, 10 μmol / L, The vertical axis represents the amount of increase in induction (reporter activity value). In the graph, the black square plot (■) is the result of the CWP1Δ strain, the black triangle plot (▲) is the result of the PDR5Δ strain, and the cross plot (×) is the result of the PDR10Δ strain, The asterisk plot (*) is the result of the PDR5Δ-CWP1Δ strain, the black circle plot (●) is the result of the PDR10Δ-CWP1Δ strain, and the vertical line plot (|) is the result of the PDR5Δ-PDR10Δ strain, The white triangle plot (Δ) is the result for the CWP1Δ-PDR5Δ-PDR10Δ strain, and the black rhombus plot (♦) is the result for the wild strain.

  As shown in Table 31 and Table 32 and FIG. 10, the wild-type strain had a detectable β-naphthoflavone concentration of 10 nmol / L or more. In contrast, induction of β-galactosidase could be confirmed for all deletion strains. Among them, when the PDR5Δ strain has a β-naphthoflavone concentration of 10 nmol / L, it can be confirmed that the induction of β-galactosidase is higher than that of the wild strain, and other strains have a β-naphthoflavone concentration of 1 nmol / L. Induction of β-galactosidase was confirmed. From this result, according to the transformed yeast of the present invention, by sufficiently deleting at least one of the PDR5 gene, the PDR10 gene, and the CWP1 gene, sufficiently high sensitivity can be obtained even when the ligand for AhR is at a low concentration. Can be obtained. In addition, all gene deletion strains showed higher reporter activity than the wild strain at detectable ligand concentrations. As a specific example, as shown in Table 32 above, at a ligand concentration of 10 nmol / L, CWP1Δ strain, PDR5Δ strain, PDR10Δ strain, PDR5Δ-CWP1Δ strain, PDR10Δ-CWP1Δ strain, PDR5Δ-PDR10Δ strain, CWP1Δ-PDR5Δ-PDR10Δ strain, The reporter activity was 16.68 times, 5.52 times, 38.73 times, 29.25 times, 12.54 times, 96.08 times, or 95.28 times that of the wild type, respectively. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. Further, as shown in Table 31 above, the EC50 was 771 nmol / L for the wild type, whereas 96.9 nmol / L for the CWP1Δ strain, 86.2 nmol / L for the PDR10Δ strain, and PDR5Δ−. CWP1Δ strain is 85.6 nmol / L, PDR10Δ-CWP1Δ strain is 94.9 nmol / L, PDR5Δ-PDR10Δ strain is 2.72 nmol / L, CWP1Δ-PDR5Δ-PDR10Δ strain is 10.9 nmol / L, It was reduced from 1/8 to 1/283.

  As described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast has improved detection sensitivity and / or detection responsiveness to the AhR ligand. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 21
In this example, a deletion yeast in which any one of the CWP1 gene, the CWP2 gene, the PDR5 gene and the PDR10 gene was deleted was prepared, and a transformed yeast into which the human AR gene was incorporated was prepared.

(1) Production of gene-deficient yeast Gene-deficient yeast strain that lacks the PDR5 gene, the PDR5 gene and the CWP1 gene, the PDR5 gene and the CWP2 gene, the CWP1 gene and the CWP2 gene, or the PDR5 gene And a gene-deleted yeast strain lacking the PDR10 gene, and a gene-deleted yeast strain lacking the CWP1 gene, PDR5 gene and PDR10 gene, or CWP1 gene, CWP2 gene and PDR5 gene.

(2) Preparation of Reporter Plasmid A plasmid into which 8 GRE sequences that are responsive to AR were inserted was used as an AR reporter plasmid.

(3) Preparation of animal nuclear receptor expression plasmid The amplified AR gene cDNA was cleaved with restriction enzymes BamHI and XhoI. The cleaved cDNA was inserted into the restriction enzyme BamHI and SalI cleaved plasmid pUdp6. In the plasmid pUdp6, the cleaved AR gene cDNA was inserted downstream of the GAL10 promoter. Thereby, an AR expression plasmid pUdp6AR having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Preparation of transformed yeast Using the gene-deleted yeast, AR reporter plasmid, linear AR expression plasmid, and animal transcription factor expression plasmid, the following seven types of transformed yeast were prepared.
1 gene deletion strain: PDR5Δ strain 2 gene deletion strain: PDR5Δ-CWP1Δ strain, PDR5Δ-CWP2Δ strain, CWP1Δ-CWP2Δ strain, PDR5Δ-PDR10Δ strain 3 gene deletion strains: CWP1Δ-PDR5Δ-PDR10Δ strain, CWP1Δ-CWP2Δ- PDR5Δ strain

[Example 22]
In this example, the transformed yeast prepared in Example 21 was used, and testosterone was used as the ligand to determine the amount of increase in induction.

  These results are shown in Tables 33 and 34 below and FIG. FIG. 11 is a graph showing the results for testosterone. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 is a plot at 100 μmol / L, and the vertical axis represents an increase in induction induced by the ligand (reporter activity value). In the graph, the black diamond plot (♦) is the result for the wild strain, the black square plot (■) is the result for the PDR5Δ strain, and the black triangle plot (▲) is the result for the CWP1Δ-PDR5Δ strain. , Cross plot (x) is the result of CWP2Δ-PDR5Δ strain, asterisk plot is (*) is the result of CWP1Δ-CWP2 strain, black circle plot (●) is the result of CWP1Δ-CWP2Δ-PDR5Δ strain Yes, the vertical line plot (|) is the result for the PDR5Δ-PDR10Δ strain, and the white triangle plot (Δ) is the result for the CWP1Δ-PDR5Δ-PDR10Δ strain.

  As shown in Table 33, Table 34 and FIG. 11, all mutant strains showed higher reporter activity than the wild strain at detectable ligand concentrations. As specific examples, as shown in Table 34 above, at a testosterone concentration of 100 nmol / L, PDR5Δ strain, CWP1Δ-PDR5Δ strain, CWP2Δ-PDR5Δ strain, CWP1Δ-CWP2Δ strain, CWP1Δ-CWP2Δ-PDR5Δ strain, PDR5Δ-PDR10Δ strain, -PDR5Δ-PDR10Δ strains have 7.7 times, 18.52 times, 3.33 times, 5.49 times, 7.42 times, 10.07 times, and 4.57 times reporter activity of the wild type strains, respectively. Indicated. From these results, it can be said that according to the transformed yeast of the present invention, the responsiveness superior to that of the wild strain is exhibited with respect to a predetermined ligand concentration. In addition, as shown in Table 33, the EC50 was 53.1 nmol / L for the wild type, whereas 70.4 nmol / L for the CWP1Δ-CWP2Δ and 7 for the CWP1Δ-CWP2Δ-PDR5Δ strain. .99 nmol / L, reduced to 1/2 and 1/7, respectively.

  As described above, by deleting either the mannan-binding protein encoding gene or the efflux pump protein encoding gene, the transformed yeast has improved detection sensitivity and / or detection response to the AR ligand. It was done. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

Example 23
In this example, a deletion yeast in which the PDR5 gene was deleted was prepared, and a transformed yeast into which the human MR gene was incorporated was prepared.

(1) Production of gene-deficient yeast A gene-deficient yeast strain lacking the PDR5 gene was used.

(2) Preparation of Reporter Plasmid A plasmid into which 8 GRE sequences that are responsive to MR were inserted was designated as an MR reporter plasmid.

(3) Preparation of Animal Nuclear Receptor Expression Plasmid The amplified MR gene cDNA was cut with the restriction enzyme AflII, smoothed, and then cut with BglII. The cut cDNA was cut with the restriction enzyme HindIII, blunted, and then inserted into the plasmid pUdp6 cut with BamHI. In the plasmid pUdp6, the cleaved MR gene cDNA was inserted downstream of the gal10 promoter. Thereby, an MR expression plasmid pUdp6MR having the nuclear receptor gene and having a promoter upstream thereof was obtained.

(4) Preparation of transformed yeast A PDR5Δ strain was prepared using the gene-deleted yeast, the MR reporter plasmid, the linear MR expression plasmid, and the animal transcription factor expression plasmid.

Example 24
In this example, the transformed yeast produced in Example 23 was used, and aldosterone was used as the ligand, and the amount of increase in induction was determined.

  These results are shown in Table 35 and Table 36 below and FIG. FIG. 12 is a graph showing the results for aldosterone. In the graph, the horizontal axis represents each ligand concentration (μmol / L), specifically, a plot at 0.001, 0.01, 0.1, 1, 10 μmol / L, and the vertical axis represents The amount of increase in induction (reporter activity value). In the graph, the black square plot (■) is the result of the PDR5Δ strain, and the black diamond plot (♦) is the result of the wild strain.

  As shown in Table 35, Table 36 and FIG. 12, the aldosterone concentration detectable for both the wild type strain and the PDR5Δ strain was 10 nmol / L or more. The PDR5Δ strain showed a reporter activity 59.1 times that of the wild strain at a ligand concentration of 100 nmol / L. The EC50 was 790 nmol / L for the wild strain, whereas it was 61.1 nmol / L for the PDR5Δ strain, which was reduced to 1/13.

  As described above, by deleting the PDR5 gene, the transformed yeast was improved in detection sensitivity and / or detection response to the MR ligand. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

  From the results of the examples of the present invention described above, by deleting either the mannan-binding protein coding gene or the efflux pump protein coding gene, the transformed yeast can be used regardless of the type of nuclear receptor gene. Detection sensitivity and / or detection responsiveness was improved for ligands of animal nuclear receptors. Therefore, it can be said that the transformed yeast of the present invention is extremely effective for assaying ligands for animal nuclear receptors.

  As described above, by using the transformed yeast of the present invention, it is possible to analyze a ligand for a nuclear receptor with high detection sensitivity. Therefore, the analysis method of the present invention is a useful method in the field of analysis of ligands such as chemical substances and proteins, and its application is not limited and is wide.

Claims (21)

A transformed yeast in which a nuclear receptor gene and a reporter gene have been introduced so that they can be expressed, and the reporter gene can be expressed by recognizing a complex of a nuclear receptor ligand and a nuclear receptor,
Furthermore, a transformed yeast, wherein at least one of a function of a cell wall protein and a function of an efflux pump protein is deleted. The transformed yeast according to claim 1, wherein the function of the cell wall protein is lost by deleting the coding gene of the cell wall protein. The transformed yeast according to claim 1 or 2, wherein the cell wall protein coding gene is a mannan-binding protein coding gene. The mannan-binding protein coding gene is at least one selected from the group consisting of CWP1 gene, CWP2 gene, CCW14 gene, CIS3 gene, KRE11 gene, PIR1 gene, PIR3 gene, TIP1 gene, TIR1 gene and TIR2 gene The transformed yeast according to claim 3. The transformed yeast according to any one of claims 1 to 4, wherein the function of the efflux pump protein is deleted by deleting the coding gene of the efflux pump protein. The transformed yeast according to any one of claims 1 to 5, wherein the gene encoding the efflux pump protein is an ABC transporter gene. The ABC transporter gene is selected from the group consisting of PDR5 gene, STE6 gene, PDR12 gene, PDR15 gene, SNQ2 gene, YOR1 gene, YDR061W gene, PDR18 gene, YOL075C gene, YBT1 gene, AUS1 gene, PDR10 gene, PDR11 gene The transformed yeast according to claim 6, wherein the transformed yeast is at least one. The transformed yeast according to any one of claims 1 to 7, wherein the coding gene for the cell wall protein and the coding gene for the efflux pump protein are deleted. The transformed yeast according to any one of claims 1 to 8, wherein the nuclear receptor is an animal nuclear receptor. The transformed yeast according to claim 9, wherein the animal nuclear receptor is a human nuclear receptor. The transformed yeast according to any one of claims 1 to 10, wherein the nuclear receptor is a homodimer, heterodimer or monomeric receptor. The transformed yeast according to claim 11, wherein the heterodimeric receptor is a receptor including a retinoid X receptor (RXR). The transformed yeast according to claim 12, wherein the heterodimeric receptor is a heterodimer of RXR and PXR. The transformed yeast according to any one of claims 1 to 13, wherein the reporter gene is a β-galactosidase gene. Having the nuclear receptor gene on the chromosome,
Having the reporter gene in a plasmid;
The transformed yeast according to any one of claims 1 to 14, wherein the plasmid comprises a nuclear receptor responsive element operatively incorporated with the reporter gene. The transformed yeast according to any one of claims 1 to 15, wherein the transformed yeast is a budding yeast ( Saccharomyces cerevisiae ). The transformed yeast according to any one of claims 1 to 16, which is used for analysis of a ligand for a nuclear receptor contained in a specimen. A method for analyzing a nuclear receptor ligand contained in a specimen, comprising:
A culture step of culturing the transformed yeast according to any one of claims 1 to 17 in a medium containing a specimen;
And a measuring step of measuring an expression product of the reporter gene produced in the medium after the culturing step. The reporter gene is a β-galactosidase gene;
The analysis method according to claim 18, wherein the medium contains at least one of galactose and glucose as a carbon source. An analysis kit for analyzing a nuclear receptor ligand contained in a specimen,
(A) a medium;
(B) the transformed yeast according to any one of claims 1 to 17;
(C) An analysis kit comprising a coloring reagent. The analysis kit according to claim 20, wherein the medium is a medium containing at least one of glucose and galactose.

Images