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WO2005110433A1 - Animal non humain dont l’expression de bach2 est artificiellement inhibée, et utilisation de celui-ci - Google Patents

Animal non humain dont l’expression de bach2 est artificiellement inhibée, et utilisation de celui-ci Download PDF

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Publication number
WO2005110433A1
WO2005110433A1 PCT/JP2005/008928 JP2005008928W WO2005110433A1 WO 2005110433 A1 WO2005110433 A1 WO 2005110433A1 JP 2005008928 W JP2005008928 W JP 2005008928W WO 2005110433 A1 WO2005110433 A1 WO 2005110433A1
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bach2
gene
cell
igm
human animal
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PCT/JP2005/008928
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Japanese (ja)
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Kazuhiko Igarashi
Tetsuhiko Muto
Masayuki Yamamoto
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Kazuhiko Igarashi
Tetsuhiko Muto
Masayuki Yamamoto
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Priority to JP2006513597A priority Critical patent/JPWO2005110433A1/ja
Publication of WO2005110433A1 publication Critical patent/WO2005110433A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention relates to a non-human animal in which the expression of Bach2 is artificially suppressed and a method for using the same. Further, the present invention provides a drug for treating or preventing inflammatory bowel disease and hyper-IgM syndrome, a drug for increasing IgM, a screening method for these drugs, and a method for testing inflammatory bowel disease and hyper-IgM syndrome. Regarding test drugs.
  • Activated B cells secrete immunoglobulin M (IgM) by dividing into plasma cells or undergo class switch recombination (CSR) to secrete other classes of immunoglobulins (See Non-Patent Documents 1 to 4).
  • IgM immunoglobulin M
  • CSR class switch recombination
  • SHM somatic hypermutation
  • V variable region exons
  • IgH immunoglobulin heavy chain
  • AID Activation-induced cytidine kinase
  • Non-patent literature 1 Snapper, C, Marcu, K. & Zelazowski, P. The immunogloDulin class switch: beyond accessibility ". Immunity 6, 217-223 (1997).
  • Non-Patent Document 2 Kinoshita, K. & Honjo, T. Linking class-switch recombination with somatic hypermutation. Nat. Rev. Mol. Cell. Biol. 2, 493—503 (2001).
  • Non-Patent Document 3 Manis, J., Tian, M. & Alt, F. Mechanism and control of class-switch recomoination. Trends. Immunol. 23, 31-39 (2002).
  • Patent Document 4 Honjo, T., Kinoshita, K. & Muramatsu, M. Molecular mechanism of class switch recombination: linkage with somatic hypermutation.
  • the present invention has been made in view of such circumstances, and has as an object to elucidate the function of Bach2 in vivo and to clarify its role in antibody production and its relevance to disease. I do. More specifically, the present invention relates to a non-human animal in which the expression of the bach2 gene is artificially suppressed and its use, a drug for treating or preventing inflammatory bowel disease and hyper-IgM syndrome, and increasing IgM. The present invention relates to a method for screening inflammatory bowel disease and hyper-IgM syndrome, and to a method for screening for such a drug. Means for solving the problem
  • bach2 gene knockout mice developed inflammatory bowel disease and dyspermic IgM syndrome, and their serum IgM levels were lower than those in controls without artificial suppression of bach2 gene expression. It was found to increase.
  • Bach2-deficient B cells failed to express AID, an essential enzyme for CSR and SHM.
  • Bach2 was accumulated in the cytoplasm of IgM secreting plasma cells and was present in the nucleus of IgG secreting cells.
  • Bach2 overexpression in B cells inhibited the formation of IgM plasma cells in vitro.
  • the present invention provides the above-mentioned non-human animal and its use, a drug for treating or preventing inflammatory bowel disease and hyper-IgM syndrome, a drug for increasing IgM, a method for screening these drugs, and Testing for inflammatory bowel disease and hyper-IgM syndrome
  • the present invention provides the following [1] to [24] regarding a method and a test agent.
  • a non-human animal wherein the expression of the bach2 gene is artificially suppressed.
  • a non-human animal characterized by having a foreign gene inserted into one or both of the bach2 gene pairs.
  • a non-human animal cell having multipotency wherein a foreign gene is inserted into one or both of the bach2 gene pair.
  • a method for producing IgM comprising the following steps (a) to (c).
  • a method for producing an antiserum containing IgM comprising the following steps (a) to (c).
  • An agent for treating or preventing inflammatory bowel disease or hyper-IgM syndrome comprising a DNA according to any one of the following (a) to (d) as an active ingredient:
  • An agent for increasing IgM comprising a DNA for suppressing the expression of Bach2 as an active ingredient.
  • test compound (a) a step of administering the test compound to the non-human animal according to [1] or [2], or the cells according to any one of [9] to [11];
  • a method for screening a Bach2 regulator comprising the following steps (a) to (c).
  • a method for screening a Bach2 regulator comprising the following steps (a) to (c).
  • (c) a step of selecting a compound that increases or decreases the activity of the Bach2 as compared to the case where the test compound is administered.
  • a method for screening a Bach2 regulator comprising the following steps (a) to (d).
  • a method for screening a Bach2 regulator comprising the following steps (a) to (d). (a) a step of providing a cell or cell extract having a DNA in which a reporter gene is functionally linked downstream of the Bach2 transcription control region
  • a method for testing inflammatory bowel disease or hyper-IgM syndrome comprising a step of measuring the expression level of the bach2 gene.
  • a method for testing inflammatory bowel disease or hyper-IgM syndrome comprising a step of detecting a mutation in the bach2 gene region.
  • a test drug for inflammatory bowel disease or IgM syndrome comprising an oligonucleotide hybridized to the bach2 gene region and having a chain length of at least 15 nucleotides.
  • An agent for detecting inflammatory bowel disease or hyper-IgM syndrome comprising an antibody that binds to Bach2.
  • FIG. La shows that the bach2 gene was disrupted by homologous recombination using a vector containing the neomycin resistance gene and the lacZ gene cassette instead of the first coded exon of bach2.
  • Figure lb is a photograph showing Southern blot analysis of DNA prepared for control and targeting embryonic stem (ES) cell cloning power. The genomic DNA treated with Sacl was hybridized with the 5 'probe shown in c.
  • FIG. Lc is a photograph showing Western blot analysis of bone marrow B cell extracts of each genotype with anti-Bach2 antiserum.
  • FIG. 2a is a photograph of a double-stained section of mouse spleen for Bach2 (center column) and MadCAM-1, IgM, or CD3 (right column). The fused column (left column) shows Bach2 (green) and MadCAM-1, IgM, or CD3 (red).
  • ⁇ 2b Representative FACS analysis of bone marrow and splenocytes of wild-type and homozygous mutant littermates.
  • FIG. 2c shows the incidence of B cell populations in bone marrow from 11 bach2 + / + (white bars) and bach2 _ / _ (hatched bars) mice.
  • FIG. 2d shows the incidence of B cell population in the spleen from 11 bach2 + / + (white bars) and bach2 _ / _ (hatched bars) mice.
  • FIG. 2e shows the relative incidence of marginal zone B (MZB) and follicle B (FOB) cells in B220 + spleen cells.
  • FIG. 2f is a photograph of a spleen section from a wild-type (left) or bach2 _ / _ (right) mouse stained with IgM (green) and MadCAM-1 (red). The lower panel shows black and white IgM staining. One scale of the scale showed 100 m.
  • FIG. 3a shows immunoglobulin levels in serum from wild-type (open circles) and bach2 _ / _ (closed triangles) mice (5 to 10 weeks old). Horizontal bars indicate average density.
  • FIG. 3b Bach2 + / + (open circles) or bach2 (closed triangles) mice were immunized with DNP-ficoll and showed anti-DNP IgM and IgG3 serum levels at days 0, 7, and 14. You. Serum levels are given in arbitrary units (absorbance at OD nm).
  • bach2 + / + (open circles) or bach2 (closed triangles) mice were immunized on day 0 and day 21 with NP-linked avian ⁇ -globulin (NP-CGG). And Anti-NP IgM and IgGl serum levels at day 28.
  • NP-CGG NP-linked avian ⁇ -globulin
  • FIG. 3d is a diagram showing the mutation occurrence rate in the ⁇ isotype Vhl86.2 transcript from MLN of NP-CGG-immunized bach2 + / + (upper panel) and bach2 _ / _ (lower panel) mice. Substitutions at specific positions (black, bars) or silent mutations (white bars) were plotted along residue numbers 1-94.
  • FIG. 4b is a photograph showing the expression level of GLT containing I ⁇ and C-exon of each isotype in splenocytes stimulated with LPS and the indicated cytodynamic force for 2 days.
  • FIG. 4c is a photograph showing the expression level of PST containing I and exon in splenocytes stimulated with LPS and the indicated cytodynamic force for 5 days.
  • FIG. 4d shows the results of measuring BrdU incorporation by culturing purified spleen B cells from wild-type (white column) and bach2 _ / _ (hatched column) mice together with LPS. The average of three independent experiments is shown. The numbers in parentheses indicate the concentration of LPS (g / ml).
  • FIG. 4e is a photograph showing the result of determining the expression level of a B cell maturation-related gene using splenocytes stimulated with LPS for 0, 2, or 5 days.
  • FIG. 5a is a photograph of LPS-stimulated wild-type splenocytes stained for Bach2, cytoplasmic IgM (upper panel), or IgG (lower panel), and B220. The fused images show Bach2 (green), IgM (red), IgG (red), and B220 (blue).
  • FIG. 5b is a diagram showing Bach2 staining in IgM-positive (174 cells) or IgG-positive (133 cells) cells.
  • Bach2 staining was classified as cytoplasmic (mainly cytoplasmic except for the nucleus), diffiise (almost evenly distributed in both compartments) (nuclear + cytoplasmic), and nucleus. Similar results were obtained in independent experiments.
  • [5c] A figure showing the results of infection of control (white), Bach2 (shaded), and Bach2 ⁇ C2 (black) viruses with purified wild-type spleen B cells, and analysis of FGFP and IgM staining by FACS. Shown are the averages from two independent experiments.
  • FIG. 6 is a photograph showing a tissue image of large intestine (hematoxylin and eosin staining) of wild-type and Bach2 knockout mice.
  • Bach2 knockout mice show vulvitis and negative abscesses.
  • FIG. 7ab is a graph showing the serum antibody titers of anti-mouse IgG antibody (a) and anti-mouse IgM antibody (b) in Bach-2 KO mice.
  • Pre ( ⁇ ) indicates the pre-immune serum antibody titer
  • 3-7 indicates the serum antibody titer at the time of 3-7 immunizations.
  • FIG. 7cd is a graph showing the serum antibody titers of anti-mouse IgG antibody (c) and anti-mouse IgM antibody (d) in Balb / c mice.
  • Pre ( ⁇ ) indicates the pre-immune serum antibody titer
  • 2-4 indicates the serum antibody titer at the time of 2-4 immunizations.
  • FIG. 8 is a graph showing the ratio of IgM among positive wells having an absorbance of 0.5 or more in Bach-2 KO mice (a) and Balb / c mice (b).
  • the present inventors have created a bach2 gene knockout mouse to elucidate the function of Bach2 in vivo. As a result, it became clear that decreased expression of Bach2 was associated with the development of inflammatory bowel disease and hyper-IgM syndrome, or increased IgM levels. The present invention is based on this finding.
  • the present invention provides a non-human animal, wherein the expression of the bach2 gene is artificially suppressed.
  • the bach2 (BTB and CNC homology 2) gene of the present invention (the protein encoded by the gene is referred to as "Bach2") is known to mice and humans! /.
  • the accession numbers of the bach2 and Bach2 sequences in each sequence are shown below.
  • the Bach2 of the present invention is not limited to the above example, but includes proteins functionally equivalent to the above Bach2.
  • Functionally equivalent proteins include proteins comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added and Z- or inserted in the amino acid sequence of Bach2 described above.
  • Other methods well known to those skilled in the art for preparing DNA encoding a protein functionally equivalent to Bach2 of the present invention include hybridization techniques under stringent conditions (see Southern EM: J Mol Biol 98: 503, 1975) and polymerase chain reaction (PCR) technology (Saiki RK, et al: Science 230: 1350, 1985, Saiki RK, et al: Science 239: 487, 1988) Method.
  • the stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5X SSC or a stringency hybridization condition equivalent thereto. Under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 ⁇ SSC, it is expected that more homologous DNA can be isolated.
  • High homology refers to sequence identity of at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more in the entire amino acid sequence.
  • the number of amino acids to be mutated in the mutant is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, further preferably within 3 amino acids, and even more preferably. No more than 2 amino acids.
  • amino acid sequence and base sequence identity were determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl. Acad Sci. USA 90: 5873, 1993) by Carlin and Artiul. Can be used to determine.
  • the expression of the bach2 gene is suppressed means that the expression of the bach2 gene is suppressed as compared to the control by artificially suppressing the expression. Means a state that is suppressed (including a state that is completely suppressed). For example, instead of the normal bach2 gene, the state in which the function as the Bach2 protein is reduced or lost, and the mutant bach2 gene encoding the mutant protein is expressed, and the state of the present invention is also referred to as "the present invention”. The expression of the bach2 gene is suppressed. "
  • Gene expression can be suppressed by a method generally known to those skilled in the art.
  • a method using a gene modification technique including a conditional gene modification technique by introducing an enzyme that promotes recombination of a target gene site, for example, Cre in Cre-lox
  • a method using an antisense DNA Alternatively, a method using RNAi technology and the like can be mentioned. Therefore, the “non-human animal in which the expression of the bach2 gene is artificially suppressed” in the present invention includes the non-human animal produced by the above method.
  • Examples of the non-human animal produced by the gene modification technique include a genetically modified non-human animal having a genetic mutation such as nucleotide insertion, deletion, or substitution in both of the bach2 gene pairs.
  • Genetically modified non-human animals in which a foreign gene has been inserted into both of the above-mentioned bach2 gene pairs can be obtained by crossing genetically modified non-human animals in which a foreign gene has been inserted into one of the bach2 gene pairs. It can be manufactured with.
  • the present invention also provides such a heterozygote.
  • the site where the gene mutation exists is not particularly limited as long as expression of the gene is suppressed, and examples thereof include an exon site and a promoter site. You can do it.
  • the species from which the non-human animal is derived in the present invention is usually a vertebrate other than a human, preferably a mammal, more preferably a mouse, rat, pig or the like.
  • non-human animal of the present invention develops inflammatory bowel disease, it can be used as an inflammatory bowel disease model animal.
  • the non-human animal of the present invention exhibits at least the characteristics of increased serum IgM levels and decreased IgG and IgA levels, and thus can be used as a hyper IgM syndrome model animal.
  • hyper IgM syndrome also called hyper-IgM immunodeficiency
  • the non-human animal of the present invention can be used for evaluation of a therapeutic agent for inflammatory bowel disease and hyper IgM syndrome.
  • the non-human animal has an increased serum IgM level as compared to a control in which the expression of the bach2 gene is not artificially suppressed.
  • the non-human animal can also be used for the production of antibodies (preferably IgM).
  • the present invention provides a method of using a non-human animal of inflammatory bowel disease model applications, hyper IgM syndrome model application or antibody production use or non-human animal or inflammatory bowel disease model hyper Ig M syndrome model, the present invention Alternatively, it provides a method used for antibody production.
  • inflammatory bowel disease includes the following 1) and 2).
  • Inflammatory bowel disease of obvious cause eg, more commonly caused by bacteria, viruses, drugs, etc.
  • Inflammatory bowel disease of unknown cause eg, ulcerative colitis, Crohn's disease, colitis associated with collagen disease, intestinal Behcet's disease
  • IBD ulcerative colitis and Crohn's disease.
  • the present invention also provides a non-human animal cell having pluripotency, wherein the expression of the bach2 gene is artificially suppressed.
  • Such cells can be used for producing the non-human animal of the present invention.
  • examples of cells having the potential for modification of the bach2 gene that may be modified include ES cells, EC cells, bone marrow stem cells, and organ stem cells.
  • the genetically modified non-human animal of the present invention can be prepared by those skilled in the art by generally known genetic engineering techniques.
  • a genetically modified mouse can be produced as follows. First, DNA containing the exon portion of the mouse bach2 gene is isolated, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector.
  • the targeting vector is introduced into a mouse ES cell line by an electoral poration method or the like, and a cell line in which homologous recombination has occurred is selected.
  • an antibiotic resistance gene such as a neomycin resistance gene is preferable.
  • a cell line in which homologous recombination has occurred can be selected only by culturing in a medium containing an antibiotic.
  • a thymidine kinase gene or the like can be linked to a targeting vector.
  • a homologous recombinant can be assayed by PCR and Southern blot to efficiently obtain a cell line in which one of the bach2 gene pair is inactivated.
  • chimera When selecting a cell line in which homologous recombination has occurred, chimera can be produced using multiple clones, since there is a risk of unknown gene disruption due to gene insertion in addition to the homologous recombination site. Preferred ⁇ .
  • the obtained ES cell line is injected into mouse blastoderm to obtain a chimeric mouse.
  • a mouse in which one of the gene pair of the bach2 gene is inactivated can be obtained.
  • a mouse in which both gene pairs of the bach2 gene are inactivated.
  • the establishment of ES cells or ES-like cells in animal species other than mice has already been reported, and such animals can be genetically modified by the same method.
  • an ES cell line in which both the bach2 gene pair was inactivated was prepared by the following method. It is also possible to obtain more. That is, by culturing an ES cell line in which one of the gene pairs has been inactivated in a medium containing a high concentration of antibiotics, a cell line in which the other of the gene pairs has been inactivated, that is, the gene of the bach2 gene, An ES cell line in which both pairs are inactivated can be obtained. Alternatively, it can also be prepared by selecting an ES cell line in which one of the gene pairs has been inactivated, introducing a targeting vector again into this cell line, and selecting a cell line in which homologous recombination has occurred. It is preferable to use a marker gene to be inserted into the targeting vector, which is different from the above-mentioned marker gene.
  • the present invention also provides a method for producing an antibody (preferably IgM) or an antiserum containing the antibody using the non-human animal.
  • the method for producing an antibody of the present invention can be used for producing a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody (sometimes expressed as a humanized antibody), or a human antibody.
  • Methods for producing an antibody derived from an immunized animal such as a mouse or a gene thereof to produce a chimeric antibody between an immunized animal and a human or a humanized antibody are known.
  • a human antibody can be obtained by modifying the bach2 gene of a transgenic mouse in which the immune system has been replaced with a human immune system, and immunizing the resulting mouse.
  • the non-human animal in the present invention also includes a non-human animal capable of producing such a human antibody.
  • the present invention also provides tissues and cells derived from non-human animals, hybridomas of cells derived from non-human animals and myeloma cells, and the like.
  • the cell derived from the non-human animal of the present invention includes a cell line established from the non-human animal.
  • a known method can be used as a method for establishing the above-mentioned cell line derived from a non-human animal. For example, in rodents, it is possible to use the method of primary culture of fetal cells (Shinsei Kagaku Kenkyusho, Vol. 18, pages 125 to 129, Tokyo Chemical Dojin, and an operation manual for mouse embryos). 262-264, Modern Publishing).
  • Known methods can be used for the immunity up to the acquisition of the antibody. Immunization of an animal with an immunogen is performed according to a known method. As a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the immunogen
  • An appropriate amount of a normal adjuvant is mixed with an appropriate amount of a suspension diluted with PBS (Phosphate-Buffered Saline) or physiological saline, if necessary, emulsified, and then administered to the animal.
  • PBS Phosphate-Buffered Saline
  • physiological saline physiological saline
  • emulsified emulsified
  • a suitable carrier can be used when immunizing the immunogen. After immunization in this manner, an increase in the level of the desired antibody in the serum is confirmed by a conventional method.
  • the blood of the immunized animal is collected after confirming that the level of the desired antibody in the serum has increased. Then, the collected blood strength is separated from the serum by a known method.
  • a serum containing the polyclonal antibody may be used. If necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used, if necessary.
  • a fraction that recognizes only the target antigen is obtained, and this fraction is further separated into protein A and protein G.
  • IgG or IgM can be prepared.
  • a cell fusion method can be used.
  • mammalian myeloma cells are used as the other parent cell to be fused with the antibody-producing cell. More preferably, a myeloma cell having a special auxotrophy or drug resistance, which serves as a selection marker for a fusion cell (hybridoma), can be mentioned.
  • the antibody-producing cells and myeloma cells can be subjected to cell fusion basically according to a known method.
  • a method for producing a monoclonal antibody using cell fusion has been established, for example, by Milstein et al. (Galfre, G. and Milstein, C, Methods Enzymol. (1981) 73, 3-46).
  • Hybridomas obtained by cell fusion are selected by culturing in a selective culture solution.
  • the selection culture solution is selected according to the characteristics of the myeloma cells used for cell fusion, and the like.
  • a HAT culture solution a culture solution containing hypoxanthine, aminopterin and thymidine
  • the hybridoma is cultured in the HAT culture medium for a time sufficient for the death of cells (non-fused cells) other than the target and hybridomas.
  • selecting hybrididomas by continuing culture for several days to several weeks Can do.
  • a conventional limiting dilution method is performed, and screening and cloning of hybridomas producing the desired antibody are performed.
  • the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, and the monoclonal antibody can be recovered as ascites of the mouse. Ascites power can also purify monoclonal antibodies.
  • Ascites power can also purify monoclonal antibodies.
  • purification of the monoclonal antibody for example, ammonium sulfate precipitation, protein A, protein G columns, DEAE ion exchange chromatography, and an affinity column to which the target antigen is coupled can be used.
  • EBV Epstein-Barr virus
  • the thus obtained monoclonal antibody can also be a recombinant antibody produced by using a genetic recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
  • Recombinant antibodies are produced by cloning DNA encoding the antibodies into antibody-producing cells such as hybridomas or sensitized lymphocytes that produce the antibodies, incorporating the DNA into an appropriate vector, and introducing it into a host.
  • the present invention includes this recombinant antibody.
  • the antibody obtained by the method of the present invention may be an antibody fragment or a modified antibody thereof.
  • the antibody fragments, Fab, F (ab ') 2, Fv or single chain heavy and light chain Fv were ligated via a suitable linker FV (SC F V) (Huston , JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).
  • SC F V linker FV
  • an antibody fragment can be obtained by treating an antibody with an enzyme, for example, papain or pepsin to generate an antibody fragment.
  • a gene encoding these antibody fragments is constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co, MS et al,
  • modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • the “antibody” of the present invention also includes these modified antibodies.
  • Such a modified antibody can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.
  • a method for obtaining a human antibody is also known.
  • a target human antibody can be obtained by immunizing a transgenic animal having the entire repertoire of human antibody genes with a target antigen (International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO
  • the antibody obtained by the method of the present invention can be a chimeric antibody having a variable region derived from a non-human antibody derived from an immunized animal and a constant region derived from a human antibody.
  • a humanized antibody having CDR (complementarity determining region) of a non-human antibody derived from an immunized animal, FR (framework region) derived from a human antibody and a constant region can also be used.
  • a chimeric antibody is an antibody comprising the variable regions of the heavy and light chains of an antibody of an immunized animal and the constant regions of the heavy and light chains of a human antibody.
  • a DNA encoding the variable region of an antibody derived from an immunized animal is ligated to a DNA encoding the constant region of a human antibody, and the DNA is inserted into an expression vector and introduced into a host to produce a chimeric antibody. Can be done.
  • a humanized antibody is a modified antibody also called a reshaped human antibody.
  • a humanized antibody is constructed by transplanting the complementarity determining region (CDR) of an antibody derived from an immunized animal into the complementarity determining region of a human antibody. Its general gene recombination technique is also known.
  • the CDR of a mouse antibody and the framework region of a human antibody (framework region;
  • the DNA sequence designed to ligate FR is also synthesized by PCR using several oligonucleotides created to have overlapping portions at the ends.
  • the obtained DNA was ligated to DNA encoding the constant region of a human antibody, and then incorporated into an expression vector in A humanized antibody can be obtained by introducing this into a host and producing it (see European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96/02576).
  • Human antibody FRs linked via CDRs are selected so that the complementarity-determining regions form favorable antigen-binding sites.
  • amino acids in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site.
  • a gene encoding an antibody can be obtained from an antibody-producing cell of an immunized animal.
  • the method for obtaining the gene encoding the antibody is not limited.
  • a gene encoding an antibody can be obtained by amplifying the gene encoding a variable region or CDR using the gene encoding PCR by PCR. Primers for amplifying antibody genes by PCR are known.
  • the desired antibody can be produced by expressing the obtained gene using an appropriate expression system.
  • the gene obtained by the present invention is used for producing the various modified antibodies described above.
  • the antibody obtained as described above can be purified to a homogeneous immu- noglobulin molecule.
  • the purification method is not particularly limited.
  • the separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out
  • Dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. can be appropriately selected and combined to separate and purify immunoglobulin (Antibodies: A
  • the concentration of the antibody obtained above can be measured by measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), or the like.
  • Columns used for affinity chromatography include a protein A column and a protein G column.
  • columns using Protein A include Hyper D, POROS, Sepharose F.F. (Pharmacia) and the like.
  • chromatography other than the affinity chromatography examples include ion exchange chromatography, hydrophobic chromatography, gel filtration, and reverse phase chromatography. (Dean R. Marshak et al "Cold Spring Harbor Laboratory Press, 1996). Strategies for Protein Purification and Characterization: A Laboratory Course Manual. It can be performed using chromatography.
  • the present invention also provides uses of the DNA encoding Bach2. Specifically, the present invention provides a drug for treating or preventing inflammatory bowel disease and hyper-IgM syndrome, which contains a DNA encoding Bach2 as an active ingredient.
  • modified Bach2 such as Bach2 ⁇ C2 is considered to be an active form because it is constitutively present in the nucleus.
  • MAZR is a Bach2 binding factor and is highly expressed in B lymphocytes, and is expected to have the same function as Bach2.
  • Maf is also a partner of Bach2 dimer formation, and its homodimer binds to the same DNA sequence as Bach2 / Maf heterodimer.
  • a modified cDNA such as Bach2 ⁇ C2 (Hoshino, H “Kobayashi, A” Yoshida, M., Kudo, N., Oyake, T "Motohashi, H., Hayashi, N., Yamamoto, M., and Igarashi, K. Oxidative Stress Abolishes Leptomycin B—sensitive Nuclear Export of Transcription Repressor Bach2 that Counteracts Activation of Maf Recognition Element. J. Biol. Chem.
  • the form of the DNA in the drug of the present invention is not particularly limited, and may be genomic DNA, cDNA, synthetic DNA, or a vector containing such DNA.
  • the genomic DNA and cDNA are not particularly limited in the organism from which they are derived. When used for treatment or prevention of human diseases, it is preferably derived from mammals, most preferably from humans.
  • Bach2 Can be prepared, for example, from spleen, bone marrow, liver, and peripheral blood lymphocytes.
  • the present invention also provides use of DNA for suppressing the expression of Bach2.
  • the present invention provides an IgM increasing agent containing, as an active ingredient, DNA for suppressing the expression of Bach2.
  • DNA for suppressing the expression of Bach2 examples include DNA encoding antisense RNA against DNA encoding Bach2, DNA encoding RNA having ribozyme activity that specifically cleaves a transcript of DNA encoding Bach2, and Bach2.
  • DNA encoding RNA that suppresses the expression of the encoding DNA by the RNAi effect.
  • Antisense nucleic acids suppress target gene expression by inhibiting various processes such as transcription, splicing, and translation.
  • Hirashima and Inoue Shinsei Kagaku Kenkyusho 2 Nucleic acid IV gene replication and expression (Japanese (Edited by the Society, Doujin Kagaku) pp.319-347, 1993).
  • the design of an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of a gene is considered to be effective in inhibiting translation of a gene.
  • a sequence complementary to the coding region or the 3′-side untranslated region can also be used.
  • the DNA containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the gene is also included in the antisense DNA used in the present invention.
  • the sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene of the organism to be transformed or a part thereof, but it is completely complementary as long as gene expression can be effectively suppressed. It does not have to be a target.
  • the transcribed RNA has preferably 90% or more, and most preferably 95% or more complementarity to the transcript of the target gene.
  • the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more. .
  • the length of commonly used antisense DNA is shorter than 5 kb, preferably shorter than 2.5 kb.
  • Ribozyme refers to an RNA molecule having catalytic activity. Research focused on ribozymes as enzymes that cleave RNA, despite the existence of ribozymes having various activities, has enabled the design of ribozymes that cleave RNA in a site-specific manner. Ribozymes include Group I introns and Ml RNA contained in RNase P.
  • the self-cleaving domain of the hammerhead ribozyme is capable of cleaving the 3 'side of C15 in the sequence G13U14C15. Its activity is based on base pairing between U14 and A9. Alternatively, it has been shown that U15 can also be cleaved (Koizumi M, et al: FEBS Lett 228: 228, 1988).
  • Hairpin ribozymes are also useful for the purpose of the present invention.
  • This ribozyme is found, for example, on the minus strand of satellite RNA of tobacco ring spot virus (Buzayan JM: Nature 323: 349, 1986). It has been shown that a hairpin-type ribozyme can also generate a target-specific RNA cleavage ribozyme (Kikuchi Y & Sasaki N: Nucl Acids Res 19: 6751, 1991, Kikuchi Hiroshi: Chemistry and Biology 30: 112, 1992) .
  • a ribozyme designed to cleave a target is linked to an appropriate promoter and transcription termination sequence. At this time, if an extra sequence is added to the 5 'end or 3' end of the transcribed RNA, the activity of the ribozyme may be lost.In such a case, the RNA containing the transcribed ribozyme It is also possible to arrange another trimming ribozyme that acts in cis on the 5 'side or 3' side of the ribozyme portion in order to accurately cut out only the ribozyme portion from the protein (Taira K, et al: Protein Eng 3: 733, 1990, Dzianott AM & Bujarski JJ: Proc Natl Acad Sci USA 86: 4823, 1989, Grosshans CA & Cech TR: Nucl Acids Res 19: 3875, 1991, Taira K, et al: Nucl Acids Res 19: 5125 , 1991).
  • the transcript of the target gene in the present invention is characterized using ribozymes. Differential cleavage can suppress the expression of the gene.
  • RNAi RNA interference
  • Endogenous gene expression can also be suppressed by RNA interference (RNAi) using double-stranded RNA having a sequence identical or similar to the target gene sequence.
  • RNAi refers to a phenomenon in which expression of a target endogenous gene is suppressed when a double-stranded RNA having a sequence identical or similar to the target gene sequence is developed in a cell. Although the details of the mechanism of RNAi are not clear, it is thought that the target gene is degraded when the double-stranded RNA is broken down into small pieces and serves as an indicator of the target gene in some way.
  • the DNA of the present invention is prepared by inserting a suitable sequence (preferably an intron sequence) between inverted repeats of a target sequence to obtain a double-stranded RNA (having a hairpin structure).
  • a suitable sequence preferably an intron sequence
  • hpRNA self-complementary 'hairpin' RNA
  • the DNA used for RNAi need not be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity. Has the property.
  • sequence identity can be determined by the above-described method.
  • a viral vector such as a retrovirus, an adenovirus, or a Sendai virus, or a non-viral vector such as a ribosome can be used. Examples of the administration method include an in vivo method and an ex vivo method.
  • the DNA can also be administered as a drug formulated by a known pharmaceutical method.
  • it can be used in the form of a sterile solution with water or another pharmaceutically acceptable liquid, or a suspension for injection.
  • a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, an emulsifier, a suspending agent, a surfactant, a stabilizer, a vehicle, a preservative, and the like, It may be formulated by mixing it in the unit dosage form required for accepted pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • a sterile composition for injection can be formulated using a vehicle such as distilled water for injection according to normal pharmaceutical practice.
  • Aqueous solutions for injection include, for example, saline, Isotonic solutions containing dextrose and other adjuvants, such as D-sorbitol, D-mannose, D-mantol, sodium salt and sodium salt, and suitable solubilizers, such as alcohol, specifically It may be used in combination with ethanol, polyalcohols such as propylene glycol, polyethylene glycol, and nonionic surfactants such as polysorbate 80 TM and HCO-50.
  • the oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer such as a phosphate buffer and a sodium acetate buffer, a soothing agent such as proforce hydrochloride, and a stabilizer such as benzyl alcohol, phenol and an antioxidant.
  • a buffer such as a phosphate buffer and a sodium acetate buffer
  • a soothing agent such as proforce hydrochloride
  • a stabilizer such as benzyl alcohol, phenol and an antioxidant.
  • the prepared injection solution is usually filled in an appropriate ampoule.
  • the dose of the drug of the present invention can be appropriately selected depending on the age and symptoms of the patient.
  • the present invention provides a method for screening a compound that substitutes for the function of Bach2.
  • a test compound is administered to the non-human animal of the present invention.
  • the test compound used in the screening method of the present invention for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, a compound library, an expression product of a gene library, and cell extraction Products, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, and the like.
  • Administration of the test compound to the non-human animal can be performed, for example, orally or parenterally.
  • test compound is a protein
  • a viral vector having a gene encoding the protein and use the infectivity to introduce the gene into the non-human animal of the present invention. It is possible.
  • the phenotype of the non-human animal is analyzed. Phenotypes include, but are not limited to, inflammatory bowel disease, hyper-IgM syndrome, stool IgA levels, serum antibody titers, and infectious diseases (such as pneumonia). Analysis of inflammatory bowel disease and hyper-IgM syndrome can be performed by the method described in Examples. Next, a compound that complements the phenotype of the non-human animal is selected in comparison with the case where the test compound is not administered.
  • the present invention also provides a screening method using cells of a genetically modified non-human animal.
  • B lymphocytes are isolated from the original spleen cells and stimulated with LPS to induce an antibody class switch.
  • a compound that complements the phenotype of the non-human animal cell is selected as compared with the case where the test compound is not administered.
  • the isolated compound can be used as an agent for treating or preventing inflammatory bowel disease and hyper-IgM syndrome.
  • the present invention also provides a method for screening for a Bach2 regulator.
  • the first embodiment of the method for screening a Bach2 controlling agent of the present invention relates to screening for a compound binding to Bach2.
  • a test compound is brought into contact with Bach2.
  • the binding between the Bach2 and the test compound is detected.
  • a test compound that binds to the Bach2 is selected.
  • the isolated compound can be used as an agent for treating or preventing inflammatory bowel disease and Neuper IgM syndrome, or as an agent for increasing IgM. Further, it is used as a test compound in the below-described screening method for IJ IJ.
  • a method for screening a polypeptide binding to Bach2 using Bach2 many methods known to those skilled in the art can be used. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows.
  • the gene encoding Bach2 is expressed in animal cells or the like by inserting it into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, or pCD8.
  • the promoter used for expression is the SV40 early promoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF- 1 promoter (Kim et al.
  • CAG promoter Niwa et al. Gene 108, p.193-200 (1991)
  • RSV LTR promoter Cullen Methods in Enzymology 152, p. 684-704 (1987), SR a promoter (Takebe et al. Mol. Cell. Biol. 8, p.466 (1988)), CMV immediate early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84, p.3365—3369 (1987), SV40 late promoter (Gheysen and Fiers J. Mol. Appl. Genet.
  • the expression of Bach2 as a fusion protein having the recognition site of the monoclonal antibody was clarified by the specificity. Can be done.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 13, 85-90 (1995)). Vectors capable of expressing a fusion protein with 13 galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP), and the like via a multicloning site are commercially available.
  • polyhistidine His-tag
  • influenza agglutinin HA human C-myc
  • FLAG Vesicular stomatitis virus glycoprotein
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7-tag human simple virus Epitopes such as viral glycoproteins (HSV-tag) and E-tag (epitopes on monoclonal phages) and monoclonal antibodies that recognize them are used as an epitope-antibody system for screening proteins that bind to Bach2 (Experimental Medicine 13, 85-90 (1995)).
  • these antibodies are added to a cell lysate prepared using an appropriate surfactant to form an immunocomplex.
  • This immune complex is Bach2, a protein capable of binding to it, and antibody.
  • immunoprecipitation using an antibody against Bach2 can also be performed.
  • Antibodies to Bach2 can be obtained, for example, by transforming the DNA encoding Bach2 into an appropriate E. coli expression vector.
  • the protein can be prepared by purifying the expressed protein in Escherichia coli, immunizing it with mouse egrets, mice, rats, goats, and birds. Alternatively, it can be prepared by immunizing the above animal with the synthesized partial peptide of Bach2.
  • the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose.
  • Bach2 is prepared as a fusion protein with, for example, an epitope such as GST, daltathione
  • a substance that specifically binds to these epitopes can be used to form an immune complex in the same manner as when Bach2 antibody is used.
  • SDS-PAGE is generally used for the analysis of immunoprecipitated proteins.
  • proteins can be analyzed by binding according to the molecular weight of the proteins.
  • the target protein can be directly purified from the SDS-polyacrylamide gel and sequenced.
  • a method for isolating a protein binding to Bach2 using Bach2 for example, the method of Skolnik et al. (Skolnik, EY et al. 'Cell (1991) 65, 83-90) Can be used. That is, a cDNA library using a phage vector (gtl, ZAP, etc.) is prepared from cells and tissues that are expected to express a protein that binds to Bach2, and expressed on LB-agarose. The protein expressed on the filter is immobilized, purified and labeled Bach2 is reacted with the filter, and the plaque expressing the protein bound to Bach2 is detected by the label.
  • Skolnik et al. Skolnik, EY et al. 'Cell (1991) 65, 83-90
  • a cDNA library using a phage vector gtl, ZAP, etc.
  • the protein expressed on the filter is immobilized, purified and labeled Bach2 is reacted with the filter, and the plaque
  • a method for labeling Bach2 a method utilizing the binding property of biotin and avidin, Bach2 or a protein fused to Bach2 (e.g., For example, a method using an antibody that specifically binds to GST), a method using a radioisotope, a method using fluorescence, and the like.
  • the screening method of the present invention includes a 2-neubrid system using cells (Fields, S "and Sternglanz, R., Trends. Genet. (1994) 10, 286-292, Dalton S, and Treisman R (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c- fos serum response element.Cell 68, 597-612, ⁇
  • MATCHMARKER Two-Hybrid System "Mammalian MATCHMAKER Two-Hybrid Assay Kit", “MATCHMAKER One-Hybrid SystemJ” (all manufactured by Clontech), "HybriZAP Two-Hybrid Vector System” (Stratagene) There are methods.
  • BB2 or its partial peptide is fused with the SRF DNA binding region or GAL4 DNA binding region and expressed in yeast cells, and the protein that binds to Bach2 is expressed.
  • a cDNA library was prepared from the cells that are expected to be expressed, and expressed in a form fused with the VP16 or GAL4 transcription activation region, and introduced into the yeast cells described above. Isolate the cDNA from the library (if the protein that binds to Bach2 is expressed in yeast cells, the binding of both will activate the reporter gene and confirm a positive clone).
  • the protein encoded by the cDNA can be obtained by introducing the isolated cDNA into E. coli and expressing it. This makes it possible to prepare a protein that binds to Bach2 or its gene.
  • Reporter genes used in the two-hybrid system include, for example,
  • HIS3 gene Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene and the like are not limited thereto. Screening by the two-hybrid method can also be performed using mammalian cells and the like in addition to yeast.
  • Screening for a compound that binds to Bach2 can also be performed using affinity chromatography.
  • Bach2 is immobilized on a carrier of an affinity column, and a test compound expected to express a protein that binds to Bach2 is applied thereto.
  • examples of the test compound include a cell extract and a cell lysate. Examination After applying the compound, the column can be washed and the protein bound to Bach2 can be prepared.
  • the obtained protein is analyzed for its amino acid sequence, an oligo DNA is synthesized based on the amino acid sequence, and a cDNA library is screened using the DNA as a probe to obtain a DNA encoding the protein. be able to.
  • a method for isolating not only proteins but also compounds that bind to Bach2 includes, for example, reacting immobilized Bach2 with a synthetic compound, a natural product bank, or a random phage peptide display library. , Bach2 binding molecules, and high-throughput screening methods using combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ., Small peptides as potent mimetics of the protein hormone erythropoietin, science (UNITED STATES) Jul 26 1996, 273 p458--64, Verdine GL., The comDinatorial chemistry of nature.Nature (ENGLAND) Nov 7 1996, 384 pi 1-13, Hogan JC Jr., Directed combinatorial chemistry. Nature (ENGLAND) Nov 7 1996, 384 pl7-9) are known to those skilled in the
  • a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound compound.
  • Biosensors using surface plasmon resonance phenomena require real-time observation of the interaction between Bach2 and the test compound as a surface plasmon resonance signal using a small amount of protein without labeling. (Eg, BIAcore, manufactured by Pharmacia).
  • the second embodiment of the method for screening a Bach2 regulator in the present invention relates to screening for a compound that regulates (increases or decreases) the activity of Bach2.
  • a test compound is brought into contact with Bach2.
  • the state of Bach2 used in the second embodiment is not particularly limited, and may be, for example, a purified state, a state expressed in a cell, a state expressed in a cell extract, or the like.
  • Cells expressing Bach2 include cells expressing endogenous Bach2 or cells expressing exogenous Bach2.
  • the cells expressing endogenous Bach2 include, but are not limited to, cultured cells.
  • the cells expressing the exogenous Bach2 can be prepared, for example, by introducing a vector containing DNA encoding Bach2 into the cells. Introduction of the vector into the cell can be performed by a method common to those skilled in the art.
  • the species from which the cell into which such exogenous Bach2 is introduced is not particularly limited, and may be any species for which a technique for expressing a foreign protein in cells has been established! ,.
  • Examples of the cell extract in which Bach2 is expressed include, for example, a cell extract contained in an in vitro transcription / translation system obtained by adding a vector containing DNA encoding Bach2 to a cell extract. it can.
  • As the in vitro transcription / translation system it is possible to use a commercially available in vitro transcription / translation kit, which is not particularly limited.
  • “contact” is performed according to the state of Bach2.
  • Bach2 when Bach2 is in a purified state, it can be performed by adding a test compound to a purified sample.
  • the expression can be carried out by adding a test compound to the cell culture or the cell extract, as long as it is expressed in the cell or in the cell extract.
  • the test compound is a protein
  • a vector containing a DNA encoding the protein is introduced into a cell expressing Bach2, or the vector is added to a cell extract expressing Bach2. It is also possible to do this.
  • a two-hybrid method using yeast or animal cells can be used.
  • the activity of Bach2 is measured as follows.
  • Bach2 activity includes transcription activity and DNA binding activity.
  • the transcription activity can be measured by, for example, a reporter assay.
  • a reporter assay By introducing a reporter gene having a Bach2 binding sequence into a cell, the effect of the test compound on its expression level can be measured.
  • the DNA binding activity can be measured, for example, by gel shift assay.
  • a DNA fragment having a Bach2 binding sequence is mixed with a cell extract, and a Bach2 / DNA complex is detected by electrophoresis.
  • the test compound is added to the cells or to the cell extract, and the effect on complex formation can be measured.
  • a test compound is administered in the following step, and a compound that increases or decreases the activity of Bach2 is selected as compared to the case where no test compound is administered.
  • Compounds that increase the activity of Bach2 are useful for treating or preventing inflammatory bowel disease and hyper-IgM syndrome. Available as a drug.
  • Compounds that decrease the activity of Bach2 can also be used as agents for increasing IgM.
  • the third embodiment of the method for screening a Bach2 regulator in the present invention relates to screening for a compound that regulates the expression level of the bach2 gene.
  • a cell or a cell extract having DNA in which a reporter gene is functionally linked downstream of the promoter region of the bach2 gene is provided.
  • “functionally linked” means that the expression of the reporter gene is induced by binding of the transcription factor to the promoter region of the bach2 gene. And are combined. Therefore, even when the reporter gene is linked to another gene and forms a fusion protein with another gene product, the binding of the transcription factor to the promoter region of the bach2 gene causes the fusion protein to bind. As long as expression of is induced, it is included in the meaning of “functionally linked”.
  • the promoter region of the bach2 gene can be extracted by comparing the bach2 cDNA sequence with the bach2 genomic sequence on a database.
  • the reporter gene is not particularly limited as long as its expression is detectable, and examples thereof include a CAT gene, a lacZ gene, a luciferase gene, a / 3-Darc port commonly used by those skilled in the art. -Dase gene (GUS) and GFP gene.
  • the reporter gene also includes a DNA encoding Bach2.
  • a test compound is brought into contact with the cells or the cell extract in the following steps: Next, the expression level of the reporter gene in the cell or the cell extract is measured.
  • the expression level of the reporter gene can be measured by a method known to those skilled in the art according to the type of the reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphene by the gene product.
  • Reporter gene power In the case of the cZ gene, a fluorescent compound is detected by detecting the color development of a dye compound catalyzed by the gene expression product, and in the case of a luciferase gene, the fluorescent compound is catalyzed by the gene expression product
  • the fluorescence Upon detection, and in the case of the j8-Darc mouth-dase gene (GUS), the luminescence of Glucuron (ICN) and the 5-bromo-4-chloro-
  • the expression level of the reporter gene can be reduced by detecting the color development of 3-indolyl-j8-Dark mouth (X-Gluc) and, if it is a GFP gene, by detecting the fluorescence of the GFP protein. Can be measured.
  • the expression level of the gene can be measured by a method known to those skilled in the art. For example, it is possible to measure the transcription level of the gene by extracting the mRNA of the gene according to a standard method and performing a Northern hybridization method or RT-PCR method using the mRNA as a type III. it can. Furthermore, the expression level of the gene can be measured using DNA array technology.
  • the translation level of a gene can also be measured by collecting a fraction containing Bach2 according to a standard method and detecting the expression of Bach2 by electrophoresis such as SDS-PAGE.
  • the translation level of the gene can be measured by detecting the expression of Bach2 by performing a Western blotting method or the like using an antibody against Bach2.
  • a test compound is administered, and a compound that increases or decreases the expression level of the reporter gene is selected as compared to the case where no test compound is administered.
  • a compound that increases the expression level of a reporter gene can be used as an agent for treating or preventing inflammatory bowel disease and hyper-IgM syndrome.
  • compounds that decrease the level of reporter gene expression can be used as an agent for increasing IgM.
  • the fourth embodiment of the method of screening for a Bach2 regulator in the present invention relates to screening for a compound that regulates transcription by Bach2.
  • the Bach2 transcription control region (Oyake, T. et al., Mol. Cell. Biol. (1996) 16, 6083-6095; Muto, A. et al, EMBO J. (1998) ) 17, 5734-5743; Hoshino, H. et al, J. Biol. Chem. (2000) 275, 15370-15376; Muto, A. et al "J. Biol. Chem. (2002) 277,
  • the Bach2 transcription control region includes the globin gene, the NF-E2 binding sequence of the antibody, and the MARE (maf recognition) of the antibody heavy chain gene. element) can be used.
  • a test compound is brought into contact with the cell or the cell extract in the presence of Bach2 in the next step.
  • a test compound can be contacted with cells or cell extracts expressing Bach2 and a reporter.
  • the expression level of the reporter gene in the cell or the cell extract is measured in the following manner.
  • a compound that increases or decreases the expression level of the reporter gene as compared to the case where the test compound is not administered is selected.
  • Compounds that increase the level of reporter gene expression can be used as agents for treating or preventing inflammatory bowel disease and hyper-IgM syndrome.
  • Compounds that reduce the level of reporter gene expression can also be used as agents for increasing IgM.
  • the present invention provides a method for testing inflammatory bowel disease and Nipper IgM syndrome, comprising a step of measuring the expression level of the bach2 gene.
  • the expression of the bach2 gene also includes the expression of the Bach2 protein as well as the expression of the bach2 mRNA.
  • the expression of the bach2 gene is reduced by the test method, it is determined that the patient has already suffered from inflammatory bowel disease and hyper-IgM syndrome, or is susceptible to the disease.
  • RNA sample of a subject is prepared.
  • the RNA sample can be extracted, for example, from a subject's spleen, peripheral blood lymphocytes, or intestinal biopsy material.
  • the amount of RNA encoding Bach2 contained in the RNA sample is measured in the following steps.
  • the measured amount of RNA is then compared to a control.
  • Examples of such a method include a Northern blotting method, a DNA array method, and an RT-PCR method.
  • the above-described test method can be carried out as follows by measuring the expression level of Bach2.
  • a protein sample is prepared from the subject.
  • the protein sample can be prepared, for example, from the subject's spleen, peripheral blood lymphocytes, and intestinal biopsy material.
  • the amount of Bach2 contained in the protein sample is measured in the following.
  • the measured amount of Bach2 is then compared to a control.
  • Such methods include SDS Polya Examples thereof include acrylamide electrophoresis, and Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence using antibodies that bind to Bach2.
  • the bach2 knockout mouse is used for inflammatory bowel disease and dyspepsia.
  • the present invention provides a method for detecting inflammatory bowel disease and Neuper IgM syndrome, comprising a step of detecting a mutation in the bach2 gene region. According to the test method, when a mutation occurs in the bach2 gene region, it is determined that inflammatory bowel disease and hyper-IgM syndrome are already affected, or are likely to be affected!
  • the bach2 gene region means the bach2 gene and a region that affects the expression of the gene.
  • the region that affects the expression of the gene is not particularly limited, and examples thereof include a promoter region.
  • examples of the type of mutation in the present invention include, but are not limited to, deletion, substitution, and insertion mutation.
  • test method including a step of detecting a mutation generated in the bach2 gene region
  • method of the present invention is not limited to these methods.
  • a DNA sample is prepared from a subject.
  • the DNA sample can be prepared, for example, based on chromosomal DNA or RNA extracted from a subject's spleen, peripheral blood lymphocytes, or intestinal biopsy.
  • the DNA containing the bach2 gene region is isolated in the next step !.
  • the gene region can be isolated by, for example, PCR using chromosomal DNA or RNA as a type II, using a primer that hybridizes to DNA containing the gene region.
  • the base sequence of the isolated DNA is determined.
  • the nucleotide sequence of the isolated DNA can be determined by a method known to those skilled in the art.
  • the determined nucleotide sequence of the DNA is then compared with a control.
  • a control refers to a DNA containing a normal (wild-type) bach2 gene region.
  • the sequence of the DNA containing the bach2 gene region of a healthy person is considered to be normal.
  • the expression "compare” usually means to compare with the sequence of DNA containing the bach2 gene region of a healthy person.
  • the mutation in the present invention can also be detected by the following method.
  • a DNA sample is prepared from a subject.
  • the prepared DNA sample is cut with a restriction enzyme.
  • the DNA fragments are separated according to their size.
  • the size of the detected DNA fragment is then compared to a control.
  • a DNA sample is prepared from a subject.
  • DNA containing the bach2 gene region is amplified.
  • the amplified DNA is cut with a restriction enzyme.
  • the DNA fragments are separated according to their size.
  • the size of the detected DNA fragment is then compared to a control.
  • Such a method includes, for example, a restriction fragment length variation (Restriction Fragment Length).
  • a DNA sample is prepared from a subject. Then
  • Amplify DNA containing bach2 gene region Furthermore, the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel. Compare the mobility of the separated single-stranded DNA on the gel with the control.
  • the method for example,
  • PCR--SSCP (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.Genomics. 1992 Jan 1; 12 (1) : 139—146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products.
  • a DNA sample is prepared from a subject. Then
  • Amplify DNA containing bach2 gene region is separated on a gel with increasing concentrations of DNA denaturant. The mobility of the separated DNA on the gel is then compared to a control.
  • Such methods include, for example, denaturant gradient gels (denaturant gradient gel electrophoresis (DGGE method) and the like.
  • a DNA containing the bach2 gene region prepared from a subject and a substrate on which a nucleotide probe that hybridizes to the DNA is immobilized are provided.
  • substrate means a plate-like material to which nucleotide probes can be immobilized.
  • nucleotides include oligonucleotides and polynucleotides.
  • the substrate of the present invention is not particularly limited as long as nucleotide probes can be immobilized, but a substrate generally used in DNA array technology can be suitably used.
  • DNA arrays are composed of thousands of nucleotides printed on a substrate at high density. Usually these DNAs are printed on the surface of a non-porous substrate.
  • a porous membrane typically a glass, can be used, for example a -trocellulose membrane.
  • examples of the method for immobilizing (array) nucleotides include oligonucleotide-based arrays developed by Aifymetrix.
  • the oligonucleotides are usually synthesized in situ.
  • photolithographic technology Aifymetrix
  • ink-jet Rasetta Inpharmatics
  • the nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect a mutation in the bach2 gene region. That is, the probe is, for example, a probe that hybridizes to DNA containing the bach2 gene region. If specific hybridization is possible, the nucleotide probe need not be completely complementary to the DNA containing the gene region.
  • the length of the nucleotide probe to be bound to the substrate when the oligonucleotide is immobilized is usually 10 to 100 bp, preferably 10 to 50 bp, and more preferably 15 to 25 bp. .
  • the substrate is brought into contact with the DNA containing the bach2 gene region in the next step.
  • DNA is hybridized with the nucleotide probe.
  • the reaction solution and reaction conditions for the hybridization and the reaction can vary depending on various factors such as the length of the nucleotide probe fixed to the substrate, but are generally performed by a method well known to those skilled in the art. I can.
  • the intensity of hybridization between the DNA containing the bach2 gene region and the nucleotide probe immobilized on the substrate is detected in the next step.
  • This detection can be performed, for example, by reading the fluorescent signal with a scanner or the like.
  • DNA array generally, DNA fixed on a slide glass is called a probe, and labeled DNA in a solution is called a target. Therefore, the above nucleotide immobilized on the substrate is referred to herein as a nucleotide probe.
  • the detected intensity of inbrids is compared with that of a control. Examples of such a method include a DNA array method (SNP gene mutation strategy, Kenichi Matsubara, Yoshiyuki Sakaki, Nakayama Shoten, pl28-135, Nature Genetics (1999) 22: 164-167) and the like.
  • an Allele Specific Oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position.
  • ASO Allele Specific Oligonucleotide
  • the MALDI-TOF / MS method (SNP polymorphism strategy, Kenichi Matsubara 'Yoshiyuki Sakaki, Nakayama Shoten, pl06-117, Trends Biotechnol (2000): 18: 77-84), TaqMan PCR method (SNP genetic polymorphism strategy, Kenichi Matsubara 'Yoshiyuki Sakaki, Nakayama Shoten, p94-105, Genet Anal. (1999) 14: 143-149), Invader method (SNP genetic polymorphism strategy, Kenichi Matsubara. Yoshiyuki Sakaki, Nakayama Shoten, p94-105, Genome Research (2000) 10: 330-343), Pyrosequencing method (Anal.
  • the present invention further provides a test agent for use in the test method of the present invention.
  • the test agent comprises an oligonucleotide that hybridizes to the bach2 gene region and has a chain length of at least 15 nucleotides.
  • the oligonucleotide specifically hybridizes to DNA (normal DNA or mutant DNA) containing the bach2 gene region.
  • “specifically hybridizes” means under ordinary hybridization conditions, preferably under stringent hybridization conditions (e.g., Sambu Norec et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, New York, USA , 2nd edition, 1989), which means that the DNA encoding other proteins does not significantly cause crossover and hybridization. If specific hybridization is possible, the oligonucleotide need not be perfectly complementary to the DNA containing the bach2 gene region!
  • Oligonucleotides that hybridize to DNA containing the bach2 gene region and have a chain length of at least 15 nucleotides are used as probes (including a substrate to which the probe is immobilized) and primers in the above-described test method of the present invention. be able to.
  • the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
  • the primer is not particularly limited as long as it can amplify at least a part of the bach2 gene region including the mutant portion.
  • the probe is not particularly limited as long as it specifically hybridizes to DNA containing the bach2 gene region.
  • the probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp or more.
  • the oligonucleotide of the present invention can be produced by, for example, a commercially available oligonucleotide synthesizer.
  • the probe can be prepared as a double-stranded DNA fragment obtained by treatment with a restriction enzyme or the like.
  • oligonucleotide of the present invention When the oligonucleotide of the present invention is used as a probe, it is preferable to appropriately label and use it. Labeling may be performed by using T4 polynucleotide kinase to label the 5 'end of the oligonucleotide by phosphorylation with 32 P, or by using a DNA polymerase such as a tarenow enzyme to form a random hexamer oligonucleotide. A method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin using a nucleotide or the like as a primer (random prime method) can be exemplified.
  • test agent of the present invention is a test reagent containing an antibody that binds to Bach2.
  • the antibody is not particularly limited as long as it can be used for the test.
  • the antibodies described in Examples can be used.
  • the antibody is labeled if necessary.
  • Frozen sections of the spleen were subjected to FITC-conjugated anti-mouse IgM, biotin-conjugated anti-mouse IgM, biotin-conjugated anti-mouse CD3 ⁇ (145-2C11), and purified anti-mouse MadCAM-1 (MECA-367) antibody (BD PharMingen) was used. Cy3-conjugated goat anti-rat HgG (ROCKLAND) and Cy3-conjugated avidin were used as secondary antibodies.
  • RBC-depleted splenocytes are cultured for 2-4 days with 20 ⁇ g / ml LPS, and site-spun on glass slides, anti-Bach2 antiserum (F69-2), PE-conjugated anti-mouse IgM (BD Pharmingen) ), Fey fragment-specific Cy3-conjugated anti-mouse IgG (subclass 1 + 2a + 2b + 3) (Jackson ImmunoResearch Lab.) And APC-conjugated anti-B220 (BD Pharmingen) Stained. Alexa Fluor 488 goat anti-Peagle IgG (Molecular Probes) was used as a secondary antibody. Nuclei were stained with 10 M Hoechst 33342. Imaging was performed by a known method (Muto, A. et al, J. Biol. Chem. (2002) 277, 20724-20733).
  • the Bach2 targeting vector (pB2TV) is a ⁇ -galatatosidase (LacZ) gene containing the SV40 polyadenylation (poly (A)) signal derived from the pCMV ⁇ vector (Clontech). It was constructed using a DNA fragment containing the gene and a neomycin resistance gene (neo) driven by a PGK promoter (PGK-neo) flanked by ⁇ sequences on both sides. These force sets were clawed between short and long arm DNA.
  • cDNA containing diphtheria toxin A (DTA), which does not contain the poly (A) signal and is driven by the PGK promoter (PGK-DTA) was subcloned beyond the short arm to complete the construct ( See figure la).
  • PGK-DTA PGK promoter
  • FACS analysis included mouse CD45R / B220, CD43 (S7), IgM (R6- 60.2), IgD, TER-119, Ly-6G / Gr-1, CD 1 lb / Mac-1, CD4, CD8, CD21, and The measurement was performed using an antibody against CD23 (BD Pharmingen). Cells were analyzed using a FACScalibur equipped with Cell Quest software (Becton Dickinson).
  • Immunoglobulin concentrations were determined using ⁇ -nitrophenyl phosphatase (Chronotyping System / AP; Southern Biotechnology Association) as a substrate for antibodies specific for each mouse Ig isotype and for alkaline phosphatase-conjugated secondary antibodies. AP; Southern Biotechnology Associates).
  • NP-Ficoll 100 ⁇ g of DNP-Ficoll (Biosearch Technologies) or 100 ⁇ g of NP- ⁇ avian gamma globulin (NP-CGG, Biosearch Technologies) mixed with alum in mice (7-12 weeks old) was immunized intraperitoneally with a phosphate buffered saline solution (PBS; Nissi).
  • PBS phosphate buffered saline solution
  • Anti-DNP and anti-NP antibody levels were measured using DNP-PB serum albumin (BSA) and NP-BSA (Biosearch Determined by an ELISA method using G.S. Somatic cell hypermutation was performed by a known method (Muramatsu, M. et al., Cell (2000) 102, 553-563).
  • RBC-dead spleen cells (1 ⁇ 10 5 cells) were cultured in a 96-well plate in a total volume of 200 ⁇ l RPMI medium.
  • Cells were harvested using 20 ⁇ g / ml LPS (0111: B4; Sigma), 10 ng / ml recombinant mouse IL-4 (BD Pharmingen), and lng / ml recombinant human TGF-j81 (R & R & D systems) in the indicated combinations.
  • Spleen B cell proliferation was measured using a cell proliferation ELISA, BrdU kit (Roche).
  • RNA preparation and cDNA synthesis were performed by known methods (Muto, A. et al., EMBO J. (1998) 17, 5734-5743). O Sequences of PCR primers are available upon request.
  • the retrovirus was prepared by a known method (Muto, A. et al., J. Biol. Chem. (2002) 277, 20724-20733). O Prior to infection, purified spleen B cells were collected at 20 ⁇ g / ml. It was preliminarily activated with LPS and 10 ng / ml IL-4 for 2 days. The cells, which had been activated beforehand, were suspended at a density of 5 ⁇ 10 5 cells / ml in a medium containing a retrovirus supplemented with 16 ⁇ g / ml polybrene, and 927 g at 90 ° C. at 32 ° C. After centrifugation for 48 minutes, incubation was performed for 48 hours. The cells were then washed and incubated for 24 hours. Viable cells were selected based on cell size and providide iodide staining (PI) for analysis of surface IgM expression using FACS.
  • PI providide iodide stain
  • Antigen-dependent terminal differentiation of B cells is determined by the spleen and lymph nodes (Kinoshita, K. & Honjo, T., Nat. Rev. Mol. Cell. Biol. (2001) 2, 493-503; Honjo, T., Kinoshita , K. &
  • the present inventors performed immunohistochemical study of Bach2 in mouse spleen.
  • Bach2 is expressed on IgM-positive cells in lymphoid follicles surrounded by marginal sinus-expressing mucosal-directed cell adhesion molecule-1 (MadCAM-1, FIG. 2a).
  • Marginal zone B cells that are IgM positive and that are outside MadCAM-1 expressing cells are Bach2 negative. I got it.
  • Bach2 was not detected in CD3 ⁇ -positive ⁇ cells in the T cell area.
  • mice were less than 30% of control littermates.
  • Immature B cells characterized as IgM high IgD_ / 1 ° w , were present at normal levels in bach2 _ / _ mice, whereas mature B cells (B220 +, IgMlQW and IgD +) _ / _ was significantly reduced in mice (Fig. 2b and d).
  • bach2 + / + and Bach2 absolute number of spleen _ mice were respectively 3.1 X 10 8 cells ⁇ 0.7 and 2.1 X 10 8 ⁇ 0.4.
  • IgM levels were 5 times higher in bach2 _ / _ mice than in wild-type mice.
  • mean IgGl, IgG2a, IgG2b, IgG3, and IgA concentrations were reduced by 31%, 5%, 33%, 4%, and 25%, respectively, in bach2 _ / _ mice compared to wild-type littermates. did.
  • This pattern of changes in serum immunoglobulin isotypes is similar to that of the hyper-IgM syndrome associated with immunodeficiency (Durandy, A. & Honjo, T., Curr. Opin.
  • bach2 _ / _ mice were challenged with T cell-independent antigens (DNP-binding ficoll, DNP-ficoll). Immunized. Pre-immune sera from bach2 _ / _ mice contained higher levels of NP-binding IgM antibodies than control mice, presumably due to higher levels of cross-reactive antibodies ( Figure 3b). After immunization with DNP-ficol, DNP-bound IgM antibodies increased in wild-type mice, but remained similarly high in bach2 _ / _ mice. The bach2 + / + mouse showed an increase in the specific IgG3 antibody response.
  • the bach _ / _ mouse showed no increase in the antibody response (FIG. 3b).
  • the T-cell dependent antibody response was examined by immunizing with a T-cell dependent antigen (NP-bound-Avian y-globulin, NP-CGG).
  • NP-bound-Avian y-globulin, NP-CGG T-cell dependent antigen
  • the bach2 + / + mice showed strong production of IgGl, but the bach2 _ / _ mice did not (Fig. 3c).
  • B cells undergo somatic hypermutation (SHM) and produce high-affinity antibodies against specific antigens in germinal centers (GCs) of peripheral lymphoid tissues (Honjo, T., Kinoshita, K & Muramatsu, M., Annu. Rev. Immunol. (2002) 20, 165-196; Papavasiliou, F. & Schatz, D., Cell (2002) 109, S35-44).
  • SHM somatic hypermutation
  • RNAs can also be extracted from NP-CGG immunized Bach2 _ / _ mice and wild-type littermates to extract the mesenchymal lymph node (MLN) power, synthesize cDNA, and stake-trowel
  • MNN mesenchymal lymph node
  • the VH186.2 exon sequence which has been well characterized as a variable region encoding a hapten, was determined (Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. & Cumano, A., EMBO J. (1989) 7, 1995-2001; Jacob, J., Kelsoe, G "Rajewsky, K.
  • CSR specificity by inducing a specific class of gene transcripts (germline IgH constant region gene transcripts, GLTs). After completion of the CSR, a post-switch transcript (PST) containing the intervention (I) and each of the switched C exons is expressed. like this
  • the PST is a proof of a switched locus (Snapper, C, Marcu, K. & Zelazowski, P., Immunity (1997) 6, 217-223; Kinoshita, K. & Honjo, T "). Nat. Rev. Mol. Cell. Biol. (2001) 2, 493-503; Manis, J., Tian, M. & Alt, F., Trends. Immunol. (2002) 23, 31-39; Honjo, T. ., Kinoshita, K. & Muramatsu, M., Annu. Rev. Immunol. (2002) 20, 165-196).
  • GLT and PST in B cells stimulated by LPS and cytoforce by RT-PCR.
  • ⁇ 2b, ⁇ 3, and a GLTs levels in bach2 _ / _ B cells were comparable to those of wild-type B cells ( Figure 4b).
  • ⁇ 1 GLT was reduced in bach2 B cells.
  • chromatin activation in the switch region is hardly affected in the absence of Bach2 except for ⁇ 1 GLT. Since Bach2 binds to the 3 ′ locus control region of the IgH gene (Muto, A. et al., EMBO J. (1998) 17, 5734-5743), changes in the expression of y1 GLT May be due to a lack of enhancer function that is particularly necessary for PSTs (Pinaud, E.
  • Bach2 may regulate the expression of genes related to CSR and SHM. This possibility was examined by comparing the expression of genes involved in plasma cell development. Using splenic B cells stimulated by LPS, the present inventors observed normal induction of transcription factors that have been reported to promote the final division of B cells into plasma cells (XBP- 1 (Reimold, A. et al., Nature (2001) 412, 300-307), Blimp-1 (Shaffer, A. et al., Immunity (2002) 17, 51-62), and IRF-4 (Mittrucker , H. et al "Science (1997) 275, 540-543), Figure 4e), suggesting that Bach2 regulates plasma cell development independently of these factors.
  • XBP- 1 Reimold, A. et al., Nature (2001) 412, 300-307
  • Blimp-1 Shaffer, A. et al., Immunity (2002) 17, 51-62
  • IRF-4 Mitsubishi , H. et al "Science (1997) 275, 540-543
  • 5216-5224 may be a substitute for Bach2 because it is expressed in B cells (Fig. 4e).
  • Bach2 is a nuclear emitter Crml / exportin 1 (Hoshino, H. et al "J. Biol. Chem. (2000) 275, 15370-15376; Muto, A. et al” J. Biol. Chem. (2002) 277, 20724-20733), we examined the subcellular localization of Bach2 in LPS-induced plasma cells by staining for Bach2, as regulated by nuclear force excretion in a dependent manner. Plasma cells were identified by strong staining of IgM or IgG in the cytoplasm. As shown in FIGS.
  • IgG plasma cells showed staining of Bach2 in the nucleus or extensively (ie, nucleus and cytoplasm). In contrast, significant parts of IgM plasma cells Minutes showed only cytoplasmic localization of Bach2. These results indicate an interesting possibility that the inactivation of Bach2 is important in the development of IgM plasma cells.
  • We used retroviral vectors to detect Bach2 or Bach2 ⁇ C2 was overexpressed and its effect on the production of IgM plasma cells was examined.
  • Bach2 AC2 lacks a nuclear export signal and therefore accumulates in the nucleus (Hoshino, H. et al, J. Biol. Chem.
  • Example 5 The bach2 gene knockout mice and littermate wild type mice were bred under free feeding under SPF conditions. After birth, bach2 gene knockout mice showed growth similar to wild type mice. However, diarrhea began to appear around 4 months after birth, and weight loss was also observed.
  • the large intestine of the bach2 gene knockout mouse was fixed in formalin, sliced, and then subjected to HE staining. Examination of the histological image of the large intestine revealed typical inflammatory bowel diseases, such as infiltration of inflammatory cells, Talibut abscess, thickening of the mucous membrane, and destruction of the villous structure ( Figure 6). This result indicates that bach2 knockout mice develop inflammatory bowel disease, and that Bach2 plays an important role in maintaining mucosal function and mucosal immunity. The simple hypothesis is that inflammatory bowel disease in bach2 knockout mice is thought to result from B lymphocyte dysfunction, but Bach2 may also function in intestinal epithelial cells themselves.
  • the binding activity was measured by ELISA using human HL-6 (Kamakura Techno Science KTS102L) as an antigen, using an anti-mouse IgG antibody or an anti-mouse IgM antibody as a secondary antibody.
  • HL-6 Kamakura Techno Science KTS102L
  • an anti-mouse IgG antibody or an anti-mouse IgM antibody as a secondary antibody.
  • B) After diluting HL-6 with a coating buffer (100 mmol / L NaHC03 pH9.6, 0.02 w / v% NaN3) to a concentration of 1 ⁇ g / mL, add 100 ⁇ L to a 96-well ELISA plate (Nunc, Maxisorp). Dispense L / well 4 The mixture was allowed to stand at ° C and solidified.
  • HRP horse radish peroxidase
  • HRP horse radish peroxidase
  • the ratio of IgM was 10% in Balb / c mice, whereas it was 67% in Bach-2 KO mice. This example shows that it is possible to generate IgM or Z and IgM-producing hybridomas with high frequency using Back-2 KO mice.
  • the first point of the industrial utility of the present invention is that a method for producing an antibody or an antiserum containing the antibody using the non-human animal of the present invention can be provided.
  • the second point is that it can provide a drug for treating or preventing inflammatory bowel disease and Neuper IgM syndrome, and a drug for increasing IgM.
  • the third point is that a screening system for these drugs can be provided.
  • the fourth point is that a test method and a test drug for the above diseases can be provided by measuring the expression level of the bach2 gene or protein.

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Abstract

Une souris knock-out pour le gène bach2 est préparée et analysée. En conséquence, il s’est avéré que la souris knock-out pour le gène bach2 développe une entéropathie inflammatoire et un syndrome d'hyper-IgM et que dans celle-ci, la quantité d’IgM dans le sérum augmente plus que les contrôles dont l'expression du gène bach2 n’est pas artificiellement inhibée.
PCT/JP2005/008928 2004-05-17 2005-05-17 Animal non humain dont l’expression de bach2 est artificiellement inhibée, et utilisation de celui-ci WO2005110433A1 (fr)

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JP5099856B2 (ja) * 2007-11-26 2012-12-19 独立行政法人科学技術振興機構 代謝症候群の治療もしくは予防剤、検査方法、検査薬、及び代謝症候群の治療薬の候補化合物のスクリーニング方法
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WO2016002760A1 (fr) * 2014-07-02 2016-01-07 学校法人東京理科大学 Procédé de fabrication de population de cellules b
JPWO2016002760A1 (ja) * 2014-07-02 2017-04-27 学校法人東京理科大学 Ｂ細胞集団の製造方法
US10428304B2 (en) 2014-07-02 2019-10-01 Tokyo University Of Science Foundation Method for producing B cell population
US10781423B2 (en) 2014-07-02 2020-09-22 Tokyo University Of Science Foundation Method for producing B cell population
WO2017082254A1 (fr) * 2015-11-09 2017-05-18 国立大学法人東北大学 Anticorps anti-bach2 phosphorylé et procédé de criblage d'immunoactivateurs antitumoraux

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