WO2005110433A1

WO2005110433A1 - Nonhuman animal having expression of bach2 artificially inhibited, and use thereof

Info

Publication number: WO2005110433A1
Application number: PCT/JP2005/008928
Authority: WO
Inventors: Kazuhiko Igarashi; Tetsuhiko Muto; Masayuki Yamamoto
Original assignee: Kazuhiko Igarashi; Tetsuhiko Muto; Masayuki Yamamoto
Priority date: 2004-05-17
Filing date: 2005-05-17
Publication date: 2005-11-24
Also published as: JPWO2005110433A1

Abstract

A bach2 gene knockout mouse was prepared and analyzed. As a result, it has been found that the bach2 gene knockout mouse develops inflammatory enteropathy and hyper-IgM syndrome and that therein, the amount of IgM in serum is increased over that of controls not having the expression of bach2 gene artificially inhibited.

Description

Specification

Non-human animal whose Bach2 expression is artificially suppressed and its use

Technical field

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.

Background art

[0002] 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). The diversity of antibody functions by CSR is important for humoral immunity. However, how the branch decision is made remains unclear.

[0003] After exposure to antigen in vivo, B cell responses are induced by somatic hypermutation (SHM) of variable (V) region exons to increase antibody affinity, and a unique after-function. It may involve CSR in the exon of the constant (C) region of the immunoglobulin heavy chain (IgH) gene to produce antibodies with the same antigen specificity. Activation-induced cytidine kinase (AID) is required for both SHM and CSR, suggesting that the mechanism between these two processes is linked. Because SHM and CSR alter DNA information and are thus possibly mutagenic, their evolution must be tightly regulated. Furthermore, CSR is central to distinguishing IgM from non-IgM plasma cells, as CSR occurs only in cells that are non-IgM plasma cells. However, we know very little about how transcription factors regulate CSR and SHM in B cells! / ヽ!

[0004] Prior art document information related to the invention of the present application is shown below.

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.

Immunol. 20, 165-196 (2002).

Disclosure of the invention

Problems to be solved by the invention

[0005] 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

[0006] The present inventors have conducted intensive studies to solve the above-mentioned problems. First, in order to elucidate the function of Bach2 in vivo, a bach2 gene knockout mouse was prepared and analyzed. As a result, 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. In addition, 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. These findings have revealed that Bach2 is an important regulator of the antibody response and provided a mechanism to distinguish between IgM and non-IgM plasma cells.

[0007] That is, 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.

[1] A non-human animal, wherein the expression of the bach2 gene is artificially suppressed. [2] A non-human animal characterized by having a foreign gene inserted into one or both of the bach2 gene pairs.

[3] the non-human animal of [1] or [2], wherein the amount of IgM is increased;

[4] the non-human animal of [3], for use in producing IgM;

[5] the non-human animal of [1] or [2], which develops hyper-IgM syndrome;

[6] The non-human animal according to [5], for use as a model animal for hyper-IgM syndrome.

[7] The non-human animal according to [1] or [2], which develops an inflammatory bowel disease.

[8] The non-human animal according to [7] for use as an inflammatory bowel disease model animal.

[9] A non-human animal cell having pluripotency, wherein the expression of the bach2 gene is artificially suppressed.

[10] A non-human animal cell having multipotency, wherein a foreign gene is inserted into one or both of the bach2 gene pair.

[11] A cell derived from the non-human animal according to [1] or [2].

[12] A method for producing IgM, comprising the following steps (a) to (c).

(a) a step of preparing a non-human animal according to [1] or [2];

(b) immunizing the prepared non-human animal with an antigen

(c) a step of collecting IgM recognizing the antigen from the immunized animal

[13] A method for producing an antiserum containing IgM, comprising the following steps (a) to (c).

(a) a step of preparing a non-human animal according to [1] or [2];

(b) immunizing the prepared non-human animal with an antigen

(c) recovering antiserum from the immunized animal

[14] 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:

(a) DNA encoding Bach2

(b) DNA encoding Bach2 ΔC2

(c) DNA encoding Bach2 binding factor MAZR (d) DNA encoding Ma! F, MafG, or MalK protein

[15] An agent for increasing IgM, comprising a DNA for suppressing the expression of Bach2 as an active ingredient.

[16] A method for screening a compound that substitutes for the function of Bach2, comprising the following steps (a) to (c).

(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];

(b) a step of analyzing the phenotype of the non-human animal or the cell

(c) a step of administering a test compound and selecting a compound that complements the phenotype of the non-human animal or the cell, as compared to a case where the compound is not administered

[17] A method for screening a Bach2 regulator, comprising the following steps (a) to (c).

(a) Step of bringing a test compound into contact with Bach2

(b) a step of detecting the binding between the Bach2 and a test compound

(c) selecting a test compound that binds to the Bach2

[18] A method for screening a Bach2 regulator, comprising the following steps (a) to (c).

(a) Step of bringing a test compound into contact with Bach2

(b) a step of measuring the activity of the Bach2

(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.

[19] A method for screening a Bach2 regulator, comprising the following steps (a) to (d).

(a) Providing a cell or cell extract having a DNA in which a reporter gene is operably linked downstream of the promoter region of the bach2 gene

(b) contacting a test compound with the cells or the cell extract

(c) measuring the expression level of the reporter gene in the cell or the cell extract

(d) a step of administering a test compound and selecting a compound that increases or decreases the level of expression of the reporter gene as compared to the case where

[20] 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

(b) a step of bringing a test compound into contact with the cells or the cell extract in the presence of Bach2

[21] A method for testing inflammatory bowel disease or hyper-IgM syndrome, comprising a step of measuring the expression level of the bach2 gene.

[22] A method for testing inflammatory bowel disease or hyper-IgM syndrome, comprising a step of detecting a mutation in the bach2 gene region.

[23] 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. [24] An agent for detecting inflammatory bowel disease or hyper-IgM syndrome, comprising an antibody that binds to Bach2.

Brief Description of Drawings

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). 圆 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).

410

[Fig. 3c] bach2 ^{+ / +} (open circles) or bach2 (closed triangles) mice were immunized on day 0 and day 21 with NP-linked avian γ-globulin (NP-CGG). And Anti-NP IgM and IgGl serum levels at day 28.

FIG. 3d is a diagram showing the mutation occurrence rate in the μ 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.

[Figure 4a] RBC-killed splenocytes from wild-type (open circles) and bach2 ^{_ / _} (closed triangles) mice were cultured with LPS and the indicated cytokin for 7 days to measure secreted immunoglobulin levels It is a figure which shows the result.

FIG. 4b is a photograph showing the expression level of GLT containing Iμ 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.

H

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 Bach2ΔC2 (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, and 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, and 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). BEST MODE FOR CARRYING OUT THE INVENTION

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.

[0010] 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.

• AL121787 (human bach2 gene sequence (SEQ ID NO: 2) and Bach2 sequence (SEQ ID NO: 3)) • AJ271878 (human bach2 cDNA sequence (SEQ ID NO: 4) and Bach2 sequence (SEQ ID NO: 3)) • NM_007521 (Mouse bach2 cDNA sequence (SEQ ID NO: 5) and Bach2 sequence (SEQ ID NO: 6); Mol. Cell. Biol. 24 (8), 3473-3484 (2004), Mol. Cell. Biol. 23 (4) , 1163-1174 (2003), J. Biochem. 132 (3), 427-431 (2002), J. Biol. Chem. 277 (23), 20724-20733 (2002), Mol. Cell. Biol. 16 ( 11), 6083-6095 (1996))

[0011] Further, 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.

[0012] In the present invention, 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 The term 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.

[0013] The 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.

[0014] In the present invention, "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. "

[0015] Gene expression can be suppressed by a method generally known to those skilled in the art. For example, 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.

[0016] 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. In the genetically modified non-human animal of the present invention, 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.

[0017] 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.

Since the non-human animal of the present invention develops inflammatory bowel disease, it can be used as an inflammatory bowel disease model animal. In addition, 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. Here, hyper IgM syndrome (also called hyper-IgM immunodeficiency)

IgM levels are normal! /, Then increase! /, But IgG and IgA levels decrease! /, Immunoglobulin deficiency, and normal or increased IgM levels! ], Which exhibit various traits such as decreased IgG and IgA levels, intermittent neutropenia, normal B cell levels, and increased susceptibility to infection (Immunology Dictionary, 2nd edition, Tokyo Kagaku Dojin 2001 p215). , Merck Manual 17th edition, 4th press 2002 pl034). Therefore, 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. Thus, the non-human animal can also be used for the production of antibodies (preferably IgM). Ie

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.

[0019] Here, inflammatory bowel disease (Inflammatory Bowel Disease: IBD) includes the following 1) and 2).

1) Inflammatory bowel disease of obvious cause (eg, more commonly caused by bacteria, viruses, drugs, etc.)

2) Inflammatory bowel disease of unknown cause (eg, ulcerative colitis, Crohn's disease, colitis associated with collagen disease, intestinal Behcet's disease)

In the narrow sense, IBD refers to ulcerative colitis and Crohn's disease.

In the present invention, of the inflammatory bowel diseases, preferred are ulcerative colitis and Crohn's disease, and most preferred are ulcerative colitis. [0020] 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. In 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. For example, 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. As the marker gene to be inserted, an antibiotic resistance gene such as a neomycin resistance gene is preferable. When an antibiotic resistance gene is inserted, a cell line in which homologous recombination has occurred can be selected only by culturing in a medium containing an antibiotic. For more efficient selection, a thymidine kinase gene or the like can be linked to a targeting vector. As a result, cell lines that have undergone heterologous recombination can be excluded. In addition, 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.

[0022] 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 ヽ. The obtained ES cell line is injected into mouse blastoderm to obtain a chimeric mouse. By mating this chimeric mouse, a mouse in which one of the gene pair of the bach2 gene is inactivated can be obtained. Furthermore, by crossing this mouse, it is possible to obtain 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.

[0023] In addition, 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.

[0024] 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. In addition, 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.

[0025] 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. As a method for establishing the above-mentioned cell line derived from a non-human animal, a known method can be used. 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).

[0026] 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. Aju For the bunt, Freund's complete adjuvant is used, for example. Further, thereafter, it is preferable to administer the immunogen mixed with an appropriate amount of incomplete Freund's adjuvant several times every 4 to 21 days. In addition, 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.

[0027] In order to obtain the desired antibody, 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. As the polyclonal antibody, 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.

[0028] For example, using an affinity column to which the target antigen is coupled, a fraction that recognizes only the target antigen is obtained, and this fraction is further separated into protein A and protein G. By utilizing and purifying, IgG or IgM can be prepared.

[0029] In order to obtain a monoclonal antibody, it is confirmed that a desired antibody level is increased in the serum of an animal sensitized with the above antigen, and then antibody-producing cells are collected from the animal and cloned. For cloning of antibody-producing cells, a cell fusion method can be used. As the other parent cell to be fused with the antibody-producing cell, for example, mammalian myeloma cells are used. 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.

[0030] 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).

[0031] 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. As the selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine) is used. 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. Usually, selecting hybrididomas by continuing culture for several days to several weeks Can do. Next, a conventional limiting dilution method is performed, and screening and cloning of hybridomas producing the desired antibody are performed.

[0032] Next, 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. For 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.

[0033] In addition to producing antibodies using hybridomas, use of cells in which antibody-producing cells such as sensitized lymphocytes that produce antibodies are immortalized by using oncogenes or viruses, etc. You can also. Epstein-Barr virus (EBV) or the like can be used as a virus for immortalizing cells.

[0034] 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.

Further, the antibody obtained by the method of the present invention may be an antibody fragment or a modified antibody thereof. For example, 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). Specifically, an antibody fragment can be obtained by treating an antibody with an enzyme, for example, papain or pepsin to generate an antibody fragment. Alternatively, 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,

J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H "Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. 1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al, Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0036] As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. 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.

[0037] 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

94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

[0038] 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. In addition, 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.

[0039] These modified antibodies can be produced using known methods. Specifically, for example, 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.

[0040] 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.

Specifically, 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. If necessary, 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. (Sato, K. et al, Cancer Res. (1993) 53, 851-856).

Further, 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. For example, 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. Alternatively, the gene obtained by the present invention is used for producing the various modified antibodies described above.

[0043] 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

Laboratory Manual. Ed Harlow ana David Lane, old Spring Harbor Laboratory, 1988). The concentration of the antibody obtained above can be measured by measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), or the like.

[0044] Columns used for affinity chromatography include a protein A column and a protein G column. For example, columns using Protein A include Hyper D, POROS, Sepharose F.F. (Pharmacia) and the like.

[0045] Examples of the chromatography other than the affinity chromatography 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.

[0047] Furthermore, modified Bach2 such as Bach2 ΔC2 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.

[0048] For the treatment of inflammatory bowel disease and Neuper IgM syndrome, a modified cDNA such as Bach2ΔC2 (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. 275, 15370-15376 (2000)), DNA encoding the Bach2 binding factor MAZR (XM_223592, AB029397, NM-019574, Kobayashi, A. et al., Mol.Cell. Biol. (2000) 20, 1733-1746), and Is a DNA encoding a small Maf protein (MalF (NM_012323, BC022952), MafG (NM—010756), MalK (NM—010757, NM—002360, NM—145673)), a partner of Bach2 dimerization. T. et al., Mol. Cell. Biol. (1996) 16, 6083-6095) The DNAs encoding MalF, MafG, and MalK may be used alone. It can also be used in combination Nag Kill only.

[0049] 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.

[0050] The present invention also provides use of DNA for suppressing the expression of Bach2. Specifically, the present invention provides an IgM increasing agent containing, as an active ingredient, DNA for suppressing the expression of Bach2. Examples of such DNA 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. Examples include DNA encoding RNA that suppresses the expression of the encoding DNA by the RNAi effect.

[0051] 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. In addition, a sequence complementary to the coding region or the 3′-side untranslated region can also be used. As described above, 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. In order to effectively suppress the expression of a target gene using an antisense sequence, 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.

[0052] Endogenous gene expression can also be suppressed using DNA encoding a ribozyme. 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. Some have a size of more than 400 nucleotides, such as a hammerhead type or a hairpin type, which has an active domain of about 40 nucleotides (Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzyme, 35: 2191, 1990) o

[0053] For example, 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). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site, it is possible to create a restriction-enzymatic RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi M , et al: FEBS Lett 239: 285, 1988; Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzyme 35: 2191, 1990;

Koizumi M, et al: Nucl Acids Res 17: 7059, 1989). For example, there are multiple target sites in the DNA encoding Bach2.

[0054] 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) .

[0055] 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). In addition, by arranging such structural units in tandem so that multiple sites in the target gene can be cleaved, the effect can be further enhanced (Yuyama N, et al: Biochem Biophys Res Commun 186: 1271). , 1992) o As described above, the transcript of the target gene in the present invention is characterized using ribozymes. Differential cleavage can suppress the expression of the gene.

[0056] 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.

[0057] 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).

Constructs that make self-complementary 'hairpin' RNA (hpRNA) (Smith, NA et al. Nature, 407: 319, 2000, Wesley, SV et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29: E55, 2001).

[0058] 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. In addition, sequence identity can be determined by the above-described method. When the above DNA is administered to a living body, 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.

[0059] The DNA can also be administered as a drug formulated by a known pharmaceutical method. For example, it can be used in the form of a sterile solution with water or another pharmaceutically acceptable liquid, or a suspension for injection. Further, for example, appropriately combined with 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.

[0060] 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 ™ and HCO-50.

[0061] 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. 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.

[0062] The present invention provides a method for screening a compound that substitutes for the function of Bach2. First, a test compound is administered to the non-human animal of the present invention. As 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. When the test compound is a protein, for example, it is also possible to construct 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. Next, 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. In the present invention, "complementary" includes not only a perfect case but also an incomplete case. Furthermore, the present invention also provides a screening method using cells of a genetically modified non-human animal. First, the non-human movement of the present invention B lymphocytes are isolated from the original spleen cells and stimulated with LPS to induce an antibody class switch. At this time, 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. By the above method, the isolated compound can be used as an agent for treating or preventing inflammatory bowel disease and hyper-IgM syndrome.

[0063] 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. In the first embodiment, first, a test compound is brought into contact with Bach2. Next, the binding between the Bach2 and the test compound is detected. Next, a test compound that binds to the Bach2 is selected. By this method, 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.

[0064] As 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. Gene 91, p.217-223 (1990)), 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. 1, p.385-394 (1982)), Adenovirus late promoter (Kauftnan et al. Mol. Cell. Biol. 9, p. 946 (1989)), any commonly used promoter such as the HSV TK promoter may be used. [0065] In order to express a foreign gene by introducing a gene into an animal cell, an electoral-portion method (Chu, G. et al. Nucl. Acid Res. 15, 1311-1326 (1987), calcium phosphate Pem method (Chen, C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)), DEAE dextran method (Lopata, MA et al. Nucl. Acids Res. 12, 5707-5717 ( 1984); Sussman, DJ and Milman, G. Mol. Cell. Biol. 4, 1642-1643 (1985), lipofectin method

(Derijard, B. Cell 7, 1025-1037 (1994); Lamb, BT et al. Nature Genetics 5, 22-30 (1993); Rabindran, SK et al. Science 259, 230-234 (1993), etc. There are, however, either way.

[0066] By introducing the recognition site (epitope) of the monoclonal antibody into the N-terminal or C-terminal of Bach2, 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. Also, in order to minimize the properties of Bach2 by making it a fusion protein, a method of preparing a fusion protein by introducing only a small epitope portion having several to a dozen or so amino acid powers in order to make it as small as possible. It has been reported. For example, polyhistidine (His-tag), influenza agglutinin HA, human C-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 genelO protein (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)).

[0067] In immunoprecipitation, 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. In addition to using an antibody against the above-mentioned epitope, 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.

[0068] For example, when the antibody is a mouse IgG antibody, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. When Bach2 is prepared as a fusion protein with, for example, an epitope such as GST, daltathione

A substance that specifically binds to these epitopes, such as Sepharose 4B, can be used to form an immune complex in the same manner as when Bach2 antibody is used.

[0069] For general methods of immunoprecipitation, see, for example, the literature (Harlow, E. and Lane, D .:

Antibodies, pp. 511-552, Cola Spring Harbor Laooratory puoiications, New York (1988)) or in accordance therewith.

[0070] SDS-PAGE is generally used for the analysis of immunoprecipitated proteins. By using a gel having an appropriate concentration, proteins can be analyzed by binding according to the molecular weight of the proteins. In addition, at this time, it is generally difficult to detect the protein bound to Bach2 by ordinary protein staining methods such as coomassie staining and silver staining, so the radioactive isotope ³⁵ S-methionine and ³⁵ S- cysteine cells are cultured inclusive culture medium, labeled protein of the intracellular, it is possible to improve the detection sensitivity by detecting this. Once the molecular weight of the protein is known, the target protein can be directly purified from the SDS-polyacrylamide gel and sequenced.

[0071] As 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. As 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.

[0072] 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.

[0073] In the two-hybrid system, Bach2 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.

[0074] Reporter genes used in the two-hybrid system include, for example,

Other than 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.

[0075] Screening for a compound that binds to Bach2 can also be performed using affinity chromatography. For example, 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. In this case, 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.

[0077] 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 art.

[0078] In the present invention, 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).

[0079] 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. In the second embodiment, first, 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. [0080] 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! ,.

[0081] 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.

Further, in the present invention, “contact” is performed according to the state of Bach2. For example, when Bach2 is in a purified state, it can be performed by adding a test compound to a purified sample. In addition, 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. When the test compound is a protein, for example, 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. Further, for example, a two-hybrid method using yeast or animal cells can be used.

[0083] In the second embodiment, 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. 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.

[0084] In the second embodiment, 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.

[0085] 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. In a third embodiment, first, 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. Here, “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.

[0086] 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.

[0087] In the third embodiment, 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.

When the bach2 gene is used as a reporter, 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.

[0089] 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. In addition, 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.

[0090] In the third embodiment, in the following, 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. In addition, compounds that decrease the level of reporter gene expression can be used as an agent for increasing IgM.

[0091] 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. As a fourth embodiment, first, 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,

20724-20733), or a cell or a cell extract having DNA to which a reporter gene is operatively linked. 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.

[0092] In the fourth embodiment, a test compound is brought into contact with the cell or the cell extract in the presence of Bach2 in the next step. For example, a test compound can be contacted with cells or cell extracts expressing Bach2 and a reporter. In a fourth embodiment, the expression level of the reporter gene in the cell or the cell extract is measured in the following manner. Next, 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.

[0093] 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. Here, the expression of the bach2 gene also includes the expression of the Bach2 protein as well as the expression of the bach2 mRNA. When 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.

[0094] Hereinafter, embodiments of the inspection method of the present invention will be exemplified, but the inspection method of the present invention is not limited thereto. In one embodiment of the above test method, first, an 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.

[0095] In the present method, 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.

[0096] The above-described test method can be carried out as follows by measuring the expression level of Bach2. First, 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.

[0097] In the present method, 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.

[0098] Further, in the present invention, the bach2 knockout mouse is used for inflammatory bowel disease and dyspepsia.

It was found to develop IgM syndrome. Therefore, it is suggested that the bach2 gene mutation causes inflammatory bowel disease in humans and the like and hyper-IgM syndrome. Thus, 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!

[0099] In the present invention, 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. In addition, examples of the type of mutation in the present invention include, but are not limited to, deletion, substitution, and insertion mutation.

[0100] Hereinafter, preferred embodiments of the test method including a step of detecting a mutation generated in the bach2 gene region will be described, but the method of the present invention is not limited to these methods.

In a preferred embodiment of the above-described test method, first, 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.

[0101] In the present method, 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. Next, in this method, 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. In the present method, the determined nucleotide sequence of the DNA is then compared with a control. In the present invention, a control refers to a DNA containing a normal (wild-type) bach2 gene region. Generally, 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.

[0102] The mutation in the present invention can also be detected by the following method. First, a DNA sample is prepared from a subject. Next, the prepared DNA sample is cut with a restriction enzyme. Next, the DNA fragments are separated according to their size. The size of the detected DNA fragment is then compared to a control. In another embodiment, first, a DNA sample is prepared from a subject. Next, DNA containing the bach2 gene region is amplified. Further, the amplified DNA is cut with a restriction enzyme. Next, 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).

Polymorphism / RFLP) and the PCR-RFLP method.

[0103] In still another method, first, 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. As 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.

Oncogene. 1991 Aug 1; 6 (8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). This method has advantages such as relatively simple operation and a small amount of a test sample, and is particularly suitable for screening a large number of DNA samples.

[0104] In still another method, first, a DNA sample is prepared from a subject. Then

Amplify DNA containing bach2 gene region. In addition, the amplified DNA 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.

[0105] In still another method, first, 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. In the present invention, “substrate” means a plate-like material to which nucleotide probes can be immobilized. In the present invention, 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. Generally, 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. For the surface layer of the substrate, a porous membrane, typically a glass, can be used, for example a -trocellulose membrane.

In the present invention, examples of the method for immobilizing (array) nucleotides include oligonucleotide-based arrays developed by Aifymetrix. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, photolithographic technology (Aifymetrix) and ink-jet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known for in situ synthesis of oligonucleotides. Can be used for the production of

[0107] 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. In the present invention, 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. .

In the present method, the substrate is brought into contact with the DNA containing the bach2 gene region in the next step. By this process, 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.

In the present method, 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. In a 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. In the present method, 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.

[0110] In addition to the above-described methods, an Allele Specific Oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and subjected to DNA hybridization with the oligonucleotide, the efficiency of hybridization is reduced when the mutation is present. It can be detected by Southern blotting or a method utilizing the property of quenching by intercalating a special fluorescent reagent into the gap of the hybrid.

Also, in the present invention, 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.

Biochem. (2000) 10: 103-110), AcycloPrime method (Genome Research (1999) 9: 492-498), SNuPE method (Rapid Vommun Mass Spectrom. (2000) 14: 950-959), etc. it can.

[0112] The present invention further provides a test agent for use in the test method of the present invention. In one embodiment, the test agent comprises an oligonucleotide that hybridizes to the bach2 gene region and has a chain length of at least 15 nucleotides. [0113] The oligonucleotide specifically hybridizes to DNA (normal DNA or mutant DNA) containing the bach2 gene region. As used herein, "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!

[0114] 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. When 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.

[0115] When the above oligonucleotide is used as a probe, 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.

[0116] 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.

[0117] 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.

[0118] Another embodiment of the test agent of the present invention is a test reagent containing an antibody that binds to Bach2. Up The antibody is not particularly limited as long as it can be used for the test. For example, the antibodies described in Examples can be used. The antibody is labeled if necessary.

[0119] In the above test agents, in addition to the oligonucleotides and antibodies as active ingredients, for example, sterilized water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers Agents (such as BSA and gelatin) and preservatives are mixed as necessary! All prior art documents cited in the specification are incorporated herein by reference.

Example

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(1) Immunofluorescent staining

Frozen sections of the spleen were subjected to FITC-conjugated anti-mouse IgM, biotin-conjugated anti-mouse IgM, biotin-conjugated anti-mouse CD3ε (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 μg / 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).

[0121] (2) Construction of targeting vector

Analysis of the genomic phage clone of mouse bach2 is known (Sun, J. et al., J. Biochem. (2001) 130, 385-392), and detailed information is available upon request. The Bach2 targeting vector (pB2TV) is a β-galatatosidase (LacZ) gene containing the SV40 polyadenylation (poly (A)) signal derived from the pCMV β 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 ΙοχΡ sequences on both sides. These force sets were clawed between short and long arm DNA. 3.0 kbp short arm DNA was isolated from genomic phage clones by PCR using Bach2-specific primers and vector sequence primers. The Bach2-specific primer had an EcoRI site at the 5 'end (B2SARev primer 5'-AATTAAgaattcGTTCAAACCCTGGAATAAAGAG-3' (SEQ ID NO: 1)), and the PCR product was cloned as EcoRI DNA. The 6.7 kbp long arm DNA also contained the EcoRI site up to the 3 'end of the phage clone end. Next, 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). pB2TV was linearized with Notl.

[0122] (3) Fluorescence activated iDani cell sorter (FACS) analysis

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).

[0123] (4) Determination of immunoglobulin level

Immunoglobulin concentrations were determined using ρ-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).

[0124] (5) In vivo immunization and SHM Atsushi

100 µg of DNP-Ficoll (Biosearch Technologies) or 100 µg of NP- ヮ 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). 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).

[0125] (6) In vitro class switch technology

RBC-dead spleen cells (1 × 10 ⁵ cells) were cultured in a 96-well plate in a total volume of 200 μl RPMI medium. Cells were harvested using 20 μg / 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).

[0126] (7) RT-PCR

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.

[0127] (8) Retroviral infection

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 μg / 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 μg / 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.

[Example 1]

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. &

It occurs in secondary lymphoid organs such as Muramatsu, M., Annu. Rev. Immunol. (2002) 20, 165-196). Anti-Bach2 antibody (Oyake, T. et al., Mol. Cell. Biol. (1996) 16,

6083-6095), 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 ε-positive Τ cells in the T cell area. These results indicate that Bach2 expression is high in follicular Β cells within lymphoid follicles. Such an expression pattern suggests a role for Bach2 in the antibody response. To examine this possibility, we disrupted the mouse bach2 gene by replacing the first codexon with a neo-resistant gene cassette (Fig. La). Using appropriately targeted embryonic stem (ES) cell clones (Figure lb), chimeric mice were obtained that harbor disrupted loci through the germline. When bach2 ^{+ / _} mice were bred, bach2 ^{_ / _} progeny were obtained with the expected incidence of medial. The bach2 ^{_ / _} progeny did not show any obvious abnormalities. There were no significant differences in bone marrow or T cell counts or cell surface expression of markers including TER-119, Gr-1, Mac-1, CD4, and CD8 (data not shown). In the immunoblotting of bone marrow extract with anti-Bach2 antibody, Bach2 was detected in wild-type mice but not in bach2 ^{_ / _} mice (Fig. Lc). After confirming that hematopoiesis was largely unaffected in the absence of Bach2, we next examined B cell development in detail. Flow cytometric analysis of bone marrow cells showed that bone marrow pro-B cells (B220 +, pan-B cell markers, and CD43 +) and pre-B cells (B220 +, CD43 ", IgM-, and IgD_) were affected in bach2 mice. In contrast, the numbers of c220 ^ω IgM + recirculating Β cells in the bone marrow of bach2 ^{_ / _} 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. In bach2 ^{_ / _} mice, marginalized B cells were less affected than follicular B cells (Fig. 2. These results suggest that Bach2 is involved in a later stage of B cell development where antigen stimulation plays a key role.We also performed immunohistochemical analysis of the spleen. In spleens from wild-type and bach2-deficient mice, IgM + B cells formed follicles surrounded by MadCAM-1 expressing marginal sinuses (Figure 2f), indicating that in the absence of Bach2 It was shown that the formation of lymphoid follicles was not affected. [Example 2]

To assess the role of Bach2 in immunoglobulin production, serum immunoglobulin concentrations were measured by isotype-specific enzyme-linked immunosorbent assay (ELISA, FIG. 3a). IgM levels were 5 times higher in bach2 ^{_ / _} mice than in wild-type mice. In contrast, 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.

Immunol. (2001) 13, 543-548).

[0130] To determine whether Bach2 is required in vivo for an antigen-dependent humoral immune response, 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). The bach2 ^{+ / +} mice showed strong production of IgGl, but the bach2 ^{_ / _} mice did not (Fig. 3c). Together, these results indicate that Bach2 is essential for both T cell-independent and -dependent production of isotype-specific antibodies, and that Bach2 is defective in IgH gene CSR in bach2 ^{_ / _} mice. Suggesting that it may be

[0131] 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). The present inventors examined whether Bach2 deficiency affected SHM efficiency. 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 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. & Weiss, U., Nature (1991) 354, 389-392) in wild-type and bach2 ^{_ / _} cells. Nucleotide substitution mutation rates were 2.2 x 10 ^_2 / bp (18 clones were sequenced and 114 substitutions were found in 5238 bases) and 1.2 x 10 ^_3 / bp (17 clones were ^isolated ). In Bach2 ^{_ / _} mice, several nucleotide substitutions were also found in the complementarity determining regions (CDRs), which are important for antigen binding. (Figure 3d) These results clearly proved that Bach2 is important not only for CSR but also for SHM.

[Example 3]

To determine whether bach2 deficiency affected CSR, we examined immunoglobulin production in vitro by treating the spleen with lipopolysaccharide (LPS) and cytodynamics . IgG and IgA concentrations in the culture medium were significantly lower in bach2 ^{_ / _} cells than in wild-type cells (Fig. 4a). In contrast, bach2 ^{_ /} -splenocytes showed relatively normal levels of IgM secretion compared to control cells, excluding the possibility that Bach2 is required for overall antibody production. These results are consistent with a decrease in serum isotype immunoglobulin levels in Bach2-deficient mice, where Bach2 is not important for the development of IgM plasma cells, but is important for the development of class-switched plasma cells. It indicates that

[0133] At the onset of CSR, the combination of the BCR and the site force-in signal is the IgH constant region (C)

H

Determining 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

H

In addition, 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). To determine the stage of CSR that requires Bach2, we examined GLT and PST in B cells stimulated by LPS and cytoforce by RT-PCR. After 2 days of culture, γ2b, γ3, and a GLTs levels in bach2 ^{_ / _} B cells were comparable to those of wild-type B cells (Figure 4b). In contrast, γ1 GLT was reduced in bach2 B cells. As described above, chromatin activation in the switch region is hardly affected in the absence of Bach2 except for γ1 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. et al, Immunity (2001) 15, 187-99). In contrast to GLTs, PSTs are expressed in bach2 ^{_ /} "B cells. These results indicate that the bach2 deletion caused a cut in the switch region and common abnormalities in Z or ligation, and cell proliferation is a prerequisite for CSR. Therefore, the proliferative activity of bach2_ ^/ "B cells was assessed by measuring BrdU incorporation of B220 + B cells purified from spleen. In the above experiment, the response to LPS used to induce plasma cells in V did not distinguish bach2 ^{_ / _} spleen B cells from wild-type spleen B cells (Fig. 4d). These results confirm that the deficiency observed for the in vitro antibody class switch is not due to a lack of reactivity to LPS in Bach2_ deficient B cells.

(Example 4)

In the simplest model, 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. , A gene required for germinal center formation (BCL-6 (Ye, B. et al., Nat. Genet. (1997) 16, 161-170) and OCA-B (Schubart, D., Rolink, A "Kosco-Vilbois, M., Botteri, F. & Matthias, P., Nature (1996) 383, 538-542; Kim, U. et. al., Nature (1996) 383, 542-547)) showed that the expression of bach2 ^{_ / _} B cells was unaffected by Pax-5 (Barberis, A., Widenhorn, K. ., Vitelli, L. & Busslinger, M., Genes. Dev.

(1990) 4, 849-859), c-Myc (Lin, Y "Wong, K. & Calame, K., Science (1997) 276, 596-599), and c-Fos (Monroe, J" J. Immunol. (1988) 140, 1454-1460) is usually down-regulated during the final sorting of B cells. Its expression was reduced in both wild type and bach2. As described above, the present inventors exerted no significant change in the expression pattern of the transcription factor gene related to the development of plasma cells. In sharp contrast, AID expression required for both SHM and CSR (Muramatsu, M. et al., Cell (2000) 102, 553-563) is severely reduced in bach2 ^{_ / _} B cells. (Figure 4e) o Given this important role of well-characterized SHM and CSR, the defects observed in bach2-deficient B cells are at least partially directly attributable to reduced AID expression. Can. Although bach2 is superior to AID, it is not known whether bach2 regulates AID directly. Residual IgG and IgA in serum in bach2 ^{_ / _} mice may be due to the presence of Bach2-independent regulation of CSR. Bachl (Oyake, T. et al., Mol. Cell. Biol. (1996) 16, 6083-6095; Sun, J. et al., EMBO J. (2002) 21,

5216-5224) may be a substitute for Bach2 because it is expressed in B cells (Fig. 4e).

After finding Bach2 to be important for CSR and SHM, we hypothesized that Bach2 could be part of a biochemical switch for performing CSR and SHM. I stood up. 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. 5a and b, 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. To determine whether Bach2 is inhibitory for the isolation of IgM plasma cells, we used retroviral vectors to detect Bach2 or Bach2 ΔC2 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. (2000) 275, 15370-15376, data not shown). As shown in FIG. 5c, when Bach2 or Bach2 AC2 was overexpressed, the number of IgM expressing cells decreased. These results, combined with earlier genetic findings, suggest that Bach2 is involved in the divergence of IgM and non-IgM plasma cells.

[0136] Plasma cell sorting repeatedly raises questions about lineage selection in hematopoietic cells, since irreversible CSR is selected only for a subset of plasma cells. Studies on lineage selection suggest that transcription factors program individual lineages. Our results suggest that Bach2 is one of the important transcription factors for non-IgM plasma cells, but is not important or even inhibitory for the production of IgM plasma cells. In addition, the subcellular localization of Bach2 appears to provide a molecular mechanism for the divergence of IgM and non-IgM plasma cells. The present inventors have proposed a model in which Bach2, which inhibits Bach2 by cytoplasmic sequestration, enables differentiation into IgM plasma cells, and promotes the division into non-IgM plasma cells by supporting CSR. Suggest. Regulation of CSR specificity (ie, C-region exons to select for CSR) power could also be overlaid on the basic CSR program guided by Bach2. Bach2 contrasts with known plasma cell transcription factors such as XBP-1 which are required for both IgM and non-IgM plasma cells (Reimold, A. et al., Nature (2001) 412, 300-307. ). Taken together, our findings demonstrate that Bach2 is a previously unknown modulator of antibody response. An understanding of the signaling cascade that regulates Bach2 activity in B cells could reveal a new and molecular link between B cell activation and antibody response.

[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.

[0138] Further, 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.

[Example 6]

Bach-2 KO mice and Balb / c mice were treated with human IL-6 (Kamakura Techno Science KTS102L) as an antigen at 40 μg / mouse together with complete freund adjuvant (DIFCO 231131) for each 3 mice, and incomplete freund after 2 Immunization was performed by ip administration of 20 μg / mouse together with adjuvant (DIFCO 263910). After confirming that the serum antibody titer had increased, the antigen was administered i.p. at a final dose of 20 g / mouse. Three days after the final immunization, the spleen was removed, spleen cells were prepared, and fosion was performed with P3U1 cells using PEG1500 (Roche Diagnostic Status 783 641), from Bach-2 KO mice and Balb / c mice. 1320 wells of the fused cells were obtained.

[0140] With the day of fosion as Day 0, the medium was replaced with a HAT medium on Days 1, 2, 3, and 5, and hybridomas were selected using the HAT medium. On Day 8, the culture supernatant was collected and the binding activity was measured by ELISA.

[0141] 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. 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 μg / mL, add 100 μL to a 96-well ELISA plate (Nunc, Maxisorp). Dispense L / well 4 The mixture was allowed to stand at ° C and solidified. Wash the plate with rinse buffer (PBS (-), 0.05% Tween 20) using micro plate washer (Bio-Rad, Model 1550), and diluent buffer (1/5 diluted Blocking One, Nacalai Tester 03953-95) ) Was added at 200 μL / well, and allowed to stand at 4 ° C for blocking. After removing the Diluent buffer, the hybridoma culture supernatant was diluted 1/4 with the diluent buffer, added to two ELISA plates that had been blocked, added at 100 μL / well, and allowed to react at room temperature for 1 hour. Wash the plate with rinse buffer. One plate contains horse radish peroxidase (HRP) -labeled anti-mouse IgG antibody (Southern Biotechnology Associates 103005), and the other plate contains 100 μL of HRP-labeled anti-mouse IgM antibody (Zymed 626820). Each well was added to each well and allowed to stand at room temperature for 1 hour. The plate is washed with a rinse buffer and the ABTS peroxidase substrate (KPL

50-62-01) with 100 μL / well, stop the reaction 20 minutes later with ABTS peroxidase stop solution (KPL 50-85-01), and then OD595 with a microplate reader (Bio-Rad Model 3550). nm was measured. Wells showing an absorbance of 0.5 or more were judged to be positive.

[0142] Among the positive wells having an absorbance of 0.5 or more, 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.

Industrial applicability

[0143] 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.

Claims

The scope of the claims

[I] A non-human animal, wherein the expression of the bach2 gene is artificially suppressed.

[2] A non-human animal characterized in that a foreign gene is inserted into one or both of the bach2 gene pair.

[3] The non-human animal according to claim 1 or 2, wherein the amount of IgM is increased.

[4] The non-human animal according to claim 3, which is used for producing IgM.

[5] The non-human animal according to claim 1 or 2, which develops hyper IgM syndrome.

[6] The non-human animal according to claim 5, which is used as a hyper IgM syndrome model animal.

[7] The non-human animal according to claim 1 or 2, which develops an inflammatory bowel disease.

[8] The non-human animal according to claim 7, which is used as an inflammatory bowel disease model animal.

[10] A non-human animal cell having pluripotency, wherein a foreign gene is inserted into one or both of the bach2 gene pair.

[II] A cell derived from the non-human animal according to claim 1 or 2.

[12] A method for producing IgM, comprising the following steps (a) to (c).

(a) producing the non-human animal according to claim 1 or 2;

(b) immunizing the prepared non-human animal with an antigen

(c) a step of collecting IgM recognizing the antigen from the immunized animal

(a) producing the non-human animal according to claim 1 or 2;

(b) immunizing the prepared non-human animal with an antigen

(c) recovering antiserum from the immunized animal

[14] An agent for treating or preventing inflammatory bowel disease or hyper-IgM syndrome, comprising the DNA described in any of (a) to (d) below as an active ingredient.

(a) DNA encoding Bach2

(b) DNA encoding Bach2 ΔC2

[15] A drug for increasing IgM, comprising as an active ingredient DNA for suppressing the expression of Bach2.

(a) administering the test compound to the non-human animal according to claim 1 or 2, or to the cell according to any one of claims 9 to: L1.

(b) a step of analyzing the phenotype of the non-human animal or the cell

(a) Step of bringing a test compound into contact with Bach2

(b) a step of detecting the binding between the Bach2 and a test compound

(c) selecting a test compound that binds to the Bach2

[18] A method for screening a Bach2 regulator, comprising the following steps (a) to (c):

(a) Step of bringing a test compound into contact with Bach2

(b) a step of measuring the activity of the Bach2

(b) contacting a test compound with the cells or the cell extract

[21] A method for testing an inflammatory bowel disease or hyper-IgM syndrome group, comprising a step of measuring the expression level of the bach2 gene.

[22] A method for testing for inflammatory bowel disease or IgA syndrome, comprising the step of detecting a mutation in the bach2 gene region.

[23] A test agent for inflammatory bowel disease or hyper-IgM syndrome, comprising an oligonucleotide hybridized to the bach2 gene region and having a chain length of at least 15 nucleotides.

[24] A test agent for inflammatory bowel disease or hyper-IgM syndrome, including an antibody that binds to Bach2.