WO2009099147A1 - Il-1 type ii receptor gene-deficient mutant of mouse - Google Patents
Il-1 type ii receptor gene-deficient mutant of mouse Download PDFInfo
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- WO2009099147A1 WO2009099147A1 PCT/JP2009/051975 JP2009051975W WO2009099147A1 WO 2009099147 A1 WO2009099147 A1 WO 2009099147A1 JP 2009051975 W JP2009051975 W JP 2009051975W WO 2009099147 A1 WO2009099147 A1 WO 2009099147A1
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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Definitions
- the present invention relates to an IL-1 type II receptor gene-deficient mutant mouse, a method for preparing an antibody preparation for treating allergy and / or inflammation using the mutant mouse, and a method for screening an antiallergic drug or anti-inflammatory drug candidate And about.
- IL-1 is a pro-inflammatory cytokine, and factors that down-regulate its production and / or activity are clinically important drug discovery targets.
- the IL-1 ligand family has three members: IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1Ra), IL-1 alpha and IL-1 beta function as agonists and IL-1Ra is known to be a specific antagonist.
- the IL-1 receptor family includes IL-1 type I receptor (IL-1R I), IL-1 type II receptor (IL-1R II), and IL-1 receptor accessory protein (IL-1RAcP).
- the IL-1 type I receptor is involved in IL-1 ligand signaling, but the IL-1 type II receptor has little intracellular domain and can bind to the IL-1 ligand. It is thought that no signal transduction occurs.
- the IL-1 type II receptor does not form a complex with the type I receptor, whereas the IL-1 receptor accessory protein forms a complex with both the IL-1 type I receptor and the IL-1 type II receptor. be able to.
- the IL-1 type II receptor is traditionally a decoy receptor that regulates IL-1 signaling by competitively inhibiting the binding of IL-1 type I receptor and IL-1 ligand. Has been considered.
- an IL-1 type II receptor binds to an IL-1 ligand, it also binds to an IL-1 receptor accessory protein to form a complex. Thus, not only an IL-1 ligand but also an IL-1 receptor acceptor is formed.
- Solly protein has also been considered to competitively inhibit binding to IL-1 type I receptor (Non-patent Document 2).
- the extracellular domain of the type II receptor is cleaved by proteolytic enzymes and exists in the blood circulation as a soluble fragment. It is also known that the soluble fragment of this type II receptor, when associated with the soluble fragment of the receptor accessory protein, has a 100-fold higher affinity for IL-1 alpha and beta and forms a stable soluble complex. (Non-Patent Document 3).
- genes for mouse IL-1 ligand and receptor family genes have been cloned one after another since the cloning of the IL-1 alpha gene in 1984.
- the cloned genes have been produced in sequence by gene-deficient mice by homologous gene recombination methods to ES cells.
- mice deficient in the IL-1 receptor family gene have been created by other groups. That is, IL-1 type I receptor (IL-1R I) and IL-1 receptor accessory protein (IL-1RAcP) gene-deficient mice were created in 1997 and 1998, respectively. However, despite the cloning of the IL-1 type II receptor gene in 1991, mice lacking the IL-1 type II receptor (IL-1R II) gene have reached the present from any laboratory in the world. There is no report that it was made until.
- IL-1R I IL-1 type I receptor
- IL-1RAcP IL-1 receptor accessory protein
- mice lacking IL-1Ra gene produced by the inventor's group spontaneously develop various autoimmune diseases including arthritis. It is used as a model animal (Non-patent Document 5).
- IL-1Ra is an antagonist of IL-1 ligand and inhibits stimulation of agonists IL-1alpha and IL-1beta.
- the IL-1 type II receptor is also a decoy receptor for IL-1 ligand and IL-1RAcP, and is thought to inhibit IL-1 alpha and IL-1 beta stimulation. Therefore, the IL-1 type II receptor gene (Il1r2 locus) ) -Deficient mice are expected to be useful disease model animals.
- the present invention provides an IL-1 type II receptor gene deletion mutant mouse having a deletion and / or insertion mutation at the IL-1 type II receptor locus on the mouse chromosome.
- the mutation may express a green fluorescent protein instead of the full-length IL-1 type II receptor protein.
- the present invention includes a step of immunizing a mouse IL-1 type II receptor protein to the mouse of the present invention to produce a monoclonal antibody against the receptor protein, and the monoclonal antibody is immunized from a wild-type mouse or a wild-type mouse.
- a method for producing an antibody preparation for treating allergy and / or inflammation which comprises the step of screening a monoclonal antibody that suppresses stimulation of the immune system by IL-1 by administration to cells.
- the present invention comprises a step of allowing IL-1 to act on an IL-1 type II receptor or a cell expressing the receptor in the presence or absence of a test substance, IL-1 type II receptor and IL-1 A method for screening an antiallergic drug or anti-inflammatory drug candidate.
- a mutant mouse deficient in an IL-1 type II receptor gene refers to a mutant mouse that cannot normally produce a wild type IL-1 type II receptor.
- the IL-1 type II receptor locus is located on the first chromosome of mouse, a 9-exon-linked mRNA is produced from a transcript of about 40.5 kb, and a polypeptide consisting of 410 amino acid residues Is translated.
- the deletion mutant of the present invention is encoded by at least a part of at least one exon of a wild type gene of IL-1 type II receptor, in addition to a mutant that does not produce any IL-1 type II receptor polypeptide. Where a polypeptide is produced, including variants that lack any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions.
- the variants of the present invention may have deletions and / or insertions in any part of any exon and / or intron of the IL-1 type II receptor locus.
- the mutant mouse of the present invention includes not only homozygous individuals in which both of the two first chromosomes contain the mutation, but also only one of the two first chromosomes contains the mutation and the other is a wild type. Some heterozygous individuals are also included.
- the mutant of the present invention may be created by chemical substances, radiation or any other method.
- a preferred method for producing the mutant of the present invention is, for example, Manipulating the Mouse Embryo: A Laboratory Manual (3rd ed., Nagy, A. et al. Cold Spring Harbor Press N.Y, explained in detail in 2003). Mutation introduction into a target gene by homologous gene recombination method into a stem cell (ES cell).
- a mutation that produces green fluorescent protein was introduced into the IL-1 type II receptor gene.
- a sequence such as Cre provided that it is a mutant lacking any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions
- Specific recombination sequences such as loxP recognized by specific recombinase are inserted, RNAi and other RNA and tRNA genes involved in gene expression control are inserted, green fluorescent protein and other fluorescent proteins, and Renilla Luciferase and other photoproteins derived from fireflies, etc., and alkaline phosphate Enzymes whose enzyme activity can be quantified or localized by enzymatic products such as oxidase, peroxidase and other enzyme reaction products, gene products that confer resistance to n
- the green fluorescent protein can be expressed using the expression control mechanism of the IL-1 type II receptor gene.
- the expression of the IL-1 type II receptor gene can be monitored using fluorescence emitted by the green fluorescent protein as an index.
- a substance that affects the expression of an IL-1 type II receptor gene comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mouse or a cell thereof that is a mutant mutant of the IL-1 type II receptor gene of the present invention.
- the substance selected by the screening method can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the expression of IL-1 type II receptor gene. Connected.
- an IL-1 type II receptor caused by the mutation of the present invention can be obtained. Active substances that compensate for dysfunction can be screened.
- a method for screening a substance that affects the expression of an IL-1 type II receptor gene comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mutant mouse or a cell thereof lacking the IL-1 type II receptor gene
- the selected substance can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the complex formation of IL-1 ligand, leading to the development of a novel immunomodulatory drug.
- Downstream of signal transduction due to stimulation of IL-1 ligand includes, for example, IL-6, procollagen, MCP (Monocyte Chemotactic Protein) -1, MIP (Macrophage Inflammatory Protein) -2, PGES (Prostaglandin E Synthase), etc.
- IL-6 procollagen
- MCP Monocyte Chemotactic Protein
- MIP Macrophage Inflammatory Protein
- PGES Prostaglandin E Synthase
- a drug discovery candidate substance is a substance that can be a new drug or a lead compound for its development, and may be either a natural substance or a synthetic substance, or a polymer or multimer of chemical substances of some unit structure. It may be a monomer or a substance having any chemical structure.
- the drug discovery candidate substance may be administered to a mouse individual or its cells using a drug delivery system according to the dissolution characteristics of the substance.
- the drug discovery candidate substance is a virus particle or nucleic acid and has a function of inducing or inhibiting the expression of some gene in mouse cells, it is expressed by virus infection, electroporation, liposome method or other methods. There is a case to let you.
- IL-1 ligand binds to a membrane-bound IL-1 type II receptor or a soluble fragment thereof, thereby inhibiting signal transduction via the IL-1 type receptor.
- the IL-1 type II receptor gene-deficient mutant mouse of the present invention showed that IL-1 stimulation was suppressed. This suggests that the IL-1 type II receptor performs a previously unknown function of promoting IL-1 stimulation in normal individuals. Therefore, a novel allergy and / or inflammation therapeutic agent can be developed by searching for a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor.
- One example of a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor is an antibody against the IL-1 type II receptor.
- the immune response against the mouse IL-1 type II receptor is not suppressed, and therefore an antibody against the mouse IL-1 type II receptor protein can be prepared.
- the monoclonal antibodies used in the present invention are described in Kohler and Milstein, Eur. J. et al. Immunol. 6: 511-519, 1976, and improved techniques thereof. These methods involve the preparation of immortal cell lines that can produce antibodies with the desired specificity.
- Such cell lines may be generated from spleen cells derived from IL-1 type II receptor-deficient mice of the present invention immunized as described above.
- the spleen cells are immortalized by various methods, and an immortal cell line having antibody-producing ability is prepared.
- the spleen cells are immortalized by, for example, fusion of the immunized IL-1 type II receptor-deficient mouse of the present invention with myeloma cells derived from the same or different animals.
- Various fusion techniques known to those skilled in the art may be used.
- the spleen cells and myeloma cells are mixed with a nonionic surfactant for several minutes and then plated at a low concentration in a selective medium that supports the growth of hybrid cells but not the growth of myeloma cells.
- a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. Hybrid colonies are observed after sufficient time, usually about 1 to 2 weeks. A single colony is selected and its culture supernatant is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.
- Monoclonal antibodies may be isolated from the supernatants of colonies of cell lines derived from selected growing hybridoma clones.
- various techniques may be used to improve yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host such as a mouse.
- Monoclonal antibodies may be recovered from the hybridoma cell ascites or blood. Contaminants may be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction.
- the antigen-binding fragment of the antibody used in the present invention includes an Fab fragment or F (ab ′) 2 fragment obtained by degrading an intact polyclonal antibody or monoclonal antibody with the proteolytic enzyme papain or pepsin, respectively, as well as natural antibody molecules.
- the recombinant antibodies used in the present invention may be prepared by expression cloning of antibody genes including transformation into a suitable bacterial host or transfection into a suitable mammalian cell host.
- the chimeric antibody used in the present invention is a fusion protein supported by the constant domain of the same or different antibody so that the antigen-binding site of the recombinant antibody of the present invention can specifically bind to the IL-1 type II receptor. is there.
- the chimeric antibody used in the present invention includes a short chain variable region antibody (scFv) comprising an antibody heavy chain variable region (V H ) operably linked to an antibody light chain variable region (V L ), a camelid ( A camelid heavy chain antibody (HCAb), or a heavy chain variable region domain (VHH) thereof, which is a class of IgG without light chain produced by animals of Camelidae, camels, dromedaries, llamas).
- the recombinant antibodies of the present invention can be prepared in large quantities using prokaryotic and eukaryotic derived gene expression systems.
- the schematic diagram which shows the structure of the targeting vector 1, and the structure of homologous recombinant chromosomal DNA by this.
- the schematic diagram which shows the structure of the targeting vector 2, and the structure of homologous recombinant chromosomal DNA by this.
- the schematic diagram which shows the structure of the targeting vector 3, and the structure of homologous recombinant chromosomal DNA by this.
- Southern blot diagram confirming mutation at IL-1 type II receptor locus by targeting vector 3.
- FIG. The bar graph which shows that the CHS model allergic reaction by DNFB application
- the bar graph which shows that the CHS model allergic reaction by TNCB application is not seen in the pinna of the IL-1 type II receptor gene deficient mouse. Electropherogram of RT-PCR product showing IL-1 type II receptor gene expression in CHS inflammatory sites.
- FIG. 1 is a schematic diagram showing the structure of targeting vector 1 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
- E2 to E8 represent the positions of the second to eighth exons, respectively. Therefore, the IL-1 type II receptor gene is transcribed from left to right in the figure.
- a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 1, the homologous region on the left side was 1.8 kb, and the homologous region on the right side was 11.2 kb. .
- the arrow of the hygromycin resistance gene indicates that the gene is transcribed from left to right.
- a diphtheria toxin gene is linked adjacent to the left homologous region.
- the second exon to the fifth exon are deleted.
- Targets 1 Isolation of homologous recombinant ES cells Transfection of targeting vector 1 was performed as follows. 20 ⁇ g of linearized targeting vector 1 per 7 ⁇ 1 ⁇ 10 7 ES cells E14.1 strain was transfected by electroporation under conditions of 500 ⁇ F and 250 V for 1 week in ES cell medium supplemented with 140 ⁇ g / mL hygromycin After culturing, ES cells incorporating the targeting vector 1 were selected. In cells in which homologous recombination has not occurred between the chromosomal DNA and the DNA of the targeting vector 1 in the homologous region between the hygromycin resistance gene and the diphtheria toxin gene, the diphtheria toxin gene is expressed and the cell is killed.
- the frequency of homologous recombination increases in colonies of ES cells resistant to hygromycin. Indeed, the inventors' group experience yields homologous recombinant clones with a frequency of 1-4% of the selected ES cells.
- FIG. 2 is a schematic diagram showing the structure of targeting vector 2 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
- a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 2
- the left homologous region was 3.2 kb and the right homologous region was 4.8 kb.
- a diphtheria toxin gene is linked adjacent to the left homologous region.
- the second exon and a part of the third exon are deleted.
- targeting vector 2 Isolation of homologous recombinant ES cells Transfection of targeting vector 2 was performed in the same manner as targeting vector 1. 749 clones of hygromycin-resistant ES cells introduced with the targeting vector 2 were isolated and confirmed by Southern blotting whether homologous recombination had occurred. However, none of the clones had undergone homologous recombination. It was.
- FIG. 3 is a schematic diagram showing the structure of targeting vector 3 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
- a neomycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 3.
- the homology region on the left side was 5.5 kb, and the homology region on the right side was 2.7 kb.
- a diphtheria toxin gene is linked adjacent to the right homologous region.
- EGFP green fluorescent protein
- targeting vector 3 was performed in the same manner as targeting vectors 1 and 2 except that 300 ⁇ g / mL G418 was added to the ES cell medium instead of hygromycin. . 1150 clones of G418-resistant ES cells into which the targeting vector 3 was introduced were isolated, and it was confirmed by Southern blotting whether homologous recombination occurred.
- genomic DNA digested with BamHI was separated by agarose gel electrophoresis and copied to a solid phase, and a KpnI-PstI fragment (about 360 bp) existing 3 ′ downstream of the fifth exon was used as a probe.
- the mutant can obtain a signal at about 7.0 kb with respect to the type of about 10.5 kb (FIG. 4). Of the 1150, only one 7A1 clone was a homologous recombinant.
- a 7A1 clone having a mutation in the IL-1 type II receptor gene locus was prepared according to a conventional method using an agglutination chimera method. Specifically, an 8-cell embryo obtained by crossing between a BDF1 male mouse and a C57BL / 6J female mouse and 5-15 ES cells were contact-cultured overnight in M16 medium (Sigma) to obtain a chimeric blastocyst. After formation, chimeric mice were born by transplanting into the uterus of ICR female mice on the third day of pseudopregnancy treatment.
- mice Male chimeric mice showing a contribution ratio of 70% or more ES cells as judged by hair color were mated with female C57BL / 6J mice, and a germline transmission test was performed.
- DNA is extracted from the tail, and whether the target locus (locus having a deletion and insertion by homologous recombination in the IL-1 type II receptor gene) is determined by the PCR method And confirmed by Southern blotting.
- Mice having the target locus were backcrossed (8 generations in the C57BL / 6J system and BALB / cA system) and used for analysis.
- Phenotype of IL-1 type II receptor gene-deficient mice IL-1 type II receptor gene-deficient mice are born according to Mendel's law in both C57BL / 6J and BALB / cA genetic backgrounds. There was no abnormality. Unlike IL-1Ra gene-deficient mice, autoimmune diseases such as rheumatoid arthritis and psoriasis-like dermatitis were not observed with aging. In addition, in IL-1 type II receptor gene-deficient mice, a phenotype indicating that the decoy receptor for the IL-1 ligand has disappeared has not been recognized at present.
- DNFB 2,4-Dinitrofluorobenzene
- TNCB 2,4,6-trinitrochlorobenzene
- FIG. 5A The result of CHS model allergic reaction by DNFB application is shown in FIG. 5A, and the result of CHS model allergic reaction by TNCB application is shown in FIG. 5B.
- FIGS. 5A and 5B As shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, the onset of contact-type hypersensitivity by hapten application was suppressed.
- IL-1 type II receptor gene expression was confirmed in wild-type mice in the CHS model allergic reaction.
- the mouse auricle in which CHS was induced was homogenized in Sepazol RNAI Super (Nacalai), mixed with chloroform, and the aqueous phase from which RNA was extracted was collected. From this, RNA was purified using 100% isopropanol and 75% ethanol. This RNA was reverse transcribed using SuperScript TM First-Strand Synthesis System for RT-PCR (Invitrogen) to obtain cDNA. Using this as a template, the expression level of IL-1R2 mRNA was examined using the RT-PCR method.
- Primer sequences used for PCR were IL-1R2 (Forward Primer; 5'-CTG GAA GGT GAA CCT GTG GT-3 '(SEQ ID NO: 1): Reverse Primer; 5'-CAT TTG CTC ACA GTG GGA TG-3 (SEQ ID NO: 2)), Gapdh (Forward Primer; 5′-ACC ACA GTC CAT GCC ATC TCT GCC A-3 ′ (SEQ ID NO: 3): Reverse Primer; 5′-TCC ACC ACC CTG TTG CTG TAG CTG TAG '(SEQ ID NO: 4)). Takara Ex Taq (Takara) was used as the thermostable DNA polymerase.
- the conditions for the PCR reaction were incubation at 94 ° C. for 2 minutes, followed by reaction for 35 cycles (94 ° C. for 15 seconds, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds), followed by 10 minutes at 72 ° C. This was performed under the condition of incubation for 1 minute.
- the IL-1 type II receptor has been thought to suppress IL-1 stimulation as a decoy receptor. Thus, it was predicted that allergic reactions would be exacerbated because IL-1 stimulation could not be suppressed in mice lacking IL-1 type II receptor gene. However, as shown in FIG. 6, although the IL-1 type II receptor gene was not expressed at all in the CHS model allergic reaction, as shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, Allergic reactions were suppressed more than wild type mice. This is a result contrary to the conventional prediction.
- IL-1 type II receptor gene-deficient mice as new IL-1 signal analysis tools Analysis using conventional gene-deficient mice reveals that IL-1 alpha signal is important for CHS exacerbation (Nakae et al., Int. Immunol. 13: 1471-1478 (2001)). Recently, in fibroblasts derived from scleroderma patients, pre-IL-1alpha present in the nucleus is involved in induction of expression of IL-6 and the like. At this time, pre-IL-1alpha is IL-1R2 It was reported that it functions as a transcription factor by forming a complex with (Kawaguchi, Y et al., Proc. Natl. Acad. Sci. USA, 103: 14151-14506 (2006)). . Thus, CHS, an IL-1alpha-dependent / IL-1beta-independent allergy model, is involved in the exacerbation of a new concept, pre-IL-1alpha / IL-1 type II receptor signal It was suggested that
- biologics targeting the IL-1 system eg, Anakinra
- Anakinra have also been put into practical use as therapeutic agents for immune diseases. From these, it is considered that the IL-1 type II receptor gene-deficient mouse is a valuable genetically modified mouse that is extremely useful and highly necessary as a signal analysis tool for IL-1.
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Abstract
To clarify the role of IL-1 type II receptor in the signal transduction by IL-1 ligand, a gene deficient mutant animal never expressing IL-1 type II receptor gene is constructed. Provided is an IL-1 type II receptor gene-deficient mutant of mouse which has deletion and/or insertion mutations at the IL-1 type II receptor locus on mouse chromosome. Also provided is a method of producing an antibody preparation for treating allergy and/or inflammation which comprises a step of immunologically sensitizing the mouse as described above with mouse IL-1 type II receptor protein and thus producing a monoclonal antibody against the receptor protein, and so on. Also disclosed is a method of screening a candidate for an antiallergic agent or an antiinflammatory agent which comprises a step of treating IL-1 type II receptor or a cell expressing the receptor with IL-1 in the presence or absence of a test substance, and so on.
Description
本発明は、ＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウスと、該変異体マウスを用いるアレルギー及び／又は炎症を治療する抗体製剤の作成方法と、抗アレルギー薬又は抗炎症薬候補のスクリーニング方法とに関する。
The present invention relates to an IL-1 type II receptor gene-deficient mutant mouse, a method for preparing an antibody preparation for treating allergy and / or inflammation using the mutant mouse, and a method for screening an antiallergic drug or anti-inflammatory drug candidate And about.
ＩＬ－１は向炎症性のサイトカインで、その産生及び／又は活性を下向き調節する因子は臨床的に重要な創薬ターゲットである。以下の非特許文献１の総説に説明されるとおり、ＩＬ－１リガンドのファミリーには、ＩＬ－１アルファ、ＩＬ－１ベータ及びＩＬ－１レセプターアンタゴニスト（ＩＬ－１Ｒａ）という３つのメンバーがあり、ＩＬ－１アルファ及びＩＬ－１ベータがアゴニストとして機能し、ＩＬ－１Ｒａは特異的なアンタゴニストであることが知られている。一方、ＩＬ－１レセプターのファミリーには、ＩＬ－１タイプＩレセプター（ＩＬ－１Ｒ Ｉ）と、ＩＬ－１タイプＩＩレセプター（ＩＬ－１Ｒ ＩＩ）と、ＩＬ－１レセプターアクセソリープロテイン（ＩＬ－１ＲＡｃＰ）という３つのメンバーがあり、ＩＬ－１タイプＩレセプターはＩＬ－１リガンドのシグナル伝達に関与するが、ＩＬ－１タイプＩＩレセプターは細胞内ドメインがほとんどなく、ＩＬ－１リガンドと結合してもシグナル伝達を行わないと考えられている。ＩＬ－１タイプＩＩレセプターは、タイプＩレセプターと複合体を形成することはないが、ＩＬ－１レセプターアクセソリープロテインは、ＩＬ－１タイプＩレセプターともＩＬ－１タイプＩＩレセプターとも複合体を形成することができる。
IL-1 is a pro-inflammatory cytokine, and factors that down-regulate its production and / or activity are clinically important drug discovery targets. As explained in the review of Non-Patent Document 1 below, the IL-1 ligand family has three members: IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1Ra), IL-1 alpha and IL-1 beta function as agonists and IL-1Ra is known to be a specific antagonist. On the other hand, the IL-1 receptor family includes IL-1 type I receptor (IL-1R I), IL-1 type II receptor (IL-1R II), and IL-1 receptor accessory protein (IL-1RAcP). The IL-1 type I receptor is involved in IL-1 ligand signaling, but the IL-1 type II receptor has little intracellular domain and can bind to the IL-1 ligand. It is thought that no signal transduction occurs. The IL-1 type II receptor does not form a complex with the type I receptor, whereas the IL-1 receptor accessory protein forms a complex with both the IL-1 type I receptor and the IL-1 type II receptor. be able to.
ＩＬ－１タイプＩＩレセプターは、従来、おとり（ｄｅｃｏｙ）レセプターであって、ＩＬ－１タイプＩレセプターとＩＬ－ｌリガンドとの結合を競合的に阻害することによって、ＩＬ－１シグナル伝達を調節すると考えられてきた。また、ＩＬ－１タイプＩＩレセプターは、ＩＬ－１リガンドと結合すると、ＩＬ－１レセプターアクセソリープロテインとも結合して複合体を形成することから、ＩＬ－１リガンドだけでなく、ＩＬ－１レセプターアクセソリープロテインについても、ＩＬ－１タイプＩレセプターとの結合を競合的に阻害するものと考えられてきた（非特許文献２）。さらにタイプＩＩレセプターの細胞外ドメインは、タンパク質分解酵素によって切断されて、可溶性断片として血液循環中に存在する。このタイプＩＩレセプターの可溶性断片は、レセプターアクセソリープロテインの可溶性断片と会合すると、ＩＬ－１アルファ及びベータへの親和性が１００倍高くなり、安定な可溶性複合体を形成することも知られている（非特許文献３）。
The IL-1 type II receptor is traditionally a decoy receptor that regulates IL-1 signaling by competitively inhibiting the binding of IL-1 type I receptor and IL-1 ligand. Has been considered. In addition, when an IL-1 type II receptor binds to an IL-1 ligand, it also binds to an IL-1 receptor accessory protein to form a complex. Thus, not only an IL-1 ligand but also an IL-1 receptor acceptor is formed. Solly protein has also been considered to competitively inhibit binding to IL-1 type I receptor (Non-patent Document 2). Furthermore, the extracellular domain of the type II receptor is cleaved by proteolytic enzymes and exists in the blood circulation as a soluble fragment. It is also known that the soluble fragment of this type II receptor, when associated with the soluble fragment of the receptor accessory protein, has a 100-fold higher affinity for IL-1 alpha and beta and forms a stable soluble complex. (Non-Patent Document 3).
表１に示すとおり、マウスＩＬ－１リガンド及びレセプターファミリー遺伝子については１９８４年にＩＬ－１アルファ遺伝子がクローニングされて以来、次々に遺伝子がクローニングされている。そして、クローニングされた遺伝子は、順次、ＥＳ細胞への相同遺伝子組換え法により遺伝子欠損マウスが作出されてきた。
As shown in Table 1, genes for mouse IL-1 ligand and receptor family genes have been cloned one after another since the cloning of the IL-1 alpha gene in 1984. The cloned genes have been produced in sequence by gene-deficient mice by homologous gene recombination methods to ES cells.
表１のうち、本発明の発明者らのグループ（Ｈｏｒａｉら、１９９８年）は、ＩＬ－１リガンドファミリー遺伝子の全てについて遺伝子欠損マウスを作出した。すなわち、ＩＬ－１アルファ及びＩＬ－１ベータ遺伝子のそれぞれの欠損マウスと、二重欠損マウスとを作出し、さらに、ＩＬ－１Ｒａ遺伝子の欠損マウスも作出した（非特許文献４）。
In Table 1, the group of the inventors of the present invention (Horai et al., 1998) created gene-deficient mice for all of the IL-1 ligand family genes. That is, a mouse deficient in each of IL-1alpha and IL-1beta genes and a double deficient mouse were created, and a mouse deficient in IL-1Ra gene was also created (Non-patent Document 4).
ＩＬ－１レセプターファミリー遺伝子の欠損マウスは他のグループによって作出されている。すなわち、ＩＬ－１タイプＩレセプター（ＩＬ－１Ｒ Ｉ）及びＩＬ－１レセプターアクセソリープロテイン（ＩＬ－１ＲＡｃＰ）の遺伝子欠損マウスについては、それぞれ、１９９７年及び１９９８年に作出されている。しかし、１９９１年にＩＬ－１タイプＩＩレセプター遺伝子がクローニングされているのに、ＩＬ－１タイプＩＩレセプター（ＩＬ－１Ｒ ＩＩ）遺伝子の欠損マウスは、世界中のいずれの研究室からも現在に至るまで作出されたという報告がない。
Mice deficient in the IL-1 receptor family gene have been created by other groups. That is, IL-1 type I receptor (IL-1R I) and IL-1 receptor accessory protein (IL-1RAcP) gene-deficient mice were created in 1997 and 1998, respectively. However, despite the cloning of the IL-1 type II receptor gene in 1991, mice lacking the IL-1 type II receptor (IL-1R II) gene have reached the present from any laboratory in the world. There is no report that it was made until.
ＩＬ－１関連遺伝子の欠損マウスのうち、発明者のグループで作出されたＩＬ－１Ｒａ遺伝子の欠損マウスは、関節炎を始めとする種々の自己免疫性疾患を自然発症することから、極めて有用な疾患モデル動物として利用されている（非特許文献５）。ＩＬ－１ＲａはＩＬ－１リガンドのアンタゴニストで、アゴニストであるＩＬ－１アルファやＩＬ－１ベータの刺激を阻害する。ＩＬ－１タイプＩＩレセプターもＩＬ－１リガンドやＩＬ－１ＲＡｃＰのデコイレセプターであり、ＩＬ－１アルファやＩＬ－１ベータの刺激を阻害すると考えられるので、ＩＬ－１タイプＩＩレセプター遺伝子（Ｉｌ１ｒ２遺伝子座）の欠損マウスも有用な疾患モデル動物となることが期待される。
Among mice lacking IL-1 related genes, mice lacking IL-1Ra gene produced by the inventor's group spontaneously develop various autoimmune diseases including arthritis. It is used as a model animal (Non-patent Document 5). IL-1Ra is an antagonist of IL-1 ligand and inhibits stimulation of agonists IL-1alpha and IL-1beta. The IL-1 type II receptor is also a decoy receptor for IL-1 ligand and IL-1RAcP, and is thought to inhibit IL-1 alpha and IL-1 beta stimulation. Therefore, the IL-1 type II receptor gene (Il1r2 locus) ) -Deficient mice are expected to be useful disease model animals.
ＩＬ－１タイプＩＩレセプターがＩＬ－１リガンドのシグナル伝達に果たす役割を解明するためには、ＩＬ－１タイプＩＩレセプター遺伝子が全く発現しない遺伝子欠損変異体動物を作出する必要がある。また、かかる動物個体におけるＩＬ－１リガンド及びレセプターの動態、複合体形成及びシグナル伝達を解析する方法を開発する必要がある。さらに、ＩＬ－１タイプＩＩレセプターを相補することができる物質の候補からＩＬ－１シグナル伝達調節活性を有する物質のスクリーニング方法を開発する必要もある。
In order to elucidate the role of IL-1 type II receptor in IL-1 ligand signaling, it is necessary to create a gene-deficient mutant animal that does not express any IL-1 type II receptor gene. There is also a need to develop methods to analyze IL-1 ligand and receptor dynamics, complex formation and signal transduction in such animals. Furthermore, it is also necessary to develop a screening method for substances having IL-1 signaling regulatory activity from candidate substances that can complement the IL-1 type II receptor.
本発明は、マウス染色体上のＩＬ?１タイプＩＩレセプター遺伝子座に欠失及び／又は挿入変異を有する、ＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウスを提供する。
The present invention provides an IL-1 type II receptor gene deletion mutant mouse having a deletion and / or insertion mutation at the IL-1 type II receptor locus on the mouse chromosome.
本発明のＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウスにおいて、該変異は、完全長ＩＬ－１タイプＩＩレセプタータンパク質の代わりに緑蛍光タンパク質が発現する場合がある。
In the IL-1 type II receptor gene-deficient mutant mouse of the present invention, the mutation may express a green fluorescent protein instead of the full-length IL-1 type II receptor protein.
本発明は、本発明のマウスにマウスＩＬ－１タイプＩＩレセプタータンパク質を免疫感作して、該レセプタータンパク質に対するモノクローナル抗体を作成するステップと、前記モノクローナル抗体を野生型マウスか野生型マウス由来の免疫細胞かに投与して、ＩＬ－１による免疫系への刺激を抑制させるモノクローナル抗体をスクリーニングするステップとを含む、アレルギー及び／又は炎症を治療する抗体製剤の作成方法を提供する。
The present invention includes a step of immunizing a mouse IL-1 type II receptor protein to the mouse of the present invention to produce a monoclonal antibody against the receptor protein, and the monoclonal antibody is immunized from a wild-type mouse or a wild-type mouse. And a method for producing an antibody preparation for treating allergy and / or inflammation, which comprises the step of screening a monoclonal antibody that suppresses stimulation of the immune system by IL-1 by administration to cells.
本発明は、試験物質存在下又は非存在下、ＩＬ－１タイプＩＩレセプターか、該レセプターを発現している細胞かにＩＬ－１を作用させるステップと、ＩＬ－１タイプＩＩレセプターとＩＬ－１との結合を測定するステップとを含む、抗アレルギー薬又は抗炎症薬候補のスクリーニング方法を提供する。
The present invention comprises a step of allowing IL-1 to act on an IL-1 type II receptor or a cell expressing the receptor in the presence or absence of a test substance, IL-1 type II receptor and IL-1 A method for screening an antiallergic drug or anti-inflammatory drug candidate.
本明細書において、ＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウスとは、野生型ＩＬ－１タイプＩＩレセプターを正常に産生することができないような変異体のマウスをいう。ＩＬ－１タイプＩＩレセプターの遺伝子座はマウスの第１番染色体に位置し、約４０．５ｋｂにわたる転写産物から９個のエクソンが連結したｍＲＮＡが産生され、４１０個のアミノ酸残基からなるポリペプチドが翻訳される。
In the present specification, a mutant mouse deficient in an IL-1 type II receptor gene refers to a mutant mouse that cannot normally produce a wild type IL-1 type II receptor. The IL-1 type II receptor locus is located on the first chromosome of mouse, a 9-exon-linked mRNA is produced from a transcript of about 40.5 kb, and a polypeptide consisting of 410 amino acid residues Is translated.
本発明の欠損変異体は、ＩＬ－１タイプＩＩレセプターのポリペプチドが全く産生されない変異体の他、ＩＬ－１タイプＩＩレセプターの野生型遺伝子の少なくとも１つエクソンの少なくとも一部にコード化されるポリペプチドが産生される場合であって、ＩＬ－１リガンドの結合、ＩＬ－１レセプターアクセソリープロテインとの会合、その他の機能のいずれか又は全てを欠いている変異体を含む。本発明の変異体は、ＩＬ－１タイプＩＩレセプター遺伝子座のうち、いずれのエクソン及び／又はイントロンのいかなる部分について欠失及び／又は挿入が生じていてもかまわない。本発明の変異体マウスには、２本の第１染色体の両方が前記変異を含むホモ接合個体だけでなく、２本の第１染色体のうち一方だけが前記変異を含み、他方は野生型である、ヘテロ接合個体も含む。
The deletion mutant of the present invention is encoded by at least a part of at least one exon of a wild type gene of IL-1 type II receptor, in addition to a mutant that does not produce any IL-1 type II receptor polypeptide. Where a polypeptide is produced, including variants that lack any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions. The variants of the present invention may have deletions and / or insertions in any part of any exon and / or intron of the IL-1 type II receptor locus. The mutant mouse of the present invention includes not only homozygous individuals in which both of the two first chromosomes contain the mutation, but also only one of the two first chromosomes contains the mutation and the other is a wild type. Some heterozygous individuals are also included.
本発明の変異体は化学物質、放射線その他のいかなる方法で作出してもかまわない。好ましい本発明の変異体の作出方法は、例えばＭａｎｉｐｕｌａｔｉｎｇ ｔｈｅ Ｍｏｕｓｅ Ｅｍｂｒｙｏ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ（３ｒｄ ｅｄ．， Ｎａｇｙ， Ａ． ｅｔ ａｌ． Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｐｒｅｓｓ Ｎ．Ｙ．， ２００３）に詳しく説明される胚性幹細胞（ＥＳ細胞）への相同遺伝子組換え法による標的遺伝子への変異導入である。
The mutant of the present invention may be created by chemical substances, radiation or any other method. A preferred method for producing the mutant of the present invention is, for example, Manipulating the Mouse Embryo: A Laboratory Manual (3rd ed., Nagy, A. et al. Cold Spring Harbor Press N.Y, explained in detail in 2003). Mutation introduction into a target gene by homologous gene recombination method into a stem cell (ES cell).
本発明の１つの実施態様では、緑蛍光タンパク質が産生されるような変異をＩＬ－１タイプＩＩレセプター遺伝子に導入した。しかし、ＩＬ－１タイプＩＩレセプターのポリペプチドが全く産生されない変異体か、ＩＬ－１タイプＩＩレセプターの野生型遺伝子の少なくとも１つエクソンの少なくとも一部にコード化されるポリペプチドが産生される場合であって、ＩＬ－１リガンドの結合、ＩＬ－１レセプターアクセソリープロテインとの会合、その他の機能のいずれか又は全てを欠いている変異体かであることを条件として、例えばＣｒｅのような配列特異的組換え酵素が認識するｌｏｘＰのような特異的組換え配列が挿入されたり、ＲＮＡｉその他遺伝子発現制御に関与するＲＮＡやｔＲＮＡの遺伝子が挿入されたり、緑蛍光タンパク質その他の蛍光タンパク質と、ウミシイタケ、ホタル等由来のルシフェラーゼその他の発光タンパク質と、アルカリフォスファターゼ、ペルオキシダーゼその他の酵素反応産物により酵素活性の定量又は局在が可能な酵素と、ネオマイシン、ハイグロマイシン、ゼオシン、ピューロマイシン、ブラストシジンＳその他の薬剤に対する耐性を付与する遺伝子産物と、栄養要求性による選択を可能にする遺伝子産物と、ＩＬ－１リガンドによるシグナル伝達機構とは明確に区別できる別のシグナル伝達機構に関与するタンパク質とを含むが、これらに限定されないいずれかのタンパク質が産生されたりしてもかまわない。
In one embodiment of the present invention, a mutation that produces green fluorescent protein was introduced into the IL-1 type II receptor gene. However, if a variant that does not produce any IL-1 type II receptor polypeptide or a polypeptide encoded by at least a part of an exon of at least one wild type gene of IL-1 type II receptor is produced A sequence such as Cre, provided that it is a mutant lacking any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions Specific recombination sequences such as loxP recognized by specific recombinase are inserted, RNAi and other RNA and tRNA genes involved in gene expression control are inserted, green fluorescent protein and other fluorescent proteins, and Renilla Luciferase and other photoproteins derived from fireflies, etc., and alkaline phosphate Enzymes whose enzyme activity can be quantified or localized by enzymatic products such as oxidase, peroxidase and other enzyme reaction products, gene products that confer resistance to neomycin, hygromycin, zeocin, puromycin, blasticidin S and other drugs, and nutritional requirements Any protein is produced that includes, but is not limited to, a gene product that allows for sexual selection and another signaling mechanism that is clearly distinguishable from that of IL-1 ligand signaling. It does not matter.
本発明の１つの実施態様で導入する変異では、ＩＬ－１タイプＩＩレセプター遺伝子の発現制御機構を用いて緑蛍光タンパク質を発現させることができる。かかる変異体マウスを用いると、緑蛍光タンパク質による蛍光発光を指標としてＩＬ－１タイプＩＩレセプター遺伝子の発現をモニターすることができる。前記変異体マウスを用いると、さまざまな創薬候補物質からＩＬ－１タイプＩＩレセプター遺伝子を上向き調節又は下向き調節する物質をハイスループットなやり方でスクリーニングすることができる。本発明のＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウス個体又はその細胞に創薬候補物質を投与及び／又は発現させるステップを含む、ＩＬ－１タイプＩＩレセプター遺伝子の発現に影響を与える物質のスクリーニング方法によって選ばれる物質は、ＩＬ－１タイプＩＩレセプター遺伝子の発現に影響を与えることを通じて、ＩＬ－１リガンドの刺激によるシグナル伝達機構を調節することができるので、新規な免疫調節医薬の開発につながる。
In the mutation introduced in one embodiment of the present invention, the green fluorescent protein can be expressed using the expression control mechanism of the IL-1 type II receptor gene. When such a mutant mouse is used, the expression of the IL-1 type II receptor gene can be monitored using fluorescence emitted by the green fluorescent protein as an index. By using the mutant mouse, it is possible to screen a substance that up-regulates or down-regulates an IL-1 type II receptor gene from various drug candidate substances in a high-throughput manner. A substance that affects the expression of an IL-1 type II receptor gene, comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mouse or a cell thereof that is a mutant mutant of the IL-1 type II receptor gene of the present invention. The substance selected by the screening method can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the expression of IL-1 type II receptor gene. Connected.
また、本発明のＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウス又はその細胞に創薬候補物質を投与及び／又は発現させることによって、本発明の変異が原因となるＩＬ－１タイプＩＩレセプターの機能不全を補償するような活性のある物質をスクリーニングすることができる。ＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウス個体又はその細胞に創薬候補物質を投与及び／又は発現させるステップを含む、ＩＬ－１タイプＩＩレセプター遺伝子の発現に影響を与える物質のスクリーニング方法によって選ばれる物質は、ＩＬ－１リガンドの複合体形成に影響を与えることを通じて、ＩＬ－１リガンドの刺激によるシグナル伝達機構を調節することができるので、新規な免疫調節医薬の開発につながる。
In addition, by administering and / or expressing a drug discovery candidate substance to a mutant mutant mouse of the IL-1 type II receptor gene of the present invention or a cell thereof, an IL-1 type II receptor caused by the mutation of the present invention can be obtained. Active substances that compensate for dysfunction can be screened. By a method for screening a substance that affects the expression of an IL-1 type II receptor gene, the method comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mutant mouse or a cell thereof lacking the IL-1 type II receptor gene The selected substance can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the complex formation of IL-1 ligand, leading to the development of a novel immunomodulatory drug.
ＩＬ－１リガンドの刺激によるシグナル伝達の下流には、例えば、ＩＬ－６、プロコラーゲン、ＭＣＰ（Monocyte Chemotactic Protein）－１、ＭＩＰ（Macrophage Inflammatory Protein）－２、ＰＧＥＳ（Prostaglandin E synthase）等を含むが、これらに限定されない遺伝子があり、ＩＬ－１リガンドによる刺激はこれらの遺伝子の発現の誘導又は亢進につながる。したがって上記の免疫調節医薬の開発において、ＩＬ－１リガンドの刺激によるシグナル伝達は、ＩＬ－６、プロコラーゲン、ＭＣＰ－１、ＭＩＰ－２、ＰＧＥＳ等を含むが、これらに限定されない遺伝子の発現量の変化に基づいて定量的に解析することができる。
Downstream of signal transduction due to stimulation of IL-1 ligand includes, for example, IL-6, procollagen, MCP (Monocyte Chemotactic Protein) -1, MIP (Macrophage Inflammatory Protein) -2, PGES (Prostaglandin E Synthase), etc. However, there are genes that are not limited to these, and stimulation with IL-1 ligand leads to induction or enhancement of the expression of these genes. Therefore, in the development of the above-described immunomodulatory drugs, signal transduction by IL-1 ligand stimulation includes IL-6, procollagen, MCP-1, MIP-2, PGES, etc., but is not limited thereto Quantitative analysis can be performed based on the change of.
本明細書において、創薬候補物質とは、新規医薬又はその開発のリード化合物となりうる物質であって、天然物質及び合成物質のいずれでもよく、なんらかの単位構造の化学物質の重合体又は多量体であっても、単量体であってもよく、いかなる化学構造の物質でもかまわない。前記創薬候補物質は、該物質の溶解特性等に応じた薬物伝達システムを用いてマウス個体又はその細胞に投与される場合がある。前記創薬候補物質がウイルス粒子又は核酸であって、マウス細胞内でなんらかの遺伝子の発現の誘導又は阻害を起こす機能を有するものであるときには、ウイルス感染、電気穿孔法、リポソーム法その他の方法で発現させる場合がある。
In this specification, a drug discovery candidate substance is a substance that can be a new drug or a lead compound for its development, and may be either a natural substance or a synthetic substance, or a polymer or multimer of chemical substances of some unit structure. It may be a monomer or a substance having any chemical structure. The drug discovery candidate substance may be administered to a mouse individual or its cells using a drug delivery system according to the dissolution characteristics of the substance. When the drug discovery candidate substance is a virus particle or nucleic acid and has a function of inducing or inhibiting the expression of some gene in mouse cells, it is expressed by virus infection, electroporation, liposome method or other methods. There is a case to let you.
ＩＬ－１リガンドは、膜結合型のＩＬ－１タイプＩＩレセプター又はその可溶性断片と結合することにより、ＩＬ－１タイプレセプターを介したシグナル伝達が阻害されると考えられてきた。しかし、本発明のＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウスでは、ＩＬ－１による刺激が抑制されるという結果が得られた。このことは、ＩＬ－１タイプＩＩレセプターは正常な個体ではＩＬ－１刺激を促進するという、これまで知られていなかった機能を果たしていることを示唆する。したがって、ＩＬ－１タイプＩＩレセプターのＩＬ－１刺激促進機能を抑制する物質を探索することにより、新規なアレルギー及び／又は炎症の治療薬を開発することができる。
It has been considered that IL-1 ligand binds to a membrane-bound IL-1 type II receptor or a soluble fragment thereof, thereby inhibiting signal transduction via the IL-1 type receptor. However, the IL-1 type II receptor gene-deficient mutant mouse of the present invention showed that IL-1 stimulation was suppressed. This suggests that the IL-1 type II receptor performs a previously unknown function of promoting IL-1 stimulation in normal individuals. Therefore, a novel allergy and / or inflammation therapeutic agent can be developed by searching for a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor.
前記ＩＬ－１タイプＩＩレセプターのＩＬ－１刺激促進機能を抑制する物質の１つの例は、ＩＬ－１タイプＩＩレセプターに対する抗体である。本発明のＩＬ－１タイプＩＩレセプター欠損マウスでは、マウスＩＬ－１タイプＩＩレセプターに対する免疫反応は抑制されないので、マウスＩＬ－１タイプＩＩレセプタータンパク質に対する抗体を作成することができる。かかる抗体には、本発明で用いられるモノクローナル抗体は、Ｋｏｈｌｅｒ及びＭｉｌｓｔｅｉｎ、Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．６：５１１－５１９、１９７６の技術と、その改良技術を用いて調製される場合がある。これらの方法は、所望の特異性を有する抗体を産生できる不死性細胞株の調製を伴う。かかる細胞株は上記のとおり免疫された本発明のＩＬ－１タイプＩＩレセプター欠損マウス由来の脾臓細胞から作成される場合がある。前記脾臓細胞は、さまざまな方法で不死化され、抗体産生能を有する不死化細胞株が調製される。前記脾臓細胞は、例えば、前記免疫された本発明のＩＬ－１タイプＩＩレセプター欠損マウスと同種又は異種の動物由来のミエローマ細胞との融合によって不死化される。当業者に周知のさまざまな融合技術を用いる場合がある。例えば、前記脾臓細胞とミエローマ細胞とは非イオン性界面活性剤と数分間混合され、それからハイブリッド細胞の増殖は支持するがミエローマ細胞の増殖は支持しない選択培地に低濃度でプレートされる。好ましい選択技術はＨＡＴ（ヒポキサンチン、アミノプテリン、チミジン）選択を用いる。通常約１ないし２週間の十分な時間の後、ハイブリッドのコロニーが観察される。シングルコロニーが選択され、その培養上清が前記ポリペプチドに対する結合活性についてテストされる。反応性及び特異性が高いハイブリドーマが好ましい。限界希釈法によるクローニングを繰り返すことにより、反応性及び特異的が高い抗体を安定的に大量に産生するハイブリドーマのクローンが選択される。モノクローナル抗体は増殖中の選択されたハイブリドーマクローン由来の細胞株のコロニーの上清から単離される場合がある。さらに、マウスのような適当な脊椎動物宿主の腹腔内に前記ハイブリドーマ細胞株を注射するような、収率を向上させるためのさまざまな技術が用いられる場合がある。モノクローナル抗体は前記ハイブリドーマ細胞腹水又は血液から回収される場合がある。汚染物は、クロマトグラフィー、ゲルろ過、沈殿及び抽出のような従来技術によって前記抗体から除去される場合がある。
One example of a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor is an antibody against the IL-1 type II receptor. In the IL-1 type II receptor-deficient mouse of the present invention, the immune response against the mouse IL-1 type II receptor is not suppressed, and therefore an antibody against the mouse IL-1 type II receptor protein can be prepared. For such antibodies, the monoclonal antibodies used in the present invention are described in Kohler and Milstein, Eur. J. et al. Immunol. 6: 511-519, 1976, and improved techniques thereof. These methods involve the preparation of immortal cell lines that can produce antibodies with the desired specificity. Such cell lines may be generated from spleen cells derived from IL-1 type II receptor-deficient mice of the present invention immunized as described above. The spleen cells are immortalized by various methods, and an immortal cell line having antibody-producing ability is prepared. The spleen cells are immortalized by, for example, fusion of the immunized IL-1 type II receptor-deficient mouse of the present invention with myeloma cells derived from the same or different animals. Various fusion techniques known to those skilled in the art may be used. For example, the spleen cells and myeloma cells are mixed with a nonionic surfactant for several minutes and then plated at a low concentration in a selective medium that supports the growth of hybrid cells but not the growth of myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. Hybrid colonies are observed after sufficient time, usually about 1 to 2 weeks. A single colony is selected and its culture supernatant is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred. By repeating the cloning by the limiting dilution method, a hybridoma clone that stably produces a large amount of highly reactive and specific antibody is selected. Monoclonal antibodies may be isolated from the supernatants of colonies of cell lines derived from selected growing hybridoma clones. In addition, various techniques may be used to improve yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host such as a mouse. Monoclonal antibodies may be recovered from the hybridoma cell ascites or blood. Contaminants may be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction.
本発明で用いられる抗体の抗原結合断片は、インタクトなポリクローナル抗体又はモノクローナル抗体をそれぞれタンパク質分解酵素パパイン又はペプシンで分解して得られるＦａｂ断片又はＦ（ａｂ’）２断片の他、天然抗体分子の抗原認識能及び結合能の多くを保持する抗原結合部位を含む非共有結合的なＶＨ及びＶＬ領域のヘテロ２量体を含むＦｖ断片を含む。
The antigen-binding fragment of the antibody used in the present invention includes an Fab fragment or F (ab ′) 2 fragment obtained by degrading an intact polyclonal antibody or monoclonal antibody with the proteolytic enzyme papain or pepsin, respectively, as well as natural antibody molecules. Includes Fv fragments containing non-covalent heterodimers of V H and V L regions that contain an antigen binding site that retains much of the ability to recognize and bind antigen.
本発明で用いられる組換え抗体は、適当な細菌宿主への形質転換か、適当なほ乳類細胞宿主へのトランスフェクションかを含む抗体遺伝子の発現クローニングによって調製される場合がある。本発明で用いられるキメラ抗体は、前記本発明の組換え抗体の抗原結合部位がＩＬ－１タイプＩＩレセプターと特異的に結合できるように同種又は異種の抗体の定常ドメインによって支持された融合タンパク質である。本発明で用いられるキメラ抗体には、抗体軽鎖可変領域（ＶＬ）に操作可能に連結された抗体重鎖可変領域（ＶＨ）を含む短鎖可変部抗体（ｓｃＦｖ）と、ラクダ科（Ｃａｍｅｌｉｄａｅ、ラクダ、ヒトコブラクダ、ラマを含む）の動物が産生する軽鎖がないＩｇＧのクラスであるラクダ重鎖抗体（ＨＣＡｂ）又はその重鎖可変部ドメイン（ＶＨＨ）とを含む。本発明の組換え抗体は原核生物及び真核生物由来の遺伝子発現システムを用いて大量に調製することができる。
The recombinant antibodies used in the present invention may be prepared by expression cloning of antibody genes including transformation into a suitable bacterial host or transfection into a suitable mammalian cell host. The chimeric antibody used in the present invention is a fusion protein supported by the constant domain of the same or different antibody so that the antigen-binding site of the recombinant antibody of the present invention can specifically bind to the IL-1 type II receptor. is there. The chimeric antibody used in the present invention includes a short chain variable region antibody (scFv) comprising an antibody heavy chain variable region (V H ) operably linked to an antibody light chain variable region (V L ), a camelid ( A camelid heavy chain antibody (HCAb), or a heavy chain variable region domain (VHH) thereof, which is a class of IgG without light chain produced by animals of Camelidae, camels, dromedaries, llamas). The recombinant antibodies of the present invention can be prepared in large quantities using prokaryotic and eukaryotic derived gene expression systems.
以下に実施例を示すが、これらは実施態様の例示を意図しており本発明の範囲を限定することは意図しない。
Examples are shown below, but these are intended to illustrate the embodiments and are not intended to limit the scope of the present invention.
ターゲティングベクター１の構造
図１は、ターゲティングベクター１の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。Ｅ２ないしＥ８はそれぞれ第２ないし第８エクソンの位置を表す。したがって、ＩＬ－１タイプＩＩレセプター遺伝子は図の左から右の向きに転写される。ターゲティングベクター１には、ターゲティングベクター１を取り込んだＥＳ細胞を選別するためのマーカーとしてハイグロマイシン耐性遺伝子を用い、その左側の相同領域は１．８ｋｂ、その右側の相同領域は１１．２ｋｂであった。ハイグロマイシン耐性遺伝子の矢印は、該遺伝子が左から右の向きに転写されることを示す。また、相同組換え体を選別するためのマーカーとして、前記左側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター１では第２エクソンから第５エクソンまでが欠失する。 Structure of TargetingVector 1 FIG. 1 is a schematic diagram showing the structure of targeting vector 1 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto. E2 to E8 represent the positions of the second to eighth exons, respectively. Therefore, the IL-1 type II receptor gene is transcribed from left to right in the figure. As the targeting vector 1, a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 1, the homologous region on the left side was 1.8 kb, and the homologous region on the right side was 11.2 kb. . The arrow of the hygromycin resistance gene indicates that the gene is transcribed from left to right. Further, as a marker for selecting a homologous recombinant, a diphtheria toxin gene is linked adjacent to the left homologous region. In targeting vector 1, the second exon to the fifth exon are deleted.
図１は、ターゲティングベクター１の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。Ｅ２ないしＥ８はそれぞれ第２ないし第８エクソンの位置を表す。したがって、ＩＬ－１タイプＩＩレセプター遺伝子は図の左から右の向きに転写される。ターゲティングベクター１には、ターゲティングベクター１を取り込んだＥＳ細胞を選別するためのマーカーとしてハイグロマイシン耐性遺伝子を用い、その左側の相同領域は１．８ｋｂ、その右側の相同領域は１１．２ｋｂであった。ハイグロマイシン耐性遺伝子の矢印は、該遺伝子が左から右の向きに転写されることを示す。また、相同組換え体を選別するためのマーカーとして、前記左側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター１では第２エクソンから第５エクソンまでが欠失する。 Structure of Targeting
相同組換え体ＥＳ細胞の単離
ターゲティングベクター１のトランスフェクションは以下のとおり行った。ＥＳ細胞Ｅ１４．１株１ｘ１０７個あたり２０μｇの直鎖化したターゲティングベクター１を５００μＦ、２５０Ｖの条件で電気穿孔法でトランスフェクションし、１４０μｇ／ｍＬのハイグロマイシンを添加したＥＳ細胞用培地で１週間培養して、ターゲティングベクター１を取り込んだＥＳ細胞を選択した。なお、ハイグロマイシン耐性遺伝子とジフテリア毒素遺伝子との間の相同領域で染色体ＤＮＡとターゲティングベクター１のＤＮＡとの間で相同組換えが起こっていない細胞では、ジフテリア毒素遺伝子が発現して細胞が死滅する場合が多く、結果的にハイグロマイシン耐性のＥＳ細胞のコロニーでは相同組換え体の頻度が上昇する。実際、発明者のグループの経験では、選択されたＥＳ細胞の１－４％の頻度で相同組換え体クローンが得られる。 Isolation of homologous recombinant ES cells Transfection of targetingvector 1 was performed as follows. 20 μg of linearized targeting vector 1 per 7 × 1 × 10 7 ES cells E14.1 strain was transfected by electroporation under conditions of 500 μF and 250 V for 1 week in ES cell medium supplemented with 140 μg / mL hygromycin After culturing, ES cells incorporating the targeting vector 1 were selected. In cells in which homologous recombination has not occurred between the chromosomal DNA and the DNA of the targeting vector 1 in the homologous region between the hygromycin resistance gene and the diphtheria toxin gene, the diphtheria toxin gene is expressed and the cell is killed. In many cases, the frequency of homologous recombination increases in colonies of ES cells resistant to hygromycin. Indeed, the inventors' group experience yields homologous recombinant clones with a frequency of 1-4% of the selected ES cells.
ターゲティングベクター１のトランスフェクションは以下のとおり行った。ＥＳ細胞Ｅ１４．１株１ｘ１０７個あたり２０μｇの直鎖化したターゲティングベクター１を５００μＦ、２５０Ｖの条件で電気穿孔法でトランスフェクションし、１４０μｇ／ｍＬのハイグロマイシンを添加したＥＳ細胞用培地で１週間培養して、ターゲティングベクター１を取り込んだＥＳ細胞を選択した。なお、ハイグロマイシン耐性遺伝子とジフテリア毒素遺伝子との間の相同領域で染色体ＤＮＡとターゲティングベクター１のＤＮＡとの間で相同組換えが起こっていない細胞では、ジフテリア毒素遺伝子が発現して細胞が死滅する場合が多く、結果的にハイグロマイシン耐性のＥＳ細胞のコロニーでは相同組換え体の頻度が上昇する。実際、発明者のグループの経験では、選択されたＥＳ細胞の１－４％の頻度で相同組換え体クローンが得られる。 Isolation of homologous recombinant ES cells Transfection of targeting
ターゲティングベクター１を導入したハイグロマイシン耐性のＥＳ細胞のクローンを１０９９個単離して、相同組換えを起こしているかどうかサザンブロット法で確認したが、相同組換えを起こしていたクローンは１個もなかった。
1099 clones of hygromycin-resistant ES cells introduced with the targeting vector 1 were isolated and confirmed by Southern blotting to determine whether homologous recombination had occurred. None of the clones had undergone homologous recombination. It was.
ターゲティングベクター２の構造
図２は、ターゲティングベクター２の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。ターゲティングベクター１には、ターゲティングベクター２を取り込んだＥＳ細胞を選別するためのマーカーとしてハイグロマイシン耐性遺伝子を用い、その左側の相同領域は３．２ｋｂ、その右側の相同領域は４．８ｋｂであった。また、相同組換え体を選別するためのマーカーとして、前記左側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター２では第２エクソンと第３エクソンの一部が欠失する。 Structure of TargetingVector 2 FIG. 2 is a schematic diagram showing the structure of targeting vector 2 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto. For the targeting vector 1, a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 2, and the left homologous region was 3.2 kb and the right homologous region was 4.8 kb. . Further, as a marker for selecting a homologous recombinant, a diphtheria toxin gene is linked adjacent to the left homologous region. In targeting vector 2, the second exon and a part of the third exon are deleted.
図２は、ターゲティングベクター２の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。ターゲティングベクター１には、ターゲティングベクター２を取り込んだＥＳ細胞を選別するためのマーカーとしてハイグロマイシン耐性遺伝子を用い、その左側の相同領域は３．２ｋｂ、その右側の相同領域は４．８ｋｂであった。また、相同組換え体を選別するためのマーカーとして、前記左側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター２では第２エクソンと第３エクソンの一部が欠失する。 Structure of Targeting
相同組換え体ＥＳ細胞の単離
ターゲティングベクター２のトランスフェクションはターゲティングベクター１と同様に行った。ターゲティングベクター２を導入したハイグロマイシン耐性のＥＳ細胞のクローンを７４９個単離して、相同組換えを起こしているかどうかサザンブロット法で確認したが、相同組換えを起こしていたクローンは１個もなかった。 Isolation of homologous recombinant ES cells Transfection of targetingvector 2 was performed in the same manner as targeting vector 1. 749 clones of hygromycin-resistant ES cells introduced with the targeting vector 2 were isolated and confirmed by Southern blotting whether homologous recombination had occurred. However, none of the clones had undergone homologous recombination. It was.
ターゲティングベクター２のトランスフェクションはターゲティングベクター１と同様に行った。ターゲティングベクター２を導入したハイグロマイシン耐性のＥＳ細胞のクローンを７４９個単離して、相同組換えを起こしているかどうかサザンブロット法で確認したが、相同組換えを起こしていたクローンは１個もなかった。 Isolation of homologous recombinant ES cells Transfection of targeting
ターゲティングベクター３の構造
図３は、ターゲティングベクター３の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。ターゲティングベクター３には、ターゲティングベクター３を取り込んだＥＳ細胞を選別するためのマーカーとしてネオマイシン耐性遺伝子を用い、その左側の相同領域は５．５ｋｂ、その右側の相同領域は２．７ｋｂであった。また、相同組換え体を選別するためのマーカーとして、前記右側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター３では第２エクソンの一部と、第３エクソン及び第４エクソンが欠失し、ＩＬ－１タイプＩＩレセプタータンパク質の代わりに緑蛍光タンパク質（ＥＧＦＰ）が発現するようにコード化される。 Structure of Targeting Vector 3 FIG. 3 is a schematic diagram showing the structure of targeting vector 3 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto. As the targeting vector 3, a neomycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 3. The homology region on the left side was 5.5 kb, and the homology region on the right side was 2.7 kb. Further, as a marker for selecting a homologous recombinant, a diphtheria toxin gene is linked adjacent to the right homologous region. In the targeting vector 3, a part of the second exon, the third exon and the fourth exon are deleted, and encoded such that green fluorescent protein (EGFP) is expressed instead of the IL-1 type II receptor protein.
図３は、ターゲティングベクター３の構造と、これに対応する野生型染色体ＤＮＡ及び相同組換え体ＤＮＡの構造とを示す模式図である。ターゲティングベクター３には、ターゲティングベクター３を取り込んだＥＳ細胞を選別するためのマーカーとしてネオマイシン耐性遺伝子を用い、その左側の相同領域は５．５ｋｂ、その右側の相同領域は２．７ｋｂであった。また、相同組換え体を選別するためのマーカーとして、前記右側の相同領域に隣接してジフテリア毒素遺伝子が連結されている。ターゲティングベクター３では第２エクソンの一部と、第３エクソン及び第４エクソンが欠失し、ＩＬ－１タイプＩＩレセプタータンパク質の代わりに緑蛍光タンパク質（ＥＧＦＰ）が発現するようにコード化される。 Structure of Targeting Vector 3 FIG. 3 is a schematic diagram showing the structure of targeting vector 3 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto. As the targeting vector 3, a neomycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 3. The homology region on the left side was 5.5 kb, and the homology region on the right side was 2.7 kb. Further, as a marker for selecting a homologous recombinant, a diphtheria toxin gene is linked adjacent to the right homologous region. In the targeting vector 3, a part of the second exon, the third exon and the fourth exon are deleted, and encoded such that green fluorescent protein (EGFP) is expressed instead of the IL-1 type II receptor protein.
相同組換え体ＥＳ細胞の単離
ターゲティングベクター３のトランスフェクションは、ハイグロマイシンの代わりに３００μｇ／ｍＬのＧ４１８をＥＳ細胞用培地に添加することを除いて、ターゲティングベクター１及び２と同様に行った。ターゲティングベクター３を導入したＧ４１８耐性のＥＳ細胞のクローンを１１５０個単離して、相同組換えを起こしているかどうかサザンブロット法で確認した。ここで、ＢａｍＨＩで消化したゲノムＤＮＡをアガロースゲル電気泳動により分離して固相に写し取り、第５エクソンの３’下流に存在するＫｐｎＩ－ＰｓｔＩ断片（約３６０ｂｐ）をプローブとして用いることにより、野生型約１０．５ｋｂに対して変異型は約７．０ ｋｂにシグナルを得ることができる（図４）。前記１１５０個のうち７Ａ１クローン１個だけが相同組換え体であった。 Isolation of homologous recombinant ES cells Transfection of targeting vector 3 was performed in the same manner as targeting vectors 1 and 2 except that 300 μg / mL G418 was added to the ES cell medium instead of hygromycin. . 1150 clones of G418-resistant ES cells into which the targeting vector 3 was introduced were isolated, and it was confirmed by Southern blotting whether homologous recombination occurred. Here, genomic DNA digested with BamHI was separated by agarose gel electrophoresis and copied to a solid phase, and a KpnI-PstI fragment (about 360 bp) existing 3 ′ downstream of the fifth exon was used as a probe. The mutant can obtain a signal at about 7.0 kb with respect to the type of about 10.5 kb (FIG. 4). Of the 1150, only one 7A1 clone was a homologous recombinant.
ターゲティングベクター３のトランスフェクションは、ハイグロマイシンの代わりに３００μｇ／ｍＬのＧ４１８をＥＳ細胞用培地に添加することを除いて、ターゲティングベクター１及び２と同様に行った。ターゲティングベクター３を導入したＧ４１８耐性のＥＳ細胞のクローンを１１５０個単離して、相同組換えを起こしているかどうかサザンブロット法で確認した。ここで、ＢａｍＨＩで消化したゲノムＤＮＡをアガロースゲル電気泳動により分離して固相に写し取り、第５エクソンの３’下流に存在するＫｐｎＩ－ＰｓｔＩ断片（約３６０ｂｐ）をプローブとして用いることにより、野生型約１０．５ｋｂに対して変異型は約７．０ ｋｂにシグナルを得ることができる（図４）。前記１１５０個のうち７Ａ１クローン１個だけが相同組換え体であった。 Isolation of homologous recombinant ES cells Transfection of targeting vector 3 was performed in the same manner as targeting
ＥＳ細胞キメラマウスの作出
ＩＬ－１タイプＩＩレセプター遺伝子座に変異を持つ７Ａ１クローンは定法に従いアグリーゲションキメラ法を用いてキメラマウスの作製を行った。すなわち、ＢＤＦ１オスマウスとＣ５７ＢＬ／６Ｊメスマウスとの交配により得られた８細胞期胚と５－１５個のＥＳ細胞とをＭ１６培地（Ｓｉｇｍａ）内で一晩接触共培養することによりキメラ胚盤胞を形成させた後、偽妊娠処理３日目のＩＣＲメスマウスの子宮内に移植することによりキメラマウスを出生させた。毛色による判定で７０％以上のＥＳ細胞の寄与率を示すオスキメラマウスは、メスＣ５７ＢＬ／６Ｊマウスとの交配を行い、生殖系列伝達試験を行った。ＥＳ細胞由来を示す野生色の仔マウスについては尻尾よりＤＮＡを抽出し、ターゲット遺伝子座（ＩＬ－１タイプＩＩレセプター遺伝子に相同組換えにより欠失及び挿入を有する遺伝子座）を持つかどうかＰＣＲ法及びサザンブロット法により確認を行った。ターゲット遺伝子座を持つマウスは、戻し交配（Ｃ５７ＢＬ／６Ｊ系及びＢＡＬＢ／ｃＡ系に８世代）行い、解析に用いた。 Production of ES cell chimeric mouse A 7A1 clone having a mutation in the IL-1 type II receptor gene locus was prepared according to a conventional method using an agglutination chimera method. Specifically, an 8-cell embryo obtained by crossing between a BDF1 male mouse and a C57BL / 6J female mouse and 5-15 ES cells were contact-cultured overnight in M16 medium (Sigma) to obtain a chimeric blastocyst. After formation, chimeric mice were born by transplanting into the uterus of ICR female mice on the third day of pseudopregnancy treatment. Male chimeric mice showing a contribution ratio of 70% or more ES cells as judged by hair color were mated with female C57BL / 6J mice, and a germline transmission test was performed. For wild-colored pups derived from ES cells, DNA is extracted from the tail, and whether the target locus (locus having a deletion and insertion by homologous recombination in the IL-1 type II receptor gene) is determined by the PCR method And confirmed by Southern blotting. Mice having the target locus were backcrossed (8 generations in the C57BL / 6J system and BALB / cA system) and used for analysis.
ＩＬ－１タイプＩＩレセプター遺伝子座に変異を持つ７Ａ１クローンは定法に従いアグリーゲションキメラ法を用いてキメラマウスの作製を行った。すなわち、ＢＤＦ１オスマウスとＣ５７ＢＬ／６Ｊメスマウスとの交配により得られた８細胞期胚と５－１５個のＥＳ細胞とをＭ１６培地（Ｓｉｇｍａ）内で一晩接触共培養することによりキメラ胚盤胞を形成させた後、偽妊娠処理３日目のＩＣＲメスマウスの子宮内に移植することによりキメラマウスを出生させた。毛色による判定で７０％以上のＥＳ細胞の寄与率を示すオスキメラマウスは、メスＣ５７ＢＬ／６Ｊマウスとの交配を行い、生殖系列伝達試験を行った。ＥＳ細胞由来を示す野生色の仔マウスについては尻尾よりＤＮＡを抽出し、ターゲット遺伝子座（ＩＬ－１タイプＩＩレセプター遺伝子に相同組換えにより欠失及び挿入を有する遺伝子座）を持つかどうかＰＣＲ法及びサザンブロット法により確認を行った。ターゲット遺伝子座を持つマウスは、戻し交配（Ｃ５７ＢＬ／６Ｊ系及びＢＡＬＢ／ｃＡ系に８世代）行い、解析に用いた。 Production of ES cell chimeric mouse A 7A1 clone having a mutation in the IL-1 type II receptor gene locus was prepared according to a conventional method using an agglutination chimera method. Specifically, an 8-cell embryo obtained by crossing between a BDF1 male mouse and a C57BL / 6J female mouse and 5-15 ES cells were contact-cultured overnight in M16 medium (Sigma) to obtain a chimeric blastocyst. After formation, chimeric mice were born by transplanting into the uterus of ICR female mice on the third day of pseudopregnancy treatment. Male chimeric mice showing a contribution ratio of 70% or more ES cells as judged by hair color were mated with female C57BL / 6J mice, and a germline transmission test was performed. For wild-colored pups derived from ES cells, DNA is extracted from the tail, and whether the target locus (locus having a deletion and insertion by homologous recombination in the IL-1 type II receptor gene) is determined by the PCR method And confirmed by Southern blotting. Mice having the target locus were backcrossed (8 generations in the C57BL / 6J system and BALB / cA system) and used for analysis.
ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスの表現型
ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスは、Ｃ５７ＢＬ／６Ｊ、ＢＡＬＢ／ｃＡいずれの遺伝的背景においてもメンデルの法則に則って出生し、外見上も特に異常は見当たらなかった。また、ＩＬ－１Ｒａ遺伝子欠損マウスと異なり、加齢と共にリウマチ様関節炎や乾癬様皮膚炎などの自己免疫疾患の発症は認められなかった。さらに、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスでは、ＩＬ－１リガンドに対するおとりレセプターが消失したことを示す表現型は、現在のところ認められていない。 Phenotype of IL-1 type II receptor gene-deficient mice IL-1 type II receptor gene-deficient mice are born according to Mendel's law in both C57BL / 6J and BALB / cA genetic backgrounds. There was no abnormality. Unlike IL-1Ra gene-deficient mice, autoimmune diseases such as rheumatoid arthritis and psoriasis-like dermatitis were not observed with aging. In addition, in IL-1 type II receptor gene-deficient mice, a phenotype indicating that the decoy receptor for the IL-1 ligand has disappeared has not been recognized at present.
ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスは、Ｃ５７ＢＬ／６Ｊ、ＢＡＬＢ／ｃＡいずれの遺伝的背景においてもメンデルの法則に則って出生し、外見上も特に異常は見当たらなかった。また、ＩＬ－１Ｒａ遺伝子欠損マウスと異なり、加齢と共にリウマチ様関節炎や乾癬様皮膚炎などの自己免疫疾患の発症は認められなかった。さらに、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスでは、ＩＬ－１リガンドに対するおとりレセプターが消失したことを示す表現型は、現在のところ認められていない。 Phenotype of IL-1 type II receptor gene-deficient mice IL-1 type II receptor gene-deficient mice are born according to Mendel's law in both C57BL / 6J and BALB / cA genetic backgrounds. There was no abnormality. Unlike IL-1Ra gene-deficient mice, autoimmune diseases such as rheumatoid arthritis and psoriasis-like dermatitis were not observed with aging. In addition, in IL-1 type II receptor gene-deficient mice, a phenotype indicating that the decoy receptor for the IL-1 ligand has disappeared has not been recognized at present.
ハプテン塗布による接触型過敏症モデル
ＩＬ－１のシグナルが増悪化に関与することが知られているハプテン塗布による接触型過敏症（ＣＨＳ）モデルをＩＬ－１タイプＩＩレセプター遺伝子欠損マウスに行なった。免疫感作するためのハプテンとしては２，４－ジニトロフルオロベンセン（ＤＮＦＢ）又は２，４，６－トリニトロクロロベンゼン（ＴＮＣＢ）を用いた。剃毛して２日後のマウスの腹部に０．５％に溶解したＤＮＦＢ又は３．０％に溶解したＴＮＣＢを５０μＬ塗布した。この時、溶媒としてオリーブオイル（和光）：アセトン（ナカライ）＝１：４の混合液を用いた。免疫感作５日後のマウスの片方の耳介に０．２％ＤＮＦＢ又は１．０％ＴＮＣＢを２５μＬ塗布し、もう一方の耳介には溶媒のみを塗布して炎症を誘導した。二度目のハプテン塗布から２４時間後にマウスの左右の耳介を直径６ｍｍのパンチで切り取りその重量差を測定した。
［耳介の腫れ（ｍｇ）］＝［ハプテンを塗布した耳介片重量（ｍｇ）］
－［溶媒を塗布した耳介片重量（ｍｇ）］。 Hapten-applied contact hypersensitivity model A hapten-applied contact hypersensitivity (CHS) model, which is known to be involved in the exacerbation of IL-1 signal, was performed on IL-1 type II receptor gene-deficient mice. 2,4-Dinitrofluorobenzene (DNFB) or 2,4,6-trinitrochlorobenzene (TNCB) was used as a hapten for immunization. 50 μL of DNFB dissolved in 0.5% or TNCB dissolved in 3.0% was applied to the abdomen of themouse 2 days after shaving. At this time, a mixed solution of olive oil (Wako): acetone (Nacalai) = 1: 4 was used as a solvent. Five days after immunization, 25 μL of 0.2% DNFB or 1.0% TNCB was applied to one pinna of the mouse, and inflammation was induced by applying only the solvent to the other pinna. 24 hours after the second hapten application, the left and right auricles of the mouse were cut with a punch having a diameter of 6 mm, and the weight difference was measured.
[Swelling of pinna (mg)] = [Weight of pinna to which hapten was applied (mg)]
-[Auricular piece weight (mg) coated with solvent].
ＩＬ－１のシグナルが増悪化に関与することが知られているハプテン塗布による接触型過敏症（ＣＨＳ）モデルをＩＬ－１タイプＩＩレセプター遺伝子欠損マウスに行なった。免疫感作するためのハプテンとしては２，４－ジニトロフルオロベンセン（ＤＮＦＢ）又は２，４，６－トリニトロクロロベンゼン（ＴＮＣＢ）を用いた。剃毛して２日後のマウスの腹部に０．５％に溶解したＤＮＦＢ又は３．０％に溶解したＴＮＣＢを５０μＬ塗布した。この時、溶媒としてオリーブオイル（和光）：アセトン（ナカライ）＝１：４の混合液を用いた。免疫感作５日後のマウスの片方の耳介に０．２％ＤＮＦＢ又は１．０％ＴＮＣＢを２５μＬ塗布し、もう一方の耳介には溶媒のみを塗布して炎症を誘導した。二度目のハプテン塗布から２４時間後にマウスの左右の耳介を直径６ｍｍのパンチで切り取りその重量差を測定した。
［耳介の腫れ（ｍｇ）］＝［ハプテンを塗布した耳介片重量（ｍｇ）］
－［溶媒を塗布した耳介片重量（ｍｇ）］。 Hapten-applied contact hypersensitivity model A hapten-applied contact hypersensitivity (CHS) model, which is known to be involved in the exacerbation of IL-1 signal, was performed on IL-1 type II receptor gene-deficient mice. 2,4-Dinitrofluorobenzene (DNFB) or 2,4,6-trinitrochlorobenzene (TNCB) was used as a hapten for immunization. 50 μL of DNFB dissolved in 0.5% or TNCB dissolved in 3.0% was applied to the abdomen of the
[Swelling of pinna (mg)] = [Weight of pinna to which hapten was applied (mg)]
-[Auricular piece weight (mg) coated with solvent].
ＤＮＦＢ塗布によるＣＨＳモデルアレルギー反応の結果を図５Ａ、ＴＮＣＢ塗布によるＣＨＳモデルアレルギー反応の結果を図５Ｂに示す。図５Ａ及び図５Ｂに示すとおり、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスでは、ハプテン塗布による接触型過敏症の発症が抑制された。
The result of CHS model allergic reaction by DNFB application is shown in FIG. 5A, and the result of CHS model allergic reaction by TNCB application is shown in FIG. 5B. As shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, the onset of contact-type hypersensitivity by hapten application was suppressed.
ＩＬ－１タイプＩＩレセプター遺伝子発現の確認
前記ＣＨＳモデルアレルギー反応においてＩＬ－１タイプＩＩレセプター遺伝子が野生型マウスでは発現していることを確認した。ＣＨＳを誘導したマウス耳介をセパゾールＲＮＡＩスーパー（ナカライ）中でホモジナイズし、クロロホルムを加えて混和した後、ＲＮＡが抽出された水相を回収した。ここから１００％イソプロパノール、７５％エタノールを用いてＲＮＡを精製した。このＲＮＡをＳｕｐｅｒＳｃｒｉｐｔ（商標）Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ Ｓｙｓｔｅｍ ｆｏｒ ＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて逆転写し、ｃＤＮＡを得た。これをテンプレートとしＲＴ－ＰＣＲ法を用いてＩＬ－１Ｒ２ｍＲＮＡ発現量を調べた。ＰＣＲに用いたプライマー配列は、ＩＬ－１Ｒ２（Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ； ５’ －ＣＴＧ ＧＡＡ ＧＧＴ ＧＡＡ ＣＣＴ ＧＴＧ ＧＴ－３’（配列番号１）： Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ； ５’ －ＣＡＴ ＴＴＧ ＣＴＣ ＡＣＡ ＧＴＧ ＧＧＡ ＴＧ－３’（配列番号２））、Ｇａｐｄｈ（Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ； ５’ －ＡＣＣ ＡＣＡ ＧＴＣ ＣＡＴ ＧＣＣ ＡＴＣ ＴＣＴ ＧＣＣ Ａ－３’（配列番号３）： Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ； ５’ －ＴＣＣ ＡＣＣ ＡＣＣ ＣＴＧ ＴＴＧ ＣＴＧ ＴＡＧ ＣＣＧ Ｔ－３’（配列番号４））である。耐熱性ＤＮＡポリメラーゼとしてＴａｋａｒａ Ｅｘ Ｔａｑ（Ｔａｋａｒａ）を用いた。ＰＣＲ反応の条件は、９４°Ｃで２分間インキュベーションした後、（９４°Ｃで１５秒間、５８°Ｃで３０秒間、７２°Ｃで３０秒間）×３５サイクル反応させ、その後７２°Ｃで１０分間インキュベーションする条件で行った。 Confirmation of IL-1 type II receptor gene expression It was confirmed that the IL-1 type II receptor gene was expressed in wild-type mice in the CHS model allergic reaction. The mouse auricle in which CHS was induced was homogenized in Sepazol RNAI Super (Nacalai), mixed with chloroform, and the aqueous phase from which RNA was extracted was collected. From this, RNA was purified using 100% isopropanol and 75% ethanol. This RNA was reverse transcribed using SuperScript ™ First-Strand Synthesis System for RT-PCR (Invitrogen) to obtain cDNA. Using this as a template, the expression level of IL-1R2 mRNA was examined using the RT-PCR method. Primer sequences used for PCR were IL-1R2 (Forward Primer; 5'-CTG GAA GGT GAA CCT GTG GT-3 '(SEQ ID NO: 1): Reverse Primer; 5'-CAT TTG CTC ACA GTG GGA TG-3 (SEQ ID NO: 2)), Gapdh (Forward Primer; 5′-ACC ACA GTC CAT GCC ATC TCT GCC A-3 ′ (SEQ ID NO: 3): Reverse Primer; 5′-TCC ACC ACC CTG TTG CTG TAG CTG TAG '(SEQ ID NO: 4)). Takara Ex Taq (Takara) was used as the thermostable DNA polymerase. The conditions for the PCR reaction were incubation at 94 ° C. for 2 minutes, followed by reaction for 35 cycles (94 ° C. for 15 seconds, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds), followed by 10 minutes at 72 ° C. This was performed under the condition of incubation for 1 minute.
前記ＣＨＳモデルアレルギー反応においてＩＬ－１タイプＩＩレセプター遺伝子が野生型マウスでは発現していることを確認した。ＣＨＳを誘導したマウス耳介をセパゾールＲＮＡＩスーパー（ナカライ）中でホモジナイズし、クロロホルムを加えて混和した後、ＲＮＡが抽出された水相を回収した。ここから１００％イソプロパノール、７５％エタノールを用いてＲＮＡを精製した。このＲＮＡをＳｕｐｅｒＳｃｒｉｐｔ（商標）Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ Ｓｙｓｔｅｍ ｆｏｒ ＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて逆転写し、ｃＤＮＡを得た。これをテンプレートとしＲＴ－ＰＣＲ法を用いてＩＬ－１Ｒ２ｍＲＮＡ発現量を調べた。ＰＣＲに用いたプライマー配列は、ＩＬ－１Ｒ２（Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ； ５’ －ＣＴＧ ＧＡＡ ＧＧＴ ＧＡＡ ＣＣＴ ＧＴＧ ＧＴ－３’（配列番号１）： Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ； ５’ －ＣＡＴ ＴＴＧ ＣＴＣ ＡＣＡ ＧＴＧ ＧＧＡ ＴＧ－３’（配列番号２））、Ｇａｐｄｈ（Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒ； ５’ －ＡＣＣ ＡＣＡ ＧＴＣ ＣＡＴ ＧＣＣ ＡＴＣ ＴＣＴ ＧＣＣ Ａ－３’（配列番号３）： Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ； ５’ －ＴＣＣ ＡＣＣ ＡＣＣ ＣＴＧ ＴＴＧ ＣＴＧ ＴＡＧ ＣＣＧ Ｔ－３’（配列番号４））である。耐熱性ＤＮＡポリメラーゼとしてＴａｋａｒａ Ｅｘ Ｔａｑ（Ｔａｋａｒａ）を用いた。ＰＣＲ反応の条件は、９４°Ｃで２分間インキュベーションした後、（９４°Ｃで１５秒間、５８°Ｃで３０秒間、７２°Ｃで３０秒間）×３５サイクル反応させ、その後７２°Ｃで１０分間インキュベーションする条件で行った。 Confirmation of IL-1 type II receptor gene expression It was confirmed that the IL-1 type II receptor gene was expressed in wild-type mice in the CHS model allergic reaction. The mouse auricle in which CHS was induced was homogenized in Sepazol RNAI Super (Nacalai), mixed with chloroform, and the aqueous phase from which RNA was extracted was collected. From this, RNA was purified using 100% isopropanol and 75% ethanol. This RNA was reverse transcribed using SuperScript ™ First-Strand Synthesis System for RT-PCR (Invitrogen) to obtain cDNA. Using this as a template, the expression level of IL-1R2 mRNA was examined using the RT-PCR method. Primer sequences used for PCR were IL-1R2 (Forward Primer; 5'-CTG GAA GGT GAA CCT GTG GT-3 '(SEQ ID NO: 1): Reverse Primer; 5'-CAT TTG CTC ACA GTG GGA TG-3 (SEQ ID NO: 2)), Gapdh (Forward Primer; 5′-ACC ACA GTC CAT GCC ATC TCT GCC A-3 ′ (SEQ ID NO: 3): Reverse Primer; 5′-TCC ACC ACC CTG TTG CTG TAG CTG TAG '(SEQ ID NO: 4)). Takara Ex Taq (Takara) was used as the thermostable DNA polymerase. The conditions for the PCR reaction were incubation at 94 ° C. for 2 minutes, followed by reaction for 35 cycles (94 ° C. for 15 seconds, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds), followed by 10 minutes at 72 ° C. This was performed under the condition of incubation for 1 minute.
図６に示すとおり、ＲＴ?ＰＣＲ法によるＩＬ－１タイプＩＩレセプター遺伝子発現解析の結果、野生型マウスでは、ＤＮＦＢ塗布によるＣＨＳモデルアレルギー反応の際にＩＬ－１タイプＩＩレセプター遺伝子は発現が亢進したが、ＩＬ－１タイプＩＩレセプター遺伝子座の欠損マウスではＩＬ－１タイプＩＩレセプター遺伝子の発現は全く検出されなかった。
As shown in FIG. 6, as a result of IL-1 type II receptor gene expression analysis by RT-PCR method, in wild type mice, expression of IL-1 type II receptor gene was enhanced during CHS model allergic reaction by DNFB application. However, no IL-1 type II receptor gene expression was detected in mice lacking the IL-1 type II receptor locus.
ＩＬ－１タイプＩＩレセプターはおとりレセプターとしてＩＬ－１刺激を抑制すると考えられてきた。そこで、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスではＩＬ－１刺激を抑制することができないからアレルギー反応は増悪すると予測された。しかし、図６に示すとおり、ＣＨＳモデルアレルギー反応ではＩＬ－１タイプＩＩレセプター遺伝子が全く発現していないにもかかわらず、図５Ａ及び図５Ｂに示すとおり、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスではアレルギー反応は野生型マウスよりも抑制された。これは、従来の予測に反する結果である。
The IL-1 type II receptor has been thought to suppress IL-1 stimulation as a decoy receptor. Thus, it was predicted that allergic reactions would be exacerbated because IL-1 stimulation could not be suppressed in mice lacking IL-1 type II receptor gene. However, as shown in FIG. 6, although the IL-1 type II receptor gene was not expressed at all in the CHS model allergic reaction, as shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, Allergic reactions were suppressed more than wild type mice. This is a result contrary to the conventional prediction.
新しいＩＬ－１シグナル解析ツールとしてのＩＬ－１タイプＩＩレセプター遺伝子欠損マウス
これまでの遺伝子欠損マウスを用いた解析から、ＣＨＳの増悪化にはＩＬ－１アルファのシグナルが重要であることが明らかになっていた（Ｎａｋａｅら、Ｉｎｔ． Ｉｍｍｕｎｏｌ．１３：１４７１－１４７８（２００１））。また、最近、強皮症患者由来の繊維芽細胞では核内に存在するｐｒｅ－ＩＬ－１アルファがＩＬ－６などの発現誘導に関与すること、このときｐｒｅ－ＩＬ－１アルファはＩＬ－１Ｒ２と複合体を形成することにより転写因子として機能していることが報告された（Ｋａｗａｇｕｃｈｉ，Ｙら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ．， １０３：１４５０１－１４５０６（２００６））。このことから、ＩＬ－１アルファ依存性／ＩＬ－１ベータ非依存性のアレルギー・モデルであるＣＨＳは、新しい概念であるｐｒｅ－ＩＬ－１アルファ／ＩＬ－１タイプＩＩレセプターシグナルが増悪化に関与していると示唆された。 IL-1 type II receptor gene-deficient mice as new IL-1 signal analysis tools Analysis using conventional gene-deficient mice reveals that IL-1 alpha signal is important for CHS exacerbation (Nakae et al., Int. Immunol. 13: 1471-1478 (2001)). Recently, in fibroblasts derived from scleroderma patients, pre-IL-1alpha present in the nucleus is involved in induction of expression of IL-6 and the like. At this time, pre-IL-1alpha is IL-1R2 It was reported that it functions as a transcription factor by forming a complex with (Kawaguchi, Y et al., Proc. Natl. Acad. Sci. USA, 103: 14151-14506 (2006)). . Thus, CHS, an IL-1alpha-dependent / IL-1beta-independent allergy model, is involved in the exacerbation of a new concept, pre-IL-1alpha / IL-1 type II receptor signal It was suggested that
これまでの遺伝子欠損マウスを用いた解析から、ＣＨＳの増悪化にはＩＬ－１アルファのシグナルが重要であることが明らかになっていた（Ｎａｋａｅら、Ｉｎｔ． Ｉｍｍｕｎｏｌ．１３：１４７１－１４７８（２００１））。また、最近、強皮症患者由来の繊維芽細胞では核内に存在するｐｒｅ－ＩＬ－１アルファがＩＬ－６などの発現誘導に関与すること、このときｐｒｅ－ＩＬ－１アルファはＩＬ－１Ｒ２と複合体を形成することにより転写因子として機能していることが報告された（Ｋａｗａｇｕｃｈｉ，Ｙら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ．， １０３：１４５０１－１４５０６（２００６））。このことから、ＩＬ－１アルファ依存性／ＩＬ－１ベータ非依存性のアレルギー・モデルであるＣＨＳは、新しい概念であるｐｒｅ－ＩＬ－１アルファ／ＩＬ－１タイプＩＩレセプターシグナルが増悪化に関与していると示唆された。 IL-1 type II receptor gene-deficient mice as new IL-1 signal analysis tools Analysis using conventional gene-deficient mice reveals that IL-1 alpha signal is important for CHS exacerbation (Nakae et al., Int. Immunol. 13: 1471-1478 (2001)). Recently, in fibroblasts derived from scleroderma patients, pre-IL-1alpha present in the nucleus is involved in induction of expression of IL-6 and the like. At this time, pre-IL-1alpha is IL-1R2 It was reported that it functions as a transcription factor by forming a complex with (Kawaguchi, Y et al., Proc. Natl. Acad. Sci. USA, 103: 14151-14506 (2006)). . Thus, CHS, an IL-1alpha-dependent / IL-1beta-independent allergy model, is involved in the exacerbation of a new concept, pre-IL-1alpha / IL-1 type II receptor signal It was suggested that
ＩＬ－１は、ＮＣＢＩが公開している医学・生物学の文献データベースであるＰｕｂＭｅｄ（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｓｉｔｅｓ／ｅｎｔｒｅｚ？ｄｂ＝ＰｕｂＭｅｄ）で検索した場合、２００８年１月現在で４３，０１４報もの論文がヒットする、極めて注目度の高い分子である。また、ＩＬ－１システムを標的とした生物製剤（例：Ａｎａｋｉｎｒａ）も免疫疾患の治療薬として実用化に至っている。これらのことから、ＩＬ－１タイプＩＩレセプター遺伝子欠損マウスはＩＬ－１のシグナル解析ツールとして、極めて有用かつ必要度の高い、貴重な遺伝子改変マウスであると考えられる。
IL-1 is searched by PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed), which is a database of medical and biological literature published by NCBI. As of January of this year, 43,014 papers are hit, and this is a very high-profile molecule. In addition, biologics targeting the IL-1 system (eg, Anakinra) have also been put into practical use as therapeutic agents for immune diseases. From these, it is considered that the IL-1 type II receptor gene-deficient mouse is a valuable genetically modified mouse that is extremely useful and highly necessary as a signal analysis tool for IL-1.
Claims (4)
- マウス染色体上のＩＬ－１タイプＩＩレセプター遺伝子座に欠失及び／又は挿入変異を有することを特徴とする、ＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウス。 An IL-1 type II receptor gene deletion mutant mouse, characterized by having a deletion and / or insertion mutation at the IL-1 type II receptor locus on the mouse chromosome.
- 前記変異は、完全長ＩＬ－１タイプＩＩレセプタータンパク質の代わりに緑蛍光タンパク質が発現することを特徴とする、請求項１に記載のＩＬ－１タイプＩＩレセプター遺伝子の欠損変異体マウス。 The IL-1 type II receptor gene-deficient mutant mouse according to claim 1, wherein the mutation expresses green fluorescent protein instead of full-length IL-1 type II receptor protein.
- 請求項１又は２に記載のマウスにマウスＩＬ－１タイプＩＩレセプタータンパク質を免疫感作して、該レセプタータンパク質に対するモノクローナル抗体を作成するステップと、前記モノクローナル抗体を野生型マウスか野生型マウス由来の免疫細胞かに投与して、ＩＬ－１による免疫系への刺激を抑制させるモノクローナル抗体をスクリーニングするステップとを含むことを特徴とする、アレルギー及び／又は炎症を治療する抗体製剤の作成方法。 A step of immunizing a mouse IL-1 type II receptor protein in the mouse according to claim 1 or 2 to produce a monoclonal antibody against the receptor protein, and said monoclonal antibody is derived from a wild type mouse or a wild type mouse. A method for producing an antibody preparation for treating allergy and / or inflammation, which comprises the step of screening for a monoclonal antibody that is administered to immune cells and suppresses stimulation of the immune system by IL-1.
- 試験物質存在下又は非存在下、ＩＬ－１タイプＩＩレセプターか、該レセプターを発現している細胞かにＩＬ－１を作用させるステップと、ＩＬ－１タイプＩＩレセプターとＩＬ－１との結合を測定するステップとを含むことを特徴とする、抗アレルギー薬又は抗炎症薬候補のスクリーニング方法。 Allowing IL-1 to act on IL-1 type II receptor or cells expressing the receptor in the presence or absence of a test substance, and binding of IL-1 type II receptor to IL-1 A method for screening an anti-allergic drug or anti-inflammatory drug candidate.
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