CANE protein

Abstract

Provision of novel NLRP3 inflammasome constituent proteins. [Solution] A protein that binds to NLRP3, selected from (a) to (c) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 2 (b) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 2, and which binds to NLRP3. (c) A protein that has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 2 and that binds to NLRP3.

Description

[Technical Field]

[0001] This invention relates to the CANE protein, an antibody against the CANE protein, and a transgenic non-human mammal expressing the CANE protein. [Background technology]

[0002] NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) (also known as "cryopyrin") is a pattern recognition receptor involved in the host's innate immune response (Non-patent Literature 1: Manji, GA et al., 2002. Journal of Biological Chemistry 277: 11570-11575; Non-patent Literature 2: Mariathasan S, et al., 2006. Nature 440: 228-232; Non-patent Literature 3: Kanneganti, T.-D. et al., 2006. Nature 440: 233-236; Non-patent Literature 4: Martinon, F. et al., 2006. Nature 440: 237-241). When NLRP3 senses DAMP (damage-associated molecular pattern) or PAMP (pathogen-associated molecular pattern), it forms a large complex called the NLRP3 inflammasome, which leads to the activation of IL-1β and IL-18 (Non-patent Literature 5: Swanson, KV et al., 2019. Nat Rev Immunol 19: 477-489). Gain-of-function mutations in CIAS1, the gene encoding NLRP3, induce abnormal activation of the NLRP3 inflammasome, thereby causing cryopyrin-associated periodic fever syndrome (CAPS) (Non-patent Literature 6: McDermott, MF et al., 1999. Cell 97: 133-144). However, the pathogenesis of CAPS is not yet fully understood. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Manji, GA et al., 2002. Journal of Biological Chemistry 277: 11570-11575. [Non-Patent Document 2] Mariathasan S, et al., 2006. Nature 440: 228-232. [Non-Patent Document 3] Kanneganti, T.-D. et al., 2006. Nature 440: 233-236. [Non-Patent Document 4] Martinon, F. et al., 2006. Nature 440: 237-241. [Non-Patent Document 5] Swanson, KV et al., 2019. Nat Rev Immunol 19: 477-489. [Non-Patent Document 6] McDermott, MF et al., 1999. Cell 97: 133-144. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] This invention was made in view of the above circumstances, and the problem it aims to solve is to provide a novel protein related to the NLRP3 inflammasome. [Means for solving the problem]

[0005] As a result of diligent research to solve the above problems, the inventors of this invention succeeded in identifying a novel protein related to the NLRP3 inflammasome, and thus completed the present invention. In other words, the present invention is as follows:

[0006] [1] A protein that binds to NLRP3, selected from (a) to (c) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 2 (b) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 2, and which binds to NLRP3. (c) A protein that has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 2 and that binds to NLRP3. [2] A polynucleotide containing the base sequence that codes for the protein described in [1] above. [3] A vector containing the polynucleotide described in [2] above. [4] A transgenic non-human mammal containing the polynucleotides described in [2] above. [5] A non-human mammalian model of dermatitis, as described in [4] above: a transgenic non-human mammal. [6] An antibody against the protein described in [1] above. [7] The antibody described in [6] above, which is a monoclonal antibody or an antigen-binding fragment thereof. [8] The antibody described in [7] above, wherein the monoclonal antibody is a chimeric antibody, a humanized antibody, or a fully human antibody. [9] A composition comprising the protein described in [1] above or the polynucleotide described in [2] above.

[10] A method for producing the protein described in [1] above, comprising the step of expressing a protein from the vector described in [3] above in a host cell or a cell-free protein synthesis system. [Effects of the Invention]

[0007] The present invention is useful for elucidating a new mechanism related to the NLRP3 inflammasome.

Brief Description of the Drawings

[0008] [Figure 1] A: Schematic diagram of screening using ALPHA. In this screening, the interaction between biotinylated NLRP3 and candidate human proteins is detected. B: Regarding the effect of 11 candidate proteins on NLRP3 inflammasome activation, NLRP3, ASC, procaspase-1, and pro-IL-1β, which are constituent proteins of the NLRP3 inflammasome, were forcibly expressed in human embryonic kidney-derived cell line (HEK: human embryonic kidney) 293T cells together with each candidate protein, and the figure shows the result of measuring the concentration of IL-1β in the culture supernatant by ELISA. C: The figure shows the result of testing the co-immunoprecipitation of 11 candidate proteins and NLRP3. D: The figure shows the result of testing the co-immunoprecipitation of FLJ40383 (CANE) and any one of NLRP3 (PYD), NLRP3 (NOD), and NLRP3 (LRR). [Figure 2] A: The figure shows the result of Western blot analysis regarding the expression of CANE protein (hereinafter, also simply referred to as "CANE") in various cell lines. B: The figure shows the result of examining the localization by forcibly expressing DsRed-CANE fused with Discosoma Red Fluorescent Protein (DsRed) and EGFP-NLRP3 fused with Enhanced Green Fluorescent Protein (EGFP) in HEK293T cells. [Figure 3]Figure showing the results of testing co-immunoprecipitation of endogenous NLRP3 and endogenous CANE in macrophages differentiated from THP-1 cells. B: Figure showing the results of imaging the localization of endogenous CANE and endogenous NLRP3 in macrophages differentiated from THP-1 cells by immunofluorescence staining with a CANE antibody (hereinafter also referred to as the "anti-CANE antibody"). C: Figure showing the results of measuring the number of speck-like light spots as a percentage (%) relative to the number of cells. [Figure 4] Figure showing the results of a knockdown test of CANE using morpholino antisense oligonucleotides. A: Figure showing the results of Western blot analysis of the processing of pro-IL-1β (p31) to cleaved IL-1β (p17), the processing of procaspase-1 (p50) to cleaved caspase-1 (p20), and the expression of CANE in the culture supernatant. B: Figure showing the results of Western blot analysis of the processing of pro-IL-1β (p31) to cleaved IL-1β (p17), procaspase-1 (p50), and the expression of CANE in the NP-40 buffer-soluble cell lysate. C: Figure showing the results of Western blot analysis of the expression of CANE in the NP-40 buffer-insoluble precipitate. D: Figure showing the results of measuring the IL-1β concentration and TNF-α concentration in the culture supernatant of THP-1 by ELISA to investigate the effect of CANE knockdown in THP-1 cells on the secretion of IL-1β and TNF-α from THP-1 cells. [Figure 5] A: Figure showing the results of confirming the expression of CANE messenger RNA in CANE transgenic mice (lines 1 and 2) by RT-PCR. B: Figure showing the results of confirming the expression of CANE protein in CANE transgenic mice by Western blotting. C: Figure showing the results of measuring the IL-1β concentration and TNF-α concentration in the culture supernatant of PBMC by ELISA to investigate the secretion of IL-1β and TNF-α from PBMC of wild-type mice and CANE transgenic mice when stimulated with LPS. [Figure 6] A: This figure shows the results of a Western blot analysis of the processing of procaspase-1 (p45) to cleavage caspase-1 (p20) in BMDM derived from CANE transgenic mice. B: This figure shows the results of an ELISA analysis of IL-1β and TNF-α secretion from BMDM derived from CANE transgenic mice. C: This figure shows the results of an ELISA measurement of IL-1β and TNF-α secretion in the culture supernatant of BMDM derived from CANE transgenic mice when poly(dA:dT) was introduced using Xfect Transfection Reagent and when flagellin was introduced using DOTAP. [Figure 7] A: Image showing the appearance of a CANE transgenic mouse. B: Histopathological image of a skin tissue section derived from a CANE transgenic mouse. C: Figure showing the results of quantitative real-time RT-PCR measurement of IL-1β mRNA and TNF-α mRNA expression in CANE transgenic mice. DG: Figure showing the results of semi-quantitative histological analysis of skin tissue. DG represents, respectively, the number of infiltrating neutrophils per tissue unit area (mm2), the number of attached neutrophils per capillary, the circumference of the capillary (μm), and the average number of capillaries. [Modes for carrying out the invention]

[0009] The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be implemented in various forms without departing from its spirit.

[0010] 1. overview Gain-of-function mutations in CIAS1, the gene encoding NLRP3, induce abnormal activation of the NLRP3 inflammasome, leading to cryopyrin-associated periodic syndrome (CAPS). However, there are cases in which the above mutation is not found, and the pathogenesis of CAPS is not yet fully understood. In response to this, the inventors succeeded in identifying a novel protein related to the NLRP3 inflammasome, thus completing the present invention. This novel protein is a protein that binds to NLRP3 and has been named CANE (cryopyrin-associated nano enhancer) by the inventors. The inventors demonstrated that CANE increases IL-1β secretion following NLRP3 inflammasome formation induced by NLRP3 agonist stimulation such as LPS and Nigericin (hereinafter also referred to as "nigericin"). Furthermore, the inventors showed that decreased expression of endogenous CANE leads to decreased IL-1β secretion induced by NLRP3 agonist stimulation. In other words, the protein CANE newly discovered by the inventors is a regulator that enhances the activation of the NLRP3 inflammasome and has been shown to be a factor contributing to the pathogenesis of CAPS.

[0011] Furthermore, the inventors developed a new CANE transgenic mouse (a mouse into which the CANE gene has been introduced) and demonstrated that IL-1β secretion is increased in this mouse. They also showed that autoinflammatory neutrophil infiltration is increased compared to wild-type (WT) mice. These phenotypes in the CANE transgenic mouse were consistent with the phenotypes of the CAPS model mouse. In other words, the CANE transgenic mouse newly developed by the inventors has been shown to be useful as a novel CAPS model mouse. Thus, the proteins, antibodies, and transgenic non-human mammals of the present invention are all extremely useful in elucidating mechanisms related to the NLRP3 inflammasome, including the pathogenesis of CAPS, and in developing new pharmaceuticals.

[0012] 2. The protein of the present invention CANE (cryopyrin-associated nano enhancer) is a protein newly discovered by the present inventors and is a regulator of the NLRP3 inflammasome. CANE increases IL-1β secretion following NLRP3 inflammasome formation. On the other hand, decreased expression of endogenous CANE leads to decreased IL-1β secretion induced by NLRP3 agonist stimulation, suggesting that CANE is a contributing factor to the pathogenesis of CAPS. As shown in the table below, the amino acid sequence of CANE is shown in SEQ ID NO: 2, and the nucleotide sequence encoding CANE is shown in SEQ ID NO: 1.

[0013] [Table 1]

[0014] The proteins of the present invention include the following (a) to (c): (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 2, (b) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 2, and that binds to NLRP3, (c) A protein that has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 2 and that binds to NLRP3. It includes proteins that bind to NLRP3, selected from among them.

[0015] Examples of "amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2" in the protein described in (b) above include: (i) an amino acid sequence in which 1 to 11 amino acids (for example, 1 to 10, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are deleted from the amino acid sequence shown in SEQ ID NO: 2 (ii) an amino acid sequence in which 1 to 11 amino acids (for example, 1 to 10, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) in the amino acid sequence shown in SEQ ID NO: 2 are replaced by other amino acids. (iii) An amino acid sequence in which 1 to 11 amino acids (for example, 1 to 10, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) are inserted into the amino acid sequence shown in SEQ ID NO: 2 (iv) An amino acid sequence in which 1 to 11 amino acids (for example, 1 to 10, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are added to the amino acid sequence shown in SEQ ID NO: 2 (v) Amino acid sequences mutated by the combinations of (i) to (iv) above These are some examples.

[0016] To prepare a protein having the above mutation, a mutagenesis kit utilizing site-directed mutagenesis methods such as the Kunkel method or the Gapped duplex method is used to introduce the mutation into the polynucleotide encoding the protein, such as QuikChange. TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor TM This can be performed using a Site-Directed Mutagenesis System (Invitrogen), a TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio), or similar systems. Alternatively, methods such as site-directed mutagenesis described in "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) can be used.

[0017] In the present invention, "binding to NLRP3" means that the protein of the present invention and NLRP3 form a reversible non-covalent bond through hydrogen bonding, hydrophobic interactions, electrostatic forces, van der Waals forces, etc. In the present invention, the "binding" between the protein of the present invention and NLRP3 is included in the "interaction" between the protein of the present invention and NLRP3. In the present invention, whether a test protein "binds to NLRP3" can be evaluated based on known methods. For example, as described in Example 3 of this specification, by checking whether the test protein and NLRP3 co-precipitate in co-immunoprecipitation, if the test protein and NLRP3 co-precipitate, the test protein can be evaluated as "binding to NLRP3".

[0018] Furthermore, the proteins of the present invention include not only proteins consisting of the amino acid sequence shown in SEQ ID NO: 2, but also proteins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and which bind to NLRP3. Such proteins include those that contain an amino acid sequence with approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, and that bind to NLRP3. Sequence identity can be checked using homology searches such as FASTA, BLAST, and PSI-BLAST on the internet-based homology search site, for example, the DNA Data Bank of Japan (DDBJ). It can also be checked using BLAST at the National Center for Biotechnology Information (NCBI).

[0019] The proteins of the present invention include proteins in which a tag or protease recognition sequence is added to the N-terminal or C-terminal amino acid of the amino acid sequence. Examples of tags include, but are not limited to, FLAG tags, Myc tags, His tags, and HA tags.

[0020] 3. The present invention provides polynucleotides The polynucleotide of the present invention is not limited to any polynucleotide that contains a base sequence encoding the protein described in "2. Protein of the present invention" above. That is, the polynucleotide of the present invention includes a protein that binds to NLRP3 selected from (a) to (c) below: (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 2, (b) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 2, and that binds to NLRP3, (c) A protein that has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 2 and that binds to NLRP3. It contains polynucleotides that include the base sequence encoding [the specified character].

[0021] Furthermore, examples of polynucleotides of the present invention include polynucleotides that encode a protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 2, and polynucleotides that contain or consist of the base sequence shown in SEQ ID NO: 1. In addition, as the polynucleotide of the present invention, a polynucleotide containing a base sequence in which the codons of the base sequence shown in SEQ ID NO: 1 are optimized according to the type of host can be used. In the present invention, "polynucleotide" includes DNA and RNA.

[0022] The polynucleotides of the present invention include, in addition to polynucleotides containing or consisting of the base sequence shown in SEQ ID NO: 1, polynucleotides containing a base sequence that hybridizes with a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions and encodes a protein that binds to NLRP3. In the present invention, "stringent conditions" may be any of low-stringent conditions, medium-stringent conditions, or high-stringent conditions. "Low-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. "Medium-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C. "Highly stringent conditions" include, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. For detailed procedures of the hybridization method, refer to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)), etc.

[0023] Furthermore, as the polynucleotide of the present invention, a polynucleotide can be used that has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in SEQ ID NO: 1, and that contains a base sequence encoding a protein that binds to NLRP3. In the present invention, whether the protein encoded by the test polynucleotide "binds to NLRP3" can be evaluated based on known methods. For example, as described in Example 3 of this specification, by checking whether the protein and NLRP3 coprecipitate in co-immunoprecipitation, if the protein and NLRP3 coprecipitate, the protein can be evaluated as "binding to NLRP3".

[0024] The method for introducing mutations into polynucleotides is the same as that described in "2. Protein of the Invention" above.

[0025] The polynucleotides and nucleic acid constructs containing the same of the present invention can be prepared, for example, by artificial synthesis, overlap extension PCR, in-fusion cloning, golden gate cloning, or the like.

[0026] 4. vector The present invention provides a vector containing the polynucleotide of the present invention. The vector of the present invention is not limited to any vector containing the polynucleotide of the present invention as described in section 3 above. In one embodiment, the vector of the present invention can be used to generate the protein encoded by this polynucleotide in a wheat germ cell-free protein synthesis system. Examples of vectors used in the present invention include plasmid vectors and viral vectors. Examples of plasmid vectors include, but are not limited to, pEU vectors, pcDNA3 vectors, pFLAG vectors, and pCAGGS vectors. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and Sendai virus vectors. The vector of the present invention may optionally include, in addition to the polynucleotide of the present invention, cis elements such as promoters and enhancers, splicing signals, poly-A addition signals, ribosome-binding sequences (SD sequences), selection marker genes, reporter genes, etc., and these may also be modified. For example, the 5' untranslated region (5'-UTR) (translational enhancer region) in the vector can be replaced with another. The promoters for expressing the polynucleotide of the present invention in a wheat germ cell-free protein synthesis system are not limited to those mentioned above, but include, for example, the T3 promoter, the T7 promoter, and the SP6 promoter. The promoters for expressing the polynucleotide of the present invention in animal cells are not limited to those mentioned above, but include, for example, virus-derived promoters such as Cytomegalovirus (CMV), Rous sarcoma virus (RSV), and Simian virus 40 (SV40), the actin promoter, the EF (elongation factor) 1α promoter, and the heat shock promoter. The promoters for expression in bacteria (e.g., Escherichia species) are not limited to those mentioned above, but include, for example, the trp promoter, the lac promoter, the λPL promoter, the tac promoter, the T3 promoter, the T7 promoter, and the SP6 promoter. Furthermore, the promoters for expression in yeast are not limited to those mentioned above, but include, for example, the GAL1 promoter, the GAL10 promoter, the PH05 promoter, the PGK promoter, the GAP promoter, and the ADH promoter. Examples of selectable marker genes include ampicillin resistance genes, neomycin resistance genes, and kanamycin resistance genes. Examples of reporter genes include green fluorescent protein (GFP) or its variants (fluorescent proteins such as EGFP, BFP, and YFP), red fluorescent protein (Ds-RED), luciferase, alkaline phosphatase, and LacZ.

[0027] 5. host cell The present invention provides a host cell comprising the polynucleotide and / or vector of the present invention. In this specification, "host cell" means a cell into which the polynucleotide and / or vector of the present invention is introduced, and which expresses and produces the protein of the present invention. In the present invention, "host cell comprising the polynucleotide and / or vector of the present invention" may also be rephrased as "transformed organism comprising the polynucleotide and / or vector of the present invention." A transformed organism comprising the polynucleotide and / or vector of the present invention can be obtained by introducing the polynucleotide described in "3." above and / or the vector described in "4." into a host cell. The polynucleotide of the present invention in the polynucleotide and / or vector of the present invention introduced into the host cell may or may not be incorporated into the genome of the host cell.

[0028] In this specification, "host cell" and "transformer" are not limited to any cell capable of producing the protein of the present invention from the polynucleotide and / or vector of the present invention. Examples of such "host cells" or "transformers" are not limited to eukaryotic cells (e.g., animal cells (e.g., HEK293T cells, CHO cells), insect cells (e.g., Sf9), plant cells, yeast (Saccharomyces, Pichia, etc.)), prokaryotic cells (e.g., Escherichia fungi), etc.

[0029] The introduction of polynucleotides and / or vectors into host cells can be carried out using known methods. Known gene transfer methods include, for example, the calcium phosphate method, methods using viral vectors, microinjection, particle gun method, DEAE-dextran method, electroporation, cationic lipid method, and methods combining these.

[0030] 6. The present invention's method for producing protein The protein of the present invention can be obtained by introducing the vector of the present invention into host cells, expressing the protein of the present invention in those cells, and recovering the expressed protein. In another embodiment, the protein of the present invention can be obtained by expressing the protein of the present invention from the vector of the present invention in a wheat germ cell-free protein synthesis system and recovering the expressed protein. The wheat germ cell-free protein synthesis system (wheat germ cell-free protein synthesis technology) is well known, and those skilled in the art can obtain the protein of the present invention in this synthesis system using a vector containing the polynucleotide of the present invention. For example, the protein of the present invention can also be obtained using the WEPRO7240 Expression Kit (CellFree Science).

[0031] In other words, the present invention provides a method for producing the protein of the present invention, comprising the following steps. (a) A step of expressing the protein of the present invention from the vector of the present invention in a host cell or a cell-free protein synthesis system. (b) Steps to recover the expressed protein

[0032] In step (a), when the protein of the present invention is expressed from the vector of the present invention in host cells, the host cells are cultured. That is, host cells are cultured to express the protein of the present invention from the vector of the present invention. The culture conditions used in the manufacturing method of the present invention, such as temperature, pH of the culture medium, and culture time, can be appropriately selected by those skilled in the art. The protein and vector of the present invention are as described in sections 2 and 4 above, respectively. Step (b) may further include a step of purifying the protein of the present invention. For example, the protein of the present invention fused with various tags can be purified by various types of chromatography. Other purification methods include, for example, salting out, solvent precipitation, dialysis, ultrafiltration, and gel filtration, but those skilled in the art can appropriately select a method suitable for the manufacturing method of the present invention.

[0033] 7. Transgenic non-human mammals The transgenic non-human mammal of the present invention contains the polynucleotide of the present invention. The transgenic non-human mammal of the present invention preferably contains the polynucleotide of the present invention in its genome.

[0034] In the present invention, non-human mammals include, but are not limited to, rodents such as mice, rats, guinea pigs, and rabbits, as well as dogs, cats, cows, horses, and monkeys. In the present invention, rodents are preferred as non-human mammals, and mice and rats are more preferred, but are not limited to these.

[0035] In one embodiment, the transgenic non-human mammal of the present invention expresses the protein of the present invention, for example, CANE. CANE is a regulator that enhances the activation of the NLRP3 inflammasome and increases the secretion of IL-1β. That is, the transgenic non-human mammal of the present invention represents a state in which the NLRP3 inflammasome is activated. In one embodiment, the transgenic non-human mammal of the present invention has dermatitis and an erect hair phenotype. Therefore, the transgenic non-human mammal of the present invention can be used as a model non-human mammal for dermatitis (e.g., subcutaneous panniculitis) caused by activation of the NLRP3 inflammasome.

[0036] 8. Methods for producing transgenic non-human mammals The transgenic non-human mammals of the present invention can be manufactured, for example, as follows, but are not limited thereto. Those skilled in the art can manufacture the transgenic non-human mammals of the present invention based on the description herein and, if necessary, publicly known information. (1) Production of transgene DNA fragments The expression vectors used to create transgenic non-human mammals are publicly known and any of them can be used. For example, the pCAGGS vector can be used as an expression vector. A polynucleotide containing the base sequence encoding the protein of the present invention, for example, cDNA encoding CANE, is inserted into the expression vector. Next, the DNA containing the base sequence encoding CANE is amplified, and the constructed construct is digested with restriction enzymes to isolate the transgene DNA fragment as an expression unit. (2) injection A purified transgene DNA fragment is microinjected into the pronucleus of a fertilized egg isolated from a non-human mammal (e.g., C57BL / 6N mouse), and the egg containing the transgene DNA is transplanted into the oviduct of a surrogate pregnant animal to create a transgenic non-human mammal. After confirming that the transgene is positive in the created transgenic non-human mammals (F0), they are crossed with wild-type non-human mammals to obtain F1 mice.

[0037] In this way, a transgenic non-human mammal containing the polynucleotide of the present invention can be obtained.

[0038] 9. antibody The present invention provides an antibody against the protein of the present invention, for example, CANE, or an antigen-binding fragment thereof. In the present invention, an antibody against the protein of the present invention means an antibody that specifically binds to the protein of the present invention. In the present invention, "specifically binds" means recognizing an epitope in the target protein and selectively or preferentially binding (reacting) to the protein that has the epitope compared to a protein that does not have the epitope.

[0039] The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.

[0040] The method for preparing the antibody of the present invention will be described below. (1) Antigen preparation As an immunogen for producing the antibodies of the present invention, the protein of the present invention, for example, CANE (a protein consisting of the amino acid sequence shown in SEQ ID NO: 2), can be used. When using CANE as an immunogen, either full-length CANE can be used, or a peptide containing a portion of the amino acid sequence of the full-length CANE sequence can be used. A description of the protein of the present invention used as an antigen or immunogen is as described in "2. Protein of the present invention" above.

[0041] When CANE is used as an immunogen or antigen, it may be a naturally occurring (i.e., three-dimensionally structured) CANE purified from human tissues or cells, or a genetically engineered CANE. When a peptide containing a portion of the full-length CANE sequence is used as an immunogen, the peptide can be used conjugated to a carrier such as Keyhole Limpet Hemocyanin (KLH) or Thyroglobulin.

[0042] (2) Production of polyclonal antibodies The CANE or partial peptide prepared as described above is administered either by itself or together with a carrier and diluent to non-human mammals, such as rabbits, dogs, guinea pigs, mice, rats, and goats, to induce immunization. The dose of antigen per animal is 0.1 to 10 mg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is mainly performed by injection intravenously, subcutaneously, or intraperitoneally. The interval between immunizations is not particularly limited, but is typically 2 to 10 times, preferably 3 to 5 times, at intervals of several days to several weeks, preferably 1 to 2 weeks. The interval between immunizations can be set by a person skilled in the art, taking into account the antibody titer obtained. It is preferable to collect test blood after 3 to 4 subcutaneous immunizations and measure the antibody titer. Antibody titers in serum can be measured by ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), radioimmunoassay (RIA), etc. After confirming that the antibody titer has risen sufficiently, the entire blood sample can be collected and the antibodies can be separated and purified by commonly used methods. Separation and purification can be performed by appropriately selecting or combining known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography. Specifically, by passing serum containing the target antibody through a column conjugated with proteins other than CANE and collecting the pass-through fraction, polyclonal antibodies with improved specificity for CANE can be obtained.

[0043] (3) Production of monoclonal antibodies (i) Collection of antibody-producing cells Similar to the production of polyclonal antibodies, CANE or partial peptides are administered to non-mammals either on their own or together with a carrier and diluent to induce immunization. The antigen dosage per animal, the type of adjuvant used, the immunization method, and the immunization interval are the same as those for the production of polyclonal antibodies. One to thirty days, preferably two to five days, after the final immunization date, individuals with observed antibody titers are selected and antibody-producing cells are collected. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, but spleen cells or lymph node cells are preferred.

[0044] (ii) cell fusion To obtain hybridomas, cell fusion is performed between antibody-producing cells and myeloma cells. The fusion procedure can be carried out according to known methods, such as the method of Kohler et al. As the myeloma cells to be fused with antibody-producing cells, commonly available cell lines from animals such as mice can be used. Preferred cell lines are those that exhibit drug selectivity, cannot survive in HAT selective medium (containing hypoxanthine, aminopterin, and thymidine) in an unfused state, and can only survive when fused with antibody-producing cells. Examples of myeloma cells include mouse myeloma cell lines such as P3-x63-Ag8U.1, SP2 / O-Ag14, PAI, P3U1, NSI / 1-Ag4-1, and NSO / 1, and rat myeloma cell lines such as YB2 / 0.

[0045] Cell fusion between the above myeloma cells and antibody-producing cells was performed in animal cell culture media such as serum-free DMEM or RPMI-1640 medium, at a rate of 1 × 10⁶ 8 ~5×10 8 A single antibody-producing cell and 2 × 10⁶ 7 ~10×10 7A mixture of antibody-producing cells and myeloma cells (with a cell ratio of 10:1 to 1:1) is used to perform the fusion reaction in the presence of a cell fusion promoter. Polyethylene glycol or Sendai virus with an average molecular weight of 1000 to 6000 daltons can be used as the cell fusion promoter. Alternatively, the antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (e.g., electroporation).

[0046] (iii) Selection and cloning of hybridomas The desired hybridomas are selected from cells after cell fusion processing. This is done by appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing 10-20% fetal bovine serum, then seeding approximately 0.3 cells / well onto a microtiter plate using the limiting dilution method. A selective medium such as HAT medium is added to each well, and the cells are cultured thereafter, with the selective medium being replaced as needed. As a result, cells that begin to grow approximately 10 days after the start of culture in the selective medium can be obtained as hybridomas.

[0047] Next, the developed hybridomas are further screened. Screening of hybridomas can be done according to standard methods and is not particularly limited. For example, a portion of the culture supernatant contained in the wells in which hybridomas are cultured can be collected and screened by enzyme immunoassay, radioimmunoassay, etc. Specifically, after adsorbing the antigen onto a 96-well plate, it is blocked with calf serum. The culture supernatant of hybridoma cells is reacted with the immobilized antigen at 37°C for 1 hour, then peroxidase-labeled anti-mouse IgG is reacted at 37°C for 1 hour, and color development is induced using orthophenylenediamine as a substrate. After stopping the reaction with acid, screening can be performed by measuring the absorbance at a wavelength of 490 nm. Hybridomas that produce monoclonal antibodies that show a positive result by the above measurement method are cloned by limiting dilution, etc. Finally, hybridomas that produce monoclonal antibodies that specifically bind to the protein of the present invention are established.

[0048] (iv) Collection of monoclonal antibodies As a method for collecting monoclonal antibodies from established hybridomas, conventional cell culture methods or ascites formation methods can be employed. In the cell culture method, hybridomas are cultured for 7 to 14 days under normal culture conditions (e.g., 37°C, 5% CO2 concentration) in animal cell culture media such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium, and antibodies are obtained from the culture supernatant. In the case of ascites formation, hybridomas are introduced into the peritoneal cavity of mammalian allogenes derived from myeloma cells, such as mice (BALB / c), at a rate of approximately 5 × 10⁻¹⁴. 6 ~2×10 7 Individual doses are administered to induce massive proliferation of hybridomas. Then, ascites fluid is collected 1 to 2 weeks later. If antibody purification is required in the above antibody collection method, it can be purified by appropriately selecting or combining known methods such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration, and affinity chromatography.

[0049] (4) Production of genetically modified antibodies One embodiment of the antibody of the present invention is a genetically modified antibody. Examples of genetically modified antibodies, though not limited to them, include chimeric antibodies, humanized antibodies, and fully human antibodies.

[0050] Chimeric antibodies (i.e., human chimeric antibodies) are antibodies in which the variable region of a mouse-derived antibody is linked (conjugated) to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984), etc.), and when creating chimeric antibodies, they can be easily constructed using genetic recombination technology to obtain antibodies in this linked state.

[0051] When producing humanized antibodies, a technique known as CDR grafting (CDR transplantation) can be employed. CDR grafting is a method of creating a reconstituted variable region by transplanting the complementarity-determining region (CDR) from the variable region of a mouse antibody into the human variable region, so that the framework region (FR) is derived from humans and the CDR is derived from mice. Next, these humanized reconstituted human variable regions are ligated to the human constant region. Such methods for producing humanized antibodies are well known in this field (see Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); Japanese Patent Publication No. 2828340, etc.).

[0052] In the present invention, fully human antibodies can be produced, for example, using mammals capable of producing human antibodies, in accordance with known methods (e.g., WO96 / 9634096, WO98 / 24893).

[0053] In the present invention, chimeric antibodies, humanized antibodies, and fully human antibodies can be produced using hybridomas or DNA or RNA extracted from said hybridomas, in accordance with the well-known methods described above. Furthermore, the antibody-fused protein of the present invention can be produced by using known genetic recombination methods to combine the variable region of the antibody with other proteins. Alternatively, the fusion protein can be produced by crosslinking a monoclonal antibody with other proteins using a crosslinker.

[0054] (5) Preparation of antigen-binding fragments of antibodies The antigen-binding fragment of the antibody against the protein of the present invention specifically binds to the protein of the present invention. Examples of antigen-binding fragments of the present invention include, but are not limited to, single-chain antibodies (scFv (single chain Fv), sc(Fv)2), double-chain antibodies (Fab, Fab', diabody (dibodies), dsFv, F(ab')2), etc. The above antibody fragments can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes as needed.

[0055] 10. composition The present invention can provide compositions comprising the protein of the present invention or the polynucleotide of the present invention. In addition to the protein of the present invention or the polynucleotide of the present invention, the compositions of the present invention may also contain known additives such as physiological saline, buffer solutions, and excipients.

[0056] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0057] <Example 1> Plasmid construction (1) The plasmids "pEU-E01-NLRP3-Biotin ligation site", "pEU-E01-FLAG-ASC (pyrin domain (PYD))", and "pEU-E01-FLAG-ASC (caspase recruitment domain (CARD))" were constructed using the methods described in the literature (Kaneko, N. et al., 2017, Eur J Inflamm 15: 85-97, Kaneko, N. et al., 2015, Journal of Immunological Methods 426: 76-81).

[0058] (2) The mammalian expression plasmid pcDNA3-NLRP3-MYC was constructed by inserting the nucleotide sequence encoding NLRP3 into the BamHI-XhoI region of pcDNA3-MYC.

[0059] (3) Plasmids encoding candidate proteins with FLAGs, namely pcDNA3-FLJ16641-FLAG, pcDNA3-FLJ96567-FLAG, pcDNA3-FLJ40383-FLAG, pcDNA3-FLJ32790-FLAG, pcDNA3-FLJ33056-FLAG, pcDNA3-FLJ26164-FLAG, pcDNA3-FLJ27267-FLAG, pcDNA3-FLJ44789-FLAG, pcDNA3-FLJ45691-FLAG, pcDNA3-FLJ23487-FLAG, and pcDNA3-FLJ40883-FLAG, were constructed by seamless cloning using InFusion (Takara Bio). The nucleotide sequences encoding each candidate protein were obtained from the FLJ full-length human cDNA database (http: / / flj.lifesciencedb.jp) (Ota, T., Y et al., 2004. Nat. Genet. 36: 40-45). In constructing the plasmid described above, the DNA containing the nucleotide sequences encoding each candidate protein was inserted into the KpnI-XhoI site of pcDNA3-FLAG.

[0060] (4) pFLAG-CMV4-ASC(PYD) and pFLAG-CMV4-ASC(CARD) were constructed according to the method described in the literature (Masumoto, J. et al., 2001., Biochem. Biophys. Res. Commun. 280: 652-655). Furthermore, pcDNA3-ASC was also constructed according to the method described in the literature (Masumoto, J. et al., 1999., Journal of Biological Chemistry 274: 33835-33838).

[0061] (5) pcDNA3-NLRP3 was constructed by inserting the nucleotide sequence encoding NLRP3 into the BamHI-XhoI region of pcDNA3. The full-length cDNA of CIAS1 was obtained from pDONR221-NLRP3 based on the methods described in the literature (Kaneko, N. et al., 2017, Eur J Inflamm 15: 85-97, Kaneko, N. et al., 2015, Journal of Immunological Methods 426: 76-81).

[0062] (6) pcDNA3-procaspase-1 and pcDNA3-proIL-1β were constructed by inserting the nucleotide sequences encoding procaspase-1 and proIL-1β, respectively, into the KpnI-XhoI region of pcDNA3. The nucleotide sequences of the full-length cDNA of procaspase-1 and full-length proIL-1β were obtained from pEU-E01-procaspase-1-FLAG and pEU-E01-proIL-1β-FLAG, respectively.

[0063] <Example 2> Human protein array screening using ALPHA To identify novel proteins that interact with NLRP3, we performed high-throughput screening using human protein arrays and ALPHA (Amplified Luminescent Proximity Homogeneous Assay).

[0064] (1) Human protein array The human protein array used in this example is an array containing 19,712 recombinant human proteins, obtained by purchasing it from CellFree Sciences (Morishita, R. et al., 2019. Sci. Rep. 9: 19349, Goshima, N. et al., 2008. Nat.Methods. 5: 1011-1017). The human proteins included in this array have a FLAG-GST tag fused to their N-terminus and were synthesized in wheat germ cell-free protein synthesis.

[0065] (2) Screening using ALPHA In ALPHA, when a "bait protein" bound to an ALPHA donor bead interacts with a "prey protein," which is a human protein being screened and bound to an ALPHA acceptor bead, the ALPHA donor bead and the ALPHA acceptor bead come into close proximity, and as a result, the ALPHA acceptor bead emits light. Specifically, when biotinylated "NLRP3" bound to streptavidin-conjugated ALPHA donor beads interacts with FLAG-GST-tagged "prey protein" (a human protein being screened) bound to protein A-conjugated ALPHA acceptor beads via an anti-DYKDDDDK-tagged antibody, the ALPHA acceptor beads emit light. (Figure 1A) By measuring the emitted chemiluminescent signal, it is possible to screen for novel proteins that interact with the bait protein NLRP3.

[0066] In this example, screening using ALPHA was performed as follows. First, 20 μl of a premix containing 0.5 μl of biotinylated NLRP3 as bait protein was added to each well of a 96-well plate using FlexDrop (PerkinElmer). Biotinylated NLRP3 was prepared using the plasmid "pEU-E01-NLRP3-Biotin ligation site" constructed in Example 1(1). This biotinylated NLRP3 is biotinylated at its C-terminus, and this biotin binds to streptavidin-conjugated ALPHA donor beads (Figure 1A). Next, 1 μl of premix containing 0.5 μl of FLAG-GST-tagged prey protein (which binds to bait protein) was added to each well using a JANUS liquid handler (PerkinElmer) equipped with a NanoHead. [As a positive control, ASC (pyrin domain (PYD)) (FLAG-tagged ASC (PYD)), which is known to bind to NLRP3 in the NLRP3 inflammasome, was used, and as a negative control, FLAG-tagged ASC (caspase recruitment domain (CARD)) was used. These controls were prepared using the plasmids "pEU-E01-FLAG-ASC (PYD)" and "pEU-E01-FLAG-ASC (CARD)" constructed in Example 1(1).] Subsequently, a 5 μl detection bead premix containing anti-DYKDDDDK tag antibody (clone 1E6 Fujifilm Wako Pure Chemical Industries) (which binds to the FLAG tag of the prey protein), streptavidin-conjugated ALPHA donor beads, and protein A-conjugated ALPHA acceptor beads (PerkinElmer) was added to each well in the dark using FlexDrop and incubated at 25°C for 24 hours. After incubation, the emitted chemiluminescent signal (ALPHA signal) was measured using an EnVision multimode plate reader (PerkinElmer).

[0067] As a result, we were able to obtain several candidate proteins that emitted ALPHA signals. Of these, the top 11 candidate proteins that emitted high ALPHA signals were selected for further screening. The selected candidate proteins are shown in Table 2 below.

[0068] [Table 2]

[0069] <Example 3> Activity screening We investigated whether 11 candidate proteins selected by screening using ALPHA affect NLRP3 inflammasome activation. Specifically, we tested the effect of the selected candidate proteins on IL-1β secretion after forced expression of NLRP3 inflammasome component proteins in HEK293T cells.

[0070] (1) Reorganization of the NLRP3 inflammasome in HEK293T cells HEK293T cells were maintained in DMEM (11995; Life Technologies) supplemented with 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin (15140 - 122; Life Technologies). Next, a total of 1×10 6 HEK293T cells were co - transfected with a predetermined amount of tag - free pcDNA3 - NLRP3, pcDNA3 - ASC, pcDNA3 - pro - caspase - 1, and pcDNA3 - proIL - 1β constructed in Example 1(5) and (6). In addition, plasmids expressing 11 FLAG - tagged candidate proteins prepared in Example 1(3) were transfected in a dose - dependent manner. The pcDNA3 vector was used as a negative control. After 8 hours, the medium used for transfection was replaced with fresh medium.

[0071] (2) Investigating the effects of candidate proteins on NLRP3 inflammasome activation. Sixteen hours after the above medium change, the IL - 1β concentration in the culture supernatant was measured using an IL - 1β ELISA kit (557953; BD Biosciences) according to the manufacturer's instructions. As a result, 10 out of 11 candidate proteins significantly increased inflammasome - mediated IL - 1β secretion in a plasmid - dose - dependent manner (Figure 1B).

[0072] (3) Co-immunoprecipitation assay with NLRP3 The interaction between the 11 candidate proteins listed in Table 2 and NLRP3 was tested by co - immunoprecipitation. Specifically, it was performed as follows. First, a total of 1×10 6Each HEK293T cell was co-introduced with the plasmid pcDNA3-NLRP3-MYC constructed in Example 1(2) and each of the plasmids encoding the 11 FLAG-tagged candidate proteins constructed in Example 1(3). NLRP3-MYC and each of the 11 FLAG-tagged candidate proteins were then co-expressed in the HEK293T cells. For gene transfer, the diluted plasmid was mixed with 10 ml of HEK293T cell culture medium and added to each 100 × 20 mm cell culture dish containing HEK293T cells. After 8 hours, the culture medium used for gene transfer was replaced with fresh medium.

[0073] Forty hours after the medium change, the soluble supernatant obtained from the whole-cell lysate of HEK293T cells co-expressing NLRP3-MYC and each candidate protein was immunoprecipitated using an anti-MYC antibody. Specifically, HEK293T cells were collected, and the collected cells were lysed in NP-40 buffer (1% Nonidet P-40, 142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.6], 0.2 mM PMSF, and 1 mM EDTA) to obtain a total cell lysate.

[0074] Next, the soluble supernatant obtained from the whole cell lysate was incubated with an anti-MYC polyclonal antibody (16286-1-AP; Proteintech) and 30 μL of protein A-conjugated agarose beads (P3476; Invitrogen) at 4°C for 5 hours. Subsequently, samples containing co-immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis. For protein detection by Western blotting, anti-FLAG M2 antibody was used as the primary antibody, and peroxidase AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment-specific antibody (115-036-072; Jackson Immunoresearch Laboratories) was used as the secondary antibody.

[0075] In this system, when the FLAG-tagged candidate protein binds to the NLRP3 portion of NLRP3-MYC to form a complex, the anti-MYC antibody bound to the protein A-conjugated agarose beads captures the [NLRP3-MYC]-[FLAG-tagged candidate protein] complex, resulting in co-immunoprecipitation.

[0076] As a result, of the 11 candidate proteins, only "FLJ40383" co-precipitated with NLRP3 (Figure 1C, arrow). The inventors named this candidate protein FLJ40383 "Cryopyrin-associated nano enhancer (CANE)". In this embodiment, a novel protein called CANE was obtained that interacts with (at least binds to) NLRP3.

[0077] (4) Investigation of NLRP3 domains that interact with CANE To identify the NLRP3 domains important for the interaction between NLRP3 and CANE, co-immunoprecipitation assays were performed using CANE and each cleavage form of NLRP3 (pyrin domain (PYD), nucleotide-binding oligomerization domain (NOD), LLR). This assay was performed in the same manner as in (3) above, except that FLJ40383-FLAG was co-expressed with either NLRP3(PYD)-MYC, NLRP3(NOD)-MYC, or NLRP3(LRR)-MYC in HEK293T cells. As a result, CANE co-precipitated with NLRP3(PYD), but not with NLRP3(NOD) or NLRP3(LRR) (Figure 1D). These results indicate that CANE interacts with (or at least binds to) the PYD of NLRP3.

[0078] In summary, this embodiment demonstrated that CANE interacts with (at least binds to) the PYD of NLRP3, and that CANE-mediated NLRP3 inflammasome formation enhances IL-1β secretion by HEK293T cells. These results suggest that CANE may promote the activation of the NLRP3 inflammasome and be involved in the development of NLRP3-related autoinflammatory diseases such as CAPS.

[0079] <Example 4> Study on the structure, distribution, and intracellular localization of CANE (1) CANE structure CANE was found to be an estimated 13 kDa protein consisting of 116 amino acids. Furthermore, the CANE gene was found to possess the Kozak consensus sequence (5'-cgggATGG-3'). Additionally, the base sequence encoding CANE is located on chromosome 7q11.21, confirming that CANE is a protein that has not been previously characterized.

[0080] (2) Detection of CANE mRNA The inventors discovered, through sequence searching using the Basic Local Alignment Search Tool (BLAST, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), that CANE is encoded by mRNA located in the 5' untranslated region (UTR) of ZNF273. ZNF273 mRNA is most highly expressed in Jurkat cells, a human T-cell leukemia cell line. Therefore, total RNA was extracted from Jurkat cells and digested with DNase I (04 716 728 001; Roche). Subsequently, reverse transcription was performed at 55°C for 60 minutes using SuperScript III Reverse Transcriptase (18080044; Thermo Fisher Scientific) with the following three different specific complementary primers a, b, and c. (1) A specific primer located at the 3' UTR of the ZNF273 coding region (primer a) :5'-GCAATAAGGAAAACTCCACTTCTGGGAGTT-3'(Sequence ID 3), (2) A specific primer located between the coding regions of CANE and ZNF273 (primer b) :5'-GCACCTCAGTGCTGCTCATGATTGCAGAGG-3' (Sequence ID 4), and (3) A specific primer located 60 bp downstream of the CANE stop codon (primer c) : 5'-GCCCGGATCCCTGCTTGGCTCCCGGAGCCC-3'(Sequence ID 5) Subsequently, each cDNA obtained by reverse transcription was amplified by PCR using the following primers. Forward primer: 5'-GGGGCCGCGTCTTCGGCAAAGTCTTCGGGG-3' (SEQ ID NO: 6) Reverse primer: 5'-GGACTGTGCCTTCCGCAAAACGGCTGCGCT-3' (SEQ ID NO: 7) Furthermore, the obtained PCR products were subjected to nested PCR using the following primers. Forward primer: 5'-TGAGAGGTCCGGCTCCTGGAGGACAGCGGG-3' (SEQ ID NO: 8) Reverse primer: 5'-CTAGGTAGGAGAAACGTTCTTCGCTTCCTG-3' (SEQ ID NO: 9) This confirmed that full-length CANE mRNA is expressed in Jurkat cells.

[0081] (3) Production of anti-CANE antibodies To identify the types of cells that constitutively express CANE, we produced mouse anti-CANE polyclonal antibodies using a standard method. Specifically, full-length CANE recombinant protein fused with a Flag tag was synthesized in E. coli or using wheat germ cell-free protein synthesis technology, and purified using an anti-Flag protein affinity gel column (Merck). 100 μg of CANE recombinant protein was dissolved in 100 μL of PBS, suspended with 100 μL of Freund's complete adjuvant, and injected intraperitoneally into Balb / c mice. After 30 days, 50 μg of CANE recombinant protein was dissolved in 50 μL of PBS and injected intraperitoneally with 50 μL of Freund's incomplete adjuvant. After 14 days, another 50 μg of CANE recombinant protein was dissolved in 50 μL of PBS and injected intraperitoneally with 50 μL of Freund's incomplete adjuvant. Blood was collected from the mice between 2 and 5 days after the procedure. Antiserum was isolated by centrifugation, and mice with antiserum that specifically recognized only the CANE protein were selected using Western blotting. Mice producing CANE antibodies were injected intraperitoneally every two weeks with 50 μg of CANE recombinant protein dissolved in 50 μL of PBS and 50 μL of Freund's incomplete adjuvant. Blood was collected between 2 and 5 days later, and the serum was used as an anti-CANE polyclonal antibody in the experiment. In this way, mouse anti-CANE polyclonal antibodies were obtained.

[0082] (4) Investigation of cell distribution in CANE Plasmids expressing EGFP-fused CANE (pEGFPC2-CANE) and DsRed-fused CANE (pDsRed-Express2-CANE) were constructed and expressed in HEK293T cells. Western blot analysis of EGFP-fused CANE and DsRed-fused CANE in HEK293T cells using the anti-CANE antibody prepared in (3) above revealed the expression of these fusion proteins. Furthermore, we performed Western blot analysis on CANE expression in various cell lines using the anti-CANE antibody of the present invention. As a result, a band with the predicted size of CANE (13 kDa) was detected in the cell lysates of various cell lines, including HEK293T, chronic myeloid leukemia-derived cell line K562, acute monocytic leukemia-derived cell line THP-1, acute promyelocytic leukemia-derived cell line HL-60, human lymphoma-derived cell line U937, acute T-cell leukemia-derived cell line Jurkat, and cervical cancer-derived cell line SiHa, confirming the expression of endogenous CANE (Figure 2A). These results indicate that CANE is expressed in a wide range of cells.

[0083] (5) Investigation of the intracellular localization of CANE To evaluate the intracellular localization of CANE, pDsRed-Express2-CANE and pEGFPC2-NLRP3 were introduced into HEK293T cells, respectively. After gene introduction, DsRed-CANE and EGFP-NLRP3 were localized in the cytoplasm of HEK293T cells (Figure 2B, left 16 panel). Next, we evaluated whether CANE co-localizes with NLRP3 in the cytoplasm. For this purpose, HEK293T cells were co-transfected with pEGFPC2-NLRP3 and pDsRed-Express2-CANE, with or without pcDNA3-ASC. Specifically, a total of 1 × 10⁻¹⁶ cells were co-transfected. 6 HEK293T cells were transiently co-transmitted with either (i) pEGFP-NLRP3 and pDsRed-Express2-CANE or (ii) pEGFP-NLRP3, pDsRed-Express2-CANE and pcDNA3-ASC. After 8 hours, the culture medium used for gene transfer was replaced with fresh medium. Sixteen hours after the medium change, immunofluorescence signals were observed using an Olympus BX53 microscope. Digital images were observed using an Olympus DP80 camera (at 400x magnification). As a result, it was revealed that in the absence of ASC, CANE and NLRP3 co-localize in the cytoplasm (Figure 2B, upper right 8 panels). On the other hand, notably, when CANE was co-introduced together with ASC and NLRP3, a "speck," a characteristic of inflammasome formation, was formed, and CANE and NLRP3 accumulated in this "speck" (Figure 2B, lower right 8 panels). These results indicate that CANE is a protein that makes up the NLRP3 inflammasome. In other words, this example demonstrates that CANE is a novel component (constituent protein) of the NLRP3 inflammasome.

[0084] (6) Investigation of the intracellular localization of endogenous CANE and NLRP3 To confirm the endogenous interaction between CANE and NLRP3 in living cells, immunoprecipitation was performed using THP-1-derived macrophages. Cell lysates were immunoprecipitated with an anti-CANE antibody, and NLRP3 was detected by Western blotting using an anti-NLRP3 antibody. Specifically, the procedure was as follows: First, based on the method described in the literature (Maeβ, MB et al., Cold. Spring.Harb. Protoc. 2011: pdb.prot5612), a total of 1 × 10 7 THP-1 cells were cultured for 48 hours in RPMI 1640 medium (11875; Life Technologies) supplemented with 10% (v / v) FBS, 1% (v / v) sodium pyruvate (11360-070; Life Technologies), 1% (v / v) MEM non-essential amino acids (11140-050; Life Technologies), 1% (v / v) penicillin / streptomycin (15140-122; Life Technologies), and 100 ng / ml PMA (162-23591; Fujifilm Wako) to differentiate into macrophages. After 48 hours, the cells were treated with 1 μg / ml LPS (L2880; Sigma) for 4 hours, followed by 10 μM nigericin (N7143; Sigma) for 1 hour, or left untreated. Nigellicin is one of the ionophores that enhances the membrane permeability of cations. In particular, it is one of the substances that enhances the membrane permeability of potassium (K) ions, which have a high affinity for nigellicin. When K ions from inside the cell leach out of the cell, the NLRP3 inflammasome is formed, and nigellicin is used to activate the NLRP3 inflammasome. Subsequently, the cells were collected, lysed in 360 μl of NP-40 buffer (1% Nonidet P-40, 142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.6], 0.2 mM PMSF, and 1 mM EDTA), and incubated with anti-CANE antibody and 30 μl of protein A-conjugated agarose beads at 4°C for 5 hours. Subsequently, the samples were subjected to SDS-PAGE and Western blot analysis. Protein detection was performed by treating the Western blot membrane with a primary antibody (anti-NLRP3 rabbit antibody (13158; Cell Signaling Technology)) and a secondary antibody (peroxidase AffiniPure F(ab')2 fragment goat anti-rabbit IgG, F(ab')2 fragment specific; 111-036-047; Jackson Immunoresearch Laboratories). As a result, endogenous NLRP3 and endogenous CANE coprecipitated in macrophages differentiated from THP-1 cells (Figure 3A). In particular, the NLRP3 band in nigelicin-treated samples was more distinct compared to the band in untreated samples (Figure 3A).

[0085] Furthermore, the co-localization of endogenous CANE and endogenous NLRP3 was confirmed by fluorescence microscopy using immunofluorescence staining. Specifically, this was done as follows. Based on the method described in the reference (Maeβ, MB et al., Cold. Spring.Harb. Protoc. 2011: pdb.prot5612), THP-1 cells were cultured for 48 hours in RPMI 1640 medium supplemented with 10% (v / v) FBS, 1% (v / v) sodium pyruvate, 1% (v / v) MEM non-essential amino acids, 1% (v / v) penicillin / streptomycin, and 100 ng / ml PMA to differentiate them into macrophages. After 48 hours, the cells were treated with 1 μg / ml LPS for 4 hours, followed by treatment with 10 μM nigericin for 1 hour, or left untreated. The cells were then fixed in 70% ethanol at -30°C for 30 minutes and dried. Next, the cells were blocked with TBS supplemented with 10% FBS and incubated with anti-CANE mouse polyclonal antibody and / or anti-NLRP3 rabbit polyclonal antibody (PA1665; Boster Biological Technology). Subsequently, they were treated with Alexa Fluor 647-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgG(H+L) (115-606-146; Jackson Immunoresearch) and Alexa Fluor 488-conjugated AffiniPure F(ab')2 fragment goat anti-rabbit IgG(H+L) (111-546-144; Jackson Immunoresearch). The nuclei were stained using ProLong Glass Antifade Mountant with NucBlue Stain (P36981; Thermo Fisher Scientific), and the immunofluorescence signal was observed and measured in the same manner as described in Example 4.

[0086] As a result, in the presence of nigericin (Figure 3B, nigericin (+)), speck formation (indicated by arrowheads), a characteristic of inflammasome formation, was observed in 24±4.6 (24±4.6%) of 100 cells across three fields of view. On the other hand, in the absence of nigericin (Figure 3B, nigericin (-)), speck formation was observed in only 3.7±2.1 (3.7±2.1%) of 100 cells (Figure 3C). These results indicate that, without nigericin treatment, most endogenous CANE and NLRP3 are diffusely distributed within the cytoplasm, but when incubated with nigericin, they co-localize to speck-like points (Figure 3B).

[0087] <Example 5> Investigation of the effects of reduced CANE expression on NLRP3 inflammasome activation. To investigate the effect of endogenous CANE on NLRP3 inflammasome activation in living cells, knockdown experiments were performed using morpholino antisense oligonucleotides in THP-1-derived macrophages. Specifically, the procedure was as follows: First, we designed morpholino antisense oligonucleotides to suppress CANE expression using Gene Tools (https: / / www.gene-tools.com). The two morpholino antisense oligonucleotides that were designed and obtained are as follows: CANE 1:5'-TCCTCCAGGAGCCGGACCTCTCACC-3'(Sequence ID 10) CANE 2:5'-CTGGGTGTGAACGGGACTCTCCCAT-3'(Sequence ID 11) On the other hand, based on the method described in the literature (Maeβ, MB et al., Cold. Spring.Harb. Protoc. 2011: pdb.prot5612), THP-1 cells were cultured for 48 hours in RPMI 1640 medium supplemented with 10% (v / v) FBS, 1% (v / v) sodium pyruvate, 1% (v / v) MEM non-essential amino acids, 1% (v / v) penicillin / streptomycin, and 100 ng / ml PMA to differentiate them into macrophages. Next, THP-1-derived macrophages were genetically modified for 24 hours using Endo-porter (Endo-PEG; Gene Tools) with CANE 1, CANE 2, or a random control morpholino (Cont.). The gene transfer reagent was prepared by mixing Endo-porter solution to a final concentration of 6 mM with each morpholino to a final concentration of 1 mM in DMEM. After 24 hours, the cells were treated with 1 μg / ml LPS for 4 hours, followed by treatment with 10 μM nigericin for 1 hour, or left untreated.

[0088] The processing of procaspase-1 and pro-IL-1β was analyzed by Western blotting of the culture supernatant, NP-40 buffer-soluble cell lysates, and NP-40 buffer-insoluble precipitates. Western blot membranes were probed with a primary antibody [anti-human IL-1β rabbit antibody (12703; Cell Signaling Technology) or anti-human caspase-1 rabbit antibody (2225; Cell Signaling Technology)] and a secondary antibody specific to these antibodies (anti-rabbit IgG, F(ab')2 fragment). Alternatively, Western blot membranes were probed with a primary antibody (anti-CANE mouse antibody) and a secondary antibody (anti-mouse IgG, F(ab')2 fragment specific antibody). The concentrations of human IL-1β and human TNF-α in the culture supernatant were measured using the IL-1β ELISA kit (557953; BD Biosciences) and the TNF ELISA kit (555212; BD Biosciences), respectively, according to the manufacturer's instructions.

[0089] As a result, Western blot analysis showed that Morpholino CANE 1 and CANE 2 reduced the processing of pro-IL-1β (p31) to cleaved IL-1β (p17) in the culture supernatant and NP-40 buffer-soluble cell lysates (Figures 4A-4C). This result was consistent with the ELISA test results. Specifically, the ELISA test also showed that Morpholino CANE 1 and CANE 2 significantly reduced IL-1β secretion (Figure 4D). On the other hand, Morpholino CANE 1 and CANE 2 did not reduce TNF-α secretion. Western blot analysis also showed that Morpholino CANE 2 reduces the processing of procaspase-1 (p50) to cleavage caspase-1 (p20) in the culture supernatant (Figure 4A). CANE expression levels were reduced by morpholino CANE 1 and CANE 2 even in untreated cells [LPS (-) and nigericin (-)] (Figure 4B). Following LPS and nigericin treatment, CANE expression levels were shown to be reduced in the NP-40 buffer-insoluble precipitate by CANE 1 and CANE 2 (Figure 4C). On the other hand, even in the absence of nigericin, small amounts of CANE were observed in the NP-40 buffer-insoluble precipitate of control or CANE 1 morpholino-transformed cells (Figure 4C). These results indicate that decreased CANE expression leads to reduced activation of caspase 1 and IL-1β, and decreased IL-1β secretion.

[0090] Furthermore, the activation of the NLRP3 inflammasome and caspase-1 is regulated in two steps. The first step is the transcription and synthesis of NLRP3 inflammasome constituent proteins and pro-IL-1β, which is NF-κB dependent. The second step is the activation of the NLRP3 inflammasome. TNF-α secretion is dependent on NF-κB activation. In this embodiment, suppressing CANE expression did not reduce TNF-α secretion, suggesting that CANE is a molecule that enhances the second stage, namely the activation of the NLRP3 inflammasome. In other words, this embodiment demonstrates that CANE functions as a regulator that enhances the activation of the NLRP3 inflammasome.

[0091] <Example 6> Production and analysis of CANE transgenic mice (1) Creation of transgenic mice (genetically modified mice) To further evaluate the in vivo role of CANE, we created transgenic mice expressing human CANE. Specifically, these mice were created as follows. First, the pCAGGS-CANE expression vector was constructed by inserting a cDNA containing the base sequence encoding CANE into the pCAGGS vector. pCAGGS included the CAG promoter (a chimeric promoter of the CMV IE enhancer and the chicken β-actin promoter), the chicken β-actin first intron, the rabbit β-globin second intron, the third exon, and the 3' flanking region. cDNA containing the CANE encoding sequence was PCR-amplified from the pcDNA3-CANE-FLAG vector using the Kozak consensus sequence. This cDNA has an EcoRI site at its 5' end and an XhoI site at its 3' end. The amplified fragments were digested with EcoRI and XhoI and inserted into the pCAGGS vector using the EcoRI-XhoI restriction site located between the β-globin intron and the 3' flanking region. The constructed construct was digested with SalI and HindIII, and linear transgene DNA fragments were isolated as 2.6 kb expression units. Purified transgene DNA fragments were microinjected into the pronuclei of fertilized eggs isolated from C57BL / 6N mice, and these eggs were transplanted into the oviducts of surrogate-pregnant mice to create transgenic mice. The following primer set was used to confirm integration into the mouse genome. pCAGGS-F1:5'-TCGACATTGATTATTGACTAGTTATTAATAGTAATC-3'(Sequence ID 12) R2275: 5'-GTCGAGGGATCTCCATAAGAGAAGAGGGACA-3'. (Sequence ID 13) Embryo injection resulted in 73 offspring. Of these, 21 mice were transgene-positive. Four founder mice (F0) were independently mated with C57BL / 6N wild-type (WT) female mice, and three founder mice were used to produce hemizygous F1 mice. All mice used in the examples described herein were raised under SPF conditions. Transgenic mice were produced by Transgenic (Kobe, Japan) with the approval of the Animal Research Committee. All experiments were conducted in accordance with the approved protocol of the Ehime University Animal Care Unit Committee (37U6-1,16) and in compliance with the International Guidelines for Animal Experimentation and the Ehime University Guidelines for Animal Experimentation.

[0092] (2) Genotyping of transgenic mice and confirmation of CANE expression The inventors obtained two CANE transgenic mouse strains labeled as Line 1 and Line 2. To confirm genotyping and CANE expression, PCR using a specific primer set and Western blotting analysis using an anti-CANE antibody were performed. Western blotting analysis confirmed CANE expression in the cell lysates of transgenic mouse myeloid cells. Mouse PBMCs were isolated by Ficol gradient centrifugation (17144002; Cytiva) according to the manufacturer's instructions. PCR results showed that the CANE gene was incorporated into the transgenic mice of line 1 and line 2 (Figure 5A). Furthermore, CANE protein expression in transgenic mice was detected in line 1 mice, detected in line 2 mice (though at a lower level than line 1 mice), and not detected in WT mice (Figure 5B). 1 × 10⁶ samples were obtained from line 1 and line 2 mice. 6 When PBMCs were stimulated with 0.1 or 1 μg / ml LPS for 8 hours, they secreted higher amounts of IL-1β and TNF-α than those derived from wild-type mice (Figure 5C). Furthermore, PBMCs obtained from Line 1 mice secreted higher amounts of IL-1β and TNF-α than PBMCs obtained from Line 2 mice. In this way, two lines of CANE transgenic mice were obtained.

[0093] (3) Investigation of caspase-1 processing and cytokine secretion from BMDM in transgenic mice. As described below, bone marrow-derived macrophages (BMDMs) were obtained from CANE transgenic mouse lines 1 and 2, as well as wild-type (WT) mice. BMDMs are a more homogeneous cell population than PBMCs. Cytokine production from BMDMs is thought to reflect the state based on the genetic background of the mouse. First, bone marrow progenitor cells obtained from the tibia, femur, and humerus were differentiated for 7 days in IMDM (12440; Life Technologies) supplemented with 10% FBS, 30% L929 cell supernatant, and 1% penicillin / streptomycin to produce BMDM. Next, the cells were collected using a cell scraper and subjected to the following experiments. BMDM was treated with 1 μg / ml LPS for 4 hours, followed by treatment with 10 μM nigericin for 1 hour, or left untreated. Procaspase-1 processing was analyzed by Western blotting of the culture supernatant. The Western blot membrane was probed with a primary antibody (anti-mouse caspase-1 rabbit antibody (24232; Cell Signaling Technology)) followed by a secondary antibody (anti-rabbit IgG, F(ab')2 fragment-specific antibody). Furthermore, the concentrations of IL-1β and TNF-α in the culture supernatant were measured by ELISA. As a result, Western blot analysis showed that the processing of procaspase-1 (p45) to cleaved caspase-1 (p20) was higher in BMDMs from Line 1 and Line 2 mice than in WT mice (Figure 6A). This result was consistent with the ELISA test results. Specifically, the ELISA test results also showed that IL-1β secretion from BMDMs from Line 1 and Line 2 mice was significantly higher compared to WT mice (Figure 6B). In contrast, TNF-α secretion from BMDM derived from these mice was not elevated compared to wild-type mice. These results indicate that CANE enhances NLRP3 inflammasome activation without activating NF-κB (i.e., without directly participating in TNF-α expression). On the other hand, as shown in Example 6(2), PBMCs derived from the transgenic mice of the present invention secreted higher amounts of IL-1β and TNF-α than those derived from WT mice. This suggests that CANE may also increase TNF-α production in response to LPS stimulation (environmental factors) through interactions between lymphocytes (T cells, B cells, and NK cells) contained in PBMCs and monocytes and dendritic cells expressing NLRP3 inflammasome components, as well as through secondary NF-κB activation by secreted IL-1β.

[0094] Next, we investigated whether the role of CANE is specific to the NLRP3 inflammasome. First, BMDM was treated with 1 μg / ml LPS for 4 hours. Then, the AIM2 agonist poly-deoxyadenylate:deoxythimidate (Poly(dA:dT)) (tlrl-patn; InvivoGen) was transfected into the BMDM at a final concentration of 1 μg for 1 hour. The transfection of Poly(dA:dT) was performed using Xfect Transfection Reagent (631318; Clontech). After treating the BMDM with 1 μg / ml LPS for 4 hours, 0.25 μg of the NLRC4 agonist flagellin (tlrl-epstfla-5; InvivoGen) was transfected into the BMDM for 4 hours. The transfection of flagellin was performed using DOTAP (11202375001; Roche). The concentrations of IL-1β and TNF-α in the culture supernatant were measured by ELISA.

[0095] As a result, the secretion of IL-1β and TNF-α in the culture supernatant of BMDM mice from line 1 and line 2 did not show significant differences compared to wild-type mice, whether incubated with the AIM2 agonist poly(dA:dT) or the NLRC4 agonist flagellin after stimulation with LPS (Figure 6C). These results further demonstrate that CANE is a regulator that specifically enhances the activation of the NLRP3 inflammasome, rather than the AIM2 or NLRC4 inflammasome.

[0096] (4) Semi-quantitative histological analysis of subcutaneous tissue from transgenic mice (4-1) Examination of phenotypic characteristics related to hair We compared the appearance of transgenic mouse lines 1, 2, and WT mice and examined their phenotypes. As a result, images of line 1 mice, which expressed higher levels of CANE than line 2 mice and WT mice, showed an edgy hair phenotype (Figure 7A). The penetration rates of the spiky hair phenotype shown in Figure 7A are summarized in the table below.

[0097] [Table 3]

[0098] This table shows the number of mice exhibiting the spiky hair phenotype and the number of mice exhibiting the normal phenotype, separated by sex, with the penetrant rate shown on the right. As shown in the table above, in line 1 males, which highly express CANE, all 17 mice (100% penetrant) showed the ruffled hair phenotype, and in line 1 females, 14 out of 19 mice (73.7% penetrant) showed the ruffled hair phenotype. On the other hand, in line 2 males, which do not express sufficient CANE protein, 3 out of 16 mice (18.8% penetrant) showed the ruffled hair phenotype, and in line 2 females, none of the 21 mice showed the ruffled hair phenotype (0% penetrant). In wild-type mice, none of the 24 males and 16 females showed the ruffled hair phenotype (0% penetrant).

[0099] (4-2) Study on subcutaneous panniculitis Furthermore, skin tissue sections were obtained from transgenic mouse lines 1 and 2, as well as WT mice, and subcutaneous inflammation was examined. Specifically, mouse skin tissue was first fixed with formalin, embedded in paraffin, and 4 μm thick sections (specimens) were prepared and then stained with H&E. Subcutaneous inflammation is (i) per tissue unit area ( / mm²) 2 (ii) the number of infiltrating neutrophils, (ii) the number of attached neutrophils per capillary measured by the pericapillary length (μm), and (iii) the number of neutrophils per tissue unit area ( / mm²). 2 The number of capillaries was evaluated using histological scoring, which involved counting the number of capillaries in the tissue.

[0100] Furthermore, the expression of IL-1β and TNF-α in the skin of transgenic mouse lines 1, 2, and WT mice was measured by quantitative real-time RT-PCR. Specifically, the procedure was as follows. Mouse epidermis and dermis were freeze-thawed, and total RNA was extracted using the Maxwell RSC Instrument (Promega). RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was synthesized using SuperScript III Reverse Transcriptase. Quantitative real-time RT-PCR of IL-1β and TNF-α was performed using StepOnePlus (Thermo Fisher Scientific) with TaqMan Gene Expression Assays (mouse TNF: Mm00443258; mouse IL-1β: Mm00434228; mouse actin: Mm02619580). IL-1β and TNF-α expression were normalized relative to actin expression.

[0101] The results are shown in Figures 7B to 7G. In Figures 7D to 7G, three unit areas (each of equal size) were extracted from each skin tissue image of five mice, and the number of neutrophils or capillaries per unit area was plotted on a graph. As shown in Figure 7B, skin tissue sections from Line 1 and Line 2 mice showed neutrophil infiltration around dilated capillaries (red arrows), as well as neutrophils attached within the capillaries (black arrows). These phenotypes are similar to those seen in CAPS model mice with NLRP3-R258W or NLRP3-A350V mutations, such as subcutaneous panniculitis. Furthermore, histological analysis of the above-mentioned skin tissue sections (Figure 7B) showed that the cause of hair loss and bristly hair in the transgenic mice of the present invention is related to neutrophil-mediated inflammation in the subcutaneous tissue.

[0102] Furthermore, as shown in Figures 7D-G, in transgenic mouse line 1, the number of infiltrating neutrophils per tissue unit area, the number of neutrophils attached to capillary walls, the circumference of capillaries, and the number of capillaries were measured, and the results showed that these numbers were statistically higher in transgenic mouse line 1 compared to WT mice. Furthermore, in transgenic mouse line 2, the number of infiltrating neutrophils per tissue unit area, the number of attached neutrophils per capillary, and the capillary circumference were also statistically higher compared to WT mice. Statistical analysis was performed using GraphPad Prism Version 9.4.1 (GraphPad Software). In contrast, quantitative real-time RT-PCR results showed no difference in IL-1β mRNA and TNF-α mRNA expression between CANE transgenic mice and WT mice in the absence of stimulation (Figure 7C). Since neutrophils are recruited by direct intercellular contact and / or paracrine mechanisms at the site of inflammation, histopathological analysis is sufficient to reveal the differences between the CANE transgenic mice of the present invention and wild-type mice, even if IL-1β or TNF-α levels are not detected by quantitative RT-PCR. As described above, the transgenic mice of the present invention showed significantly higher histopathological scores for neutrophil recruitment and vasodilation, which are markers of skin inflammation, compared to wild-type mice. From the above, it was shown that the expression of exogenous CANE induced dermatitis in the transgenic mice of the present invention. In other words, it was shown that the transgenic mice of the present invention are useful as NLRP3 inflammasome-related disease model mice, for example, as disease model mice for inflammation (particularly dermatitis) related to the NLRP3 inflammasome.

[0103] statistical analysis For in vitro analysis, all results were presented as the mean ± standard deviation of three wells (triplicate wells) for each sample. Because parametric tests may not be optimal for small sample sizes, non-parametric tests were used, and the significance of the differences was assessed using the Mann-Whitney U test. For histological analysis, all results were expressed as the mean ± standard deviation of neutrophil count, capillary perimeter, or capillary count per unit area at three different locations in each mouse (n=5 mice). As a preliminary test, one-way analysis of variance (ANOVA) was used to detect overall differences between groups. Subsequently, post hoc tests were used to compare the differences between groups. Specifically, after confirming significant differences using one-way ANOVA, Tukey's multiple comparison post hoc test was performed. [Industrial applicability]

[0104] This invention is useful for elucidating novel mechanisms related to the NLRP3 inflammasome. [Sequence Listing Free Text]

[0105] Sequence IDs 3-13: Synthetic DNA

Claims

1. A protein that binds to NLRP3, selected from (a) to (c) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 2 (b) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 2, and which binds to NLRP3. (c) A protein that has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 2 and that binds to NLRP3.

2. A polynucleotide comprising a base sequence encoding the protein described in claim 1.

3. A vector comprising the polynucleotide described in claim 2.

4. A transgenic non-human mammal comprising the polynucleotide described in claim 2.

5. A transgenic non-human mammal according to claim 4, which is a model non-human mammal for dermatitis.

6. An antibody against the protein described in claim 1.

7. The antibody according to claim 6, which is a monoclonal antibody or an antigen-binding fragment thereof.

8. The antibody according to claim 7, wherein the monoclonal antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

9. A composition comprising the protein described in claim 1 or the polynucleotide described in claim 2.

10. A method for producing the protein according to claim 1, comprising the step of expressing a protein from the vector according to claim 3 in a host cell or a cell-free protein synthesis system.

Images