Regulating protein of NLRP3 inflammasome and application thereof

Abstract

The invention specifically discloses a regulatory protein of NLRP3 inflammasome and application thereof, and relates to the technical field of animal model construction. The invention provides application of COPB2 protein, nucleic acid molecules encoding the protein or an accelerant of the nucleic acid molecules in preparation of an animal inflammation model, expression of the COPB2 protein is conveniently controlled by gene editing means such as a hydrodynamic injection method and crisp knock-in, NLRP3 protein activation is caused, and accordingly inflammation occurrence and development of different tissues and organs are caused. The NLRP3 is not activated by an exogenous stimulant, so that the activation result of the NLRP3 is simplified, and inflammation of a specific part can be caused by specific expression of tissues and organs. Therefore, understanding of the NLRP3-mediated inflammation is accelerated, and development of drugs for the NLRP3-mediated inflammation is accelerated.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to the regulatory protein of the NLRP3 inflammasome and its applications. Background Technology

[0002] NLRP3, an inflammatory protein, plays a crucial role in Alzheimer's disease, cancer, and various inflammatory conditions, and has therefore been a key target for drug therapy. However, the activation factors of NLRP3 are numerous, involving potassium flux, mitochondria, lysosomes, and other influencing factors, and these remain unclear and controversial.

[0003] Animal inflammation models are used not only to study the pathogenesis of inflammation-related diseases but also to evaluate drug efficacy. For example, mesenchymal stem cells (MSCs) have shown potential in the treatment of various diseases. Studying the therapeutic effects of MSCs in LPS-induced animal models provides new insights into the treatment of inflammation-related diseases. Some research institutions have developed novel large animal models. For instance, a research team at the University of Michigan used a pig model to simulate human sepsis and acute respiratory distress syndrome, which better reflects clinical relevance and compensates for the limitations of small animal models.

[0004] However, on the one hand, there are differences in operating procedures and experimental conditions between different laboratories, which leads to inconsistent model construction results; on the other hand, existing multi-target inflammatory animal models are often based on the simple concept of "multi-factor superposition" and lack precise target organ response control mechanisms, which easily lead to "non-targeted spread" of inflammatory response, local over-excitation or insufficient activation of some target organs. The models exhibit high instability and poor reproducibility. Summary of the Invention

[0005] (1) Technical problems solved

[0006] Therefore, one of the main objectives of this invention is to provide the application of the COPB2 protein, the nucleic acid molecule encoding the protein, or its promoter in the preparation of animal inflammation models. COPB2 protein expression can be conveniently manipulated using gene editing techniques such as crisp knock-in, leading to NLRP3 protein activation and consequently causing inflammation in different tissues and organs. NLRP3 activation is independent of exogenous stimuli, thus streamlining the NLRP3 activation outcome and allowing for tissue- and organ-specific expression to induce inflammation at specific sites. This accelerates the understanding of NLRP3-mediated inflammation and the development of drugs targeting NLRP3-mediated inflammation.

[0007] (2) Technical solution

[0008] To achieve the above objectives, the present invention provides the application of COPB2 protein, nucleic acid molecules encoding the protein, or promoters thereof in the preparation of animal inflammation models.

[0009] In one embodiment, the promoter is selected from: COPB2 protein or an overexpression vector encoding the COPB2 protein sequence; exogenous COPB2 protein; naked DNA encoding the COPB2 protein sequence; liposome-encapsulated DNA encoding the COPB2 protein sequence; COPB2 protein precursor protein, conjugate, or complex that can be converted into COPB2 protein in vivo.

[0010] In one embodiment, the inflammation is liver inflammation.

[0011] In another aspect, the present invention provides the use of the COPB2 protein, the nucleic acid molecule encoding the protein, or a promoter thereof in the preparation of an animal liver inflammation model.

[0012] In one embodiment, the animal includes both mammals and non-mammals.

[0013] In one embodiment, the animal is a mammal.

[0014] In one embodiment, the mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs.

[0015] In one embodiment, the mammal is a mouse.

[0016] In another aspect, the present invention provides the use of the COPB2 protein, the nucleic acid molecule encoding the protein, or a promoter thereof in the preparation of a mouse liver inflammation model.

[0017] In one embodiment, the promoter is an overexpression vector encoding the COPB2 protein sequence.

[0018] In another aspect, this invention provides the application of an overexpression vector encoding the COPB2 protein sequence in the preparation of a mouse liver inflammation model.

[0019] In another aspect, the present invention also provides a method for constructing an animal inflammation model, the method comprising: administering the animal a COPB2 protein, a nucleic acid molecule encoding the protein, or a promoter thereof.

[0020] In one embodiment, the inflammation includes liver inflammation.

[0021] In one embodiment, the promoter is selected from: COPB2 protein or an overexpression vector encoding the COPB2 protein sequence; exogenous COPB2 protein; naked DNA encoding the COPB2 protein sequence; liposome-encapsulated DNA encoding the COPB2 protein sequence; COPB2 protein precursor protein, conjugate, or complex that can be converted into COPB2 protein in vivo.

[0022] In one embodiment, the promoter is an overexpression vector encoding the COPB2 protein sequence.

[0023] In one embodiment, the construction method specifically includes administering an overexpression vector encoding the COPB2 protein to the animal.

[0024] In one embodiment, the overexpression vector includes a viral vector or a non-viral vector.

[0025] In one embodiment, the overexpression vector is a non-viral vector.

[0026] In one embodiment, the non-viral vector includes one or a combination of DNA vectors, nanoparticles, cationic polymers, extracellular vesicles, or liposomes.

[0027] In one embodiment, the non-viral vector is a DNA vector.

[0028] In one embodiment, the DNA vector includes one or a combination of plasmid vectors, granular vectors, phage vectors, and so on.

[0029] In one embodiment, the DNA vector is a plasmid vector.

[0030] In one embodiment, the construction method specifically includes administering a plasmid vector encoding the COPB2 protein to the animal.

[0031] In one embodiment, the animal includes both mammals and non-mammals.

[0032] In one embodiment, the animal is a mammal.

[0033] In one embodiment, the mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs.

[0034] In one embodiment, the mammal is a mouse.

[0035] In one embodiment, the construction method specifically includes administering a plasmid vector encoding the COPB2 protein to mice.

[0036] In one embodiment, the route of administration includes intranasal (e.g., by inhalation), intrathecal (into the spinal canal or subarachnoid space), intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, sublingual, oral (by ingestion), intracerebral, transdermal (by absorption, e.g., through a skin catheter), or local (e.g., at the lesion site).

[0037] In one embodiment, the approach given is local.

[0038] In one embodiment, the region includes the liver.

[0039] In one embodiment, the administration is performed by hydrodynamic injection.

[0040] In one embodiment, the construction method specifically includes administering a plasmid vector encoding the COPB2 protein sequence to a mouse liver via hydrodynamic injection.

[0041] In another aspect, the present invention also provides an animal inflammation model, which is obtained by the above-described construction method.

[0042] In another aspect, the present invention also provides an animal liver inflammation model, which is obtained by the above-described construction method.

[0043] In another aspect, the present invention also provides the application of the above-mentioned animal inflammation model in exploring the pathogenesis of inflammation and / or evaluating anti-inflammatory drugs.

[0044] In another aspect, the present invention also provides the application of the above-mentioned animal liver inflammation model in exploring the pathogenesis of liver inflammation and / or evaluating anti-hepatitis drugs.

[0045] In another aspect, the present invention also provides the application of the above-mentioned animal inflammation model in the preparation of drugs for the prevention and / or treatment of inflammation.

[0046] In another aspect, the present invention also provides the application of the above-mentioned animal liver inflammation model in the preparation of drugs for the prevention and / or treatment of liver inflammation.

[0047] (3) Beneficial effects

[0048] This invention provides a regulatory protein of the NLRP3 inflammasome and its applications. Compared with the prior art, it has the following advantages:

[0049] 1. Gene editing techniques such as crisp knock-in can be used to conveniently manipulate COPB2 protein expression, leading to NLRP3 protein activation and consequently causing inflammation in various tissues and organs. This method activates NLRP3 independently of exogenous stimuli, thus streamlining the NLRP3 activation outcome and inducing inflammation at specific sites through organ-specific expression. This accelerates the understanding of NLRP3-mediated inflammation and the development of drugs targeting NLRP3-mediated inflammation.

[0050] 2. The inflammation sites in the mouse model are controllable and not limited to a single tissue or organ; the mechanisms of inflammation occurrence and development are clearly understood. It can be manipulated using various techniques such as hydrodynamics and crispf, offering diverse approaches. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 High expression of COPB2 enhances the stability of NLRP3 protein, primarily by inhibiting its degradation via the proteasome pathway.

[0053] Figure 2 COPB2 high expression enhances its stability by removing K48-Ub from NLRP3.

[0054] Figure 3 Overexpression of COPB2 can induce ASC oligomerization.

[0055] Figure 4 This is a Western blot diagram showing the activation of downstream proteins after COPB2 overexpression.

[0056] Figure 5 The high expression of COPB2 in mouse liver was detected by immunofluorescence, which showed the aggregation of NLRP3 protein.

[0057] Figure 6 The liver slices of mice with high expression of COPB2 were stained with H&E.

[0058] Figure 7 The results showed that COPB2 was highly expressed in mouse livers via liver RNA-seq. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] Terms and Definitions

[0061] As used herein, the terms "COPB2 protein" and "COPB2" are used interchangeably and refer to the β2 subunit of the coating protein complex. The COPB2 protein disclosed in this invention may be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animals, insects, and mammalian cells) using recombinant technology. Preferably, the COPB2 protein disclosed in this invention is encoded by the mouse COPB2 protein gene or its homologous genes or family genes.

[0062] This invention discloses variants of the COPB2 protein including (but not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the COPB2 protein. Similarly, adding one or more amino acids to the C-terminus and / or N-terminus typically does not alter the function of the COPB2 protein.

[0063] Depending on the host used in the recombinant production protocol, the COPB2 protein disclosed in this invention may be glycosylated or non-glycosylated. The term also includes active fragments and active derivatives of the COPB2 protein.

[0064] The variants of this polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, COPB2 proteins encoded by sequences that hybridize with the COPB2 protein-coding sequence under high or low stringency conditions, and COPB2 proteins obtained using antiserum against the COPB2 protein. Other COPB2 proteins, such as fusion proteins comprising the COPB2 protein or fragments thereof, may also be used in this invention. In addition to the nearly full-length COPB2 protein, this disclosure also includes soluble fragments of the COPB2 protein. Typically, this fragment has at least about 10 consecutive amino acids of the COPB2 protein sequence, typically at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0065] As used herein, the terms “COPB2 gene,” “COPB2 encoding gene,” “COPB2 protein encoding gene,” or “nucleic acid molecule encoding COPB2” are used interchangeably and all refer to a nucleotide sequence encoding the COPB2 protein disclosed in this invention.

[0066] The full-length nucleotide sequence or fragments of the COPB2 gene disclosed in this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this disclosure, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0067] It should be understood that the COPB2 gene disclosed in this invention is preferably obtained from mice. Other genes obtained from other animals that are highly homologous to the mouse gene (e.g., having more than 50%, preferably more than 55%, 60%, 65%, 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity) are also within the scope of this preferred consideration. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0068] As used herein, the terms "promoter" or "promoter of COPB2 protein or its coding sequence" are used interchangeably and refer to substances that can increase the level or activity of COPB2 protein or its encoding nucleic acid molecule. Promoters that can be used in this invention include, but are not limited to: COPB2 protein expression vectors, exogenous COPB2 protein, naked nucleic acid molecules encoding the COPB2 protein sequence, liposome-encapsulated nucleic acid molecules encoding the COPB2 protein sequence, COPB2 protein precursor proteins or conjugates or complexes that can be converted into COPB2 protein in vivo.

[0069] The COPB2 protein or its coding sequence disclosed in this invention or its promoters can directly activate the NLRP3 inflammatory protein, thereby triggering inflammatory cell death, leading to the release of inflammatory cytokines, and ultimately causing the development of inflammation.

[0070] As used herein, the terms "inhibitor" or "inhibitor of COPB2 protein or its encoding nucleic acid molecule" are used interchangeably and refer to a substance that can reduce the level or activity of COPB2 protein or its encoding nucleic acid molecule. Inhibitors that can be used in this disclosure include, but are not limited to: antibodies against COPB2 protein or nucleic acid molecules encoding the protein, siRNA, miRNA, antisense oligonucleotides, antagonists, and blocking agents.

[0071] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0072] Methods well known to those skilled in the art can be used to construct expression vectors containing the COPB2 protein-coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0073] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0074] Vectors containing the appropriate DNA sequence and suitable promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells.

[0075] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0076] As used in this paper, the following approaches can be adopted: (1) direct naked DNA or protein injection; (2) linking the cDNA, mRNA, and protein of COPB2 protein with a transferrin / poly-L-lysine complex to enhance its biological effects; (3) forming complexes of cDNA, mRNA, and protein with positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA, and protein in liposomes to mediate their entry into the cell, which facilitates the smooth entry of macromolecules and protects them from various extracellular enzymes. Hydrolysis; (5) cDNA, mRNA and protein bind to cholesterol, increasing their cytoplasmic retention time by 10 times; (6) Using immunoliposomes to transport cDNA, mRNA and protein can specifically transport them to target tissues and target cells; (7) Transfecting cDNA, mRNA and protein in vitro to regenerating cells (such as fibroblasts) can also effectively load COPB2 protein-related drugs into target cells; (8) Electroporation, which uses electric current to introduce cDNA, mRNA and protein into target cells.

[0077] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0078] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0079] Example 1: In vitro experiment:

[0080] 1. COPB2 protein overexpression:

[0081] Transfection was performed using PEI transfection reagent (taking iBMDM cell transfection as an example).

[0082] 1.1. Culture iBMDM cells in six-well plates until the cell density reaches 60%-70%.

[0083] 1.2. Prepare the following systems in a 1.5 ml sterile centrifuge tube: 100 μL serum-free DME M, 3 μg plasmid (Copb2 cDNA was amplified from mouse total cDNA by PCR and cloned into the pLV3-CMV vector) and 6 μL PEI. Mix gently and let stand for 10 minutes to prepare the DNA-PEI complex.

[0084] 1.3. Add the DNA-PEI complex dropwise into the cells and mix gently.

[0085] 1.4. Incubate cells in a 37℃ incubator for 24-48 hours, collect cells to extract proteins for subsequent experiments.

[0086] 2. Inhibiting protein synthesis using actinomycetes:

[0087] 2.1. After COPB2 protein is overexpressed, actinomycin is added to make the final concentration 1 μm.

[0088] 2.2. Cells were collected immediately after being inhibited with actinomycin for 0, 2, 4, and 6 hours, respectively, for Western blot (WB) experiments.

[0089] 3. Inhibition of the proteasome using cyclooxygenase:

[0090] 3.1. After COPB2 protein was overexpressed, cyclooxygenase was added to make the final concentration 50 nm.

[0091] 3.2. Cells were collected immediately after 0, 2, 4, and 6 hours of inhibition with cyclooxygenase for Western blot (WB) experiments.

[0092] 4. Inhibition of lysosomes using bafloxacin A1:

[0093] 4.1. After COPB2 protein is overexpressed, cyclooxygenase is added to make the final concentration 30 nm.

[0094] 4.2. Cells were immediately harvested for Western blot (WB) experiments after being inhibited with bafloxacin A1 for 0, 2, 4, and 6 hours, respectively.

[0095] like Figure 1 As shown, the use of cyclohexime ketone (CHX), epoxomicin, and bafomycin A1 (BafA1) demonstrated that high expression of COPB2 protein can significantly enhance the stability of NLR P3 protein.

[0096] 5. Immunofluorescence assay:

[0097] 5.1. Seed the appropriate number of iBMDM cells onto a 24-well cell slide according to the cell seeding requirements. After culturing the cells overnight, transfect them according to the method described above.

[0098] 5.2. The cell smears were washed three times with PBS for 5 minutes each time.

[0099] 5.3. Add 1 mL of methanol to fix the cells for 15 min, and wash the cell smears three times with PBS for 5 min each time.

[0100] 5.4. Add 500 μL of 5% BSA and block at room temperature for 1 h.

[0101] 5.5. Remove the BSA and add the primary antibody diluted according to the instructions. Incubate overnight at 4C.

[0102] 5.6. Remove the primary antibody, add PBST to wash the cell slide three times, 5 min each time.

[0103] 5.7. Add the immunofluorescent secondary antibody corresponding to the species of the primary antibody according to the instructions, and incubate at 37°C in the dark for 1 hour.

[0104] 5.8. Remove the secondary antibody, wash the cells three times with PBST for 5 minutes each time (avoid light).

[0105] 5.9. Add DAPI and incubate in the dark for 5 min for cell nuclear staining. Wash with PBST 3 times, 5 min each time.

[0106] 5.10. Aspirate the liquid from the stem cell slide and place the slide onto a glass slide with an anti-fluorescence quenching mounting medium. Use a confocal microscope to capture images of cell fluorescence.

[0107] like Figure 2 As shown, COPB2 protein can significantly reduce ubiquitin linkage on NLRP3 protein.

[0108] 6. CO-IP experiment:

[0109] 6.1. Discard the cell culture medium and wash the cells three times with 2 mL of pre-cooled PBS.

[0110] 6.2. After completely removing PBS, add an appropriate amount of IPbuffe containing protease inhibitors, lyse cells on ice, scrape off the cells at the bottom of the culture plate with a cell scraper, transfer the lysis buffer to a 15 mL EP tube, vortex for 1 min, and continue to lyse the cells on ice for 20 min.

[0111] 6.3. Centrifuge at 14000 rpm and 4℃ for 20 min.

[0112] 6.4. Take 40 μL of cell lysis supernatant as the input group and add 10 μL of 5xSDS protein loading buffer. Mix well, heat at 100℃ for 5 min, centrifuge at 12000 rpm for 2 min to allow the sample to settle to the bottom, and store at -20℃.

[0113] 6.5. Add the required antibody to the remaining supernatant, mix by inversion in a 4°C chromatography freezer for 2 hours, then add 40 μL of protein A / G agarose beads, mix by inversion in a 4°C chromatography freezer overnight.

[0114] 6.6. On the second day, centrifuge the IP sample at 1000 rpm for 5 min at 4°C. Gently aspirate the supernatant (avoiding the beads). Add 400 μL of IP buffer, mix by inverting, and centrifuge at 1000 rpm for 5 min at 4°C. Repeat 5 times. Aspirate the supernatant, add 40 μL of 1xSDS protein loading buffer, heat at 100°C for 5 min, and centrifuge to allow the sample to settle at the bottom of the tube. Store at -80°C.

[0115] like Figures 3-4 As shown, overexpression of Copb2 protein can directly lead to oligomerization of the ASC linker, which is one of the core indicators of NLRP3 protein activation leading to inflammatory cell death. Western blotting experiments also showed that high expression of Copb2 protein resulted in IL-1β cleavage, triggering inflammatory cell death.

[0116] Example 2: In vivo experiment:

[0117] 1.1. In the experimental group, the target plasmid and Sleeping Beauty transposase plasmid were mixed in 2 ml of physiological saline at a mass ratio of 4:1. In the control group, the empty vector and Sleeping Beauty transposase plasmid were mixed in 2 ml of physiological saline at a mass ratio of 4:1.

[0118] 1.2. Rapidly inject physiological saline into the tail vein of the mouse.

[0119] 1.3. Two weeks after injection, liver tissue was harvested for sectioning and sequencing analysis.

[0120] like Figure 5 As shown, overexpression of copb2 protein can directly induce NLRP3 oligomerization (green fluorescence represents F4 / 80, which is a macrophage marker; red represents NLRP3 protein; and pink represents Copb2 protein), indicating NLRP3 activation, which triggers inflammatory cell death, leading to the release of inflammatory cytokines and ultimately causing the development and progression of inflammation.

[0121] like Figure 6 As shown, H&E staining results indicate monocyte aggregation and the development of inflammation.

[0122] like Figure 7 As shown, high expression of the Copb2 gene in mouse liver led to the enrichment of multiple inflammatory pathways, suggesting the occurrence and development of inflammation.

[0123] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of COPB2 protein, nucleic acid molecules encoding the protein, or their promoters in the preparation of animal inflammation models.

2. The application according to claim 1, characterized in that, The promoter includes one or a combination of COPB2 protein or an overexpression vector encoding the COPB2 protein sequence, exogenous COPB2 protein, naked DNA encoding the COPB2 protein sequence, liposome-encapsulated DNA encoding the COPB2 protein sequence, COPB2 protein precursor protein or conjugate or complex that can be converted into COPB2 protein in vivo.

3. A method for constructing an animal inflammation model, characterized in that, The construction method includes administering the COPB2 protein, a nucleic acid molecule encoding the protein, or a promoter thereof to the animal.

4. The construction method according to claim 3, characterized in that, The promoter includes one or a combination of COPB2 protein or an overexpression vector encoding the COPB2 protein sequence, exogenous COPB2 protein, naked DNA encoding the COPB2 protein sequence, liposome-encapsulated DNA encoding the COPB2 protein sequence, COPB2 protein precursor protein or conjugate or complex that can be converted into COPB2 protein in vivo.

5. An animal inflammation model, characterized in that, The animal inflammation model is obtained by the construction method described in any one of claims 3-4.

6. The application of the animal inflammation model of claim 5 in exploring the pathogenesis of inflammation and / or evaluating anti-inflammatory drugs.

7. The use of the animal inflammation model of claim 5 in the preparation of drugs for the prevention and / or treatment of inflammation.

8. The application according to claim 1, 2, 6, or 7, or the construction method according to claim 3 or 4, or the animal inflammation model according to claim 5, characterized in that, The inflammation includes liver inflammation.

9. The application according to claim 1, 2, 6, or 7, or the construction method according to claim 3 or 4, or the animal inflammation model according to claim 5, characterized in that, The animals mentioned include both mammals and non-mammals.

10. The application according to claim 1, 2, 6, or 7, or the construction method according to claim 3 or 4, or the animal inflammation model according to claim 5, characterized in that, The animal in question is a mouse.

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