A method for constructing a SARS-CoV-2 antigen-induced cytokine storm mouse model and its application

Abstract

The present invention discloses a method for constructing a SARS‑CoV‑2 antigen-induced cytokine storm mouse model and its application. The construction method is simple to operate, does not require an animal P3 laboratory, and has relatively low requirements on operators, laboratory hardware and levels. It is suitable for major scientific research institutes and enterprises to develop effective drugs against the novel coronavirus-induced cytokine storm, and is also suitable for clinical screening of effective drugs for the treatment of novel coronavirus patients, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing a cytokine storm mouse model induced by a SARS-CoV-2 antigen and an application thereof. Background Art

[0002] Cytokine storm, also known as cytokine release syndrome (CRS), is an overreaction of the body to infection. When the body is infected by certain viruses or the immune function is abnormal, the cytokine balance in the body is broken, pro-inflammatory cytokines continue to be produced in large quantities, and more immune cells are activated to gather at the site of inflammation. Excessive immune cells and multiple pro-inflammatory cytokines can cause tissue congestion, edema, fever, and damage, and may also cause other secondary infections and even lead to "systemic inflammatory response syndrome" (sepsis), causing the patient to die from multiple organ failure.

[0003] The main cause of death in patients with moderate to severe COVID-19 is the occurrence and development of cytokine storms, and the development of drugs that can alleviate cytokine storms is urgent. The virus-induced cytokine storm model (virus-induced model) is the most direct and effective choice for studying COVID-19, but virus research is dangerous, especially the SARS-CoV-2 virus is highly contagious, and has extremely high requirements for laboratory levels and personnel qualifications, which limits the choice of this virus-induced cytokine storm model to a certain extent.

[0004] Animal models are essential for viral pathology, vaccine development, and drug screening. Nonhuman primate models are commonly used in preclinical experiments, but they are expensive, inconvenient to use, and require relatively complex facilities, which limits the application of this animal model in SARS-CoV-2. Mouse models are a more ideal preclinical experimental model because they are cheap, more accessible, and have simple breeding conditions. Several human angiotensin-converting enzyme 2 (hACE2) transgenic mice have been generated using different strategies and used to simulate SARS-CoV infection. In general, lung pathology caused by SARS-CoV-2 in transgenic mice is associated with the level of hACE2 expression in the lung. However, this model is expensive and requires the use of SARS-CoV-2 virus induction, making it difficult to promote universally.

[0005] Animal P3 laboratories are required to conduct animal research on the new coronavirus, but there are not many institutions with animal P3 laboratories among major scientific research institutes and enterprises in my country, which makes it difficult to directly study the new coronavirus and to establish an animal model of cytokine release syndrome caused by the new coronavirus. Even if the corresponding animal model is established, it is difficult to promote and use it widely due to the lack of P3 laboratories or high costs. The lack of a simple, effective, and low-cost animal model of the new coronavirus-induced massive release of cytokines has led to a slow development of drugs for the new coronavirus-induced cytokine release. In addition, some researchers are using alternative viruses or substances such as LPS to construct inflammation models, but non-new coronavirus-induced models cannot truly reflect the actual situation in which the new coronavirus causes the body to release a large number of cytokines. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a SARS-CoV-2 antigen-induced cytokine storm mouse model and a construction method thereof in the art to solve the above-mentioned technical problems existing in the prior art.

[0007] The present invention constructs for the first time an in vivo mouse model of massive cytokine release induced by the novel coronavirus antigen, which can be carried out without an animal P3 laboratory. The model construction method is simple to operate, has relatively low requirements for laboratory hardware and level, and has relatively low requirements for operators. It is suitable for major scientific research institutes and enterprises to develop effective drugs to fight against the novel coronavirus-induced cytokine storm, and is also suitable for clinical screening of effective drugs for the treatment of new coronavirus patients.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention provides a method for constructing a SARS-CoV-2 antigen-induced cytokine storm mouse model.

[0010] Furthermore, the method comprises the following steps: administering coronavirus antigens, TNF-α and IFN-γ to mice to construct the cytokine storm mouse model.

[0011] Furthermore, the administration method is intraperitoneal injection.

[0012] Further, the mice include C57BL / 6 mice, BALB / c mice, ICR mice, KM mice, NIH mice or LACA mice;

[0013] Optionally, the mouse is a C57BL / 6 mouse.

[0014] Further, the coronavirus antigen is SARS-CoV-2 spike protein or SARS-CoV-2 nucleoprotein;

[0015] Optionally, the coronavirus antigen is the SARS-CoV-2 spike protein.

[0016] Furthermore, the administration dose of the SARS-CoV-2 spike protein is 1.0-5.0 μg / mouse;

[0017] The administration dose of TNF-α is 0.5-2.5 μg / mouse;

[0018] The IFN-γ administration dose is 0.5-2.5 μg / mouse.

[0019] Furthermore, the administration dose of the SARS-CoV-2 spike protein is 2.76 μg / mouse;

[0020] The administration dose of TNF-α is 1.5 μg / mouse;

[0021] The IFN-γ administration dose was 1.5 μg / mouse.

[0022] Furthermore, the cytokine storm mouse model was constructed after coronavirus antigen, TNF-α and IFN-γ were administered to mice simultaneously for 6 hours.

[0023] In the present invention, the coronavirus is a large class of viruses widely present in nature. Coronavirus belongs to the genus Coronavirus of the family Coronaviridae of the order Nidovirales in systematic classification. The virus of the genus Coronavirus is an RNA virus with an envelope and a linear single-stranded positive-strand genome. The diameter of the coronavirus is about 80-120nm, the 5′ end of the genome has a methylated cap structure, the 3′ end has a poly (A) tail, and the total length of the genome is about 27-32kb, which is the largest genome among the known RNA viruses.

[0024] In some embodiments, the coronavirus includes but is not limited to: new coronavirus (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV).

[0025] In a specific embodiment of the present invention, the coronavirus is the new coronavirus SARS-CoV-2 or its related variants.

[0026] In the present invention, the cytokine storm is also called cytokine release syndrome, abbreviated as CRS, which is an overreaction of the body to infection. At present, the specific pathophysiological mechanism of cytokine storm is still unclear, and there is no specific corresponding treatment drug.

[0027] In some embodiments, the cytokine storm may be a cytokine storm caused by various viral infections, macromolecular antibody therapy, organ transplantation, or CAR-T therapy.

[0028] In a preferred embodiment, the cytokine storm is a cytokine storm caused by viral infection.

[0029] In a more preferred embodiment, the cytokine storm is a cytokine storm caused by coronavirus infection.

[0030] In a specific embodiment of the present invention, the cytokine storm is a cytokine storm caused by SARS-CoV-2 infection.

[0031] In the present invention, for the first time, a SARS-CoV-2 antigen is combined with TNF-α and IFN-γ and injected into the peritoneal cavity of mice to construct a cytokine storm mouse model. The model can be successfully established in only 6 hours using the construction method of the present invention as described above, and the cytokine storm mouse model constructed by the construction method of the present invention as described above produces a larger amount of cytokines, which can not only stimulate the production of cytokine storm-related cytokines in the peritoneal cavity of mice, but also promote the production of cytokine storm-related cytokines in the blood of mice, and can further simulate the phenomenon that SARS-CoV-2 induces the human body to release a large amount of cytokines, thereby providing a new cytokine storm mouse model with better cytokine storm simulation effect and can be effectively used for screening anti-coronavirus infection drugs in the field.

[0032] The new cytokine storm mouse model provided by the present invention can be used to screen drugs that inhibit or reduce the cytokine storm caused by SARS-CoV-2, which is conducive to the development of drugs related to inhibiting cytokine storm. In addition, the new cytokine storm mouse model provided by the present invention can also be used for drug screening for clinical treatment of SARS-CoV-2 infected patients, while improving the treatment effect on the infected patients, reducing unnecessary medication and reducing treatment costs.

[0033] Although TNF-α is often used as an inducer of mouse inflammation model, its dosage is usually large (10 μg per mouse). Although this dosage can effectively induce inflammation in mice, it will also cause the mice to die quickly. The present invention found that even if 5 μg TNF-α is injected intraperitoneally per mouse, all mice will die within 30 hours, while the model mice constructed by the construction method provided by the present invention can still survive until 48 hours.

[0034] In the present invention, the TNF-α is tumor necrosis factor α, also known as cachectin or TNFSF1A, which is a ligand of the TNF superfamily. It is a pleiotropic cell molecule that plays a central role in apoptosis and immune system development. TNF-α is secreted by macrophages, monocytes, neutrophils, CD4+T cells, and NK cells. TNF-α can coordinate the production of other cytokines, cell survival and death to coordinate the homeostasis of tissues.

[0035] TNF-α exists in two biologically active forms: transmembrane (tmTNF-α) and secreted (sTNF-α). Its expression is increased in many pathological conditions, including sepsis, malignancies, heart failure, and chronic inflammatory diseases. TNF-α can also be produced by malignant cells and immune cells in the tumor-associated microenvironment, where it acts as an endogenous tumor promoter and promotes the progression of malignant diseases by generating an inflammatory niche.

[0036] In the present invention, the IFN-γ is interferon-γ, which is a type of cytokine, mainly a pro-inflammatory cytokine produced by T cells and NK cells. It has different immune action mechanisms and produces immunomodulatory responses in multiple signaling pathways; IFN-γ has broad-spectrum antiviral and immunomodulatory functions. The role of IFN-γ in the tumor microenvironment is very complex. It can not only play an anti-tumor role, but also promote tumorigenesis under certain conditions.

[0037] Interferon can be divided into IFN-α, IFN-B and IFN-γ according to the differences in interferon cell sources, physicochemical properties and biological activities; IFN-γ is also called subtype interferon, which is mainly produced by activated T cells and NK cells. The mature molecule of human IFN-γ exists in the form of homodimeric glycoprotein.

[0038] In some embodiments, the concentration of the SARS-CoV-2 viral antigen injected into mice is 200 nM (27.6 ng / μL) of spike protein, the injection volume is 100 μL per mouse, and each mouse is injected with 2.76 μg of spike protein.

[0039] In some embodiments, the injected dose of TNF-α per mouse is 0.5-2.5 μg, preferably 1.5 μg per mouse.

[0040] In some embodiments, the injection dose of IFN-γ per mouse is 0.5-2.5 μg, preferably 1.5 μg per mouse.

[0041] In some embodiments, the mode of administration includes, but is not limited to, intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, and / or intracerebroventricular injection.

[0042] In a preferred embodiment, the administration is by intraperitoneal injection or intravenous injection.

[0043] In a more preferred embodiment, the administration is by intraperitoneal injection.

[0044] In some embodiments, the mouse can be selected from any one of the mouse strains commonly used in the art.

[0045] In a preferred embodiment, the mouse is an immunocompetent mouse, including but not limited to: C57BL / 6 mouse, BALB / c mouse, ICR mouse, KM mouse, NIH mouse or LACA mouse. The use of immunocompetent mice can more truly reflect the real changes in the immune response of the body after being stimulated by viral antigens.

[0046] In a more preferred embodiment, the mouse is a C57BL / 6 mouse, which has the advantages of not requiring any genetic modification, being cheap, easy to raise, and convenient for promotion.

[0047] In a specific embodiment of the present invention, the weight of the C57BL / 6 mice used in the present invention is kept consistent, and the age is about 8 weeks. If the age is too large, the inflammatory response may be stronger, but the risk of death increases, which is not conducive to long-term observation. Most of the mice commonly used in the prior art are genetically engineered mice, which are not only costly, but also require the use of SARS-CoV-2 virus when inducing cytokine storms, which has special requirements for laboratory levels and operators, and is not convenient for promotion and application. The use of C57BL / 6 described in the present invention can overcome the above-mentioned problems in the prior art.

[0048] In some embodiments, the coronavirus antigens include, but are not limited to, spike protein (S), nucleoprotein (N), envelope protein (E), membrane protein (M) and / or hemagglutinin-esterase glycoprotein (HE).

[0049] In a preferred embodiment, the coronavirus antigen is the spike protein (S) or the nucleoprotein (N).

[0050] In a more preferred embodiment, the coronavirus antigen is the spike protein (S).

[0051] In a specific embodiment of the present invention, the present invention uses the SARS-CoV-2 spike protein instead of the SARS-CoV-2 virus. Therefore, the method for constructing a cytokine storm mouse model as described above provided by the present invention has low requirements for laboratory level and operators, and is easy to promote.

[0052] In addition, the concentration of TNF-α used in the present invention is relatively low, and the problem of rapid and massive damage to the internal organs of mice and rapid death of mice due to excessive TNF-α dosage will not be caused.

[0053] A second aspect of the present invention provides a SARS-CoV-2 antigen-induced cytokine storm mouse model.

[0054] Furthermore, the cytokine storm mouse model is a cytokine storm mouse model constructed using the method described in the first aspect of the present invention.

[0055] In some embodiments, the cytokine storm mouse model can be successfully established after the coronavirus antigen, TNF-α and IFN-γ are simultaneously administered to the mouse for 6 hours using the method described in the first aspect of the present invention.

[0056] The third aspect of the present invention provides the use of a cytokine storm mouse model constructed by the method described in the first aspect of the present invention in screening drugs that inhibit cytokine storm.

[0057] In some embodiments, the drugs for inhibiting cytokine storm include, but are not limited to: any small molecule compounds, polypeptides, proteins, antibodies, vaccines, nucleic acid molecules, polysaccharides, Chinese herbal medicine extracts, etc. that may play a role in inhibiting cytokine storm.

[0058] The fourth aspect of the present invention provides a method for screening drugs that inhibit cytokine storm.

[0059] Furthermore, the method comprises the following steps: using the cytokine storm mouse model constructed by the method described in the first aspect of the present invention to screen candidate drugs that can inhibit cytokine storm.

[0060] In some embodiments, the candidate drugs include, but are not limited to: any small molecule compounds, polypeptides, proteins, antibodies, vaccines, nucleic acid molecules, polysaccharides, Chinese herbal medicine extracts, etc. that may be able to inhibit cytokine storm.

[0061] In some embodiments, the method includes the following steps: administering a suitable dose of a candidate drug to the cytokine storm mouse model constructed by the method described in the first aspect of the present invention, and determining whether the candidate drug can become an effective drug for inhibiting the cytokine storm by detecting the release of cytokine storm-related inflammatory factors in the cytokine storm mouse model after administration of the candidate drug.

[0062] In some embodiments, if the candidate drug can inhibit the release of cytokine storm-related inflammatory factors in the cytokine storm mouse model, the candidate drug is an effective drug that can inhibit cytokine storm.

[0063] In some embodiments, ELISA or CBA is used to detect the effect of the candidate drug on the release of cytokine storm-related inflammatory factors in the cytokine storm mouse model.

[0064] In some embodiments, the ELISA is an enzyme-linked immunosorbent assay (ELISA). The ELISA method is the most classic method for detecting cytokines, and is also the most widely used and most mature immunoassay technology. The double antibody sandwich method is commonly used. The ELISA method can directly detect cytokines secreted outside the cell in a free state. It has high sensitivity, simple operation, and is economical. The laboratory can complete the entire experiment with a microplate reader.

[0065] In some embodiments, the CBA is a cytokine microsphere detection technology (Cytometric Bead Array, CBA), which is a multiple protein quantitative detection method based on a flow cytometric detection system, which can simultaneously detect multiple indicators in a single sample. Its working principle is to use microspheres with different fluorescence intensities, which are equipped with antibodies (Capture Antibody) that can recognize specific proteins. After interacting with samples (such as serum, plasma, culture supernatant, cell lysate, etc.) and PE detection antibodies (Detection Antibody), flow cytometry is used for analysis. According to the different PE fluorescence intensities, software is used for analysis and comparison with standard products, and specific proteins in the sample can be quantified. Compared with ELISA, it saves more samples and is more convenient to operate.

[0066] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0067] The present invention discloses for the first time that a cytokine storm mouse model can be successfully induced by combining a novel coronavirus antigen with TNF-α and IFN-γ. The model construction method is simple to operate, does not require an animal P3 laboratory, and has relatively low requirements for operators, laboratory hardware and levels. It is suitable for major scientific research institutes and enterprises to develop effective drugs against cytokine storms induced by the novel coronavirus, and is also suitable for clinical screening of effective drugs for the treatment of novel coronavirus patients, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The spike protein combined with TNF-α and IFN-γ can stimulate the mouse peritoneal cavity to produce cytokine storm-related cytokines; C57BL / 6 mice were intraperitoneally injected with PBS, spike protein (S), TNF-α+IFN-γ, and spike protein (S)+TNF-α+IFN-γ, and the mice were killed 6 hours later, and the peritoneal cavity was lavaged with 1 mL PBS, and the lavage fluid was collected and the concentrations of various cytokines were detected by CBA;

[0069] Figure 2 The spike protein combined with TNF-α and IFN-γ can stimulate the mouse serum to produce cytokine storm-related cytokines; C57BL / 6 mice were intraperitoneally injected with PBS, spike protein (S), TNF-α+IFN-γ, and spike protein (S)+TNF-α+IFN-γ, and the mice were killed 6 hours later, blood was collected from the eyeballs, and serum was collected, and the concentrations of various cytokines were detected by CBA;

[0070] Figure 3 The spike protein combined with IL-2 could not stimulate the mouse peritoneal cavity to produce a large number of cytokine storm-related cytokines; C57BL / 6 mice were intraperitoneally injected with PBS, spike protein (S), IL-2, and spike protein (S) + IL-2, and the mice were killed 6 hours later. The peritoneal cavity was lavaged with 1 mL PBS, and the lavage fluid was collected and the concentrations of various cytokines were detected by CBA;

[0071] Figure 4 The combined effect of spike protein with TNF-α and IFN-γ was independent of the dose of spike protein. C57BL / 6 mice were intraperitoneally injected with different concentrations of spike protein (low-dose S: ​​2.76 μg / mouse; high-dose S: ​​5.52 μg / mouse) mixed with TNF-α and IFN-γ. The mice were killed 6 hours later, and the peritoneal cavity was lavaged with 1 mL PBS. The lavage fluid was collected and the concentrations of various cytokines were detected by CBA.

[0072] Figure 5The survival rate of mice in the inflammatory model induced by the spike protein combined with TNF-α and IFN-γ was high; C57BL / 6 mice were divided into two groups, with 3 mice in each group. One group of mice was intraperitoneally injected with a mixture of spike protein, low-dose TNF-α (1.5μg / mouse) and IFN-γ, and the other group was intraperitoneally injected with a high-dose TNF-α (5μg / mouse). The survival of the mice was recorded every 6 hours for a total of 48 hours. DETAILED DESCRIPTION

[0073] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0074] The main experimental materials used in the examples of the present invention are shown in Table 1 below.

[0075] Table 1 Experimental materials

[0076]

[0077] Example Construction of a mouse model of cytokine storm induced by novel coronavirus antigen

[0078] 1. Preparation of drugs

[0079] 1. Preparation of SARS-CoV-2 spike protein working solution: Preparation of spike protein storage solution: Dissolve 50 μg of SARS-CoV-2 spike protein in 1811.6 μL of sterile PBS solution to prepare a working solution with a concentration of 200 nM (27.6 ng / μL), and dispense it into 200 μL EP tubes, 100 μL per tube.

[0080] 2. Preparation of IL-2 working solution: Use PBS to prepare IL-2 into a 50 μg / mL working solution.

[0081] 3. Preparation of TNF-α: Prepare TNF-α into a 30 μg / mL working solution.

[0082] 4. Preparation of IFN-γ: Prepare IFN-γ into a 30 μg / mL working solution.

[0083] 2. Establishment of in vivo animal model

[0084] 1. Animal requirements: SFP grade C57BL / 6 mice, aged more than 8 weeks, of the same gender, weighing 20-25g.

[0085] 2. Animal grouping:

[0086] ①PBS group: 200 μL PBS.

[0087] ② Spike protein group: 100μL spike protein working solution + 100μL PBS.

[0088] ③TNF-α+IFN-γ group: 50μL TNF-α working solution + 50μL IFN-γ working solution + 100μL PBS.

[0089] ④TNF-α+IFN-γ group+spike protein group: 50μL TNF-α working solution + 50μL IFN-γ working solution + 100μL spike protein working solution.

[0090] ⑤IL-2 group: 100μL IL-2 working solution + 100μL PBS.

[0091] ⑥IL-2+spike protein group: 100μL IL-2 working solution + 100μL spike protein working solution.

[0092] 3. Injection method: intraperitoneal injection.

[0093] 4. Culture conditions: SFP environment to prevent other bacterial microorganisms from causing infection in mice.

[0094] 5. Blood collection from the eyeball: 6 hours after drug injection, fix the mouse with one hand; gently press the eye skin on the blood collection side to make the eyeball congested and protruding; quickly remove the eyeball with elbow forceps, and use a sterile 1.5mL EP tube to collect blood; at the same time, press the mouse heart with the middle finger of the left hand to speed up the heart's blood pumping speed. After the blood is collected, place it at room temperature to allow the blood to coagulate and the serum to precipitate. Transfer the precipitated serum to a new EP tube and use a pipette to measure the volume of the recovered liquid. For short-term storage, it can be placed in a 4°C refrigerator, and for long-term storage, it can be placed in a -20 or -80°C refrigerator.

[0095] 6. Take the peritoneal lavage fluid: Slowly cut the mouse peritoneal cavity, gently rinse the peritoneal cavity with 1 mL of sterile PBS, and try to suck back all the lavage fluid, centrifuge at 3000 rpm for 5 minutes at room temperature, collect the supernatant with a pipette and measure the supernatant volume. For short-term storage, place it in a 4℃ refrigerator, and for long-term storage, place it in a -20℃ or -80℃ refrigerator.

[0096] 7. ELISA or CBA was used to detect cytokine release syndrome-related cytokines IL-1β and IL-6.

[0097] 3. Experimental Results

[0098] The mouse inflammation model constructed by the present invention can induce the mouse peritoneal cavity to produce a large amount of IL-6 and IL-1β related to cytokine storm within 6 hours (see Figure 1 ). This effect was much stronger than that of the spike protein alone and the TNF-α+IFN-γ group.

[0099] The mouse inflammation model constructed by the present invention can induce the mouse serum to produce a large amount of IL-6 and IL-1β related to cytokine storm within 6 hours (see Figure 2 ). This effect was much stronger than that of the spike protein alone and the TNF-α+IFN-γ group.

[0100] The inventor of the present invention previously applied for an authorized patent entitled "Establishment and Application of a SARS-CoV-2 Antigen-Induced Cytokine Storm Model" (Patent No.: ZL202310628453.8), which used SARS-CoV-2 spike protein combined with IL-2 to stimulate human peripheral blood mononuclear cells to produce cytokines, thereby constructing an in vitro cell model of cytokine storm. This example further constructed a mouse model induced by SARS-CoV-2 spike protein combined with IL-2 (such as the IL-2+spike protein group described above). The results showed that SARS-CoV-2 spike protein combined with IL-2 did not have the effect of synergistically inducing the production of IL-6 and IL-1β (see Figure 3 ), it can be seen that a simple combination or replacement of cytokines is not sufficient to successfully induce a cytokine storm mouse model, nor is a cytokine combination that can successfully induce a cytokine storm cell model in vitro necessarily sufficient to successfully induce a cytokine storm mouse model in vivo. That is, the technical solution of the present invention of using a spike protein in combination with TNF-α+IFN-γ to induce a cytokine storm mouse model has achieved unexpected technical effects.

[0101] In order to analyze whether the mouse model induced by the combination of spike protein and TNF-α+IFN-γ is spike protein dose-dependent, the inventors increased the dose of spike protein and found that the secretion of IL-6 and IL-1β did not increase after doubling the dose of spike protein (see Figure 4 ). This indicates that increasing the dose of spike protein may not necessarily induce more CRS-related cytokine release.

[0102] TNF-α is a commonly used inducer of mouse inflammation model, and the infusion method is intraperitoneal injection or intravenous injection, with a dose of 5-20 μg / mouse. Due to the large dose of TNF-α, it is difficult for mice to survive for more than 24 hours. The inventors compared the method of the present invention with the conventional method of inducing high-dose TNF-α (5 μg / mouse) and found that the model mice induced by high-dose TNF-α all died within 30 hours, while the inflammation model mice induced by the present invention all survived within 48 hours (see Figure 5 ), this result indicates that the survival rate of the cytokine storm mouse model constructed by the method provided by the present invention is significantly better than the cytokine storm mouse model constructed by the conventional method reported in the prior art.

[0103] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a SARS-CoV-2 antigen-induced cytokine storm mouse model, characterized in that: The method comprises the following steps: administering coronavirus antigen, TNF-α and IFN-γ to mice to construct the cytokine storm mouse model; The coronavirus antigen is the SARS-CoV-2 spike protein; The administration method is intraperitoneal injection.

2. The method according to claim 1, characterized in that The mice include C57BL / 6 mice, BALB / c mice, ICR mice, KM mice, NIH mice or LACA mice.

3. The method according to claim 2, characterized in that The mice are C57BL / 6 mice.

4. The method according to claim 1, characterized in that: The SARS-CoV-2 spike protein is administered at a dose of 1.0-5.0 μg / mouse; The administration dose of TNF-α is 0.5-2.5 μg / mouse; The IFN-γ administration dose was 0.5-2.5 μg / mouse.

5. The method according to claim 4, characterized in that The SARS-CoV-2 spike protein was administered at a dose of 2.76 μg / mouse; The TNF-α administration dose was 1.5 μg / mouse; The IFN-γ was administered at a dose of 1.5 μg per mouse.

6. The method according to claim 1, characterized in that The cytokine storm mouse model was established after coronavirus antigen, TNF-α and IFN-γ were administered to mice simultaneously for 6 hours.

7. Use of the cytokine storm mouse model constructed by the method according to any one of claims 1 to 6 in screening drugs that inhibit cytokine storm.

8. A method for screening drugs that inhibit cytokine storm, characterized in that: The method comprises the following steps: using the cytokine storm mouse model constructed by the method according to any one of claims 1 to 6 to screen candidate drugs capable of inhibiting cytokine storm.

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