CN112237630A - Application of MASP-2, coronavirus N protein or their combined substances in the preparation of drugs for diseases caused by coronavirus - Google Patents

Abstract

The invention provides application of a substance aiming at MASP-2, coronavirus N protein or a combination thereof in preparing a medicament for treating diseases caused by coronaviruses. The invention proves that SARS-CoV N protein and MASP-2 have interaction through the research of the interaction between N protein and MASP-2 and related molecular mechanism, and the interaction area is highly homologous with MERS-CoV N protein and SARS-CoV-2N protein; the N protein is found to promote the downstream complement activation of the lectin pathway to be intensified; the mortality rate of the Masp2 gene knockout mice is obviously reduced and recovered more quickly after infection; in vivo infection experiments prove that the death rate of mice can be effectively reduced by targeting MASP-2 or N protein or the combination of the MASP-2 and N protein. Proves that the inhibitor aiming at MASP-2 and N protein or combination of MASP-2 and N protein has value in being used as candidate drugs for preventing and treating SARS-CoV virus infection.

Description

Application of substance aiming at MASP-2, coronavirus N protein or combination thereof in preparation of drugs for diseases caused by coronaviruses

Technical Field

The invention relates to the application of a substance aiming at MASP-2, coronavirus N protein or a combination thereof in preparing a medicament for treating diseases caused by coronaviruses.

Background

Coronaviruses are positive-stranded RNA viruses of the order nested viruses, with genomes about 3 kilobases in length. Among them, SARS coronavirus (SARS-CoV) causing Severe Acute Respiratory Syndrome (SARS), MERS coronavirus causing middle east respiratory syndrome (MERS-CoV), and pneumonia (COVID-19) caused by 2019 novel coronavirus which has outbreak in 2019, and novel coronavirus (SARS-CoV-2) which is a pathogen thereof can cause severe acute respiratory infectious diseases. Although SARS-CoV has disappeared for many years, due to the existence of the SARS-like coronavirus bank in the bat body, and the RNA virus itself is relatively mutable, there is a possibility that the virus-like virus undergoes host changes to infect humans. The homology between SARS-CoV-2 and SARS-CoV is about 80% by genome sequencing analysis, and it may be from the same bat coronavirus. In addition, SARS and similar coronavirus droplet transmission characteristics, and lack of specific drugs, once outbreak is epidemic, the control is difficult, and the health of human beings is threatened. Improving the prevention and control capability of the virus and similar virus infection, and developing and storing the prevention and control medicine aiming at SARS coronavirus has important significance.

SARS-CoV, MERS-CoV and SARS-CoV-2 belong to the genus beta coronavirus of the family Coronaviridae, are single-stranded positive-strand RNA viruses, are enveloped by an envelope, and are named because the spinous process proteins present a crown shape. Its genome is about 29kb in size, expressing 4 structural proteins and several non-structural proteins. The structural proteins include surface spinous process (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein. The N protein is wrapped outside the viral genome RNA to form the nucleocapsid, so that the viral RNA can be protected from being attacked by a host and the replication of the viral genome can be assisted in the viral replication process, and simultaneously, the N protein plays a plurality of roles of promoting cell apoptosis, inhibiting interferon generation and the like. The protein is more conserved than the envelope protein because it is located inside the virus.

Mannose-binding lectin binding-associated serine protease-2 (MASP-2) is a key enzyme of the complement lectin activation pathway, consisting of 686 amino acid residues (royal qian et al. study progress of mannose-binding lectin binding-associatedserine protease 2. journal of cellular and molecular immunology 2015,31 (1)). MASP-2 is encoded by MASP2 gene, and has the main functions of combining with substrate C4 to hydrolyze into two fragments of C4a and C4b, combining with C2 under the condition of combining with C4b to hydrolyze into C2a and C2b, finally forming C4b2a, namely C3 convertase, starting a series of cascade activation processes of a downstream complement system, and exciting natural immune response.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare a medicament for preventing and/or treating diseases caused by coronavirus or coronavirus infection, and/or how to prepare a coronavirus inhibitor.

In order to solve the technical problems, the invention provides the application of the substance for inhibiting the activity of MASP-2 and/or reducing the expression level of MASP-2 genes and/or reducing the content of MASP-2 in the preparation of the medicine for preventing and/or treating diseases caused by coronavirus or coronavirus infection.

The invention also provides the application of the substance for inhibiting the activity of MASP-2 and/or reducing the expression level of MASP-2 gene and/or reducing the content of MASP-2 in the preparation of coronavirus inhibitors.

In the application, the coronavirus belongs to the beta coronavirus with the N protein and the amino acid homology of the 107-125 site amino acid residue of the SARS-CoV N protein being more than 75 percent, and can be SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.

In the above application, the disease caused by coronavirus may be respiratory infection and/or digestive infection. The respiratory system infection is respiratory tract infection and/or lung infection, the respiratory tract infection can be nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis and/or bronchitis, and the lung infection can be pneumonia. The digestive system infection may be diarrhea. Patients with coronavirus infection exhibit symptoms of atypical pneumonia characterized by high fever, dyspnea, lymphopenia, rapid progression of chest radiograph visible lung shadows, acute lung injury caused by the virus-induced cytokine storm, acute respiratory distress syndrome in critically ill patients, and even respiratory failure.

In the above application, the substance may be a reagent.

In the above applications, the reagent may be an organic molecule such as C1INH (Complement C1 Esterase Inhibitor, complete C1-Esterase Inhibitor) which can only inhibit the activity of MASP-2 and/or reduce the expression level of MASP-2 gene and/or reduce the content of MASP-2, and may also contain a carrier or excipient.

The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these are in particular water-soluble carrier materials. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa.

In the above applications, the reagent may be an antibody or an antigen-binding fragment thereof, such as an antibody against MASP-2, which inhibits the activity of MASP-2 and/or reduces the expression level of a gene of MASP-2 and/or reduces the amount of MASP-2, and may further contain the above-mentioned carrier or excipient.

In the above applications, the reagent may be only the polynucleotide targeting the gene of MASP-2, or may further comprise the above vector or excipient.

The invention also provides the application of the substance for inhibiting the combination of the MASP-2 and the N protein of the coronavirus in the medicine for preventing and/or treating the diseases caused by the coronavirus or the coronavirus infection.

The invention also provides the application of the substance for inhibiting the combination of the MASP-2 and the N protein of the coronavirus in preparing the coronavirus inhibitor.

In the application, the coronavirus belongs to the beta coronavirus with the N protein and the amino acid homology of the 107-125 site amino acid residue of the SARS-CoV N protein being more than 75 percent, and can be SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.

In the above application, the substance is a reagent.

In the above applications, the agent may be only an organic molecule that inhibits the binding of MASP-2 to the N protein of coronaviruses, or may further comprise the above-mentioned carrier or excipient.

In the above applications, the reagent may be only an antibody or an antigen-binding fragment thereof that binds to MASP-2 and the N protein of coronavirus, and may further comprise the above-mentioned carrier or excipient.

In the above applications, the agent may be only the polynucleotide targeting the combination of MASP-2 and the N protein of coronavirus, or the agent may further comprise the above-mentioned vector or excipient.

The invention provides application of substances for inhibiting the activity of N protein of coronavirus and/or reducing the expression quantity of the gene of the N protein of coronavirus and/or reducing the content of the N protein of coronavirus in preparing a medicament for preventing and/or treating diseases caused by coronavirus or coronavirus infection.

The invention provides an application of a substance for inhibiting the activity of an N protein of coronavirus and/or reducing the expression quantity of a gene of the N protein of coronavirus and/or reducing the content of the N protein of coronavirus in preparation of a coronavirus inhibitor.

In the application, the coronavirus belongs to the beta coronavirus with the N protein and the amino acid homology of the 107-125 site amino acid residue of the SARS-CoV N protein being more than 75 percent, and can be SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.

In the above application, the substance is a reagent.

In the above applications, the agent may be only an organic molecule that inhibits the activity of the N protein of coronavirus and/or reduces the expression level of the gene of the N protein of coronavirus and/or reduces the content of the N protein of coronavirus, and may further contain the above-mentioned carrier or excipient.

In the above applications, the agent may be an antibody or an antigen-binding fragment thereof, such as an anti-N antibody, which inhibits the activity of the N protein of coronavirus and/or reduces the expression level of the gene of the N protein of coronavirus and/or reduces the content of the N protein of coronavirus, and may further contain the above-mentioned carrier or excipient.

In the above application, the agent may be a polynucleotide comprising a gene targeting the N protein of coronavirus, or may further comprise the above vector or excipient.

In the present application, MASP-2 is human mannose-binding lectin-binding associated serine protease-2, or MASP-2 from other animals with over 70% homology to human MASP-2.

The homology mentioned above refers to the homology of amino acid sequences. Homology of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing calculation by searching for homology to an amino acid sequence, the value (%) of homology (identities) can be obtained. The 70% or more homology may be at least 70%, 80%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% homology.

The invention discovers the molecular mechanism of the host over-stimulated immune response caused by the N protein by researching the interaction between the SARS-CoV N protein and the host protein MASP-2 and the related molecular mechanism: the interaction between SARS-CoV N protein and MASP-2 is proved by immunoprecipitation and immunoblotting experiment, and the interaction area is found to be highly homologous with MERS-CoV N protein and SARS-CoV-2N protein, and MERS-CoV N protein has the same effect; the complement deposition experiment shows that both SARS-CoV N protein and MERS-CoV N protein can promote the activation of agglutinin pathway and cause the aggravation of downstream complement activation; the mortality rate of the mouse infected with the virus with the knockout of the Masp2 gene is obviously reduced and recovered more quickly; in vivo infection experiments prove that the death rate of SARS-CoV mouse adapted strain infected mice can be effectively reduced by targeting MASP-2 medication, such as application of MASP-2 inhibitor C1INH or antibody, or targeting N protein, such as application of N protein monoclonal antibody. The above results suggest that MASP-2 and N protein interaction may be a therapeutic target. The MASP-2 inhibitor or antibody and N protein antibody may be used as candidate medicine for preventing and treating SARS-CoV and SARS-CoV-like virus infection. The invention has application value in the treatment of SARS-CoV infection and similar virus.

Drawings

FIG. 1 shows SARS-CoV infection in Masp2 knockout mice in example 1^MALine graphs of survival versus weight recovery. A in FIG. 1 is the survival rate of the mouse, B in FIG. 1 is the weight ratio of the survival mouse to the original weight, KO is amask 2 knockout mouse, and WT is a littermate-negative mouse thereof.

FIG. 2 is a line graph showing survival and weight recovery of mice after various infections and treatments in example 2. A in FIG. 2 is the survival rate of mice, the left graph shows the injection treatment on the day of infection, and the right graph shows theinjection treatment 2 days after infection; b in figure 2 is the weight of the surviving mice in the proportion of the original weight, the left figure is the injection treatment on the day of infection, and the right figure is theinjection treatment 2 days after infection; in the figure, n.s. means that the difference is not significant (P ≧ 0.05), x means that the difference is significant (P <0.05), and x means that the difference is significant (P < 0.01).

FIG. 3 is a graph showing the results of immunoblotting inmechanism experiment 1. In FIG. 3, A is the immunoblot after co-immunoprecipitation of SARS-CoV N protein and MASP-2, and B is the immunoblot after co-immunoprecipitation of MERS-CoV N protein and MASP-2 in FIG. 3. The "+" in the table on each band in the figure indicates the addition of the substance and the "-" indicates the absence of the addition of the substance.

FIG. 4 is the homology comparison between SARS-CoV N protein, MERS-CoV N protein, SARS-CoV-2N protein and MASP-2 interaction core region.

FIG. 5 is a line graph of complement deposition of SARS-CoV N protein. The left panel in FIG. 5 shows the result of C4b deposition, the middle panel in FIG. 5 shows the result of activated C3 deposition, and the right panel in FIG. 5 shows the result of C5b-9 deposition, in which SARS N represents the treatment with SARS-CoV N protein, Control represents the Control without SARS-CoV N protein, and 229E N represents the treatment with human coronavirus 229EN protein.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

1 cell line, plasmid and gene

The biological material can be obtained by the HEK293 cell (human embryo kidney cell 293, ATCC CRL-1573) and VeroE6 cell (African green monkey kidney cell, ATCC CRL-1586) public from the military medical research institute of military science institute of the people's liberation army of China, and is only used for repeating the relevant experiments of the invention, and can not be used for other purposes.

pEGFPC1-NP is a product of Beijing Yi Qiao Shen science and technology Co., Ltd, with the product number VG 40143-ACGLN. pCDNA3.0 is a product of Invitrogen corporation.

MASP2 gene (HG18035-UT), the full-length gene of SARS-CoV N protein, SARS-CoV N protein (40143-V08B), and the full-length gene of MERS-CoV N protein are all available from Beijing Yi Qian Shen science and technology Co.

All primer synthesis and sequencing in the examples below was done by the company Ooding, Beijing.

2 molecular biological reagents and antibodies

C1INH (cat. SRP3318) is a product of Merck; the Transfection reagent Lipofectamine 3000Transfection Kit is a product of Invitrogen company, and the product number is L3000-015; complement C4 (cat No. 80295-48-3), complement C4b (cat No. 204897), and C1 q-deficient serum (cat No. 234401) are all products of Sigma; the complement deposition kit (human MBL/MASP-2 assay kit, Hycult, HK327-02) is a product of HBT company; protease inhibitor Cocktail (EDTA-free, cat # 04693132001) is a product of Roche; DMEM medium (cat No. 11965-092), Opti-MEM^TMThe culture medium (cat # 31985088), pancreatin (cat # 25200072), and 1 XPBS (cat # 10010023) were all products of GIBCO Co; ECL chemiluminescence color developing liquid (the goods number is 45-002-.

An HRP-labeled anti-Flag antibody (cat # A8592) and an HRP-labeled anti-GFP antibody (cat # AB16901) are products of Sigma company; anti-MASP-2 antibody (cat # sc-17905, goat polyclonal antibody, available for human mouse rat MASP-2 detection), anti-NP antibody (cat # sc-52906, mouse monoclonal antibody, available for human SARS coronavirus N protein detection), anti-C4 b antibody (cat # sc-25815, rabbit polyclonal antibody, available for human mouse C4 detection), and anti-activated C3 antibody (sc-47687) are all products of Santa Cruz company; anti-Flag agarose beads (cat # F2426) were from Sigma.

4 mice

Wild-type SPF-grade BALB/c mice were standard strains purchased from Witongliwawa and Spubeft.

All data in the following examples were analyzed for significance using the IBM SPSS 22.

The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged.

EXAMPLE 1 SARS-CoV Virus infection Masp2 Gene knockout mice

1 materials and methods

The following experiments were performed in BSL-3 and ABSL-3 laboratories.

1.1 viruses

The virus is SARS-CoV mouse adapted strain, SARS-CoV (striinnv 2163) (C.W.day et al, virology395,210(Dec20,2009)), hereinafter referred to as SARS-CoV^MA. Passaging for three times in VeroE6 cell in high-sugar DMEM medium containing 2% fetal calf serum when SARS-CoV^MAThe titer reaches 10⁷PFU/mL, collecting culture supernatant, centrifuging at 3000rpm for 2min, and collecting supernatant to obtain a product containing SARS-CoV^MAThe cell culture supernatant of (1).

1.2 mice

C57 mouse (MASP-2) with knockout of Masp2 gene^-/-Mice) and their littermate negative Mice (W.J. Schwaeble et al, Proc Natl Acad Sci U S A108,7523 (May3,2011)) were divided into two groups, KO and WT, respectively. The KO group was C57 mice in which the Masp2 gene was knocked out, 10 mice, 8-12 weeks old, and each mouse weighed 25. + -.3 g. WT group was homozygous progeny of littermate-negative mice of C57 mice in which the Masp2 gene was knocked out (i.e., Masp2 gene was not knocked out, Masp-2^+/+Mice), 10, 8-12 weeks old, 25 + -3 g per body weight.

1.3 methods of infection

Both groups are inoculated with SARS-CoV-containing vaccine by nasal drip^MAThe cell culture supernatant of (1), specifically 100. mu.L per nasal drop (i.e., 10) after ether anesthesia⁶PFU/only). Mice were then observed every 1-2 days for survival and body weight was monitored.

2 results

The specific results are shown in FIG. 1, wherein A in FIG. 1 is the survival rate of the mice, and B in FIG. 1 is the weight ratio of the survival mice to the original weight. From FIG. 1, it can be seen that the mice knockout the Masp2 gene have a significantly lower mortality rate and faster recovery than littermate negative mice infected with the virus.

EXAMPLE 2SARS-CoV Virus infection in mice and treatment

1 materials and methods

1.1 laboratory

The following experiments were performed in BSL-3 and ABSL-3 laboratories.

1.2 viruses

The virus is SARS-CoV mouse adapted strain, SARS-CoV (strain v2163) (C.W.day et al, Virology395,210(Dec20,2009)), hereinafter referred to as SARS-CoV^MA. Passaging for three times in Vero E6 cell in high-sugar DMEM medium containing 2% fetal calf serum when SARS-CoV^MAThe titer reaches 10⁷PFU/mL, collecting culture supernatant, centrifuging at 3000rpm for 2min, and collecting supernatant to obtain a product containing SARS-CoV^MAThe cell culture supernatant of (1).

1.3 inhibitors and antibodies

C1INH, anti-MASP-2 antibody, anti-NP antibody.

1.4 mice

The mice used were wild-type SPF-grade BALB/c mice from Wintonlihua and Spubeft. The experiment was performed in two batches.

1.4.1 evaluation of therapeutic efficacy on the day of infection (treatment at initial stage of infection):

female mice of 10-12 weeks of age and approximately 20g in weight were selected and randomly divided into 4 groups of 8-10 mice per group: day of Medium + Saline infection treatment group (healthy control), SARS-CoV^MA+ saline day of infection treatment group (infection control), SARS-CoV^MA+ C1INH day of infection treatment group, SARS-CoV^MA+ anti-MASP-2 day of infection treatment group.

1.4.2 evaluation of efficacy of 2-day treatment of infection (post-infection acute phase treatment):

female mice of 10-12 weeks of age and approximately 20g in weight were selected and randomly divided into 5 groups of 8-10 mice per group:day 2 treatment group after Medium + Saline infection (healthy control), SARS-CoV^MA+ treatment group onday 2 after saline infection (infection control), SARS-CoV^MA+day 2 treatment group after C1INH infection, SARS-CoV^MA+ anti-MASP-2 day post infection treatment group, SARS-CoV^MA+ anti-N treatment group onday 2 post infection.

1.5 infection and treatment

1.5.1 treatment sessions

The 9 groups of mice were provided with 5 infection and treatment modes and 2 treatment times. Against SARS-CoV^MA+ anti-N sets only one treatment time forday 2 treatment after infection, and the other treatments set two treatment times forday 2 treatment after infection.

1.5.2 infections and methods of treatment

Treatment groups on the day of Medium + saline infection: after ether anesthesia of mice, 100. mu.L of VeroE6 cell culture supernatant without virus inoculation (subculture medium is high-glucose DMEM medium containing 2% (w/v) fetal bovine serum) was dropped into each nose, and after 30 minutes, 100. mu.L of physiological saline was injected into each mouse.

Treatment group 2 days after Medium + saline infection: after ether anesthesia of mice, 100. mu.L of VeroE6 cell culture supernatant without virus inoculation (subculture medium is high-sugar DMEM medium containing 2% (w/v) fetal bovine serum) was dropped into each nose, and after 2 days, 100. mu.L of physiological saline was injected into each mouse.

SARS-CoV^MASense of + saltDay-of-stain treatment groups: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of the virus (i.e., infectious dose of 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 30 minutes, each mouse was injected with 100. mu.L of physiological saline.

SARS-CoV^MA+ treatment group onday 2 post-saline infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (1) (i.e., infectious dose is 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 2 days, each mouse was injected with 100. mu.L of physiological saline.

SARS-CoV^MATreatment groups on day of + C1INH infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (1) (i.e., infectious dose is 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 30 minutes, 100. mu.L of a C1 INH-containing solution was injected into each mouse, and the injection dose of C1INH was 4mg/kg body weight. The C1 INH-containing liquid is a liquid obtained by dissolving C1INH in physiological saline.

SARS-CoV^MATreatment group onday 2 post C1INH infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (1) (i.e., infectious dose is 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 2 days, 100. mu.L of the above liquid containing C1INH was injected into each mouse, and the injection dose of C1INH was 4mg/kg body weight.

SARS-CoV^MATreatment groups on day of + anti-MASP-2 infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (1) (i.e., infectious dose is 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 30 minutes, 100. mu.L of a liquid containing anti-MASP-2 antibody was injected into each mouse, and the injection dose of the anti-MASP-2 antibody was 200. mu.g/kg body weight. The liquid containing the anti-MASP-2 antibody is obtained by dissolving the anti-MASP-2 antibody in physiological saline.

SARS-CoV^MA+ anti-MASP-2 treatment group onday 2 post infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (i.e., infection)The dosage is 10⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 2 days, 100. mu.L of the above liquid containing anti-MASP-2 antibody was injected into each mouse, and the injection dose of anti-MASP-2 antibody was 200. mu.g/kg body weight.

SARS-CoV^MATreatment group onday 2 post-anti-N infection: after ether anesthesia of mice, 100 mu L of SARS-CoV-containing solution 1.2 per nose drop^MACell culture supernatant of (1) (i.e., infectious dose is 10)⁶PFU/single), nasal drip inoculation of SARS-CoV^MAAfter 2 days, each mouse was injected with 100. mu.L of the anti-NP antibody-containing solution at a dose of 200. mu.g/kg body weight. The anti-NP antibody-containing solution is obtained by dissolving an anti-NP antibody in physiological saline.

SARS-CoV^MAThe virus nasal drop method was the same as in example 1.

The injection is tail vein injection, diluted with physiological saline and injected, each injection is 100 μ L, and the administration is carried out once. Mice were then observed every 1-2 days for survival and body weight was monitored.

2 results

The specific results are shown in fig. 2, wherein a in fig. 2 is the survival rate of the mice, the left side is the treatment on the day of infection, and the right side is the treatment on the 2 nd day after infection; in fig. 2, B is the weight of the surviving mice relative to the original weight, with treatment on the day of infection on the left and treatment onday 2 after infection on the right. As can be seen from fig. 2:

the mice infected with the anti-MASP-2 antibody or the C1INH on the same day have remarkably reduced lethality rate and good weight recovery compared with the infected control, wherein the weight recovery of the mice injected with the anti-MASP-2 antibody on the 8 th day after infection is not significantly different from that of the healthy control. Specifically, in the experiment of the treatment group on the day, the survival rate (survival rate) of the treatment group on the day of Medium + saline infection was 100%, and SARS-CoV^MAThe survival rate of the treatment group on the day of + saline infection was 13%, SARS-CoV^MAThe survival rate of the treatment group on the day of + C1INH infection was 88%, SARS-CoV^MAThe survival rate of the treatment group on the day of + anti-MASP-2 infection was 75%.

② mice injected with any one of anti-MASP-2 antibody, anti-NP antibody andC1INH 2 days after infection (acute onset period) are compared with infection controlThe time of collective death of mice (the number of deaths of mice in 1 day reaches more than 50% of the total number of deaths in the group) can be delayed for 1-4 days, and the mortality of infected mice can be effectively reduced, wherein the body weight of the mice injected with anti-NP antibody or C1INH is well restored. In particular, the survival rate of the treatment group atday 2 after Medium + Saline infection was 100%, SARS-CoV^MAThe survival rate of the treatment group was 20% onday 2 post-salt infection, and 50% of the dead mice died on day 3; SARS-CoV^MASurvival of the treatment group was 40% onday 2 post + C1INH infection, 50% of the dead mice died (delayed by 4 days) on day 7; SARS-CoV^MAThe survival rate of the treatment group atday 2 after + anti-MASP-2 infection was 30%, and 50% of the dead mice died at day 7 (delayed by 4 days). SARS-CoV^MAThe survival rate of the treatment group was 60% onday 2 after + anti-N infection, and 50% of the dead mice died (delayed by 1 day) onday 4.

Mechanism experiment 1 immunoprecipitation and immunoblotting detection of interaction of MASP-2 with N protein

1 materials and methods

1.1 reagents

Cell lysis solution: 50mmol/L Tris-HCl pH7.4, 150mmol/L NaCl, 2mmol/L CaCl₂1 tablet/50 ml of the protease inhibitor Cocktail (EDTA-free, cat # 04693132001), 1% NP 40.

Cell lysate without protease inhibitor: 50mmol/L Tris-HCl pH7.4, 150mmol/L NaCl, 2mmol/L CaCl₂，1％NP40。

1 × transfer membrane buffer: Tris-HCl 24mM, glycine 5mM, 20% (v/v) methanol.

1.2 plasmids

pcDNA3.0-MASP-2-Flag is an expression vector for expressing protein MASP-2-Flag, and MASP-2-Flag is a fusion protein of human MASP-2 and Flag.

The coronavirus N protein and mutant expression plasmid thereof are specifically as follows: 5 kinds of pcDNA3.0-Flag-SARS N (WT), pcDNA3.0-Flag-SARS N (Δ 321-323), pcDNA3.0-Flag-SARS N (Δ 116-124), pcDNA3.0-Flag-MERS N (WT), and pcDNA3.0-Flag-MERS N (Δ 104-112). Wherein pcDNA3.0-Flag-SARS N (WT) is an expression vector for expressing the protein Flag-SARS N (WT), Flag-SARS N (WT) is a fusion protein of SARS N (WT) and Flag, and the rest expression vectors are analogized. SARS N (WT) is the full length of SARS-CoV N protein, Flag is the protein tag; SARS N (delta 321-323) is a mutant protein of deletion of 321-323 amino acid residues of SARS-CoV N (WT) protein, SARS N (delta 116-124) is a mutant protein of deletion of 116-124 amino acid residues of SARS-CoV N (WT) protein, MERS N (WT) is the full length of MERS-CoV N protein, and MERS N (delta 104-112) is a mutant protein of deletion of 104-112 amino acid residues of MERS-CoV N (WT) protein.

1.2.1 plasmid pcDNA3.0-MASP-2-Flag

A Flag gene fragment (gattacaaggacgacgatgacaag) was ligated to the 3' -end stop codon of the gene encoding human MASP-2 protein (i.e., MASP2 gene) to obtain a DNA named MASP-2-Flag gene, which was used to replace the fragment between the recognition sites for restriction endonucleases HindIII and KpnI (small fragments including the recognition site for HindIII and the recognition site for KpnI) of pCDNA3.0(Invitrogen) vector, and the remaining sequences of the pCDNA3.0 vector were kept unchanged to obtain a recombinant expression vector for MASP-2-Flag protein, which was named pcDNA3.0-MASP-2-Flag.

1.2.2 plasmid pcDNA3.0-Flag-SARS N (WT)

After inserting a Flag gene fragment (gattacaaggacgacgatgacaag) after the start codon at the 5' end of the full-length gene of SARS-CoV N protein to obtain a DNA named as Flag-SARS N (WT) gene, the DNA was used to replace the fragment (small fragment including the recognition site for BamHI and the recognition site for EcoRI) between the restriction endonucleases BamHI and EcoRI of the pCDNA3.0(Invitrogen) vector, and the other sequences of pCDNA3.0 were kept unchanged to obtain a recombinant expression vector of Flag-SARSN protein (also named as SARSN (WT)) named as pcDNA3.0-Flag-SARS N (WT).

1.2.3 plasmid pcDNA3.0-Flag-SARS N (. DELTA.321-

After the start codon of 5' end of SARS N (delta 321-323) gene is inserted Flag gene fragment (gattacaaggacgacgatgacaag) to obtain DNA named as Flag-SARS N (delta 321-323) gene, the DNA is used to replace the fragment between restriction endonuclease BamHI and EcoRI recognition sites of pCDNA3.0(Invitrogen) vector (small fragment including BamHI recognition site and EcoRI recognition site), and the other sequences of pCDNA3.0 are maintained to obtain recombinant expression vector of SARSN protein deletion 321-323 amino acid deletion mutant (also named as SARS N (delta 321-323)), named as pcDNA3.0-Flag-SARS N (delta 321-323).

1.2.4 plasmid pcDNA3.0-Flag-SARS N (. DELTA.116-

After inserting Flag gene fragment (gattacaaggacgacgatgacaag) after the initiation codon at the 5' end of the SARSN (delta 116-124) gene, DNA named as Flag-SARS N (delta 116-124) gene is obtained, the DNA is used to replace the fragment (small fragment including the recognition site of BamHI and the recognition site of EcoRI) between restriction endonucleases BamHI and EcoRI of pCDNA3.0(Invitrogen) vector, and the other sequences of pCDNA3.0 are kept unchanged, so as to obtain the recombinant expression vector of deletion mutant (also named as SARS N (delta 116-124)) of SARSN protein deletion 116-124 amino acid, named as pcDNA3.0-Flag-SARS N (delta 116-124)).

1.2.5 plasmid pcDNA3.0-Flag-MERS N (WT)

After inserting a Flag gene fragment (gattacaaggacgacgatgacaag) after the start codon at the 5' end of the full-length gene of MERS-CoV N protein to obtain a DNA named as Flag-MERSN (WT) gene, the DNA was used to replace a fragment (a small fragment including a recognition site for BamHI and a recognition site for EcoRI) between restriction endonucleases BamHI and EcoRI of pCDNA3.0(Invitrogen) vector, and the other sequences of pCDNA3.0 were kept unchanged to obtain a recombinant expression vector of MERSN protein named as pcDNA3.0-Flag-MERSN N (WT).

1.2.6 plasmid pcDNA3.0-Flag-MERS N (. DELTA.104-112)

After inserting a Flag gene fragment (gattacaaggacgacgatgacaag) after the start codon at the 5' end of the MERSN (. DELTA.104-112) gene, a DNA named as Flag-MERSN (. DELTA.104-112) gene was obtained, the DNA was used to replace a fragment (a small fragment including the recognition site for BamHI and the recognition site for EcoRI) between the restriction endonucleases BamHI and EcoRI of the pCDNA3.0(Invitrogen) vector, and the other sequences of pCDNA3.0 were kept unchanged to obtain a recombinant expression vector of a deletion mutant (also named as MERSN N (. DELTA.104-112)) of the MERSN protein deletion amino acid at position 104-112, which was named as pcDNA3.0-Flag-MERS N (. DELTA.104-112).

1.3 transfection

1.3.1 preparation of HEK293/pcDNA3.0-MASP-2-Flag cells

HEK293 cells were transfected when cultured to 70-90% confluence. Lipofectamine, Thermo corporation, was used^TM3000transfection reagents, plasmid transfection according to the instructions: mu.g of pcDNA3.0-MASP-2-Flag plasmid and 10. mu. L P3000^TMAdd 250. mu.L of Opti-MEM^TMDiluting in a culture medium to prepare a DNA premix; according to the plasmid: lipofectamine^TM3000transfection reagents 1:3 ratio, 15. mu.L Lipofectamine^TM3000transfection reagent was added to 250. mu.L of Opti-MEM^TMDiluting the culture medium, adding the diluted DNA premix, uniformly mixing, incubating at room temperature for 10-15min to obtain a DNA-lipid complex, adding the DNA-lipid complex into HEK293 cells, transfecting for 36-48h, removing culture supernatant, washing the cells for 2 times by using 5mL of precooled 1 XPBS, adding 5mL of 1 XPBS into each dish, scraping the cells by using a cell scraper, uniformly blowing and beating the cells into a 15mL centrifuge tube, centrifuging for 3min at 4 ℃ and 2000 Xg, and collecting the cells to obtain the HEK293/pcDNA3.0-MASP-2-Flag cells.

1.3.2 preparation of HEK293/pcDNA3.0-Flag-MERS N (WT) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pcDNA3.0-Flag-MERS N (WT) plasmid, and transfection was performed in the same manner as described above to obtain HEK293/pcDNA3.0-Flag-MERS N (WT) cells.

1.3.3 preparation of HEK293/pcDNA3.0-Flag-SARS N (. DELTA.321-323) cells

The pcDNA3.0-Flag-SARS N (. DELTA.321-MASP-2-Flag) plasmid in 1.3.1 was replaced by pcDNA3.0-Flag-SARS N (. DELTA.321-323) plasmid, and transfection was carried out in the same manner as described above to obtain HEK293/pcDNA3.0-Flag-SARS N (. DELTA.321-323) cells.

1.3.4 preparation of HEK293/pcDNA3.0-Flag-SARS N (. DELTA.116-124) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pcDNA3.0-Flag-SARS N (. DELTA.116-124) plasmid and transfected by the same procedure as described above to obtain HEK293/pcDNA3.0-Flag-SARS N (. DELTA.116-124) cells.

1.3.5 preparation of HEK293/pcDNA3.0-Flag-MERS N (WT) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pcDNA3.0-Flag-MERS N (WT) plasmid, and transfection was performed in the same manner as described above to obtain HEK293/pcDNA3.0-Flag-MERSN (WT) cells.

1.3.6 preparation of HEK293/pcDNA3.0-Flag-MERS N (. DELTA.104-112) cells

The pcDNA3.0-Flag-MERS N (. DELTA.104-112) plasmid was used in place of the pcDNA3.0-MASP-2-Flag plasmid in 1.3.1, and transfection was carried out in the same manner as described above to obtain HEK293/pcDNA3.0-Flag-MERS N (. DELTA.104-112) cells.

1.4 cleavage

Adding the HEK293/pcDNA3.0-MASP-2-Flag cell into 500 μ L cell lysate, performing ice lysis for 10min, and centrifuging at 16000rpm for 10min at 4 deg.C; the supernatant was transferred to a new 1.5mL centrifuge tube to obtain a lysate of HEK293/pcDNA3.0-MASP-2-Flag cells.

5 kinds of lysates containing coronavirus N protein cells (i.e., HEK293/pcDNA3.0-Flag-SARS N (WT) cells, HEK293/pcDNA3.0-Flag-SARS N (Δ 321-) -323 cells, HEK293/pcDNA3.0-Flag-SARS N (Δ 116-) -124 cells, HEK293/pcDNA3.0-Flag-MERS N (WT) cells, HEK293/pcDNA3.0-Flag-MERS N (Δ 104-) -112) cells were lysed in the same manner as described above to obtain 5 kinds of lysates containing coronavirus N protein cells (i.e., HEK293/pcDNA3.0-Flag-SARS N (WT) cells, HEK293/pcDNA3.0-Flag-SARS N (Δ 321-), HEK 293/pcDNA293/pcDNA3.0-Flag-SARS N (Δ 321-), SARS 293/WT 293/pcDNA3.0-FLA-MERS) cells, and HEK 293/pcDNA3.0-MEMS (WT) cells, HEK293/pcDNA3.0-Flag-MERS N (. DELTA.104-112) cell lysate.

1.5 Co-immunoprecipitation

Adding 20 mu L of anti-Flag agarose beads into a lysis solution of HEK293/pcDNA3.0-MASP-2-Flag cells, carrying out rotary incubation for 2h at 4 ℃ for carrying out co-immunoprecipitation, then centrifuging at 4 ℃ and 3000rpm for 5min, washing the beads 3 times by using 1mL of cell lysis solution without protease inhibitor, centrifuging to remove supernatant, subpackaging and storing at-70 ℃ for later use, and obtaining the MASP-2-combined agarose beads.

Sepharose beads containing MASP-2 were added to lysates containing coronavirus N protein cells (any of the above-mentioned lysates of HEK293/pcDNA3.0-Flag-SARS N (WT) cells, HEK293/pcDNA3.0-Flag-SARS N (Δ 321-) -323 cells, HEK293/pcDNA3.0-Flag-SARS N (Δ 116-) -124 cells, HEK293/pcDNA3.0-Flag-MERS N (WT) cells, and HEK293/pcDNA3.0-Flag-MERS N (Δ 104-) -112 cells) and incubated at 4 ℃ for 2 hours for immunoprecipitation. Then centrifuging at 4 deg.C and 3000rpm for 5min, collecting precipitate, washing the beads with 1mL cell lysate containing no protease inhibitor for 3 times, centrifuging to remove supernatant, adding 40-60 μ L1 xSDS loading buffer, boiling at 100 deg.C for 5min, centrifuging at 4 deg.C and 16000 × g for 10min, and collecting appropriate amount of supernatant for SDS-PAGE electrophoresis and immunoblotting.

And adding 10 mu L of supernatant sample into an SDS-PAGE gel hole for electrophoresis, stopping electrophoresis when bromophenol blue migrates to the bottom of the separation gel, and preparing for membrane transferring operation. The PVDF membrane was activated with methanol for 30s, and then the PVDF membrane and the filter paper were soaked together in 1 Xtrans-membrane buffer for 30 min. After the electrophoresis, the membrane was placed on a semi-dry membrane converter in the order of filter paper-PVDF membrane-SDS gel-filter paper from bottom to top, and the membrane was converted at 20V for about 1.5 hours. After the membrane transfer is finished, sealing the PVDF membrane for 1 hour at room temperature by using 1 xTBST sealing liquid containing 5% (mass percentage content) of skimmed milk powder, and then washing for 5 minutes each time for 3 times by using 1 xTBST sealing liquid; adding an HRP-marked anti-Flag antibody and an HRP-marked anti-GFP antibody respectively, incubating the PVDF membrane at normal temperature for 1h, washing for 3 times, and performing ECL development analysis by using ECL chemiluminescence color development liquid.

2 results

As a result, as shown in FIG. 3, SARS-CoV N protein (i.e., SARS N (WT), SARS N (Δ 321-.

While the SARS-CoV-2N protein is highly homologous with SARS-CoV N protein and MERS-CoV N protein in the interaction region with MASP-2, as shown in FIG. 4, and SARS-CoV-2N protein interacts with MASP-2.

Mechanism experiment 2SARS-CoV N protein promoting complement deposition experiment

1 materials and methods

1.1 reagents

High salt binding buffer: 10mM Tris-HCl pH7.4, 1M NaCl, 0.5mM MgCl₂0.05% (v/v) Tween-20, and 0.1% (w/v) gelatin (gelatin),2mM CaCl₂。

binding buffer: 10mM Tris-HCl pH7.4, 150mM NaCl, 0.5mM MgCl₂0.05% (v/v) Tween-20, and 0.1% (w/v) gelatin (gelatin), 2mM CaCl₂。

1.2 complement deposition

The C4b deposition experiment was performed using the complement deposition kit from HBT. Human C1q depleted serum was used, with high salt binding buffer (10mM Tris-HCl pH7.4, 1M NaCl, 0.5mM MgCl)₂0.05% (v/v) Tween-20, and 0.1% (w/v) gelatin (gelatin), 2mM CaCl₂) After dilution, the cells were added to the mannan-precoated ELISA plates (10. mu.g/well) and incubated overnight at 4 ℃. After washing the plate three times with 1 XPBST, binding buffer and purified complement C4b (Sigma, 204897) and SARS-CoV N protein (Chinesen, Yi-Qiao, 40143-V08B) were added (Control without SARS-CoV N protein and Control treatment with human coronavirus 229EN was also set), and incubation was carried out at 37 ℃ for 1.5 h. Washing the plate for three times, adding anti-C4 b antibody, incubating for 1h, washing the plate for three times, adding HRP secondary antibody, incubating for 1h, washing the plate for three times, adding TMB color development solution, and adding 2M H after 15-30min₂SO₄The reaction was stopped and the OD read at 450 nm.

C3 deposition experiments, human C1q serum deletion with binding buffer dilution and pre-coated mannan ELISA plate, 4 ℃ after 1 hours of incubation, do not wash the plate, at 37 ℃ incubation for 1.5 h. Washing the plate for three times, adding activated C3 antibody (Santa Cruz, sc-47687) and corresponding HRP secondary antibody, incubating for 1h, washing the plate for three times, adding TMB color developing solution, adding 2M H after 15-30min₂SO₄The reaction was stopped and the OD read at 450 nm.

The C5b-9 deposition experiment was performed according to the method of the C3 deposition experiment described above, with the anti-C3 antibody being replaced by the anti-C5 b-9 antibody only.

2 results

Specific results see FIG. 5, SARS-CoV N protein was found to promote lectin pathway activation and lead to increased downstream complement activation by complement deposition experiments.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

Translated fromChinese

1.抑制MASP-2的活性和/或降低MASP-2的基因的表达量和/或降低MASP-2的含量的物质的应用，所述应用为下述任一种：1. Application of a substance that inhibits the activity of MASP-2 and/or reduces the expression level of MASP-2 gene and/or reduces the content of MASP-2, the application is any one of the following:U1、抑制MASP-2的活性和/或降低MASP-2的基因的表达量和/或降低MASP-2的含量的物质在制备预防和/或治疗冠状病毒所致疾病或冠状病毒感染的药物中的应用；U1, the substances that inhibit the activity of MASP-2 and/or reduce the expression level of MASP-2 gene and/or reduce the content of MASP-2 are in the preparation of medicines for preventing and/or treating diseases caused by coronavirus or coronavirus infection Applications;U2、抑制MASP-2的活性和/或降低MASP-2的基因的表达量和/或降低MASP-2的含量的物质在制备冠状病毒抑制剂中的应用。U2, the application of substances that inhibit the activity of MASP-2 and/or reduce the expression level of the MASP-2 gene and/or reduce the content of MASP-2 in the preparation of coronavirus inhibitors.2.根据权利要求1所述的应用，其特征在于：所述冠状病毒为其N蛋白与SARS-CoVN蛋白107-125位氨基酸残基氨基酸同源性在75％以上的β冠状病毒属病毒；具体地，所述冠状病毒为SARS-CoV和/或MERS-CoV和/或SARS-CoV-2。2. application according to claim 1, is characterized in that: described coronavirus is betacoronavirus whose N protein and SARS-CoV N protein 107-125 amino acid residue amino acid homology is more than 75%; Specifically, the coronavirus is SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.3.根据权利要求1或2任一项所述的应用，其特征在于：所述物质为试剂，所述试剂包含如下1)和/或2)：3. The application according to any one of claims 1 and 2, wherein the substance is a reagent, and the reagent comprises the following 1) and/or 2):1)抑制MASP-2的活性和/或降低MASP-2的基因的表达量和/或降低MASP-2的含量的有机分子；1) Organic molecules that inhibit the activity of MASP-2 and/or reduce the expression of MASP-2 genes and/or reduce the content of MASP-2;2)抑制MASP-2的活性和/或降低MASP-2的基因的表达量和/或降低MASP-2的含量的抗体或其抗原结合片段。2) An antibody or antigen-binding fragment thereof that inhibits the activity of MASP-2 and/or reduces the expression level of MASP-2 gene and/or reduces the content of MASP-2.4.根据权利要求3所述的应用，其特征在于：所述有机分子为C1INH。4. The application according to claim 3, wherein the organic molecule is C1INH.5.抑制MASP-2和冠状病毒的N蛋白结合的物质的应用，其特征在于：所述应用为下述任一种：5. the application of the substance that suppresses the N protein binding of MASP-2 and coronavirus, is characterized in that: described application is any of following:M1、抑制MASP-2和冠状病毒的N蛋白结合的物质在预防和/或治疗冠状病毒所致疾病或冠状病毒感染的药物中的应用；M1. The use of substances that inhibit the binding of MASP-2 and the N protein of coronavirus in drugs for preventing and/or treating diseases caused by coronavirus or coronavirus infection;M2、抑制MASP-2和冠状病毒的N蛋白结合的物质在制备冠状病毒抑制剂中的应用。M2. The application of a substance inhibiting the binding of MASP-2 and the N protein of coronavirus in the preparation of a coronavirus inhibitor.6.根据权利要求5所述的应用，其特征在于：所述冠状病毒为其N蛋白与SARS-CoVN蛋白107-125位氨基酸残基氨基酸同源性在75％以上的β冠状病毒属病毒；具体地，所述冠状病毒为SARS-CoV和/或MERS-CoV和/或SARS-CoV-2。6. The application according to claim 5, wherein the coronavirus is a betacoronavirus whose amino acid homology between the N protein and the 107-125 amino acid residues of the SARS-CoV N protein is more than 75%; Specifically, the coronavirus is SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.7.根据权利要求5或6任一项所述的应用，其特征在于：所述物质为试剂，所述试剂包含如下A和/或B：7. The application according to any one of claims 5 or 6, wherein the substance is a reagent, and the reagent comprises the following A and/or B:A抑制MASP-2和冠状病毒的N蛋白结合的有机分子；A Organic molecule that inhibits the binding of MASP-2 and the N protein of coronavirus;B抑制MASP-2和冠状病毒的N蛋白结合的抗体或其抗原结合片段。B Antibodies or antigen-binding fragments thereof that inhibit the binding of MASP-2 to the N protein of coronavirus.8.抑制冠状病毒的N蛋白的活性和/或降低冠状病毒的N蛋白的基因的表达量和/或降低冠状病毒的N蛋白的含量的物质的应用，其特征在于：所述应用为下述任一种：8. the application of the substance that suppresses the activity of the N protein of coronavirus and/or reduces the expression of the gene of the N protein of coronavirus and/or reduces the content of the N protein of coronavirus, it is characterized in that: described application is following Either:S1、抑制冠状病毒的N蛋白的活性和/或降低冠状病毒的N蛋白的基因的表达量和/或降低冠状病毒的N蛋白的含量的物质在制备预防和/或治疗冠状病毒所致疾病或冠状病毒感染的药物中的应用；S1, suppress the activity of the N protein of coronavirus and/or reduce the expression level of the gene of the N protein of coronavirus and/or reduce the substance of the content of the N protein of coronavirus in the preparation of prevention and/or treatment of diseases caused by coronavirus or The use of medicines for coronavirus infections;S2、抑制冠状病毒的N蛋白的活性和/或降低冠状病毒的N蛋白的基因的表达量和/或降低冠状病毒的N蛋白的含量的物质在制备冠状病毒抑制剂中的应用。S2, the application of the substance that suppresses the activity of the N protein of coronavirus and/or reduces the expression amount of the gene of the N protein of coronavirus and/or reduces the content of the N protein of coronavirus in the preparation of a coronavirus inhibitor.9.根据权利要求8所述的应用，其特征在于：所述冠状病毒为其N蛋白与SARS-CoVN蛋白107-125位氨基酸残基氨基酸同源性在75％以上的β冠状病毒属病毒；具体地，所述冠状病毒为SARS-CoV和/或MERS-CoV和/或SARS-CoV-2。9. The application according to claim 8, wherein the coronavirus is a betacoronavirus whose amino acid homology between the N protein and the amino acid residues at positions 107-125 of the SARS-CoV N protein is more than 75%; Specifically, the coronavirus is SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.10.根据权利要求8或9任一项所述的应用，其特征在于：所述物质为试剂，所述试剂包含如下I和/或II：10. The application according to any one of claims 8 or 9, characterized in that: the substance is a reagent, and the reagent comprises the following I and/or II:I抑制冠状病毒的N蛋白的活性和/或降低冠状病毒的N蛋白的基因的表达量和/或降低冠状病毒的N蛋白的含量的有机分子；I inhibit the activity of the N protein of the coronavirus and/or reduce the expression of the gene of the N protein of the coronavirus and/or reduce the organic molecule of the content of the N protein of the coronavirus;II抑制冠状病毒的N蛋白的活性和/或降低冠状病毒的N蛋白的基因的表达量和/或降低冠状病毒的N蛋白的含量的抗体或其抗原结合片段。II inhibits the activity of the N protein of the coronavirus and/or reduces the expression level of the gene of the N protein of the coronavirus and/or reduces the content of the N protein of the coronavirus antibody or its antigen-binding fragment.

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