CN112023045A

CN112023045A - Application of substance for inhibiting MASP-2 and downstream complement activation effect thereof in preparation of drugs for treating diseases caused by coronavirus

Info

Publication number: CN112023045A
Application number: CN202010241620.XA
Authority: CN
Inventors: 曹诚; 高婷; 靳彦文; 刘萱; 李平; 胡勇; 周志文; 李忠
Original assignee: Institute of Pharmacology and Toxicology of AMMS; Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Staidson Beijing Biopharmaceutical Co Ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-12-04

Abstract

The invention provides application of a substance for inhibiting MASP-2 and a downstream complement activation effect thereof in preparation of a medicine for treating diseases caused by coronavirus. According to the invention, through the research on the molecular mechanisms related to the activation of coronavirus N protein, MASP-2 and downstream complements thereof, the interaction of SARS-CoV N protein, MERS-CoV N protein, SARS-CoV-2N protein and MASP-2 is proved, the activation of MASP-2 downstream complements C4, C3, C5 and the like can be aggravated, and the remarkable increase of complement fragment C5a in the serum of SARS-CoV-2 severe patients is found; the mortality rate of the Masp2 gene knockout mice is obviously reduced and recovered more quickly after infection; the death rate of mice can be effectively reduced by targeting MASP-2. Proves that the inhibitor aiming at MASP-2 or the downstream complement activation effect thereof has value in being used as candidate drugs for preventing and treating SARS-CoV virus infection.

Description

Application of substance for inhibiting MASP-2 and downstream complement activation effect thereof in preparation of drugs for treating diseases caused by coronavirus

Technical Field

The invention relates to an application of a substance for inhibiting MASP-2 and a downstream complement activation effect thereof in preparing a medicine for treating diseases caused by coronavirus.

Background

During coronavirus infection, viral surface glycosyl groups activate the complement lectin pathway mannan-binding lectin (MBL), which leads to activation of its downstream mannan-binding lectin associated serine protease-2 (MASP-2). MASP-2 is a key enzyme of MBL and consists of 686 amino acid residues (Royal, et al. Studies on mannose-binding lectin binding-associated serine protease 2. J. mol. Immunol 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, forming C4b2a, namely C3 convertase, further activating C3 and C5 to form C5b-9 membrane attack complex, inducing virus elimination and cell lysis, and simultaneously generating anaphylatoxin C3a, C5a and the like which can recruit inflammatory cells, activate and amplify inflammatory reaction. The generated complement fragments such as C4b, C3b and the like can play roles in opsonophagocytosis and antigen presentation.

The anaphylatoxin C3a, C5a induces inflammatory reaction after binding with its specific receptor C3aR, C5 aR. Anaphylatoxin induces smooth muscle contraction, mast cells release histamine, and vascular permeability is increased; and mediates chemotaxis, inflammatory response, and the generation of cytotoxic oxygen radicals, therefore, anaphylatoxins have important effects on the induction and regulation of inflammatory response, with the strongest effect of C5 a. C3a and C5a are small molecular peptides which are composed of 76 amino acids and have the molecular weight of about 10kD, and the small molecular peptides all have carboxyl terminals containing arginine residues, can be rapidly decomposed by serum carboxypeptidase to generate de-arginine derivatives, and show that the biological activity is remarkably reduced.

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 (MERS-CoV) causing middle east respiratory syndrome (MERS-CoV) cause severe acute respiratory infectious disease. 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. 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. 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.

Disclosure of Invention

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

In order to solve the technical problems, the invention provides the application of the substance for inhibiting the downstream complement activation effect of MASP-2 in preparing the medicine for preventing and/or treating the diseases caused by the coronavirus or the coronavirus infection.

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 for inhibiting the downstream complement activation effect of MASP-2 can be any one or more of the following X1-X7:

x1, a substance that inhibits the activity of complement downstream of MASP-2;

x2, a substance that reduces the amount of complement downstream of MASP-2;

x3, an agent that inhibits the activity of a complement fragment downstream of MASP-2;

x4, a substance that reduces the amount of complement fragments downstream of MASP-2;

x5, a substance that inhibits the binding of the downstream complement fragment of MASP-2 to its corresponding specific receptor;

x6, an agent that inhibits specific receptor activity of a complement fragment downstream of MASP-2;

x7, a substance that reduces the level of specific receptors of the complement fragments downstream of MASP-2.

In the above applications, the MASP-2 downstream complement of X1 or X2 may be MASP-2 downstream complement of the complement activation lectin pathway, and may be at least one of complement C2, C3, C4 and C5.

In the above application, the MASP-2 downstream complement fragment in X3, X4, and X5 may be MASP-2 downstream complement fragment in complement activation lectin pathway, specifically at least one of complement fragments C2a, C2b, C3a, C3b, C4a, C4b, C5a, and C5 b-9; more specifically, it may be at least one of C3a, C4a, and C5a having an anaphylatoxin action.

In the above applications, the receptor specific for the complement fragment downstream of MASP-2 in X5, X6, X7 may be a receptor specific for a complement fragment downstream of MASP-2 in the pathway of complement activating lectin, specifically C3aR and/or C5 aR.

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

In the above applications, the agent may be an antibody or antigen-binding fragment thereof that inhibits the downstream complement activation effects of MASP-2, or may further comprise a carrier or excipient. Specifically, the antibody may be at least one of an antibody against C3a, an antibody against C5a, an antibody against C3aR, and an antibody against C5 aR.

In the above applications, the agent may also be only an organic molecule that inhibits the downstream complement activation effects of MASP-2, such as a binding fragment with a neutralizing effect, specifically a binding peptide fragment, for example: c3a antisense peptide, C5a antisense peptide, C3a mutant having C3a receptor binding activity, C5a mutant having C5a receptor binding activity, etc., and may further contain the above-mentioned 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.

The invention also provides application of a substance specifically bound with the complement fragment C5a or a substance specifically bound with the complement fragment C5a gene in preparing a product for screening or assisting in screening patients with severe coronavirus infection.

The substance specifically binding to the complement fragment C5a is a reagent or a kit containing an antibody against the complement fragment C5a or an antigen-binding fragment thereof, and the substance specifically binding to the complement fragment C5a gene is a reagent or a kit containing a specific primer for PCR amplification of the complement fragment C5a gene.

In order to solve the technical problems, the invention also provides the application of the substance for inhibiting the activity of MASP-2 and/or reducing the expression level of the gene of MASP-2 and/or reducing the content of MASP-2 in the preparation of 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 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 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.

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 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 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 host over-stimulated immune reaction caused by N protein by researching the interaction of SARS-CoV N protein, MERS-CoV N protein, SARS-CoV-2N protein and host protein MASP-2 and related molecular mechanism: the interaction of SARS-CoV N protein, MERS-CoV N protein, SARS-CoV-2N protein and MASP-2 is proved by immunoprecipitation and immunoblotting experiment, and the three are highly homologous with the interaction region of MASP-2; the complement deposition experiments show that SARS-CoV N protein, MERS-CoV N protein and SARS-CoV-2N protein can promote the activation of agglutinin pathway and can aggravate the activation of MASP-2 downstream complements C4, C3, C5 and the like; the complement fragment C5a in the serum of SARS-CoV-2 severe patients is found to be obviously increased, which indicates that the material specifically combined with the complement fragment C5a or the gene thereof can be used for preparing products for screening or assisting in screening patients with severe coronavirus infection; 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. These results suggest that MASP-2 or its downstream complement activation effects may be a therapeutic target. The MASP-2 inhibitor or the inhibitor of the downstream complement activation effect thereof can be used as candidate drugs for preventing and treating coronavirus infection. The invention has application value in screening and treating the infection coronavirus.

Drawings

FIG. 1 is a schematic diagram showing the homology comparison between 5 coronavirus N proteins and the interaction core region of MASP-2.

FIG. 2 is a graph showing the results of immunoblotting in example 1. In FIG. 2, 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. FIG. 2C is the immunoblot of the SARS-CoV-2N protein after co-immunoprecipitation with MASP-2. 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. 3 is a line graph of complement deposition of SARS-CoV N protein. A in fig. 3 is the result of deposition of C4 promoted by SARS-CoV N protein in example 2, B in fig. 3 is the result of deposition of C3 promoted by SARS-CoV N protein in example 2, C in fig. 3 is the result of deposition of C5B-9 promoted by SARS-CoV N protein in example 2, D in fig. 3 is the result of deposition of C4 promoted by MERS-CoV N protein in example 3, E in fig. 3 is the result of deposition of C4 promoted by SARS-CoV-2N protein in example 4, F in fig. 3 is the result of detection of C5a in serum of SARS-CoV-2 infection, severe patient and normal human in example 5, "×" indicates that severe patient and normal human infected with SARS-CoV-2 have a very significant difference (P < 0.001). In the figure, SARS N represents the treatment with the addition of SARS-CoV N protein, MERS N represents the treatment with the addition of MERS-CoV N protein, SARS-CoV-2N represents the treatment with the addition of SARS-CoV-2N protein, 229E N represents the treatment with human coronavirus 229E N protein, and Control represents the Control without coronavirus N protein.

FIG. 4 is a line graph showing the survival rate of Masp2 knockout mice in example 6 after LPS treatment in LPS + N protein expressing adenovirus induced pneumonia model. KO is a Masp2 knockout mouse, WT is a littermate negative mouse.

FIG. 5 is a line graph showing survival and weight recovery of mice after various infections and treatments in example 7. FIG. 5A is the mouse survival rate after LPS-induced pneumonia of pre-infected adenovirus expressing SARS-CoV N protein (Ad-SARS N); b in FIG. 5 is the mouse survival rate after LPS-induced pneumonia of pre-infected adenovirus expressing MERS-CoV N protein (Ad-MERS N); antibody and inhibitor treatments were given 30 minutes prior to LPS injection as shown. 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).

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 is available to the public of HEK293 cells (human embryonic kidney cells 293, ATCC CRL-1573) from the military medical research institute of the national people's liberation military academy of sciences, and is only used for repeating the relevant experiments of the present invention, and is not 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, the full-length gene of MERS-CoV N protein, the full-length gene of SARS-CoV-2N protein, SARS-CoV N protein (40143-V08B), MERS-CoV N protein (40068-V08B), and SARS-CoV-2N protein (40588-V08B) are all available from Beijing Yi Qian Shen science and technology Co Ltd.

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 3000 Transfection 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-2assay kit, Hycult, HK327-02) is a product of HBT company; the 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; an anti-HA antibody (cat # h6533) produced by (Sigma); 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 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) are from Sigma; the human complement C5a ELISA kit (DY2037) is a product of R & D, and the C5a antibody (bdb001) is a product of Staidson Biopharmaceutical co.

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 immunoprecipitation and immunoblotting detection of interaction of MASP-2 with coronavirus N protein

Comparing the sequences of the N protein of 5 kinds of coronavirus such as SARS-CoV, MERS-CoV, SARS-CoV-2, Bat coronavirus (HKU5) and Bat SARS-like coronavirus (Bat-SL-CoV-ZC45) with the interaction region of MASP-2, see in particular FIG. 1, it can be seen that the sequences of the N protein of the 5 kinds of coronavirus and the interaction region of MASP-2 are highly homologous. Interaction of MASP-2 with coronavirus N protein was detected by immunoprecipitation and immunoblotting as follows:

1 materials and methods

1.1 reagents

Cell lysis solution: 50mmol/L Tris-HCl pH7.4, 150mmol/L NaCl, 2mmol/L CaCl ₂1 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: pEGFPC1- -SARS N (WT), pEGFPC1-SARS N (. DELTA.321-. Wherein pEGFPC1-SARS N (WT) is an expression vector for expressing protein GFP-SARS N (WT), GFP-SARS N (WT) is a fusion protein of SARS N (WT) and GFP, HA-SARS-CoV-2N is a fusion protein of SARS-CoV-2N and HA, and the rest expression vectors are analogized. SARS N (WT) is the full length of SARS-CoV N protein, GFP is protein label, SARS N (Delta 321-323) is mutant protein of deletion of amino acid residue at 321-323 of SARS-CoV N (WT) protein, SARS N (Delta 116-124) is mutant protein of deletion of amino acid residue at 116-124 of SARS-CoV N (WT) protein, MERS N (WT) is the full length of MERS-CoV N protein, MERS N (Delta 104-112) is mutant protein of deletion of amino acid residue at 104-112 of MERS-CoV N (WT) protein, SARS-CoV-2N is the full length of SARS-CoV-2N protein, and HA is protein label.

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 pEGFPC1-SARS N (WT)

The full-length gene of SARS-CoV N protein was inserted between restriction endonuclease EcoRI and BamHI recognition sites of pEGFPC1(Invitrogen) vector, and the other sequences of pEGFPC1 were kept unchanged to obtain a recombinant expression vector of GFP-SARS N protein (also called SARS N (WT)), which was named pEGFPC1-SARS N (WT).

1.2.3 plasmid pEGFPC1-SARS N (. DELTA.321-

The SARS N (. DELTA.321-323) gene was inserted between restriction endonuclease EcoRI and BamHI recognition sites of pEGFPC1(Invitrogen) vector, and the other sequences of pEGFPC1 were kept unchanged to obtain a recombinant expression vector of a deletion mutant (also called SARS N (. DELTA.321-323)) of the 321-323 amino acid deletion site of SARS N protein, which was designated as pEGFPC1-SARS N (. DELTA.321-323).

1.2.4 plasmid pEGFPC1-SARS N (. DELTA.116-124)

The SARS N (. DELTA.116-124) gene was inserted into the restriction endonuclease EcoRI and BamHI recognition sites of pEGFPC1(Invitrogen) vector, and the other sequences of pEGFPC1 were kept unchanged to obtain a recombinant expression vector of a deletion mutant (also called SARS N (. DELTA.116-124)) of SARS N protein deletion 116-124 amino acid, which was designated as pEGFPC1-SARS N (. DELTA.116-124).

1.2.5 plasmid pEGFPC1-MERS N (WT)

The full-length gene of MERS-CoV N protein was inserted between restriction endonuclease EcoRI and BamHI recognition sites of pEGFPC1(Invitrogen) vector, and the other sequences of pEGFPC1 were kept unchanged to obtain a recombinant expression vector of MERS N protein, which was designated as pEGFPC1-MERS N (WT).

1.2.6 plasmid pEGFPC1-MERS N (. DELTA.104-

The MERS N (. DELTA.104-112) gene was inserted into the restriction endonuclease EcoRI and BamHI recognition sites of the pEGFPC1(Invitrogen) vector, and the other sequences of pEGFPC1 were kept unchanged to obtain a recombinant expression vector of a deletion mutant (also called MERS N (. DELTA.104-112)) of the MERS N protein deletion 104-112 amino acids, which was designated as pEGFPC1-MERS N (. DELTA.104-112).

1.2.7 plasmid pcDNA3.1-HA-SARS-CoV-2N

The HA gene fragment (TACCCATACGACGTACCAGATTACGCT) was inserted before the stop codon at the 3' end of the full-length gene of SARS-CoV-2N protein to obtain the DNA named HA-SARS-CoV-2N gene, which was inserted between the recognition sites for restriction endonucleases KpnI and XbaI of pCDNA3.1(Invitrogen) vector while keeping the other sequences of pCDNA3.1 unchanged to obtain a recombinant expression vector for HA-SARS-CoV-2N protein, which was named pcDNA3.1-HA-SARS-CoV-2N.

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^TM3000 transfection 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^TM3000 transfection reagents 1:3 ratio, 15. mu.L Lipofectamine^TM3000 transfection 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/GFP-SARS N (WT) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pEGFPC1-SARS N (WT) plasmid and transfected by the same procedure as above to obtain HEK293/GFP-SARS N (WT) cells.

1.3.3 preparation of HEK293/GFP-SARS N (. DELTA.321-323) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with the pEGFPC1-SARS N (. DELTA.321-323) plasmid and transfected in the same manner as described above to obtain HEK293/GFP-SARS N (. DELTA.321-323) cells.

1.3.4 preparation of HEK293/GFP-SARS N (. DELTA.116-124) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with the pEGFPC1-SARS N (. DELTA.116-124) plasmid and transfected in the same manner as described above to obtain HEK293/GFP-SARS N (. DELTA.116-124) cells.

1.3.5 preparation of HEK293/GFP-MERS N (WT) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pEGFPC1-MERS N (WT) plasmid and transfected by the same procedure as above to obtain HEK293/GFP-MERS N (WT) cells.

1.3.6 preparation of HEK293/GFP-MERS N (. DELTA.104-112) cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with the pEGFPC1-MERS N (. DELTA.104-112) plasmid and transfected in the same manner as described above to obtain HEK293/GFP-MERS N (. DELTA.104-112) cells.

1.3.7 preparation of HEK 293/HA-SARS-CoV-2N cells

The pcDNA3.0-MASP-2-Flag plasmid in 1.3.1 was replaced with pcDNA3.1-HA-SARS-CoV-2N plasmid, and transfection was performed by the same procedure as above to obtain HEK 293/HA-SARS-CoV-2N 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.

6 kinds of cells containing coronavirus N protein (i.e., HEK293/GFP-SARS N (WT) cells, HEK293/GFP-SARS N (Δ 321-, HEK293/GFP-MERS N (. DELTA.104-112) cell lysate, HEK 293/HA-SARS-CoV-2N 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 bound with MASP-2 were added to lysates containing coronavirus N protein cells (any of the above-mentioned lysates of HEK293/GFP-SARS N (WT) cells, HEK293/GFP-SARS N (Δ 321-. 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

The results are shown in FIG. 2, and SARS-CoV N protein (i.e., SARS N (WT), SARS N (Δ 321-.

EXAMPLE 2 SARS-CoV N protein accelerated complement deposition assay

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 C4 deposition experiment was performed using the HBT complement deposition kit. Human C1 q-depleted serum was used, diluted with high salt binding buffer and added to the mannan pre-coated 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 C4(Sigma, 204897) and SARS-CoV N protein (Chinesen, Yi-Qiao, 40143-V08B) were added (Control without SARS-CoV N protein and Control with human coronavirus 229E N was also added), and incubation was carried out at 37 ℃ for 1.5 h. Washing the plate for three times, adding anti-C4 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 assay, human C1q serum depleted by dilution with binding bufferAdd to the pre-coated mannan ELISA plate, after 1h incubation at 4 ℃, do not wash the plate, incubate for 1.5h at 37 ℃. 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 A of FIG. 3, B of FIG. 3, and C of FIG. 3, and it was found by complement deposition experiments that SARS-CoV N protein promotes lectin pathway activation and leads to increased downstream complement activation.

Example 3 MERS-CoV N protein accelerated complement deposition assay

1 materials and methods

1.1 reagents

1.2 complement deposition

The C4 deposition experiment was performed using the HBT complement deposition kit. Human C1q depleted serum was used, with high salt binding buffer, 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 C4(Sigma, 204897) and MERS-CoV N protein (Casino, Chinesen, 40068-V08B) (Control without MERS-CoV N protein) were added and incubated at 37 ℃ for 1.5 h. Washing the plate for three times, adding anti-C4 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.

2 results

Specific results are shown in figure 3D, and MERS-CoV N protein was found to promote lectin pathway activation and lead to increased downstream complement activation by complement deposition experiments.

EXAMPLE 4 SARS-CoV-2N protein accelerated complement deposition assay

1 materials and methods

1.1 reagents

1.2 complement deposition

The C4 deposition experiment was performed using the HBT complement deposition kit. Human C1q depleted serum was used, with high salt binding buffer, 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 C4(Sigma, 204897) and SARS-CoV-2N protein (40588-V08B, Chiense) (Control without SARS-CoV-2N protein) were added and incubated at 37 ℃ for 1.5 h. Washing the plate for three times, adding anti-C4 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 2MH after 15-30min₂SO₄The reaction was stopped and the OD read at 450 nm.

Example 5 serum Allergoxin C5a detection of SARS-CoV-2 infected patients

The following experiments are carried outWuhan dynastyThe clinical detection laboratory of the general hospital in the middle war zone.

1 materials and methods

Detecting the content of anaphylatoxin C5a (complement fragment C5a) in the serum of severe patients and mild patients infected by SARS-CoV-2, and using the content of anaphylatoxin C5a in the serum of normal people as control. Serum from SARS-CoV-2 infected severe patients was from 18 different severe patients, serum from SARS-CoV-2 infected mild patients was from 12 different mild patients, serum from 14 different normal persons, serum from each person was set to 3 replicate samples, and the results were averaged and subjected to significance analysis using IBM SPSS 22.

Sera were collected by the clinical laboratory with inclusion criteria as follows:

the inclusion criteria for normal persons were: the body behaves normally.

The inclusion criteria for severe patients with SARS-CoV-2 infection are: fever and at least one of the following symptoms: respiratory frequency >30 beats/minute, severe respiratory distress, or hypoxemia SpO2< 90%, serum from (7 men and 11 women, mean age 72.4 ± 10.1 years, mean course 40.15 ± 12.74 days).

The inclusion criteria for patients with mild symptoms of SARS-CoV-2 infection were: pneumonia without severe symptoms, and serum derived from 6 men and 6 women (average age 56 + -12.1 years, average course of disease 26.3 + -9.1 days).

Detection using R&Human complement C5a ELISA kit from company D. Diluting the serum with PBS buffer solution, adding into ELISA plate pre-coated with C5a antibody, washing the plate for three times after 1 hr, incubating for detecting antibody for 1 hr, washing the plate for three times, adding TMB color developing solution, adding stop solution 2M H after 15-30min₂SO₄Solution, OD was read at 450 nm.

2 results

Complement deposition results see FIG. 3E, and the SARS-CoV-2N protein was found to promote lectin pathway activation and to contribute to the exacerbation of downstream complement activation by complement deposition experiments. The detection result of C5a in the serum of SARS-CoV-2 patient is shown in F in FIG. 3, the anaphylatoxin C5a in the serum of SARS-CoV-2 infected severe patients is (209.3 + -21.8) ng/mL, which is much higher than that of SARS-CoV-2 infected mild patients (49.2 + -24.2 ng/mL) and normal persons (18.6 + -2.2 ng/mL), the SARS-CoV-2 infected severe patients and normal persons have very significant difference (P <0.001), the SARS-CoV-2 infected severe patients and SARS-CoV-2 infected mild patients have no significant difference (P > 0.05), and the SARS-CoV-2 infected mild patients and normal persons have no significant difference (P > 0.05). The experimental result shows that the substance specifically bound with the complement fragment C5a or the gene thereof can be used for preparing products for screening or assisting in screening patients with severe coronavirus infection. Indicating excessive complement activation in the patient, and the anaphylatoxin C5a has the functions of recruiting inflammatory cells, activating and amplifying inflammatory responses. Therefore, the substance targeting C5a has value in being used as a candidate drug for preventing and treating SARS-CoV-like virus infection.

EXAMPLE 6 MASP2 knockout mice that express SARS-CoV N protein or express MERS-CoV N protein and infect LPS-induced pneumonia

1 materials and methods

The following experiments were performed in BSL-2 and ABSL-2 laboratories.

1.1 viruses

The adenovirus expressing SARS-CoV N protein and the adenovirus expressing MERS-CoV N protein were synthesized by Beijing Baiaochuan Biotechnology GmbH.

1.2 mice

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

1.3 methods of infection

One group of KO group and one group of WT group are inoculated with adenovirus (Ad-SARS N) expressing SARS-CoV N protein by tail vein injection, and each group is injected with 100 μ L (i.e. 10 μ L)⁹PFU/one), once daily, twice daily, and on day 6 via tail vein injection of LPS (lipopolysaccharide solution), 5 mg/kg. Mice were then observed every 1-3 hours for survival.

The KO group and the WT group were inoculated with adenovirus expressing MERS-CoV N protein (Ad-MERS N) by tail vein injection, each at 100. mu.L (i.e., 10)⁹PFU/one), once daily, twice daily, and on day 6 via tail vein injection of LPS (lipopolysaccharide solution), 5 mg/kg. Mice were then observed every 1-3 hours for survival.

2 results

The specific results are shown in fig. 4, and it can be seen that the death time of mice with the Masp2 gene knocked out is obviously delayed and the survival rate is obviously increased compared with littermate negative mice after virus infection through LPS induced pneumonia.

Example 7 treatment of mice with LPS-Ad-SARS or LPS-Ad-MERS N induced pneumonia

1 materials and methods

1.1 laboratory

The following experiments were performed in BSL-2 and ABSL-2 laboratories.

1.2 viruses

The adenovirus expressing SARS-CoV N protein (LPS + Ad-SARS N) and the adenovirus expressing MERS-CoV N protein (LPS + Ad-MERS N) were synthesized by Beijing Baiaochuan Biotechnology GmbH.

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 LPS+Ad-SARS N

Female mice of 10-12 weeks of age and approximately 20g in weight were selected and randomly divided into 9 groups of 8-10 mice per group: the salan is a healthy control group, the Ad-null is an adenovirus vector negative control group which does not express N protein, the Ad-SARS N is an infectious control group, the Ad-SARS N + C1INH, the Ad-SARS N + anti-MASP-2 and the Ad-SARS N + anti-N are treatment groups, the Ad-SARS N delta 116-124, the Ad-SARS N delta 321-323 are SARS N mutant control groups, and the Ad-229E N are weak pathogenic coronavirus N protein control groups.

1.4.2 LPS+Ad-MERS N

Female mice of 10-12 weeks of age and approximately 20g in weight were selected and randomly divided into 6 groups of 8-10 mice per group: salin is a healthy control group, Ad-null is an adenovirus vector negative control group which does not express N protein, Ad-MERS N is an infected control group, Ad-MERS N + C1INH, Ad-MERS N + anti-MASP-2 is a treatment group, and Ad-MERS N delta 104-112 is an MERS N mutant control group.

1.5 infection and treatment

1.5.1 infections and methods of treatment

1.5.1.1 LPS + Ad-SARS N pneumonia model construction and treatment implementing method

A salt group: LPS is not injected, and only physiological saline is injected;

ad-null group: injecting adenovirus that does not express SARS N protein;

Ad-SARS N group: injecting adenovirus expressing SARS N protein;

Ad-SARS N + C1INH group: injecting adenovirus expressing SARS N protein, and treating with C1 INH;

Ad-SARS N + anti-MASP-2 group: injecting adenovirus expressing SARS N protein, and treating with anti-MASP-2;

Ad-SARS N + anti-N group: injecting adenovirus expressing SARS N protein, and treating with anti-N;

Ad-SARS N.DELTA.116-124 group: injecting adenovirus expressing SARS N delta 116-124 protein;

Ad-SARS N.DELTA.321-323 group: injecting adenovirus expressing SARS N delta 321-323 protein.

All the above adenoviruses are according to 10⁹PFU/single injection per tail vein was injected continuously for 3 days, the other groups were injected with an equal volume of physiological saline, and all mice were injected with LPS 5mg/kg via tail vein on day 6. All drug treatments were injected via tail vein 30min prior to LPS injection. Wherein the dosage of the C1INH is 4mg/kg, and the anti-MASP-2 antibody and the anti-N antibody are both 200 mu g/kg.

Mice were then observed every 1-3 hours for survival.

1.5.1.2 LPS + Ad-MERS N pneumonia model construction and treatment implementation method

A salt group: LPS is not injected, and only physiological saline is injected;

ad-null group: injecting an adenovirus that does not express MERS N protein;

Ad-MERS N group: injecting an adenovirus expressing MERS N protein;

Ad-MERS N + C1INH group: injecting adenovirus expressing MERS N protein, and treating with C1 INH;

Ad-MERS N + anti-MASP-2 group: injecting adenovirus expressing MERS N protein, and treating with anti-MASP-2;

Ad-MERS N Δ 104-112 group: injection of an adenovirus expressing MERS N.DELTA.104-112 protein.

Mice were then observed every 1-3 hours for survival.

2 results

The specific results are shown in FIG. 5, wherein A in FIG. 5 is the survival rate of mice treated by the LPS-Ad-SARS N model; b in FIG. 5 is the LPS-Ad-MERS N model and the survival rate of the treated mice. As can be seen from fig. 5:

in an LPS + Ad-SARS N pneumonia model, a mouse injected with anti-MASP-2 antibody anti-N antibody or C1INH has a very obviously reduced lethality rate compared with an infection control. Specifically, the survival rates (survival rates) of the Saline and Ad-null infected groups after LPS injection are equal to or greater than 80%, all the Ad-SARSN groups die within 12 hours after LPS injection, and the survival rates of the antibody and inhibitor treatment group and the adenovirus pre-infected group expressing the weakly pathogenic N protein mutant or 229E N are both greater than 80% within 12 hours after LPS injection and greater than 40% within 24 hours.

② in LPS + Ad-MERS N induced pneumonia model, injecting anti-MASP-2 antibody anti-N antibody or C1INH mouse, compared with control of infection, significantly reducing lethality rate. Specifically, the survival rate (survival rate) of the Saline and Ad-null infected group after LPS injection is larger than or equal to 80 percent; the Ad-MERS N group died 80% of the mice within 12 hours and all within 24 hours after LPS injection. The survival rates of the antibody and inhibitor treatment group and the adenovirus pre-infection group expressing the N protein mutant without interaction with MASP-2 are respectively over 70 percent within 12 hours and over 30 percent within 24 hours after LPS injection

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

1. Use of an agent that inhibits the effects of MASP-2 downstream complement activation, said use being any one of:

y1, and the application of the substance for inhibiting the downstream complement activation effect of MASP-2 in the preparation of the medicine for preventing and/or treating the diseases caused by coronavirus or coronavirus infection;

y2, and the application of the substance for inhibiting the downstream complement activation effect of MASP-2 in preparing coronavirus inhibitor.

2. Use according to claim 1, characterized in that: the substance for inhibiting the downstream complement activation effect of MASP-2 is any one or more of the following X1-X7:

x1, a substance that inhibits the activity of complement downstream of MASP-2;

x2, a substance that reduces the amount of complement downstream of MASP-2;

3. Use according to claim 2, characterized in that: the MASP-2 downstream complement in X1 or X2 is at least one of complement C2, C3, C4 and C5; the complement fragment downstream of MASP-2 in X3, X4, X5 is at least one of complement fragments C2a, C2b, C3a, C3b, C4a, C4b, C5a and C5 b-9; specific receptors for the complement fragment downstream of MASP-2 in X5, X6, X7 are C3aR and/or C5 aR.

4. Use according to any one of claims 1 to 3, characterized in that: the agent that inhibits the downstream complement activation effects of MASP-2 is an agent comprising the following I and/or II:

i an antibody or antigen-binding fragment thereof that inhibits a downstream complement activation effect of MASP-2;

II inhibits MASP-2 downstream complement activation effect of organic molecules.

5. Use according to claim 4, characterized in that: the antibody or antigen binding fragment thereof that inhibits the downstream complement activation effects of MASP-2 is at least one of an antibody against C3a, an antibody against C5a, an antibody against C3aR, an antibody against C5 aR; the organic molecule for inhibiting the downstream complement activation effect of MASP-2 is at least one of C3a antisense peptide, C5a antisense peptide, C3a mutant with C3a receptor binding activity and C5a mutant with C5a receptor binding activity.

6. The application of the material specifically combined with the complement fragment C5a or the material specifically combined with the complement fragment C5a gene in the preparation of products for screening or assisting in screening patients with severe coronavirus infection.

7. Use according to claim 6, characterized in that: the substance specifically binding to the complement fragment C5a is a reagent or a kit containing an antibody against the complement fragment C5a or an antigen-binding fragment thereof, and the substance specifically binding to the complement fragment C5a gene is a reagent or a kit containing a specific primer for PCR amplification of the complement fragment C5a gene.

8. Use of a substance that inhibits the activity of MASP-2 and/or reduces the amount of expression of a gene of MASP-2 and/or reduces the amount of MASP-2, said use being any of the following:

u1, and the application of substances 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 medicines for preventing and/or treating diseases caused by coronavirus or coronavirus infection;

u2, and 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 inhibitor.

9. Use according to claim 8, characterized in that: the substance is a reagent comprising the following 1) and/or 2):

1) an organic molecule which inhibits the activity of MASP-2 and/or reduces the expression level of a gene of MASP-2 and/or reduces the content of MASP-2; the organic molecule is specifically C1 INH;

2) an antibody or antigen-binding fragment thereof that inhibits the activity of MASP-2 and/or reduces the expression level of a gene of MASP-2 and/or reduces the level of MASP-2.

10. Use according to any one of claims 1 to 9, characterized in that: the coronavirus is a beta coronavirus which has N protein and SARS-CoVN protein 107-125 site amino acid residue amino acid homology of more than 75 percent; in particular, the coronavirus is SARS-CoV and/or MERS-CoV and/or SARS-CoV-2.