Antibody against coronavirus n protein and application thereof

Abstract

The application discloses an antibody against coronavirus N protein and application thereof, and the antibody comprises three CDRs of a heavy chain variable region and three CDRs of a light chain variable region; wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are as shown in SEQ ID NO: 2, 3 and 4, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are as shown in SEQ ID NO: 10, 11 and 12; meanwhile, the amino acid sequences of four framework regions of the heavy chain variable region and four framework regions of the light chain variable region, nucleic acid molecules, expression vectors, host cells and pharmaceutical compositions related to the antibody and application thereof are disclosed. The antibody has high binding activity to the coronavirus and can accurately detect the coronavirus or a variant strain thereof.

Description

Technical Field

[0001] This invention belongs to the fields of cell biotechnology and immunology, and relates to an antibody against coronavirus N protein and its application. Background Technology

[0002] SARS-CoV-2 has continuously evolved and mutated during its ongoing transmission, resulting in multiple variants. Among them, the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) variants are more infectious and resistant to vaccines and treatments, posing new challenges to COVID-19 prevention and control. These variants have been identified as variants of concern by the World Health Organization. SARS-CoV-2 is primarily transmitted through respiratory droplets and close contact, exhibiting extremely high infectivity. Some patients are asymptomatic yet capable of infecting others. Accurate diagnosis of COVID-19 is crucial for epidemic control, early detection, isolation, and treatment of patients.

[0003] Currently, nucleic acid testing is the most commonly used method for detecting the novel coronavirus. The testing method depends on the difficulty of clinical sampling and patient acceptance; the most commonly used specimen in clinical practice is the oropharyngeal swab, followed by the nasopharyngeal swab. Nucleic acid testing detects the genetic material (RNA) of the novel coronavirus. If the test result is positive, it can directly confirm an infection case. Real-time fluorescent RT-PCR is the most commonly used method for nucleic acid testing and is considered the "gold standard" for diagnosing COVID-19. RT-PCR is the most widely used nucleic acid testing method during the pandemic due to its speed, simplicity, low cost, and high specificity. However, the test results are easily affected by many factors, which may lead to "false negatives" and missed diagnoses.

[0004] Because nucleic acid testing has a high false negative rate, is time-consuming, and requires sophisticated facilities, equipment, and technical personnel, immunological testing has gained increasing attention due to its speed, on-site testing capability, relatively accurate diagnosis, and ability to assist in clinical confirmation. High-titer antibody plasma has also been shown to be helpful in treating severe COVID-19 patients. The "Diagnosis and Treatment Protocol for Novel Coronavirus-Infected Pneumonia (Trial Version 7)" for the first time included serological testing as one of the diagnostic criteria; therefore, serological immunological testing has certain clinical significance as a diagnostic indicator for the novel coronavirus. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide an antibody against the coronavirus N protein, said antibody exhibiting high activity in binding to the N protein. This invention is achieved through the following scheme:

[0006] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising complementarity-determining regions CDR1, CDR2, CDR3 of the heavy chain variable region and / or complementarity-determining regions CDR1, CDR2, CDR3 of the light chain variable region; the amino acid sequences of the heavy chain variable regions CDR1, CDR2, CDR3 are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, respectively.

[0007] Preferably, the antibody or its antigen-binding fragment is an antibody against coronavirus N protein or its antigen-binding fragment.

[0008] Preferably, the coronavirus is selected from SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0009] Preferably, the coronavirus is SARS-CoV-2.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody includes the sequence shown in SEQ ID NO:9 or a sequence having at least 90% identity with it; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO:17 or a sequence having at least 90% identity with it.

[0011] Preferably, the antigen-binding fragment of the antibody includes Fab, Fab′, F(ab′)2, Fv, or a single-chain antibody.

[0012] A second aspect of the present invention provides an isolated nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0013] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0014] Preferably, the carrier further comprises a signal peptide operatively linked to the antibody or its antigen-binding fragment as described in the first aspect of the invention.

[0015] A fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention.

[0016] Preferably, the host cell is selected from prokaryotes or eukaryotes.

[0017] Preferably, the host cell is selected from eukaryotic organisms.

[0018] Preferably, the host cell is selected from mammalian cells.

[0019] Preferably, the host cell is selected from HEK293 cells.

[0020] A fifth aspect of the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, an expression vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, and / or a pharmaceutically acceptable vector.

[0021] The sixth aspect of the present invention provides a detection product comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and / or the host cell described in the fourth aspect of the present invention.

[0022] Preferably, the product includes a reagent kit.

[0023] Preferably, the kit includes a colloidal gold immunoassay kit, a chemiluminescence immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay (ELISA) kit, a fluorescence immunoassay kit, or a microfluidic chip.

[0024] A seventh aspect of the present invention provides a method comprising any one of the following:

[0025] 1) A method for generating an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising culturing a host cell as described in the fourth aspect of the present invention and recovering the antibody or antigen-binding fragment thereof;

[0026] 2) A method for detecting coronavirus infection, the method comprising contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, and observing the immune response between the sample to be tested and the antibody or the antigen-binding fragment thereof, so as to determine the expression level of N protein in the sample or the status of coronavirus infection.

[0027] Preferably, the method is a non-diagnostic detection method.

[0028] An eighth aspect of the present invention provides an application, the application comprising any of the following:

[0029] 1) The application of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the detection product described in the sixth aspect of the present invention in the detection of coronavirus infection;

[0030] 2) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the pharmaceutical composition described in the fifth aspect of the present invention in inhibiting coronavirus infection;

[0031] 3) The application of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the detection product described in the sixth aspect of the present invention in the preparation of products for diagnosing coronavirus infection-related diseases;

[0032] 4) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a medicament for the prevention / treatment of diseases related to coronavirus infection.

[0033] Preferably, the coronavirus infection-related illnesses include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms, organ failure, and septic shock.

[0034] Preferably, the coronavirus infection-related disease is selected from pneumonia.

[0035] Preferably, the coronavirus is selected from the SARS-CoV-2 virus.

[0036] The beneficial effects of this invention are:

[0037] This invention provides an antibody against the coronavirus SARS-CoV-2, which is characterized by high purity and high expression level. It has good binding activity with the N protein or coronavirus variants and can be used as an effective detection method for screening coronaviruses. It has a high detection accuracy for both coronavirus protein infection and its variants. Attached Figure Description

[0038] Figure 1 This is an electrophoresis image for detecting monoclonal antibody OP2;

[0039] Figure 2 This is an HPLC chromatogram for detecting the monoclonal antibody OP2;

[0040] Figure 3 This is a graph showing the binding activity of the monoclonal antibody OP2 detected by ELISA. Detailed Implementation

[0041] Before describing the method of the present invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used in this invention is for the purpose of describing specific embodiments only and is not intended to be restrictive, as the scope of the invention is limited only by the appended claims.

[0042] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0043] The term "antibody" as used in this invention includes whole-antibody molecules and functional fragments thereof capable of binding to the antigenic portion of a target polypeptide, such as Fab, F(ab')2, Fv, single-chain antibodies ("SCA" or ScFv"), or any combination thereof. Antibodies are preferably monospecific, such as monoclonal antibodies or antigen-binding fragments thereof. The term "monospecific antibody" refers to an antibody that exhibits single binding specificity and affinity for a specific target (e.g., epitope). This term includes "monoclonal antibody" or "monoclonal antibody composition," which in this invention refers to the preparation of a single-molecule antibody or fragment thereof. Antibodies can be human antibodies, chimeric antibodies, recombinant antibodies, humanized antibodies, monoclonal antibodies, or polyclonal antibodies. Antibodies can be complete immunoglobulins, such as IgA, IgG, Ig, IgD, IgM, or their subtypes, or functional fragments of complete antibody molecules (e.g., compounds with biological or chemical functions). The anti-N protein antibody used in this invention is named OP2. OP2 can specifically bind to any variant of the coronavirus containing the N protein or coronavirus antigen. Coronavirus variants include, but are not limited to, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) variants.

[0044] The terms "antibody fragment," "antibody or antigen-binding fragment thereof," "antigen-active fragment of an antibody," and "multiple antibody fragments" are used interchangeably in this invention and refer to a portion of a complete antibody including an antigen-binding site or variable region. This portion does not include the constant heavy chain domain (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, bivalent antibodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region. "CDR" in this invention refers to a "complementarity-determining region" within the variable sequence of the antibody. There are three CDRs in each variable region of the heavy and light chains, which are specifically designated as "CDR1," "CDR2," and "CDR3" for each variable region.

[0045] In this invention, "frame" (FR) or "frame sequence" refers to the sequence of the variable region minus the CDR. Because the precise definition of the CDR sequence can be determined by different systems, the meaning of the frame sequence is subject to correspondingly different interpretations. The frame region in the heavy chain variable region consists of six CDRs: three complementarity-determining regions CDR1, CDR2, and CDR3; and six complementarity-determining regions CDR1, CDR2, and CDR3 in the light chain variable region. This also divides the frame region in the heavy and light chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Where no specific sub-region is designated as FR1, FR2, FR3, and FR4, the frame region, as otherwise indicated, represents the FRs combined within the variable region of a single, naturally occurring immunoglobulin chain. As used in this invention, FR represents one of the four sub-regions, and FR represents two or more of the four sub-regions constituting the frame region.

[0046] coronavirus

[0047] The term "coronaviruses" as used in this invention refers to single-stranded positive-sense RNA viruses belonging to the subfamily Orthocoronavirinae of the family Coronaviridae in the order Nidovirales. These viruses can infect various species, including humans, bats, pigs, rats, cattle, horses, goats, and monkeys. Seven types of coronaviruses (HCoVs) are known to infect humans, including Middle East Respiratory Syndrome-related coronavirus (MERSr-CoV) and Severe Acute Respiratory Syndrome-related coronavirus (SARSr-CoV), specifically HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-nCoV, and SARS-CoV-2. In this specific embodiment, the coronavirus described in this invention is SARS-CoV-2.

[0048] identity

[0049] As used herein, the term "identity" refers to sequence similarity to the amino acid sequences used in this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Functionally equivalent variants of the amino acid sequences shown in SEQ ID NO:1-18 provided by this invention, wherein the variants have one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) with the amino acid sequences shown in SEQ ID NO:1-18, or have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO:1-18, are all within the scope of protection of this invention.

[0050] expression carrier

[0051] The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, lentiviruses, or other vectors well known in the art, such as plasmid pcDNA, pcDNA3.3 TOPO (Life Technologies, New York), plasmid pTT3, plasmid pEF-BOS, plasmid pEM5.1, etc. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements. In a specific embodiment of this invention, the pEM5.1 vector is used.

[0052] This invention provides an isolated nucleic acid molecule, wherein the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in a linear or circular form in single-stranded and / or double-stranded form, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (e.g., dsDNA, ssDNA, cDNA), RNA (e.g., dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (e.g., PNA). Preferably, the nucleic acid is DNA or RNA. The term "isolated" generally refers to being largely free of components (e.g., viruses, nucleic acids, or proteins) that are normally accompanied or interact with in their natural environment. The term "isolated nucleic acid molecule" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue thereof, isolated from or synthesized artificially from its natural environment, which has been isolated from at least about 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials that are naturally found with the nucleic acid molecule when the total nucleic acid is isolated from the source cell. In some embodiments, the isolated nucleic acid molecules are substantially free of any other contaminating nucleic acid molecules or other molecules found in the natural environment of nucleic acids that could interfere with their use in polypeptide production or their therapeutic, diagnostic, preventative, or research uses.

[0053] This invention provides a host cell comprising an expression vector. In this invention, "recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other terms representing microorganisms or higher-level eukaryotic cell lines cultured as single-cell entities are used interchangeably. These can be used, or have been used, as recipients of recombinant vectors or other transfected DNA, and include the original progeny of transfected original cells. The expression vector is used to express the antibody against the N protein described in this invention or the antibody or functional fragment thereof described in this invention. Host cells useful in this invention include, but are not limited to, eukaryotic host cells. Preferred eukaryotic host cells include, but are not limited to, mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algal host cells, nematode host cells, protozoan host cells, and fish host cells. Preferably, mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells, young hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, or MDCK cells. Preferred fungal host cells include *Aspergillus*, *Neurospora*, *Saccharomyces*, *Pichia*, *Hansenula*, *Schizosaccharomyces*, *Kluyveromyces*, *Yarrowia*, and *Candida*. In embodiments of the present invention, the host cell is HEK293 cells.

[0054] The term "pharmaceutically acceptable carrier" as used in this invention refers to an auxiliary material widely used in the pharmaceutical manufacturing field. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for enabling the active ingredient to dissolve at a desired rate or to promote the effective absorption of the active ingredient in the body of the administered subject after administration of the drug to a subject. A pharmaceutically acceptable carrier can be an inert filler or an active ingredient that provides a function (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition) for any pharmaceutical composition, such as an antibody against the anti-N protein or its antigen-binding fragment, nucleic acid molecules, expression vectors, host cells, etc. Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

[0055] As used in this invention, the terms "transformation" and "transfection" have the meanings commonly understood by those skilled in the art, namely, the process of introducing exogenous DNA into a host. The methods of transformation and transfection include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0056] As used in this invention, "treatment" means, after a coronavirus infection has caused related illness, exposing (e.g., administering medication) a subject to the anti-N protein antibody of this invention or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell, pharmaceutical composition, etc., thereby alleviating the symptoms of the disease compared to when there is no exposure, without implying the necessity of completely suppressing the symptoms of the disease. In one specific example, the disease related to novel coronavirus infection is novel coronavirus pneumonia (COVID-19).

[0057] As used in this invention, "prevention" means: exposing (e.g., administering medication) the anti-N protein antibody of this invention or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell, etc., to a subject prior to coronavirus infection, thereby reducing the incidence of disease caused by coronavirus infection compared to non-exposure. In one specific example, the disease related to novel coronavirus infection is novel coronavirus pneumonia (COVID-19).

[0058] As described in this invention, the terms "biological subject," "subject," and "individual" are used interchangeably in this invention to refer to animal subjects, particularly vertebrate subjects, and more particularly mammalian subjects. Suitable vertebrates falling within the scope of this invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, apes, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovids (e.g., cattle), sheep (e.g., sheep), goats (e.g., ibex), swine (e.g., pigs), equines (e.g., horses), canids (e.g., dogs), felines (e.g., cats), birds (e.g., chickens; ducks; geese; companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish. Preferred subjects are primates (e.g., humans, apes, monkeys, chimpanzees) and rodents (e.g., mice, rats, guinea pigs).

[0059] The most reliable results are likely to be obtained when samples are processed in a laboratory setting. For example, samples may be obtained from subjects in a physician's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide immediate results in a clinician's office or allow subjects to perform the test at home. In some cases, the need for portable, pre-packaged, disposable tests that can be used by subjects without assistance or guidance is more important than high accuracy. In many cases, especially with physician follow-up, preliminary testing, even tests with reduced sensitivity and / or specificity, may suffice. Therefore, assays provided in product form may involve detecting and measuring the N protein in relatively small amounts of subject samples to reduce the complexity and cost of the assay. Any form of sample assay capable of detecting the coronavirus N protein in a sample, as described in this invention, can be used. Typically, the assay quantifies antibodies in the sample to a certain extent, such as whether their concentration or amount is above or below a predetermined threshold. Such kits include colloidal gold immunoassay kits, chemiluminescent kits, radioimmunoassay kits, enzyme-linked immunosorbent assay (ELISA) kits, fluorescent immunoassay kits, or microfluidic chips. It should be noted that the detection methods and kits described in this invention are not only applicable to the detection of patient (human, animal) samples, but also to the detection of environmental samples in the environment for non-diagnostic purposes, including but not limited to water samples, food samples, air samples, microbial samples, animal cell samples, surface materials, soil samples, crystal samples and industrial samples.

[0060] In this invention, the term "sample" is used in its broadest sense. In some cases, a sample includes cells (e.g., bacteria, yeast, and fungi), organisms, specimens or cultures obtained from any source, and biological and environmental samples. Biological samples can be obtained from animals (including humans) and refer to biological materials or compositions found therein, including but not limited to bone marrow, interstitial fluid, urine, cerebrospinal fluid, nucleic acids, DNA, blood, serum, platelets, plasma, tissues, and their purified or filtered forms; environmental samples include but are not limited to water samples, food samples, air samples, microbial samples, animal cell samples, surface materials, soil samples, crystal samples, and industrial samples.

[0061] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0062] Example 1: Screening of Monoclonal Antibodies

[0063] 1. Synthesis of recombinant SARS-CoV-2 N protein

[0064] The N protein sequence of SARS-CoV-2 virus was synthesized and constructed into the pEM5.1 vector; plasmid for transfection was extracted; the cells were transfected into HEK293 cells and cultured for 7 days; the supernatant was harvested, purified by Ni column, and concentrated and replaced with buffer to obtain recombinant SARS-CoV-2 N protein. The recombinant SARS-CoV-2 N protein sequence was derived from Uniprot P0DTC9, and the sequence is shown in Table 1.

[0065] Table 1. Sequence of recombinant SARS-CoV-2 N protein

[0066]

[0067] 2. Immunity

[0068] The mice were first immunized with Freund's complete adjuvant at a dose of 100 μg per mouse via intraperitoneal injection, with a total dose of 0.5 ml per mouse. A second immunization was performed 3 weeks later. From the second immunization onwards, the mice were immunized with Freund's incomplete adjuvant at a dose of 50 μg / 0.5 ml per mouse. A third immunization was performed 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0069] To obtain feeder cells, use 10 5 / Hole application, lay 10 plates the day before fusion. 5 100 μl / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μl / well.

[0070] 3. Screening and cloning of hybridoma cells

[0071] Positive wells were screened using ELISA, and recombinant N protein expression cells were seeded and incubated overnight. The cells were washed, blocked with skim milk powder, and incubated at 37°C for 1 hour. After washing, 100 μl of 96-well culture supernatant was added, and the cells were incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse secondary antibody was added, and the cells were incubated at 37°C for 30 minutes. After washing, chromogenic buffer was added, and the cells were incubated for 10 minutes. Stop buffer was added, and the OD450 value was recorded. High-expression cell lines were selected for subcloning.

[0072] 4. Sequencing

[0073] Cells were collected, and total RNA was extracted using Trizol. cDNA was generated by reverse transcription using oligo(dT)20 primers. Then, the variable regions of the heavy and light chains were amplified by PCR using specific primers. After electrophoresis purification, the PCR products were transformed into a TA clone insertion vector, and positive clones were selected for sequencing.

[0074] 5. Results

[0075] The monoclonal antibody OP2 against SARS-CoV-2 was screened out, and its sequence is shown in Table 2.

[0076] Table 2. Sequence of monoclonal antibody OP2

[0077]

[0078]

[0079] Example 2: Functional Study of Monoclonal Antibody OP2

[0080] 1. Expression and purification of monoclonal antibodies

[0081] 1) The selected sequences were chemically synthesized and cloned into a eukaryotic expression vector;

[0082] 2) Plasmid amplification and extraction;

[0083] 3) Transfect the plasmid encoding the antibody transiently into HEK293 mammalian cells;

[0084] 4) Collect the supernatant and purify it using affinity chromatography to obtain the monoclonal antibody;

[0085] 5) As a result, the purified antibody expression level was 536 mg / L.

[0086] 2. Detection of the physicochemical properties of monoclonal antibodies

[0087] 2.1 Gel electrophoresis to detect the purity of monoclonal antibodies

[0088] 1) Instruments and equipment

[0089] Table 3. Instruments and equipment used for determining the purity of monoclonal antibody OP2 by gel electrophoresis.

[0090] name Manufacturer model Chemiluminescence imaging instrument Tanon Tanon-5200 Electrophoresis apparatus BIO-RAD poweerpac basic Electrophoresis tank BIO-RAD DYC-Mini4

[0091] 2) Main reagents

[0092] Table 4. Reagents used for determining the purity of monoclonal antibody OP2 by gel electrophoresis.

[0093]

[0094] 3) Sample preparation

[0095] Take 20 μl of sample and mix it with 5 μl of 5× reduction buffer. Heat at 95 °C for 5 min and then cool.

[0096] 4) Electrophoresis

[0097] Prepare the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0098] 5) Dyeing and bleaching

[0099] After electrophoresis, place the gel in an appropriate amount of Coomassie Brilliant Blue staining solution and stain at room temperature for 1 hour or longer. Pour off the staining solution, add an appropriate amount of Coomassie Brilliant Blue destaining solution, and destain at room temperature for 4-24 hours. After destaining, soak in ddH2O, compare with the unstained gel using the marker protein as a reference, and cut off the gel containing the desired protein component. Collect the gel. Then, separate the protein to be purified from the gel.

[0100] 6) Results

[0101] The results are as follows Figure 1 As shown, the bands from left to right are the marker and the reduction band, respectively; the detection purity of the monoclonal antibody is greater than 95%.

[0102] 2.2 HPLC determination of monoclonal antibody purity

[0103] 1) Instruments and equipment

[0104] Table 5. Instruments and equipment used for HPLC purity determination of monoclonal antibody OP2

[0105]

[0106] 2) Main reagents

[0107] Table 6. Reagents used for HPLC purity determination of monoclonal antibody OP2

[0108] name Manufacturer Specification Item number Potassium dihydrogen phosphate trihydrate Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10017592 Potassium dihydrogen phosphate Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10017692 Potassium chloride Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10016392

[0109] 3) Preparation of mobile phase

[0110] Add potassium dihydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride to approximately 900 ml of purified water, stir to dissolve, and bring the volume to 1 L. Measure the pH using a pH meter to ensure it is between 6.2 and 0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0111] 4) Sample preparation

[0112] System suitability test sample: MIL62 standard diluted to 2 mg / ml with mobile phase.

[0113] Test sample: The sample to be tested was diluted with the mobile phase to 2 mg / ml.

[0114] 5) Chromatographic conditions

[0115] Table 7. Chromatographic conditions for HPLC purity determination of monoclonal antibody OP2

[0116]

[0117] 6) Results

[0118] The results are as follows Figure 2 As shown, the detection purity of the monoclonal antibody is greater than 95%.

[0119] 3. Detection of monoclonal antibody binding activity

[0120] 1) Instruments and equipment

[0121] Table 8. Instruments and equipment used for the assay of monoclonal antibody OP2 binding activity.

[0122]

[0123]

[0124] 2) Solutions and reagents used

[0125] Coating solution: 1.5g Na2CO3, 2.93g NaHCO3, diluted to 1000ml;

[0126] DPBS (1x): 0.2g KCl, 0.2g KH2PO4, 8.0g NaCl, 2.9g Na2HPO4·12H2O, bring to a final volume of 1L;

[0127] Developing solution: TMB (Huzhou Yingchuang Biotechnology Co., Ltd. TMB-S-001 1000ml);

[0128] Stop solution: 2N H2SO4 (56ml H2SO4 + 944ml H2O);

[0129] Blocking solution: 5% high-protein skimmed high-calcium milk powder dissolved in 10ml DPBS;

[0130] Washing solution: DPBS containing 3‰ Tween (TWEEN 20, Sigma reagent V900548-500ml)

[0131] The antigens used for coating were: N-HIS6X-terminator (where N is the synthesized N protein mentioned above) and SARS-COV2-N-B1617 (sequence shown in Table 9).

[0132] Table 9. Antigens and sequences used for coating.

[0133]

[0134] 3) Experimental steps

[0135] ① Coating: Dilute antigen N protein to 2 μg / ml with coating solution, mix well, add 100 μl / well to a 96-well coated plate, seal with membrane, and incubate overnight at 4°C.

[0136] ② Wash the board three times with the washing machine. After the last wash, there should be no liquid residue left on the board. Pat the surface of the board dry with absorbent paper.

[0137] ③ Blocking: Add blocking solution, 300 μl / well, incubate at 37℃ for 1 h, and wash the plate 3 times as in step 2).

[0138] ④ Dilute the antibody in a gradient, resulting in 11 concentration gradients, 100 μl / well, react at 37℃ for 1 h, and wash the plate 3 times as in step 2).

[0139] ⑤ Add secondary antibody: Dilute with DPBS at a ratio of 1:3000, add 100 μl / well to a 96-well plate, react at 37°C for 1 h, and wash the plate 3 times as in step 2).

[0140] ⑥ Color development: Add TMB, 100 μl / well, and develop color at room temperature in the dark for 10 min.

[0141] ⑦ Termination: Add 2N H2SO4, 100μl / well.

[0142] ⑧ Measure OD450 with an ELISA reader within 10 minutes.

[0143] ⑨ Result

[0144] The specific substances used and results of the monoclonal antibody binding activity assay are shown in Table 10.

[0145] Table 10. Substances and Results Used in Monoclonal Antibody Binding Activity Assay

[0146]

[0147] Combined with activity results such as Figure 3 As shown, the monoclonal antibody OP2 can specifically bind to the antigen N protein, with an EC50 of 0.01134 μg / ml. It can also specifically bind to the SARS-CoV-2 variant B1617, with an EC50 of 0.01664 μg / ml.

[0148] In summary, the antibody OP2 in this invention has good binding activity with the coronavirus N protein antigen or coronavirus variants, and can be used for clinical detection of coronavirus SARS-CoV2, improving the detection accuracy of coronavirus protoprotein or its variants.

[0149] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention. sequence list <110> Jiangsu Dongkang Biomedical Technology Co., Ltd. <120> An antibody against coronavirus N protein and its application <141> 2022-06-22 <160> 18 <170> SIPOSequenceListing 1.0 <210> 20 <211> 419 <212> PRT <213> Artificial Sequence <400> 20 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Gly Ser Thr Phe Ser Asn Phe Gly Met 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <400> 3 Ser Ser Ala Ser Ser Ile Ile 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <400> 4 Ala Arg Ser Ala Met Asp Tyr 1 5 <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <400> 5 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser 20 25 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <400> 6 His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr 1 5 10 15 Ile <210> 7 <211> 38 <212> PRT <213> Artificial Sequence <400> 7 Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Pro Lys Asn Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <400> 8 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 114 <212> PRT <213> Artificial Sequence <400> 9 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Asn Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Ala Ser Ser Ile Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 100 105 110 Ser Ser <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <400> 10 Tyr Ser Gly Asp Ser Tyr 1 5 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <400> 11 Ala Ala Ser 1 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Gln Gln Ser Glu Glu Gln Pro Tyr Thr 1 5 <210> 13 <211> 30 <212> PRT <213> Artificial Sequence <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asn 20 25 30 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <400> 14 Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 15 <211> 36 <212> PRT <213> Artificial Sequence <400> 15 Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <400> 16 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 17 <211> 111 <212> PRT <213> Artificial Sequence <400> 17 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asn Tyr Ser 20 25 30 Gly Asp Ser Tyr Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Glu 85 90 95 Glu Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 18 <211> 419 <212> PRT Artificial Sequence <400> 18 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Met Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Tyr Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala

Claims

1. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment includes complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region and complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region; the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment against the coronavirus N protein, and the coronavirus is SARS. CoV 2.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody includes the sequence shown in SEQ ID NO:9; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO:

17.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The antigen-binding fragments of the antibody include Fab, Fab′, F(ab′)2, and Fv.

5. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 5.

7. The expression vector according to claim 6, characterized in that, The carrier further comprises a signal peptide operatively linked to the antibody or its antigen-binding fragment as described in any one of claims 1-4.

8. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 5 or the expression vector of any one of claims 6-7.

9. The host cell according to claim 8, characterized in that, The host cells are selected from prokaryotes or eukaryotes.

10. The host cell according to claim 9, characterized in that, The host cells are selected from eukaryotic organisms.

11. The host cell according to claim 10, characterized in that, The host cell is selected from mammalian cells.

12. The host cell according to claim 8, characterized in that, The host cells were selected from HEK293 cells.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the nucleic acid molecule as described in claim 5, the expression vector as described in any one of claims 6-7, the host cell as described in any one of claims 8-12, and / or a pharmaceutically acceptable vector.

14. A testing product, characterized in that, The detection product includes the antibody or its antigen-binding fragment as described in any one of claims 1-4, the nucleic acid molecule as described in claim 5, the expression vector as described in any one of claims 6-7, and / or the host cell as described in any one of claims 8-12.

15. The testing product according to claim 14, characterized in that, The product includes a reagent kit.

16. The testing product according to claim 15, characterized in that, The kits include colloidal gold immunoassay kits, chemiluminescence kits, radioimmunoassay kits, enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or microfluidic chips.

17. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1-4, the method comprising culturing a host cell according to any one of claims 8-12 and recovering the antibody or the antigen-binding fragment thereof.

18. A method for detecting coronavirus infection for non-diagnostic purposes, the method comprising contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-4, and observing the immune response of the sample to the antibody or the antigen-binding fragment thereof to determine the expression level of N protein in the sample or the status of coronavirus infection.

19. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of any one of claims 6-7, the host cell of any one of claims 8-12, and the detection product of any one of claims 14-16 in the preparation of products for detecting coronavirus infection.

20. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of any one of claims 6-7, the host cell of any one of claims 8-12, and the detection product of any one of claims 14-16 in the preparation of products for diagnosing coronavirus infection-related diseases.

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