Monoclonal antibody 31A8 against novel coronavirus N protein and products and applications thereof

Abstract

The application discloses an anti-novel coronavirus N protein monoclonal antibody 31A8 and products and applications thereof, and belongs to the fields of cell biology technology and immunology. The light chain of the anti-novel coronavirus N protein monoclonal antibody 31A8 belongs to kappa, the heavy chain belongs to IgG1, the amino acid sequences of three complementarity determining regions (LCDR1-3) of the light chain variable region are respectively shown as SEQ ID No. 1-3, the amino acid sequences of HCDR1-3 of the heavy chain variable region are respectively shown as SEQ ID No. 10-13, and the amino acid sequences of four framework regions (LFR1-4) of the light chain variable region and four framework regions (HFR1-4) of the heavy chain variable region are disclosed. Experiments prove that the monoclonal antibody 31A8 provided in the application has good binding activity with the novel coronavirus, is specific, and can be used for clinically detecting the novel coronavirus SARS-COV-2.

Description

Technical Field

[0001] This invention belongs to the fields of cell biotechnology and immunology, and specifically relates to a monoclonal antibody against the N protein of the novel coronavirus (named 31A8), its products, and applications. Background Technology

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subtype of SARS-CoV-2 belonging to the genus *Betacoronavirus* and the species *SARS-CoV-2*. During its replication process, continuous adaptation leads to constant mutations, resulting in multiple variants that alter its transmissibility, pathogenicity, and immunogenicity. Therefore, effective and rapid detection of SARS-CoV-2 remains one of the effective means of protecting public health.

[0003] There are two main methods for COVID-19 testing that are widely known to the public: nucleic acid testing and antigen testing. Nucleic acid testing is time-consuming and labor-intensive, and it has high requirements for operators, testing equipment, and the testing environment, thus limiting its application in routine rapid virus detection. Antigen testing, on the other hand, has advantages such as fast testing speed, low cost, relatively simple operation, and the ability to perform at-home testing. However, the sensitivity of currently available antigen test kits is slightly lower than that of nucleic acid testing. Furthermore, due to the continuous mutation of the COVID-19 virus, there is an urgent need for antibodies with better affinity and broader spectrum for antigen testing. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody 31A8 against the N protein of the novel coronavirus, its products and applications, in order to solve the problems of low N protein binding activity and poor affinity of existing monoclonal antibodies against the novel coronavirus.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] A first aspect of the present invention provides a monoclonal antibody 31A8 against the novel coronavirus N protein, the monoclonal antibody 31A8 comprising a light chain and a heavy chain, wherein the light chain belongs to kappa and the heavy chain belongs to IgG1; wherein the amino acid sequences of the three complementarity-determining regions (LCDR1-3) of the variable region of the light chain of the monoclonal antibody 31A8 are as shown in SEQ ID No. 1-3, and the amino acid sequences of HCDR1-3 of the variable region of the heavy chain are as shown in SEQ ID No. 10-12.

[0007] Furthermore, the light chain variable region framework regions LFR1-4 of the monoclonal antibody 31A8 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 4-7, and the heavy chain variable region framework regions HFR1-4 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 13-16.

[0008] Furthermore, the light chain variable region of the monoclonal antibody 31A8 has a VL with at least 90% sequence identity, preferably 95% sequence identity, of the amino acid sequence shown in SEQ ID No. 8, and the heavy chain variable region has a VH with at least 90% sequence identity, preferably 95% sequence identity, of the amino acid sequence shown in SEQ ID No. 17.

[0009] Furthermore, the light chain variable region of the monoclonal antibody 31A8 has a VL region that is at least 90% sequence identical to the nucleotide sequence shown in SEQ ID No. 9, preferably 95% sequence identical, and the heavy chain variable region has a VH region that is at least 90% sequence identical to the nucleotide sequence shown in SEQ ID No. 18, preferably 95% sequence identical.

[0010] A second aspect of the present invention provides a product for novel coronavirus infection, comprising the monoclonal antibody or a functional fragment thereof described in the first aspect, or a nucleic acid molecule, recombinant expression vector, or host cell encoding the monoclonal antibody or a functional fragment thereof described in the first aspect of the present invention; wherein:

[0011] a) Nucleic acid molecule: The nucleic acid molecule encodes the monoclonal antibody or its functional fragment as described in the first aspect of the present invention;

[0012] b) Recombinant expression vector: the recombinant expression vector comprises the nucleic acid molecule described in a);

[0013] c) Host cell: The host cell contains the recombinant expression vector described in b);

[0014] d) Drug conjugates comprising the anti-novel coronavirus N protein monoclonal antibody as described in the first aspect of this invention or based on a functional fragment thereof.

[0015] Furthermore, the recombinant expression vector has a signal peptide operatively linked to an antibody;

[0016] Furthermore, the recombinant expression vector further includes transcriptional regulatory elements.

[0017] Furthermore, the product also includes reagents for performing antigen-antibody reactions or reagents for detecting reactions;

[0018] Furthermore, reagents used to perform antigen-antibody reactions include buffers, salts, diluents, etc.

[0019] Furthermore, the drug conjugate also includes a conjugation motif selected from the group consisting of: detectable markers, drugs, toxins, cytokines, or enzymes.

[0020] The third aspect of the present invention provides a method according to any one of the following:

[0021] 1) A method for preparing the monoclonal antibody according to the first aspect of the present invention, the method comprising: culturing the host cell according to the second aspect of the present invention, optionally isolating the monoclonal antibody from the host cell and / or the culture medium in which the host cell is grown;

[0022] 2) A method for detecting N protein in a sample, the method comprising contacting the sample to be tested with the monoclonal antibody as described in the first aspect of the present invention to determine the presence or level of N protein in the sample to be tested.

[0023] 3) Further, the method described in 1) also includes purifying the monoclonal antibody;

[0024] 4) Furthermore, the host cell is selected from mammalian cells;

[0025] 5) Further, the cells are selected from 293T cells, CHO cells, or Expi293F cells. TM cell.

[0026] The fourth aspect of the invention provides for any of the following applications:

[0027] 1) The application of the monoclonal antibody described in the first aspect of the present invention and the substance described in the second aspect of the present invention in the detection of N protein;

[0028] 2) The use of the monoclonal antibody described in the first aspect of the present invention and the substance described in the second aspect of the present invention in the preparation of products for detecting novel coronavirus infection;

[0029] 3) The use of the monoclonal antibody described in the first aspect of the present invention and the substance described in the second aspect of the present invention in the preparation of products for diagnosing diseases related to novel coronavirus infection;

[0030] 4) The use of the monoclonal antibody described in the first aspect of the present invention, the substance described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of diseases related to novel coronavirus infection.

[0031] Furthermore, the product includes a reagent kit.

[0032] Furthermore, the kit includes: a colloidal gold immunoassay kit, a chemiluminescence immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, and a microfluidic chip.

[0033] Furthermore, the novel coronavirus is SARS-CoV-2.

[0034] Furthermore, the novel coronavirus infection-related disease is COVID.

[0035] Furthermore, the novel coronavirus infection-related disease is COVID-19.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention discloses a monoclonal antibody against the N protein of the novel coronavirus, named 31A8, and explicitly provides the amino acid sequences of the complementarity-determining regions (CDRs) of its light and heavy chains. The light chain belongs to kappa, and the heavy chain belongs to IgG1. The amino acid sequences of the three CDRs (LCDR1-3) of the light chain variable region are shown in SEQ ID No. 1-3, and the amino acid sequences of the HCDR1-3 of the heavy chain variable region are shown in SEQ ID No. 10-13. The amino acid sequences of the four frame regions (LFR1-4) of the light chain variable region and the four frame regions (HFR1-4) of the heavy chain variable region are also disclosed. Experiments have shown that the monoclonal antibody 31A8 provided in this invention has good binding activity and high specificity against the novel coronavirus, and can be used for clinical detection of SARS-CoV-2. Attached Figure Description

[0038] Figure 1 These are the results of antibody titer assays in mice immunized with N protein;

[0039] Figure 2 This is the result of the monoclonal antibody 31A8 subtype identification;

[0040] Figure 3 This is the result of ELISA verification of the specificity of monoclonal antibody 31A8;

[0041] Figure 4 This is an ELISA graph showing the binding activity of monoclonal antibody 31A8. Detailed Implementation

[0042] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0043] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification and claims of this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such processes, methods, products, or devices.

[0044] The term "antibody" used in this invention, also known as immunoglobulin (Ig), refers to a class of immunoglobulins synthesized and secreted by immune cells after B cells differentiate and mature into plasma cells following activation by antigens. These antibodies are capable of specifically binding to corresponding antigens and possessing immune functions. They include whole-antibody molecules and their functional fragments capable of binding to the antigenic portion of target proteins, such as Fab, F(ab')2, Fv, single-chain antibodies, or any combination thereof. The basic structure of an antibody consists of two identical peptide chains with relatively small molecular weights (called light chains, L chains) and two identical peptide chains with relatively large molecular weights (called heavy chains, H chains). The regions of the L and H chains near the N-terminus where the amino acid sequence changes significantly are called the variable region (V), and the regions with more conserved amino acid sequences at the C-terminus are called the constant region (C). The amino acid composition and sequence of certain local regions in the VL and VH regions exhibit a higher degree of variability and are antigen-binding sites; therefore, they are called hypervariable regions (HVRs) or complementarity-determining regions (CDRs). Both VL and VH include three CDRs: CDR1, CDR2, and CDR3, with CDR3 exhibiting the highest degree of variability. The amino acid composition and sequence of non-CDR regions within the V region are relatively conserved and are called framework regions (FRs). Both VL and VH have four framework regions: FR1, FR2, FR3, and FR4.

[0045] The term "monospecific antibody" as used in this invention refers to an antibody that exhibits single binding specificity and affinity for a specific target (e.g., an epitope). This term includes "monoclonal antibody" or "monoclonal antibody composition," which in this invention refers to the preparation of an antibody or fragment thereof that is a single molecule. Antibodies can be complete immunoglobulins, such as IgA, IgG, IgM, or other 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 31A8, and 31A8 can specifically bind to any variant of the N protein or the novel coronavirus antigen.

[0046] The terms "antibody fragment," "antibody or antigen-binding fragment thereof," "antigen-active fragment of an antibody," and "multiple antibody fragments" used in this invention are interchangeable and refer to a portion of a complete antibody that includes an antigen-binding site or variable region. This portion includes the constant region heavy chain domain (such as CH2, CH3, or CH4, depending primarily 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 retaining only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0047] The term "novel coronavirus" as used in this invention refers to a single-stranded positive-sense RNA virus, belonging to the order Nematovirales, family Coronaviridae, subfamily Orthocoronaviruses. The novel coronavirus described in this invention is SARS-CoV-2, a type of severe acute respiratory syndrome-associated coronavirus (SARSr-Cov).

[0048] The term "identity" as used in this invention refers to the sequence similarity to the amino acid sequences used in this invention. Identity can be evaluated visually or using computer software. When 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. 1-8 and SEQ ID No. 10-17 provided by this invention, which have n (n = 1-10) amino acid substitutions, deletions, or additions to the amino acid sequences shown in SEQ ID No. 1-8 and SEQ ID No. 10-17, or have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 1-8 and SEQ ID No. 10-17, are all within the scope of protection of this invention.

[0049] The term "expression vector" as used in this invention refers to bacterial plasmids, bacteriophages, yeast extracts, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, lentiviruses, or other vectors well-known in the art, such as plasmid pcDNA, plasmid pTT3, plasmid pEF, plasmid pFUSE, 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 regulatory elements. In specific embodiments of this invention, the pFUSE series of vectors are used.

[0050] 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 single-stranded and / or double-stranded linear or circular form, or a mixture thereof. Thus, the nucleic acids of this 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 means substantially 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 separated from at least 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials 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.

[0051] 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 interchangeable. These can be used, or have been used, as recipients of recombinant vectors or other transfected DNA, and include the original progeny of already transfected primitive cells. The expression vector is used to express the antibody against the N protein described in this invention or the antibody or its functional fragment described in this invention. Useful host cells 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, and protozoan host cells. Preferably, mammalian host cells include, but are not limited to, CHO cells, COS cells, Vero cells, SP2 / 0 cells, HEK293 cells, and Expi293F. TM Cells or NIH3T3 cells. Preferred fungal host cells include Aspergillus, yeast, Pichia pastoris, and Candida. In embodiments of the present invention, the host cell is Expi293F. TM cell.

[0052] 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 can provide a function (such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient in the composition) for any composition of the anti-N protein antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell, etc. Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, disintegrants, lubricants, flow aids, enhancers, adsorbents, chelating agents, buffers, sweeteners, etc.

[0053] As used in this invention, the terms "transformation" and "transfection" refer to the process of introducing exogenous DNA into a host. The methods of transformation and transfection include any method of introducing nucleic acids into host cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride precipitation, polyethylene glycol method, and cationic liposome method.

[0054] As used in this invention, the term "treatment" refers to the reduction of symptoms of a disease following infection with the novel coronavirus, resulting from exposure (e.g., administration of medication) to the anti-N protein antibody, antigen-binding fragment, nucleic acid molecule, expression vector, host cell, pharmaceutical composition, etc., of this invention, compared to the absence of exposure. In one specific embodiment, the novel coronavirus infection-related disease is COVID-19.

[0055] The terms "biological subject," "subject," and "individual" used in this invention are interchangeable and refer to animal subjects, particularly vertebrate subjects, preferably mammalian subjects. Suitable vertebrate subjects within the scope of this invention include, but are not limited to, any member of the subphylum Chordata, including primates (such as humans, apes, monkeys, and chimpanzees), rodents (such as mice, rats, and guinea pigs), lagomorphs (such as domestic rabbits and hares), and sheep (such as sheep). Preferred subjects are primates (such as humans, apes, monkeys, and chimpanzees) and rodents (such as mice, rats, and guinea pigs).

[0056] Assays provided in product form can involve detecting and measuring the N protein in relatively small amounts of subject samples, reducing the complexity and cost of the assay. Any form of sample assay described in this invention capable of detecting the N protein of the novel coronavirus in a sample can be tested. Typically, the assay quantifies antibodies in the sample to a certain level, such as whether the concentration or amount is above or below a predetermined threshold. Such kits include colloidal gold immunoassay kits, chemiluminescence immunoassay kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits, fluorescence immunoassay kits, and microfluidic chips. It is important to note that the detection methods and kits described in this invention are applicable not only to the detection of patient samples but also to the non-diagnostic detection of environmental samples, including but not limited to water samples, food samples, air samples, animal cell samples, soil samples, and industrial samples.

[0057] The term "sample" as used in this invention is used in its broadest sense. In some cases, a sample includes cells, organisms, and biological and environmental samples. Among them, biological samples are biological materials or compositions obtained from animals (including humans) and intended to be found therein, including but not limited to bone marrow, urine, cerebrospinal fluid, nucleic acids, blood, serum, tissues, and their purified or filtered forms; environmental samples include but are not limited to water samples, food samples, air samples, animal cell samples, soil samples, and industrial samples.

[0058] 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.

[0059] 1. Preparation of the novel coronavirus N protein

[0060] The gene sequence of the SARS-CoV-2 N protein was synthesized into a PUC vector, and then the target gene was cloned into the pET-32a vector. The plasmid was extracted, identified by double enzyme digestion, and sequenced for alignment. The constructed prokaryotic expression plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium, incubated at 37°C until the OD value reached 0.5–1, and then inducing expression overnight at 16°C after the addition of IPTG. The bacterial cells were collected, sonicated, centrifuged, and the supernatant was collected. The protein purified by Ni column affinity chromatography was dialyzed and concentrated to obtain a high concentration of SARS-CoV-2 N protein.

[0061] 2. Obtaining hybridoma cells

[0062] Step 1: Immunize mice with N protein

[0063] Three female Balb / c mice aged 5-8 weeks were used. After one week of acclimatization, the mice were given the first immunization by mixing 50 μg of N protein with Freund's complete adjuvant at a 1:1 ratio, emulsifying it completely, and then injecting it subcutaneously at four points. Three weeks later, the mice were given the second immunization by mixing 50 μg of N protein with Freund's incomplete adjuvant at a 1:1 ratio, emulsifying it completely, and then injecting it subcutaneously at four points. Three weeks later, the mice were given the third immunization by mixing 50 μg of N protein with an appropriate amount of physiological saline and then injecting it intraperitoneally.

[0064] Step 2: ELISA detection of antibody production in immunized mice

[0065] Three weeks after the third immunization, tail blood was collected from mice, and serum was collected by centrifugation. ELISA was used to detect antibody production in immunized mice. The specific steps were as follows: N protein was coated onto the ELISA plate (100 ng / well) and incubated overnight at 4°C; PBST was used to wash three times for 2 min each time, and the plate was then patted dry; 3% BSA was used to fill the wells and the plate was blocked at 37°C for 2 h; PBST was used to wash three times for 2 min each time, and the plate was then patted dry; serum diluted serially according to a certain ratio (100 μL / well) was added and incubated at 37°C for 1 h; PBST was used to wash three times for 2 min each time, and the plate was then patted dry; goat anti-mouse IgG-HRP was added (100 μL / well) and incubated at 37°C for 40 min; PBST was used to wash three times for 2 min each time, and the plate was then patted dry; TMB chromogenic solution was added (100 μL / well) and incubated at room temperature (RT) for 10 min, then the incubation was stopped with stop solution. The absorbance was measured on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. The results are shown below. Figure 1 Compared to normal mice without immunization, the antibody titers of the three immunized mice all reached 1:1,024,000 or higher, indicating that all three immunized mice can be used for subsequent experiments.

[0066] Step 3: Cell Fusion

[0067] After resuscitation, SP2 / 0 myeloma cells were acclimated to 20% fetal bovine serum for one week. Three days prior to fusion, 50 μg of N protein was mixed with an appropriate amount of physiological saline and injected intraperitoneally to complete a booster immunization in mice (sample number 2). On the day of fusion, the mice were euthanized, and the spleens were aseptically harvested, ground, and washed twice with serum-free medium. The spleens were then mixed with SP2 / 0 cells at a ratio of 10:1, centrifuged at 1200 rpm for 10 min, and the supernatant was discarded. The bottom of the centrifuge tube was gently tapped to loosen the cell pellet. Within 30 seconds, 1 mL of 45% PEG solution preheated to 37°C was slowly added. After incubation at room temperature for 90 seconds, incomplete culture medium preheated to 37°C was slowly added, with 1 mL added in the first minute, 2 mL in the second minute, 3 mL in the third minute, and 4 mL in the fourth minute, until 25 mL was added. The mixture was then thoroughly mixed, centrifuged at 800 rpm for 6 min, and the supernatant was discarded. Add 40 mL of preheated (37°C) complete culture medium, mix well, and then add to a 96-well cell culture plate already inoculated with peritoneal macrophages. Incubate at 37°C in a 5% CO2 incubator. From day 2 to 7 post-fusion, supplement with HAT and HT every other day to screen fused cells.

[0068] Step 4: ELISA detection of positive hybridoma cells and cloning of hybridoma cells

[0069] 7-10 days after fusion, observe the growth of hybridoma cells. When the cells in the wells exceed 1 / 3 of the bottom, the secretion of hybridoma antibodies can be detected. The detection steps are as follows: Coat the ELISA plate with N protein, 100 ng / well, overnight at 4°C; wash 3 times with PBST for 2 min each time, and pat dry; block the wells with 3% BSA at 37°C for 2 h; wash 3 times with PBST for 2 min each time, and pat dry; add hybridoma cell supernatant, 100 μL / well, and incubate at 37°C for 1 h; wash 3 times with PBST for 2 min each time, and pat dry; add goat anti-mouse IgG-HRP, 100 μL / well, and incubate at 37°C for 40 min; wash 3 times with PBST for 2 min each time, and pat dry; add TMB chromogenic solution, 100 μL / well, incubate at RT for 10 min, and then stop the incubation with the stop solution. Measure the absorbance on a microplate reader. The measurement wavelength is 450 nm, and the reference wavelength is 630 nm. Clones with high OD values ​​were selected for subcloning and screened again by ELISA after 7-10 days. After three rounds of cloning, one positive clone was finally selected and named 31A8.

[0070] 3. Monoclonal antibody subtype identification

[0071] N protein was used to coat ELISA plates, 100 ng / well, and incubated overnight at 4°C. The plates were washed three times with PBST for 2 min each time, and then patted dry. 3% BSA was used to fill the wells, and the plates were blocked at 37°C for 2 h. The plates were washed three times with PBST for 2 min each time, and then patted dry. Hybridoma cell supernatant was added, 100 μL / well, and incubated at 37°C for 1 h. The plates were washed three times with PBST for 2 min each time, and then patted dry. HRP-labeled rabbit anti-mouse secondary antibody (IgG1, IgG2a, IgG2b, IgG3, IgM, kappa) was added, 100 μL / well, and incubated at 37°C for 40 min. The plates were washed three times with PBST for 2 min each time, and then patted dry. TMB chromogenic solution was added, 100 μL / well, and incubated at RT for 10 min. The incubation was terminated with stop solution, and the absorbance was measured using a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. The experimental results are as follows: Figure 2 As shown, the OD values ​​of 31A8 IgG1 and kappa were significantly higher than those of the negative control. Therefore, the heavy chain of the 31A8 monoclonal antibody is of the IgG type, and the light chain is of the kappa type.

[0072] 4. Sequencing of monoclonal antibody 31A8

[0073] Total RNA was extracted from hybridoma cells and cDNA was obtained by reverse transcription. The variable regions of the antibody heavy and light chains were amplified using the 5' RACE method. The amplified fragments were subcloned into the pEASY-Blunt vector, and the plasmid was extracted and sequenced. The antibody light and heavy chain sequences were obtained (SEQ ID No. 8-9 and 17-18), and the CDR region of the antibody amino acid sequence was labeled using the Kabat method.

[0074] 31A8 antibody light chain variable region (VL) gene sequence (SEQ ID No. 9):

[0075] ATGATGAGTCCTGCCCAGTTTCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGCTACACATTTTCTTCAGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAA

[0076] Amino acid sequence of the variable region of the light chain (VL) of the 31A8 antibody (SEQ ID No. 8):

[0077] MMSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQATHFLQTFGGGTKVEIK

[0078] Gene sequence of the variable region of the heavy chain (VH) of the 31A8 antibody (SEQ ID No. 18):

[0079] ATGGGATGGAGCTGGATCTTTCTCTTTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACCAAATACACCATGCACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGT GGATTGGAGGTATTAATCCTAACAATGGTGGTAATAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTGCAACGCCGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0080] 31A8 antibody heavy chain variable region (VH) amino acid sequence (SEQ ID No. 17):

[0081] MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFT KYTMHWVKQSHGKSLEWIGGINPNNGGNSYNQKFKGKATLTVDKSSSTAY MELRSLTSEDSAVYYCATPDYWGQGTTLTVSS.

[0082] 5. ELISA verification of the specificity of monoclonal antibody 31A8

[0083] A MERS-N protein expression vector was constructed and transformed into BL-21(DE3) competent cells. The cells were shaken and IPTG was used to induce expression. The cells were collected, sonicated, centrifuged, and the supernatant was collected and subjected to Ni column affinity chromatography. The obtained protein was dialyzed, concentrated, and the protein concentration was determined.

[0084] N protein, MERS-N protein, and BSA were coated onto ELISA plates at 100 ng / well and incubated overnight at 4°C. The plates were washed three times with PBST for 2 min each time and then patted dry. 3% BSA was applied to fill all wells and the plates were blocked at 37°C for 2 h. The plates were then washed three times with PBST for 2 min each time and then patted dry. 31A8 hybridoma cell supernatant was added at 100 μL / well and incubated at 37°C for 1 h. The plates were washed three times with PBST for 2 min each time and then patted dry. Goat anti-mouse IgG-HRP was added at 100 μL / well and incubated at 37°C for 40 min. The plates were washed three times with PBST for 2 min each time and then patted dry. TMB chromogenic solution was added at 100 μL / well and incubated at RT for 10 min. The incubation was terminated with stop solution, and the absorbance was measured using a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm.

[0085] The experimental results are shown in Table 1 below. Figure 3 As shown:

[0086] Table 1. ELISA validation of the specificity of monoclonal antibody 31A8

[0087]

[0088] It can be seen that monoclonal antibody 31A8 can specifically recognize the N protein of SARS-CoV-2 virus.

[0089] 6. Recombinant 31A8 monoclonal antibody and its effects

[0090] Step 1: Construction of antibody expression vector

[0091] The antibody heavy and light chain variable regions, identified by sequencing, were cloned into the antibody expression vectors pFUSE-CHIg-mG1 and pFUSE2-CLIg-mk, respectively, and labeled as 31A8mG1 and 31A8mk. After enzyme digestion and identification, the samples were sent for sequencing. Large quantities of correctly sequenced plasmids were extracted and used for cell transfection to prepare recombinant antibodies.

[0092] Step 2: ELISA verification of the effect of recombinant 31A8

[0093] The constructed recombinant antibody expression plasmids 31A8mG1, 31A8mk, 31A8mG1+31A8mk, and mG1+mk (empty vector control) were transfected into Expi293F culture cells cultured at transfection density. TMCells were cultured for 72 h. N protein was coated onto ELISA plates (100 ng / well) and incubated overnight at 4°C. The plates were washed three times with PBST for 2 min each time, and then patted dry. 3% BSA was applied to fill all wells, and the plates were blocked at 37°C for 2 h. The plates were washed three times with PBST for 2 min each time, and then patted dry. 100 μL of the transfected cell supernatant was added to each well, with hybridoma cell supernatant used as a positive control. The plates were incubated at 37°C for 1 h. The plates were washed three times with PBST for 2 min each time, and then patted dry. 100 μL of goat anti-mouse IgG-HRP was added to each well, and the plates were incubated at 37°C for 40 min. The plates were washed three times with PBST for 2 min each time, and then patted dry. 100 μL of TMB chromogenic solution was added to each well, and the incubation was stopped with stop solution after 10 min. The absorbance was measured using a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. The experimental results (Table 2) show that transfection with either the heavy chain 31A8mG1 or the light chain 31A8mk of the 31A8 antibody alone cannot recognize the N protein; however, co-transfection with the heavy and light chain recombinant expression plasmids 31A8mG1 and 31A8mk results in the detection of significant signals. This fully demonstrates the effectiveness of the 31A8 monoclonal antibody in recognizing N protein N on Expi293F. TM The cells successfully expressed the recombinant monoclonal antibody, which was secreted into the cell supernatant. This recombinant monoclonal antibody recognizes the N protein and exhibits biological activity.

[0094] Table 2. ELISA validation of the efficacy of recombinant 31A8 monoclonal antibody

[0095]

[0096]

[0097] Step 3: ELISA detection of the binding activity of recombinant monoclonal antibody 31A8

[0098] The constructed recombinant antibody expression plasmid 31A8mG1+31A8mk was transfected into Expi293F. TM After culturing cells for 72 h, the supernatant was collected and purified using protein A affinity chromatography to obtain recombinant monoclonal antibody 31A8, and its concentration was determined. N protein was used to coat ELISA plates (100 ng / well) and incubated overnight at 4°C; the plates were washed three times with PBST for 2 min each time, and then patted dry; 3% BSA was used to fill the wells and the plates were blocked at 37°C for 2 h; the plates were washed three times with PBST for 2 min each time, and then patted dry; serially diluted recombinant 31A8 was added and incubated at 37°C for 1 h; the plates were washed three times with PBST for 2 min each time, and then patted dry; 100 μL of goat anti-mouse IgG-HRP was added per well and incubated at 37°C for 40 min; the plates were washed three times with PBST for 2 min each time, and then patted dry; 100 μL of TMB chromogenic solution was added per well, and the incubation was stopped at RT for 10 min, followed by termination with stop solution. The absorbance was measured on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. Experimental results ( Figure 4 This indicates that the recombinant monoclonal antibody 31A8 can bind to the N protein well in a concentration-dependent manner, and EC50 is within the range specified. 50 It is 0.007 μg / mL.

[0099] In summary, the monoclonal antibody 31A8 provided in this invention has good binding activity with the novel coronavirus and high specificity, and can be used for clinical detection of the novel coronavirus SARS-CoV-2.

[0100] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2, characterized in that, It includes a light chain and a heavy chain, wherein the light chain belongs to kappa and the heavy chain belongs to IgG1; wherein: The amino acid sequences of the complementarity-determining regions LCDR1 and LCDR3 of the light chain variable region are shown in SEQ ID No. 1 and SEQ ID No. 3, respectively; the amino acid sequence of the complementarity-determining region LCDR2 of the light chain variable region is LVS. The amino acid sequences of the three complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, respectively.

2. The monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2 according to claim 1, characterized in that, The amino acid sequences of the light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 of this monoclonal antibody against the N protein of the novel coronavirus SARS-CoV-2 are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7, respectively. The amino acid sequences of the heavy chain variable region framework regions HFR1, HFR2, HFR3 and HFR4 of this monoclonal antibody against the N protein of the novel coronavirus SARS-CoV-2 are shown in SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16, respectively.

3. The monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2 according to claim 1 or 2, characterized in that, The light chain variable region of this monoclonal antibody against the N protein of the novel coronavirus has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 8; the heavy chain variable region of this monoclonal antibody against the N protein of the novel coronavirus has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.

17.

4. The monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2 according to claim 3, characterized in that, The light chain variable region of this monoclonal antibody against the N protein of the novel coronavirus has 95% sequence identity with the amino acid sequence shown in SEQ ID No. 8; the heavy chain variable region of this monoclonal antibody against the N protein of the novel coronavirus has 95% sequence identity with the amino acid sequence shown in SEQ ID No.

17.

5. A product for detecting novel coronavirus SARS-CoV-2 infection, characterized in that, include: a) A nucleic acid molecule encoding a monoclonal antibody against the N protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1 to 4; b) A recombinant expression vector comprising the nucleic acid molecule described in a); c) A host cell containing the recombinant expression vector described in b); d) A composition comprising at least two of a), b), and c) above.

6. The use of the monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4 in the preparation of products for detecting novel coronavirus SARS-CoV-2 infection, characterized in that, The products include testing reagents, kits, or test strips.

7. The use of the monoclonal antibody against the N protein of novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4, and the product for detecting novel coronavirus SARS-CoV-2 infection according to claim 5, in the preparation of products for diagnosing diseases related to novel coronavirus SARS-CoV-2 infection, characterized in that, The products include diagnostic reagents, kits, or diagnostic test strips; the disease associated with novel coronavirus SARS-CoV-2 infection is COVID-19.

8. The application as described in claim 6 or 7, characterized in that, The kits include: colloidal gold immunoassay kit, chemiluminescence immunoassay kit, radioimmunoassay kit, enzyme-linked immunosorbent assay kit, and fluorescence immunoassay kit.

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