Antibody or antigen-binding fragment thereof against novel coronavirus membrane protein and application thereof

Abstract

The present invention belongs to the field of antibody technology, and discloses an antibody or antigen-binding fragment thereof against novel coronavirus membrane protein and its application; the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; the heavy chain of the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region, which comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; the light chain of the antibody or antigen-binding fragment thereof comprises: a light chain variable region, which comprises CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region. The antibody or antigen-binding fragment thereof can be used to detect the presence or level of novel coronavirus membrane protein in a sample, detect novel coronavirus, and diagnose diseases related to novel coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the field of antibody technology, and specifically relates to an antibody or an antigen-binding fragment thereof against a novel coronavirus membrane protein and an application thereof. Background Art

[0002] Diseases caused by the novel coronavirus (SARS-CoV-2) are prevalent to varying degrees around the world. Rapid screening of infected people and blocking the transmission route are the most effective ways to control its spread, so it is particularly important to develop convenient, fast and accurate detection methods.

[0003] SARS-CoV-2 is a membrane-bound single-stranded RNA virus with four structural proteins, namely spike (S), membrane (M), envelope (E), and nucleocapsid (N). M protein is the most abundant structural protein in SARS-CoV-2, consisting of 222 amino acids and a molecular weight of 25-30 kDa. As a transmembrane glycoprotein, it has three main domains: an N-terminal glycosylated ectodomain, three transmembrane domains (TM1-TM3), and a C-terminal endodomain. M protein is the main component of the envelope and plays an important role in forming the viral envelope and maintaining its structural shape. M protein forms the envelope during the budding process of the virus, interacts with other structural proteins to stabilize the nucleocapsid and the inner core of the virus particle, and helps complete the assembly of the virus particle. M protein plays an important role in intracellular protein transport and regulation, and can induce specific humoral responses. Antigenic epitopes have been identified in the TM1 and TM2 regions, so M protein can be used as a potential immunogen in therapeutic applications.

[0004] At present, the detection methods of the new coronavirus include nucleic acid detection, antibody detection, and antigen detection. Although nucleic acid detection is the main method for detecting the new coronavirus, the method has high sensitivity, specificity, and accuracy. The disadvantage is that the detection conditions are high and time-consuming. Antibody detection is that the body can detect antibodies only after a certain period of time after viral infection. Antibodies may not be detected in the early stage of viral infection. In addition, people who have been vaccinated can also detect antibodies to the new coronavirus, and there are false positives, so antigen detection is more meaningful. Antigen detection is generally used in the acute infection period, that is, sample detection within 7 days of the onset of symptoms in suspected people. Antigen-positive results can be used for early diversion and rapid management of suspected people. The method is simple and convenient to operate, and the test results can be obtained within 15 to 20 minutes. Residents can complete sampling and self-testing at home. Compared with the "gold standard" of nucleic acid detection, although there is a certain probability of missed detection, compared with the high risk, high prevalence, and clustered infection faced during the detection process, antigen detection is still an effective detection method that can detect new coronavirus infection early.

[0005] There are three common rapid detection methods for novel coronavirus antigens: colloidal gold method, latex method, and fluorescent immunoassay. For general screening, the colloidal gold detection method has very low requirements for personnel, sites, and equipment, and is suitable for large-scale rapid detection. This method does not require special specimen processing, and can qualitatively detect novel coronavirus in nasal swab samples in vitro. The test results can be obtained by naked eye observation within 15 minutes. People under home quarantine observation, close contacts, and secondary close contacts can complete the test by themselves to avoid cross-infection between doctors and patients. It breaks through the limitations of existing detection technologies on personnel and places, and has the advantages of strong specificity, high sensitivity, simplicity and rapidity, naked eye interpretation, and high stability. In order to achieve rapid detection of novel coronavirus antigens, it is of great significance to study and obtain monoclonal antibodies against the novel coronavirus M protein. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes an antibody or an antigen-binding fragment thereof against a novel coronavirus membrane protein, which can be used to detect the level of novel coronavirus membrane protein in a sample.

[0007] The invention also provides a recombinant protein.

[0008] The present invention also proposes a biological material related to the antibody or antigen-binding fragment thereof against the novel coronavirus membrane protein described in the first aspect of the present invention or the recombinant protein belonging to the second aspect.

[0009] The present invention also provides a conjugate.

[0010] The present invention also provides a solid phase carrier.

[0011] The present invention also provides an application.

[0012] The invention also provides a product.

[0013] The present invention also provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the recombinant protein as described in the second aspect.

[0014] According to the first aspect of the present invention, an antibody or an antigen-binding fragment thereof against a novel coronavirus membrane protein (M protein) is provided, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain:

[0015] The heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0016] a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of said heavy chain variable region;

[0017] The light chain of the antibody or antigen-binding fragment thereof comprises:

[0018] a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of said light chain variable region;

[0019] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, the amino acid sequence of CDR-L2 is: KVS, and the CDRs are defined according to the IMGT definition scheme; or

[0020] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:17, respectively, and the CDRs are defined according to the Kabat definition scheme; or

[0021] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 17, respectively, and the CDRs are defined according to the Chothia definition scheme; or

[0022] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and the CDRs are defined according to the Contact definition scheme.

[0023] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises:

[0024] a1) SEQ ID NO: 2; or

[0025] a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 2 and which has the same function as the protein shown in SEQ ID NO: 2; or

[0026] a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homologous to SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2.

[0027] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof comprises:

[0028] b1) SEQ ID NO: 4; or

[0029] b2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 4 and which has the same function as the protein shown in SEQ ID NO: 4; or

[0030] b3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homologous to SEQ ID NO:4 and has the same function as the protein shown in SEQ ID NO:4.

[0031] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, and a multispecific antibody.

[0032] In some embodiments of the present invention, the heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region.

[0033] In some embodiments of the present invention, the light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.

[0034] According to a second aspect of the present invention, a recombinant protein is provided, comprising: the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0035] Optional tag sequence to facilitate expression and / or purification.

[0036] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, and a FLAG tag.

[0037] According to the third aspect of the present invention, a biological material related to the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention is provided, wherein the biological material comprises at least one of h1) to h8):

[0038] h1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or the recombinant protein according to claim 3;

[0039] h2) an expression cassette comprising the nucleic acid molecule described in h1);

[0040] h3) a vector comprising the nucleic acid molecule described in h1);

[0041] h4) a vector comprising the expression cassette described in h2);

[0042] h5) a transgenic cell line comprising the nucleic acid molecule described in h1);

[0043] h6) a transgenic cell line comprising the expression cassette described in h2);

[0044] h7) a transgenic cell line comprising the vector described in h3);

[0045] h8) A transgenic cell line comprising the vector described in h4).

[0046] In some embodiments of the invention, the transgenic cell line does not contain reproductive material.

[0047] In some embodiments of the present invention, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof against the novel coronavirus membrane protein described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the anti-novel coronavirus membrane protein described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the anti-novel coronavirus membrane protein described in the first aspect of the present invention.

[0048] In some preferred embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the anti-novel coronavirus membrane protein described in the first aspect of the present invention comprises:

[0049] a211) the nucleotide sequence as shown in SEQ ID NO: 1; or

[0050] a212) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 1 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 1; or

[0051] a213) has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology with SEQ ID NO:1, and has a nucleotide sequence with the same function as the nucleic acid molecule shown in SEQ ID NO:1.

[0052] In some preferred embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the anti-novel coronavirus membrane protein described in the first aspect of the present invention comprises:

[0053] a221) the nucleotide sequence shown in SEQ ID NO: 3; or

[0054] a222) a nucleotide sequence in which one or more nucleotides in SEQ ID NO: 3 are substituted and / or deleted and / or added, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 3; or

[0055] a223) has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology with SEQ ID NO:3, and has a nucleotide sequence with the same function as the nucleic acid molecule shown in SEQ ID NO:3.

[0056] According to a fourth aspect of the present invention, a conjugate is provided, comprising: at least one of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention;

[0057] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein.

[0058] In some embodiments of the invention, the detectable label is a fluorescent or luminescent label.

[0059] In some preferred embodiments of the present invention, the detectable marker is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.

[0060] In some embodiments of the present invention, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188.

[0061] In some embodiments of the present invention, the drug is other drugs for preventing or treating novel coronavirus infection and / or diseases related to novel coronavirus infection (such as antiviral drugs: favipiravir, remdesivir, or interferon, etc.).

[0062] In some embodiments of the present invention, the disease associated with novel coronavirus infection comprises novel coronavirus infection.

[0063] According to the fifth aspect of the present invention, a solid phase carrier is proposed, the surface of which is coupled with the antibody or its antigen-binding fragment against the novel coronavirus membrane protein described in the first aspect of the present invention and / or the recombinant protein described in the second aspect.

[0064] According to the sixth aspect of the present invention, the use of the antibody or antigen-binding fragment thereof against the novel coronavirus membrane protein as described in the first aspect of the present invention, the recombinant protein as described in the second aspect, the biomaterial as described in the third aspect, the conjugate as described in the fourth aspect, and / or the solid phase carrier as described in the fifth aspect in the preparation of a product is proposed.

[0065] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0066] In some preferred embodiments of the present invention, the drug has at least one of the functions i1) to i2):

[0067] i1) Prevention and treatment of novel coronavirus infection;

[0068] i2) Prevent and treat diseases related to novel coronavirus infection.

[0069] In some preferred embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3):

[0070] j1) Detecting the presence or level of novel coronavirus membrane protein in a sample;

[0071] j2) Detection of novel coronavirus;

[0072] j3) Diagnosis of diseases related to novel coronavirus infection.

[0073] In some more preferred embodiments of the present invention, the disease associated with novel coronavirus infection comprises novel coronavirus infection.

[0074] According to a seventh aspect of the present invention, a product is provided, the product comprising at least one of k1) to k4):

[0075] k1) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0076] k2) the recombinant protein according to the second aspect of the present invention;

[0077] k3) the conjugate according to the fourth aspect of the present invention;

[0078] k4) The solid phase carrier described in the fifth aspect of the present invention.

[0079] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0080] In some preferred embodiments of the present invention, the drug has at least one of the functions i1) to i2):

[0081] i1) Prevention and treatment of novel coronavirus infection;

[0082] i2) Prevent and treat diseases related to novel coronavirus infection.

[0083] In some preferred embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3):

[0084] j1) Detecting the presence or level of novel coronavirus membrane protein in a sample;

[0085] j2) Detection of novel coronavirus;

[0086] j3) Diagnosis of diseases related to novel coronavirus infection.

[0087] In some more preferred embodiments of the present invention, the disease associated with novel coronavirus infection comprises novel coronavirus infection.

[0088] According to the eighth aspect of the present invention, a method for preparing an antibody or an antigen-binding fragment thereof against the novel coronavirus membrane protein as described in the first aspect of the present invention or the recombinant protein as described in the second aspect is proposed, which is obtained by culturing the transgenic cell line described in the third aspect of the present invention.

[0089] The present invention has at least the following beneficial effects:

[0090] The present invention provides an antibody or an antigen-binding fragment thereof against the novel coronavirus membrane protein. The antibody or the antigen-binding fragment thereof has good specificity for the novel coronavirus and / or the membrane protein of the novel coronavirus, and can be used to detect the presence or level of the novel coronavirus membrane protein in a sample, detect the novel coronavirus, and diagnose diseases related to novel coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0092] Figure 1 The result of SDS-PAGE of the eluted sample in Example 1 is shown below: wherein M is a protein molecular weight standard (Marker), 1 is a purified novel coronavirus M protein;

[0093] Figure 2 This is a graph showing the results of the serum titer test of mice after immunization in Example 2;

[0094] Figure 3 This is a graph showing the results of the titer test of mouse ascites after injection of cell line S9 in Example 3;

[0095] Figure 4 The figure is the result of SDS-PAGE of the purified novel coronavirus M protein monoclonal antibody S9 in Example 3: wherein M is a protein molecular weight standard (Marker), and 1 is the purified novel coronavirus M protein monoclonal antibody S9;

[0096] Figure 5 This is a graph showing the results of specific detection of the novel coronavirus M protein monoclonal antibody S9 in Example 3. DETAILED DESCRIPTION

[0097] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0098] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0099] Example 1 Obtaining the novel coronavirus M recombinant protein

[0100] In this example, a recombinant protein of the novel coronavirus M was prepared. The specific preparation method includes the following steps:

[0101] 1. Construction of recombinant expression vector

[0102] Using the cDNA sequence of SARS-CoV-2 as a template, primers were designed and synthesized according to the NCBI sequence number (No. MT123290.1), and the gene sequence of SARS-CoV-2M was amplified (the amplified product was 26529-27194 bp of MT123290.1). The product was identified by agarose gel electrophoresis, and the vector pMAL-c6T and the PCR amplification product were double-digested with NotI and BamHI. After agarose gel electrophoresis, the target gene and the vector large fragment were recovered by a gel recovery kit, connected in a conventional manner, transformed into Escherichia coli BL21 (DE3) competent cells, and a single clone was picked and expanded for culture. After PCR amplification, restriction digestion and sequencing, the expression vector pMAL-c6TM was obtained.

[0103] 2. Expression and purification of SARS-CoV-2M protein

[0104] After the positive BL21 (DE3) clone bacteria containing the recombinant expression vector pMAL-c6TM were identified and amplified, the plasmid was extracted and transformed according to the conventional method. A single colony was picked and expanded in LB medium containing ampicillin resistance. When the bacterial solution concentration reached A600 of 0.5, 0.1mM IPTG, 200rpm, and 16°C were used to induce expression for 18h. The bacteria were collected by centrifugation at 4°C and 4000rpm for 20min, resuspended and washed with 20mL PBS, and Binding buffer was added to ice-bath ultrasonic disruption for 3min. The ultrasonic power was 30W. After ultrasonication for 2s, the ultrasonication was repeated at intervals of 2s until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4°C and 12000rpm for 20min for purification.

[0105] The target protein (M recombinant protein) was purified by nickel column affinity chromatography: 50% NI-NTA was loaded on the column, 4 mL of deionized water was used to wash the column, and then 5 mL of Binding buffer was added to balance the column. The sample was loaded, and the lysate containing the M recombinant protein was added to the column. The flow-through solution was collected and loaded again. The unbound impurities were washed with Binding Buffer, and 20 mL of Washing buffer was added to wash the impurities. The protein was eluted with 250 mM imidazole Elution buffer. The eluted sample was subjected to SDS-PAGE electrophoresis to detect the purity of the sample. The results are shown in the figure. Figure 1 As shown: The purity of M protein obtained by affinity chromatography is relatively high. The concentration of purified M recombinant protein is determined by BCA method.

[0106] Example 2 Establishment of a novel coronavirus M protein monoclonal antibody cell line

[0107] This example prepares a novel coronavirus M protein monoclonal antibody cell line, and the specific preparation method includes the following steps:

[0108] 1. Mouse Immunization

[0109] The M recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (the first immunization day was counted as the first day), Freund's complete adjuvant and the immunogen were emulsified at a volume ratio of 1:1, with a dose of 100 μg / mouse, and 5 immunization points were subcutaneously injected on the back and abdomen; on the 15th day, a second immunization was performed, and Freund's incomplete adjuvant and the immunogen were emulsified at a volume ratio of 1:1, with a dose of 50 μg / mouse, and the immunization method was the same as before; on the 29th day, a third immunization was performed, and the immunization method was the same as the second immunization; on the 36th day, a trace amount of tail blood was collected for ELISA determination, and the antibody titer was greater than 1:10000, and then antigen impact immunization and spleen cell fusion could be performed. If the antibody titer was less than 1:10000, four immunizations were performed on the 43rd day in the manner of three immunizations; 3 days before cell fusion, intraperitoneal impact immunization was performed, and 100 μg of the immunogen was directly injected intraperitoneally without adjuvant.

[0110] 2. Mouse serum titer detection

[0111] (1) Antigen coating: The M recombinant protein obtained in Example 1 was used as a coating source and adjusted to 1 μg / mL in the coating buffer. 100 μL was added to each well of the ELISA plate and incubated at 4° C. overnight.

[0112] (2) Washing: On the second day, discard the liquid in the wells, pat dry, and wash twice with PBST in a plate washer.

[0113] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 1 h.

[0114] (4) Preparation of serum (primary antibody): Blood was collected by tail cutting of mice, and serum from non-immunized mice was used as a negative control.

[0115] (5) Adding primary antibody: Use blocking solution to dilute the serum samples to be tested in multiples. Use non-immune mouse serum at a dilution of 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37°C for 1 h, discard the liquid in the wells, pat dry, wash twice with PBST, and pat dry.

[0116] (6) Add enzyme-labeled secondary antibody: dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking solution, 100 μL / well, incubate at 37°C for 1 h, discard the liquid in the well, and pat dry.

[0117] (7) Color development and colorimetry: Add 50 μL / well of TMB colorimetric solution, color at 37°C in the dark for 15 min, add 100 μL / well of 2M sulfuric acid to terminate the reaction, and measure the A value at 450 nm using an enzyme marker. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well is P / N ≥ 2.1 is the titer / titer of the serum. Mice with a titer exceeding 1:100,000 are prepared for the next step of fusion. The results are as follows: Figure 2 As shown, the reaction of recombinant M protein with immune serum was identified by indirect ELISA, and the titer of mouse serum was 1:160,000.

[0118] 3. Cell Fusion

[0119] Aseptically take spleen cells from immunized mice and mix them with SP2 / 0 mouse myeloma cells in a ratio of about 5:1 in a 50mL centrifuge tube, wash twice with culture medium and discard the supernatant, slowly add 0.9mL preheated PEG-1500 within 50s to disperse the cells as evenly as possible in PEG, and let it stand for 1min. Slowly drop 20mL of serum-free DMEM culture medium preheated at 37℃, add 2mL in the first two minutes, add 18ml in the last two minutes, add all within 4min, let it stand for 3min, centrifuge at 800rpm for 5min, discard the supernatant, add preheated FBS and HAT culture medium, mix gently by blowing, transfer to a 96-well culture plate, 200μL per well, and culture in an incubator.

[0120] 4. Screening of positive hybridoma cells

[0121] On the tenth day after cell fusion, when the fused cells covered more than 50% of the wells, the hybridoma cells were screened by the indirect ELISA method.

[0122] 5. Subcloning of positive hybridoma cells

[0123] The positive wells obtained by screening were subcloned by limiting dilution method. The number and position of cell clusters in the positive wells were observed under an inverted microscope. The cell clusters were aspirated with a 200μL pipette tip in the clean bench, and the number of cells was diluted to 1 to 2 cells in 100μL. The prepared feeder cells were taken out, and the diluted cells were added to a 96-well plate, 100μL per well, marked, and cultured at 37℃ in a 5% CO2 incubator for 9 days. After 3 subclones, until there was 1 cell per well, the titer of the cell supernatant was detected by indirect ELISA method, and the positive rate reached 100%. The culture was expanded and the cell line S9 was preserved.

[0124] Example 3 Preparation and identification of monoclonal antibodies against the novel coronavirus M protein

[0125] In this example, a novel coronavirus M protein monoclonal antibody was prepared and subclass identification was performed. The specific steps are as follows:

[0126] 1. Preparation of ascites fluid of monoclonal antibodies against the novel coronavirus M protein and determination of ascites fluid titer

[0127] 12-16 week old BALB / c female mice were intraperitoneally injected with 0.5 mL of sterilized liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of the cell suspension of the cell line S9 preserved in Example 2 (5×10 5 =100 / mouse, the day of injection of cell suspension was counted as day 1); on day 7, the abdominal cavity of the mouse was obviously swollen, and the ascites was collected and centrifuged at 3000rpm for 20min to remove the fat tissue, and the supernatant was aspirated and stored at -20℃ for later use. The titer of ascites was determined by indirect ELISA method, and the results were as follows Figure 3 Shown: Ascites antibody titer 1:1280000.

[0128] 2. Purification of Monoclonal Antibodies

[0129] Ascites was collected, and the novel coronavirus M protein monoclonal antibody S9 was purified by caprylic acid-ammonium sulfate precipitation method, as follows: ascites was taken, centrifuged at 4°C and 12000rpm for 5min, the supernatant was taken, 2 volumes of 0.06M acetate buffer (pH 4.0) were added, and the pH value was adjusted to 4.5; 33μL of caprylic acid was added per milliliter of ascites, stirred at room temperature for 30min, and allowed to stand at 4°C for 1h to fully precipitate the impurities, centrifuged at 4°C and 1000g for 30min, 0.277g of ammonium sulfate powder was added per milliliter of supernatant, the beaker was placed on a magnetic stirrer, stirred for 1h, centrifuged at 4°C and 10000g for 20min, the supernatant was discarded, the precipitate was dissolved with PBS, and the purity of the monoclonal antibody was identified by SDS-PAGE. The results are as follows Figure 4 As shown: High-purity novel coronavirus M protein monoclonal antibody S9 was obtained.

[0130] 3. Identification of monoclonal antibody types and subclasses

[0131] The experimental operation was carried out according to the instructions of the mouse subtype identification kit. After identification, the type and subclass of the novel coronavirus M protein monoclonal antibody S9 were IgG1 and kappa light chain.

[0132] At the same time, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to sequence the monoclonal antibody S9 produced by the cell line S9 preserved in Example 2 (i.e., the novel coronavirus M protein monoclonal antibody S9 purified in step 2 of Example 3), and the results are as follows:

[0133] The nucleotide sequence of the heavy chain variable region of the novel coronavirus M protein monoclonal antibody S9 is: 5'-CAAGTTACTCTA AAAGAGTCTGGCCCTGGGATTTTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTCTGGGTGTTGGCTGGTTTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCTCACATTTGGTGGGATGATGATACGTACTATAATCCAACCCTGCAGAGCCGGCTCACAGTCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAATATCACCAGTGTGGACACTGCAGATAATGCCACTTACTACTGTGCTCAAAGTCCAAACTGGGGCCACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA-3' (SEQ ID NO: 1),

[0134] The corresponding amino acid sequence is: QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGLGVGWFRQPSGKGLEWLAHIWWDDDTYYNPTLQSRLTVSKDTSRNQVFLNITSVDTADNATY YCAQSPNWGHWGQGTSVTVSS (SEQ ID NO: 2);

[0135] The nucleotide sequence of the light chain variable region is: 5'-GATGTTGTGATGACCCAAACTCCACTCTCCCT GCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCTGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAATCGATTTTCTGGGGT CCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCATCAGAGTGGAGGCTGAAGATCTGGGAGTTTATTTCTGCTCTCAAACTACACGTGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA-3'(SEQID NO: 3),

[0136] The corresponding amino acid sequence is: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYL HWYLQKPGLSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIIRVEAEDLGVYFCSQTT RVPYTFGGGTKLEIK (SEQ ID NO: 4).

[0137] The CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the novel coronavirus M protein monoclonal antibody S9 defined by different definition schemes are shown in Table 1.

[0138] Table 1 CDR-H1, CDR-H2, CDR-H3 defined in different definition schemes in the heavy chain variable region

[0139]

[0140] The CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the novel coronavirus M protein monoclonal antibody S9 defined by different definition schemes are shown in Table 2.

[0141] Table 2 CDR-L1, CDR-L2, CDR-L3 defined in different definition schemes in the light chain variable region

[0142]

[0143]

[0144] 4. Specificity detection of monoclonal antibodies

[0145] Inactivated 2019-nCoV, human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), influenza A (H1N1) virus, influenza A (H3N2) virus, influenza B virus (Victoria), and influenza B virus (Yamagata) were used as antigens (the inactivated viruses tested were all reference materials obtained from the China Food and Drug Inspection Institute or the Chinese Center for Disease Control and Prevention);

[0146] The ELISA plate was coated under the same conditions, the monoclonal antibody S9 purified in step 2 of Example 3 was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The A 450 nm The specificity of the monoclonal antibody was detected by indirect ELISA (see Example 2). The results are shown in Figure 2. Figure 5As shown, it can be seen that the monoclonal antibody S9 produced by the cell line S9 preserved in Example 2 (i.e., the novel coronavirus M protein monoclonal antibody S9 purified in step 2 of Example 3) has good specificity and can be used to detect the novel coronavirus and / or diseases caused by the novel coronavirus.

[0147] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antibody or antigen-binding fragment thereof against the membrane protein of the novel coronavirus SARS-CoV-2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; The heavy chain of the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of said heavy chain variable region; The light chain of the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of said light chain variable region; The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, the amino acid sequence of CDR-L2 is: KVS, and the CDRs are defined according to the IMGT definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:17, respectively, and the CDRs are defined according to the Kabat definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 17, respectively, and the CDRs are defined according to the Chothia definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and the CDRs are defined according to the Contact definition scheme.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: a1) SEQ ID NO: 2; or a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 2 and which has the same function as the protein shown in SEQ ID NO: 2; or a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homologous to SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2; The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof comprises: b1) SEQ ID NO: 4; or b2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 4 and which has the same function as the protein shown in SEQ ID NO: 4; or b3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homologous to SEQ ID NO:4 and has the same function as the protein shown in SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

4. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region; and / or The light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.

5. A recombinant protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; and An optional tag sequence to facilitate expression and / or purification.

6. A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5, wherein the biological material comprises at least one of h1) to h8): h1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) A transgenic cell line comprising the vector described in h4).

7. A solid phase carrier, the surface of which is coupled with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and / or the recombinant protein according to claim 5.

8. Use of at least one of (1) to (4) in the preparation of a product; (1) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; (2) The recombinant protein according to claim 5; (3) The biomaterial according to claim 6; (4) The solid phase carrier according to claim 7; The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; The drug has the function of preventing and treating SARS-CoV-2 infection: The reagent, detection plate, detection chip or kit has at least one function among j1) to j2): j1) detecting the presence or level of SARS-CoV-2 membrane protein in a sample; j2) Detection of SARS-CoV-2.

9. A product, comprising at least one of k1) to k3): k1) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; k2) The recombinant protein according to claim 5; k3) The solid phase carrier according to claim 7.

10. The product according to claim 9, characterized in that: The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

11. The product according to claim 10, characterized in that The drug has the function of preventing and treating SARS-CoV-2 infection.

12. The product according to claim 10, characterized in that The reagent, detection plate, detection chip or kit has at least one function among j1) to j2): j1) detecting the presence or level of SARS-CoV-2 membrane protein in a sample; j2) Detection of SARS-CoV-2.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the recombinant protein according to claim 5, which is obtained by culturing the transgenic cell line according to claim 6.

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