6G3 monoclonal antibody and application thereof

Abstract

The invention discloses a 6G3 monoclonal antibody and application thereof, the 6G3 monoclonal antibody is used for specifically binding a 6HB structural domain in a spike protein S2 subunit of SARS-CoV-2, the 6G3 monoclonal antibody comprises VHCDR1-3 of a heavy chain variable region and VLCDR1-3 of a light chain variable region, the amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are respectively shown as SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 are respectively shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6. The invention further discloses a preparation method of the 6G3 monoclonal antibody. The 6G3 monoclonal antibody can neutralize SARS-CoV-2 infection, and has important application value in serological detection of new coronavirus, preparation of a reagent for new coronavirus infection detection and a reagent for new coronavirus antigen or antibody detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a 6G3 monoclonal antibody and its uses. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) belongs to the coronavirus family, specifically the β-coronavirus genus. It is a single-stranded RNA virus with a genome sequence of 29903 bp. SARS-CoV-2 contains an envelope structure, primarily composed of four structural proteins. Among them, the S2 subunit of the spike protein (S) can form a novel thermostable trimeric domain six-helix bundle (6HB), thereby shortening the distance between the viral membrane and the target cell membrane. This is an important form of viral-target cell membrane binding. Currently, various drugs and vaccines are used for the prevention and supportive treatment of SARS-CoV-2 infection, but there is still no specific drug for targeted treatment. Convalescent plasma contains high concentrations of specific antigen-neutralizing antibodies. When infused into patients, these antibodies can neutralize the SARS-CoV-2 pathogen and mediate an effective immune response. Currently, most neutralizing antibodies target the S1 subunit, and the high variability of S1 limits the application of such monoclonal antibodies. Developing monoclonal antibody drugs with high neutralizing activity and a stable targeting structure with broad-spectrum applications will provide an effective treatment for patients infected with COVID-19, reduce mortality, and protect patient lives. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the present invention aims to provide a 6G3 monoclonal antibody and its uses.

[0004] The specific technical solution of this invention is as follows:

[0005] The first aspect of this invention provides a 6G3 monoclonal antibody or an antibody fragment thereof, wherein the 6G3 monoclonal antibody is used to specifically bind to the 6HB domain in the S2 subunit of the SARS-CoV-2 spike protein, which includes a V-region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively;

[0006] The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

[0007] Furthermore, the SARS-CoV-2 is at least one of the SARS-CoV-2 mutant strain B1.351 and SARS-CoV-2OmicronXBB.1.5.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the 6G3 monoclonal antibody is shown in SEQ ID NO.7.

[0009] Preferably, the amino acid sequence of the light chain variable region of the 6G3 monoclonal antibody is shown in SEQ ID NO.8.

[0010] A second aspect of the present invention provides a polynucleotide sequence encoding the 6G3 monoclonal antibody or an antibody fragment thereof.

[0011] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the 6G3 monoclonal antibody is shown in SEQ ID NO.9;

[0012] And / or, the nucleotide sequence encoding the light chain variable region of the 6G3 monoclonal antibody of claim 1 is shown in SEQ ID NO. 10.

[0013] A third aspect of the present invention provides a biomaterial expressing the 6G3 monoclonal antibody or an antibody fragment of the monoclonal antibody, or containing the polynucleotide sequence described above, wherein the biomaterial comprises at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

[0014] The fourth aspect of this invention provides the 6G3 monoclonal antibody described above:

[0015] (1) Prepare reagents for detecting and / or diagnosing SARS-CoV-2 infection;

[0016] (2) Preparation of reagents that specifically bind to the Spike protein of SARS-CoV-2 virus;

[0017] (3) To prepare drugs for the prevention and / or treatment of SARS-CoV-2 virus infection;

[0018] (4) Prepare drugs for the prevention and / or treatment of symptoms caused by SARS-CoV-2 virus infection;

[0019] (5) Prepare reagents for detecting SARS-CoV-2 antibodies.

[0020] A fifth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the 6G3 monoclonal antibody or an antibody fragment thereof.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a 6G3 monoclonal antibody and demonstrates its application in recognizing and binding to the SARS-CoV-2 6HB antigen. Furthermore, this invention provides the application of the 6G3 monoclonal antibody binding to the SARS-CoV-2 Spike protein and demonstrates its ability to recognize the SARS-CoV-2 Spike protein for SARS-CoV-2 virus-related detection. This invention screened and obtained a hybridoma cell line capable of efficiently and stably secreting and expressing a SARS-CoV-2 6HB monoclonal antibody, as well as the SARS-CoV-2 6HB monoclonal antibody secreted by it. The monoclonal antibody of this invention exhibits high neutralizing activity; a concentration of 1.5 nM or less can inhibit more than 50% of SARS-CoV-2 pseudovirus activity, representing the best neutralizing activity reported so far for SARS-CoV-2 monoclonal antibodies. The monoclonal antibody produced and expressed by the monoclonal cell line of this invention has high titer and good specificity. Furthermore, the monoclonal cell line of this invention is produced using mouse ascites fluid, and the monoclonal antibody acquisition process is simple to operate, can be artificially intervened, and has relatively low cost. It is easy to express, collect, and purify, and the purity can reach more than 90%. The hybridoma cell line or SARS-CoV-26HB monoclonal antibody provided by this invention can specifically react with the live SARS-CoV-2 virus, and has important application value in the serological detection of SARS-CoV-2 and in the preparation of reagents or drugs for the diagnosis, prevention, or treatment of SARS-CoV-2 infection and in the preparation of reagents for the detection of SARS-CoV-2 antigens or antibodies.

[0023] The development of the 6G3 monoclonal antibody in this invention provides experimental evidence for clinical research on drugs for treating SARS-CoV-2 infection and related symptoms. Specifically:

[0024] This invention first evaluated the affinity of the 6G3 monoclonal antibody for the 6HB antigen and the Spike protein. Further, this invention selected the full-length Spike protein and its S2 subunit. Enzyme-linked immunosorbent assay (ELISA) showed that the EC50 of the 6G3 monoclonal antibody binding to the full-length Spike protein was 27.83 ng / mL, and the EC50 binding to the S2 subunit was 46.52 ng / mL, revealing that the 6G3 monoclonal antibody can bind to the Spike protein, and the binding site may be located on the S2 subunit.

[0025] In the affinity evaluation of 6HB antigen, full-length Spike protein, and S2 subunit, enzyme-linked immunosorbent assay (ELISA) showed that the 6G3 monoclonal antibody had a high binding titer of 100,000:1 for recombinant 6HB protein in vitro. This indicates that the 6G3 monoclonal antibody has good affinity for 6HB.

[0026] In the epitope identification of the 6G3 antibody, the amino acid sequence of the SARS-CoV-2 Spike protein was used to obtain the HR1P and HR2P polypeptide sequences in the 6-HB domain. HR1P and HR2P were used as antigens to coat 96-well plates, and enzyme-linked immunosorbent assay (ELISA) was performed using the 6G3 antibody as the primary antibody. The results showed that the 6G3 antibody could bind to both HR1P and HR2P. The EC50 for binding to HR1P was 167.4 ng / mL, and the EC50 for binding to HR2P was 239 ng / mL, suggesting that the 6G3 antibody may bind to the hydrophobic groove of the 6HB structure formed in the S2 subunit of the Spike protein.

[0027] In pseudovirus experiments, the 6G3 monoclonal antibody exhibited strong antiviral activity, significantly inhibiting the infection of ACE2-293T cells by SARS-CoV-2B1.351 E484K mutant and omicron XBB.1.5. The IC50 for inhibiting SARS-CoV-2B1.351 E484K mutant pseudovirus was 0.09883 μg / mL, and the IC50 for inhibiting omicron XBB.1.5 pseudovirus was 0.2242 μg / mL.

[0028] In live virus experiments, the 6G3 monoclonal antibody, used as the primary antibody, showed significant fluorescence in the virus group, indicating that the 6G3 antibody reacted with the S protein in virus-infected Vero cells. This method can be used for IFA detection of SARS-CoV-2 infection. Simultaneously, the fluorescence data were scanned and quantified. The 6G3 antibody showed an inhibitory IC50 of 0.01595 μg / mL against wild-type SARS-CoV-2, indicating that the 6G3 antibody has a good inhibitory effect on infection caused by live SARS-CoV-2 virus and can be considered a candidate drug for the development of COVID-19 antibodies. Attached Figure Description

[0029] Figure 1 This is an SDS-PAGE electrophoresis image of the 6G3 monoclonal antibody.

[0030] Figure 2 A graph showing the binding titer of 6G3 monoclonal antibody to 6HB antigen for enzyme-linked immunosorbent assay (ELISA).

[0031] Figure 3 This is a curve showing the binding rates of the full-length Spike protein and its S2 subunit as detected by enzyme-linked immunosorbent assay (ELISA).

[0032] Figure 4 The image shows the results of ELISA identification of the epitopes of 6G3 monoclonal antibody binding to HR1P and HR2P. HR1P and HR2P are peptides obtained from the 6-HB domain using the amino acid sequence of the SARS-CoV-2 Spike protein.

[0033] Figure 5 The graph shows the inhibitory effect of the 6G3 monoclonal antibody on SARS-CoV-2 pseudovirus infection. The groups are the SARS-CoV-2B1.351 E484K mutant group and the omicron XBB.1.5 group, respectively.

[0034] Figure 6 This study demonstrates the inhibitory effect of the 6G3 monoclonal antibody on real SARS-CoV-2 infection. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0036] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0037] Unless otherwise specified in the embodiments, any techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0038] Example 1: Preparation of 6G3 monoclonal antibody

[0039] 1.6G3 monoclonal antibody

[0040] Purified recombinant 6HB protein was used as an immunogen to immunize 8-12 week old BALB / c mice subcutaneously and intraperitoneally at multiple sites, with booster immunizations every 2 weeks for a total of three immunizations. Blood was collected from immunized mice, and antibody titers were screened by ELISA. Mice with the highest antibody titers were selected for fusion. Seven days before fusion, a booster immunization was performed. Spleen cells from these mice were fused with myeloma cells SP2 / 0. After multiple clonal screenings using limiting dilution, a monoclonal cell line was obtained, and the 6G3 monoclonal antibody, an IgG subtype, was identified. The 6G3 monoclonal antibody specifically binds to the 6HB domain in the S2 subunit of the SARS-CoV-2 receptor, with epitopes HR1920-970 and HR21163-1202. The 6G3 monoclonal antibody consists of a heavy chain (H chain) and a light chain (L chain), each including a heavy chain variable region (V). H ) and light chain variable region (V L ), heavy chain variable region (VH This includes the heavy chain complementarity determinant region (V). H CDR1, V H CDR2 and V H CDR3), light chain variable region (V L This includes the light chain complementarity determinant region (V). L CDR1, V L CDR2 and V L CDR3). Among them, V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0041] The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the 6G3 monoclonal antibody, SEQ ID NO.1:

[0042] GYSITSGYS

[0043] The amino acid sequence of the light chain complementarity-determining region CDR1 of the 6G3 monoclonal antibody, SEQ ID NO.2:

[0044] QNVGIA

[0045] The amino acid sequence of the heavy chain complementarity-determining region CDR2 of the 6G3 monoclonal antibody, SEQ ID NO.3:

[0046] IHYSGST

[0047] The amino acid sequence of the light chain complementarity-determining region CDR2 of the 6G3 monoclonal antibody, SEQ ID NO.4:

[0048] SAS

[0049] The amino acid sequence of the heavy chain complementarity-determining region (CDR3) of the 6G3 monoclonal antibody is SEQ ID NO. 5:

[0050] ARSGGNYYAMDY

[0051] The amino acid sequence of the light chain complementarity-determining region CDR3 of the 6G3 monoclonal antibody, SEQ ID NO. 6:

[0052] QQYINYPLT

[0053] The amino acid sequence of the variable region of the heavy chain of the 6G3 monoclonal antibody, SEQ ID NO.7:

[0054] VKLVESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSGGNYYAMDYWGQGTSVTVSS

[0055] The amino acid sequence of the variable region of the light chain of the 6G3 monoclonal antibody, SEQ ID NO. 8:

[0056] TQSQKFMSTSVGDRVSITCKASQNVGIAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYINYPLTFGGGTR

[0057] The nucleic acid sequence encoding the variable region of the heavy chain of the 6G3 monoclonal antibody is SEQ ID NO.9:

[0058] GACGTGAAGCTCGTGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGGTAGCACTAACTA CAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATCGGGTGGTAACTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAG

[0059] The nucleic acid sequence encoding the variable region of the light chain of the 6G3 monoclonal antibody is SEQ ID NO.10:

[0060] GAAAATGTGCTCACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTATTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCAATCG GTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATATGCAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATATCAACTATCCTCTCACGTTCGGAGGGGGGACCAGACTGGAAATAAAC

[0061] Preparation and purification of 2.6G3 monoclonal antibody

[0062] In this embodiment, the ascites solution was prepared by intraperitoneal injection of 0.5 mL / mouse of Freund's incomplete adjuvant in 8-week-old male BALB / c mice. Seven days later, the mice were intraperitoneally injected with 5 × 10⁻⁶ PBS diluted with PBS. 5 0.5 mL of 6G3 monoclonal hybridoma cell suspension was collected. Mice were continuously observed. After about 10 days, when the mice's abdomens became significantly enlarged and they showed signs of slow movement, the mice were euthanized by cervical dislocation. The ascites was removed, centrifuged to remove insoluble matter, and the supernatant was stored at -80°C for later use.

[0063] Purification of 6G3 monoclonal antibody:

[0064] In this study, the purification of murine antibodies was performed using Protein G affinity chromatography. After thawing the ascites fluid, floating flocculent insoluble matter was scraped off with a pipette tip, and 5 volumes of Protein G binding buffer (25 mM NaH2PO3 pH 7.5, 150 mL NaCl) were added. Then, 5 mL of Protein G resin equilibrated with the binding buffer was added, and the mixture was incubated on ice with a shaker for 2 hours. After incubation, the mixture was transferred to a gravity chromatography column, the permeate was discarded, and at least 10 column volumes of binding buffer were added to wash away non-specifically bound packing material. Finally, 5 column volumes of 0.1 M glycine pH 3.0 elution buffer were added, and the collection tube was pre-filled with 1 / 10 elution volume of 1 M Tris-HCl pH 8.5 neutralization buffer. Samples were taken for SDS-PAGE electrophoresis, and Coomassie brilliant blue staining was used to observe the purity of the purified antibody. The mAb was concentrated using an ultrafiltration tube and the buffer was replaced with sterile PBS. Protein concentration was detected using the A280 method. The product was stored at -80°C for later use.

[0065] Figure 1 This is a Coomassie brilliant blue staining image of the purified 6G3 monoclonal antibody. The molecular weights of the heavy chain (HC) and light chain (LC) of the 6G3 monoclonal antibody are 53 kDa and 27 kDa, respectively.

[0066] Example 2: Determination of 6G3 monoclonal antibody titer and evaluation of affinity with Spike protein and S2 subunit

[0067] This embodiment uses enzyme-linked immunosorbent assay (ELISA) to analyze the binding titer of 6G3 monoclonal antibody to 6HB antigen and the recognition of the full-length Spike protein and its S2 subunit. The specific steps include the following:

[0068] ① Assay for the titer of binding to 6HB antigen: 100 μg of recombinant SARS-CoV-2-6HB was dissolved in 10 mL of 0.05 M carbonate coating buffer (pH 9.6), and added to a 96-well plate at 100 μL / well. The plate was coated overnight at 4°C. The plate was washed three times with PBST, and blocked at 37°C for 1 h with 150 μL / well of PBS containing 5% skim milk blocking buffer. After washing three times with PBST, 100 μL of purified 6G3 antibody (initial dilution 5000) was added to each well, and the plate was serially diluted down to at least 6 concentrations. The plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL / well of horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody was added and the plate was incubated at 37°C for 1 h. After washing three times with PBST, TMB was used for color development at 100 μL / well. The plate was incubated at room temperature in the dark for 15 min, and the reaction was terminated by adding 100 μL / well of 2 M H2SO4. The absorbance at 450 nm was measured.

[0069] ② Binding affinity with full-length Spike protein and S2 subunit: 10 μg of full-length Spike protein or S2 subunit was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, and added to a 96-well plate at 200 μL / well. The plate was coated overnight at 4°C. The plate was washed three times with PBST, and blocked with 200 μL / well of PBS containing 5% skim milk blocking buffer at 37°C for 1 h. After washing three times with PBST, 100 μL each of serially diluted 6HB recombinant protein and 6G3 monoclonal antibody (adjusted to 2500 ng / mL with PBST, and down-diluted 10 times in a 2-fold gradient) was added to each well, and the plate was incubated at 37°C for 1 h. After washing the plate three times with PBST, add 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody to each well and incubate at 37°C for 1 h. After washing three times with PBST, perform TMB color development at 100 μL per well and incubate at room temperature in the dark for 15 min. Terminate the reaction with 100 μL of 2M H₂SO₄ per well and measure the absorbance at 450 nm. (OD) 实验 -OD 空白 ) / OD 实验 *100 calculates the binding rate.

[0070] Experimental results are as follows Figure 2 and 3 As shown. Figure 2 The enzyme-linked immunosorbent assay (ELISA) yield curves show the binding titer of 6G3 monoclonal antibody to 6HB antigen, with an antibody titer reaching 100,000:1. Figure 3 The enzyme-linked immunosorbent assay (ELISA) curves were used to detect the binding rates of 6G3 monoclonal antibody to the full-length Spike protein and the S2 subunit. After fitting and calculation, the EC50 for binding to the full-length Spike protein was 27.83 ng / mL, and the EC50 for binding to the S2 protein was 46.52 ng / mL, indicating that the 6G3 monoclonal antibody has a strong affinity for both 6HB and Spike proteins.

[0071] Example 3: Epitope identification of 6G3 monoclonal antibody binding to 6HB-HR1 and 6HB-HR2

[0072] This embodiment uses enzyme-linked immunosorbent assay (ELISA) to analyze antibody epitopes. HR1 and HR2 peptides were coated onto 96-well half-well plates. The next day, unbound wells were blocked with 5% skim milk. Then, different concentrations of 6G3 monoclonal antibody were added and incubated at 37°C for 1 hour. The bound 6G3 monoclonal antibody was detected using HRP-labeled goat anti-mouse IgG. After color development, the absorbance of the 96-well half-well plate at 450 nm was measured using a microplate reader, expressed as (OD). 实验 -OD 空白 ) / OD 实验 *100 calculates the binding rate.

[0073] Experimental results are as follows Figure 4 As shown. Figure 4 The image shows the results of ELISA identification of the binding of the 6G3 monoclonal antibody to the 6HB epitope. The EC50 of the 6G3 monoclonal antibody binding to HR1P was 167.4 ng / mL, and the EC50 of binding to HR2P was 239 ng / mL. HR1P and HR2P are polypeptides obtained from the 6-HB domain of the SARS-CoV-2 Spike protein using amino acid sequences, and the 6G3 monoclonal antibody bound to both.

[0074] Example 4: Evaluation of the inhibitory effect of 6G3 monoclonal antibody against SARS-CoV-2 pseudovirus

[0075] One day prior to infection, ACE2-293T cells were seeded into 96-well plates. The selected 6G3 monoclonal antibody was diluted to 50 μg / mL, then to 10 μg / mL, followed by serial dilutions of 2-fold. 50 μL of each diluted monoclonal antibody was mixed with an equal volume of SARS-CoV-2 pseudovirus and incubated at 37°C for 1 hour. The mixture was then used to infect ACE2-293T cells, with three replicates per monoclonal antibody concentration. After 12 hours of infection, the culture medium was replaced with fresh medium, and luciferase activity was measured after 48 hours. Cells were lysed with Glo lysis buffer (Promega), followed by the addition of luciferase substrate (Bright-Glo luciferase assay substrate, Promega). Luciferase activity was measured using a GloMax 96-well microplate spectrophotometer (Promega).

[0076] Experimental results are as follows Figure 5 As shown. Figure 5 Figure showing the results of the 6G3 monoclonal antibody inhibiting SARS-CoV-2 B1.351 E484K mutant and SARS-CoV-2 OmicronXBB.1.5. The monoclonal antibody SARS-CoV-2 6G3 (10 μg / mL) inhibited the infection of both pseudoviruses by almost 80%.

[0077] Example 5: Effect of 6G3 monoclonal antibody on real SARS-CoV-2 infection

[0078] This embodiment uses indirect immunofluorescence assay to detect the effect of 6G3 monoclonal antibody on SARS-CoV-2 real virus infection. Specifically, it includes the following steps:

[0079] One day prior to the incubation, Vero cells were digested and seeded into 24-well plates at a specific cell density. When cell confluence reached 80-90%, cells were infected with 0.02 MOI SARS-CoV-2 virus at 37°C for 1 hour. Unbound virus was discarded, and the cells were cultured with the appropriate concentration of antibody. When cell pathogenesis was observed, IFA (Infectious Disease Analysis) was performed. Cells were washed once with PBS and fixed with 4% paraformaldehyde for 30 minutes. 0.1% Triton-X-100 was added and the cells were incubated at room temperature for 10 minutes for permeabilization. 6G3 monoclonal antibody was used as the primary antibody, and the cells were incubated at 37°C for 2 hours. Cells were then washed three times with PBS for 3 minutes each time. Diluted CoraLite 488Goat anti-mouse IgG (H+L) fluorescent secondary antibody was added, and the cells were incubated at 37°C in the dark for 1 hour. The secondary antibody was discarded, and the cells were washed three times with PBS for 3 minutes each time. After washing, the cell nuclei were stained with DAPI for 10 minutes. Wash the cells three times with PBS, 3 minutes each time. After the last wash, blot dry the PBS and observe and photograph the cells under a fluorescence microscope.

[0080] Experimental results are as follows Figure 6 As shown, immunofluorescence of the 6G3 monoclonal antibody as the primary antibody showed obvious fluorescence in the virus group, indicating that the 6G3 monoclonal antibody reacted with the S protein in the virus-infected Vero cells. This method can be used for IFA detection of SARS-CoV-2 infection.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification are covered by the claims of the present invention.

Claims

1. A 6G3 monoclonal antibody or an antibody fragment thereof, characterized in that, The 6G3 monoclonal antibody is used to specifically bind to the 6HB domain in the S2 subunit of the SARS-CoV-2 spike protein, which contains the V region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively; The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

2. The 6G3 monoclonal antibody or an antibody fragment thereof according to claim 1, characterized in that, The SARS-CoV-2 mentioned is at least one of the SARS-CoV-2 mutant strain B1.351 and SARS-CoV-2Omicron XBB.1.

5.

3. The 6G3 monoclonal antibody or an antibody fragment thereof according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the 6G3 monoclonal antibody is shown in SEQ ID NO.7; And / or, the amino acid sequence of the light chain variable region of the 6G3 monoclonal antibody is shown in SEQ ID NO.

8.

4. A polynucleotide sequence, characterized in that, The polynucleotide sequence encodes the 6G3 monoclonal antibody as described in any one of claims 1-3 or an antibody fragment thereof.

5. The polynucleotide sequence according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the 6G3 monoclonal antibody of claim 1 is shown in SEQ ID NO. 9; And / or, the nucleotide sequence encoding the light chain variable region of the 6G3 monoclonal antibody of claim 1 is shown in SEQ ID NO.

10.

6. A biomaterial, characterized in that, The biological material expresses the 6G3 monoclonal antibody or an antibody fragment of the monoclonal antibody as described in any one of claims 1-3, or contains the polynucleotide sequence as described in claim 4 or 5, and the biological material includes at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

7. Use of the 6G3 monoclonal antibody according to any one of claims 1-3, or an antibody fragment thereof, in any of the following: (1) Prepare reagents for detecting and / or diagnosing SARS-CoV-2 infection; (2) Preparation of reagents that specifically bind to the Spike protein of SARS-CoV-2 virus; (3) To prepare drugs for the prevention and / or treatment of SARS-CoV-2 virus infection; (4) Prepare drugs for the prevention and / or treatment of symptoms caused by SARS-CoV-2 virus infection; (5) Prepare reagents for detecting SARS-CoV-2 antibodies.

8. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the 6G3 monoclonal antibody as described in any one of claims 1-3 or an antibody fragment thereof.

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