An antibody or antigen-binding fragment thereof against novel coronavirus envelope protein and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an antibody against the envelope protein of the novel coronavirus or its antigen-binding fragment and its applications. Background Technology

[0002] The disease caused by the novel coronavirus (SARS-CoV-2) is prevalent to varying degrees worldwide and has become one of the deadliest epidemics in human history. Currently, rapid screening of infected individuals and blocking transmission routes are the most effective methods to control its spread. Therefore, researching convenient, rapid, and accurate testing methods is particularly important for epidemic prevention and control.

[0003] SARS-CoV-2 is a membrane-bound, single-stranded RNA virus with four structural proteins: spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein. The E protein is the smallest structural protein in SARS-CoV-2, composed of only about 80 amino acids. As an important transmembrane protein in the viral life cycle, it has three domains: a short hydrophilic N-terminal domain (NTD) of 7-12 amino acids, a hydrophobic transmembrane domain (TMD) of about 25 amino acids, and a relatively long hydrophilic C-terminal domain (CTD). The E protein has multiple functions at different stages of infection; it not only participates in the assembly and release of progeny viruses but also affects viral replication and proliferation, and can serve as a target antigen in prevention and treatment.

[0004] Currently, detection methods for the novel coronavirus include nucleic acid testing, antibody testing, and antigen testing. While nucleic acid testing is the primary method for detecting the novel coronavirus due to its high sensitivity, specificity, and accuracy, its drawbacks include demanding testing conditions and a long testing time. Antibody testing only detects antibodies after a certain period following viral infection; antibodies may not be detectable in the early stages of infection. Furthermore, antibodies can be detected in vaccinated individuals, leading to false positives. Therefore, antigen testing is more meaningful. Antigen testing is generally used during the acute infection phase, specifically for samples from suspected cases within 7 days of symptom onset. Positive antigen results can be used for early triage and rapid management of suspected cases. This method is simple and convenient, providing results within 15 to 20 minutes, allowing residents to complete sampling and self-testing at home. Compared to nucleic acid testing, the "gold standard," although it has a certain probability of false negatives, considering the high risk, high prevalence, and potential for cluster infections during the testing process, antigen testing remains an effective detection method for early detection of novel coronavirus infection.

[0005] Common rapid detection methods for novel coronavirus antigens include colloidal gold assay, latex assay, and fluorescence immunochromatography. For routine screening, the colloidal gold assay has very low requirements for personnel, facilities, and equipment, making it suitable for large-scale rapid testing. This method requires no special sample treatment and can qualitatively detect the novel coronavirus in nasal swab samples in vitro. Results are obtained within 15 minutes through visual observation. Individuals under home quarantine, close contacts, and secondary close contacts can perform the test themselves, avoiding cross-infection between medical staff and patients. This method overcomes the limitations of existing testing technologies in terms of personnel and facilities, and has advantages such as high specificity, high sensitivity, simplicity, speed, visual interpretation, and high stability. To achieve rapid detection of novel coronavirus antigens, researching and obtaining monoclonal antibodies against the novel coronavirus E protein is of great significance. Summary of the Invention

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

[0007] This invention also proposes a recombinant protein.

[0008] The present invention also proposes a biomaterial related to an antibody against the envelope protein of the novel coronavirus as described in the first aspect of the present invention or an antigen-binding fragment thereof, or a recombinant protein as described in the second aspect of the present invention.

[0009] The present invention also proposes a coupling agent.

[0010] The present invention also proposes a solid-phase support.

[0011] The present invention also proposes an application.

[0012] The present invention also proposes a product.

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

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

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

[0016] Heavy chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3;

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

[0018] Light chain variable region, wherein the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0019] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the antibody or its antigen-binding fragment 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 WVS, and the CDR is 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 its antigen-binding fragment 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, wherein the CDR is defined according to the Kabat 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 its antigen-binding fragment 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, wherein the CDR is defined according to the Chothia scheme; or

[0022] The amino acid sequences of the antibody or its antigen-binding fragments CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 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, wherein the CDR is defined using 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 its antigen-binding fragment comprises:

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

[0025] a2) An amino acid sequence of SEQ ID NO: 2 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 2; or

[0026] a3) has 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% homology with 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 its antigen-binding fragment comprises:

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

[0029] b2) A sequence of amino acids in SEQ ID NO: 4 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 4; or

[0030] b3) has 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% homology with 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 its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody.

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

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

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

[0035] Optional tag sequences to assist in 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: His tag, GGGS sequence, FLAG tag.

[0037] According to a third aspect of the present invention, biomaterials relating to the antibodies or antigen-binding fragments thereof described in the first aspect of the present invention, or the recombinant proteins described in the second aspect of the present invention, are provided, said biomaterials comprising at least one of h1) to h8):

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

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

[0040] h3) A carrier containing the nucleic acid molecule described in h1);

[0041] h4) A carrier containing the expression box described in h2);

[0042] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0043] h6) Transgenic cell lines containing the expression cassette described in h2);

[0044] h7) A transgenic cell line containing the vector described in h3);

[0045] h8) is a transgenic cell line containing the vector described in h4).

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

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

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

[0050] a212) A nucleotide sequence of SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and 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 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 envelope protein according to 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 of SEQ ID NO: 3 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and 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 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 described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention;

[0057] And a coupling portion comprising at least one of a detectable marker, drug, toxin, electron-dense marker, biotin / avidin, spin marker, antibody, antibody Fc fragment, antibody scFv fragment, radionuclide, enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, and viral capsid proteins.

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

[0059] In some preferred embodiments of the present invention, the detectable marker is selected from any one of acridine ester, acridine 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 the prevention and treatment of novel coronavirus infection and / or diseases related to novel coronavirus infection (e.g., antiviral drugs: favipiravir, remdesivir, or interferon, etc.).

[0062] In some embodiments of the present invention, the disease related to novel coronavirus infection includes novel coronavirus infection.

[0063] According to a fifth aspect of the present invention, a solid-phase support is provided, the surface of which is coupled with an antibody against the envelope protein of the novel coronavirus as described in the first aspect of the present invention or an antigen-binding fragment thereof and / or a recombinant protein as described in the second aspect of the present invention.

[0064] According to a sixth aspect of the present invention, the use of an antibody against the envelope protein of the novel coronavirus or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect, a biomaterial as described in the third aspect, a conjugate as described in the fourth aspect, and / or a solid-phase support 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 detection plate, a reagent kit, and a detection chip.

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

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

[0068] i2) Prevention and control of diseases related to novel coronavirus infection.

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

[0070] j1) Detect the presence or level of the novel coronavirus envelope protein in the sample;

[0071] j2) Detection of novel coronavirus;

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

[0073] In some preferred embodiments of the present invention, the disease related to novel coronavirus infection includes 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 as described in the first aspect of the present invention;

[0076] k2) The recombinant protein described in the second aspect of the present invention;

[0077] k3) The coupling described in the fourth aspect of the present invention;

[0078] k4) The solid support 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 detection plate, a reagent kit, and a detection chip.

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

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

[0082] i2) Prevention and control of diseases related to novel coronavirus infection.

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

[0084] j1) Detect the presence or level of the novel coronavirus envelope protein in the sample;

[0085] j2) Detection of novel coronavirus;

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

[0087] In some preferred embodiments of the present invention, the disease related to novel coronavirus infection includes novel coronavirus infection.

[0088] According to an eighth aspect of the present invention, a method for preparing an antibody against the envelope protein of the novel coronavirus as described in the first aspect of the present invention or an antigen-binding fragment thereof, or a recombinant protein as described in the second aspect of the present invention, is provided, 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] This invention provides an antibody against the envelope protein of the novel coronavirus or its antigen-binding fragment. The antibody or its antigen-binding fragment has good specificity for the novel coronavirus and / or its envelope protein, and can be used to detect the presence or level of the novel coronavirus envelope protein in a sample, detect the novel coronavirus, and diagnose diseases related to novel coronavirus infection. Attached Figure Description

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

[0092] Figure 1 The image shows the SDS-PAGE results of the eluted sample in Example 1: where M is the protein molecular weight standard (Marker), 1 is the bacterial cell without induced expression, 2 is the bacterial cell after induced expression, and 3 is the purified novel coronavirus E protein.

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

[0094] Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of cell line 23172N in Example 3;

[0095] Figure 4 This is the result of SDS-PAGE analysis of the purified novel coronavirus E protein monoclonal antibody 23172N in Example 3: where M is the protein molecular weight standard (Marker) and 1 is the purified novel coronavirus E protein monoclonal antibody 23172N.

[0096] Figure 5 This is a graph showing the results of the specificity detection of the novel coronavirus E protein monoclonal antibody 23172N in Example 3. Detailed Implementation

[0097] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0098] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0099] Example 1: Obtaining the recombinant protein E of the novel coronavirus

[0100] This embodiment prepared the recombinant E protein of the novel coronavirus. The specific preparation method includes the following steps:

[0101] 1. Construction of recombinant expression vectors

[0102] Using the SARS-CoV-2 cDNA sequence as a template, primers were designed and synthesized according to the NCBI sequence number (No. MT123290.1) to amplify the SARS-CoV-2E gene sequence (the amplification product is 26248–26475 bp of MT123290.1). The product was identified by agarose gel electrophoresis. The vector pMAL-c6T and the PCR amplification product were double-digested with NotI and BamHI. The target gene and the large fragment of the vector were recovered by a gel recovery kit after agarose gel electrophoresis, ligated according to conventional methods, transformed into E. coli BL21(DE3) competent cells, single clones were selected, expanded cultured, and confirmed to be correct by PCR amplification, enzyme digestion, and sequencing to obtain the expression vector pMAL-c6T E.

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

[0104] After confirming the correct BL21(DE3) clone containing the recombinant expression vector pMAL-c6TE, plasmid was extracted and transformed using standard methods. Single colonies were then picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600 When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16 °C. The cells were collected by centrifugation at 4 °C and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated on ice for 3 min with Binding buffer at 30 W. The sonication was repeated for 2 seconds with a 2-second interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4 °C and 12000 rpm for 20 min for purification.

[0105] The target protein (recombinant protein E) was purified by nickel-column affinity chromatography: A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The sample was loaded, and the lysate containing recombinant protein E was added to the column. The permeate was collected and loaded again. Unbound contaminants were washed away with binding buffer, followed by 20 mL of washing buffer. The protein was then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown, the E protein obtained by affinity chromatography had high purity. The concentration of the purified recombinant E protein was determined by BCA method.

[0106] Example 2: Establishment of a monoclonal antibody cell line for the novel coronavirus E protein

[0107] This embodiment prepared a monoclonal antibody cell line against the novel coronavirus E protein. The specific preparation method includes the following steps:

[0108] 1. Mouse immunization

[0109] The recombinant E protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, administered subcutaneously at five sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg per mouse, following the same method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing. If the antibody titer was greater than 1:10000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10000, a fourth immunization was performed on day 43, following the same method. Three days before cell fusion, an intraperitoneal pulse immunization was performed, with 100 μg of immunogen injected directly into the peritoneum without adjuvant.

[0110] 2. Mouse serum titer detection

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

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

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

[0114] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0115] (5) Add primary antibody: Use blocking buffer to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking buffer as a blank control. Incubate at 37℃ for 1 hour, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

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

[0117] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution per well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2M sulfuric acid per well to terminate the reaction. Measure the A value at 450 nm using a microplate reader. 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 (P / N) ≥ 2.1 is taken as the serum titer. Mice with a dilution greater than 1:100,000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction between recombinant E protein and immune serum was identified by indirect ELISA, with a mouse serum titer of 1:160000.

[0118] 3. Cell fusion

[0119] Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible in the PEG. The mixture was allowed to stand for 1 min. Then, 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a rate of 200 μL per well and incubated in an incubator.

[0120] 4. Screening of positive hybridoma cells

[0121] Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0122] 5. Subcloning of positive hybridoma cells

[0123] Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in 96-well plates, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was detected using an indirect ELISA method. The positive rate reached 100%. The culture was expanded, and cell line 23172N was preserved.

[0124] Example 3: Preparation and Identification of Monoclonal Antibody for Novel Coronavirus E Protein

[0125] This embodiment prepared a monoclonal antibody against the novel coronavirus E protein and identified its subclass. The specific steps are as follows:

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

[0127] 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of cell suspension (5 × 10⁻⁶) of cell line 23172N preserved in Example 2. 5 Each mouse was injected with cell suspension (day 1 was counted as one cell per mouse); on day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, the supernatant was collected, and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0128] 2. Purification of monoclonal antibodies

[0129] Ascites fluid was collected, and the monoclonal antibody 23172N, the E protein of the novel coronavirus, was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. It was then incubated at 4°C for 1 h to allow for complete precipitation of impurities. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. The purity of the monoclonal antibody was determined by SDS-PAGE. The results are shown below. Figure 4 As shown: High-purity monoclonal antibody 23172N against the novel coronavirus E protein was obtained.

[0130] 3. Identification of Monoclonal Antibody Types and Subclasses

[0131] The mouse subtype identification kit was used for experimental procedures. The novel coronavirus E protein monoclonal antibody 23172N was identified as IgG1, kappa light chain.

[0132] Meanwhile, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to sequence the monoclonal antibody 23172N (i.e., the novel coronavirus E protein monoclonal antibody 23172N purified in step 2 of Example 3) produced by the cell line 23172N preserved in Example 2. The results are as follows:

[0133] The nucleotide sequence of the heavy chain variable region of the novel coronavirus E protein monoclonal antibody 23172N is: 5'-CAGATCCAG TTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTACTGGTTATACCTTCACAGACTATCCACTTCACTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAATACTGAGACTGGTGAACCAGCATATGCAGATGACTTCCAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCAATGCCTATTTGCAGATTAACAACCTCAAAAATGAGGACACGGCGACATATTTCTGTGCTCGCGGTAGCGACAACTACTATGCTATGGACTACTGGGGTCTAGGAACCTCAGTCACCGTCTCCTCA-3' (SEQ ID NO: 1),

[0134] Its corresponding amino acid sequence is: QIQLVQSGPELKKPGETVKISCKATGYTFTDYPLHWVK QAPGKGLKWMGWINTETGEPAYADDFQGRFAFSLETSASNAYLQINNLKNEDTATYF CARGSDNYYAMDYWGLGTSVTVSS (SEQ ID NO: 2);

[0135] The nucleotide sequence of the light chain variable region is: 5'-GACATTGTGATGTCACAGTCTCCATCCTCCCTA GCTGTGTCAGTTGGAGAGGGTTACTGTGAACTGCAAGTCCAGTCAGAGCCTTTTATATAGTACCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCCACACTGCTGATTTACTGGGTATCTATTAGGGAATCTGGG GTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATCGCTATCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3'(SEQ ID NO: 3),

[0136] The corresponding amino acid sequence is: DIVMSQSPSSLAVSVGERVTVNCKSSQSLLYSTNQKNYL AWYQQKPGQSPTLLIYWVSIRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQY YRYPPTFGGGTKLEIK (SEQ ID NO: 4).

[0137] The heavy chain variable regions of the novel coronavirus E protein monoclonal antibody 23172N, defined as CDR-H1, CDR-H2, and CDR-H3 using different definition schemes, are shown in Table 1.

[0138] Table 1. CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region defined by different schemes.

[0139]

[0140] Table 2 shows the CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the novel coronavirus E protein monoclonal antibody 23172N, defined by different definition schemes.

[0141] Table 2. CDR-L1, CDR-L2, and CDR-L3 of the variable region of the light chain defined by different schemes.

[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 (all inactivated viruses used in the tests were reference materials obtained from the National Institutes for Food and Drug Control or the Chinese Center for Disease Control and Prevention).

[0146] The ELISA plate was coated under the same conditions, using the monoclonal antibody 23172N purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450 nm The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5As shown, the monoclonal antibody 23172N produced by the cell line 23172N preserved in Example 2 (i.e., the novel coronavirus E protein monoclonal antibody 23172N 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 have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. An antibody or antigen-binding fragment thereof against the envelope protein of the novel coronavirus SARS-CoV-2, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The heavy chain of the antibody or its antigen-binding fragment comprises: Heavy chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3; The light chain of the antibody or its antigen-binding fragment comprises: Light chain variable region, wherein the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3; The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the antibody or its antigen-binding fragment 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 WVS, and the CDR is 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 its antigen-binding fragment 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, wherein the CDR is defined according to the Kabat 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: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 17, respectively, wherein the CDR is defined according to the Chothia scheme; or The amino acid sequences of the antibody or its antigen-binding fragments CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 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, wherein the CDR is defined using the Contact definition scheme.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes: a1) SEQ ID NO: 2; or a2) An amino acid sequence of SEQ ID NO: 2 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 2; or a3) An amino acid sequence that has at least 90% homology with 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 its antigen-binding fragment includes: b1) SEQ ID NO: 4; or b2) An amino acid sequence of SEQ ID NO: 4 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 4; or b3) An amino acid sequence that has at least 90% homology with SEQ ID NO:4 and has the same function as the protein shown in SEQ ID NO:

4.

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

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

5. A recombinant protein, characterized in that, The recombinant protein is the antibody or its antigen-binding fragment as described in any one of claims 1 to 4; and Optional tag sequences to assist in expression and / or purification.

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

7. A solid-phase support having an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and / or a recombinant protein as described in claim 5, coupled to its surface.

8. The application of at least one of (1) to (4) in the preparation of the product; (1) The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; (2) The recombinant protein according to claim 5; (3) The biomaterial as described in claim 6; (4) The solid support as described in claim 7; The product is at least one of the following: drug, reagent, test plate, reagent kit, and test chip; The drug has the function of preventing and treating infection with the novel coronavirus SARS-CoV-2; The reagent, detection plate, detection chip, or kit has at least one function of j1) to j2): j1) Detect the presence or level of the SARS-CoV-2 envelope protein in the sample; j2) Detection of the novel coronavirus SARS-CoV-2.

9. A product comprising at least one of k1) to k3): k1) The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; k2) The recombinant protein according to claim 5; k3) The solid support as described in claim 7; The product is at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

10. The product according to claim 9, characterized in that: The drug has the function of preventing and treating infection with the novel coronavirus SARS-CoV-2; The reagent, detection plate, detection chip, or kit has at least one function of j1) to j2): j1) Detect the presence or level of the SARS-CoV-2 envelope protein in the sample; j2) Detection of the novel coronavirus SARS-CoV-2.

11. The method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or the recombinant protein as described in claim 5, obtained by culturing the transgenic cell line as described in claim 6.

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