Antibody for resisting novel coronavirus envelope protein or antigen binding fragment thereof and application of antibody or antigen binding fragment

Abstract

The invention belongs to the technical field of antibodies, and discloses an antibody for resisting novel coronavirus envelope protein or an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof comprises a heavy chain and a light chain; a heavy chain of the antibody or the antigen binding fragment thereof comprises: a heavy chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3; a light chain of the antibody or the antigen binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3. The antibody or the antigen binding fragment thereof can be used for detecting the existence or the level of the novel coronavirus envelope protein in a sample, detecting the novel coronavirus and diagnosing 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 antigen-binding fragment thereof against the novel coronavirus envelope protein and its application. Background Art

[0002] The disease caused by the novel coronavirus (SARS-CoV-2) has spread to varying degrees around the world 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 for controlling its spread. Therefore, the development of convenient, rapid, and accurate detection 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), membrane (M), envelope (E), and nucleocapsid (N). The E protein is the smallest structural protein in SARS-CoV-2, consisting of only approximately 80 amino acids. As a transmembrane protein crucial 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 approximately 25 amino acids, and a longer, 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 influences viral replication and proliferation, making it a target antigen for prevention and treatment.

[0004] Currently, novel coronavirus detection methods include nucleic acid testing, antibody testing, and antigen testing. While nucleic acid testing is the primary method for novel coronavirus detection, offering high sensitivity, specificity, and accuracy, its drawbacks are the complex testing requirements and time required. Antibody testing requires time after viral infection to detect antibodies, and may not be detected in the early stages of infection. Furthermore, novel coronavirus antibodies can be detected in vaccinated individuals, but this can result in false positives. Therefore, antigen testing is more valuable. Antigen testing is generally used during the acute phase of infection, i.e., within seven 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, with results available within 15 to 20 minutes, allowing residents to collect samples and self-test at home. While there is a certain rate of missed detection compared to the gold standard nucleic acid testing, antigen testing remains an effective method for early detection of novel coronavirus infection in situations characterized by high risk, high prevalence, and clusters of infections.

[0005] Common rapid detection methods for novel coronavirus antigens include colloidal gold method, latex method, and fluorescent immunoassay. For general screening, the colloidal gold detection method has very low requirements for personnel, venues, and equipment, and is suitable for large-scale rapid testing. 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 visual observation within 15 minutes. People under home quarantine, 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 venues, and has the advantages of strong specificity, high sensitivity, simplicity and rapidity, visual 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 E 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 antigen-binding fragment thereof against the novel coronavirus envelope protein, which can be used to detect the level of the novel coronavirus envelope 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 envelope 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 present invention also provides a product.

[0013] The present invention also provides a method for preparing 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.

[0014] According to the first aspect of the present invention, an antibody or antigen-binding fragment thereof against the novel coronavirus envelope protein (E protein) is proposed, wherein the antibody or 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;

[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;

[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: WVS, 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 antigen-binding fragment thereof 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 obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO: 2 and having 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% identical 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 obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 4 and having 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% identical 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 according to 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 a third aspect of the present invention, a biomaterial 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 biomaterial 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 comprise 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 envelope 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 envelope 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 envelope 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 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 obtained by substituting and / or deleting and / or adding one or more nucleotides in SEQ ID NO: 1 and having 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 to 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 in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 3, 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 to SEQ ID NO: 3, and has a nucleotide sequence that 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 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 comprising 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 present invention, the detectable label is a fluorescent or luminescent label.

[0059] In some preferred embodiments of the present invention, the detectable label 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 antigen-binding fragment thereof against the novel coronavirus envelope 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 envelope 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 envelope 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 includes novel coronavirus infection.

[0074] According to a seventh aspect of the present invention, a product is provided, 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 envelope 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 includes novel coronavirus infection.

[0088] According to the eighth aspect of the present invention, a method for preparing an antibody or antigen-binding fragment thereof against the novel coronavirus envelope 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 antigen-binding fragment thereof against the novel coronavirus envelope protein. The antibody or antigen-binding fragment thereof has good specificity for the novel coronavirus and / or the novel coronavirus 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0092] Figure 1 The figure is the result of SDS-PAGE of the eluted sample in Example 1: wherein M is a protein molecular weight standard (Marker), 1 is a bacterial cell without induced expression, 2 is a bacterial cell after induced expression, and 3 is a purified novel coronavirus E protein;

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

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

[0095] Figure 4 This is the result of SDS-PAGE of the purified novel coronavirus E protein monoclonal antibody 23172N in Example 3: wherein M is a protein molecular weight standard (Marker), and I is the purified novel coronavirus E protein monoclonal antibody 23172N;

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

[0097] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

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

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

[0101] 1. Construction of recombinant expression vector

[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 amplified product was 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. After agarose gel electrophoresis, the target gene and vector large fragment were recovered using a gel recovery kit. They were ligated according to the conventional method and transformed into Escherichia coli BL21 (DE3) competent cells. A single clone was picked and expanded. The expression vector pMAL-c6T E was obtained by PCR amplification, enzyme digestion, and sequencing.

[0103] 2. Expression and Purification of SARS-CoV-2E Protein

[0104] After the positive BL21 (DE3) clones containing the recombinant expression vector pMAL-c6T E were amplified and cultured, the plasmid was extracted and transformed according to the conventional method. A single colony was picked and cultured in LB medium containing ampicillin resistance until the bacterial solution concentration reached A 600 When the concentration was 0.5, the expression was induced with 0.1 mM IPTG, 200 rpm, and 16°C for 18 h. The bacteria were collected by centrifugation at 4°C and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and added with Binding buffer for ultrasonic disruption in an ice bath for 3 min. The ultrasonic power was 30 W. After ultrasonication for 2 s, the ultrasonication was repeated at intervals of 2 s 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 (E recombinant protein) was purified by nickel column affinity chromatography: 50% NI-NTA was loaded onto the column, 4 mL of deionized water was used to wash the column, and then 5 mL of Binding buffer was added to equilibrate the column. The sample was loaded onto the column, and the lysate containing the E recombinant protein was loaded onto the column. The flow-through was collected and loaded onto the column again. Unbound proteins were washed away with Binding Buffer, and 20 mL of Washing Buffer was added to wash the proteins. The protein was eluted with 250 mM imidazole Elution Buffer. The purity of the eluted sample was detected by SDS-PAGE electrophoresis. The results are shown in the figure below. Figure 1 As shown: The E protein obtained by affinity chromatography is of high purity. The concentration of the purified E recombinant protein was determined by BCA assay.

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

[0107] This example prepared a novel coronavirus E protein monoclonal antibody cell line, and the specific preparation method included the following steps:

[0108] 1. Mouse Immunization

[0109] The E recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (the day of the first immunization was counted as day 1), Freund's complete adjuvant was emulsified with the immunogen in a volume ratio of 1:1, with a dose of 100 μg / mouse, and five immunization points were injected subcutaneously on the back and abdomen; on the 15th day, a second immunization was performed, using Freund's incomplete adjuvant and the immunogen in a volume ratio of 1:1 emulsified at 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. If the antibody titer was greater than 1:10,000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10,000, four immunizations were performed on the 43rd day in the manner of three immunizations; three days before cell fusion, intraperitoneal pulse immunization was performed, and 100 μg of immunogen was directly injected into the peritoneal cavity without adding adjuvant.

[0110] 2. Mouse Serum Titer Detection

[0111] (1) Antigen coating: The E recombinant protein obtained in Example 1 was used as a coating agent 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 from mice by tail amputation, and serum from non-immunized mice was used as a negative control.

[0115] (5) Add primary antibody: dilute the serum samples to be tested in blocking solution in multiple ratios. Use non-immune mouse serum diluted 1:2500 as negative control and blocking solution as 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, develop color at 37°C in the dark for 15 minutes, add 100 μL / well of 2M sulfuric acid to terminate the reaction, and 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 is P / N ≥ 2.1 is the titer / titer of the serum. Mice with a dilution ratio 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 E 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 remove spleen cells from immunized mice and mix them with SP2 / 0 mouse myeloma cells in a ratio of approximately 5:1 in a 50mL centrifuge tube. Wash twice with culture medium and discard the supernatant. Slowly add 0.9mL of preheated PEG-1500 over 50 seconds to disperse the cells as evenly as possible in the PEG. Let it stand for 1 minute. Slowly add 20mL of serum-free DMEM medium preheated at 37°C dropwise, adding 2mL in the first two minutes and 18mL in the last two minutes. Add the entire amount within 4 minutes. Let it stand for 3 minutes. Centrifuge at 800rpm for 5 minutes and discard the supernatant. Add preheated FBS and HAT medium, gently pipette to mix, transfer 200μL per well to a 96-well culture plate, and culture in an incubator.

[0120] 4. Screening of positive hybridoma cells

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

[0122] 5. Subcloning of positive hybridoma cells

[0123] Positive wells screened were subcloned using the limiting dilution method. The number and location of cell clusters in the positive wells were first observed under an inverted microscope. Within a clean bench, the cell clusters were aspirated using a 200μL pipette tip and diluted to a concentration of 1–2 cells per 100μL. The prepared feeder cells were removed and added to a 96-well plate at a volume of 100μL per well. Label the plate and incubate in a 5% CO2 incubator at 37°C for 9 days. After three subcloning cycles, the plate was subcloned to a single cell per well. The titer of the cell supernatant was determined by indirect ELISA, indicating a 100% positive rate. The plate was then expanded and the cell line 23172N was established.

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

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

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

[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 (5×10 5 =100 cells / mouse, with the day of cell suspension injection counted as day 1); on day 7, after the abdominal cavity of the mice was significantly distended, ascites was collected and centrifuged at 3000 rpm for 20 min. The adipose tissue was removed, and the supernatant was aspirated and frozen at -20°C for later use. The titer of ascites was determined by indirect ELISA. The results were as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0128] 2. Purification of Monoclonal Antibodies

[0129] Ascites was collected and the novel coronavirus E protein monoclonal antibody 23172N was purified by caprylic acid-ammonium sulfate precipitation method as follows: ascites was taken, centrifuged at 4°C and 12000rpm for 5 minutes, 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; 33 μL of caprylic acid was added per milliliter of ascites, stirred at room temperature for 30 minutes, and allowed to stand at 4°C for 1 hour to allow the impurities to precipitate completely, centrifuged at 4°C and 1000g for 30 minutes, 0.277g of ammonium sulfate powder was added per milliliter of supernatant, the beaker was placed on a magnetic stirrer, stirred for 1 hour, and centrifuged at 4°C and 10000g for 20 minutes, 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: A high-purity novel coronavirus E protein monoclonal antibody 23172N 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 E protein monoclonal antibody 23172N were IgG1, kappa light chain.

[0132] At the same time, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to sequence 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). 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] The 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 CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the novel coronavirus E protein monoclonal antibody 23172N defined by 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 definition schemes

[0139]

[0140] 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 are shown in Table 2.

[0141] Table 2 CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region defined by different definition 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 tested were reference materials obtained from the China Food and Drug Administration or the Chinese Center for Disease Control and Prevention);

[0146] The same conditions were used to coat the ELISA plate, and the monoclonal antibody 23172N 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 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 novel coronaviruses and / or diseases caused by novel coronaviruses.

[0147] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. An antibody or antigen-binding fragment thereof against a novel coronavirus envelope protein, 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; 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; 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: WVS, 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 antigen-binding fragment thereof 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, wherein 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 obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO: 2 and having 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% identical to SEQ ID NO: 2 and has the same function as the protein set forth 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 obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 4 and having 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% identical to SEQ ID NO: 4 and has the same function as the protein set forth in SEQ ID NO: 4; Preferably, the antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody; Preferably, 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.

3. A recombinant protein comprising: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; and Optional tag sequence to facilitate expression and / or purification.

4. A biomaterial related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or the recombinant protein according to claim 3, wherein the biomaterial 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 2, or the recombinant protein according to claim 3; 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).

5. A conjugate comprising: at least one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and the recombinant protein according to claim 3; and a coupling moiety, the coupling moiety comprising 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; Preferably, the detectable marker is a fluorescent or luminescent marker; Preferably, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase; Preferably, 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.

6. 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 2, and / or the recombinant protein according to claim 3.

7. Use of at least one of (1) to (5) in the preparation of a product; (1) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; (2) the recombinant protein according to claim 3; (3) The biomaterial according to claim 4; (4) The conjugate according to claim 5; (5) The solid phase carrier according to claim 6; Preferably, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions i1) to i2): i1) Prevention and treatment of novel coronavirus infection; i2) Preventing and treating diseases related to novel coronavirus infection; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3): j1) Detecting the presence or level of novel coronavirus envelope protein in a sample; j2) Detection of novel coronavirus; j3) Diagnosis of diseases related to novel coronavirus infection.

8. A product comprising at least one of k1) to k4): k1) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; k2) the recombinant protein according to claim 3; k3) the conjugate according to claim 5; k4) The solid phase carrier according to claim 6.

9. The product according to claim 8, characterized in that: The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions i1) to i2): i1) Prevention and treatment of novel coronavirus infection; i2) Preventing and treating diseases related to novel coronavirus infection; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3): j1) Detecting the presence or level of novel coronavirus envelope protein in a sample; j2) Detection of novel coronavirus; j3) Diagnosis of diseases related to novel coronavirus infection.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or the recombinant protein according to claim 3, which is obtained by culturing the transgenic cell line according to claim 4.

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