Monoclonal antibody recognizing a conformational epitope of neutralizing porcine acute diarrhea syndrome coronavirus and use thereof

Abstract

This invention belongs to the field of biotechnology and discloses a monoclonal antibody that recognizes a conformational epitope to neutralize porcine acute diarrhea syndrome coronavirus and its applications. Specifically, it discloses an antibody or its antigen-binding fragment. The antibody provided by this invention can neutralize 200 TCID at a concentration of 31.75 μg / mL. 50 The SADS-CoV virus particles were analyzed using the BLI assay, which showed that the antibody had a strong affinity for the S protein and was not easily dissociated after binding, providing important material for the development of SADS-CoV neutralizing antibody antiviral drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody that recognizes conformational epitopes and neutralizes porcine acute diarrhea syndrome coronavirus and its applications. Background Technology

[0002] In 2017, porcine acute diarrhea syndrome coronavirus (SADS-CoV) spread to four large-scale pig farms in Guangdong Province in just three months, causing the deaths of approximately 25,000 piglets. The mortality rate in infected piglets under five days old can be as high as 90%, making it one of the important pathogens causing diarrhea in piglets. Furthermore, the virus has the potential risk of cross-species transmission. However, there are currently no vaccines or antiviral agents for SADS-CoV available in clinical practice. Therefore, the development of antiviral agents for SADS-CoV is of great importance.

[0003] In coronaviruses, the S protein, in the form of a trimer, covers the viral surface and plays a crucial role in determining the host range and cell tropism of the coronavirus. The S protein of SADS-CoV (1130 amino acids in length) is smaller than that of other coronavirus S proteins and shows low homology with other coronavirus S genes. Studies have shown that the SADS coronavirus S protein has a unique structure; and evolutionary analysis indicates that the SADS-CoV S protein belongs to the β-coronavirus group, suggesting that SADS-CoV may have originated from a mixed infection involving α-coronaviruses and β-coronaviruses. Therefore, further research on the SADS coronavirus S protein is of great significance.

[0004] Neutralizing antibodies are antibodies that, upon binding to a pathogen, can significantly reduce or even completely eliminate the pathogen's infectivity. They are a major factor in the body's antiviral immunity. Compared to other therapeutic drugs, they can precisely bind to key functional regions of viral proteins, blocking their invasion of host cells. Furthermore, they possess advantages such as high specificity and sensitivity. Therefore, neutralizing antibodies are a key focus in antiviral drug development. However, currently, there are no monoclonal antibodies with high neutralizing titers. Summary of the Invention

[0005] The first aspect of the present invention is to provide an antibody or an antigen-binding fragment thereof.

[0006] A second aspect of the present invention is to provide a recombinant protein.

[0007] A third aspect of the present invention aims to provide biological materials related to the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention.

[0008] A fourth aspect of the present invention is to provide a protein conjugate.

[0009] The fifth aspect of this invention aims to provide the application of the antibody or antigen-binding fragment thereof of the first aspect of this invention, the recombinant protein of the second aspect of this invention, the biomaterial of the third aspect of this invention, or the protein conjugate of the fourth aspect of this invention.

[0010] The sixth aspect of this invention aims to provide a product.

[0011] The seventh aspect of this invention is to provide a medicine.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] Porcine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) can cause vomiting, severe diarrhea, and death in newborn piglets. Its spike (S) protein plays a crucial role in promoting viral invasion and inducing the production of neutralizing antibodies. Therefore, the inventors immunized mice with the S protein and, in their research on preparing monoclonal antibodies against the SADS-CoV S protein, obtained a cell line that stably secretes anti-(SADS-CoV), named the 8E4 hybridoma cell line. By injecting this hybridoma cell line into the peritoneal cavity of mice, a monoclonal antibody was obtained, named the 8E4 monoclonal antibody (8E4 antibody).

[0014] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, characterized in that the antibody or the antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each contain complementarity-determining regions CDR1, CDR2, and CDR3, respectively.

[0015] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively.

[0016] The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively.

[0017] 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:

[0018] a1) The amino acid sequence as shown in SEQ ID NO:2; or

[0019] a2) has at least 90% homology with SEQ ID NO:2 and has the same functional amino acid sequence as the protein shown in SEQ ID NO:2;

[0020] The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes:

[0021] a3) The amino acid sequence as shown in SEQ ID NO:10; or

[0022] a4) has at least 90% homology with SEQ ID NO:10 and has the same functional amino acid sequence as the protein shown in SEQ ID NO:10.

[0023] In some embodiments of the present invention, the antibody or its antigen-binding fragment includes at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, chimeric antibody, bispecific antibody, and multispecific antibody.

[0024] The term "chimeric antibody" refers to a protein molecule designed by recombining the variable regions of the light and heavy chains of immunoglobulins from non-human species with the constant regions of human immunoglobulin molecules. Although this invention provides only one mouse antibody, those skilled in the art can analyze the corresponding CDR sequences based on the sequences of the heavy and light chain variable regions, and replace other parts with constant regions and framework regions (FRs) from other animal sources based on the CDR sequences, thereby obtaining antibodies from other animal sources, and anticipating similar or identical technical effects to those of this invention. Preferably, the other animal-derived antibodies include at least one of human antibodies and porcine antibodies.

[0025] In some embodiments of the present invention, the antibody or its antigen-binding fragment is a mouse antibody.

[0026] In some embodiments of the present invention, the antibody or its antigen-binding fragment is a neutralizing antibody.

[0027] The antibody provided by this invention can neutralize 200 TCID at a concentration of 31.75 μg / mL. 50 The SADS-CoV virus particles were analyzed using the BLI assay, which showed that the antibody had a strong affinity for the S protein and was not easily dissociated after binding, providing important material for the development of SADS-CoV neutralizing antibody antiviral drugs.

[0028] A second aspect of the invention provides a recombinant protein comprising an antibody or antigen-binding fragment thereof of the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0029] 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, Myc tag, HA tag; further, His tag; and even further, 6×His tag.

[0030] A third aspect of the present invention provides biological materials related to the antibody or antigen-binding fragment thereof of the first aspect of the present invention and the recombinant protein of the second aspect of the present invention, said biological material being any one of b1) to b12):

[0031] b1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the first aspect of the present invention or a recombinant protein of the second aspect of the present invention;

[0032] b2) An expression cassette containing the nucleic acid molecule described in b1);

[0033] b3) A recombinant vector containing the nucleic acid molecule described in b1);

[0034] b4) A recombinant vector containing the expression cassette described in b2);

[0035] b5) Recombinant microorganisms containing the nucleic acid molecules described in b1);

[0036] b6) Recombinant microorganisms containing the expression cassette described in b2);

[0037] b7) Recombinant microorganisms containing the recombinant vector described in b3);

[0038] b8) Recombinant microorganisms containing the recombinant vector described in b4);

[0039] b9) Transgenic cell lines containing the nucleic acid molecules described in b1);

[0040] b10) Transgenic cell lines containing the expression cassette described in b2);

[0041] b11) Transgenic cell lines containing the recombinant vector described in b3);

[0042] b12) Transgenic cell lines containing the recombinant vector described in b4).

[0043] In some embodiments of the present invention, the nucleic acid molecule is any one of c1) to c2) and contains a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region;

[0044] c1) The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:1; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:9;

[0045] The nucleotide sequence defined in c2) has 80%, 85%, or 90% or more homology with the nucleotide sequence defined in c1) and encodes the nucleic acid molecule of the antibody or its antigen-binding fragment.

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

[0047] In some embodiments of the present invention, the expression cassette includes a 5' transcriptional control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR), and a transcription termination signal.

[0048] In some embodiments of the present invention, the 5' transcriptional control region includes a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeper" promoter may be used), a transcription start site, an enhancer, and / or a silencing element.

[0049] In some embodiments of the invention, the 3'UTR may encode AU-rich elements, which are common regulators of mRNA stability via the 3'-5' exogenous pathway and are typically located in the 3'UTR. AU-rich elements may comprise one or more repeats of the sequence AUUUA. It may also comprise one or more so-called US2B elements having the sequence AUAUAU.

[0050] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0051] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0052] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0053] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0054] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0055] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0056] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0057] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0058] In some embodiments of the present invention, the cell lines include cell lines for expressing antigenic epitope peptides, such as Expi293F; and cell lines for preparing hybridomas, such as Sp-2 / 0. Those skilled in the art will expect that other common engineered cell lines in the art can also achieve the same or similar technical effects as the present invention.

[0059] In some embodiments of the present invention, the microorganisms include Escherichia coli Transetta (DE3). Those skilled in the art will expect that other common microorganisms in the art, such as engineered bacteria like Escherichia coli DH5α, Escherichia coli BL21, Bacillus subtilis, and yeast, can also achieve the same or similar technical effects as the present invention.

[0060] A fourth aspect of the present invention provides a protein conjugate comprising:

[0061] (A) at least one of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and the recombinant protein of the second aspect of the present invention; and

[0062] (B) Functional molecules that are connected to (A) (including but not limited to covalent linkage, coupling, attachment, and adsorption).

[0063] In some embodiments of the present invention, the functional molecule comprises at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0064] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0065] In some embodiments of the present invention, the functional molecule is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0066] A fifth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the recombinant protein of the second aspect of the present invention, the biological material of the third aspect of the present invention, or the protein conjugate of the fourth aspect of the present invention in c1) to c4):

[0067] c1) Prepare products for detecting porcine acute diarrhea syndrome coronavirus;

[0068] c2) Prepare products for the prevention of porcine acute diarrhea syndrome coronavirus infection;

[0069] c3) Prepare products for the treatment and / or prevention of diseases caused by porcine acute diarrhea syndrome coronavirus infection;

[0070] c4) Prepare a sample to detect the presence or level of the S protein of porcine acute diarrhea syndrome coronavirus.

[0071] In some embodiments of the present invention, the product includes at least one of reagents, reagent kits, drugs (including vaccines), detection plates, and detection chips.

[0072] A sixth aspect of the present invention provides a product comprising an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a recombinant protein of the second aspect of the present invention, a biomaterial of the third aspect of the present invention, or a protein conjugate of the fourth aspect of the present invention.

[0073] 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.

[0074] A seventh aspect of the present invention provides a medicament comprising an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a recombinant protein of the second aspect of the present invention, a biological material of the third aspect of the present invention, or a protein conjugate of the fourth aspect of the present invention.

[0075] In some embodiments of the present invention, the drug includes a vaccine.

[0076] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0077] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0078] In some embodiments of the present invention, the drug further includes other active ingredients; other active ingredients include, but are not limited to, other drugs for treating or preventing acute diarrhea syndrome in pigs or other drug components with synergistic effects.

[0079] In some embodiments of the present invention, the dosage form of the drug includes at least one of capsules, tablets, microcapsule preparations, lyophilized powder injections, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0080] The beneficial effects of this invention are:

[0081] This invention provides an antibody or its antigen-binding fragment thereof, wherein the nucleotide sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:2; and the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:10. Neutralization assay results show that this antibody can completely neutralize 200 TCID at a concentration of 31.75 μg / mL. 50 The antibody contains SADS-CoV viral particles. It provides valuable research material for basic research on the molecular mechanisms of SADS-CoV invasion and for applied research on SADS-CoV detection and treatment.

[0082] Specifically, the isotype of the antibody (8E4 antibody) was determined, showing that the heavy chain of the 8E4 antibody is IgG1 and the light chain is κ. Indirect immunofluorescence assay revealed that the 8E4 antibody can react with SADS-CoV; however, Western blot results showed that the 8E4 antibody did not react with SADS-CoV particles or the S protein. Further studies indicated that the antigenic epitope of this antibody is a conformational epitope, and the binding ability of the antibody to the S protein was detected by BLI assay, showing that the 8E4 monoclonal antibody has a strong affinity for the S protein and is not easily dissociated after binding. The structure of the complex of 8E4 Fab and SADS-CoV S protein was resolved by cryo-electron microscopy. The electron density map showed that the 8E4 Fab binding site is located in the top region of the SADS-CoV S protein trimer, and this binding site is located in the CTD. The CTD is the main binding region of the surface receptor of SADS-CoV invading cells. Therefore, this 8E4 antibody can be used to develop drugs for the prevention and treatment of SADS-CoV, as well as for basic research on the molecular mechanism of SADS-CoV invasion. Attached Figure Description

[0083] Figure 1 The reaction of 8E4 antibody with SADS-CoV was detected by IFA; where A represents healthy control cells and B represents SADS-CoV infected cells.

[0084] Figure 2 The reaction of 8E4 antibody with SADS-CoV was detected by Western blot. In the figure, 1 represents the purified S protein expressed in eukaryotes, and 2 represents the cell lysate infected with SADS-CoV.

[0085] Figure 3 For the subtype identification of the purified 8E4 antibody.

[0086] Figure 4 This indicates the neutralizing activity of the 8E4 antibody.

[0087] Figure 5 To detect the affinity of the 8E4 antibody for the S protein.

[0088] Figure 6 The figure shows the PCR amplification of the heavy and light chains of the 8E4 antibody. In the figure, 1 represents the amplification band of the heavy chain of the 8E4 antibody, and 2 represents the amplification band of the light chain of the 8E4 antibody.

[0089] Figure 7 The expression and purification of 8E4 antibody Fab is shown in the figure. 1 represents the unreduced 8E4 antibody Fab protein, and 2 represents the reduced 8E4 antibody Fab protein.

[0090] Figure 8 Electron density diagram of the SADS-CoV S protein complex with 8E4 Fab. Detailed Implementation

[0091] The present invention will be further described in detail below through specific embodiments.

[0092] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0093] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0094] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0095] Cells, experimental animals, antibodies, and reagents: The SADS-CoV strain was isolated and cultured in our laboratory (GenBank accession number MG557844); SP2 / 0 and Vero cell lines were preserved by the New Detection Technology Team of the Guangdong Provincial Institute of Laboratory Animal Monitoring; Balb / c mice were purchased by the Laboratory Animal Center of our institute (permitted by the Laboratory Animal Use and Management Committee of the Guangdong Provincial Institute of Laboratory Animal Monitoring, IACUC2021167). The SBA mouse monoclonal antibody typing kit (HRP marker) SBA clonotyping System-HRP was purchased from Southern Biotech.

[0096] Example 1

[0097] The S protein of coronaviruses plays a crucial role in viral invasion of cells and is also a major target protein for neutralizing antibodies, making it an important target for the prevention and control of SADS-CoV. Therefore, in this example, mice were immunized with the S protein to obtain a hybridoma cell line with strong neutralizing activity against SADS-CoV, named the 8E4 hybridoma cell line.

[0098] Determination of the stability of monoclonal antibodies secreted by the 1.8E4 cell line

[0099] Positive hybridoma cells frozen for 3 months were revived, and the cell supernatant was tested for positivity to determine whether the revived positive cells had the ability to secrete antibodies. Hybridoma cells with a 100% positivity rate after 3 subcloning were continuously passaged in vitro for 3 months, and the cell supernatant was subjected to IFA testing every 2 weeks.

[0100] The IFA assay procedure was as follows: Vero cells were seeded in 96-well plates. After forming a monolayer, the cells were infected with SADS-CoV at a fold increase (MOI) of 0.1. When cytopathic effect (CPE) occurred, the cells were fixed with 4% paraformaldehyde at 4°C for 10 min and blocked with 2 wt% bovine serum albumin for 2 h. The cells were then incubated with the culture supernatant of 8E4 hybridoma cell line at 37°C for 1 h. After washing three times, Alexa Fluor 488 conjugated with goat anti-mouse IgG (diluted 1:2000) was incubated at 37°C for 45 min. Finally, the cell nuclei were stained with DAPI (Sigma), and cell images were acquired using a confocal laser scanning microscope (Zeiss, JENA, Germany).

[0101] like Figure 1 As shown, IFA screening of the culture supernatant of 8E4 hybridoma cell line revealed that the monoclonal antibody supernatant produced by 8E4 hybridoma cell line could react with cells inoculated with SADS-CoV, but could not react with negative serum. Furthermore, the antibody secretion ability of hybridoma cells was stable and showed good expression even after continuous passage.

[0102] 2. Western blot analysis of monoclonal antibodies

[0103] SADS-CoV S protein (amino acid sequence as SEQ ID NO:23) and SADS-CoV viral particles were subjected to SDS-PAGE electrophoresis and then semi-dry-transferred onto a PVDF membrane. The PVDF membrane was blocked with 5 wt% skim milk and incubated at room temperature with 8E4 hybridoma cell culture supernatant as the primary antibody and goat anti-mouse IgG-HRP (diluted 1:2000) as the secondary antibody. Finally, specific proteins were imaged using electrochemiluminescence (ECL) reagents and an Azure c600 bioanalytical imaging system (Azure Biosystems, USA).

[0104] like Figure 2 As shown, the culture supernatant of the 8E4 hybridoma cell line was used as the primary antibody for Western blot detection. The results showed that the monoclonal antibody produced by the 8E4 hybridoma cell line could not react with the eukaryotic S protein and viral particles expressed by SDS-PAGE gel electrophoresis. Therefore, the monoclonal antibody produced by the 8E4 hybridoma cell line could not be used for Western blot detection, proving that the binding epitope of the monoclonal antibody produced by the 8E4 hybridoma cell line is a conformational epitope.

[0105] 3. Preparation and purification of monoclonal antibody ascites fluid

[0106] Eight-week-old BALB / c mice were intraperitoneally injected with Freund's incomplete adjuvant (0.5 mL per mouse). Seven days later, hybridoma cells in good growth condition that could secrete 8E4 antibodies were injected at a rate of approximately 5 × 10⁶ cells per mouse. 5 One cell was injected into the peritoneal cavity of BALB / c mice. Around day 7, the mice's abdomens began to swell significantly. Ascites fluid was repeatedly aspirated, centrifuged at 2000 rpm for 10 minutes to remove cellular components and other precipitates, and the supernatant was collected. The ascites fluid was purified according to the HiTrap Protein G HP (GE Healthcare) antibody purification instructions, then aliquoted and stored at -80°C to obtain the anticlonal antibody, named 8E4 monoclonal antibody (8E4 antibody).

[0107] The sequence of the 8E4 antibody was identified.

[0108] The nucleotide sequence of the heavy chain variable region of the 8E4 antibody is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2; the nucleotide sequence of CDR1 in its heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4; the nucleotide sequence of CDR2 is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; the nucleotide sequence of CDR3 is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The nucleotide sequence of the light chain variable region of the 8E4 antibody is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10; the nucleotide sequence of CDR1 in its heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12; the nucleotide sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14; the nucleotide sequence of CDR3 is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16.

[0109] Nucleotide sequence of the variable region of the 8E4 antibody heavy chain:

[0110] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGTTATGCTATTCACTGGGTGAAGCAGAGTCCTGGGCAGGGCCTTGAGTGGATTGGATATGTTAATCCTTACAATGATGGTACTGAATACAA TGGGAAATTCAGAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAGCGCTATGATGGTTACTACAGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:1);

[0111] CDR1: GGATACACATTCACTAGTTATGCT (SEQ ID NO: 3);

[0112] CDR2: GTTAATCCTTACAATGATGGTACT (SEQ ID NO: 5);

[0113] CDR3: GCAAGAGAGCGCTATGATGGTTACTACAGGTTTACTTAC (SEQ ID NO:7);

[0114] Amino acid sequence of the heavy chain variable region of the 8E4 antibody:

[0115] EVQLQQSGPELVKPGASVKMSCKASGYTFTSYAIHWVKQSPGQGLEWIGYVNPYNDGTEYNGKFRGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARERYDGYYRFTYWGQGTLVTVSA (SEQ ID NO:2);

[0116] CDR1: GYTFTSYA (SEQ ID NO:4);

[0117] CDR2: VNPYNDGT (SEQ ID NO:6);

[0118] CDR3: ARERYDGYYRFTY (SEQ ID NO:8);

[0119] Nucleic acid sequence of the light chain variable region of the 8E4 antibody:

[0120] GACATTGTGTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATTCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCCATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGCTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:9);

[0121] CDR1: GAAAGTGTTGATAGTTATGGCCATAGTTTT (SEQ ID NO:11);

[0122] CDR2: CGTGCATCC (SEQ ID NO:13);

[0123] CDR3: CAGCAAAGTAATGAGGATCGGACG (SEQ ID NO: 15);

[0124] The amino acid sequence of the variable region of the 8E4 antibody light chain:

[0125] DIVLTQSPASLAVSLGQRATIFCRASESVDSYGHSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDRTFGGGTKLEIK(SEQ ID NO:10);

[0126] CDR1:ESVDSYGHSF(SEQ ID NO:12);

[0127] CDR2: RAS (SEQ ID NO:14);

[0128] CDR3: QQSNEDRT (SEQ ID NO:16).

[0129] Example 2: Identification of 8E4 antibody subtypes

[0130] The monoclonal antibodies obtained in the above experiments were identified according to the instructions of the subtype identification kit (SBA mouse monoclonal antibody typing kit (HRP labeling) SBAclonotyping System-HRP purchased from Southern Biotech).

[0131] like Figure 3 As shown, the 8E4 antibody was identified as an IgG1 subtype, and the light chain was a κ chain.

[0132] Example 3: Assay of 8E4 antibody neutralizing activity

[0133] 8E4 antibody ascites fluid was filtered through a 0.22 μm filter and then purified using a Protein G purification column. The purified antibody was diluted to 2 mg / mL, followed by a 2-fold serial dilution with DMEM culture medium; SADS-CoV was diluted to 200 TCID50 per 50 μL of viral fluid. 50 Mix 50 μL of virus solution with 50 μL of antibody dilution solution in equal volumes, react at 37°C for 1 hour, add trypsin to a final concentration of 8 μg / mL, inoculate cells, and culture in a cell culture incubator for 3 days to observe cytopathic effects.

[0134] Neutralization assays showed that the 8E4 antibody at 31.75 μg / mL could completely neutralize 200 TCIDs. 50The antibody has high neutralizing activity against SADS-CoV virus particles. Figure 4 ).

[0135] Example 4: Bio-Layer Interferometry (BLI)

[0136] First, the 8E4 antibody was diluted to 11 μg / mL. The purified S protein was serially diluted from 200 nM to 3.125 nM in a 2-fold gradient. After dilution, the samples were then subjected to the following procedures: the biosensor Protein A was soaked in PBST containing 1 mg / mL BSA for 10 min; the sensor was incubated in the wells containing the antibody to be bound for 600 s, followed by equilibration in binding buffer for 120 s to wash away unbound antibody; the sensor was incubated with different concentrations of diluted S protein for 120 s to detect antibody-S protein binding; finally, the sensor was dissociated in binding buffer for 300 s. The dissociation of antibody and S protein was observed. The affinity of the 8E4 antibody for the S protein was detected by the BLI assay.

[0137] The binding characteristics of 8E4 antibody to SADS-CoV S protein were analyzed by BLI affinity assay. The results showed that 8E4 antibody has a high affinity for the S protein, with an equilibrium dissociation constant of less than 10. -12 It has high affinity and will not be dissociated by the dissociation fluid after binding. Figure 5 ).

[0138] Example 5: Construction of 8E4 antibody IgG eukaryotic expression plasmid

[0139] 1. Constructing the 8E4 antibody IgG eukaryotic expression plasmid

[0140] RNA was extracted from hybridoma cell line 8E4 according to the Simply P Total RNA Extraction Kit instructions and then processed using PrimeScript. TM RT Master Mix reverse transcription reagent was used to obtain cDNA. Using cDNA as a template, PCR was performed according to the La Taq enzyme instructions to amplify the light and heavy chain variable region gene sequence of the 8E4 antibody. The PCR product was ligated into the pMD18-T vector. After successful sequencing, the amplified variable region gene was inserted into the pcDNA3.4 vector containing the heavy and light chain constant region backbone of the mouse antibody IgG, and then sequenced.

[0141] Amplification heavy chain primer VH-F: 5'-TTTTAAAAGGTGTCCAGTGT-3' (SEQ ID NO:17);

[0142] VH-R: 5'-AGAAGGTGTGCACACCGCTGGA-3' (SEQ ID NO: 18).

[0143] Amplification of light chain primer VL-F: 5'-GTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID NO:19);

[0144] VL-R: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID NO: 20).

[0145] PCR amplification of the 8E4 antibody yielded amplified fragments of the heavy and light chains. The PCR products were ligated into the pMD18-T vector. After successful sequencing, the amplified variable region gene was inserted into the pcDNA3.4 vector containing the heavy and light chain constant region backbones of mouse antibody IgG, resulting in eukaryotic IgG sequences expressing the 8E4 heavy and light chains. Figure 6 ).

[0146] Construction, expression, and purification of the antigen-binding fragment (Fab) of the 2.8E4 antibody.

[0147] Primers were designed for the correctly sequenced heavy chain IgG eukaryotic expression plasmid. Using this plasmid as a template, PCR was performed according to the La Taq enzyme instructions to construct a plasmid expressing the 8E4 Fab-His tag for the heavy chain, which was then sequenced. After successful sequencing, the antibody heavy chain Fab and light chain IgG were co-transfected into Expi293F cells for expression and purification. Specific procedures: The antibody IgG heavy chain Fab and light chain expression plasmid were transfected into Expi293F cells using PEI transfection reagent to express the antibody Fab. The transfected cells were then cultured in a shaker containing 8% CO2 at 33°C and 120 rpm. Five days after transfection, the cells were centrifuged at 4000g for 10 min, and the cell culture supernatant was collected. The cell supernatant was filtered through a 0.22 μM filter membrane and then purified using the AKTA protein purification system and a HiTrapTALON crude column Fab chromatography column. The specific steps involve binding the sample to the chromatography column, then washing the column with 5mM phosphate buffer (pH 8.0) containing 5mM imidazole, and finally eluting with a linear gradient of 500mM-5mM imidazole phosphate buffer (pH 8.0). The purified mAb Fab protein is identified by SDS-PAGE and stored at -80°C.

[0148] Primers for amplifying the Fab region of the 8E4 antibody heavy chain:

[0149] H-Fab-F: 5'-GGACACCACCACCACCATCATTGATTCTAGAGTCGACAATCA-3' (SEQ ID NO: 21);

[0150] H-Fab-R: 5'-TCAATGATGGTGGTGGTGGTGTCCGCCGCCGCAGTCGCGGGGCAC-3' (SEQ ID NO: 22).

[0151] The heavy chain Fab and light chain plasmids expressing antibody IgG were transfected into Expi293F to express antibody Fab. The results are shown in Figure 7, and Fab of 8E4 antibody was successfully obtained.

[0152] Example 6: Cryo-electron Microscopy Sample Preparation and Sieving

[0153] (1) Carry the network Au-300 mesh R1.2 / R1.3 was subjected to hydrophilic treatment (current and time were set to 15mA and 30s, respectively).

[0154] (2) The SADS-CoV S protein and 8E4 antibody Fab (as determined in Example 5) were mixed in a molar ratio of 1:1 and incubated on ice for 1 min. The final concentration of S protein was 1.5 mg / mL and the final concentration of 8E4 antibody Fab was 0.5 mg / mL.

[0155] (3) Pre-cool the metal vessel with liquid nitrogen. After pre-cooling, introduce gaseous ethane into the copper cup to liquefy it at the bottom of the copper cup. When the liquid ethane level is about 3-5 mm away from the mouth of the copper cup, turn off the ethane switch and remove the tube.

[0156] (4) Turn on the Vitrobot instrument. When the sample preparation temperature is 4℃ and the humidity is 100%, add filter paper to start sample preparation. Set the filter paper adsorption time (blot time) to 2.5s and the adsorption force (blot force) to 4.

[0157] (5) Use Vitrobot’s special tweezers to pick up the net and then fix the tweezers to Vitrobot’s robotic arm.

[0158] (6) Take 3 μL of the above antigen-antibody complex, load it onto the grid, start the Vitrobot program to remove excess sample from the grid, and quickly freeze it in liquid ethane.

[0159] (7) Quickly transfer the carrier from liquid ethane to liquid nitrogen and place the carrier in the sample box in the predetermined order.

[0160] (8) After all samples are prepared, use the “O” ring and the “C” ring to perform a snap ring operation.

[0161] (9) After the sample is clamped, it is placed in a special storage box for cryo-electron microscopy samples and frozen in liquid nitrogen for later use.

[0162] (10) The samples were screened by a 200keV Talos Arctica cryo-electron microscope.

[0163] (11) Data were collected from qualified samples using a 200keV Talos Arctica cryo-electron microscope.

[0164] (12) The electron microscopy data were processed using cryoSPARC 4.0 and RELION 4.0 software to obtain high-resolution cryo-electron microscopy density maps.

[0165] (13) The electron density map with ideal resolution obtained is used to build, correct and analyze the model using software such as Chimera, Coot and PHENIX, and finally obtain the atomic resolution structural information of the target protein.

[0166] Electron microscopy images collected by a 200 keV cryo-electron microscope were imported into the CryoSPARC processing system for processing. After a series of processes including particle extraction, 2D classification, and 3D classification, an electron density map with a resolution of 3.2 angstroms was obtained. Figure 8 Using UCSF ChimeraX 1.6.1, the domains of the 8E4 antibody were divided and mapped. It can be seen that the top of the CTD binds to the Fab. From a top view, the Fab of the 8E4 antibody can completely cover the CTD region after binding to the CTD, indicating that the antigenic epitope of the 8E4 antibody is located at the CTD site of the S protein.

[0167] 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, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An antibody or antigen-binding fragment thereof that neutralizes porcine acute diarrhea syndrome coronavirus, characterized in that, The antibody or its antigen-binding fragment has a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each contain complementarity-determining regions CDR1, CDR2 and CDR3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively.

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) The amino acid sequence as shown in SEQ ID NO:2; or a2) It has at least 90% homology with SEQ ID NO:2 and has the same amino acid sequence 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: a3) The amino acid sequence as shown in SEQ ID NO:10; or a4) has at least 90% homology with SEQ ID NO:10 and has the same amino acid sequence as the protein shown in SEQ ID NO:

10.

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

4. A recombinant protein comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; and a tag sequence for assisting expression and / or purification.

5. A biological material relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4, wherein the biological material is any one of b1) to b5): b1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3 or the recombinant protein as described in claim 4; b2) An expression cassette containing the nucleic acid molecule described in b1); b3) A recombinant vector containing the nucleic acid molecule described in b1); b4) Recombinant microorganisms containing the nucleic acid molecules described in b1); b5) Transgenic cell lines containing the nucleic acid molecules described in b1).

6. The biomaterial according to claim 5, characterized in that, The biomaterial is any one of b6) to b8): b6) A recombinant vector containing the expression cassette described in b2); b7) Recombinant microorganisms containing the expression cassette described in b2); b8) Transgenic animal cell lines containing the expression cassette described in b2).

7. The biomaterial according to claim 5, characterized in that, The biomaterial is either b9 or b10. b9) Recombinant microorganisms containing the recombinant vector described in b3); b10) Transgenic animal cell lines containing the recombinant vector described in b3).

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the recombinant protein as described in claim 4, or the biological material as described in any one of claims 5 to 7 in c1) to c4): c1) Prepare products for detecting porcine acute diarrhea syndrome coronavirus; c2) Prepare products for the prevention of porcine acute diarrhea syndrome coronavirus infection; c3) Prepare products for the treatment and / or prevention of diseases caused by porcine acute diarrhea syndrome coronavirus infection; c4) Prepare a product for detecting the presence or level of the S protein of porcine acute diarrhea syndrome coronavirus in a sample.

9. A reagent comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the recombinant protein as described in claim 4, or the biological material as described in any one of claims 5 to 7.

10. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the recombinant protein as described in claim 4, or the biological material as described in any one of claims 5 to 7.

11. A detection plate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, a recombinant protein as described in claim 4, or a biological material as described in any one of claims 5 to 7.

12. A detection chip comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, a recombinant protein as described in claim 4, or a biological material as described in any one of claims 5 to 7.

13. A drug comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the recombinant protein as described in claim 4, or the biological material as described in any one of claims 5 to 7.

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