Monoclonal antibody capable of recognizing conformational epitope and neutralizing porcine acute diarrhea syndrome coronavirus and application of monoclonal antibody

Abstract

The invention belongs to the technical field of biology, discloses a monoclonal antibody capable of recognizing conformational epitopes and neutralizing porcine acute diarrhea syndrome coronavirus and application of the monoclonal antibody, and particularly discloses an antibody or an antigen binding fragment thereof. The antibody provided by the invention can neutralize 200 SADS-CoV virions of TCID50 under the concentration of 31.75 mu g / mL, the antibody is detected to have very strong affinity with S protein through a BLI test, the antibody is not easy to dissociate after being combined, and an important material is provided for research and development of SADS-CoV neutralizing antibody antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological industry, and specifically relates to a monoclonal antibody for neutralizing porcine acute diarrhea syndrome coronavirus and an application thereof, which recognizes a conformational epitope. Background Art

[0002] Porcine acute diarrhea syndrome coronavirus (SADS CoV) broke out in four large-scale pig farms in Guangdong Province in just three months in 2017, causing the death of about 25,000 piglets. The mortality rate of infected piglets under five days old can be as high as 90%. It is one of the important pathogens causing diarrhea in piglets, and the virus has a potential risk of cross-species transmission. However, there are currently no vaccines and antiviral agents for SADS-CoV in clinical practice. Therefore, it is crucial to develop antiviral agents for SADS-CoV.

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

[0004] Neutralizing antibodies usually refer to antibodies that can significantly reduce or even completely eliminate the pathogen's ability to infect after binding to the pathogen. They are the main factor in the body's antiviral immunity. Compared with other therapeutic drugs, they can accurately bind to important functional areas of viral proteins to block their invasion of host cells. They also have the advantages of strong specificity and high sensitivity. Therefore, neutralizing antibodies are the focus of antiviral drug development. However, there are currently no monoclonal antibodies with good neutralizing titers. Summary of the invention

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

[0006] The second aspect of the present invention aims to provide a recombinant protein.

[0007] The 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] The fourth aspect of the present invention aims to provide a protein conjugate.

[0009] The purpose of the fifth aspect of the present invention is to provide an application 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 biomaterial of the third aspect of the present invention or the protein conjugate of the fourth aspect of the present invention.

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

[0011] The seventh aspect of the present invention aims to provide a medicine.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] Porcine acute diarrhea syndrome coronavirus can cause vomiting, severe diarrhea and death in newborn piglets. Its spike (S) protein plays a vital role in promoting virus invasion and inducing the production of neutralizing antibodies. Therefore, the inventors immunized mice with S protein, and obtained a cell line that can stably secrete anti-(SADS CoV) in the study of preparing monoclonal antibodies against SADS-CoV S protein, named 8E4 hybridoma cell line. By injecting the hybridoma cell line into the peritoneal cavity of mice, a monoclonal antibody was obtained, named 8E4 monoclonal antibody (8E4 antibody).

[0014] The first aspect of 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, and the heavy chain variable region and the light chain variable region each contain complementary determining regions CDR1, CDR2, and CDR3;

[0015] The amino acid sequences of CDR1, CDR2, and CDR3 of 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 of 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 antigen-binding fragment thereof comprises:

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

[0019] a2) 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;

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

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

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

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

[0024] The definition of "chimeric antibody" refers to a protein molecule designed by recombining the variable regions of the light and heavy chains of immunoglobulins of non-human species with the constant regions of human immunoglobulin molecules. Although the embodiment of the present invention only provides a mouse antibody, those skilled in the art can still analyze the corresponding CDR sequence based on the sequence of the heavy and light chain variable regions, and replace the other parts with the constant regions and framework regions (framework region, FR) of other animal sources based on the CDR sequence, thereby obtaining antibodies of other animal sources, and it can be expected to have similar or identical technical effects to the present invention. Preferably, the antibodies of other animal origin include at least one of human antibodies and porcine antibodies.

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

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

[0027] The antibody provided by the present invention can neutralize 200 TCID at a concentration of 31.75 μg / mL. 50 The BLI test showed that the antibody has a strong affinity with the S protein and is not easy to dissociate after binding, providing important materials for the development of SADS-CoV neutralizing antibody antiviral drugs.

[0028] The second aspect of the present invention provides a recombinant protein, comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and an optional tag sequence for facilitating 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; further 6×His tag.

[0030] The third aspect of the present invention provides a biological material 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, wherein the biological material is any one of b1) to b12):

[0031] b1) a nucleic acid molecule encoding 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;

[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) a recombinant microorganism containing the nucleic acid molecule described in b1);

[0036] b6) a recombinant microorganism containing the expression cassette described in b2);

[0037] b7) a recombinant microorganism containing the recombinant vector described in b3);

[0038] b8) a recombinant microorganism containing the recombinant vector described in b4);

[0039] b9) a transgenic cell line containing the nucleic acid molecule described in b1);

[0040] b10) a transgenic cell line containing the expression cassette described in b2);

[0041] b11) a transgenic cell line containing the recombinant vector described in b3);

[0042] b12) A transgenic cell line 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 comprises 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] c2) A nucleic acid molecule that has 80%, 85% or 90% or more homology with the nucleotide sequence defined in c1) and encodes the antibody or antigen-binding fragment thereof.

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

[0047] In some embodiments of the present invention, the expression cassette comprises a 5' transcription 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' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0049] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0050] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

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

[0052] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

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

[0054] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, a F cosmid, 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 species of plants or animals.

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

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

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

[0061] (A) 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; and

[0062] (B) a functional molecule connected to (A) (including but not limited to covalently linked, coupled, attached, adsorbed).

[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 a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0065] In some embodiments of the present invention, the functional molecules are selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents or enzymes capable of producing detectable products, radionuclides, biological toxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0066] The 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 biomaterial 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) Preparation of products for detecting porcine acute diarrhea syndrome coronavirus;

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

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

[0070] c4) Preparation for detecting the presence or level of S protein of porcine acute diarrhea syndrome coronavirus in a sample.

[0071] In some embodiments of the present invention, the product includes at least one of a reagent, a test kit, a drug (including a vaccine), a test plate, and a test chip.

[0072] The sixth aspect of the present invention provides a product, comprising 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 biomaterial of the third aspect of the present invention, or the 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 test plate, a test kit, and a test chip.

[0074] The seventh aspect of the present invention provides a drug, comprising 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 biomaterial of the third aspect of the present invention, or the protein conjugate of the fourth aspect of the present invention.

[0075] In some embodiments of the invention, the medicament comprises a vaccine.

[0076] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

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

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

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

[0080] The beneficial effects of the present invention are:

[0081] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the nucleotide sequence of the heavy chain variable region of the antibody or the antigen-binding fragment thereof is shown in SEQ ID NO: 2; the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 10. The neutralization test results show that the antibody can completely neutralize 200 TCID 50 The antibody can be used in basic research on the molecular mechanism of SADS-CoV invasion and provide good scientific research materials for applied research on SADS-CoV detection and treatment.

[0082] Specifically, by determining the subtype of the antibody (8E4 antibody), the results showed that the heavy chain of the 8E4 antibody was IgG1 and the light chain was κ type. Indirect immunofluorescence detection found that the 8E4 antibody could react with SADS CoV; while Western blot results showed that the 8E4 antibody did not react with SADS CoV particles or S protein; further studies showed that the antigenic epitope of the antibody was a conformational epitope, and the binding ability of the antibody to the S protein was detected by BLI test. The results showed that the 8E4 monoclonal antibody had a strong affinity with the S protein and was not easy to dissociate after binding. The complex structure of 8E4 Fab and SADS-CoV S protein was analyzed by cryo-electron microscopy. The electron density map showed that the 8E4 Fab binding site was located in the top region of the SADS-CoV S protein trimer, and the binding site was located in CTD. CTD is the main binding region of the surface receptor of SADS-CoV invading cells, so the 8E4 antibody can be used for the development of preventive and therapeutic drugs for SADS CoV, as well as basic research on the molecular mechanism of SADS CoV invasion. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 IFA was used to detect the reaction between 8E4 antibody and SADS-CoV; A is a healthy control cell and B is a SADS-CoV infected cell.

[0084] Figure 2 Western blot was used to detect the reaction between 8E4 antibody and SADS-CoV. In the figure, 1 represents the eukaryotic expressed and purified S protein, and 2 represents the cell lysate infected with SADS-CoV.

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

[0086] Figure 4 The neutralizing activity of 8E4 antibody.

[0087] Figure 5 This is a test of the affinity of 8E4 antibody to S protein.

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

[0089] Figure 7 This is the expression and purification of 8E4 antibody Fab. In the figure, 1 represents the non-reduced 8E4 antibody Fab protein, and 2 represents the reduced 8E4 antibody Fab protein.

[0090] Figure 8 This is the electron density map of the SADS-CoV S protein and 8E4 Fab complex. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below through specific examples.

[0092] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0093] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0094] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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

[0096] Example 1

[0097] The S protein of coronavirus plays a key role in the process of virus invasion of cells, is also the main target protein of neutralization, and is an important target for the prevention and control of SADS-CoV. Therefore, in this embodiment, mice were immunized with S protein to obtain a SADS-CoV monoclonal antibody hybridoma cell line with strong neutralizing activity, named 8E4 hybridoma cell line.

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

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

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

[0101] like Figure 1 As shown, the culture supernatant of 8E4 hybridoma cell line was subjected to IFA screening test and it was found that the monoclonal antibody supernatant produced by 8E4 hybridoma cell line could react with cells infected with SADS-CoV, but could not react with negative serum. In addition, the hybridoma cells had stable antibody secretion ability and good expression after continuous passaging.

[0102] 2. Western-blot analysis of monoclonal antibodies

[0103] After SDS-PAGE electrophoresis, the S protein of SADS-CoV (amino acid sequence such as SEQ ID NO: 23) and SADS-CoV virus particles were semi-dry transferred to a PVDF membrane, and the PVDF membrane was blocked with 5wt% skim milk, and then incubated with 8E4 hybridoma cell line culture supernatant as the first antibody and goat anti-mouse IgG-HRP (dilution 1: 2000) as the second antibody at room temperature. Finally, electrochemiluminescence (ECL) reagents and Azure c600 bioanalysis imaging system (Azure Biosystems, USA) were used to image specific proteins.

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

[0105] 3. Preparation and Purification of Monoclonal Antibody Ascites

[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 that were growing well and could secrete 8E4 antibodies were inoculated at a rate of about 5 × 10 5 The cells were injected into the peritoneal cavity of BALB / c mice. Around 7 days, the abdomen of the mice began to swell significantly. The ascites was repeatedly extracted and centrifuged at 2 000r / min for 10 minutes to remove the cell components and other precipitates, and the supernatant was collected. The ascites was purified according to the HiTrap Protein G HP (GE Healthcare) antibody purification instructions, then aliquoted and stored at -80°C to obtain the anti-clonal antibody, which was 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 of the 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 of the 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 heavy chain variable region of the 8E4 antibody:

[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 light chain variable region of the 8E4 antibody:

[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 subtypes of 8E4 antibody

[0130] The subtype identification of the monoclonal antibody obtained in the above experiment was performed according to the operating instructions of the subtype identification kit (SBA mouse monoclonal antibody typing kit (HRP labeled) SBAclonotyping System-HRP purchased from Southern Biotech).

[0131] like Figure 3 As shown, the 8E4 antibody was subjected to subtype identification, and the results showed that the 8E4 antibody was of IgG1 subtype, and the light chain was a κ chain.

[0132] Example 3 8E4 Antibody Neutralization Activity Determination

[0133] The 8E4 antibody ascites was filtered through a 0.22 μm filter and purified using a protein G purification column. The purified antibody was diluted to 2 mg / mL and then diluted 2-fold with DMEM culture medium; SADS-CoV was diluted to contain 200 TCID per 50 μL virus solution. 50 , 50 μL of virus solution and 50 μL of antibody diluent were mixed in equal volumes, reacted at 37°C for 1 h, trypsin was added, the final concentration of trypsin was 8 μg / mL, cells were inoculated, cultured in a cell culture incubator for 3 days, and the cytopathic effect was observed.

[0134] Neutralization assay showed that 8E4 antibody could completely neutralize 200 TCID 50SADS-CoV virus particles, the antibody has a high neutralizing activity ( Figure 4 ).

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

[0136] First, the 8E4 antibody was diluted to 11 μg / mL, and the purified S protein was diluted from 200 nM to 3.125 nM in a 2-fold gradient dilution. After the sample was diluted, it was operated on the machine. The specific operation steps were as follows: the biosensor Protein A was soaked with PBST containing 1 mg / mL BSA for 10 minutes; the sensor was incubated in the antibody well to be bound for 600 seconds, and then balanced in the binding buffer for 120 seconds to wash away the unbound antibody; incubated with dilutions of different concentrations of S protein for 120 seconds to detect the binding of the antibody and S protein, and finally the sensor was dissociated in the binding buffer for 300 seconds. Observe the dissociation of the antibody and S protein. The affinity of the 8E4 antibody to the S protein was detected by BLI experiment.

[0137] The binding characteristics of 8E4 antibody to SADS-CoV S protein were detected by BLI affinity test. The experimental results showed that 8E4 antibody has high affinity for S protein, and the equilibrium dissociation constant is less than 10 -12 , high affinity, and will not be dissociated by the dissociation solution after binding ( Figure 5 ).

[0138] Example 5 Construction of 8E4 Antibody IgG Eukaryotic Expression Plasmid

[0139] 1. Construction of 8E4 antibody IgG eukaryotic expression plasmid

[0140] The RNA of hybridoma cell line 8E4 was extracted according to the instructions of Simply P total RNA extraction kit and then stained with PrimeScript TM cDNA was obtained by reverse transcription using RT Master Mix reverse transcription reagent, and used as a template to perform PCR reaction according to the instructions of La Taq enzyme to amplify the light and heavy chain variable region gene sequences of 8E4 antibody, and the PCR products were connected to the pMD18-T vector. After the sequencing was correct, the amplified variable region genes were inserted into the pcDNA3.4 vector containing the heavy and light chain constant region skeletons of mouse antibody IgG for sequencing.

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

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

[0145] The heavy chain and light chain amplified fragments were obtained by PCR amplification of the 8E4 antibody, and the PCR products were connected to the pMD18-T vector. After the sequencing was correct, the amplified variable region genes were inserted into the pcDNA3.4 vector containing the heavy chain and light chain constant region skeleton of the mouse antibody IgG to obtain the eukaryotic expression of the 8E4 heavy chain and light chain IgG sequence ( Figure 6 ).

[0146] 2. Construction, expression and purification of the antigen-binding fragment (Fab) of the 8E4 antibody

[0147] Primers were designed for the heavy chain IgG eukaryotic expression plasmid with correct sequencing, and it was used as a template. PCR reaction was carried out according to the instructions of La Taq enzyme to construct a plasmid expressing the heavy chain 8E4 Fab-His tag and sequenced. After the sequencing was correct, the heavy chain Fab and light chain IgG of the antibody were co-transfected into Expi293 F cells for expression and purification. Specific operation method: The heavy chain Fab and light chain expression plasmids of the antibody IgG were transfected into Expi293F using PEI transfection reagent to express the antibody Fab, and the transfected cells were placed in a shaker containing 8% CO2, 33°C, and 120rpm for culture and expression. Five days after transfection, the cell culture supernatant was collected by centrifugation at 4000g for 10 minutes. The cell supernatant was filtered with a 0.22μM filter membrane, and then purified using the AKTA protein purification system and HiTrapTALON crude column chromatography column Fab. The specific steps are as follows: the sample is combined with the chromatography column, and then the chromatography column is washed with 5mM phosphate buffer (pH 8.0) containing 5mM imidazole, and finally eluted with a linear gradient of imidazole 500mM-5mM phosphate buffer (pH 8.0). The purified mAb Fab protein is identified by SDS-PAGE and stored in a -80°C refrigerator.

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

[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 FIG7 , and the Fab of 8E4 antibody was successfully obtained.

[0152] Example 6 Cryo-EM Sample Preparation and Screening

[0153] (1) Load the network Au-300 mesh-R1.2 / R1.3 (Au 300mesh R1.2 / 1.3) was subjected to hydrophilization treatment (the current and time were set to 15 mA and 30 s, respectively).

[0154] (2) SADS-CoV S protein and 8E4 antibody Fab (according to Example 5) were mixed at 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) Precool the metal kettle with liquid nitrogen. After precooling, 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 to 5 mm away from the mouth of the copper cup, turn off the ethane switch and remove the catheter.

[0156] (4) Turn on the Vitrobot instrument, and when the sample preparation temperature reaches 4°C and the humidity reaches 100%, add filter paper to start sample preparation; set the filter paper adsorption time (blot time) to 2.5 s and the adsorption force (blot force) to 4.

[0157] (5) Use the Vitrobot-specific tweezers to pick up the carrier mesh, and then fix the tweezers to the Vitrobot's robotic arm.

[0158] (6) 3 μL of the antigen-antibody complex was aspirated and loaded onto the grid. The Vitrobot program was started to remove excess sample on the grid and the sample was quickly frozen in liquid ethane.

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

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

[0161] (9) The sample with the ring is placed in a special storage box for cryo-EM samples and frozen in a liquid nitrogen tank for later use.

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

[0163] (11) The qualified samples were collected 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-EM density maps.

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

[0166] The electron micrographs collected by the 200keV cryo-EM were imported into the CryoSPARC processing system for processing. After a series of particle extraction, 2D classification, 3D classification and other processes, an electron density map with a resolution of 3.2 angstroms was obtained ( Figure 8 ). Using UCSF ChimeraX 1.6.1 to divide and map its domains, it can be seen that the top of the CTD is bound to the Fab. From a bird's-eye 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 in the CTD site of the S protein.

[0167] The above and the accompanying drawings have been described in detail the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises 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 complementary determining regions CDR1, CDR2, and CDR3; The amino acid sequences of CDR1, CDR2, and CDR3 of 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 of 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 antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: a1) the amino acid sequence shown in SEQ ID NO: 2; or a2) 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 antigen-binding fragment thereof comprises: a3) the amino acid sequence shown in SEQ ID NO: 10; or a4) an amino acid sequence that has at least 90% homology with SEQ ID NO: 10 and has the same function as the protein shown in SEQ ID NO:

10.

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

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

5. A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the recombinant protein according to claim 4, wherein the biological material is any one of b1) to b12): b1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the recombinant protein according to 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) a recombinant vector containing the expression cassette described in b2); b5) a recombinant microorganism containing the nucleic acid molecule described in b1); b6) a recombinant microorganism containing the expression cassette described in b2); b7) a recombinant microorganism containing the recombinant vector described in b3); b8) a recombinant microorganism containing the recombinant vector described in b4); b9) a transgenic cell line containing the nucleic acid molecule described in b1); b10) a transgenic cell line containing the expression cassette described in b2); b11) a transgenic cell line containing the recombinant vector described in b3); b12) A transgenic cell line containing the recombinant vector described in b4).

6. A protein conjugate comprising: (A) at least one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and the recombinant protein according to claim 4; as well as (B) a functional molecule linked to (A); Preferably, the functional molecule comprises at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

7. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4, the biomaterial according to claim 5 or the protein conjugate according to claim 6 in c1) to c4): c1) preparing a product for detecting porcine acute diarrhea syndrome coronavirus; c2) Preparation of products for the prevention of porcine acute diarrhea syndrome coronavirus infection; c3) Preparation of products for the treatment and / or prevention of diseases caused by porcine acute diarrhea syndrome coronavirus infection; c4) Preparation for detecting the presence or level of S protein of porcine acute diarrhea syndrome coronavirus in a sample.

8. A product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4, the biomaterial according to claim 5 or the protein conjugate 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.

10. A drug comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4, the biomaterial according to claim 5 or the protein conjugate according to claim 6.

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