Pig acute diarrhea syndrome coronavirus spike protein antigen epitope peptide and monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of biological industry, and particularly relates to a spike protein antigen epitope peptide of porcine acute diarrhea syndrome coronavirus as well as a monoclonal antibody and application of the spike protein antigen epitope peptide. The invention provides an antibody or an antigen binding fragment thereof. A nucleotide sequence of a heavy chain variable region of the antibody or the antigen binding fragment thereof is as shown in SEQ ID NO. 2; the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 4. The amino acid sequence of the minimum binding epitope is as shown in SEQ ID NO.22. The antigen epitope is highly conserved in a plurality of different subtypes of SADS-CoV. The antibody can be applied to fundamental research on an SADS-CoV invasive molecular mechanism and provides a good scientific research material for application research on SADS-CoV detection and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-industry, and specifically relates to a spike protein antigen epitope peptide of porcine acute diarrhea syndrome coronavirus, a monoclonal antibody thereof and an application thereof. Background Art

[0002] Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered porcine enteric coronavirus in 2017, belonging to the genus Alphacoronavirus of the family Coronaviridae. The clinical symptoms of sick pigs are similar to those of other porcine enteric coronaviruses, and the clinical manifestations of infected sows are mild diarrhea. Newborn piglets infected within 5 days of age develop acute diarrhea and vomiting, leading to acute death, with a mortality rate of up to 90%, so the prevention and control of SADS-CoV is crucial.

[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 tissue 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 shows that the SADS-CoV S protein belongs to the beta coronavirus group, and SADS-CoV may have originated from a mixed infection involving alpha coronavirus and beta coronavirus. Therefore, further research on the SADS coronavirus S protein is of great significance. However, there is currently no monoclonal antibody that can be used for western blotting detection of SADS-CoV S protein. Summary of the invention

[0004] The first aspect of the present invention aims to provide an antibody or antigen-binding fragment.

[0005] The second aspect of the present invention aims to provide a recombinant protein.

[0006] 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 and the recombinant protein of the second aspect of the present invention.

[0007] The fourth aspect of the present invention aims to provide an antigen epitope peptide.

[0008] The fifth aspect of the present invention aims to provide a protein conjugate.

[0009] The sixth aspect of the present invention aims to provide an application.

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

[0011] The purpose of the eighth aspect of the present invention is to provide a method for preparing 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.

[0012] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof;

[0014] 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;

[0015] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs. 30 to 32, respectively;

[0016] The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NOs. 36 to 38. Preferably, the antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region;

[0017] Preferably, 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.3; or

[0019] a2) an amino acid sequence having 90%, 95% or more homology with SEQ ID NO.3 and having the same function as the protein shown in SEQ ID NO.3;

[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.5; or

[0022] a4) an amino acid sequence that has 90%, 95% or more homology with SEQ ID NO.5 and has the same function as the protein shown in SEQ ID NO.5.

[0023] Preferably, the antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, mouse antibody, rat antibody, rabbit antibody, sheep antibody, monkey antibody, humanized antibody, chimeric antibody (e.g., human-mouse chimeric antibody) or bispecific antibody, multispecific antibody.

[0024] Preferably, the antibody or antigen-binding fragment thereof is selected from any one of Fab, Fab', F(ab')2, Fv, scFv, diabody, fusion antibody and intact antibody.

[0025] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof specifically binds to an amino acid sequence as shown in any one of SEQ ID NO. 6, 8, 11, 14, 15, 18, 19, 20, 21, and 22. Those skilled in the art will appreciate that antigens including the minimal epitope sequence (SEQ ID NO. 22) can bind to the monoclonal antibody.

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

[0027] Preferably, the antibody or antigen-binding fragment thereof is a neutralizing antibody.

[0028] In the first aspect of the present invention, the definition of "chimeric antibody" refers to a protein molecule designed by recombining the variable regions of immunoglobulins of non-human species with the constant regions of human immunoglobulin molecules. The definition of "humanization" refers to a protein molecule designed by recombining and designing only the complementary determining regions (CDRs) of the variable regions of immunoglobulin molecules from non-human species, and the remaining main variable regions and all constant regions are from human sources. 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 chain 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.

[0029] The second aspect of the present invention provides a recombinant protein comprising a tag and the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0030] Preferably, the tag molecules include but are not limited to His, GST, HA, Flag, and Myc.

[0031] 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 b5);

[0032] b1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention;

[0033] b2) an expression cassette containing the nucleic acid molecule described in b1);

[0034] b3) a recombinant vector containing the nucleic acid molecule described in b1) or the expression cassette described in b2);

[0035] b4) a recombinant microorganism containing the nucleic acid molecule described in b1) or the expression cassette described in b2) or the recombinant vector described in b3);

[0036] b5) A recombinant cell line containing the nucleic acid molecule described in b1) or the expression cassette described in b2) or the recombinant vector described in b3).

[0037] Preferably, the nucleic acid molecule is a DNA molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the recombinant protein described in the second aspect of the present invention.

[0038] Preferably, the DNA 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;

[0039] c1) the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.2; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4;

[0040] c2) A DNA 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.

[0041] Preferably, the recombinant cell line comprises a hybridoma cell.

[0042] Preferably, the hybridoma cell classification is named hybridoma cell line 5D6, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 6, 2024, with the deposit number GDMCC NO: 64961.

[0043] In some embodiments of the present invention, the vector includes a eukaryotic expression vector pCDNA3.1 and a prokaryotic expression vector pGEX-6p-1. Those skilled in the art can expect that other vectors in the art can also achieve the same or similar technical effects of the present invention.

[0044] 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.

[0045] 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.

[0046] The fourth aspect of the present invention provides an antigenic epitope peptide, which is derived from the S protein fragment of swine acute diarrhea syndrome coronavirus (SADS-CoV), including NTD-2b-6, and its amino acid sequence is shown in SEQ ID NO.22.

[0047] In some embodiments of the present invention, the antigen epitope peptide also includes S1, NTD, NTD-2, NTD-2b, NTD-2c, NTD-2b-2, NTD-2b-3, NTD-2b-4, NTD-2b-5, and NTD-2b-6, whose amino acid sequences are SEQ ID NO. 6, 8, 11, 14, 15, 18, 19, 20, 21, and 22, respectively.

[0048] In the first aspect of the present invention, the S protein includes the extracellular domain portion of the S protein, and its amino acid sequence is shown in SEQ ID NO. 1. Those skilled in the art will appreciate that in the amino acid sequence of the S protein, mutations or variations (including but not limited to, substitutions, deletions, and / or additions, such as S proteins of different genotypes or genotypes) may be naturally generated or artificially introduced without affecting its biological function, including the sequence shown in SEQ ID NO. 1 and other natural or artificial variants having 95%, 96%, 97%, 98%, 99%, 100% homology.

[0049] In the first aspect of the present invention, the "epitope peptide" refers to a peptide segment on an antigen that can be used as an epitope. In some embodiments of the present invention, a single epitope peptide can be specifically bound / recognized by an antibody against the epitope. In other embodiments of the present invention, other sequences containing the epitope peptide can also bind to the antibody or antigen-binding fragment described in the first aspect of the present invention.

[0050] In the first aspect of the present invention, the minimum epitope sequence of the antigenic epitope peptide capable of binding to an antibody is 136 STSHAAD 142(SEQ ID NO.22), the protein sequence with this epitope can be used as SADS-CoV antigen for the development and application of subsequent products such as antibodies and vaccines. For example, the amino acid sequences of SEQ ID NO.6, 8, 11, 14, 18, 19, 20, 21, and 22 can also be used for the development of subsequent products such as SADS-CoV antibodies and vaccines.

[0051] The fifth aspect of the present invention provides a protein conjugate, which comprises a conjugated portion and 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.

[0052] Preferably, the coupling part is selected from one or more of a label molecule, a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier, a liposome, and biotin.

[0053] In the fifth aspect of the present invention, the conjugate is used for tracing, purification, or enhancement. For example, by adding label molecules and biotin, subsequent protein purification is facilitated; by adding radionuclides and fluorescein, it is used to indicate the location of the antibody or antigen-binding fragment in the organism; by coupling with other drugs and cytokines, the immune effect is improved.

[0054] The sixth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the recombinant protein described in the second aspect of the invention, or the protein conjugate described in the fifth aspect of the invention in d1) to d3):

[0055] d1) Preparation of products for detecting porcine acute diarrhea syndrome coronavirus;

[0056] d2) Preparation of products for the prevention of porcine acute diarrhea syndrome coronavirus;

[0057] d3) Preparation of products for treating porcine acute diarrhea syndrome coronavirus.

[0058] Preferably, the product includes at least one of a reagent, a kit, a medicine, and a vaccine.

[0059] A seventh aspect of the present invention provides a product, comprising at least one of e1) to e4):

[0060] e1) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0061] e2) the recombinant protein according to the second aspect of the present invention;

[0062] e3) The biomaterial according to the third aspect of the present invention;

[0063] e4) the protein conjugate according to the fifth aspect of the present invention;

[0064] The product is selected from at least one of drugs, reagents, test plates, chips, test papers and test kits.

[0065] Preferably, the medicament comprises a pharmaceutically acceptable carrier and / or excipient.

[0066] Preferably, the medicament comprises a vaccine.

[0067] Preferably, the carrier comprises a solid carrier or a liquid carrier.

[0068] Preferably, the liquid carrier comprises: saline, buffer, glucose solution, aqueous solution, glycerol, ethanol.

[0069] Preferably, the solid carrier comprises at least one of low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, sucrose, sodium chloride and sorbitol.

[0070] Preferably, 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.

[0071] Preferably, 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.

[0072] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0073] Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, a carrier, a lubricant, a sweetener and a flavoring agent.

[0074] The eighth aspect of the present invention 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 of the present invention, comprising the following steps:

[0075] The method is obtained by culturing the recombinant microorganism or recombinant cell described in the third aspect of the present invention.

[0076] In some embodiments of the present invention, the steps of the method for preparing the recombinant cell are:

[0077] Administering an effective dose of the antigenic epitope peptide according to the fourth aspect of the present invention to an animal to induce an anti-SADS-CoV immune response in the animal;

[0078] Immune cells are separated from the animal, fused with myeloma cells, and positive cells are screened to obtain.

[0079] Preferably, the animal is a non-human animal, such as a rat, a mouse, a rabbit, a monkey, or a goat.

[0080] Preferably, the immune cells are spleen cells, and further are B cells.

[0081] Preferably, the myeloma cells have the characteristics of being sensitive to HAT, having strong antibody secretion ability, being easy to fuse, etc. In some embodiments of the present invention, mouse myeloma cells are used, such as MOP-21, MC-11, SP-2 / 0, X63-Ag8-653 cell lines; preferably, SP-2 / 0 cell line.

[0082] Preferably, the fusion method is specifically as follows: administering an immunogen (adjuvant may be added) to inject a suitable host animal, and the injection method may be subcutaneous multi-point injection or intraperitoneal injection. Adjuvants include Freund's adjuvant, Freund's incomplete adjuvant, Addavax adjuvant, etc., which can enhance the immunogenicity of the antigen in the host. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. The relevant lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG), and positive cells are screened to obtain hybridoma cells.

[0083] Preferably, the hybridoma cells are inoculated into a suitable culture medium for growth, wherein the culture medium contains one or more substances that can inhibit the growth of unfused, parental myeloma cells, such as hypoxanthine, aminopterin, thymidine (HAT medium).

[0084] In some embodiments of the present invention, the 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 of the present invention comprises:

[0085] The recombinant cell line according to the third aspect of the present invention is cultured, and the antibody or antigen-binding fragment is isolated from the culture of the recombinant cell line.

[0086] Preferably, the recombinant cell line comprises a hybridoma cell.

[0087] Preferably, the hybridoma cell classification is named hybridoma cell line 5D6, and was deposited in Guangdong Provincial Microbiological Culture Collection Center (5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on August 6, 2024, with the deposit number GDMCC NO: 64961.

[0088] In some embodiments of the present invention, the hybridoma cells are injected into the peritoneal cavity of mice for culture, and the monoclonal antibodies are separated and purified from the culture (ascites). Further, specifically: BALB / c mice aged 6 to 10 weeks are taken, and 1×10 5 ~1×10 6 5D6 hybridoma cells were centrifuged and purified.

[0089] Preferably, the purification method includes but is not limited to gel electrophoresis, affinity chromatography, dialysis, affinity purification.

[0090] In addition, antibodies or antigen-binding fragments thereof can also be obtained by genetic engineering recombination technology. PCR amplification is performed using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody, and DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody are isolated from hybridoma cells, and the DNA molecules are inserted into expression vectors, and then transfected into suitable host cells to obtain recombinantly expressed target antibodies. The nucleotide sequence of the heavy chain variable region of the antibody or 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.4.

[0091] The beneficial effects of the present invention are:

[0092] 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.4. The amino acid sequence of the minimum binding epitope of the antibody or the antigen-binding fragment thereof is shown in SEQ ID NO.22, and the antigen epitope is highly conserved in various subtypes of SADS-CoV. The results of the neutralization test showed 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The following are the identification results of SADS-CoV S protein, where A is the SDS-PAGE Coomassie Brilliant Blue staining result; B is the Western blotting result.

[0094] Figure 2The following are the identification results of 5D6 monoclonal antibody, where A is the Western blotting result; B is the IFA detection result of 5D6 monoclonal antibody and SADS-CoV reaction.

[0095] Figure 3 A is the Western blotting result of the reaction of purified 5D6 monoclonal antibody with S protein; B is the SDS-PAGE Coomassie Brilliant Blue staining result for detecting the heavy chain and light chain of 5D6 monoclonal antibody, and the subtype identification result.

[0096] Figure 4 The results are for the neutralizing activity of 5D6 monoclonal antibody.

[0097] Figure 5 The figure shows the result of PCR using heavy chain and light chain primers for 5D6 monoclonal antibody, where 1 is the heavy chain PCR band and 2 is the light chain PCR band.

[0098] Figure 6 Schematic diagram of truncation of antigenic epitopes.

[0099] Figure 7 This is the result of identifying the minimal antigen epitope of the 5D6 monoclonal antibody.

[0100] Figure 8 This is the sequence alignment of the minimal antigenic epitope in the S protein of different SADS-CoV strains. DETAILED DESCRIPTION

[0101] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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.

[0102] The 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; Expi293F cells were purchased from Thermo Fisher Scientific (catalog number A14527), and Balb / c mice were purchased by the Laboratory Animal Center of our institute. The relevant animal experiments were approved by the Laboratory Animal Use and Management Committee of Guangdong Provincial Laboratory Animal Monitoring Institute (IACUC2021167). DMEM culture medium (Gibco); Expi293F expression medium (Thermo Fisher Scientific), Addavax adjuvant (InvivoGen,); Freund's incomplete adjuvant, HT and HAT medium, PEG (P7306) were all purchased from Sigma; horseradish peroxidase (HRP) was purchased from Sigma; Simply P total RNA extraction kit: purchased from Bio-Ray Biotechnology Co., Ltd.; LATaq enzyme, pMD TM 19-T vector was purchased from TaKaRa

[0103] Example 1 Protein expression and purification

[0104]

[0105] The S protein was expressed by transiently transfecting 1 mg of DNA into 1 L of Expi293F cells using PEI transfection reagent. Three days after transfection, the cell culture supernatant was collected. The S protein of SADS-CoV was purified by Ni-NTA affinity chromatography column with His tag. The specific operation was carried out according to the instructions of Ni-NTA affinity chromatography column. The purified S protein was identified by SDS-PAGE and western blotting and stored at -80°C.

[0106] The pCDNA-SADS-CoV-S recombinant plasmid was transfected into Expi293F cells and expressed in Expi293F. The protein was purified from the supernatant. Figure 1 As shown, the recombinant pCDNA3.1-SADS-CoV-S protein was successfully expressed and verified by SDS-PAGE and western blot analysis.

[0107] Example 2 Preparation of a monoclonal cell line expressing anti-SADS-CoV S protein

[0108] 1. Immunization of BALB / c mice with recombinant protein

[0109] The purified SADS-CoV S protein was mixed with an equal volume of Addavax and inoculated subcutaneously into the abdomen of three 6-week-old BALB / c mice, with each inoculated with an antigen dose of 100 μg. Two weeks later, the same dose of Addavax adjuvant-treated antigen was injected. Three days before cell fusion, an equal amount of antigen without adjuvant (200 μg per mouse) was injected intraperitoneally for booster immunization once. Three days later, the mice were killed and the spleen cells of the immunized mice were collected for cell fusion.

[0110] 2. Preparation of monoclonal antibody cell lines stably expressing SADS-CoV S protein

[0111] The cell counting method was used to obtain 1×10 8 The spleen cells of immune mice were 2×10 7 The SP2 / 0 cells were mixed at a ratio of 5:1 and 0.8 mL of PEG was added for fusion, and the cells were cultured in a 96-well culture plate at 37°C in a CO2 incubator. The fused cells were first cultured in hypoxanthine-aminopterin-thymidine (HAT, Sigma, USA) medium for six days, and then the medium was replaced with hypoxanthine-thymidine (HT, Sigma, US) to select the fused cells.

[0112] When the hybridoma cells grow to 1 / 10 of the bottom area of ​​the well, the supernatant is aspirated and the positive hybridoma cells are screened by indirect immunofluorescence test (IFA, see Example 4 for details). After Vero cells are plated on a 96-well plate to form a monolayer, the cells are infected with a 0.1-fold infection multiple (MOI), and the cell lesions are observed after 24 hours. When 50% of the cells show lesions, the cells are fixed with 4wt% paraformaldehyde, and the supernatant of the fused cells is used as the primary antibody, and goat anti-mouse IgG (H+L) labeled with Alexa Flur 488 is used as the secondary antibody. After 45 minutes of reaction, it is observed under an inverted fluorescence microscope, and the positive wells with green fluorescence are selected, and then the positive wells are subcloned three times in a row by limiting dilution method until all monoclonal wells are positive. The monoclonal clones finally obtained are expanded and cultured to build a library and frozen. The hybridoma cell line that stably secretes specific antibodies finally obtained is named 5D6 cell line.

[0113] 3. Determination of the stability of monoclonal antibodies secreted by 5D6 hybridoma cell line

[0114] The 5D6 hybridoma cells frozen for 3 months were revived and cultured in 24-well cell plates. The 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 once every 2 weeks by IFA.

[0115] The 5D6 hybridoma cells prepared in this example have stable antibody secretion ability and have good expression after continuous passage.

[0116] The hybridoma cell line 5D6 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 6, 2024, with the deposit number GDMCC NO: 64961.

[0117] Example 3 Preparation of monoclonal antibodies

[0118] 1. Establishment of monoclonal antibody screening method

[0119] The indirect IFA method was established according to the conventional method. After the Vero cells formed a monolayer, the cells were infected with a 0.1-fold infection multiplicity (MOI), and the cell lesions were observed after 24 hours. When 50% of the cells showed lesions, the cells were fixed with 4wt% paraformaldehyde and stored at 4℃ or -20℃ for later use. Mouse negative serum, positive serum or hybridoma cells to be tested later were used as test samples, and goat anti-mouse IgG (H+L) labeled with Alexa Flur 488 was used as the secondary antibody. After 45 minutes of reaction, the cells were observed under an inverted fluorescence microscope, and the cell wells with positive cells were recorded.

[0120] 2. Preparation and purification of monoclonal antibody ascites

[0121] Eight-week-old BALB / c mice were intraperitoneally injected with Freund's incomplete adjuvant (0.5 mL per mouse). Seven days later, the hybridoma cell line 5D6, which was growing well, was inoculated with approximately 5 × 10 5 BALB / c mice were injected intraperitoneally. Around 7 days, the abdomen of the mouse began to swell significantly, and the ascites was repeatedly extracted. The cell components and other precipitates were removed by centrifugation at 2000g / min for 10 minutes, and the supernatant was collected. After the supernatant was purified, it was packaged and stored at -80°C. The ascites was diluted incrementally with PBS, and the titer of the monoclonal antibody was determined by IFA. Then, the ascites was purified according to the HiTrap Protein G HP (GE Healthcare) antibody purification instructions to obtain a monoclonal antibody, which was named 5D6 antibody (monoclonal antibody).

[0122] Example 4 Identification of 5D6 Antibody

[0123] 1. Western-blot analysis of monoclonal antibodies

[0124] After SDS-PAGE electrophoresis, the purified S protein and SADS-CoV virus particles were semi-dry transferred to a PVDF membrane. The PVDF membrane was blocked with 5wt% skim milk and then incubated with a purified monoclonal antibody (5D6, 2mg / mL) diluted 1:1000 as the primary antibody and goat anti-mouse IgG-HRP as the secondary antibody at room temperature. Finally, electrochemiluminescence (ECL) reagents and the Azure c600 bioanalysis imaging system (Azure Biosystems, USA) were used to image specific proteins.

[0125] 2. Identification of monoclonal antibody subtypes

[0126] 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 labeling, SBA clonotyping System-HRP, purchased from Southern Biotech).

[0127] 3. IFA detection of monoclonal antibodies

[0128] 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 cytopathic effect (CPE) appeared, the cells were fixed with 4wt% paraformaldehyde at 4°C for 10 min and blocked with 2wt% bovine serum albumin for 2 h. The cells were then incubated with a monoclonal antibody (5D6) at 37°C for 1 h, washed three times, and incubated with Alexa Flur 488-labeled goat anti-mouse IgG (H+L) as a secondary antibody (dilution 1:2000) at 37°C for 45 min. Finally, the nuclei were stained with DAPI (Sigma), and cell images were acquired using a confocal laser scanning microscope (Zeiss, JENA, Germany).

[0129] 4. Determination of monoclonal antibody neutralization activity

[0130] The 5D6 monoclonal antibody ascites was filtered through a 0.22 μm filter membrane 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, and trypsin was added with a final concentration of 8 μg / mL. Vero cells were inoculated and placed in a cell culture incubator for 3 days to observe the cytopathic effect.

[0131] 5. Sequence identification of 5D6 antibody

[0132] According to the instructions of Simply P total RNA extraction kit, RNA of 5D6 hybridoma cell line was extracted, cDNA was reverse transcribed, PCR reaction was carried out according to the instructions of La Taq enzyme, the target band was recovered by gel, and then ligated to pMD TM 19-T Vector was used for sequencing.

[0133] Amplification heavy chain primer VH-F: CGTTCCTGGTATCCTGTCT (SEQ ID NO. 24);

[0134] VH-R: AGAAGGTGTGCACACCGCTGGA (SEQ ID NO. 25).

[0135] Amplification light chain primer VL-F: CAGTTCCTGTTTCTGTTAGTGCTCTGG (SEQ ID NO. 26);

[0136] VL-R: TGGGAAGATGGATACAGTT (SEQ ID NO. 27).

[0137] 6. Results

[0138] Western blotting and IFA results of 5D6 monoclonal antibody are shown in Figure 2 As shown, the test found that the 5D6 monoclonal antibody supernatant could react with cells infected with SADS-CoV, but not with healthy control cells.

[0139] The purified S protein was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking with skim milk, the membrane was incubated with a 1:2000 diluted purified 5D6 monoclonal antibody. Figure 3 A in the middle shows that the purified monoclonal antibody can react with the S protein, and then SDS-PAGE was used to identify that the purification effect of the monoclonal antibody was good. The monoclonal antibody purified in this experiment was subjected to subtype identification. Figure 3 The results in Figure B showed that the 5D6 monoclonal antibody was of IgG1 subtype and its light chain was a κ chain.

[0140] The results of the monoclonal antibody neutralization test were as follows Figure 4 As shown, 5D6 monoclonal antibody can completely neutralize SADS-CoV strain at 250 μg / ml, and the antibody has neutralizing activity.

[0141] The PCR amplification results of the light chain and heavy chain of 5D6 antibody are as follows Figure 5 As shown, the sequence results of 5D6 antibody are as follows:

[0142] Among them, the nucleotide sequence of the 5D6 heavy chain variable region is shown in SEQ ID NO.2: gatgtgcagcttcaggagtcgggacc tggcctggtgaaaccttctcagtttctgtccctcacctgcactgtcactggctactccatcaccagtgattttgcctggaactggatccggcagtttccaggaaacaaactggagtggatgggctacataagctacagtggtaccactagctacaacccat ctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttcaattctgtgactgaggacacagccacatattactgtgcaaccagtgattggtggagggactactggggccaaggcaccactctcacagtctcctca. The amino acid sequence of the 5D6 heavy chain variable region is shown in SEQ ID NO.3: DVQLQESGPGLVKPSQFLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGTTSYNPSLKSRISITR DTSKNQFFLQFNSVTTEDTATYYCATSDWWRDYWGQGTTLTVSS.

[0143] The nucleotide sequence of VH CDR1 of 5D6 antibody is ggctactccatcaccagtgattttgcc (SEQ ID NO.28), the nucleotide sequence of VH CDR2 is ataagctacagtggtaccact (SEQ ID NO.29), and the nucleotide sequence of VH CDR3 is gcaaccagtgattggtggagggactac (SEQ ID NO.30). The corresponding amino acid sequences of VH are shown in CDR1: GYSITSDFA (SEQ ID NO.31), CDR2: ISYSGTT (SEQ ID NO.32), and ATSDWWRDY (SEQ ID NO.33).

[0144] The nucleotide sequence of the 5D6 light chain variable region is shown in SEQ ID NO.4: gatgttgtgatgacccagactccactcactttgtc ggttaccattggacaaccagcctccatctcttgcaagtcacgtcagagcctcttacatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatccatctggtgtctaagctggactctgga gtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggaatttattattgctggcaaggtacacattttcctctcacgttcggtgctgggaccaaggtggagctgaaac. The amino acid sequence of the 5D6 light chain variable region is shown in SEQ ID NO.5: DVVMTQTPLTLSVTIGQPASISCKSRQSLLHSDGKTYLNWLLQRPGQSPKRLIHLVSKLDSGVPDRFTGSGTDFTLKISRVEAEDLGIYYCWQGTHFPLTFGAGTKVELK.

[0145] The nucleotide sequence of VL CDR1 of 5D6 antibody is cagagcctcttacatagtgatggaaagacatat (SEQ ID NO.34), the nucleotide sequence of VL CDR2 is ctggtgtct (SEQ ID NO.35), and the nucleotide sequence of VL CDR3 is tggcaaggtacacattttcctctcacg (SEQ ID NO.36). The corresponding amino acid sequences of VH are CDR1: QSLLHSDGKTY (SEQ ID NO.37), CDR2: LVS (SEQ ID NO.38), and CDR3: WQGTHFPLT (SEQ ID NO.39).

[0146] Example 5 Identification of the antigenic epitope recognized by the 5D6 antibody

[0147] 1. Identification of monoclonal antibody epitopes

[0148] In order to identify the antigenic epitope recognized by 5D6, Figure 6As shown, the expression gene of SADS-CoV S protein was subjected to a series of overlapping truncations, and the truncated gene was inserted into the pGEX-6p-1 vector to construct a prokaryotic expression plasmid, and expressed as a fusion protein containing a GST tag. The correctly sequenced recombinant plasmid was transformed into Escherichia coli Transetta (DE3), and the expression was induced by incubation with IPTG at 16°C for 8 hours, and Western blotting was performed using anti-GST monoclonal antibody and monoclonal antibody 5D6.

[0149] The truncated sequences are as follows:

[0150] Among them, the amino acid sequence of S1 is shown in SEQ ID NO.6: MKLFTVFTLLASIRVLYGCESVDFNL FNTIFSTHRLGLSNTTSVITGAYPSTNKSDWSCNTRTGHLSGSGFGIGLYVQTPREQYQYDGSGAGGYTIAVSPIHVTNLTWELWIHRKWGVNSVVTVRLCRWWQFMSFNSTSHAADAGPTNAFECLING SYPTHRNTGYMFGVTWYNDLVRIVFPPTVLEMQLDGLQWERVQFNSPVNAGHATRFNVVKDISTVLVETNSGGSVFRYSYCADGFVNGLQCKLRLFDIPPGVYSNSEVEYPTALYTVVHNMSACPERPDS YCGSNSCPFKRAVFSNCIVNYTTWVNPDQRDFQHLILSNGKFNPFTECNGLNRIVDGCVPGFVLRVGRGKAVNRTIVTPYLKPYECFGWSWNDNQDSIYDWWIADFVSTGAFVCESNPEAPKTGVCVTYT VEKVTFQGVLYESNFTFAQYYNLLYVGSQLRYVRILGKVYEVSSCFEASYDVLYRNNQSFGLLYRSFDCNQLHIKSARFVDRLLPSHNGTATVLGCLFNASYAPNDTMVNCTNPLGDGFCADLLGNVAV.

[0151] The amino acid sequence of S2 is shown in SEQ ID NO.7: VNCTNPLGDGFCADLLGNVAVRRMTFEKHDTTYVAPVTNERYTEMPLDHQLILTEQFLQTTMPKFSVSCETYICDVSKACKNLLFRYGGFCQKVEADIRGAGILLDGDVSSLYSTIAAKTSSVVPTTDRFNVSQFFLPKTQSSANKYESRSAIEDLLFSKIETTGPGFYGDYYNCKKNAIQDLTCAQYHNGILVIPPIMDAETLGMYGGIAAASVTLGIFGGQAGMATWSVAMAGRLNALGVVQNALVDDVNKLANGFNQLTASVSKLALTTSSALQAIQAVVNQNAAQVESLVSGITENFGAISTNFKVISQRLDKLEADVQMDRLINGRMNVLQLFVTNYKLKIAELRNTHRYVQSLINECVYAQSLRNGFCGQGLHVLSLMQNAPSGIMFFHYSLIPNNTITVKTTPGLCESDELGSKCIVAKDGVLVSANLSYWQWSPRNLYKPENLTFANVIAVSRGANYTTLNKTFDIPELNSTFPIEEEFREYFQNMSSELQVLKNLTADMSKLNISAEIQLINEIAHNVSNMRVEVEKFQRYVNYVK。

[0152] The amino acid sequence of NTD is shown in SEQ ID NO.8: MKLFTVFTLLASIRVLYGCESVDFNLFNTIFSTHRGLSNTTSVITGAYPSTNKSDWSCNTRTGHLSGSGFGIGLYVQTPREQYQYDGSGAGGYTIAVSPIHVTNLTWELWIHRKWGVNSVVTVRLCRWWQFMSFNSTSHAADAGPTNAFECLINGSYPTHRNTGYMFGVTWYNDLVRIVFPPTVLEMQLDGLQWERVQFNSPVNAGHATRFNVVKDISTVLVETNSGGSVFRYSYCADGFVNGLQCKLRLFDIPPGVYSNSEVEYPTALYTVVHN。

[0153] The amino acid sequence of CTD is shown in SEQ ID NO.9: YPTALYTVVHNMSACPERPDSYCGSNSCPF KRAVFSNCIVNYTTWVNPDQRDFQHLILSNGKFNPFTECNGLNRIVDGCVPGFVLRVGRGKAVNRTIVTPYLKPYECFGWSWNDNQDSIYDWWIADFVSTGAFVCESNPEAPKTGVCVTYTVEKVTFQGV LYESNFTFAQYYNLLYVGSQLRYVRILGKVYEVSSCFEASYDVLYRNNQSFGLLYRSFDCNQLHIKSARFVDRLLPSHNGTATVLGCLFNASYAPNDTMVNCTNPLGDGFCADLLGNVAVRRMTFEKHDT.

[0154] The amino acid sequence of NTD-1 is shown in SEQ ID NO.10: MKLFTVFTLLASIRVLYGCESVDFNLFNTIFSTHRGLSNTTSVITGAYPSTNKSDWSCNTRTGHLSGSGFGIGLYVQTPREQYQYDGSGAGG YTIAVSPI.

[0155] The amino acid sequence of NTD-2 is shown in SEQ ID NO. 11: YDGSGAGGYTIAVSPIHVTNLTWELWIHR, KWGVNSVVTVRLCRWWQFMSFNSTSHAADAGPTNAFECLINGSYPTHRNTGYMFGVTW YNDLVRIVFPPTVL.

[0156] The amino acid sequence of NTD-3 is shown in SEQ ID NO.12: LVRIVFPPTVLEMQLDGLQWERVQFNSPVNAGHATRFNVVKDISTVLVETNSGGSVFRYSYCADGFVNGLQCKLRLFDIPPGVYSNSE VEYPTALYTVVHN.

[0157] The amino acid sequence of NTD-2a is shown in SEQ ID NO.13: MYDGSGAGGYTIAVSPIHVTNLTWELWIHRKWGV.

[0158] The amino acid sequence of NTD-2b is shown in SEQ ID NO.14: TWELWIHRKWGVNSVVTVRLCRWWQFMSFNSTSHAADAGPTN.

[0159] The amino acid sequence of NTD-2c is shown in SEQ ID NO.15: STSHAADAGPTNAFECLINGSYPTHRNTGYMFGVTWYNDLVR.

[0160] The amino acid sequence of NTD-2d is shown in SEQ ID NO.16: SYPTHRNTGYMFGVTWYNDLVRIVFP PTVL.

[0161] The amino acid sequence of NTD-2b-1 is shown in SEQ ID NO.17: TSHAADAGPTN.

[0162] The amino acid sequence of NTD-2b-2 is shown in SEQ ID NO.18: STSHAADAGPT.

[0163] The amino acid sequence of NTD-2b-3 is shown in SEQ ID NO.19: STSHAADAGP.

[0164] The amino acid sequence of NTD-2b-4 is shown in SEQ ID NO.20: STSHAADAG.

[0165] The amino acid sequence of NTD-2b-5 is shown in SEQ ID NO.21: STSHAADA.

[0166] The amino acid sequence of NTD-2b-6 is shown in SEQ ID NO.22: STSHAAD.

[0167] The amino acid sequence of NTD-2b-7 is shown in SEQ ID NO.23: STSHAA.

[0168] 2. Conservative analysis of epitope peptides recognized by monoclonal antibodies

[0169] In order to determine the conservation of the monoclonal antibody 5D6 epitope, 15 SADS-CoV strains from 2016 to 2023 were downloaded from GenBank, and the identified epitope regions were compared and analyzed among the 15 SADS-CoV strains using the Jotun Hein method in the software program MegAlign.

[0170] 3. Experimental results

[0171] Western Blotting results of antigen epitope identification are as follows Figure 7As shown. Antigen epitopes S1, NTD, NTD-2, NTD-2b, NTD-2c, NTD-2b-2, NTD-2b-3, NTD-2b-4, NTD-2b-5, and NTD-2b-6 can all bind specifically to the 5D6 antibody. The smallest antigen epitope recognized by the 5D6 monoclonal antibody is located at 136 STSHAAD 142 .

[0172] The epitope peptide region recognized by the 5D6 antibody was compared with 15 SADS-CoV strains obtained between 2016 and 2023. Figure 8 As shown, the epitope peptide region recognized by this antibody is highly conserved among SADS-CoV strains. It can be used as a diagnostic antibody. (Black dots indicate completely matching residues).

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 NOs. 30 to 32, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs. 36 to 38, 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.3; or a2) an amino acid sequence having 90%, 95% or more homology with SEQ ID NO.3 and having the same function as the protein shown in SEQ ID NO.3; 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.5; or a4) an amino acid sequence having 90%, 95% or more homology with SEQ ID NO.5 and having the same function as the protein shown in SEQ ID NO.5; The antibody or antigen-binding fragment thereof is selected from any one of Fab, Fab', F(ab')2, Fv, scFv, diabody, fusion antibody and complete antibody.

3. A recombinant protein comprising a tag and the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

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, characterized in that: The biological material is any one of b1) to b5); b1) 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; b2) an expression cassette containing the nucleic acid molecule described in b1); b3) a recombinant vector containing the nucleic acid molecule described in b1) or the expression cassette described in b2); b4) a recombinant microorganism containing the nucleic acid molecule described in b1) or the expression cassette described in b2) or the recombinant vector described in b3); b5) a recombinant cell line containing the nucleic acid molecule described in b1) or the expression cassette described in b2) or the recombinant vector described in b3); 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; c1) the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.2; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4; c2) A DNA 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.

5. The biomaterial according to claim 4, characterized in that: The recombinant cell lines include hybridoma cells; The hybridoma cell classification is named hybridoma cell line 5D6, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 6, 2024, with the deposit number GDMCC NO: 64961.

6. An antigen epitope peptide, characterized in that: The antigen epitope peptide is derived from the S protein fragment of porcine acute diarrhea syndrome coronavirus, and its amino acid sequence is shown in SEQ ID NO.22; The antigen epitope peptide specifically binds to the antibody or antigen binding fragment thereof according to claim 1 or 2, or the recombinant protein according to claim 3.

7. A protein conjugate, characterized in that: The protein conjugate comprises a conjugated portion, and the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the recombinant protein according to claim 3; The coupling part is selected from one or more of a label molecule, a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier, a liposome, and biotin.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the recombinant protein according to claim 3, or the protein conjugate according to claim 7 in d1) to d3): d1) Preparation of products for detecting porcine acute diarrhea syndrome coronavirus; d2) Preparation of products for the prevention of porcine acute diarrhea syndrome coronavirus; d3) Preparation of products for treating porcine acute diarrhea syndrome coronavirus.

9. A product, characterized in that: Including at least one of e1) to e4): e1) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; e2) the recombinant protein according to claim 3; e3) The biomaterial according to any one of claims 4 to 5; e4) the protein conjugate according to claim 7; The product is selected from at least one of drugs, reagents, test plates, chips, test papers and test kits.

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, comprising the following steps: The method is obtained by culturing the recombinant microorganism or recombinant cell according to any one of claims 4 to 5.

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