A spike protein antigen epitope peptide of porcine acute diarrhea syndrome coronavirus, monoclonal antibody thereof and application

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a spike protein epitope peptide of porcine acute diarrhea syndrome coronavirus and its monoclonal antibody and applications. Background Technology

[0002] Porcine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) is a newly discovered porcine enteric coronavirus belonging to the genus Alphacoronavirus in the family Coronaviridae. The clinical symptoms in infected pigs are similar to other porcine enteric coronaviruses, with infected sows exhibiting mild diarrhea. Newborn piglets infected within 5 days of age develop acute diarrhea and vomiting, leading to acute death, with a mortality rate as high as 90%. Therefore, prevention and control of SADS-CoV are crucial.

[0003] In coronaviruses, the S protein, in the form of a trimer, covers the viral surface and plays a crucial role in determining the host range and tissue orientation of the coronavirus. The S protein of SADS-CoV (1130 amino acids in length) is smaller than that of other coronavirus S proteins and shows low homology with other coronavirus S genes. Studies have shown that the SADS coronavirus S protein has a unique structure; and evolutionary analysis indicates that the SADS-CoV S protein belongs to the β-coronavirus group, suggesting that SADS-CoV may have originated from a mixed infection involving α-coronaviruses and β-coronaviruses. Therefore, further research on the SADS coronavirus S protein is of great significance. However, there is currently no monoclonal antibody that can be used for the western blotting detection of the SADS-CoV S protein. Summary of the Invention

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

[0005] A second aspect of the present invention is to provide a recombinant protein.

[0006] A third aspect of the present invention aims to provide biomaterials 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 this invention aims to provide an antigenic epitope peptide.

[0008] The fifth aspect of this invention is to provide a protein conjugate.

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

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

[0011] The object 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 and the recombinant protein of the second aspect of the present invention.

[0012] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0013] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof;

[0014] The antibody or its antigen-binding fragment has a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each contain complementarity-determining regions CDR1, CDR2 and CDR3, respectively.

[0015] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO. 30-32, respectively.

[0016] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO. 36-38, respectively. Preferably, the antibody or its antigen-binding fragment 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 its antigen-binding fragment comprises:

[0018] a1) The amino acid sequence as shown in SEQ ID NO.3; or

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

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

[0021] a3) The amino acid sequence as shown in SEQ ID NO. 5; or

[0022] a4) has 90%, 95% or more homology with SEQ ID NO.5 and has the same amino acid sequence 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 or multispecific antibody.

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

[0025] In some embodiments of the present invention, the antibody or its antigen-binding fragment specifically binds to amino acid sequences such as those shown in SEQ ID NO. 6, 8, 11, 14, 15, 18, 19, 20, 21, and 22. Those skilled in the art will understand that antigens including the minimal epitope sequence (SEQ ID NO. 22) are capable of binding to the monoclonal antibody.

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

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

[0028] In a first aspect of this invention, the term "chimeric antibody" is defined as a protein molecule recombinantly designed from the variable regions of the light and heavy chains of immunoglobulins from non-human species and the constant regions of human immunoglobulin molecules. The term "humanized" refers to a protein molecule recombinantly designed where only a portion of the complementarity-determining region (CDR) of the variable region of an immunoglobulin molecule from a non-human species is retained, while the remaining major variable regions and all constant regions are derived from human sources. Although this invention provides only a mouse antibody, those skilled in the art can analyze the corresponding CDR sequences based on the sequences of the heavy and light chain variable regions, and replace other portions with constant regions and framework regions (FRs) from other animal sources based on the CDR sequences, thereby obtaining antibodies from other animal sources, and anticipating similar or identical technical effects to those of this invention. Preferably, the other animal-derived antibodies include at least one of human antibodies and porcine antibodies.

[0029] A second aspect of the present invention aims to provide a recombinant protein comprising a tag and an antibody or antigen-binding fragment thereof as described in 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] A third aspect of the present invention provides biological materials related to the antibody or antigen-binding fragment thereof of the first aspect of the present invention and the recombinant protein of the second aspect of the present invention, said biological materials being any one of b1) to b5);

[0032] b1) A nucleic acid molecule that encodes the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention;

[0033] b2) An expression cassette containing the nucleic acid molecules 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) Recombinant microorganisms containing the nucleic acid molecules described in b1), 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), 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 its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention.

[0038] Preferably, the DNA molecule is any one of c1) to c2), and contains a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region;

[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] The nucleotide sequences defined in c2) and c1) have 80%, 85% or 90% or more homology and encode the DNA molecule of the antibody or its antigen-binding fragment.

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

[0042] Preferably, the hybridoma cell is classified as hybridoma cell line 5D6 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 6, 2024, with accession number GDMCC NO: 64961.

[0043] In some embodiments of the present invention, the vector includes the eukaryotic expression vector pCDNA3.1 and the 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 as the present invention.

[0044] In some embodiments of the present invention, the cell lines include cell lines for expressing antigenic epitope peptides, such as Expi293F; and cell lines for preparing hybridomas, such as Sp-2 / 0. Those skilled in the art will expect that other common engineered cell lines in the art can also achieve the same or similar technical effects as the present invention.

[0045] In some embodiments of the present invention, the microorganisms include Escherichia coli Transetta (DE3). Those skilled in the art will expect that other common microorganisms in the art, such as engineered bacteria like Escherichia coli DH5α, Escherichia coli BL21, Bacillus subtilis, and yeast, can also achieve the same or similar technical effects as the present invention.

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

[0047] In some embodiments of the present invention, the antigenic epitope peptide further 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 as shown in SEQ ID NO. 6, 8, 11, 14, 15, 18, 19, 20, 21, and 22, respectively.

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

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

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

[0051] A fifth aspect of the present invention provides a protein conjugate comprising a conjugation portion, and an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention.

[0052] Preferably, the coupling portion is selected from one or more of the following: tag molecules, radionuclides, drugs, toxins, cytokines, enzymes, luciferins, carriers, liposomes, and biotin.

[0053] In a fifth aspect of the invention, the conjugate is used for tracing, purification, or enhancement. For example, by adding a tag molecule or biotin, it facilitates subsequent protein purification; by adding a radionuclide or fluorescein, it indicates the location of the antibody or antigen-binding fragment in the organism; and by conjugating it with other drugs or cytokines, it enhances the immune response.

[0054] A 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 days 1 to 3):

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

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

[0057] d3) Prepare products for the treatment of porcine acute diarrhea syndrome coronavirus.

[0058] Preferably, the product includes at least one of reagents, reagent kits, pharmaceuticals, and vaccines.

[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 as described in the first aspect of the present invention;

[0061] e2) The recombinant protein described in the second aspect of the present invention;

[0062] e3) The biomaterials described in the third aspect of this invention;

[0063] e4) Protein conjugates of the fifth aspect of this invention;

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

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

[0066] Preferably, the drug includes a vaccine.

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

[0068] Preferably, the liquid carrier includes: saline, buffer solution, glucose solution, aqueous solution, glycerol, and 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 includes other active ingredients; other active ingredients include, but are not limited to, other drugs for treating or preventing acute diarrhea syndrome in pigs, or other drug components with synergistic effects.

[0071] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, lyophilized powder for injection, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0072] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0073] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, carriers, lubricants, sweeteners, and flavorings.

[0074] The eighth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention, comprising the following steps:

[0075] It is obtained by culturing the recombinant microorganisms or recombinant cells described in the third aspect of the present invention.

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

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

[0078] Immune cells were isolated from the animals and fused with myeloma cells. Positive cells were then screened to obtain the desired result.

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

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

[0081] Preferably, the myeloma cells are characterized by HAT sensitivity, strong antibody secretion capacity, and ease of fusion. In some embodiments of the present invention, murine myeloma cells are used, such as MOP-21, MC-11, SP-2 / 0, and X63-Ag8-653 cell lines; preferably, the SP-2 / 0 cell line is used.

[0082] Preferably, the fusion method specifically involves: administering an immunogen (with an adjuvant added) to a suitable host animal via 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 immunization, the animal produces lymphocytes that secrete antibodies specifically binding to the immunogen. These lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (e.g., PEG). Positive cells are then screened to obtain hybridoma cells.

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

[0084] In some embodiments of the present invention, the preparation methods of the antibody or its antigen-binding fragment described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention include:

[0085] Cultivate the recombinant cell line described in the third aspect of the present invention, and isolate antibody or antigen-binding fragments from the culture of the recombinant cell line.

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

[0087] Preferably, the hybridoma cell is classified as hybridoma cell line 5D6 and was deposited on August 6, 2024 at Guangdong Provincial Center for Microbial Culture Collection (5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with accession 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 antibody is isolated and purified from the culture (ascites). More specifically, 6-10 week old BALB / c mice are injected intraperitoneally with 1×10- 5 ~1×10 6 5D6 hybridoma cells were collected individually, centrifuged, and purified.

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

[0090] In addition, antibodies or their antigen-binding fragments can be obtained through recombinant genetic engineering. PCR amplification is performed using nucleic acid primers that specifically bind to the heavy and light chain genes of a monoclonal antibody. DNA molecules encoding the heavy and light chain genes of the monoclonal antibody are isolated from hybridoma cells. These DNA molecules are then inserted into an expression vector and transfected into suitable host cells to obtain the recombinantly expressed target antibody. The nucleotide sequence of the heavy chain variable region of the antibody or its antigen-binding fragment 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 this invention are:

[0092] This invention provides an antibody or its antigen-binding fragment thereof, wherein the nucleotide sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.2; and the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.4. The amino acid sequence of the minimal binding epitope of the antibody or its antigen-binding fragment is shown in SEQ ID NO.22, and this epitope is highly conserved in various subtypes of SADS-CoV. Neutralization assay results show that the antibody can completely neutralize 200 TCID at a concentration of 250 μg / ml. 50 The antibody contains SADS-CoV viral particles. It provides valuable research material for basic research on the molecular mechanisms of SADS-CoV invasion and for applied research on SADS-CoV detection and treatment. Attached Figure Description

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

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

[0095] Figure 3 Image A shows the Western blotting results of the reaction between the purified 5D6 monoclonal antibody and the S protein; Image B shows the results of SDS-PAGE Coomassie Brilliant Blue staining for detection of the heavy and light chains of the 5D6 monoclonal antibody and the subtype identification results.

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

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

[0098] Figure 6 This is a schematic diagram of the truncated antigenic epitope.

[0099] Figure 7 The results show the identification of the smallest antigenic epitope for the 5D6 monoclonal antibody.

[0100] Figure 8 Sequence alignment of the minimal antigenic epitope in the S protein of different SADS-CoV strains. Detailed Implementation

[0101] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0102] The SADS-CoV strain was isolated and cultured in our laboratory (GenBank accession number MG557844); SP2 / 0 and Vero cell lines were preserved by the New Detection Technology Team of the Guangdong Provincial Institute of Laboratory Animal Monitoring; Expi293F cells were purchased from Thermo Fisher Scientific (catalog number A14527); Balb / c mice were purchased by our Laboratory Animal Center; and the relevant animal experiments were approved by the Laboratory Animal Use and Management Committee of the Guangdong Provincial Institute of Laboratory Animal Monitoring (IACUC2021167). DMEM culture medium (Gibco); Expi293F expression medium (Thermo Fisher Scientific); Addavax adjuvant (InvivoGen); Freund's incomplete adjuvant, HT and HAT media, and PEG (P7306) were all purchased from Sigma-Aldrich; horseradish peroxidase (HRP) was purchased from Sigma-Aldrich; Simply P total RNA extraction kit was purchased from Bozhi Biotechnology Co., Ltd.; LATaq enzyme and pMD were also used. TM The 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 SADS-CoV S protein was purified using a Ni-NTA affinity chromatography column with the His tag, following the instructions for the 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, enabling its expression in Expi293F cells. The protein was purified from the supernatant. Results are as follows... Figure 1 As shown, the recombinant pCDNA3.1-SADS-CoV-S protein has been 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] Purified SADS-CoV S protein was mixed with an equal volume of Addavax and subcutaneously inoculated into the abdomen of three 6-week-old BALB / c mice, with each mouse receiving 100 μg of antigen. Two weeks later, the same dose of Addavax-adjuvanted antigen was injected. Three days before cell fusion, a booster immunization was administered intraperitoneally with an equal volume of unadjuvanted antigen (200 μg per mouse). The mice were sacrificed three days later, and spleen cells from the immunized mice were collected for cell fusion.

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

[0111] 1×10⁻⁶ cells were obtained using cell counting methods. 8 Immunized mouse spleen cells and 2×10 7 SP2 / 0 cells were mixed at a ratio of 5:1 and 0.8 mL of PEG was added for fusion. The mixture was then cultured in 96-well plates 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 for fused cells.

[0112] When the hybridoma cells reached 1 / 10 of the well bottom area, the supernatant was aspirated, and positive hybridoma cells were screened by indirect immunofluorescence assay (IFA, see Example 4 for details). Vero cells were seeded into a monolayer in a 96-well plate and infected at a fold increase of 0.1 times (MOI). Cytopathic effects were observed after 24 hours. When 50% of the cells showed cytopathic effects, the cells were fixed with 4 wt% paraformaldehyde. The supernatant of the fused cells was used as the primary antibody, and Alexa Flur 488-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody. After 45 minutes of reaction, the cells were observed under an inverted fluorescence microscope. Positive wells with green fluorescence were selected, and then subcloning was performed three times consecutively using the limiting dilution method until all single-clone wells were positive. The finally obtained single clones were expanded and cultured, then a library was constructed and cryopreserved. The hybridoma cell line that stably secreted specific antibodies was finally obtained and named the 5D6 cell line.

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

[0114] 5D6 hybridoma cells frozen for 3 months were revived, cultured in 24-well cell plates, and the supernatant was tested for positivity to determine whether the revived positive cells had the ability to secrete antibodies. Hybridoma cells with a 100% positivity rate after 3 subcloning were continuously passaged in vitro for 3 months, and the cell supernatant was tested with IFA every 2 weeks.

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

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

[0117] Example 3: Preparation of Monoclonal Antibodies

[0118] 1. Establishment of monoclonal antibody screening methods

[0119] An indirect IFA method was established using standard procedures. After Vero cells formed a monolayer, they were infected with an infection modulus (MOI) of 0.1. Cytopathic effects were observed after 24 hours. When 50% of the cells showed cytopathic effects, the cells were fixed with 4 wt% paraformaldehyde and stored at 4°C to -20°C for later use. Mouse negative serum, positive serum, or hybridoma cells to be tested were used as samples. Alexa Flur 488-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody. After 45 minutes of reaction, the cells were observed under an inverted fluorescence microscope, and the cell wells containing 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 well-growing hybridoma cell line 5D6 was injected at a rate of approximately 5 × 10⁶ cells per mouse. 5 BALB / c mice were intraperitoneally injected. Around day 7, the mice's abdomens began to swell significantly. Ascites fluid was repeatedly aspirated, centrifuged at 2000g / min for 10 min to remove cellular components and other precipitates, and the supernatant was collected. The supernatant was purified, aliquoted, and stored at -80℃. The ascites fluid was serially diluted with PBS, and the monoclonal antibody titer was determined by IFA. The ascites fluid was then purified according to the HiTrap Protein G HP (GE Healthcare) antibody purification instructions to obtain the monoclonal antibody, named 5D6 antibody (monoclonal antibody).

[0122] Example 4 Identification of 5D6 antibody

[0123] 1. Western blot analysis of monoclonal antibodies

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

[0125] 2. Monoclonal antibody subtype identification

[0126] The monoclonal antibodies obtained in the above experiments were identified according to the instructions of the subtype identification kit (SBA mouse monoclonal antibody typing kit - HRP labeling, SBAclonotypingSystem-HRP, purchased from Southern Biotech).

[0127] 3. IFA detection of monoclonal antibodies

[0128] Vero cells were seeded in 96-well plates and, after forming a monolayer, were infected with SADS-CoV at a fold increase (MOI) of 0.1. When cytopathic effect (CPE) occurred, cells were fixed with 4 wt% paraformaldehyde at 4°C for 10 min and blocked with 2 wt% bovine serum albumin for 2 h. 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 (diluted 1:2000) at 37°C for 45 min. Finally, cell nuclei were stained with DAPI (Sigma), and cell images were acquired using a confocal laser scanning microscope (Zeiss, JENA, Germany).

[0129] 4. Monoclonal antibody neutralizing activity assay

[0130] 5D6 monoclonal antibody ascites fluid 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 serially diluted 2-fold with DMEM culture medium; SADS-CoV was diluted to 200 TCID50 per 50 μL of viral fluid. 50 Mix 50 μL of virus solution with 50 μL of antibody dilution solution in equal volumes, react at 37°C for 1 hour, add trypsin to a final concentration of 8 μg / mL, inoculate Vero cells, place in a cell culture incubator for 3 days, and observe the cytopathic effect.

[0131] 5. Sequence identification of 5D6 antibody

[0132] RNA was extracted from 5D6 hybridoma cells according to the Simply P Total RNA Extraction Kit instructions. cDNA was reverse transcribed, and PCR was performed using La Taq polymerase according to the instructions. The target band was recovered from the gel and ligated into pMD. TM Sequencing was performed using the 19-T vector.

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

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

[0135] Amplification of 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 the 5D6 monoclonal antibody are as follows: Figure 2 As shown, the test revealed that the 5D6 monoclonal antibody supernatant could react with cells inoculated with SADS-CoV, but not with healthy control cells.

[0139] The purified S protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with skim milk, and then incubated with a 1:2000 diluted purified 5D6 monoclonal antibody. The results were as follows: Figure 3 As shown in Figure A, the purified monoclonal antibody can react with the S protein. SDS-PAGE analysis confirmed that the purification effect of the monoclonal antibody was good. The purified monoclonal antibody in this experiment was then subjected to subtype identification. Figure 3 The results of the B test showed that the 5D6 monoclonal antibody was an IgG1 subtype with a κ light chain.

[0140] Results of monoclonal antibody neutralization assay as follows Figure 4 As shown, the 5D6 monoclonal antibody can completely neutralize the SADS-CoV strain at 250 μg / ml, indicating that the antibody has neutralizing activity.

[0141] The PCR amplification results of the light and heavy chains of the 5D6 antibody are as follows: Figure 5 As shown, the sequence results of the 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 variable region of the 5D6 heavy chain is shown in SEQ ID NO.3: DVQLQESGPGLVKPSQFLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGTTSYNPSLKSRISITR DTSKNQFFLQFNSVTTEDTATYYCATSDWWRDYWGQGTTLTVSS.

[0143] The nucleotide sequences of VH CDR1 of the 5D6 antibody are ggctactccatcaccagtgattttgcc (SEQ ID NO. 28), VH CDR2 are ataagctacagtggtaccact (SEQ ID NO. 29), and VH CDR3 are gcaaccagtgattggtggagggactac (SEQ ID NO. 30). The corresponding amino acid sequences of VH are as follows: 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 variable region of the 5D6 light chain is shown in SEQ ID NO.5: DVVMTQTPLTLSVTIGQPASISCKSRQSLLHSDGKTYLNWLLQRPGQSPKRLIHLVSKLDSGVPDRFTGSG SGTDFTLKISRVEAEDLGIYYCWQGTHFPLTFGAGTKVELK.

[0145] The nucleotide sequences of VL CDR1 of the 5D6 antibody are cagagcctcttacatagtgatggaaagacatat (SEQ ID NO.34), VL CDR2 is ctggtgtct (SEQ ID NO.35), and 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 antigenic epitopes recognized by 5D6 antibody

[0147] 1. Identification of antigenic epitopes of monoclonal antibodies

[0148] To identify the antigenic epitopes recognized by 5D6, such as Figure 6As shown, the expression gene of SADS-CoV S protein was truncated in a series of overlapping steps, and the truncated gene was inserted into the pGEX-6p-1 vector to construct a prokaryotic expression plasmid, which was expressed as a fusion protein containing a GST tag. The correctly sequenced recombinant plasmid was transformed into E. coli Transetta(DE3), and expression was induced by incubation with IPTG at 16°C for 8 hours. 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: VNCTNPLGDGFCADLLGNVAVRRMTFEKHD TTYVAPVTNERYTEMPLDHQLILTEQFLQTTMPKFSVSCETYICDVSKACKNLLFRYGGFCQKVEADIRGAGILLDGDVSSLYSTIAAKTSSVVPTTDRFNVSQFFLPKTQSSANKYESRSAIEDLLFSKIETTGPGFYGDYYNCKKNAIQDLTCAQYHNGILVIPPIMDAETLGMYGGIAAASVTLGIFGGQAGMATWSVAMAGRLNALGVVQNALVDDVNKLANGFNQLTASVSKLALTTSSALQAIQAVVNQNAAQVESLVSGITENFGAISTNFKVISQRLDKLEADVQMDRLINGRMNVLQLFVTNYKLKIAELRNTHRYVQSLINECVYAQSLRNGFCGQGLHVLSLMQNAPSGIMFFHYSLIPNNTITVKTTPGLCESDELGSKCIVAKDGVLVSANLSYWQWSPRNLYKPENLTFANVIAVSRGANYTTLNKTFDIPELNSTFPIEEEFREYFQNMSSELQVLKNLTADMSKLNISAEIQLINEIAHNVSNMRVEVEKFQRYVNYVK。

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

[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: LVRIVFPPTVLEMQLDGLQWERVQFN SPVNAGHATRFNVVKDISTVLVETNSGGSVFRYSYCADGFVNGLQCKLRLFDIPPGVYSNSE 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. Conservation analysis of monoclonal antibody recognition epitope peptides

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

[0170] 3. Experimental Results

[0171] Western blotting results of antigenic epitopes are as follows: Figure 7As shown, 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 specifically bind to the 5D6 antibody. The smallest 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, and the results are as follows: 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 perfectly matching residues).

Claims

1. An antibody or antigen-binding fragment thereof against SADS-CoV S protein, characterized in that: the antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region, each of which contains complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO.31-33; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as SEQ ID NO.37-39. 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 is shown as SEQ ID NO.3; the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown as SEQ ID NO.5; the antibody or antigen-binding fragment thereof is selected from any one of Fab, Fab', F(ab')2, Fv or scFv. 3.A recombinant protein consisting of a tag and the antibody or antigen-binding fragment thereof according to any one of claims 1-2. 4.A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1-2, or the recombinant protein according to claim 3, characterized in that: the biological material is any one of b1)-b5); b1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-2, or the recombinant protein according to claim 3; b2) an expression cassette containing the nucleic acid molecule in b1); b3) a recombinant vector containing the nucleic acid molecule in b1) or the expression cassette in b2); b4) a recombinant microorganism containing the nucleic acid molecule in b1) or the expression cassette in b2) or the recombinant vector in b3); b5) a recombinant cell line containing the nucleic acid molecule in b1) or the expression cassette in b2) or the recombinant vector in b3); the nucleic acid molecule encodes the heavy chain variable region and the light chain variable region; the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown as SEQ ID NO.2; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO.

4. 5.The biological material according to claim 4, characterized in that: the recombinant cell line comprises a hybridoma cell; the hybridoma cell is classified and named as hybridoma cell strain 5D6, which was preserved in Guangdong Microbial Culture Collection Center on August 6, 2024, and the preservation number is GDMCC NO: 64961. 6.Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, or the recombinant protein according to claim 3 in the preparation of a product for detecting porcine acute diarrhea syndrome coronavirus; the product is at least one selected from reagents, detection plates, chips, test papers and kits. 7.A product, characterized in that: it comprises at least one of e1)-e2): e1) the antibody or antigen-binding fragment thereof according to any one of claims 1-2; e2) the recombinant protein of claim 3; The product is at least one selected from the group consisting of reagent, detection plate, chip, test paper and kit.

8. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-2, the recombinant protein of claim 3, comprising the following steps: obtained by culturing the recombinant microorganism or recombinant cell line of any one of claims 4-5.

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