Monoclonal antibody against porcine delta coronavirus n protein and application thereof

Abstract

The application discloses a kind of anti-porcine delta coronavirus (PDCoV) N protein monoclonal antibody and application.The heavy chain variable region of the monoclonal antibody includes CDR1-3 shown in SEQ ID No.1-3;Light chain variable region includes CDR1-3 shown in SEQ ID No.4-6;The monoclonal antibody prepared in the application can recognize PDCoV, and the conservation is good, provides material for the research of PDCoV pathogenesis and for the development of PDCoV diagnostic kit;And can specifically recognize and combine the region of N protein 294-307aa, provides reliable research tool for exploring the function of N protein;Meanwhile, it can be obtained using conventional genetic engineering or protein engineering method, avoids the loss of antibody in long-term frozen hybridoma cell, also be conducive to antibody optimization on gene and protein level, to improve the specificity and affinity of antibody.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostics, specifically relating to a monoclonal antibody against porcine deltacoronavirus N protein and its applications. Background Technology

[0002] Porcine Deltacoronavirus Disease (PDCoV) is an acute, highly contagious enteric infectious disease caused by porcine deltacoronavirus (PDCoV). The main symptoms are vomiting, watery diarrhea, dehydration, and high mortality in piglets. It is a newly discovered porcine enteric coronavirus disease that has become widespread in many pig-producing countries worldwide, causing significant economic losses to the pig industry. PDCoV can infect pigs of all ages. Adult pigs typically exhibit subclinical infection or mild diarrhea and transient anorexia, but it is highly pathogenic to suckling piglets (especially those under one week old), with a mortality rate of 40%-100%. It is also frequently co-infected with other enteroviruses such as PEDV and TGEV, further exacerbating the disease.

[0003] PDCoV belongs to the genus *δ-coronavirus* of the family Coronaviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 25.4 kb. Its genome has a cap structure at the 5' end and a polyadenylated tail at the 3' end, encoding eight open reading frames (ORFs): ORF1a, ORF1b, and proteins encoding the nucleocapsid protein (N), membrane protein (M), spike protein (S), envelope protein (E), and accessory proteins (NS6 and NS7). Among these, the PDCoV N protein plays a crucial role in viral particle assembly and RNA synthesis. It is the most highly expressed structural protein after viral infection of host cells, stimulating the body to produce high levels of specific antibodies and exhibiting high conservation among different PDCoV strains. Based on these characteristics of the N protein, researchers have widely applied it to the development of PDCoV serological detection methods. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a monoclonal antibody against PDCoV N protein and its applications, specifically including the following: In a first aspect, the present invention provides an antigenic epitope peptide of PDCoV N protein, wherein the antigenic epitope peptide is located at 294-307aa of PDCoV N protein, and its amino acid sequence is shown in SEQ ID No. 11.

[0005] Preferably, the amino acid sequence of the N protein is as shown in SEQ ID No. 12.

[0006] In a second aspect, the present invention provides the use of the antigenic epitope peptide described in the first aspect in the preparation of anti-PDCoV N protein monoclonal antibodies.

[0007] Thirdly, the present invention provides a monoclonal antibody against PDCoV N protein, said monoclonal antibody comprising an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No. 6.

[0008] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 8.

[0009] Fourthly, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody described in the third aspect above.

[0010] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID No. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID No. 10.

[0011] Fifthly, the present invention provides an expression cassette, expression vector, and recombinant bacteria containing the nucleic acid described in the fourth aspect above.

[0012] In a sixth aspect, the present invention provides the application of the monoclonal antibody described in the third aspect above in the preparation of reagents for detecting PDCoV.

[0013] In a seventh aspect, the present invention provides the application of the monoclonal antibody described in the third aspect above in the detection of PDCoV infection for non-disease diagnosis purposes or in the study of PDCoV N protein function.

[0014] Eighthly, the present invention provides a PDCoV detection kit, the kit comprising the monoclonal antibody described in the third aspect above.

[0015] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0016] Ninthly, the present invention provides a method for preparing the monoclonal antibody described in the third aspect above. The method comprises: cloning the PDCoV N gene into the pET-28a vector to construct a prokaryotic expression vector, inducing expression, purifying the protein by elution with different concentrations of imidazole, using the purified recombinant N protein as an antigen to immunize Balb / c mice, fusing mouse spleen cells with myeloma cells SP2 / 0 to prepare hybridoma cells; verifying the cell supernatant by indirect ELISA and indirect immunofluorescence, and screening for positive clones; after three subcloning processes, injecting the hybridoma cells into mice to prepare ascites fluid, and finally purifying the obtained ascites fluid to obtain the PDCoV N protein monoclonal antibody.

[0017] The beneficial effects of this invention are as follows: This invention utilizes a prokaryotic expression system to express and purify recombinant PDCoV N protein, which is then used as an immunogen to immunize mice. Through cell fusion and subcellular screening, a monoclonal antibody targeting the N protein was successfully obtained. By truncating the N gene, it was found that the antibody specifically recognizes and binds to the N protein region 294-307aa, providing a reliable research tool for exploring the function of the N protein.

[0018] The monoclonal antibody prepared in this invention can recognize PDCoV and is well conserved, providing materials for the study of the pathogenesis mechanism of PDCoV and the development of PDCoV diagnostic kits.

[0019] The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 Results of PCR amplification of the PDCoV N gene and double enzyme digestion identification of the pET-28a plasmid; In Figure A, M is the relative molecular mass standard of the 250 bp DNA ladder, and 1 is the N gene, approximately 1026 bp in size; In Figure B, M is the relative molecular mass standard of the 250 bp DNA ladder, and 1 is the pET-28a recombinant plasmid used for... Nco I and Xho I double enzyme digestion Figure 2pET-28a-N recombinant protein expression and purification results; In Figure A, M is the protein marker, 1 is the uninduced whole bacterial culture, and 2 is the induced whole bacterial culture; In Figure B, M is the protein marker, 1 is the supernatant after sonication, and 2 is the precipitate after sonication; In Figure C, M is the protein marker, and 1 is the purified recombinant N protein.

[0022] Figure 3 Results of serum titer assay for N protein after three immunizations in mice; 1 is the serum from the first immunization, 2 is the serum from the second immunization, 3 is the serum from the third immunization, and NC is the negative control.

[0023] Figure 4 Results of monoclonal antibody titer assay.

[0024] Figure 5 IFA is used to identify the reactivity of monoclonal antibodies; A is used to identify the reactivity of monoclonal antibodies with PDCoV strains using IFA; B is used to identify the reactivity of monoclonal antibodies with uninfected cells using IFA.

[0025] Figure 6 Western blot was used to identify the reactivity of monoclonal antibodies; M is the protein marker, 1 is uninfected VERO cells, and 2 is PDCoV-infected VERO cells.

[0026] Figure 7 Identification results of monoclonal antibody 5B7 subclass.

[0027] Figure 8Figure 5 shows the identification results of the N protein region recognized by monoclonal antibody 5B7; Figure A is a schematic diagram of truncated N protein expression; Figure B shows the SDS-PAGE identification results after C1 and C2 induction, M is the protein marker, 1 is whole bacteria without C1 expression, 2 is whole bacteria with C1 expression, 3 is whole bacteria without C2 expression, and 4 is whole bacteria with C2 expression; Figure C shows the Western blot identification results of monoclonal antibody 5B7, M is the protein marker, 1 is whole bacteria with induced N protein expression, 2 is whole bacteria with C1 expression, and 3 is whole bacteria with C2 expression; Figure D shows a schematic diagram of truncated C2 expression; Figure E shows the SDS-PAGE identification results after induction of D3, D4, and D5 genes, M is the protein marker, 1 is whole bacteria without D3 expression, 2 is whole bacteria with D3 expression, 3 is whole bacteria without D4 expression, 4 is whole bacteria with D4 expression, 5 is whole bacteria without D5 expression, and 7 is whole bacteria with D5 expression; Figure F shows the Western blot identification results of monoclonal antibody 5B7. Figure 1 shows the Western blot results for monoclonal antibody 5B7. M represents the protein marker, 1 represents whole bacteria induced by D3, 2 represents whole bacteria induced by D4, and 3 represents whole bacteria induced by D5. Figure G shows a schematic diagram of D5 truncated expression. Figure H shows the SDS-PAGE results after expression induced by D6, D7, and D8. M represents the protein marker, 1 represents whole bacteria without D6 expression, 2 represents whole bacteria induced by D6 expression, 3 represents whole bacteria without D7 expression, 4 represents whole bacteria induced by D7 expression, 5 represents whole bacteria without D8 expression, and 6 represents whole bacteria induced by D8 expression. Figure I shows the Western blot results for monoclonal antibody 5B7. M represents the protein marker, 1 represents whole bacteria induced by D6 expression, 2 represents whole bacteria induced by D7 expression, and 3 represents whole bacteria induced by D8 expression.

[0028] Figure 9 ELISA epitope identification results; further, the D7 region was truncated and peptided into D9 (ALNTVVNQTYETPT), D10 (QTYETPTKPTKDKKD), D11 (KPTKDKKPDKQDQS) and D12 (PDKQDQSAKPKQQK).

[0029] Figure 10 PCR amplification results of variable regions; M is a 250 bp DNA ladder, 1 is PCR amplification of the variable region of the 5B7 heavy chain monoclonal antibody, and 2 is PCR amplification of the variable region of the 5B7 light chain monoclonal antibody. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustration and explanation of the present invention and should not be regarded as limiting the scope of protection of the present invention. In addition, where specific technical operation steps or conditions are not specified in the embodiments, they are all carried out in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially. Example 1: Construction of PDCOV N protein recombinant plasmid

[0031] 1.1 Primer Design PCR primers were designed targeting the PDCoV N protein (the amino acid sequence of which is shown in SEQ ID No. 12), as detailed in Table 1. The underlined portions in Table 1 represent enzyme restriction sites. The primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0032] Table 1 PCR Primers

[0033] 1.2 PDCoV gene amplification The PDCoV N fragment was amplified. The PCR amplification system and procedure are shown in Tables 2 and 3. The PCR products were detected by 1% agarose gel electrophoresis. The results of the PCR products after 1% agarose gel electrophoresis are shown in Tables 2 and 3. Figure 1 As shown in Figure A, the N gene fragment is approximately 1026 bp in size.

[0034] Table 2 PCR amplification system

[0035] Table 3 PCR amplification program

[0036] 1.3 Construction of Recombinant Plasmids pET-28a vector Nco I and Xho After double digestion with enzyme I, the double-digested vector was ligated with the N gene PCR gel-recovered product and transformed, then transformed into... DH5α In competent cells, the constructed recombinant plasmid was subjected to bacterial culture PCR. The positive bacterial cultures were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0037] The results after double enzyme digestion are as follows Figure 1 As shown in Figure B, the vector was then constructed, and sequencing results confirmed that the sequence was correct and there were no base mismatches. The recombinant plasmid was named pET-28a-N. Example 2: Induction, expression, purification, and serum titer determination of recombinant protein

[0038] 2.1 Induction and purification of recombinant proteins The recombinant plasmid pET-28a-N prepared in Example 1 was transformed into Escherichia coli BL21(DE3). A single colony was picked and incubated overnight in 1 mL of kanamycin-resistant LB medium. The next day, the bacterial culture was inoculated into 10 mL of kanamycin-resistant medium at a ratio of 1:100 and incubated at 37°C and 220 rpm / min for approximately 3 hours until OD was achieved. 600 When the value was between 0.6 and 0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression. After 6 h of induction, the precipitate was collected by centrifugation at 8000 rpm for 5 min. The precipitate was resuspended in 10 mL of PBS and sonicated for 10 min (3 s sonication, 3 s pause). After sonication, the precipitate was centrifuged at 12000 rpm for 30-60 min at 4 °C. The supernatant and precipitate were collected separately. The precipitate was dissolved in approximately 10 mL of 8 M urea at room temperature for 3 h. 2× loading buffer was added to the supernatant and precipitate respectively, and the mixture was placed on an electric oven at 100 °C for 10 min before SDS-PAGE identification.

[0039] Following the method described above, the induced expression level of pET-28a-N was increased. The supernatant after sonication was collected, filtered through a 0.2µm filter, and incubated overnight with a nickel column for purification. The specific operational steps are as follows: a. Column packing: Add 2 mL of nickel NTA resin to the purification tube, wash the column once with 5 column volumes of distilled water, and then equilibrate the column twice with 5 column volumes of equilibration buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris base, pH=8.0). b. Sample loading: Protein samples need to be filtered through a 0.45 μm filter before loading, then mixed with the nickel column, loaded into a 50 mL centrifuge tube and incubated overnight at 4°C. The next day, purification is performed, and the eluents of various gradients of the sample are collected. Finally, the column is washed twice with 5 column volumes of equilibration buffer, and 20% ethanol is added to the nickel column and stored at 4°C. c. Protein elution: Protein samples were eluted with different concentrations of imidazole (10 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1 M and 2 M) using equilibration buffer, 10 mL each time. The purpose was to determine the optimal elution concentration of imidazole and the protein elution concentration. d. SDS-PAGE identification: After processing the sample, run a protein gel to determine the optimal elution concentration.

[0040] e. Protein dialysis: The purified protein is subjected to gradient dialysis, with the dialysis gradient of the equilibration buffer set to (1.5M, 1M, 0.5M, 0M) imidazole.

[0041] 2.2 ELISA method for detecting polyclonal antibody titers in mouse serum The obtained protein was coated onto an ELISA plate at a concentration of 200 ng / well for 2 hours at 37°C. The plate was then washed four times with PBS, patted dry, and blocked with 5% skim milk powder at 37°C for 2 hours. Positive and negative sera were serially diluted with PBS, with a dilution gradient from 1:1000 to 1:128000, for a total of 8 gradients. The plates were incubated at 37°C for 1 hour, washed four times with PBS, and HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added. The plates were washed four times with PBS, patted dry, and incubated with TMB for 15 minutes. Stop solution was then added, and the plates were placed on a microplate reader to measure OD. 450nm .

[0042] The recombinant plasmid pET-28a-N was transformed into Escherichia coli BL21(DE3), and expression was induced by adding IPTG at a final concentration of 1 mmol / L. The induced product was sonicated and then identified by SDS-PAGE. The results showed that pET-28a-N was successfully expressed. Figure 2 As shown in Figure A), and expressed in large quantities in the supernatant ( Figure 2 As shown in Figure B), this indicates that the N recombinant protein exists in a soluble protein form. The supernatant after ultrasonic centrifugation was then purified using a nickel column purification method. Figure 2 As shown in Figure C, high-purity N recombinant protein was obtained. The purified N recombinant protein was used to immunize BALB / c mice three times, followed by antibody titer detection. An ELISA plate was coated with N recombinant protein, and antibody titers were measured. Serum from unimmunized mice was used as a negative control (NC). The results are as follows. Figure 3 As shown, when serum is diluted 1:128000, the OD of immunized mice... 450nm / NC≥2.1 indicates that the antibody titer can reach 1:128000 or higher. Example 3: Preparation, Reactivity, and Variable Region Sequencing of Monoclonal Antibodies

[0043] 3.1 Animal Immunization Three 6-week-old female BALB / c mice were selected, and the purified N recombinant protein from Example 2 was immunized in mice at a dose of 50 μg / mouse. For the first immunization, the N protein was mixed with an equal volume of Bio-Drone water adjuvant and injected subcutaneously into two points on the back of the mice. A booster immunization was performed 21 days after the first immunization, and a total of three immunizations were performed. Blood samples were collected from the eyeballs 14 days after the third immunization to measure antibody titers. Before fusion, a booster immunization was performed, and the N recombinant protein was injected intraperitoneally into the mice to be subjected to cell fusion at a dose of 30 μg / mouse.

[0044] 3.2 Preparation of Monoclonal Antibodies The specific steps are as follows: (1) Cell fusion: Spleen cells and an appropriate amount of SP2 / 0 cells were fused using the fusion agent PEG. The fused cells were seeded in 96-well plates, and the culture medium contained PEG-enriched PEG.

[0045] (2) Screening of positive clones: a) Indirect ELISA detection: The purified N recombinant protein is coated onto an ELISA plate to detect the antibody secretion of fusion cells.

[0046] b) Indirect immunofluorescence (IFA) detection: The supernatant from the ELISA antibody-positive cell wells was added to PDCoV-infected VERO cells, followed by the addition of FITC-labeled goat anti-mouse IgG. After the reaction was complete, the results were observed under a fluorescence microscope.

[0047] (3) Subcloning of positive hybridoma cells: Wells that were positive for both ELISA and IFA were selected, and the positive hybridoma cells were subcloned using the limiting dilution method. After 10 days, the wells with only a single clone were marked under an inverted microscope. The supernatant was collected, and antibody detection was performed using the ELISA and IFA methods described above. Positive cells were then used in the next round of subcloning, for a total of three times.

[0048] (4) Preparation of ascites fluid: Take 10-12 week old female BALB / c mice, and prepare 0.5 mL of special adjuvant from the peritoneal fluid of each mouse. After 10 days, inject 5 × 10 mL of the adjuvant into each mouse intraperitoneally. 5 After 7–10 days, the abdominal cavity of the mouse showed obvious bulging. Ascites fluid was collected and stored at -80°C.

[0049] (5) Purification of ascites fluid: Affinity chromatography was performed according to the ProteinIso® Protein G Resin from TransGen Biotech, and the antibody purity was identified by SDS-PAGE electrophoresis.

[0050] 3.3 Identification of Monoclonal Antibody Reactivity (1) Reactivity verification: including ELISA, IFA and Western blot verification.

[0051] (a) ELISA validation of purified antibody titer: The purified N recombinant protein was coated onto an ELISA plate at 200 μL / well and incubated at 37°C for 2 h. Serially diluted 5B7 monoclonal antibody was added, and the plate was incubated at 37°C for 1 h. The plate was washed four times with PBS'T, and HRP-labeled goat anti-mouse IgG (1:8000 dilution) was added. The plate was washed four times with PBS'T, and TMB was added for 15 min for color development. Stop solution was then added, and the plate was placed on a microplate reader to measure OD. 450nm .

[0052] A monoclonal antibody specifically recognizing the PDCoV N protein was obtained through cell fusion technology and named 5B7. ELISA results are as follows: Figure 4 As shown, this monoclonal antibody reacts with the N recombinant protein and has a high titer.

[0053] (b) IFA verification of antibody reactivity: VERO cells infected with PDCoV strain and uninfected VERO cells were fixed with paraformaldehyde at 37°C for 10 min, and then permeabilized with 1% Triton X-100 at 37°C for 10 min. The purified antibody was diluted 1:300 with 5% skim milk powder and added to the cell plate. The cells were incubated at 37°C for 1 h, washed three times with PBS, and then incubated at 37°C with FITC-anti-mouse secondary antibody (1:300 dilution) for 1 h. The cells were washed three times with PBS and observed under a fluorescence microscope.

[0054] IFA assessment results are as follows: Figure 5 As shown, by Figure 5 As can be seen from section A, the monoclonal antibody 5B7 prepared in this application can detect a specific green fluorescent signal in VERO cells infected with the PDCoV strain. Figure 5 As can be seen from B, uninfected VERO cells do not react with the monoclonal antibody 5B7 prepared in this application.

[0055] (c) Western blot verification of antibody reactivity: PDCoV-infected and uninfected VERO cells were collected and run on SDS-PAGE. The protein gel was then transferred to an NC membrane for Western blot validation. The primary antibody was a diluted 5B7 monoclonal antibody, and the secondary antibody was HRP-labeled IgG.

[0056] Western blot validation results are as follows: Figure 6 As shown, the monoclonal antibody 5B7 prepared in this application can specifically bind to PDCoV-infected VERO cell samples, but does not react with uninfected VERO cell samples.

[0057] (2) Identification of monoclonal antibody subclasses: The obtained monoclonal antibodies were identified according to the instructions of Proteintech's Mouse Monoclonal Antibody Isotyping Kit.

[0058] The monoclonal antibody 5B7 prepared in this application was subjected to subtype identification using a mouse monoclonal antibody subtype identification kit. The identification results are as follows: Figure 7 As shown, the heavy chain constant region of the monoclonal antibody 5B7 prepared in this application is of the IgG1 type, and their light chain constant region is of the Kappa type.

[0059] (3) Epitope identification: The N gene was truncated and cloned into a prokaryotic expression vector. After induction of expression, SDS-PAGE was performed and the gene was transferred to an NC membrane. The reactivity of the monoclonal antibody was verified by Western blot, thereby determining the antigenic epitope recognized by the monoclonal antibody. The truncation was continued, and the antigen recognition site was further shortened using the same method.

[0060] To identify the region of the monoclonal antibody that recognizes the N recombinant protein, the N recombinant protein was subjected to the following process: Figure 8 Genes were truncated as shown in Figure A and named C1 and C2, respectively. These were then inserted into the prokaryotic expression vector pET-28a, and the results are as follows: Figure 8 As shown in Figure B, both regions C1 and C2 are expressed. Western blot results are as follows... Figure 8 As shown in Figure C, the monoclonal antibody 5B7 prepared in this application reacts with the C2 region but not with the C1 region, indicating that the region of the N protein recognized by the monoclonal antibody 5B7 prepared in this application is the C2 region, i.e., 145-342aa. Further truncating the C2 region for expression ( Figure 8 As shown in Figure D), the monoclonal antibody 5B7 prepared in this application recognizes the D5 region but cannot recognize the D3 and D4 regions. This indicates that the monoclonal antibody 5B7 prepared in this application recognizes the D5 region of the N protein, namely 255-342aa. Figure 8 (As shown in E and F). The expression of the D5 region is further truncated ( Figure 8 As shown in Figure G), the monoclonal antibody 5B7 prepared in this application, constructed into the pMAL-C2X vector, can recognize D7 but cannot recognize D6 and D8. This indicates that the monoclonal antibody 5B7 prepared in this application recognizes the D7 region of the N protein, namely 280-314aa. Figure 8 (As shown in H and I).

[0061] Further, the D8 region (280-314aa) was truncated and polypeptides were synthesized: D9 (ALNTVVNQTYETPT), D10 (QTYETPTKPTKDKKD), D11 (KPTKDKKPDKQDQS), and D12 (PDKQDQSAKPKQQK). Epitopes were identified by ELISA, and the results are as follows: Figure 9 As shown, the monoclonal antibody 5B7 described in this application recognizes the D11 region of the N protein, namely 294-307aa, and the recognized antigenic epitope sequence is shown in SEQ ID No. 11.

[0062] 3.4 PCR amplification and sequencing of the variable region gene of monoclonal antibody First, RNA was extracted from monoclonal antibody hybridoma cells, and then reverse transcribed into cDNA using the Novozymes HiScript II Q RTSuperMix for qPCR reverse transcription kit.

[0063] The antibody variable region gene was amplified using nested PCR. First, using the aforementioned cDNA as a template, the antibody variable region gene was amplified using the first round of mouse antibody IgG1 and κ light chain primers. Then, using the first-round product as a template, the antibody variable region gene was amplified using the second antibody IgG1 and κ light chain primers. The PCR reaction system and procedure are shown in Tables 4 and 5.

[0064] Primers for antibody variable region gene amplification were referenced in the literature (Von Boehmer L, Liu C, Ackerman S, et al. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016;11(10):1908-1923.). After amplification, 1% agarose gel electrophoresis was performed. The size of the heavy and light chain variable regions was approximately 300 bp. The target fragment was excised and recovered. The recovered target fragment was inserted into the pMD-19T vector and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0065] Table 4 PCR amplification antibody variable regions

[0066] Table 5 PCR amplification program

[0067] The PCR amplification results of the variable region of the monoclonal antibody 5B7 described in this application are as follows: Figure 10 As shown, a PCR product of approximately 300 bp in size was amplified from cDNA of 5B7 hybridoma cells. Figure 10 The results of amplifying the 5B7 heavy chain variable region gene and the light chain variable region gene were consistent with the expected amplification product size; after gel extraction and recovery, the product was cloned into the pMD19-T vector and sequenced.

[0068] The sequencing results were compared with the antibody gene library (IMGT). The sequencing results confirmed that the amplified sequence was the complementarity determining region (CDR) sequence of the heavy chain variable region and the light chain variable region of the monoclonal antibody, as shown in Table 6.

[0069] Table 6 Antibody variable region sequences

[0070] The amino acid sequence of the heavy chain variable region is: ESGPGLVQPSQSLSMTCTVSGFSLTRYGVHWVRQSPGKGLEWLGVMWRRGITDYNAAFISRLTITKDNSRSQVFFKMNSLQPNDTGKYFCARKRDGYAMDYWGQGTSVIVSSP (SEQ ID No. 7). The amino acid sequence of the light chain variable region is: QSPLSLPVTLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK (as shown in SEQ ID No. 8); The gene sequence encoding the heavy chain variable region is: GAGTCTGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATGACCTGCACAGTCTCTGGTTTCTCTTTAACTCGGTATGGTGTACACTGGGTTCGTCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATGTGGCGTCGTGGAATCACAGACTATAA TGCGGCTTTCATATCCAGACTGACCATCACCAGGACAATTCCAGGAGTCAAGTTTTCTTAAATGAACAGTCTGCAACCTAATGACACAGGCAAATATTTCTGTGCCAGAAAACGAGACGGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCATCGTCTCCTCACCC (SEQ IDNo.9 shown); The gene sequence encoding the light chain variable region is: CAATTCCACTCTCCCTGCCTGTCACTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACTTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCGGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ Shown as ID No.10); The N protein monoclonal antibody can specifically recognize the region of the porcine epidemic diarrhea virus N protein as: KPTKDKKPDKQDQS (shown in SEQ ID No. 11). PDCoV N protein amino acid sequence: MAAPVVPTTDASWFQVLKAQNKKATHPQFRGNGVPLNSAIKPVENHGYWLRYTRQKPGGTPIPPSYAFYYTGTGPRGNLKYGELPPNDTPATTRVTWVKGSGADTSIKPHVAKRNPNNPKHQLLPLRFPTGDGPAQGFRVDPFNARGRPQERGSGPRSQSVNSRGTGN Question Shown as IDNo.12).

[0071] In summary, this invention provides a monoclonal antibody against the N protein of porcine deltacoronavirus (PDCoV). This monoclonal antibody specifically recognizes and binds to amino acids 294-307 of the N protein, providing a reliable tool for exploring the function of the N protein. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention provides materials for studying the pathogenic mechanism of PDCoV and for developing PDCoV diagnostic kits.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of the present invention should be included within the scope of the present invention.

Claims

1. An antigenic epitope peptide of the porcine deltacoronavirus N protein, characterized in that, The antigenic epitope peptide is located at 294-307aa of the porcine deltacoronavirus N protein, and its amino acid sequence is shown in SEQ ID No.

11.

2. The use of the antigenic epitope peptide as described in claim 1 in the preparation of monoclonal antibodies against porcine deltacoronavirus N protein.

3. A monoclonal antibody against porcine deltacoronavirus N protein, characterized in that, The monoclonal antibody 5B7 comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No.

6.

4. The monoclonal antibody as described in claim 3, characterized in that, The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No.

8.

5. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody of claim 3 or 4.

6. The nucleic acid as described in claim 5, characterized in that, The nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID No. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID No.

10.

7. An expression cassette, expression vector, or recombinant bacteria containing the nucleic acid of claim 5 or 6.

8. The use of the monoclonal antibody as described in claim 3 or 4 in the preparation of reagents for detecting porcine deltacoronavirus.

9. The use of the monoclonal antibody as described in claim 3 or 4 in the detection of porcine deltacoronavirus infectivity for non-disease diagnosis purposes or in the study of the function of the porcine deltacoronavirus N protein.

10. A porcine deltacoronavirus detection kit, characterized in that, The kit includes the monoclonal antibody as described in claim 3 or 4.

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