Porcine delta coronavirus spike protein monoclonal antibody, antigen epitope peptide and application

Abstract

The application discloses a porcine delta coronavirus spike protein monoclonal antibody, an antigen epitope peptide and application, and belongs to the technical field of biology. The antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 3. The application first identifies a highly conserved linear B cell epitope (the amino acid sequence is DFGEARLD) of a PDCoV spike protein receptor binding domain (S-RBD) and a neutralizing monoclonal antibody capable of specifically combining the epitope. The epitope peptide and the monoclonal antibody provided by the application can be used for immunological detection and serological investigation of PDCoV.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody against the spike protein of a porcine delta coronavirus, an antigenic epitope peptide, and their applications, belonging to the field of biotechnology. Background Technology

[0002] Since its inception, monoclonal antibody technology has become an indispensable core tool in life science research and medical applications. These antibodies possess significant advantages such as high specificity, good uniformity, and the ability to be mass-produced, and are widely used in disease diagnosis, targeted therapy, pathogen detection, and basic research. With the continuous advancement of hybridoma technology and genetically engineered antibody technology, the application scope of monoclonal antibodies has expanded to fields such as agriculture, food safety, and environmental monitoring.

[0003] Porcine deltacoronavirus (PDCoV), also known as swine deltacoronavirus (PDCoV), was previously called swine type D coronavirus or swine coronavirus HKU15. It belongs to the order Nidovirales, family Coronaviridae, subfamily Coronavirinae, and genus Deltacoronavirus. This virus was first detected in pig herds in Hong Kong, China in 2012, and was subsequently formally identified and named during a swine diarrhea outbreak in several US states in 2014. Etiological studies have shown that PDCoV can cause acute diarrhea, vomiting, dehydration, and even death in piglets, with morbidity and mortality rates as high as 50%–100%, seriously endangering the healthy development of the pig industry and causing significant economic losses. Genome alignment shows that the strain isolated from the United States is highly homologous to the Hong Kong strain HKU15 in the M and N gene sequences, with a similarity exceeding 99%, further confirming its taxonomic status.

[0004] Similar to other coronaviruses, the spike protein (S protein) of PDCoV is located on the surface of the viral particle, with a molecular weight of approximately 200 kDa. It exhibits high homology (over 90%) among different strains and is a key protein mediating viral invasion of host cells, playing a central role in receptor recognition, membrane fusion, and neutralizing antibody induction. Therefore, the S protein has become an important target for the development of diagnostic reagents, subunit vaccines, and therapeutic antibodies. However, the identification of PDCoV S protein epitopes remains limited, especially targeting the S protein receptor-binding domain (RBD). High-affinity, high-specificity monoclonal antibodies targeting this receptor-binding domain are extremely scarce, hindering the development of related detection technologies and immune intervention strategies.

[0005] Therefore, it is urgent to identify immunogenic epitopes in the PDCoV S-RBD protein and to develop specific monoclonal antibodies with good reactivity, so as to provide key technical support for establishing effective pathogen detection methods, serological evaluation systems and the development of neutralizing antibody drugs. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a monoclonal antibody against the spike protein of porcine delta coronavirus, its specifically binding antigenic epitope peptide, and its applications.

[0007] Technical solution: The present invention provides a monoclonal antibody against the spike protein of a porcine delta coronavirus, the antibody comprising a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID No. 3.

[0008] Furthermore, the heavy chain and the light chain are connected by disulfide bonds.

[0009] Furthermore, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 2, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 4.

[0010] Furthermore, the heavy chain of the monoclonal antibody is IgG1.

[0011] The present invention discloses a porcine delta coronavirus antigenic epitope peptide, which can specifically bind to the aforementioned monoclonal antibody; the amino acid sequence of the antigenic epitope peptide is DFGEARLD.

[0012] The present invention relates to the application of monoclonal antibodies and porcine delta coronavirus antigenic epitope peptides in the detection of porcine delta coronavirus for non-disease treatment and diagnostic purposes.

[0013] The present invention discloses a kit for detecting porcine delta coronavirus, wherein the kit contains the aforementioned porcine delta coronavirus spike protein monoclonal antibody and / or the aforementioned porcine delta coronavirus antigenic epitope peptide.

[0014] Furthermore, the kit also contains RBD recombinant protein, coating solution, blocking solution, diluent, PBST, TMB substrate chromogenic solution, and H2SO4 stop solution.

[0015] Further, the method of using the kit includes the following steps: washing and blocking with RBD recombinant protein or the above-mentioned porcine δ-coronavirus epitope peptide as the coating antigen; adding diluted test serum, incubating, and washing; adding diluted porcine δ-coronavirus spike protein monoclonal antibody as the enzyme-labeled antibody, incubating, and washing; adding chromogenic solution, incubating, adding stop solution, and reading OD.450 value.

[0016] Furthermore, the RBD recombinant protein coating concentration is 10 ng / well, and the enzyme-labeled antibody dilution is 1:50,000.

[0017] Furthermore, the method for judging the test results is to calculate the inhibition rate (PI). A PI ≥ 31.45% is judged as positive; a PI < 31.45% is judged as negative.

[0018] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention is the first to identify a highly conserved linear B-cell epitope (with the amino acid sequence DFGEARLD) of the PDCoV spike protein receptor-binding domain (S-RBD) and a neutralizing monoclonal antibody capable of specifically binding to this epitope. The epitope peptide and monoclonal antibody provided by this invention can be used for the immunological detection and serological survey of PDCoV. Attached Figure Description

[0019] Figure 1 This is an indirect immunofluorescence diagram illustrating the reactivity of porcine delta coronavirus (PDCoV) monoclonal antibodies 1C12 and 4C9 with LLC-PK1 cells infected with three different PDCoV strains, as described in this invention.

[0020] Figure 2 This is a schematic diagram illustrating the protein expression and purification of the S, S1, and RBD proteins of PDCoV in this invention. Figure 2 A is a schematic diagram of the extracellular domain topology of the PDCoV S protein; Figure 2 B is an SDS-PAGE image of the S, S1, and RBD proteins of PDCoV; Figure 2 C is a Western blot plot of the S protein, S1 protein and RBD protein of PDCoV detected using 1C12 monoclonal antibody; Figure 2 B and Figure 2 Lanes 1, 2, and 3 in C represent the S protein, S1 protein, and RBD protein of PDCoV, respectively.

[0021] Figure 3 This is a schematic diagram of the antigenic epitope screening of the PDCoV monoclonal antibody 1C12 in this invention.

[0022] Figure 4 This is a schematic diagram illustrating the sequence alignment of the S protein of different PDCoV strains in this invention.

[0023] Figure 5 This diagram illustrates the optimal antigen coating concentration and optimal enzyme-labeled antibody dilution screening degree for blocking ELISA in this invention.

[0024] Figure 6This is a schematic diagram of the ROC curve (A) and detection scatter plot (B) of the blocking ELISA in this invention.

[0025] Figure 7 This is a schematic diagram illustrating the specificity verification of blocking ELISA in this invention.

[0026] Figure 8 This is a schematic diagram illustrating the analytical sensitivity verification of blocking ELISA in this invention. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1: Preparation and screening of monoclonal antibodies against porcine delta coronavirus (PDCoV) 1. Preparation and identification of monoclonal antibodies against porcine delta coronavirus (PDCoV) 1.1 Preparation of monoclonal antibodies against porcine delta coronavirus (PDCoV) In the preparation and immunogenicity evaluation of monoclonal antibodies, the PDCoV strain (titer 10) preserved by the Animal Emerging and Major Diseases Comprehensive Prevention and Control Team of the College of Veterinary Medicine, Yangzhou University, was used. 7.3 TCID 50 A concentrated viral solution (300 μL / molar) was used as the immunogen. The solution was emulsified with an equal volume of Freund's complete adjuvant and used for the primary immunization. Three booster immunizations were performed using an equal volume of Freund's incomplete adjuvant. The second immunization was two weeks after the first, and the three booster immunizations were one week apart. Five 4-6 week old female BALB / c mice (purchased from the Experimental Animal Center of Yangzhou University, approximately 20 g / mouse) were injected intraperitoneally with the emulsified immunogen. Serum was collected 7-10 days after the fourth immunization, and the titer was detected by indirect immunofluorescence assay (IFA). Preliminary results (Table 1) showed that the immunized group met the requirements for cell fusion experiments.

[0029] The specific procedure for indirect immunofluorescence assay is as follows: LLC-PK1 cells are inoculated at 2×10⁻⁶ cells per cell line. 4 Cells were seeded per well in 96-well plates and cultured for 24 hours to form a monolayer. PDCoV virus solution was then inoculated into each well, with normal cells used as a negative control. After 36 hours of culture, the cells were fixed with 80% acetone. The test samples were diluted twice with PBS (1:200–1:102400), and 100 μL was added to each well. The wells were incubated at 37°C for 30 minutes, followed by detection using FITC-labeled rabbit anti-mouse IgG secondary antibody diluted 1:800. Results were determined by fluorescence microscopy: no fluorescence was observed in the negative control, and specific green fluorescence was visible in the cytoplasm of the sample wells, indicating a positive result. The highest dilution producing a clear positive signal was determined as the antibody titer.

[0030] Table 1. Serum IFA titer detection in mice immunized with PDCoV strain

[0031] Mice-2 and mice-5 were selected and intraperitoneally injected with unadjuvanted, unconcentrated PDCoV virus solution as an immunogen 3 days before fusion, at a dose of 300 μL / mouse for shock immunization. Three days after immunization, mouse spleen cells and SP2 / 0 cells were collected for cell fusion. Hybridoma cells were screened using the IFA method. After multiple screenings and subcloning, two positive hybridoma cell lines were obtained. One positive hybridoma cell line from mouse-2 was named 1C12, and one positive hybridoma cell line from mouse-5 was named 4C9. Subsequently, six 8-week-old BALB / c mice (purchased from the Experimental Animal Center of Yangzhou University, with three mice inoculated with each hybridoma cell line) were selected and intraperitoneally injected with Freund's incomplete adjuvant (0.2–0.3 mL per mouse). Three days later, the corresponding hybridoma cells were injected intraperitoneally to sensitize the mice (1–3 × 10⁶ cells per mouse). 6 (10 cells, suspended in 0.3 mL of culture medium). Starting on day 5 post-injection, the abdominal distension of mice was observed. If significant swelling and abdominal wall tension were felt upon palpation, ascites fluid was collected using a 16-gauge needle. Each mouse could be collected 2-3 times consecutively. The collected ascites fluid was centrifuged at 2000 rpm for 5 minutes to remove the supernatant and red blood cells from the precipitate. The ascites fluid antibody titer was determined using indirect immunofluorescence (IFA). The results showed that the ascites fluid titer for strain 1C12 reached 1:51200, and that for strain 4C9 was 1:12800. The ascites supernatant was aliquoted and stored at –80°C. Thus, two anti-PDCoV monoclonal antibodies were successfully obtained.

[0032] 1.2 Subtype identification of porcine delta coronavirus (PDCoV) monoclonal antibodies The two obtained monoclonal antibodies were identified as having antibody subclasses using a monoclonal antibody subclass identification kit from Bio-Tech. The specific method was performed according to the manufacturer's instructions. Table 2 shows that the heavy chain subclass of both monoclonal antibodies was IgG1; the light chain subclass of both monoclonal antibodies was Kappa.

[0033] Table 2. Monoclonal antibody subtype validation

[0034] 2. Screening for monoclonal antibodies against porcine delta coronavirus (PDCoV) 2.1 Validation of the reactivity of monoclonal antibodies against porcine delta coronavirus (PDCoV) We verified the reactivity of the monoclonal antibody to the virus strain using an IFA experiment. First, LLC-PK1 cells were divided into groups of 2 × 10⁶ cells per well. 4Cells were inoculated into 96-well plates and cultured for 24 hours to form a monolayer. Then, PDCoV virus solutions isolated from pathogens from three different provinces were inoculated into each well, with normal cells used as a negative control. Thirty-six hours after infection, cells were fixed with 80% acetone. Monoclonal antibody ascites fluid was diluted 1:1000 with PBS, and 100 μL was added to each well, incubated at 37°C for 30 minutes. Subsequently, FITC-labeled rabbit anti-mouse IgG secondary antibody diluted 1:800 was added for detection. Results were interpreted under a fluorescence microscope: negative control wells should show no fluorescence signal, and sample wells were considered positive if specific green fluorescence was observed in the cytoplasm.

[0035] Three PDCoV strains isolated from different provinces were preserved and provided by the Animal Emerging and Major Diseases Comprehensive Prevention and Control Team of the College of Veterinary Medicine, Yangzhou University. Figure 1 As shown, two monoclonal antibodies were found to have good reactivity with three prevalent strains.

[0036] 2.2 Verification of virus neutralizing activity of porcine delta coronavirus (PDCoV) monoclonal antibody Furthermore, we determined the virus neutralizing activity of the two monoclonal antibodies. The three PDCoV strains isolated from different provinces were provided by the Animal Emerging and Major Diseases Comprehensive Prevention and Control Team of the College of Veterinary Medicine, Yangzhou University. The specific neutralizing titers are shown in Table 3. Through screening, we found that only the 1C12 monoclonal antibody had neutralizing activity.

[0037] The specific procedure for the virus neutralization experiment is as follows: LLC-PK1 cells were seeded in 96-well plates. Once the cells reached confluence or approximately 90% confluence, the two prepared monoclonal antibodies were filtered and sterilized, then inactivated in a 56°C water bath for 30 min. The two monoclonal antibodies were serially diluted (1:2~1:256) using DMEM. For each dilution, 300 μL of the diluted antibody was vortexed with 300 μL of virus suspension to prepare an antibody-virus mixture, which was incubated at 37°C for 1 h. After incubation, the supernatant from the LLC-PK1 cell culture in the 96-well plate was aspirated using a multi-row pipette, and the cells were washed twice with sterile PBS. 100 μL of the antibody-virus mixture was added to the LLC-PK1 cell monolayer in the 96-well plate. Four replicates were performed for each dilution, and four positive control wells were included (containing only 100 μL / 200 TCID45). 50 The virus suspension and four negative control wells (containing only 100 μL of cell maintenance medium) were incubated in a 37°C, 5% CO2 incubator. The culture medium was replaced after 2 hours. The results were observed daily until the cells stopped producing CPE after 2–4 days.

[0038] Table 3. Verification of monoclonal antibody neutralization activity

[0039] Based on the above screening, the monoclonal antibody strain 1C12 was selected for further research.

[0040] 2.3 Sequencing of the light and heavy chain variable regions of porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed: C H -F: 5'-ACTACTTGACGTGCTCTAGGTCACTTTACTTTCCCT-3' C H -R: 5'-CGGAGCTTTCCAGCGCCARCCCATATACTGRTGG-3' Design primer sequences for the light chain variable region: C L -F: 5'-GCCATCTAGRAWCATTKWCYCAAGTCTTT-3' C L -R: 5'-CGGAGCCTTACTGCCTGTAAGAAGATGGA-3' Hybridoma cells of strain 1C12 were collected, RNA was extracted, and cDNA was reverse transcribed using the RNA as a template. The variable region sequence was amplified using the primers described above, and the amplified product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. Results: The amino acid sequence of the light chain variable region of monoclonal antibody 1C12 was determined as shown in SEQ ID No. 1: DIVLTQSPASLAVSPRQRATISCKSAVSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPVRFSGSGSGTDFSLNIHPVEEERAATYYCQQSNEDPFTFGSGTKLEIKR; The nucleotide sequence of the light chain variable region is shown in SEQ ID No. 2: gacattgtgctgacccaatctccagcttctttggctgtgtctccacgacagagggccaccatctcctgcaagtcggcggtaagtgttgattatgatggtgatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcatccaatctagaatctgggatcccagtaaggtttagtggcagtgggtctgggacagacttctcgctcaacatccatcctgtggaggaagagcgggctgcaacctattactgtcagcaaagtaatgaagatccattcacgttcggctcggggacaaagttggaaataaaacgg; The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.3: EVQLQQSGPILVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNGGTSYNQKFKGKATLTVDKSSSTSYMELRSLTSEDSAVYYCARDGYDTRYSYVMDYWGQGTSVTVSSAKTTPPS; The base nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.4: gaggtccagctgcaacagtctggacctatactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggatacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggtattaatcctaacaatggtggtactagttacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacatcatacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagagatggttacgacacgcggtatagctatgttatggactactggggtcaaggaacctcagtcaccgtctcctctgccaaaacaacacccccatct。

[0041] Example 2: Screening of antigenic epitopes for porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 1. Expression and purification of porcine delta coronavirus (PDCoV) S, S1 and RBD proteins In this embodiment, we describe in detail a method for expressing S, S1, and RBD proteins in ExpiCHO-S cells. First, we obtained the required ExpiCHO-S cells, expression medium, and transfection reagents from Thermo Fisher Scientific and cultured them at 32 °C with 5% CO2. According to... Figure 2 Based on the extracellular domain topology of the PDCoV S protein in strain A, and referencing the coding sequences of the S, S1, and RBD domains of the PDCoV CZ2020 strain (GenBank accession No. OK546242) published by NCBI, we added a signal peptide (MDAMKRGLCCVLLLCGAVFVSAS) to the N-terminus of the reference sequence to ensure secretory expression of the protein. The sequence was optimized and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the genome was cloned into the pcDNA3.1(+) vector. Subsequently, we transfected the recombinant plasmids pcDNA3.1-S, pcDNA3.1-S1, and pcDNA3.1-RBD into ExpiCHO-S cells and cultured them under the same conditions for 10-12 days.

[0042] After the protein expression phase, we collected the culture supernatant and clarified it by centrifugation at 5,000 × g for 30 min at 4 °C, followed by filtration through a 0.22 μm sterile membrane. The target protein was captured by affinity purification using Ni-NTA agarose resin at 4 °C for 2 h. Next, the protein was washed with 1× phosphate-buffered saline (PBS) to remove weakly bound impurities, and the target protein was eluted with 1× PBS containing 500 mM imidazole. The fractions containing the target protein were pooled, and their concentration was determined using a BCA assay kit. Finally, the high-purity protein was aliquoted and stored at -80 °C for future use. This method allows for the production of... Figure 2 B. SDS-PAGE images show that we successfully expressed S, S1, and RBD proteins in ExpiCHO-S cells and effectively purified and stored them, providing high-quality protein samples for subsequent experimental studies.

[0043] 2. Screening of epitopes for porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 Epitope screening is of great value for vaccine design and diagnostic reagent development. To determine the minimal antigenic epitope recognized by the 1C12 monoclonal antibody, this study performed a systematic analysis using Western blotting (WB). First, purified S, S1, and RBD proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was then blocked with 5% bovine serum albumin (BSA) for 2 hours, washed five times with PBST (PBS buffer containing 0.05% Tween-20) at pH 7.2, and incubated with anti-His monoclonal antibody (diluted 1:2000 with blocking buffer) at room temperature for 2 hours. After washing five times with PBST, the membrane was incubated with HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (diluted 1:10000) at room temperature for 1 hour. After thorough washing with PBST, the membrane was analyzed using Clarity assay. TM Western chemiluminescence (ECL) was used to develop the chemiluminescent substrate, and images were acquired using a VILBERFusion FX7 chemiluminescence imaging system. For example... Figure 2 As shown in C, the purified protein was detected by Western blot using the 1C12 monoclonal antibody. The results showed that the antibody could specifically react with S, S1 and RBD proteins, indicating that its recognition epitope is located in the RBD region, that is, the 300-419 amino acid range of the S protein.

[0044] To screen for epitopes recognized by monoclonal antibodies, the RBD region (amino acid 300-419 of the S protein) was further truncated and expressed. First, using the pcDNA3.1-RBD plasmid as a template, the full-length RBD (300-419 aa) sequence was amplified using the primer pairs listed in Table 4 (EGFP-δ-RBD-F / R). Three truncated mutants were then amplified using corresponding primers: RBD (300-349 aa), RBD (340-389 aa), and RBD (380-419 aa). Simultaneously, using the pEGFP-C1 vector as a template, the vector was linearized using the EGFP-F / R primers listed in Table 4, and the amplified sequences were constructed into the pEGFP-C1 vector. Each recombinant plasmid was transfected into HEK293T cells, and total cellular protein was collected after 24 hours of culture. Western blot analysis preliminarily located the epitope identified by monoclonal antibody 1C12 within the 300-349 amino acid range. Figure 3 A).

[0045] To further screen the epitopes recognized by the monoclonal antibody, the pEGFP-RBD (300-349 aa) plasmid was used as a template and further truncated using the primers in Table 4 to construct expression plasmids pEGFP-RBD (300-319 aa), pEGFP-RBD (310-329 aa), pEGFP-RBD (320-339 aa), and pEGFP-RBD (330-349 aa). Each of the constructed recombinant plasmids was transfected into HEK293T cells, and total cell protein was collected after 24 hours of culture. Western blot analysis narrowed the recognition epitope to the 310-329 amino acid region (…). Figure 3 B).

[0046] To definitively determine the minimal recognition epitope, the pEGFP-RBD (300-319aa) plasmid was used as a template. Using primers from Table 4, the region was progressively truncated from the amino and carboxyl ends, resulting in the construction of eight expression plasmids: pEGFP-RBD (313-329aa), pEGFP-RBD (316-329aa), pEGFP-RBD (319-329aa), pEGFP-RBD (322-329aa), pEGFP-RBD (310-326aa), pEGFP-RBD (310-323aa), pEGFP-RBD (310-320aa), and pEGFP-RBD (310-317aa). Each recombinant plasmid was then transfected into HEK293T cells, and total cellular protein was collected after 24 hours of culture. Western blot analysis showed that the minimal linear B-cell epitope recognized by the 1C12 monoclonal antibody is located in the 316-323 amino acid range. Figure 3 C), whose core sequence is DFGEARLD. In the figure, red indicates a reaction with monoclonal antibodies, and green indicates no reaction with monoclonal antibodies.

[0047] Sequence alignment was performed using MEGA12 software, and it was found that " 316 DFGEARLD 323 "Epitope sequences are completely conserved in representative PDCoV strains reported in different countries." Figure 4 ).

[0048] In this embodiment, the primers for the truncated protein to be expressed are shown in Table 4. The primers were synthesized by Shanghai Sangon Biotech. The polymerase chain reaction (PCR) system and amplification procedure are as follows: Amplification was performed using a 50 μL PCR system according to the standard procedure in the manufacturer's manual. The reaction system contained: 25 μL 2×Hieff Canace ®The following PCR premix (containing dye) was prepared: 2 μL each of forward and reverse primers (10 μM), 1 ng template DNA (diluted with ddH2O), and the total volume was brought to 50 μL with ddH2O. The amplification program was set as follows: first, pre-denaturation at 94℃ for 3 minutes; then 30 cycles, each cycle including denaturation at 98℃ for 10 seconds, annealing at 56℃ for 20 seconds, and extension at 72℃ (30 sec / kb); after the cycles, a final extension at 72℃ for 5 minutes; after the reaction was terminated, the product was stored at 4℃.

[0049] Table 4 Primer sequences for PCR amplification of truncated protein genes EGFP-F GCTCAAGCTTCGAATTCTGCA EGFP-R TCGAGATCTGAGTCCGGACTTG EGFP-δ-RBD-F agtccggactcagatctcgaAAATTACCAGAATTAGAAGTTGTTCAACT EGFP-δ-RBD-R tgcagaattcgaagcttgagcTCACACGGAGGTGGTGCCT GFP-RBD(300-349aa)-F agtccggactcagatctcgaAAATTACCAGAATTAGAAGTTGTTCAACT GFP-RBD(300-349aa)-R tgcagaattcgaagcttgagcTCATGTGCACATGAAATTAGTTTCG GFP-RBD(340-389aa)-F agtccggactcagatctcgaTTTCGACTCGAAACTAATTTCATGTGCACA GFP-RBD(340-389aa)-R tgcagaattcgaagcttgagcTCAGATCTTCATCTCACAGGCCCCGCTCTCTGT GFP-RBD(380-419aa)-F agtccggactcagatctcgaACAGAGAGCGGGGCCTGTGAGATGAAGATC GFP-RBD(380-419aa)-R tgcagaattcgaagcttgagcTCACACGGAGGTGGTGCCT GFP-RBD(300-319aa)-F agtccggactcagatctcgaAAATTACCAGAATTAGAAGTTGTTCAACT GFP-RBD(300-319aa)-R gaattcgaagcttgagctcaTTCGCCGAAGTCCATGTGTG GFP-RBD(310-329aa)-F agtccggactcagatctcgaAATATATCAGCACACATGGACTTCG GFP-RBD(310-329aa)-R gaattcgaagcttgagctcaGCCGTTTATTGTGACTGAGTCTAGTC GFP-RBD(320-339aa)-F agtccggactcagatctcgaGCTCGACTAGACTCAGTCACAATAAAC GFP-RBD(320-339aa)-R gaattcgaagcttgagctcaATAAGGTTTTGTAACGCAGTAGGATG GFP-RBD(330-349aa)-F agtccggactcagatctcgaAACACATCCTACTGCGTTACAAAAC GFP-RBD(330-349aa)-R tgcagaattcgaagcttgagcTCACACGGAGGTGGTGCCT GFP-RBD(313-329aa)-F AGATCTCGAGCACACATGGACTTCGGCGAAGCT GFP-RBD(313-329aa)-R CATGTGTGCTCGAGATCTGAGTCCGGACTTGTA GFP-RBD(316-329aa)-F TCAGATCTCGAGACTTCGGCGAAGCTCGACTAG GFP-RBD(316-329aa)-R CGAAGTCTCGAGATCTGAGTCCGGACTTGTACA GFP-RBD(319-329aa)-F AGATCTCGAGAAGCTCGACTAGACTCAGTCACAA GFP-RBD(319-329aa)-R TCGAGCTTCTCGAGATCTGAGTCCGGACTTGTA GFP-RBD(322-329aa)-F CAGATCTCGACTAGACTCAGTCACAATAAACGGCTG GFP-RBD(322-329aa)-R GAGTCTAGTCGAGATCTGAGTCCGGACTTGTAC GFP-RBD(310-326aa)-F CTCAGTCACATGAGCTCAAGCTTCGAATTCTGC GFP-RBD(310-326aa)-R TGAGCTCATGTGACTGAGTCTAGTCGAGCTTCG GFP-RBD(310-323aa)-F CGACTAGACTGAGCTCAAGCTTCGAATTCTGCA GFP-RBD(310-323aa)-R TTGAGCTCAGTCTAGTCGAGCTTCGCCGAAGTC GFP-RBD(310-320aa)-F CGGCGAAGCTTGAGCTCAAGCTTCGAATTCTGCAGTC GFP-RBD(310-320aa)-R AGCTTGAGCTCAAGCTTCGCCGAAGTCCATGTG GFP-RBD(310-317aa)-F CATGGACTTCTGAGCTCAAGCTTCGAATTCTGC GFP-RBD(310-317aa)-R TGAGCTCAGAAGTCCATGTGTGCTGATATATTTCG Example 3: Establishment, evaluation, and application of a universal blocking ELISA antibody detection method based on the prepared monoclonal antibody. In this embodiment, PDCoV antibody-positive and negative swine serum and clinical swine serum samples were provided by the Animal Emerging and Major Diseases Comprehensive Prevention and Control Team of the College of Veterinary Medicine, Yangzhou University, and their antibody positivity and positivity were verified by virus neutralization test.

[0050] 1. The establishment of the blocking ELISA procedure based on 1C12 monoclonal antibody is as follows: 1) Serially dilute the purified RBD recombinant protein expressed in Example 2 to 5, 10, and 20 ng / well (diluent: 1× carbonate buffer at pH 9.6; 20× carbonate buffer ratio: 29 g NaHCO3 + 16 g NaCO3, bring to 1 L, and filter after thorough dissolution). Dilute serum samples 1:1 (diluent: PBS at pH 7.2). Use a horseradish peroxidase (HRP) labeling kit purchased from Suzhou Bio-Long Technology Co., Ltd. to label monoclonal antibody 1C12 with HRP, following the instructions. HRP-1C12 was diluted to 1:10,000, 1:30,000, 1:50,000, and 1:80,000 (using PBST at pH 7.2 and PBS buffer containing 0.05% Tween-20). The optimal antigen coating concentration and optimal enzyme-labeled antibody dilution ratio were screened using a checkerboard method with 5 PDCoV antibody-positive swine sera and 3 PDCoV antibody-negative swine sera validated by a virus neutralization assay.

[0051] 2) Coat the ELISA plate with carbonate coating buffer containing RBD recombinant protein at a volume of 100 μL / well and incubate at 4 °C for 24 h.

[0052] 3) Wash the plate once with PBST at a volume of 300 μL / well, then add 150 μL of 2.5% BSA (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to each well and block at 4 ℃ for 24 h.

[0053] 4) Incubate the diluted serum samples at 37 °C for 60 min. After washing four times with PBST, add diluted HRP-1C12 and incubate at 37 °C for 30 min. After washing four times with PBST, add 100 μL TMB to each well to start the colorimetric reaction.

[0054] 5) Add 50 μL of 2 M H2SO4 stop solution to each well, and terminate the reaction at 37 °C for 15 min. Then measure the OD. 450 The absorbance value at nm is used to calculate the absorbance value of the negative sample / the absorbance value of the positive sample (N / P value). Based on the principle that the maximum N / P value is the optimal condition, the optimal antigen coating amount and the optimal dilution ratio of the enzyme-labeled antibody are selected.

[0055] according to Figure 5 The results showed that the coating amount of RBD protein was set at 10 ng / well, the serum dilution was 1:1, and the optimal dilution ratio of enzyme-labeled antibody was set at 1:50,000.

[0056] Ultimately, it was determined that the optimal sample reaction time was 60 min, the optimal enzyme-labeled reagent reaction time was 30 min, and the optimal TMB color development time was 15 min when using 10 ng / well of RBD recombinant protein coating, serum sample dilution was set at 1:1, enzyme-labeled antibody dilution was set at 1:50,000.

[0057] 2. The optimized ELISA conditions are as follows: 1) Coating: RBD recombinant protein was used as the coating antigen, with a coating amount of 10 ng / well; coating was carried out in carbonate coating buffer at 4 ℃ for 24 h. 2) Washing: Wash once with phosphate-buffered saline (PBST) containing 0.05% Tween-20 at pH 7.2, gently shake for 1 min and then discard. 3) Blocking: Block with blocking solution containing 2.5% BSA in PBST buffer at pH 7.2, at 4 ℃ for 24 h; 4) Serum reaction conditions: Add the serum dilution mixture (serum: PBS = 1:1) to each well, incubate at 37 ℃ for 60 min, then spin dry and wash 4 times with PBST; 5) Enzyme-labeled antibody incubation conditions: Dilute HRP-1C12 secondary antibody with PBST at pH 7.2 at a ratio of 1:50,000, add 100 μL to each well, incubate at 37 ℃ for 30 min, then spin dry and wash 4 times with PBST. 6) Substrate color development: 100 μL of TMB substrate color development solution per well, develop color at 37 ℃ in the dark for 15 min; 7) Termination of reaction: Add 50 μL of 2 M H2SO4 stop solution to each well to terminate the colorimetric reaction; read the data using an ELISA reader at an absorbance of 450 nm.

[0058] 3. Determination of the cutoff value for blocking ELISA Determination of the cut-off value: 81 antibody-positive serum samples and 89 antibody-negative serum samples from clinically infected PDCoV pigs were stored in the laboratory. The optimized ELISA conditions were used for detection, and ROC curves were plotted. The optimal cut-off value was 31.45% obtained by calculating the Youden index (sensitivity + specificity - 1). When the PI value = [1 - (sample OD...]... 450 nm value / negative control OD 450 A value of [nm value (×100%)] above 31.45% was considered positive. ROC curve analysis showed that the diagnostic sensitivity of this method was 98.77% and the diagnostic specificity was 94.38%. The specific ROC curve and scatter plot are shown below. Figure 6 As shown.

[0059] 4. Evaluation of the specificity of blocking ELISA Using the blocking ELISA method established in this embodiment, antibody-positive sera for common swine diseases (PCV3 / PCV2 / PRRSV / PRV / PoRV / ASFV / CSFV / PEDV) were detected. All sera were provided by the Comprehensive Prevention and Control Team for Emerging and Major Animal Diseases, College of Veterinary Medicine, Yangzhou University. The test results show that the established method has good specificity and no cross-reactivity occurred with antibody-positive sera for other pathogens. Figure 7 As shown.

[0060] 5. Sensitivity evaluation of blocking ELISA analysis The blocking ELISA method established in this embodiment was used to detect PDCoV antibody-positive pig serum at serially diluted levels. According to the established criteria, a cut-off value greater than 31.45% was considered positive. Figure 8 The results showed that serum, at a 1:256 dilution, was the limit of detection in the RBD-coated plate. This indicates that the ELISA detection method established in this invention has high analytical sensitivity.

[0061] 6. Validation of the repeatability and reproducibility of blocking ELISA To evaluate the accuracy of the established ELISA for detecting PDCoV antibodies, this study assessed its repeatability in a single experiment (intra-plate repeatability) and its reproducibility across different experiments (inter-batch repeatability). For repeatability assessment, three porcine serum samples with strong, moderate, and weak positive reactions were selected and tested eight times on a single ELISA plate to evaluate intra-plate repeatability. Simultaneously, in different experiments, three ELISA plates from the same batch (manufactured by Xiamen Yijiamei Experimental Equipment Co., Ltd.) were used to test each serum sample eight times to evaluate inter-batch repeatability. The OD values ​​for each serum sample were calculated. 450 The standard deviation (SD) and mean of nm values The ratio of the two values, i.e., the coefficient of variation (CV), is used to quantify the accuracy of the test results. As shown in Table 5, the test method exhibits good intra-assay and inter-assay repeatability, with intra-assay CVs all less than 10% and inter-assay CVs all less than 10%. This indicates that the ELISA test method of the present invention has high accuracy and stability.

[0062] Table 5. Validation of repeatability and reproducibility of blocking ELISA

[0063] 7. Validation of the diagnostic sensitivity of blocking ELISA To further validate the diagnostic sensitivity of this method, 150 clinical samples were tested, and the neutralizing antibody titers of these samples were also measured. Neutralizing antibodies are considered the gold standard for diagnosing PDCoV antibodies. We compared the positive and negative results of the established blocking ELISA method with those of neutralizing antibodies. The analysis showed that the positive rate of the blocking ELISA was 53.3%, and the negative rate was 46.7%, while the positive rate of the neutralizing antibody detection (VNT) was 50.7%, and the negative rate was 49.3%. Comparing the results of the blocking ELISA with the neutralizing antibody titer detection, statistical analysis showed that the overall concordance rate between the blocking ELISA and neutralizing antibody methods was 93.3%. Detailed results are shown in Table 6.

[0064] Table 6. Validation of the diagnostic sensitivity of blocking ELISA

[0065] 8. Application of the blocking ELISA method Using the universally established method of this invention, 2002 clinical swine serum samples from 175 farms in 28 cities across 10 provinces and autonomous regions in China were tested in 2025. Among them, 273 serum samples were positive, with an overall antibody positivity rate of 13.6%. The positivity rate of pig farms in each province is shown in Table 7. The overall positivity rate of PDCoV antibodies in pig farms reached 30.3% (53 / 175). The majority of samples came from pig farms in Henan Province, with a positive antibody rate of 32.4% (47 / 145).

[0066] Table 7. Serological analysis of PDCoV in pig farms in 10 provinces of China in 2025 .

Claims

1. A monoclonal antibody against the spike protein of a porcine delta coronavirus, characterized in that, The antibody includes a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

3.

2. The gene encoding the porcine delta coronavirus spike protein monoclonal antibody of claim 1, characterized in that, The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 2, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

4.

3. The porcine delta coronavirus spike protein monoclonal antibody according to claim 1, characterized in that, The heavy chain of the monoclonal antibody is IgG1.

4. The use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a kit for detecting porcine delta coronavirus.

5. A kit for detecting porcine delta coronavirus, characterized in that, The kit contains a porcine delta coronavirus spike protein monoclonal antibody as described in any one of claims 1 to 3.

6. The reagent kit according to claim 5, characterized in that, The kit also includes RBD recombinant protein, coating solution, blocking solution, diluent, PBST, TMB substrate chromogenic solution, and H2SO4 stop solution.

Images