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

Abstract

The invention 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 as shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.3. According to the invention, a highly conservative 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 being specifically bound to the epitope are identified for the first time. The epitope peptide and the monoclonal antibody provided by the invention can be used for immunological detection and serological investigation of the PDCoV.

Description

TECHNICAL FIELD

[0001] The present application relates to a porcine delta coronavirus spike protein monoclonal antibody, an antigen epitope peptide and an application, belonging to the technical field of biotechnology. BACKGROUND

[0002] Since the advent of monoclonal antibody technology, it has become one of the indispensable core tools in life science research and medical applications. This kind of antibody has high specificity, good uniformity, can be mass-produced, etc. It is widely used in disease diagnosis, targeted therapy, pathogen detection and basic research, etc. With the continuous progress of hybridoma technology and genetic engineering antibody technology, the application range of monoclonal antibodies has expanded to the fields of agriculture, food safety and environmental monitoring.

[0003] Porcine deltacoronavirus (PDCoV), also known as porcine delta coronavirus (PDCoV), was also called porcine coronavirus HKU15 in early stage, which is a member of Nidovirales, Coronaviridae, Coronavirinae and Deltacoronavirus. The virus was first detected in pig herds in Hong Kong, China in 2012, and was officially identified and named in the process of pig diarrhea outbreak in multiple states of the United States in 2014. Pathogenicity studies have shown that PDCoV can cause acute diarrhea, vomiting, dehydration and even death in piglets, with a high incidence and mortality rate of 50% to 100%, which seriously endangers the healthy development of the pig industry and causes significant economic losses. Genome alignment shows that the strains isolated from the United States and the HKU15 strain in Hong Kong are highly homologous in M and N gene sequences, with a similarity of more than 99%, further confirming its classification status.

[0004] Similar to other coronaviruses, the spike protein (S protein) of PDCoV is located on the surface of the virus particle, with a molecular weight of about 200 kDa, and has high homology (more than 90%) between different strains. It is a key protein that mediates viral invasion of host cells, and plays a core role in receptor recognition, membrane fusion and neutralizing antibody induction. Therefore, 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 antigen epitopes is still limited, especially for the receptor binding domain (RBD) of S protein. High-affinity and high-specificity monoclonal antibodies targeting the receptor binding domain of the protein are very scarce, which restricts the development of related detection techniques and immunization intervention strategies.

[0005] Therefore, it is urgent to identify the immunodominant antigen epitope in the PDCoV S-RBD protein, and to develop specific monoclonal antibodies with good reactivity, so as to provide key technical support for the establishment of effective pathogen detection methods, serological evaluation system and the development of neutralizing antibody drugs. SUMMARY

[0006] The purpose of the application is to provide a porcine delta coronavirus spike protein monoclonal antibody, and an antigen epitope peptide specifically combined therewith and application thereof.

[0007] The technical scheme is as follows: the porcine delta coronavirus spike protein monoclonal antibody provided by the application 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.

[0008] Further, the heavy chain and the light chain are connected through a disulfide bond.

[0009] Further, the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 2, and the nucleotide sequence for coding the heavy chain variable region is shown as SEQ ID No. 4.

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

[0011] The porcine delta coronavirus antigen epitope peptide provided by the application can be specifically combined with the above-mentioned monoclonal antibody; and the amino acid sequence of the antigen epitope peptide is DFGEARLD.

[0012] The application of the monoclonal antibody and the porcine delta coronavirus antigen epitope peptide to the detection of porcine delta coronavirus for non-disease treatment and diagnosis purposes.

[0013] The kit for detecting porcine delta coronavirus provided by the application comprises the porcine delta coronavirus spike protein monoclonal antibody and / or the porcine delta coronavirus antigen epitope peptide.

[0014] Further, the kit further comprises an RBD recombinant protein, a coating liquid, a blocking liquid, a dilution liquid, a PBST, a TMB substrate developing liquid and an H2SO4 termination liquid.

[0015] Further, the use method of the kit comprises the following steps: washing and blocking with the RBD recombinant protein or the porcine delta coronavirus antigen epitope peptide as a coating antigen; adding, incubating and washing after diluting the serum to be detected; diluting and adding the porcine delta coronavirus spike protein monoclonal antibody as an enzyme-labeled antibody, incubating and washing; adding a developing liquid, incubating, adding a termination liquid and reading OD.450 Values.

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

[0017] Further, the test result judgment method is to calculate the inhibition rate PI, and PI≥31.45% is judged as positive; and PI<31.45% is judged as negative.

[0018] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application first identifies a highly conserved linear B cell epitope (the amino acid sequence of which is DFGEARLD) of the S-RBD of PDCoV spike protein and a neutralizing monoclonal antibody capable of specifically binding to the epitope. The epitope peptide and monoclonal antibody provided by the present application can be used for immunological detection and serological investigation of PDCoV. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 An indirect immunofluorescence schematic diagram for verifying the reactivity of the porcine delta coronavirus (PDCoV) monoclonal antibodies 1C12 and 4C9 of the present application to infection of LLC-PK1 cells by three different PDCoV strains.

[0020] Figure 2 A protein expression and purification schematic diagram of the S protein, the S1 protein and the RBD protein of PDCoV in the present application. Figure 2 A is a schematic diagram of the extracellular domain topology of the S protein of PDCoV; Figure 2 B is a SDS-PAGE diagram of the S protein, the S1 protein and the RBD protein of PDCoV; Figure 2 C is a Western-blot diagram of detecting the S protein, the S1 protein and the RBD protein of PDCoV by using the 1C12 monoclonal antibody; Figure 2 B and Figure 2 Lane 1, 2 and 3 in C respectively represent the S protein, the S1 protein and the RBD protein of PDCoV.

[0021] Figure 3 An antigen epitope screening schematic diagram of the PDCoV monoclonal antibody 1C12 in the present application.

[0022] Figure 4 A sequence alignment schematic diagram of the S protein of different strains of PDCoV in the present application.

[0023] Figure 5 A schematic diagram of screening the optimal antigen coating concentration and the optimal enzyme-labeled antibody dilution of the blocking ELISA in the present application.

[0024] Figure 6ROC curve (A) and detection scatter plot (B) of the blocking ELISA in the present application.

[0025] Figure 7 Specificity verification diagram of the blocking ELISA in the present application.

[0026] Figure 8 Analysis sensitivity verification diagram of the blocking ELISA in the present application. DETAILED DESCRIPTION

[0027] The technical solutions of the present application are further described below in combination with the drawings.

[0028] Example 1: Preparation and screening of porcine delta coronavirus (PDCoV) monoclonal antibody 1. Preparation and identification of porcine delta coronavirus (PDCoV) monoclonal antibody 1.1 Preparation of porcine delta coronavirus (PDCoV) monoclonal antibody In the preparation and immune titer evaluation of the monoclonal antibody, the concentrated virus liquid of the PDCoV strain (titer 10 7.3 TCID 50 / mL) preserved by the Animal New and Major Epidemic Disease Comprehensive Prevention and Control Team of the College of Veterinary Medicine of Yangzhou University was used as the immunogen, and the immunogen was emulsified with an equal volume of Freund's complete adjuvant for primary immunization, and emulsified with an equal volume of Freund's incomplete adjuvant for three booster immunizations. The second immunization was separated from the first immunization by two weeks, and the three booster immunizations were separated by one week. Five 4-6 week old female BALB / c mice (purchased from the Experimental Animal Center of Yangzhou University, about 20 g each) were injected intraperitoneally with the emulsified immunogen (300 μL each), and serum was collected 7-10 days after the fourth immunization. The titer was detected by indirect immunofluorescence (IFA). The preliminary results (Table 1) showed that the immune group met the requirements for cell fusion experiments.

[0029] The specific process of indirect immunofluorescence method is as follows: LLC-PK1 cells were seeded in a 96-well plate at 2×10 4 cells / well, cultured for 24 hours to form a monolayer, inoculated with PDCoV virus liquid, and normal cells were set up as negative controls, and cultured for 36 hours and then fixed with 80% acetone. The test sample was diluted with PBS according to a 2-fold ratio (1:200~1:102400), added to the well at a volume of 100 μL per well, incubated at 37°C for 30 minutes, and then detected using a 1:800 diluted FITC-labeled rabbit anti-mouse IgG secondary antibody. The results were determined by observing under a fluorescence microscope: no fluorescence in the negative control, and specific green fluorescence appeared in the cytoplasm of the sample well, which was positive. The highest dilution that produced 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. 4After inoculating 96-well plates and forming monolayers for 24 hours, three strains of PDCoV virus liquid isolated from different provinces were inoculated, and normal cells were set as negative controls. After 36 hours of infection, the cells were fixed with 80% acetone. The monoclonal antibody ascites was diluted with PBS at 1:1000, 100 μL was added to each well, and incubated at 37°C for 30 minutes. Then, a 1:800 diluted FITC-labeled rabbit anti-mouse IgG secondary antibody was added for detection. The results were determined under a fluorescence microscope: the negative control wells should have no fluorescence signal, and the sample wells were considered positive if specific green fluorescence was observed in the cytoplasm.

[0035] Among them, three strains of PDCoV virus isolated from different provinces were provided by the Animal Emerging and Major Epidemic Disease Comprehensive Prevention and Control Team of the College of Animal Medicine of Yangzhou University. As shown in Table 1, two monoclonal antibodies were found to have good reactivity with three epidemic strains. Figure 1

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

[0037] The specific process of the virus neutralization experiment is as follows: LLC-PK1 cells were plated in 96-well plates, and when the cells were fully grown or grew to about 90%, the prepared 2 monoclonal antibodies were filtered and sterilized, and placed in a 56 ℃ water bath for 30 min; DMEM was taken to prepare a 2-fold dilution of the two monoclonal antibodies (1:2~1:256), 300 μL of each dilution 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 LLC-PK1 cell culture supernatant in the 96-well plate was removed with a multi-pipette, and then washed with sterile PBS for 2 times; 100 μL of the antibody-virus mixture was added to the LLC-PK1 cell monolayer in the 96-well plate, 4 parallel samples were prepared for each dilution, 4 positive control wells (only 100 μL / 200 TCID 50 of virus suspension was added) and 4 negative control wells (only 100 μL of cell maintenance solution was added) were set, and incubated at 37°C in a 5% CO2 incubator. The results were observed every day, and the CPE was no longer produced by the cells for 2-4 days.

[0038] Table 3 Verification of neutralization activity of monoclonal antibodies

[0039] ​In combination with the above-mentioned screening, the monoclonal antibody 1C12 strain is preferably selected for further study.

[0040] 2.3 Sequencing of the light and heavy chain variable regions of the porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 According to the sequence characteristics of the mouse-derived monoclonal antibody, the heavy chain variable region primer sequence is designed as follows: C H -F: 5'-ACTACTTGACGTGCTCTAGGTCACTTTACTTTCCCT-3' C H -R: 5'-CGGAGCTTCCAGCGCCARCCCATATACTGRTGG-3' The light chain variable region primer sequence is designed as follows: C L -F: 5'-GCCATCTAGRAWCATTKWCYCAAGTCTTT-3' C L -R: 5'-CGGAGCCTTACTGCCTGTAAGAAGATGGA-3' The 1C12 strain hybridoma cells are collected, and after RNA extraction and reverse transcription, the cDNA is used as a template for amplification of the variable region sequence using the above primers. The amplification product is sent to Suzhou Jinweizhi Biological Technology Co., Ltd. for sequencing. The results show that the amino acid sequence of the light chain variable region of the monoclonal antibody 1C12 is as shown in SEQ ID No. 1: DIVLTQSPASLAVSPRQRATISCKSAVSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPVRFSGSGSGTDFSLNIHPVEEERAATYYCQQSNEDPFTFGSGTKLEIKR; The base nucleotide sequence of the light chain variable region is as shown in SEQ ID No. 2: gacattgtgctgacccaatctccagcttctttggctgtgtctccacgacagagggccaccatctcctgcaagtcggcggtaagtgttgattatgatggtgatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcatccaatctagaatctgggatcccagtaaggtttagtggcagtgggtctgggacagacttctcgctcaacatccatcctgtggaggaagagcgggctgcaacctattactgtcagcaaagtaatgaagatccattcacgttcggctcggggacaaagttggaaataaaacgg; The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 3: EVQLQQSGPILVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNGGTSYNQKFKGKATLTVDKSSSTSYMELRSLTSEDSAVYYCARDGYDTRYSYVMDYWGQGTSVTVSSAKTTPPS; The base nucleotide sequence of the heavy chain variable region is shown as SEQ ID No. 4: gaggtccagctgcaacagtctggacctatactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggatacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggtattaatcctaacaatggtggtactagttacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacatcatacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagagatggttacgacacgcggtatagctatgttatggactactggggtcaaggaacctcagtcaccgtctcctctgccaaaacaacacccccatct.

[0041] Example 2: Antigenic epitope screening of porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 1. Expression and purification of porcine delta coronavirus (PDCoV) S, S1 and RBD proteins In this example, we describe a method for expressing S, S1, RBD proteins in ExpiCHO-S cells in detail. First, we obtained the required ExpiCHO-S cells, expression medium and transfection reagent from Thermo Fisher Scientific and cultured them at 32 °C under 5% CO2 conditions. According to the manufacturer's instructions, we transfected the ExpiCHO-S cells with the recombinant plasmids pcDNA3.1-S, pcDNA3.1-S1 and pcDNA3.1-RBD, respectively, and cultured them under the same conditions for 10-12 days. Figure 2 A, the extracellular domain topology of PDCoV S protein, and according to the sequence of the PDCoV CZ2020 strain (GenBank accession No. OK546242) strain published by NCBI, we referred to the coding sequence of the S, S1 and RBD domains of the strain, and added a signal peptide (MDAMKRGLCCVLLLCGAVFVSAS) at the N-terminus of the reference sequence to ensure the secretory expression of the protein. The sequence was optimized and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the gene 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, respectively, and cultured them under the same culture conditions for 10-12 days.

[0042] After the protein expression stage was completed, we collected the culture supernatant and clarified it by centrifugation at 5,000 × g for 30 minutes at 4 °C, followed by filtration through a 0.22 μm sterile membrane. The target protein was captured using Ni-NTA agarose resin for 2 hours at 4 °C. Next, the weakly bound impurities were removed by washing with 1 × phosphate buffered saline (PBS), 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 by BCA assay kit. Finally, the high-purity protein was aliquoted and stored at -80 °C for future use. By this method, as shown in Figure 2 B, SDS-PAGE showed that we successfully expressed S, S1, RBD proteins in ExpiCHO-S cells and effectively purified and stored them, providing high-quality protein samples for subsequent experimental studies.

[0043] 2. Antigenic epitope screening of porcine delta coronavirus (PDCoV) monoclonal antibody 1C12 Screening of epitope region is of great value for vaccine design and development of diagnostic reagents. To determine the minimum epitope recognized by the 1C12 monoclonal antibody, a systematic analysis was performed by Western Blot (WB) experiment. First, the purified S, S1 and RBD proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. Then, the membrane was blocked with 5% bovine serum albumin (BSA) for 2 hours, washed 5 times with PBST (PBS buffer containing 0.05% Tween-20) at pH = 7.2, and then incubated with anti-His monoclonal antibody (diluted with blocking solution at a ratio of 1:2000) at room temperature for 2 hours. After washing 5 times with PBST, the membrane was incubated with HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (diluted at a ratio of 1:10000) at room temperature for 1 hour. After thorough washing with PBST, the Clarity TM Western ECL chemiluminescent substrate was used for color development, and the image was collected by VILBER Fusion FX7 chemiluminescence imaging system. As shown in FIG. C, the 1C12 monoclonal antibody was used to detect the above purified proteins by WB, and the results showed that the antibody could specifically react with S, S1 and RBD proteins, indicating that it recognized the epitope in the RBD region, i.e., the 300-419 amino acid region of S protein. Figure 2 C, the 1C12 monoclonal antibody was used to detect the above purified proteins by WB, and the results showed that the antibody could specifically react with S, S1 and RBD proteins, indicating that it recognized the epitope in the RBD region, i.e., the 300-419 amino acid region of S protein.

[0044] To screen the epitope recognized by the monoclonal antibody, the RBD segment (300-419 amino acid region of S protein) was further truncated and expressed. First, the RBD (300-419 aa) full-length sequence was amplified from the pcDNA3.1-RBD plasmid using the primers listed in Table 4, and three truncated mutants: RBD (300-349 aa), RBD (340-389 aa) and RBD (380-419 aa) were further amplified by the corresponding primers. At the same time, the pEGFP-C1 vector was linearized using the EGFP-F / R primers in Table 4, and the above amplified sequences were constructed into the pEGFP-C1 vector. The constructed recombinant plasmids were transfected into HEK293T cells, and the total protein was collected after 24 hours of culture. Western Blot analysis showed that the epitope recognized by the 1C12 monoclonal antibody was located in the 300-349 amino acid segment (FIG. A). Figure 3 A).

[0045] To further screen the epitope recognized by the monoclonal antibody, the pEGFP-RBD (300-349 aa) plasmid was used as a template, and further truncation was performed using the primers in Table 4 to construct the pEGFP-RBD (300-319 aa), pEGFP-RBD (310-329 aa), pEGFP-RBD (320-339 aa), and pEGFP-RBD (330-349 aa) expression plasmids. The constructed recombinant plasmids were respectively transfected into HEK293T cells, and total cell proteins were collected after 24 hours of culture. Western Blot analysis showed that the recognized epitope was narrowed down to the 310-329 amino acid segment Figure 3 B}.

[0046] To finally determine the minimum recognized epitope, the pEGFP-RBD (300-319 aa) plasmid was used as a template, and the segment was expressed by stepwise truncation from the amino terminal and carboxyl terminal using the primers in Table 4, respectively, to construct the pEGFP-RBD (313-329 aa), pEGFP-RBD (316-329 aa), pEGFP-RBD (319-329 aa), pEGFP-RBD (322-329 aa), pEGFP-RBD (310-326 aa), pEGFP-RBD (310-323 aa), pEGFP-RBD (310-320 aa), and pEGFP-RBD (310-317 aa) eight expression plasmids. The constructed recombinant plasmids were respectively transfected into HEK293T cells, and total cell proteins were collected after 24 hours of culture. Western Blot analysis showed that the minimum linear B cell epitope recognized by the 1C12 monoclonal antibody was located in the 316-323 amino acid interval Figure 3 C}, and the core sequence was DFGEARLD. In the figure, red indicates a reaction with the monoclonal antibody, and green indicates no reaction with the monoclonal antibody.

[0047] Sequence alignment using MEGA12 software found that the “DFGEARLD” epitope sequence was completely conserved in representative PDCoV strain sequences reported in different countries 316 DFGEARLD 323 Figure 4 .

[0048] In this embodiment, the primers for the truncated proteins to be expressed are shown in Table 4, and the primers were synthesized by Shanghai Shengong. The PCR system and amplification program are as follows: according to the standard process in the instructions, 50 μL of the PCR system was used for amplification. The reaction system contains: 25 μL 2×Hieff Canace ® ​Plus PCR Master Mix (with dye) (Sangon Biotech), 2 μL of forward and reverse primers (10 μM), 1 ng of template DNA (dilute plasmid with ddH2O), and use ddH2O to make up to a total volume of 50 μL. The amplification program is set as follows: first 94 °C pre-denaturation for 3 min; then 30 cycles, each cycle including 98 °C denaturation for 10 s, 56 °C annealing for 20 s, and 72 °C extension (30 sec / kb); after the cycles, 72 °C final extension for 5 min; after the reaction is terminated, the product is stored at 4 °C.

[0049] Table 4 PCR amplification primer sequences of truncated protein genes Primer name Sequence (5' to 3') 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 tgcagaattcgaagcttgagcTCAGATCTTCATCTCACAGGCCCCGCTCTGT 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 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 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 universal blocking ELISA antibody detection method based on prepared monoclonal antibody In this embodiment, PDCoV antibody positive and negative pig serum and clinical pig serum samples were provided by the Animal Emerging and Major Disease Comprehensive Prevention and Control Team of the College of Animal Medicine of Yangzhou University, and their antibody positive and negative were verified by virus neutralization test.

[0050] 1. Establishment of blocking ELISA process based on 1C12 monoclonal antibody, specifically: 1) The RBD recombinant protein expressed and purified in Example 2 was serially diluted to 5, 10 and 20 ng / well (diluent was 1x carbonate buffer with pH = 9.6, 20x carbonate buffer was prepared by mixing 29 g NaHCO3 + 16 g NaCO3, and then diluted to 1 L, and then filtered after fully dissolved), the serum sample was diluted 1:1 (diluent was PBS with pH = 7.2), and the horseradish peroxidase (HRP) labeling kit purchased from Suzhou Bioon Biotech Co., Ltd. was used to label the monoclonal antibody 1C12 with HRP, and the operation was performed according to the instruction. HRP-1C12 was diluted to 1:10,000, 1:30,000, 1:50,000 and 1:80,000 (diluent was PBST with pH = 7.2, containing 0.05% Tween-20 PBS buffer), and 5 PDCoV antibody positive pig sera and 3 PDCoV antibody negative pig sera verified by virus neutralization test were used to screen the best antigen coating concentration and the best enzyme-labeled antibody dilution ratio by chessboard method.

[0051] 2) According to the volume of 100 μL / well, carbonate coating buffer containing RBD recombinant protein was coated on the ELISA plate, and incubated 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 Solayebio Technology Co., Ltd.) per well, and seal at 4 °C for 24 h.

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

[0054] 5) Add 50 μL of 2 M H2SO4 per well to terminate the reaction after 15 min at 37 °C, and measure the absorbance at OD 450 nm. Calculate the negative sample absorbance / positive sample absorbance (N / P value), and select the optimal antigen coating amount and enzyme-labeled antibody dilution ratio according to the principle of maximum N / P value and optimal conditions.

[0055] According to Figure 5 Based on the results shown, the coating amount of RBD protein is set to 10 ng / well, the serum dilution is 1: 1, and the optimal dilution ratio of enzyme-labeled antibody is 1: 50,000.

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

[0057] 2、The optimized ELISA conditions are: 1) Coating: Use RBD recombinant protein as the coating antigen, and the coating amount is 10 ng / well; coat in carbonate coating buffer at 4 °C for 24 h; 2) Washing: Wash once with phosphate buffered saline (PBST) containing 0.05% Tween-20 by volume at pH 7.2, discard after gentle shaking for 1 min; 3) Blocking: Block with blocking solution containing 2.5% BSA in PBST buffer at pH 7.2, and seal at 4 °C for 24 h; 4) Serum action conditions: Add the serum dilution mixture (serum: PBS = 1: 1) per well, and incubate at 37 °C for 60 min, then shake dry, and wash 4 times with PBST; 5) The action conditions of enzyme-labeled antibody: dilute HRP-1C12 secondary antibody by 1:50,000 with PBST of pH 7.2, add 100 μL per well, shake dry after being acted for 30 min at 37 °C, and wash 4 times with PBST; 6) Substrate color development: 100 μL of TMB substrate color development solution per well, color development for 15 min at 37 °C in the dark; 7) Reaction termination: terminate the color development reaction by adding 50 μL of 2 M H2SO4 termination solution per well; and read the data at an absorbance of 450 nm by using an enzyme-labeled instrument.

[0058] 3. Determination of the blocking ELISA critical value Determination of the critical value (cut-off value): 81 positive serum samples of PDCoV-infected pigs and 89 negative serum samples were preserved in the laboratory, and were detected by using the above-optimized ELISA conditions, and a ROC curve was drawn, and the optimal cut-off value was obtained by calculating the Youden index (sensitivity + specificity - 1) as 31.45%. When the PI value = [1- (sample OD 450 nm value / negative control OD 450 nm value) x 100%] is greater than 31.45%, it is determined to be positive. According to the ROC curve analysis, at this time, the diagnostic sensitivity of the method is 98.77%, and the diagnostic specificity is 94.38%. The specific ROC curve and scatter plot are shown in Figure 6 .

[0059] 4. Specificity evaluation of the blocking ELISA The blocking ELISA method established in this embodiment was used to detect antibody positive sera of common pig diseases (PCV3 / PCV2 / PRRSV / PRV / PoRV / ASFV / CSFV / PEDV), which were provided by the Animal New and Major Epidemic Disease Comprehensive Prevention and Control Team of the College of Animal Medicine of Yangzhou University. As shown by the detection results, the method has good specificity, and no cross-reaction occurs with antibody positive sera of other pathogens, as shown in Figure 7 .

[0060] 5. Analysis sensitivity evaluation of the blocking ELISA The blocking ELISA method established in this embodiment was used to detect gradient-diluted PDCoV antibody positive pig serum. According to the set determination standard, the cut-off value greater than 31.45% is determined to be positive. According to the results of Figure 8 , the detection lower limit of the serum in the RBD-coated plate is 1:256 dilution. This shows that the ELISA detection method established in the present application has high analysis 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 method of the present application, 2002 clinical pig sera from 175 farms in 28 cities of 10 provinces and autonomous regions in China in 2025 were detected, of which 273 were positive sera, the overall antibody positive rate was 13.6%, and the positive rate of each province was as shown in Table 7. The overall positive rate of PDCoV antibody in pig farms was 30.3% (53 / 175), and the number of samples from pig farms in Henan Province was the largest, and the antibody positive rate of pig farms in Henan Province accounted for 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 porcine delta coronavirus spike protein monoclonal antibody according to 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. A porcine delta coronavirus antigenic epitope peptide, characterized in that, The antigenic epitope peptide can specifically bind to the monoclonal antibody according to claim 1 or 2; the amino acid sequence of the antigenic epitope peptide is DFGEARLD.

5. The use of the monoclonal antibody according to any one of claims 1 to 3 and the porcine delta coronavirus antigenic epitope peptide according to claim 4 in the detection of porcine delta coronavirus for non-disease treatment and diagnostic purposes.

6. A kit for detecting porcine delta coronavirus, characterized in that, The kit contains the porcine delta coronavirus spike protein monoclonal antibody as described in any one of claims 1 to 3 and / or the porcine delta coronavirus antigenic epitope peptide as described in claim 4.

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

8. The method of using the reagent kit according to any one of claims 6 to 7, characterized in that, The process includes the following steps: washing and blocking with RBD recombinant protein or the porcine δ-coronavirus epitope peptide of claim 4 as the coating antigen; adding diluted test serum, incubating, and washing; adding diluted porcine δ-coronavirus spike protein monoclonal antibody of any one of claims 1 to 3 as the enzyme-labeled antibody, incubating, and washing; adding chromogenic solution, incubating, adding stop solution, and reading OD. 450 value.

9. The method according to claim 8, characterized in that, The RBD recombinant protein coating concentration was 10 ng / well, and the enzyme-labeled antibody was diluted 1:50,000.

10. The method according to claim 8, characterized in that, The test results are judged by calculating the inhibition rate (PI). A PI ≥ 31.45% is considered positive, and a PI < 31.45% is considered negative.

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