An antigen epitope polypeptide for detecting antibodies produced by porcine delta coronavirus infection, products and applications thereof

Abstract

This invention relates to the field of biotechnology, and in particular to an antigenic epitope peptide, product, and application for detecting antibodies produced by porcine deltacoronavirus infection. This invention identified short peptide epitopes in PDCoV longitudinal challenge serum samples, obtaining 12 short peptides, which were then prepared into a porcine deltacoronavirus antibody detection kit. Finally, six field serum samples were selected for antibody detection, and the effectiveness was verified using a virus neutralization experiment. The results showed that the porcine deltacoronavirus antibody detection kit provided by this invention is effective. Furthermore, the porcine deltacoronavirus antibody detection kit provided by this invention, as a novel detection method, improves sensitivity and specificity while simultaneously providing multiple detection results, namely, determining whether viral infection has occurred, identifying the stage of infection, and achieving DIVA (Differentiation, Infection, and Variability) analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antigenic epitope polypeptide for detecting antibodies produced by porcine deltacoronavirus infection, the product thereof, and its application. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is a novel porcine enteric coronavirus. It can cause severe atrophic enteritis in piglets, accompanied by diarrhea, vomiting, and dehydration, with a mortality rate of 40%-80% in suckling piglets, resulting in enormous losses. Furthermore, experiments have shown that PDCoV can enter the cells of pigs, humans, and poultry through receptors, and can also infect chickens, calves, and mice in experiments. As a newly emerging coronavirus, it not only exists in livestock but also has an impact on humans. In 2021, it was reported that three children in Haiti were infected. This phenomenon indicates that the virus may have spilled over from animals to humans, posing a threat to current national public health security (including veterinary care).

[0003] PDCoV causes mild symptoms in adult pigs and sows, and they may recover without treatment. However, it is significantly harmful to piglets, thus causing substantial economic losses. Traditional methods for detecting PDCoV include PCR (detecting DNA) and ELISA (detecting antigen (sELISA) or antibody (iELISA)).

[0004] Traditional detection methods have the following drawbacks: 1) There is a certain window period. The window period for PCR is because in the early stages of infection, the viral genetic material may not be sufficient to be detected, and a certain amount of time is needed for the viral genetic material to reach a detectable level. The window period for ELISA (antigen or antibody) testing is determined by the time required for the human immune system to respond to a newly invading virus, as well as the sensitivity and specificity of the detection technology.

[0005] 2) Inability to determine the stage of infection (recent or past infection). PCR methods are insufficient for determining recent or past infections, while ELISA requires collecting samples at multiple time points to observe the trends in IgG and IgM levels. This method is time-consuming and yields non-unique results.

[0006] 3) It cannot differentiate between animal immunity and infection (DIVA). Vaccines are an effective means of preventing diseases, but DIVA also presents a significant challenge compared to traditional methods.

[0007] 4) Single test result. PCR indicates the presence of the virus by detecting viral DNA. ELISA, on the other hand, indicates the presence or absence of the virus by detecting antigens or antibodies.

[0008] In summary, current diagnostic methods primarily focus on viral DNA and structural proteins. However, research on viral short peptide epitopes is scarce. In recent years, protein-peptide hybrid microarrays (PPHMs) have emerged as a next-generation serological diagnostic / detection technology and have been applied to multiple viruses. For example, in 2020, short peptide epitopes in PPHMs enabled the differentiation between immunity and infection (Differentiating infected from vaccinated, DIV) of petit ruminants virus (PPRV). Furthermore, in 2022, a PPHM for PCV2 was obtained, successfully differentiating between immunity and infection (Differentiating infected from vaccinated animals, DIVA) of five PCV2 vaccines. Therefore, the PPHM developed for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can achieve highly sensitive and specific diagnosis. Summary of the Invention

[0009] The purpose of this invention is to provide an antigenic epitope polypeptide for detecting antibodies produced by porcine deltacoronavirus infection, the product thereof, and its application, in order to solve the problems existing in the prior art.

[0010] To achieve the above objectives, the present invention provides the following solution: This invention provides an antigenic epitope polypeptide for detecting antibodies produced by porcine deltacoronavirus infection, characterized in that the amino acid sequence of the antigenic epitope polypeptide is shown in SEQ ID NO.1-SEQ ID NO.12.

[0011] This invention provides the application of the above-mentioned antigenic epitope peptide in the preparation of products for detecting antibodies produced by porcine deltacoronavirus infection.

[0012] Optionally, the product includes reagents, reagent kits, and chips.

[0013] The present invention provides a reagent for detecting antibodies produced by porcine deltacoronavirus infection, the reagent containing the above-mentioned antigenic epitope polypeptide.

[0014] The present invention provides a chip for detecting antibodies produced by porcine deltacoronavirus infection, the chip comprising a solid-phase carrier containing the aforementioned antigenic epitope polypeptide.

[0015] The present invention provides a kit for detecting antibodies produced by porcine deltacoronavirus infection, the kit comprising the aforementioned chip.

[0016] Optionally, the kit also includes 20× concentrated washing solution, sample diluent, enzyme-labeled antibody solution, and luminescent substrate solution.

[0017] Optionally, the 20× concentrated cleaning solution includes Tris-HCl, NaCl, and Tween 20; The sample diluents include PBS, BSA, PVP, and Tween 20.

[0018] Optionally, the concentration of Tris-HCl in the 20× concentrated cleaning solution is 0.4 M, the concentration of NaCl is 2.74 M, and the volume percentage of Tween 20 is 2%. The concentration of PBS in the sample diluent is 0.05 M, the mass percentage of BSA is 1 wt%, the mass percentage of PVP is 0.2 wt%, and the volume percentage of Tween 20 is 0.5%.

[0019] This invention provides the application of the above-described antigenic epitope peptide, the above-described reagent, the above-described chip, or the above-described kit in the preparation of products for evaluating intestinal diseases.

[0020] The present invention discloses the following technical effects: This invention identified short peptide epitopes of PDCoV through longitudinal challenge serum samples, obtaining 12 short peptides, which were then prepared into a porcine deltacoronavirus antibody detection kit. Finally, six field serum samples were selected for antibody detection, and the effectiveness was verified using a virus neutralization experiment. The results showed that the porcine deltacoronavirus antibody detection kit provided by this invention is effective.

[0021] Furthermore, the present invention also has the following advantages: 1) High sensitivity and high specificity detection. This embodiment employs a multi-index, multi-value method to simultaneously improve the sensitivity and specificity of the detection. The porcine deltacoronavirus antibody detection kit provided by this invention contains multiple short peptide detection probes (12 short peptides) and uses a binary method of Digital Microarray Index (DMI) (i.e., DMI=2) to determine the detection results.

[0022] 2) It can determine whether an infection is recent or past. Based on the combined results of the "Porcine Delta Coronavirus Antibody Detection Kit" and the virus neutralization experiment, we can determine the stage of infection in a single serum sample.

[0023] 3) DIVA can be achieved. It has been reported that short peptide antibodies are transiently produced IgGs (TPIs). They initiate a response when a virus invades and subside when the virus disappears. However, the response is restarted upon reinfection. Therefore, for vaccine-immunized samples, short peptide antibodies typically recede below the detection threshold in about 90 days. The presence of a short peptide antibody response after 90 days indicates infection.

[0024] 4) It can achieve multifunctional detection. The porcine deltacoronavirus antibody detection kit provided by this invention is a new detection method that, while improving sensitivity and specificity, can simultaneously provide multiple detection results, such as determining whether the virus is infected, judging the stage of infection, and achieving DIVA. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 For preliminary screening of challenge serum; where a represents the four structural proteins of PDCoV; b represents the experimental design of the PDCoV challenge group: three 27-day-old piglets were selected and administered 10 mL of serum orally on day 0, with a viral load of 10... 6.75 PDCoV copies / mL, blood was collected every 3-4 days from 0 to 93 dpc after challenge, and every 6-8 days from 93 to 142 dpc (33 blood collections per pig); c is a schematic diagram of PPHM-#1 composition and screening results of Pig1 challenge serum at 24 dpc and 49 dpc; Figure 2Determination of pig candidate epitopes numbered 1; among which, a is the classification heatmap display of Pig1 anti-probe IgGs (2 proteins + 150 short peptides) assisted by the IsDAED method, and all probes can be divided into three types: SI (red), NSI (blue), and Null (gray) according to the classification criteria of the IsDAED method; b is the heatmap display of anti-probe IgGs after high-level classification of 11 SI probes of the pig numbered 1; c is the IsDAED curve display of anti-probe IgGs after high-level classification of SI probes of the pig numbered 1. Figure 3 Optimization of PDCoV candidate epitopes; among which, a is the antigenicity test of candidate epitopes with 50 PDCoV-negative sera, the S protein belongs to the high-antigenicity epitope (RR≥50%), the N protein belongs to the medium-antigenicity epitope (20%<RR<50%), and the others belong to the low-antigenicity epitope (RR≤20%); b is the PDCoV diagnostic specificity test of PPHM <( Figure 4 Time dynamics of DMI and neutralizing antibodies during the infection process; among which, a is the development trend chart of neutralizing antibodies and DMI after the virus invades the body, the left model is the determination model of the infection stage, and the right side is the judgment basis; b-e are the virus load changes (PCR, black), neutralizing antibodies (left ordinate) and PPHM PDCoV DMI (right ordinate) test results of Pig 4, 5, 6, and 7 longitudinal serum samples; f is the virus load, neutralizing antibodies and PPHM PDCoV DMI test results in the serum samples of 6 randomly selected 175-day-old pigs in the field. Detailed implementation manners

[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1 1. Experimental reagents and instruments Porcine IgG was purchased from Millipore; iPDMS nanomembrane was purchased from Suzhou Siju Biomaterials Co., Ltd.; glycerol, acetonitrile, mannitol, triaton, N-hydroxybutyramide, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd.; PB-10 pH meter was purchased from Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; MXM-X3-80 spin incubator was purchased from Suzhou Siju Biomaterials Co., Ltd.; and SmartArrayer 48 microarray chip spotting system was purchased from Bio-Rad Laboratories.

[0033] 2. Chip fabrication This embodiment selects PDCoV of the Coronaviridae family as the research object. From the perspective of viral composition, the PDCoV genome contains four structural proteins, namely spike [S], envelope [E], membrane [M], and nucleocapsid [N] ( Figure 1(a) To systematically cover all possible linear B-cell epitopes of the four structural proteins, this embodiment uses the amino acid sequence information of each protein and employs a shingled cleavage method to obtain a series of short peptide fragments of 20 amino acids in length (20-mer). To ensure that epitope regions are not missed due to the limitation of cleavage length, a 10-amino acid overlap is set between adjacent short peptides. This design strategy can increase the probability of recognizing linear epitopes with strong immunogenicity. After completing the design and sequence information processing of all short peptide fragments, a total of 178 short peptides were obtained, but some were not synthesized (13) + synthesis failed (10+2+2+1) = 28, so 150 were finally successfully synthesized.

[0034] (1) Chip fabrication method 1) Short peptide synthesis: A total of 150 short peptides were obtained through chemical synthesis methods. The final number of short peptides obtained for each structural protein is as follows: S protein yielded 92 short peptides (the order of all short peptides is S1 to S115; among them, S60, S61, S68, S69, S72, S75, S76, S78, S87, S90, S91, S108, and S109 were not synthesized due to high homology with the laboratory short peptide library; S1, S25, S26, S37, S49, S59, S74, S84, S112, and S113 failed to synthesize). E protein yielded 6 short peptides (the order of short peptides is E1 to E8; among them, E2 and E3 failed to synthesize). M protein yielded 19 short peptides (the order of short peptides is M1 to M21; among them, M2 and M8 failed to synthesize). N protein yielded 33 short peptides (the order of short peptides is N1 to N34; among them, N26 failed to synthesize).

[0035] 2) Short peptide dissolution: Dissolve the synthesized short peptide powder (1 mg / tube) in 1 mL of 30% acetonitrile solution to prepare a stock solution with a concentration of 1 mg / mL, and store it at -20℃ for a short period. When spotting, take out the stock solution and prepare it to the specific working concentration as needed.

[0036] 3) Reagent Preparation: First, the spotting solution is prepared by mixing equal volumes of 0.02% Triton, 1.5% mannitol, and 0.1% glycerol and adding them to PBS solution. This spotting solution maintains the wetness of the chip surface and helps preserve the bioactivity of short peptides. Its function is to prevent the drift of short peptide spots during the spotting process and to avoid the formation of hollow spots. Second, the iPDMS activation solution is prepared by dissolving 0.3 g of EDC in 10 mL of ultrapure water, followed by dissolving 0.2 g of N-hydroxysuccinimide in 10 mL of water. Then, these two solutions are mixed in equal volumes to obtain the activation solution for modifying the silica membrane surface.

[0037] 4) Short peptide spotting: Immerse the iPDMS membrane in the pre-prepared activation solution and let it stand at room temperature for 30 minutes. Then, gently remove the membrane with tweezers and gently dry the membrane surface using a vacuum compressed air canister. To ensure the activity of the carboxyl groups on the membrane surface is maintained, the spotting operation must be completed within 4 hours.

[0038] In an environment with a temperature of 25°C and a relative humidity of over 50%, the prepared short peptide solution was precisely spotted onto the surface of a silica membrane using a microarray spotter.

[0039] After spotting, the iPDMS membrane was left to stand overnight to allow the peptides to be fully immobilized on the membrane surface. The next day, the peptide microarray chip was inspected using an electron microscope to confirm that the membrane was intact and that the peptides were not drifting. The iPDMS membrane was then assembled into the reactor, placed in a vacuum-sealed bag, and stored at 4°C for later use.

[0040] (2) Chip type PPHM-#1 consists of PPHM-#1-1 and PPHM-#1-2. The detection probe of PPHM-#1-1 consists of 134 peptides, including 6 peptides from the E protein, 19 peptides from the M protein, 33 peptides from the N protein, and 76 peptides from the S protein. The detection probe of PPHM-#1-2 consists of the remaining 16 peptides derived from the S protein, as well as the N and S proteins (accession number OK546242 for the whole genome). All probes were spotted onto activated iPDMS nanomembranes using a non-contact spotting instrument, sciFLEXARRAYER S1 (Scienion, Berlin, Germany). The peptide concentration was 100 μg / mL, and the S and N protein concentrations were 50 μg / mL. PPHM-#1 also includes seven positive controls with porcine IgG at concentrations of 2, 4, 6, 8, 10, 12, and 14 μg / mL, and a negative control prepared with buffer, with 40 drops spotted at each spot. The negative control solution used was glycerol, Triton X-100 and mannitol, and was brought to a final volume of 5 mL with 0.3 M PB. The final concentrations of glycerol, Triton X-100 and mannitol were 0.2% (v / v), 0.01% (v / v) and 1.5 wt%.

[0041] 3. Collection of serum samples The animal experiment protocol was reviewed and approved by the Laboratory Animal Ethics Committee of Jiangsu Academy of Agricultural Sciences (Nanjing, China) (Approval No.: IACUC-LE-2023-05-013) and was conducted in strict accordance with relevant guidelines and regulations.

[0042] 3.1 Obtaining porcine serum samples Three 27-day-old piglets (Pig 1, 2, and 3) were selected and administered 10 mL of the solution orally on day 0. The viral load was 10. 6.0 TCID 50 / mL of PDCoV CZ2020 (this strain is disclosed in the literature "Pathogenicity, infective dose and altered gut microbiota in piglets infected with porcine deltacoronavirus" and "Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cellculture and intestinal contents in 27-day-old piglets") was collected. Serum samples were taken from 3 experimental pigs at intervals of 3-4 days from 0 to 93 days (dpc), and at intervals of 6-8 days from 93 to 142 days (dpc), for a total of 33 serum samples collected from each pig. Figure 1 (b) In this embodiment, the serum samples are high-frequency longitudinal cohort serums of PDCoV obtained by "challenge", and the longitudinal cohort samples of challenge can cover the complete antibody development range.

[0043] 3.2 Serum Sample Processing: Collect 3 mL of whole blood from the superior vena cava of the pig. Strict aseptic technique must be followed during blood collection to avoid sample contamination. The collected whole blood should be immediately placed in a disposable sterile container and rapidly transferred to a 4°C environment for 2 hours to promote the natural separation of cellular components from the serum. The low temperature also helps maintain the stability of bioactive substances in the sample.

[0044] After settling, transfer the sample to a centrifuge and centrifuge at 2000 rpm for 15 minutes. After centrifugation, carefully remove the supernatant (i.e., serum) and place it in a 56°C water bath for 30 minutes for heat inactivation.

[0045] Finally, the inactivated serum samples were aliquoted into sterile cryovials and quickly placed in an ultra-low temperature freezer at -70°C for storage.

[0046] Taking pig number 1 as an example, 33 serum samples were collected to screen for PPHM-#1 and obtain the signal values ​​of the response probes. PPHM-#1 consists of 150 short peptides of 20 mers synthesized from the amino acid sequences of 4 structural proteins, and 2 proteins: S and N proteins. The screening results show that the response probe signals gradually disappeared over time (between 24 dpc and 49 dpc). Figure 1 (c in the text)

[0047] 4. The IsDAED method for identifying candidate diagnostic epitopes Based on the reported IgG sero-dynamics curves aid epitope discovery (IsDAED) method (referenced in "Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts"), this embodiment categorizes the development curves of each short peptide antibody over time into three types: specific interaction (SI), non-specific interaction (NSI), and null, and displays them using a classification heatmap. Figure 2 (a) However, within the SI category, short peptide antibodies exhibit different response patterns. Therefore, we need to manually classify Pig 1 SI probes into four categories based on their response patterns in immune and non-immune response regions. This classification method is called high-level classification. The high-level classification heatmap and IsD curve respectively illustrate the characteristics of the four types of probes (a). Figure 2(b and c in the text). Type 1 is the proteome (N and S): Unlike the common anti-protein IgG development pattern, PDCoV's anti-N IgG and anti-S IgG did not show sustained response in the immune response zone. Instead, similar to the development trend of anti-peptide IgG, they exhibited an upward and downward process, initiating a response again after 60 dpc. Type 2 is the "standard" anti-peptide IgG response group (including 6 short peptides: M21 / N5 / N8 / S4 / S15 / S102), i.e., responding in the immune response zone (0~60 dpc) and not responding in the non-immune response zone (60~142 dpc), exhibiting a complete antibody development pattern. Type 3 is the early-initiating anti-peptide IgG response group (M17): This short peptide is characterized by a response at 0 dpc. Type 4 is a "non-standard" anti-peptide IgG response group (including two short peptides: N33 / S114): they exhibit a clear pattern of antibody development and progression within the immune response region, but show a low-level sustained response in the non-immune response region. This grouping method allows us to directly obtain the optimal candidate epitopes of Pig1 (Type 1, 2, 3) and directly exclude Type 4.

[0048] 5. PPHM PDCoV The determination Candidate epitopes were also identified in pigs numbered 2 (Pig 2) and 3 (Pig 3) according to the above analysis procedure. Combining the screening results of the three pigs (Type 1, 2, 3), this embodiment can preliminarily identify 12 short peptides and 2 proteins as PDCoV candidate epitopes to form PPHM-#2 (PPHM-#2: This chip consists of S protein, N protein, and 12 short peptides (M17, M21, N5, N8, N29, S4, S15, S28, S38, S65, S80, S102), 3 positive porcine IgGs, and 1 negative spotting solution. The spotting concentration of the short peptides was set at 100 μg / mL, and the spotting concentration of the 3 positive porcine IgGs was 50 μg / mL, with 40 drops for each spot).

[0049] 6. Virus neutralization test The high antigenicity of the probe is one of the main reasons for false positives in diagnosis. Since asymptomatic infections of PDCoV exist, it is more difficult to obtain negative sera. The virus neutralization test, as one of the gold standards for diagnosis, can help determine PDCoV negative sera. Here, to test the antigenicity of 12 short peptides and 2 proteins, this example selected 50 samples (blood samples from 50 normal pigs) and used PDCoV negative sera determined by the virus neutralization test to screen PPHM-#2. According to the response rate, the antigenicity of the candidate epitopes was divided into three groups: high (RR ≥ 50%), medium (20% < RR < 50%), and low (RR ≤ 20%). The screening results showed that the response rate of the S protein was 74%, belonging to a high antigenicity epitope (aggregate). The response rate of the N protein was 26%, belonging to a medium antigenicity epitope (aggregate). The response rates of the remaining 12 short peptides were all lower than 20%, belonging to low antigenicity epitopes ( Figure 3 a) in Figure 3 In diagnosis, to avoid false positives in test results, we should first exclude high and medium antigenicity epitopes and retain the 12 short peptide epitopes with low antigenicity. After statistics, when DMI ≥ 2, only 2 out of 50 negative samples were positive (response rate 4%), and the specificity was 96% ( PDCoV b) in

[0050] This threshold setting is consistent with previous studies (the literature "Immunoglobin G Sero-Dynamics AidedHost Specific Linear Epitope Identification and Differentiation of Infectedfrom Vaccinated Hosts" and the literature "Previously Unrecognized Nonreproducible Antibody−Probe Interactions"). So finally, these 12 short peptides (antigenic epitope polypeptides) were selected as the diagnostic epitopes of PDCoV to form PPHM PDCoV (Table 1).

[0050] Table 1 PDCoV diagnostic epitopes determined in the challenge group The sequences of the 12 epitope peptides in Table 1 are as follows: M17: VIVSPSDTFHYTFKKPVESN, SEQ IDNO.1; M21: HTITTSKAGDARLYKYM, SEQ IDNO.2; N5: KPVENHGYWLRYTRQKPGGT, SEQ IDNO.3; N8: TGTGPRGNLKYGELPPNDTP, SEQ IDNO.4; N29: LNTVVNQTYEPPTKPTKDKK, SEQ IDNO.5; S4: AHRFLHKLTSNSSSLYSRAN, SEQ IDNO.6; S15: SVEVVGQHGENYVFVCSEQF, SEQ IDNO.7; S28: RSSHEVEDGFYSDPKSAVRA, SEQ IDNO.8; S38: MSFSQFCLSTESGACEMKII, SEQ IDNO.9; S65: QTSTQSLQLANITNFKGDYN, SEQ IDNO.10; S80: ALSSVNDAIQQTSEALSTVA, SEQ IDNO.11; S102: IIPDVIDVNQTVSDIIDNLP, SEQ IDNO.12; The epitope peptide sequences used in subsequent embodiments were synthesized by GenScript Biotech.

[0051] Example 2: Preparation of a porcine deltacoronavirus antibody detection kit Porcine Delta Coronavirus Antibody Detection Kit Contains PPHM PDCoV Chip, 20× concentrated cleaning solution, sample diluent, enzyme-labeled antibody solution, and luminescent substrate solution; PPHM PDCoV Chip: The chip consists of 12 short peptides (M17 / 21, N5 / 8 / 29, S4 / 15 / 28 / 38 / 65 / 80 / 102), 3 positive porcine IgG antibodies, and 1 negative spotting solution. The spotting concentration of the short peptides was set at 100 μg / mL, and the spotting concentration of the 3 positive porcine IgG control spots was 50 μg / mL, with 40 drops for each spot. The specific preparation method is as follows: the 12 short peptides, porcine IgG antibodies, and PB buffer were respectively spotted onto a solid-phase carrier used in conventional ELISA (the solid-phase carrier used in this example is an iPDMS nanomembrane (manufacturer: Suzhou Siju Biomaterials Co., Ltd.)).

[0052] 20× Concentrated Cleaning Solution (TBST): 0.4 M Tris-HCl, 2.74 M NaCl, 2% (v / v) Tween 20, pH 7.2±0.2; Sample dilution: 0.05 M PBS, 1 wt% BSA, 0.2 wt% PVP (polyvinylpyrrolidone), 0.5% (v / v) Tween 20, pH 7.2 ± 0.2; Enzyme-labeled antibody solution: rabbit anti-pig IgG-HRP (Sigma, A5420), diluted 1:7500; Luminescent substrate solution: Luminescent substrate solution (Thermo, Prod#37074).

[0053] Example 3: Procedure for using the porcine deltacoronavirus antibody detection kit (1) Dilute the 20× concentrated cleaning solution (TBST: 0.4 M Tris-HCl, 2.74 M NaCl, 2% (v / v) Tween 20, pH 7.2±0.2) with purified water at a volume ratio of 1:20 to obtain the cleaning solution. To ensure complete wetting of the detection chip surface, add approximately 100 μL of the cleaning solution to the detection chip surface using a pipette and soak the detection chip for 5 min.

[0054] (2) Dilute the serum sample to be tested with sample diluent (0.05 M PBS, 1 wt% BSA, 0.2 wt% PVP, 0.5% (v / v) Tween 20, pH 7.2 ± 0.2) at a volume ratio of 1:50.

[0055] (3) For the detection chip that has been cleaned, add 100 μL of diluted serum sample to the detection chip while the surface is completely wet.

[0056] (4) Incubate the detection chip in a constant temperature shaker at 500 rpm for 30 min at a temperature of 37 ℃.

[0057] (5) Discard the serum sample and clean the surface of the detection chip with cleaning solution three times.

[0058] (6) After cleaning, add 100 μL of enzyme-labeled antibody solution (rabbit anti-pig IgG-HRP, dilution ratio 1:7500, Sigma, A5420) to the detection chip, and incubate the detection chip in a constant temperature shaker at 500 rpm for 30 min at 37℃.

[0059] (7) Discard the enzyme-labeled antibody solution and wash the chip surface three times with the cleaning solution to clean it thoroughly.

[0060] (8) After cleaning, spread 15 μL / well of luminescent substrate solution (Thermo, Prod#37074) evenly on the surface of the detection chip.

[0061] (9) Use a gel imaging system to perform chemiluminescence imaging on the detection chip and determine the results.

[0062] (10) Result determination: For each serum sample, a signal value of 2000 or higher for the antigen epitope polypeptide spot on the antibody detection chip indicates a “response”; otherwise, it indicates a “no response”. When two or more of the 12 antigen epitope polypeptide spots show a response (DMI≥2), it is determined that PDCoV antibody is detected; otherwise (DMI<2), it is determined that PDCoV antibody is not detected.

[0063] Example 4: Actual Sample Detection The development trend graphs of neutralizing antibodies and DMI after viral invasion of the body were constructed according to the method in the literature "Transiently produced IgGs enable universal SARS-CoV-2 diagnosis and differentiation recent from past infections". The results are as follows: Figure 4 As shown in 'a' in the figure. Simultaneously, four pigs (Pig 4, 5, 6, and 7) from a pig farm were randomly selected to validate this trend chart, following the same validation method as the literature "Transiently produced IgG-senable universal SARS-CoV-2 diagnosis and differentiation recent from pastinfections". The results are as follows: Figure 4 As shown in be in the diagram. Results show that when both neutralizing antibody and PPHM are positive, or when neutralizing antibody is negative and PPHM is positive, according to... Figure 4 The model for determining 'a' in the data indicates a recent infection. A positive result for neutralizing antibodies and a negative result for PPHM indicate a past infection.

[0064] In conclusion, Figure 4 The trend chart provided by 'a' in the chart can be used to accurately determine the stage of infection in pigs (recent infection or past infection).

[0065] Subsequently, serum samples from six 175-day-old field pigs (Pig 8, 9, 10, 11, 12, and 13) were randomly selected. None of these six pigs had been vaccinated against PDCoV. This example used both the "virus neutralization experiment" (one of the gold standard methods) and the "porcine deltacoronavirus antibody detection kit" constructed in Example 2 for testing. The virus neutralization experiment results showed that all six serum samples were positive for neutralizing antibodies. The porcine deltacoronavirus antibody detection kit also showed that all six serum samples were positive for antibodies (DMI ≥ 2). Figure 4 As shown in f, the DMI values ​​were 5, 2, 6, 2, 4, and 3, respectively, achieving a 100% compliance rate. Therefore, the porcine deltacoronavirus antibody detection kit provided by this invention has good practical value.

[0066] Example 5: Sensitivity and Specificity Serum samples from 3 pigs with a 0 dpc (3 samples in total) from step "3.1, Obtaining Pig Serum Samples" in Example 1 were used as negative samples, and serum samples with a 9-15 dpc (9 samples in total, and confirmed by qPCR to show viral shedding) were used as positive samples. The "Porcine Delta Coronavirus Antibody Detection Kit" constructed in Example 2 was used for testing. Calculations showed that when the DMI was set to 2, the sensitivity was 88.9% and the specificity was 100%.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antigenic epitope polypeptide for detecting antibodies produced by porcine deltacoronavirus infection, characterized in that, The amino acid sequences of the antigenic epitope polypeptide are shown in SEQ ID NO.1-SEQ ID NO.

12.

2. The use of the antigenic epitope polypeptide of claim 1 in the preparation of a product for detecting antibodies produced by porcine deltacoronavirus infection.

3. The application according to claim 2, characterized in that, The products include reagents, reagent kits, and chips.

4. A reagent for detecting antibodies produced by porcine deltacoronavirus infection, characterized in that, The reagent contains the antigenic epitope polypeptide as described in claim 1.

5. A chip for detecting antibodies produced by porcine deltacoronavirus infection, characterized in that, The chip includes a solid-phase carrier containing the antigenic epitope polypeptide of claim 1.

6. A kit for detecting antibodies produced by porcine deltacoronavirus infection, characterized in that, The kit includes the chip described in claim 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes 20× concentrated washing solution, sample diluent, enzyme-labeled antibody solution, and luminescent substrate solution.

8. The reagent kit according to claim 7, characterized in that, The 20× concentrated cleaning solution includes Tris-HCl, NaCl, and Tween 20; The sample diluents include PBS, BSA, PVP, and Tween 20.

9. The reagent kit according to claim 8, characterized in that, The concentration of Tris-HCl in the 20× concentrated cleaning solution is 0.4 M, the concentration of NaCl is 2.74 M, and the volume percentage of Tween 20 is 2%. The concentration of PBS in the sample diluent is 0.05M, the mass percentage of BSA is 1wt%, the mass percentage of PVP is 0.2wt%, and the volume percentage of Tween 20 is 0.5%.

10. The use of the antigenic epitope polypeptide of claim 1, the reagent of claim 4, the chip of claim 5, or the kit of claim 6 in the preparation of products for evaluating intestinal diseases.

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