Porcine epidemic diarrhea virus s1 recombinant protein, kit and application thereof

Abstract

The disclosure provides a porcine epidemic diarrhea virus S1 recombinant protein, a kit and application thereof, and belongs to the field of veterinary biological technology. The recombinant protein has good antigenicity, hydrophilicity and stability. The recombinant protein provided by the disclosure is used for preparing an anti-PEDV immunoglobulin antibody detection kit, and has the advantages of high specificity and sensitivity, no cross reaction, good repeatability within and between batches, strong thermal stability and the like. Moreover, the kit can be used for detecting various samples such as oral swabs, anal swabs, serum or colostrum, and can distinguish natural infection from vaccine immunization pig groups. The recombinant protein and the ELISA kit provided by the disclosure provide a good platform for the diagnosis, vaccine and therapeutic drug research and development of porcine epidemic diarrhea, and have important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary biotechnology, and particularly relates to a porcine epidemic diarrhea virus S1 recombinant protein, a kit and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease characterized by watery diarrhea, vomiting and dehydration caused by porcine epidemic diarrhea virus (PEDV) infection. The mortality rate of PEDV-infected suckling piglets can reach 100%, and pigs of all ages can be infected. The rapidly spreading new high virulence PEDV strain in China has caused a large number of piglet deaths, with a mortality rate close to 100%, causing huge economic losses to the pig industry in China.

[0003] The serological detection of PEDV mainly includes neutralization test (SNT), indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). Among them, the ELISA method is easy to operate, specific, and can be used for high-throughput detection, and is a commonly used serological antibody detection method. At present, there is no effective treatment drug for PED, and passive immunization conferred by maternal antibodies obtained from colostrum is needed. The most important immune factors in colostrum are IgA and IgG, which perform different immune functions. Secretory IgA (sIgA) can anchor on the mucosa to protect the intestinal tract and other mucosal tissues of piglets from pathogenic microorganisms.

[0004] The existing anti-PEDV immunoglobulin detection kit can only detect anti-PEDV immunoglobulin in pig serum and colostrum, and requires collection of pig blood and sow colostrum samples for detection. Under the background of the African swine fever epidemic, blood collection is not conducive to the prevention and control of African swine fever, and colostrum detection, even if the antibody level is low, does not have time to adjust the immune program to change the antibody level in colostrum, and the results have a lag effect on the guidance of PED prevention and control. SUMMARY

[0005] To solve at least one of the above problems, the present disclosure provides a recombinant protein, a kit and application thereof.

[0006] According to a first aspect of the present disclosure, a recombinant protein is provided, which includes any one or more fragments of the following:

[0007] a first fragment having an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 60% sequence identity thereto;

[0008] a second fragment having an amino acid sequence as set forth in SEQ ID NO: 3, or an amino acid sequence having at least 60% sequence identity thereto;

[0009] a third fragment having an amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence having at least 60% sequence identity thereto;

[0010] a fourth fragment having an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence having at least 60% sequence identity thereto; and / or

[0011] a fifth fragment having an amino acid sequence as set forth in SEQ ID NO: 6, or an amino acid sequence having at least 60% sequence identity thereto.

[0012] In some embodiments, the first fragment, the second fragment, the third fragment, the fourth fragment, and / or the fifth fragment are each independently linked by a linker peptide,

[0013] In some embodiments, the linker peptide comprises a flexible linker peptide,

[0014] In some embodiments, the flexible linker peptide comprises (G m S t G w ) n , (G n S) m , (G) n , (EA3K) n or (XP) n , wherein n, m, t, w are each independently selected from an integer from 0 to 5. In some embodiments, the flexible linker peptide has an amino acid sequence as set forth in SEQ ID NO: 7.

[0015] In some embodiments, the linker peptide between the first fragment, the second fragment, the third fragment, the fourth fragment, and / or the fifth fragment is the same or different.

[0016] In some embodiments, the recombinant protein comprises 1 to 10 copies of any of the fragments.

[0017] In some embodiments, the recombinant protein comprises 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, or 10 copies of any of the fragments.

[0018] In some embodiments, the recombinant protein comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 60% sequence identity thereto and retaining immunogenicity of the PEDV S1 protein.

[0019] According to a second aspect of the present disclosure, there is provided a nucleic acid molecule, the nucleotide sequence of which is capable of encoding the recombinant protein of the first aspect.

[0020] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 8, or a nucleotide sequence having at least 60% sequence identity thereto.

[0021] According to a third aspect of the present disclosure, there is provided a recombinant vector comprising the nucleic acid molecule of the second aspect.

[0022] In some embodiments, the vector comprises one or more of, but not limited to, the following: a pFastBacl plasmid, a pGEX-4T2 plasmid, a pVL1393 plasmid, and a psPAX2 plasmid.

[0023] According to a fourth aspect of the present disclosure, there is provided a host cell comprising the nucleic acid molecule of the second aspect or the recombinant vector of the third aspect.

[0024] In some embodiments, the cell comprises a eukaryotic or prokaryotic cell.

[0025] In some embodiments, the prokaryotic cell comprises a bacterium.

[0026] In some embodiments, the bacterium comprises Escherichia Coli.

[0027] In some embodiments, the Escherichia Coli comprises one or more of, but not limited to, the following: a DH10Bac cell, a BL21(DE3) cell, a BW25113 cell, a MG1655 cell, a W3110 cell, or a W cell.

[0028] In some embodiments, the eukaryotic cell comprises one or more of the following: a yeast, an insect, a plant, or a mammalian cell.

[0029] In some embodiments, the insect cell comprises a lepidopteran insect cell.

[0030] In some embodiments, the insect includes, but is not limited to, one or more of Bombyx mori Linnaeus, Mamestra brassicae Linnaeus, Spodoptera frugiperda, Trichoplusia ni, trichogrammatid.

[0031] In some embodiments, the insect cell includes, but is not limited to, one or more of sf9 insect cell, sf21 insect cell, SF+ cell line, or High Five cell.

[0032] According to a fifth aspect of the present disclosure, a kit for detecting anti-PEDV immunoglobulin is provided, the kit comprising the recombinant protein of the first aspect.

[0033] In some embodiments, the kit further comprises a secondary antibody of the anti-PEDV immunoglobulin labeled with a label, and a substrate and / or a control.

[0034] In some embodiments, the kit further comprises a sample diluent, a washing solution, and / or a termination solution.

[0035] In some embodiments, the recombinant protein is immobilized on an enzyme-labeled plate.

[0036] In some embodiments, the recombinant protein is immobilized on an enzyme-labeled plate in a square array.

[0037] In some embodiments, the recombinant protein is immobilized on an enzyme-labeled plate at a concentration of 0.1-5 ug / mL.

[0038] In some embodiments, the recombinant protein is immobilized on the enzyme- labeled plate at a concentration of 0.1 ug / mL, 0.2 ug / mL, 0.3 ug / mL, 0.4 ug / mL, 0.5 ug / mL, 0.6 ug / mL, 0.7 ug / mL, 0.8 ug / mL, 0.9 ug / mL, 1.0 ug / mL, 1.1 ug / mL, 1.2 ug / mL, 1.3 ug / mL, 1.4 ug / mL, 1.5 ug / mL, 1.6 ug / mL, 1.7 ug / mL, 1.8 ug / mL, 1.9 ug / mL, 2 ug / mL, 2.1 ug / mL, 2.2 ug / mL, 2.3 ug / mL, 2.4 ug / mL, 2.5 ug / mL, 2.6 ug / mL, 2.7 ug / mL, 2.8 ug / mL, 2.9 ug / mL, 3 ug / mL, 3.1 ug / mL, 3.2 ug / mL, 3.3 ug / mL, 3.4 ug / mL, 3.5 ug / mL, 3.6 ug / mL, 3.7 ug / mL, 3.8 ug / mL, 3.9 ug / mL, 4 ug / mL, 4.1 ug / mL, 4.2 ug / mL, 4.3 ug / mL, 4.4 ug / mL, 4.5 ug / mL, 4.6 ug / mL, 4.7 ug / mL, 4.8 ug / mL, 4.9 ug / mL, or 5 ug / mL.

[0039] In some embodiments, the label comprises an enzyme.

[0040] In some embodiments, the enzyme comprises horseradish peroxidase (HRP) or alkaline phosphatase.

[0041] In some embodiments, the substrate comprises tetramethylbenzidine (TMB) or 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS).

[0042] In some embodiments, the immunoglobulin comprises one or more of IgA type, IgD type, IgE type, IgG type, and IgM type.

[0043] In some embodiments, the sample diluent comprises one or more of serum protein, surfactant, and PBS buffer.

[0044] In some embodiments, the sample diluent comprises 0.5% to 5% serum protein.

[0045] In some embodiments, the sample diluent comprises 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5% serum protein.

[0046] In some embodiments, the serum protein comprises bovine serum protein.

[0047] In some embodiments, the washing solution comprises one or more of a surfactant and a PBS buffer.

[0048] In some embodiments, the termination solution comprises H2SO4.

[0049] In some embodiments, the surfactant comprises Tween.

[0050] In some embodiments, the Tween comprises, but is not limited to, one or more of Tween 20, Tween 40, Tween 60, and Tween 80.

[0051] In some embodiments, the control comprises a positive control and / or a negative control.

[0052] In some embodiments, the positive control comprises anti-PEDV immunoglobulin positive serum.

[0053] In some embodiments, the negative control comprises anti-PEDV immunoglobulin negative serum.

[0054] In some embodiments, the kit can be stored at 4°C for more than 1 year.

[0055] According to a sixth aspect of the present disclosure, a method for detecting anti-PEDV immunoglobulin in a sample to be tested using the kit of the fifth aspect is provided, the method comprising the following steps:

[0056] b1, adding the sample to be tested to the enzyme-labeled plate coated with the recombinant protein for reaction;

[0057] b2, adding the labeled anti-PEDV immunoglobulin for reaction to obtain a reaction product;

[0058] b3, the reactant reacts with the substrate to produce a signal, and whether the anti-PEDV immunoglobulin exists in the sample to be tested is determined according to the signal, wherein the intensity of the signal is related to the concentration of the anti-PEDV immunoglobulin in the sample to be tested.

[0059] In some embodiments, the step b1 comprises adding the sample to be tested, the positive control and the negative control into an enzyme-labeled plate coated with the recombinant protein for reaction.

[0060] In some embodiments, the sample to be tested, the positive control and the negative control in the step b1 need to be diluted, and the dilution ratio includes 10-200 times.

[0061] In some embodiments, the dilution ratio of the sample to be tested, the positive control and the negative control includes 50-200 times.

[0062] In some embodiments, the sample to be tested, the positive control, the negative control need to be diluted, and the dilution factor includes 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 folds.

[0063] In some embodiments, the reaction temperature of step b1 is room temperature.

[0064] In some embodiments, the reaction condition of step b1 is avoiding light.

[0065] In some embodiments, the reaction time of step b1 includes 20-60 min.

[0066] In some embodiments, the reaction time in step b1 includes, but is not limited to, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min.

[0067] In some embodiments, the dilution fold of the marker-labeled anti-PEDV immunoglobulin in step b2 includes 5000-15000 folds.

[0068] In some embodiments, the dilution fold of the marker-labeled anti-PEDV immunoglobulin in step b2 includes, but is not limited to, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 folds.

[0069] In some embodiments, the temperature of the reaction in step b2 includes 30-45 °C.

[0070] In some embodiments, the temperature of the reaction in step b2 includes 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C.

[0071] In some embodiments, the time of the reaction in step b2 includes 30-60 min.

[0072] In some embodiments, the time of the reaction in step b2 includes, but is not limited to, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min.

[0073] In some embodiments, the time for the substrate to react in step b2 comprises 10-30 min.

[0074] In some embodiments, the time for the substrate to react in step b2 comprises, but not limited to, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min.

[0075] In some embodiments, the sample to be tested comprises a sample of a body fluid of a pig.

[0076] In some embodiments, the sample of a body fluid of a pig comprises one or more of detection of blood, plasma, serum, milk, nasopharyngeal swab, buccal swab, feces, anal swab or their processed products.

[0077] In some embodiments, the sample of a body fluid of a pig comprises one or more of a buccal swab, anal swab, blood or milk of a pig.

[0078] In some embodiments, the milk comprises colostrum.

[0079] According to a seventh aspect of the present disclosure, there is provided use of the recombinant protein of the first aspect or the nucleic acid molecule of the second aspect in any one or more of the following (1)-(3):

[0080] (1) preparation of a PEDV vaccine;

[0081] (2) preparation of an anti-PEDV immunoglobulin detection kit;

[0082] (3) preparation of a therapeutic drug for preventing or treating PEDV infection (e.g. epidemic diarrhea of a pig).

[0083] Advantages of the present disclosure:

[0084] The present disclosure selects a target fragment with good antigenicity and hydrophilicity in the S1 protein gene, performs truncated expression and repeated expression, and the obtained recombinant protein has good antigenicity, hydrophilicity and stability.

[0085] The anti-PEDV immunoglobulin detection kit prepared from the recombinant protein provided by the present disclosure has high specificity and high sensitivity, does not produce cross-reactions, has good batch and batch repeatability, and has strong thermal stability. And can be used for the detection of various types of samples such as oral swabs, anal swabs, serum or colostrum, and can distinguish between naturally infected and vaccinated pig populations. It provides a good platform for the detection of porcine epidemic diarrhea and the research and development of vaccines and therapeutic drugs, and has important application value. DETAILED DESCRIPTION

[0086] The present application carries out a large number of sequence alignment and analysis on PEDV S1 protein sequence in the database, and selects the target fragment with good antigenicity and hydrophilicity, as shown in Table 1.

[0087] Table 1. Amino acid sequence of PEDV S1 protein and recombinant protein

[0088]

[0089]

[0090] The fragments described in Table 1 are connected using flexible amino acids to construct a recombinant protein, which has good antigenicity, hydrophilicity and stability.

[0091] The kit for detecting anti-PEDV immunoglobulin prepared from the recombinant protein further comprises an enzyme-labeled anti-PEDV immunoglobulin and a substrate.

[0092] In some embodiments, the kit further comprises one or more of a sample diluent, a washing solution, a termination solution, or a control.

[0093] Using the kit to detect anti-PEDV immunoglobulin in a test sample comprises the following steps:

[0094] b1, adding the test sample to the enzyme-labeled plate coated with the recombinant protein for reaction;

[0095] b2, adding an enzyme-labeled anti-PEDV immunoglobulin for reaction;

[0096] b3, reacting with a substrate to develop color, and determining whether anti-PEDV immunoglobulin exists in the sample, wherein the signal intensity is related to the concentration of PEDV immunoglobulin in the test sample.

[0097] In some embodiments, the step b1 comprises mixing the test sample, the positive control, and the negative control with the recombinant protein for reaction, respectively.

[0098] In some embodiments, the judging method comprises comparing the OD value with a threshold value, and judging as positive when the signal value is ≥ the threshold value, and judging as negative when the signal value is < the threshold value.

[0099] In some embodiments, the signal value comprises a corrected signal value.

[0100] In some embodiments, the corrected signal value is calculated as follows:

[0101]

[0102] In some embodiments, the threshold value is selected from 0.2-0.5.

[0103] In some embodiments, the threshold value is selected from 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5.

[0104] Definitions

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied, for the purposes of interpretation of this specification, and shall have the indicated meaning throughout this specification and claims unless otherwise defined.

[0106] As used herein, the expressions "a" and "an" include plural referents unless the context clearly indicates otherwise.

[0107] The expression "about" as used herein is as understood by one of ordinary skill in the art and varies in its scope depending on the context in which it is used. If one of ordinary skill in the art is not apprised of the context in which the term is used, "about" will mean at most plus or minus 10% of the particular value.

[0108] In the present context, the term "Porcine epidemic diarrhea virus (PEDV)" has a genome size of about 28 kb, with a cap structure (Cap) and a Poly(A) tail at both ends, the genome comprises 4 structural proteins (S protein, E protein, M protein, N protein) and 3 non-structural proteins (replicase la, replicase lb and ORF3), ORF3 encodes a product of unknown function and has polymorphisms. S is the largest structural protein and contains neutralizing antibody epitopes and specific receptor binding sites for viral entry.

[0109] In the present context, the term "nucleic acid" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term also means, unless specifically limited, oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and

[0110] In the present context, the term "expression" is the transcription or / and translation of a foreign gene in a host.

[0111] In the present context, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description in terms of a polypeptide equally applies to a description in terms of a peptide and to a description in terms of a protein and vice versa. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more of the amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e. antigens), in which the amino acid residues are connected via covalent peptide bonds.

[0112] The term "recombinant protein" as used herein refers to a protein molecule, e.g. a protein of interest, which is foreign to the cell in which the polypeptide is produced, expressed using a recombinant DNA molecule.

[0113] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host and which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector which primarily functions to insert DNA or RNA into a cell, a vector which primarily functions to replicate DNA or RNA, and a vector which primarily functions for transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide which, when introduced into a suitable host cell, is capable of being transcribed and translated into a polypeptide. Generally, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

[0114] In the present context, the term "host cell" means a cell which comprises a polynucleotide of the present application, regardless of the method by which the insertion is achieved, e.g., direct uptake, transduction, conjugation, or other means known in the art. The exogenous polynucleotide can be maintained as a non-integrated vector, e.g., a plasmid, or can be integrated into the host genome.

[0115] In the present disclosure, the term "sequence identity" refers to the "percent sequence identity" or "percent identity" between two polynucleotides, i.e., the number of identical matching positions in the comparison window, accounting for any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is found in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted since they are not nucleotides. Likewise, gaps present in the reference sequence are not counted since they are not counted from the nucleotides of the target sequence. At least 60% sequence identity includes a contiguous stretch of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the length of the sequence having sequence identity.

[0116] Methods for alignment of comparison sequences are well known in the art, for example, by existing software BLASR, DALIGN, BLAST, BLAST-2, ALIGN, ALIGN-2 BWA, BOWTIE, or Megalign ((DNASTAR)), etc.

[0117] The term "linker peptide" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. The individual fragments in the recombinant protein of the present application can be connected by a linker, in some embodiments, the linker peptide between the first fragment, the second fragment, the third fragment, the fourth fragment and / or the fifth fragment is the same or different. The linker should have a length suitable for connecting two or more monomeric domains in this way, the linker is able to ensure that the different domains it connects are correctly folded and properly presented, thereby functioning to exert their biological activity. In some embodiments, the linker has a flexible conformation. The flexible linker has, for example, glycine, glutamine and / or serine residues.

[0118] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made in combination with the embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation to the present application. The actual protection scope of the present application is set forth in the claims. In the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following embodiments are not mentioned the source, are commercially available in the market, or are obtained by conventional methods known to those skilled in the art.

[0119] Embodiments

[0120] Example 1. Preparation of PEDV S1 full-length protein and PEDV S1 recombinant protein and comparison of detection effects

[0121] Preparation and identification of recombinant PEDV S1 protein includes the following steps:

[0122] (1) Construction of recombinant expression plasmid: The present application performs a large number of sequence alignments on the S1 protein sequences in the database, selects the target fragments with good antigenicity and hydrophilicity, performs truncated expression and repeated expression, the peptide segment amino acid sequences are shown as SEQ ID NO: 2-6, the flexible amino acids (amino acid sequence is shown as SEQ ID NO: 7) are used to connect between the peptide segments, the S1 recombinant protein (amino acid sequence is shown as SEQ ID NO: 1) with good antigenicity, hydrophilicity and stability is finally screened by comparison and detection with the S1 full-length protein. The sequence is shown in Table 1.

[0123] (2) Preparation of recombinant bacmid:

[0124] The nucleic acid sequence encoding S1 full-length protein and S1 recombinant protein (with histidine tag 6xHis, nucleic acid sequence as shown in SEQ ID NO: 8) were inserted into pFastBacl plasmid through restriction enzyme sites BamHI and SacI, respectively, to obtain plasmid pFastBacl-S1 full-length and plasmid pFastBacl-S1 recombinant, respectively. DH10Bac TM In the competent cells, recombinant bacmids containing nucleic acid encoding S1 full-length protein and S1 recombinant protein were generated, respectively.

[0125] (3) Construction of recombinant baculovirus: The recombinant bacmid DNA described above was transfected into Sf9 insect cell line, respectively, to generate recombinant baculovirus containing nucleic acid sequence encoding S1 full-length protein and S1 recombinant protein, respectively.

[0126] (4) Expression of S1 full-length protein and S1 recombinant protein: According to the manual of baculovirus expression system, the two recombinant baculoviruses described above were inoculated into Sf9 cells and cultured continuously for two generations, respectively. TM The virus liquid was collected and inoculated into High Five cells for expression of S1 full-length protein and S1 recombinant protein, respectively.

[0127] (5) Purification of S1 full-length protein and S1 recombinant protein: S1 full-length protein and S1 recombinant protein were purified by Ni 2+ affinity chromatography, respectively.

[0128] (6) Identification of S1 recombinant protein:

[0129] The purified S1 recombinant protein was subjected to SDS-PAGE electrophoresis, and the results showed that specific protein bands appeared at about 110 kDa.

[0130] ​(7) Comparison of detection effects of PEDV S1 recombinant protein and S1 full-length protein on PEDV IgA, and the detection effect of the protein was evaluated according to the principle of the maximum average P / N value, and the specific steps were as follows: the purified PEDV S1 recombinant protein and S1 full-length protein were respectively diluted to 1 ug / mL with carbonate buffer (pH 9.6), and 100 uL per well was added to the enzyme-labeled plate, and 4℃ coating was carried out for 24h; 2 times of washing with PBS (pH 7.4) containing 0.05% Tween-20 (volume ratio) (PBST) was carried out, and blocking was carried out at 37℃ for 1h with blocking solution (Changsha Yijie Biological Technology), and the plate was washed with PBST and air-dried at room temperature. Four positive sera and four negative sera from clinical samples of porcine epidemic diarrhea virus were diluted at 1:50, and 100 uL per well was added to the enzyme-labeled plate, and 37℃ incubation was carried out for 30min; after 4 times of washing with PBS (pH 7.4) containing 0.05% Tween-20 (volume ratio) (PBST), the plate was dried, HRP-labeled goat anti-pig IgA was diluted at 1:10000, 100 uL per well was added to the enzyme-labeled plate, and 37℃ incubation was carried out for 30min; 37℃ incubation was carried out for 30min; after 4 times of washing with PBS (pH 7.4) containing 0.05% Tween-20 (volume ratio) (PBST), the plate was dried, 50 uL per well was added, TMB was colored for 15min in the dark, 2mol / L H2SO4 was used to terminate the reaction, and the OD 450nm value was determined, and the average P / N value (ratio of positive serum to negative serum) was calculated. The determination results are shown in Table 2: P1, P2, P3, P4 represent positive samples 1, 2, 3, 4 in turn; N1, N2, N3, N4 represent negative samples 1, 2, 3, 4 in turn.

[0131] Table 2. Comparison results of detection effects of PEDV S1 recombinant protein and S1 full-length protein

[0132]

[0133] As shown in Table 2, by analyzing the detection results after coating different proteins, it is determined that, relative to S1 full-length protein, PEDV S1 recombinant protein is a better antigen for detection effect, and therefore it is determined as a coating antigen for subsequent experiments.

[0134] Example 2. Optimization of ELISA conditions for detecting PEDV IgA

[0135] In this example, the ELISA conditions for detecting PEDV IgA were verified respectively:

[0136] (1) Determination of optimal coating concentration of antigen and optimal dilution of serum

[0137] The purified PEDV S1 recombinant protein was diluted to 0.25, 0.5, 1 ug / mL with carbonate buffer (pH 9.6) by chessboard method, and 100 uL was added to each well of the enzyme-labeled plate for coating at 4°C for 24 h. Four positive and four negative swine epidemic diarrhea virus sera from clinical samples were each diluted by 1:50, 1:100, and 1:200 for square array titration. HRP-labeled goat anti-swine IgA was diluted by 1:10,000, and TMB was used for color development in the dark for 15 min. The reaction was terminated by 2 mol / L H2SO4, and the OD 450nm value, the P / N value (ratio of positive serum to negative serum), the antigen concentration at the maximum P / N value, and the serum dilution were used as the optimal antigen working concentration and serum dilution. The determination results are shown in Table 3: P1, P2, P3, and P4 represent positive samples 1, 2, 3, and 4, respectively; and N1, N2, N3, and N4 represent negative samples 1, 2, 3, and 4, respectively.

[0138] Table 3. Determination of optimal coating concentration of antigen and optimal dilution of serum

[0139]

[0140] By comparing the P / N values under different conditions, the condition with the highest P / N value was selected as the optimal condition. The antigen coating concentration of 0.5 ug / mL and the optimal serum dilution of 1:50 were determined as the optimal reaction conditions.

[0141] (2) Determination of sample incubation time

[0142] Based on the optimal experimental conditions determined above, the sample incubation time was screened. The clinical samples diluted by 1:50 were added to each well, and the reaction was carried out at room temperature in the dark for 20 min, 30 min, 45 min, and 60 min, respectively. ELISA tests were performed on 3 negative sera and 5 positive sera from clinical samples, and the OD 450nm values were determined. The results are shown in Table 4.

[0143] Table 4. Determination of optimal incubation time of samples

[0144]

[0145] The PEDV positive and negative sera were incubated with the antigen plate at 37°C for different times under the optimal antigen coating concentration and serum dilution. The results showed (Table 4) that the average P / N value was the highest (18.60) when the serum was incubated for 30 min, and therefore the optimal incubation time of the serum was determined to be 30 min.

[0146] (3) Determination of optimal dilution of enzyme-labeled secondary antibody and reaction time

[0147] The square matrix titration method was used to determine the optimal enzyme-labeled secondary antibody dilution multiple and action time. Five PEDV positive sera and three PEDV negative sera were selected for detection according to the determined optimal conditions. The HRP-labeled enzyme-labeled secondary antibody goat anti-pig IgA (KPL company) was diluted according to 1:5000, 1:10000, 1:15000, and then acted at 37°C for 30 min, 45 min, and 60 min, respectively. After the detection was completed, the average P / N value was calculated, and the optimal enzyme-labeled secondary antibody dilution multiple and action time were determined according to the size of the average P / N value.

[0148] Table 5. Determination of optimal secondary antibody dilution multiple and action time

[0149]

[0150]

[0151] The results showed (Table 5) that when the enzyme-labeled secondary antibody was diluted 10000 times and the action time was 30 min, the average P / N value was the highest (23.91), so the optimal dilution multiple of the enzyme-labeled secondary antibody was determined to be 10000 times, and the action time was 30 min.

[0152] (4) Determination of optimal substrate action time

[0153] According to the optimal antigen coating concentration determined in the above experiment, the antigen plate was prepared, 5 PEDV positive sera and 3 PEDV negative sera were selected for incubation according to the optimal serum dilution multiple and serum incubation time, and the enzyme-labeled secondary antibody and action time were performed according to the optimal conditions. TMB (tetramethyl benzene diamine) was used as the substrate to act for 10 min, 15 min and 20 min, respectively. After the reaction was terminated, the reading was performed at OD 450nm wavelength, the average P / N value was calculated, and the optimal substrate action time was determined according to the size of the average P / N value.

[0154] Table 6. Determination of optimal substrate action time

[0155]

[0156] The results showed (Table 6) that when the substrate acted for 15 min, the average P / N value was the highest (19.59), so the optimal substrate action time was determined to be 15 min.

[0157] (5) Determination of ELISA critical value

[0158] After the ELISA reaction conditions were determined, 49 serum samples that were negative for antibody detection by the commercial IDEXX PEDV IgA antibody detection kit were selected, and negative and positive control wells were set. Then, under the optimal working conditions described above, ELISA detection was performed, and the average S / P value (S / P value = (sample detection OD 450nm value - negative control serum OD 450nm value) / (positive control serum OD 450nm - negative control serum OD 450nm value) and the standard deviation s value of the 49 negative sera were calculated, and the cut-off value was calculated according to the formula cut-off value = (average S / P value of negative samples + 3 x s value) to determine the result determination standard. The average S / P value of the negative sera was calculated to be 0.0515, and the standard deviation was 0.0518. Therefore, the detection cut-off value was set to 0.207. That is, when the S / P value of the sample to be tested is ≥ 0.207, it is determined to be positive, and when the S / P value is < 0.207, it is determined to be negative.

[0159] Example 3. Assembly of an ELISA kit for detecting PEDV IgA

[0160] The porcine epidemic diarrhea virus S1 recombinant protein IgA antibody indirect ELISA kit, abbreviated as PEDV-S1-iELISA kit, was assembled according to the following ingredients.

[0161] (1) Antibody detection plate strip: each kit contains 2 ELISA plates, each plate contains a detachable ELISA plate strip coated with detection antigen, with a specification of 8 holes x 12 strips.

[0162] Preparation of ELISA plate strip (antigen plate): The purified PEDV S1 recombinant protein obtained in Example 1 was diluted with 0.05 M carbonate buffer (pH 9.6), and the optimal coating concentration was determined to be 0.5 ug / mL using a square array method. 100 uL was added to each well of a detachable 96-well enzyme-labeled plate, and after coating at 4°C for 24 hours, it was washed twice with PBS (pH 7.4) containing 0.05% Tween-20 (volume ratio) (PBST), blocked at 37°C for 1 hour with 5% skim milk (mass / volume ratio), and washed thoroughly with PBST, and air-dried at room temperature.

[0163] (2) Negative and positive control sera (1.5 mL each):

[0164] a. Negative control serum: healthy piglet serum that has not been vaccinated and is PEDV negative by both fluorescent PCR and ELISA detection;

[0165] b. Positive control serum: PEDV antibody strongly positive serum (inactivated) was diluted to the working concentration according to the proportion with diluent.

[0166] The negative serum is the serum of healthy pigs without PEDV infection, and the positive serum is the serum collected from pigs after inoculation with PEDV virus.

[0167] (3) Sample diluent (60 mL): universal diluent from Changsha Jyei Biological Technology Co., Ltd., product number: SEZH01.

[0168] (4) Enzyme-labeled secondary antibody IgA (15 mL): goat anti-pig IgA (KPL company) labeled with horseradish peroxidase (KPL company) was diluted 10,000 times with 1% BSA-containing PBST as the enzyme-labeled secondary antibody with a direct use concentration.

[0169] (5) Washing solution (75 mL): 10-fold concentrated 1M PBS (pH 7.4) containing 0.5% Tween-20.

[0170] (6) Substrate developing solution (15 mL): 200 mg of tetramethyl benzidine (TMB) was weighed, dissolved in 100 mL of anhydrous ethanol or DMSO, and then made up to 1000 mL with double distilled water to prepare developing solution A. 9.33 g of citric acid, 14.6 g of sodium hydrogen phosphate (Na2HPO4·12H2O), and 6.4 mL of 0.75% hydrogen peroxide urea were mixed, the pH value was adjusted to 5.0-5.4, and then made up to 1000 mL with double distilled water to prepare developing solution B. Equal volumes of developing solution A and B were mixed to obtain the substrate developing solution.

[0171] (7) Stop solution (15 mL): 2 mol / L H2SO4.

[0172] Example 4. Evaluation of the detection effect of the PEDV-S1-iELISA kit

[0173] (1) Specificity experiment

[0174] The 92 clinical samples which were detected as negative for porcine epidemic diarrhea virus antibody by the commercial IDEXX PEDV IgA kit (including the following samples: porcine fever positive serum, porcine reproductive and respiratory syndrome positive serum, porcine circovirus type 2 positive serum, pseudorabies positive serum) and 96 clinical samples which were detected as positive for porcine epidemic diarrhea virus antibody by the commercial IDEXX PEDV IgA kit were detected by the PEDV-S1-iELISA kit assembled in Example 3 under the optimal detection conditions determined in Example 2. The detection results of the PEDV-S1-iELISA kit are as follows (Table 7): 89 of the 92 negative sera were detected as negative, and 3 were detected as positive; 94 of the 96 porcine epidemic diarrhea virus positive sera were detected as positive, and 2 were detected as negative. Therefore, the specificity was 96.7%, and the sensitivity was 97.9%. At the same time, the porcine fever virus (CSFV) antibody positive serum, the porcine reproductive and respiratory syndrome virus (PRRSV) antibody positive serum, the porcine circovirus 2 (PCV2) antibody positive serum, and the pseudorabies virus (PRV) antibody positive serum were all negative, indicating that the method has good specificity (Table 8).

[0175] Table 7. Comparison of detection results of PEDV-S1-iELISA kit and commercial kit

[0176]

[0177] Table 8. Detection results of serum cross reaction

[0178]

[0179] (2) Sensitivity test

[0180] Under the optimal detection conditions determined by the PEDV-S1-iELISA kit assembled in Example 3, one porcine milk sample which was detected as positive for porcine epidemic diarrhea virus antibody by the IDEXX PEDV IgA kit was diluted by 2 times successively from 1:500, and the OD 450nm and the S / P value was calculated, and the results are shown in Table 9: when the dilution of the positive milk sample was 512000 times, the S / P value detected by the PEDV-S1-iELISA kit was greater than 0.207, and it was still positive; indicating that the detection method provided by the present disclosure has high sensitivity.

[0181] Table 9. Sensitivity test

[0182]

[0183] (3) Reproducibility test

[0184] Seven porcine clinical serum samples, both positive and negative for porcine epidemic diarrhea virus (PEDV) antibodies as detected by the IDEXX PEDVIgA kit, were selected. Intra-batch reproducibility was evaluated by performing three tests on antigen-coated plates from the same batch of the PEDV-S1-iELISA kit; inter-batch reproducibility was evaluated by performing tests on antigen-coated plates from three different batches. The mean OD of each serum sample was calculated. 450nm value and standard deviation (SD), coefficient of variation

[0185] The test results are shown in Table 10. Intra-batch variation analysis showed that the coefficients of variation for the seven serum samples ranged from 0.16% to 6.88%, with an average of 2.87%. Inter-batch variation analysis showed that the coefficients of variation ranged from 0.78% to 11.36%, with an average of 4.83%. These results indicate that the coefficients of variation were <15% for both intra-batch and inter-batch repeated tests, demonstrating that this kit exhibits good intra-batch and inter-batch repeatability.

[0186] Table 10. Results of PEDV-S1-ELISA repeatability tests

[0187]

[0188] (4) Stability test

[0189] Different batches of PEDV-S1-iELISA kits were prepared. One batch of PEDV-S1-iELISA kit was randomly selected and placed in a 37℃ incubator for a 15-day thermostability test. The results were compared with those of kits stored at 4℃. The degradation rate of OD values ​​of positive control and clinical samples on day 4 and day 15 was calculated, as shown in Tables 11 and 12.

[0190] Table 11. Degradation rate of PEDV-S1-iELISA kit on day 4 of stability test

[0191] Sample No. 4℃ 37℃ Degradation rate Positive control 1.302 1.158 11.060％ Negative sample 0.054 0.057 -5.556％ Clinical sample 1 3.234 2.943 8.998％ Clinical sample 2 3.37 3.074 8.783％ Clinical sample 3 1.7 1.427 16.059％ Clinical sample 4 1.754 1.622 7.526％ Clinical sample 5 0.357 0.365 -2.241％ Clinical sample 6 0.736 0.661 10.190％ Clinical sample 7 0.182 0.181 0.549％

[0192] Table 12. Degradation rate of PEDV-S1-iELISA kit on day 15 of stability test

[0193] Sample No. 4℃ 37℃ Degradation rate Positive control 1.283 1.078 15.978％ Negative sample 0.051 0.052 -1.961％ Clinical sample 1 3.084 2.385 22.665％ Clinical sample 2 3.067 2.516 17.965％ Clinical sample 3 1.44 1.107 23.497％ Clinical sample 4 1.457 1.156 20.659％ Clinical sample 5 0.293 0.295 -0.683％ Clinical sample 6 0.651 0.459 29.493％ Clinical sample 7 0.178 0.162 8.989％

[0194] Stability assays of the PEDV-S1-iELISA kit on days 4 and 15, and OD values ​​of positive control and clinical samples. 450nm The degradation rate is less than 30%. According to the thermal stability test, the test kit can be stored at 4℃ for more than 1 year.

[0195] Example 5. PEDV-S1-iELISA kit for detection of oral swab samples

[0196] PEDV-S1-iELISA kit assembled in Example 3 was used to detect PEDV antibody levels in oral swabs of vaccinated but uninfected pigs, naturally infected pigs, and negative pigs. When S / P value was ≥0.207, it was determined as positive, and when S / P value was <0.207, it was determined as negative. The results showed (Table 13) that the average S / P value of the vaccinated but uninfected group was 0.862, and the positive rate was 60%; the average S / P value of the naturally infected group was 3.293, and the positive rate was 100%; the average S / P value of the negative pig group was 0.050, and the positive rate was 0. IgA antibodies in oral swabs could be detected, and the IgA antibody level in oral swabs of naturally infected pigs was significantly higher than that of vaccinated pigs.

[0197] Table 13. IgA antibody levels in oral swabs of pigs with different immune backgrounds

[0198]

[0199] Example 6. PEDV-S1-iELISA kit for detection of anal swab samples

[0200] PEDV-S1-iELISA kit assembled in Example 3 was used to detect PEDV antibody levels in anal swabs of vaccinated but uninfected pigs, naturally infected pigs, and negative pigs. When S / P value was ≥0.207, it was determined as positive, and when S / P value was <0.207, it was determined as negative. The results showed (Table 14) that the average S / P value of the vaccinated but uninfected group was 0.095, and the positive rate was 10%; the average S / P value of the naturally infected group was 2.639, and the positive rate was 100%; the average S / P value of the negative pig group was 0.048, and the positive rate was 0. PEDV-S1-iELISA kit could detect IgA antibodies in anal swabs, and the IgA antibody level in anal swabs of naturally infected pigs was significantly higher than that of vaccinated pigs.

[0201] Table 14. IgA antibody levels in anal swabs of pigs with different immune backgrounds

[0202]

[0203] Example 7. PEDV-S1-iELISA kit for detection of serum samples

[0204] The PEDV-S1-iELISA kit assembled in Example 3 was used to simultaneously detect PEDV antibody levels in the serum of vaccinated but uninfected pigs, naturally infected pigs, and negative-positive pigs. A positive result was defined as an S / P value ≥ 0.207, and a negative result as an S / P value < 0.207. The results (Table 15) showed that the average S / P value in the vaccinated but uninfected group was 0.511, with a positive rate of 35%; the average S / P value in the naturally infected group was 3.921, with a positive rate of 100%; and the average S / P value in the negative-positive group was 0.012, with a positive rate of 0%. The PEDV-S1-iELISA kit can detect IgA antibodies in serum, and the IgA antibody levels in the serum of naturally infected pigs were significantly higher than those in vaccinated pigs.

[0205] Table 15. IgA antibody levels in porcine serum under different immune backgrounds

[0206]

[0207]

[0208] Example 8. PEDV-S1-iELISA kit for colostrum sample detection

[0209] The PEDV-S1-iELISA kit assembled in Example 3 was used to simultaneously detect PEDV antibody levels in colostrum from vaccinated but uninfected pigs, naturally infected pigs, and negative-positive pigs. A positive result was defined as an S / P value ≥ 0.207, and a negative result as an S / P value < 0.207. The results (Table 16) showed that the average S / P value in the vaccinated but uninfected group was 1.636, with a positive rate of 95%; the average S / P value in the naturally infected group was 4.258, with a positive rate of 100%; and the average S / P value in the negative-positive group was 0.067, with a positive rate of 0%. The PEDV-S1-iELISA kit can detect IgA antibodies in colostrum, and the results indicate that the serum IgA antibody level in naturally infected pigs was significantly higher than that in vaccinated pigs.

[0210] Table 16. IgA antibody levels in porcine colostrum under different immune backgrounds

[0211]

[0212] The PEDV-S1-iELISA kit and detection method provided in this disclosure can distinguish between naturally infected pig herds and vaccinated but uninfected pig herds. It can detect IgA antibodies in pig serum, oral swabs, anal swabs, and colostrum samples. According to the results analysis, the PEDV IgA antibody level in naturally infected pig herds is much higher than that in PEDV-vaccinated pig herds.

[0213] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, Its nucleotide sequence encodes the recombinant protein as described in claim 1.

3. A recombinant vector, characterized in that, It includes the nucleic acid molecules as described in claim 2.

4. A host cell, characterized in that, It comprises the nucleic acid molecule as described in claim 2, or the recombinant vector as described in claim 3.

5. The host cell according to claim 4, characterized in that, The cells include eukaryotic or prokaryotic cells.

6. The host cell according to claim 5, characterized in that, The prokaryotic cells include bacteria.

7. The host cell according to claim 6, characterized in that, The bacteria include Escherichia coli.

8. The host cell according to claim 5, characterized in that, The eukaryotic cells include one or more of yeast, insect, plant, or mammalian cells.

9. The host cell according to claim 8, characterized in that, The insect cells include Lepidoptera insect cells.

10. A kit for detecting anti-PEDV immunoglobulin, characterized in that, The kit comprises the recombinant protein of claim 1.

11. The reagent kit according to claim 10, characterized in that, The kit also includes a marker-labeled secondary antibody, a substrate, and / or a control.

12. The reagent kit according to claim 10, characterized in that, The kit also includes sample diluent, washing solution and / or stop solution.

13. The kit according to claim 11, characterized in that, The markers include enzymes.

14. The kit according to claim 13, characterized in that, The enzymes include horseradish peroxidase or alkaline phosphatase.

15. The kit according to claim 11, characterized in that, The substrates include tetramethylbenzidine or 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid.

16. The kit according to claim 11, characterized in that, The reference standards include positive and / or negative controls.

17. The kit according to claim 10, characterized in that, The immunoglobulins include one or more of the following types: IgA, IgD, IgE, IgG, and IgM.

18. The use of the recombinant protein as described in claim 1 or the nucleic acid molecule as described in claim 2 in any one or more of the following (1) to (3): (1) Preparation of PEDV vaccine; (2) Preparation of anti-PEDV immunoglobulin detection kit; (3) Prepare drugs for the prevention or treatment of PEDV infection.

19. The application according to claim 18, characterized in that, The kit is used to detect anti-PEDV immunoglobulin in the sample to be tested.

20. The application according to claim 19, characterized in that, The samples to be tested include bodily fluid samples from pigs.

21. The application according to claim 20, characterized in that, The bodily fluid samples of the pig include one or more of the following: plasma, serum, milk, nasopharyngeal swabs, oral swabs, feces, anal swabs, or their processed products.

22. The application according to claim 20, characterized in that, The bodily fluid samples from the pigs included blood.

23. The application according to claim 20, characterized in that, The bodily fluid samples from the pig include one or more of the following: oral swabs, anal swabs, blood, or milk.

24. The application according to claim 20, characterized in that, The bodily fluid sample from the pig was colostrum.

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