Porcine epidemic diarrhea virus S1 recombinant protein, kit and application thereof

Abstract

The invention provides a porcine epidemic diarrhea virus S1 recombinant protein, a kit and application of the porcine epidemic diarrhea virus S1 recombinant protein, and belongs to the technical field of veterinary biology. The recombinant protein disclosed by the invention has good antigenicity, hydrophilicity and stability. The anti-PEDV immunoglobulin antibody detection kit prepared from the recombinant protein provided by the invention has the advantages of high specificity and sensitivity, no cross reaction, good intra-batch and inter-batch repeatability, strong thermal stability and the like. And the kit can be used for detecting various samples such as oral swabs, anus swabs, serum or colostrum and the like, and can be used for distinguishing naturally infected swines from vaccine-immunized swines. The recombinant protein and the ELISA kit provided by the invention provide a good platform for diagnosis of porcine epidemic diarrhea and research and development of vaccines and therapeutic drugs, and have important application value.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biotechnology, and in particular relates to a porcine epidemic diarrhea virus S1 recombinant protein, a kit and applications thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by infection with porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting, and dehydration. The mortality rate of suckling piglets infected with PEDV is as high as 100%, and it can be infected in all age groups. The new highly virulent PEDV strain that spread rapidly in my country has caused the death of a large number of piglets, with a mortality rate close to 100%, causing huge economic losses to my country's pig industry.

[0003] 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, has good specificity, and can perform high-throughput detection. It is a commonly used serological antibody detection method. At present, there is no effective treatment for the onset of PED, and passive immunity conferred by maternal antibodies needs to be obtained through colostrum. 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 intestines and other mucosal tissues of piglets from invasion by pathogenic microorganisms.

[0004] The existing anti-PEDV immunoglobulin test kit can only detect anti-PEDV immunoglobulin in pig serum and colostrum, and requires the collection of pig blood and sow colostrum samples for testing. In the context of the African swine fever epidemic, blood sampling is not conducive to the prevention and control of African swine fever, and for colostrum testing, even if the antibody level is low, there is no time to adjust the immunization program to change the antibody level in colostrum, and the results have a lagging guiding role in PED prevention and control. Summary of the invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a recombinant protein, a kit and applications thereof.

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

[0007] A first fragment having an amino acid sequence as shown 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 shown 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 shown in SEQ ID NO: 4, or an amino acid sequence having at least 60% sequence identity thereto;

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

[0011] The fifth fragment has the amino acid sequence shown 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 connected by a connecting peptide.

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

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

[0015] In some embodiments, the connecting peptides between the first fragment, the second fragment, the third fragment, the fourth fragment and / or the fifth fragment are the same or different.

[0016] In some embodiments, the recombinant protein comprises 1 to 10 copies of any one 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 the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 60% sequence identity thereto and retaining the immunogenicity of the PEDV S1 protein.

[0019] According to a second aspect of the present disclosure, a nucleic acid molecule is provided, the nucleotide sequence of which can encode the recombinant protein described in the first aspect.

[0020] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown 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, a recombinant vector is provided, which includes the nucleic acid molecule described in the second aspect.

[0022] In some embodiments, the vector includes, but is not limited to, one or more of the following: pFastBac1 plasmid, pGEX-4T2 plasmid, pVL1393 plasmid, and psPAX2 plasmid.

[0023] According to a fourth aspect of the present disclosure, a host cell is provided, which comprises the nucleic acid molecule described in the second aspect or the recombinant vector described in 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 bacteria comprises Escherichia Coli.

[0027] In some embodiments, the Escherichia coli includes, but is not limited to, one or more of DH10Bac cells, BL21 (DE3) cells, BW25113 cells, MG1655 cells, W3110 cells or W cells.

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

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

[0030] In some embodiments, the insects include but are not limited to one or more of Bombyx mori Linnaeus, Mamestra brassicae Linnaeus, Spodopterafrugiperda, Trichoplusia ni, and trichogrammatid.

[0031] In some embodiments, the insect cells include, but are not limited to, one or more of sf9 insect cells, sf21 insect cells, SF+ cell lines, or High Five cells.

[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 described in the first aspect.

[0033] In some embodiments, the kit further comprises a secondary antibody against PEDV immunoglobulin labeled with a marker and a substrate and / or a control substance.

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

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

[0036] In some embodiments, the recombinant protein is immobilized on the ELISA plate using a square array method.

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

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

[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'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS).

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

[0043] In some embodiments, the sample diluent includes one or more of serum protein, a surfactant, and a 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 albumin.

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

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

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

[0050] In some embodiments, the Tween includes 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 binding in a sample to be tested using the kit described in the fifth aspect is provided, the method comprising the following steps:

[0056] b1, adding the sample to be tested to the ELISA plate coated with the recombinant protein to react;

[0057] b2, adding anti-PEDV immunoglobulin labeled with a marker to react and obtain a reactant;

[0058] b3, the reactant reacts with the substrate to generate a signal, and the presence of the anti-PEDV immunoglobulin in the sample to be tested is determined based on 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 includes adding the sample to be tested, the positive control substance, and the negative control substance to the ELISA plate coated with the recombinant protein for reaction.

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

[0061] In some embodiments, the dilution multiples of the test sample, positive control, and negative control are 50 to 200 times.

[0062] In some embodiments, the test sample, positive control, and negative control need to be diluted first, and the dilution multiple 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, 6、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 6, 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 times.

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

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

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

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

[0067] In some embodiments, the dilution factor of the anti-PEDV immunoglobulin labeled with the marker in step b2 is 5000 to 15000 times.

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

[0069] In some embodiments, the reaction temperature in step b2 is 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, and 45°C.

[0071] In some embodiments, the reaction time in step b2 is 30 to 60 minutes.

[0072] In some embodiments, the reaction time 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 reacting with the substrate in step b2 is 10 to 30 minutes.

[0074] In some embodiments, the time for acting with the substrate in step b2 includes but is 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 includes a body fluid sample from a pig.

[0076] In some embodiments, the bodily fluid sample of the pig includes one or more of blood, plasma, serum, milk, nasopharyngeal swab, oral swab, feces, anal swab, or processed materials thereof.

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

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

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

[0080] (1) preparing PEDV vaccines;

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

[0082] (3) Preparation of therapeutic drugs for preventing or treating PEDV infection (e.g. porcine epidemic diarrhea).

[0083] Beneficial effects of the present disclosure:

[0084] The present invention 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 by using the recombinant protein provided by the present disclosure has the advantages of high specificity and high sensitivity, no cross-reaction, good intra-batch and inter-batch repeatability, strong thermal stability, etc. It 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 vaccine-immunized pigs. It provides a good platform for the detection of porcine epidemic diarrhea, as well as the research and development of vaccines and therapeutic drugs, and has important application value. DETAILED DESCRIPTION

[0086] The present invention has carried out a large number of sequence comparisons and analyses on the PEDV S1 protein sequences in the database, and selected target fragments with good antigenicity and hydrophilicity, as shown in Table 1.

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

[0088]

[0089]

[0090] The fragments described in Table 1 were connected using flexible amino acids to construct recombinant proteins, so that they have good antigenicity, hydrophilicity and stability.

[0091] The recombinant protein is used to prepare a kit for detecting anti-PEDV immunoglobulin, and the kit also includes 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 stop solution or a control substance.

[0093] Using the kit to detect anti-PEDV immunoglobulin in the sample to be tested includes the following steps:

[0094] b1, adding the sample to be tested to the ELISA plate coated with the recombinant protein to react;

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

[0096] b3, and then react with the substrate to develop color to determine whether anti-PEDV immunoglobulin exists in the sample, wherein the signal intensity is related to the concentration of PEDV immunoglobulin in the sample to be tested.

[0097] In some embodiments, the step b1 includes mixing the sample to be tested, the positive control substance, and the negative control substance with the recombinant protein for reaction.

[0098] In some embodiments, the determination method includes comparing the OD value with a threshold value, and determining it as positive when the signal value is ≥ the threshold value, and determining it 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 is selected from 0.2 to 0.5.

[0103] In some embodiments, the threshold 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] definition

[0105] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

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

[0107] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0108] In this article, the term "Porcine epidemic diarrhea virus (PEDV)" has a gene size of about 28kb, with cap structures (Cap) and Poly (A) tails at both ends. The genome contains 4 structural proteins (S protein, E protein, M protein, N protein) and 3 non-structural proteins (replicase 1a, replicase 1b and ORF3), ORF3 encodes a product of unknown function and polymorphism. S is the largest structural protein, containing neutralizing antibody epitopes and specific receptor binding sites for virus entry.

[0109] As used herein, the term "nucleic acid" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0110] Herein, the term "expression" refers to the transcription and / or translation of foreign genes in a host.

[0111] In this article, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides are equally applicable to describing peptides and describing proteins and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. 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 linked via covalent peptide bonds.

[0112] As used herein, the term "recombinant protein" refers to a protein molecule, such as a target protein, expressed using a recombinant DNA molecule, which is foreign to the cell in which the polypeptide is produced.

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

[0114] As used herein, the term "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may 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 shared by the sequences within a comparison window, taking into account 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 present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides are counted and nucleotides from the reference sequence are not counted, gaps present in the reference sequence are not counted. At least 60% sequence identity includes a contiguous segment having 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% sequence identity over the entire length of the sequence.

[0116] Methods for comparing sequences are well known in the art and can be, for example, performed using existing software such as BLASR, DALIGN, BLAST, BLAST-2, ALIGN, ALIGN-2BWA, BOWTIE or Megalign (DNASTAR).

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

[0118] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. The actual protection scope of the present invention is set forth in the claims. In the following description, the description of 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 examples are not mentioned in terms of their sources, and are all commercially available on the market or obtained by conventional methods known to those skilled in the art.

[0119] Example

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

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

[0122] (1) Construction of recombinant expression plasmid: The present invention has conducted a large number of sequence comparisons on the S1 protein sequence in the database, selected the target fragment with good antigenicity and hydrophilicity, and carried out truncated expression and repeated expression. The peptide amino acid sequence is shown in SEQ ID NO: 2 to 6, and the peptides are connected by flexible amino acids (amino acid sequence is shown in SEQ ID NO: 7). By comparing and detecting with the full-length S1 protein, the S1 recombinant protein with good antigenicity, hydrophilicity and stability (amino acid sequence is shown in SEQ ID NO: 1) was finally screened. The sequence is shown in Table 1.

[0123] (2) Preparation of recombinant protein bacmid:

[0124] The coding nucleic acid sequences of S1 full-length protein and S1 recombinant protein (with histidine tag 6×His, the nucleic acid sequence is shown in SEQ ID NO:8) were inserted into the pFastBac1 plasmid through the restriction enzyme cutting sites BamHI and SacI, respectively, to obtain plasmid pFastBac1-S1 full-length and plasmid pFastBac1-S1 recombinant. The plasmid pFastBac1-S1 full-length and plasmid pFastBac1-S1 recombinant were transformed into MAX DH10Bac TM In competent cells A, recombinant bacmids containing nucleic acids encoding the full-length S1 protein and the recombinant protein are generated respectively.

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

[0126] (4) Expression of S1 full-length protein and S1 recombinant protein: refer to Baculovirus expression system operation manual, the above two recombinant baculoviruses were inoculated into Sf9 cells and cultured for two generations. The virus fluids were collected and inoculated into High Five TM The cells expressed the full-length S1 protein and the recombinant S1 protein.

[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+ Purification by affinity chromatography.

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

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

[0130] (7) The detection effects of PEDV S1 recombinant protein and S1 full-length protein on PEDVIgA were compared. The detection effect of the protein was evaluated based on the principle of maximizing the average P / N value. The specific steps were as follows: the purified PEDV S1 recombinant protein and S1 full-length protein were diluted to 1 ug / mL with carbonate buffer (pH 9.6), and 100 uL per well was added to the ELISA plate and coated at 4°C for 24 h; washed twice with PBS (pH 7.4) washing solution (PBST) containing 0.05% Tween-20 (volume ratio), blocked with blocking solution (Changsha Jingyue Biotechnology) at 37°C for 1 h, washed thoroughly with PBST, and air-dried at room temperature. Four copies of porcine epidemic diarrhea virus positive serum and negative serum from clinical samples were diluted at 1:50, and 100uL was added to each well of the ELISA plate and incubated at 37°C for 30min; washed 4 times with PBS (pH7.4) washing solution (PBST) containing 0.05% Tween-20 (volume ratio) and patted dry, and HRP-labeled sheep anti-swine IgA was diluted at 1:10000 and added to each well of the ELISA plate, incubated at 37°C for 30min; incubated at 37°C for 30min; washed 4 times with PBS (pH7.4) washing solution (PBST) containing 0.05% Tween-20 (volume ratio) and patted dry, and 50uL of TMB was added to each well for color development in the dark for 15min, and 2mol / LH2SO4 was used to terminate the reaction, and the OD was measured. 450nm The average P / N value (ratio of positive serum to negative serum) was calculated. The results are shown in Table 2: P1, P2, P3, P4 represent positive samples 1, 2, 3, 4 respectively; N1, N2, N3, N4 represent negative samples 1, 2, 3, 4 respectively.

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

[0132]

[0133] As shown in Table 2, by analyzing the test results after coating with different proteins, it was determined that PEDVS1 recombinant protein was an antigen with better detection effect than S1 full-length protein, so it was determined as the coating antigen for subsequent experiments.

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

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

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

[0137] The purified PEDV S1 recombinant protein was diluted to 0.25, 0.5, and 1ug / mL with carbonate buffer (pH 9.6) according to the chessboard method, and then 100uL was added to each well of the ELISA plate and coated at 4°C for 24h. Four samples of porcine epidemic diarrhea virus positive and negative sera from clinical samples were diluted at 1:50, 1:100, and 1:200, respectively, and then the square array titration was performed; HRP-labeled sheep anti-swine IgA was diluted at 1:10000, and TMB was used to develop color in the dark for 15min, and 2mol / LH2SO4 was used to terminate the reaction, and the OD was measured. 450nm The antigen concentration and serum dilution when the P / N value (ratio of positive serum to negative serum) is the largest are taken as the optimal antigen working concentration and serum dilution. The results are shown in Table 3: P1, P2, P3, P4 represent positive samples 1, 2, 3, 4 respectively; N1, N2, N3, N4 represent negative samples 1, 2, 3, 4 respectively.

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

[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 0.5ug / mL and the optimal serum dilution of 1:50 were determined to be 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. A 1:50 diluted clinical sample was added to each well and reacted at room temperature in the dark for 20 min, 30 min, 45 min, and 60 min. ELISA tests were performed on 3 negative sera and 5 positive sera from clinical samples to determine the OD 450nm The results are shown in Table 4.

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

[0144]

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

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

[0147] The square array 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 sheep anti-swine IgA (KPL Company) was diluted at 1:5000, 1:10000, and 1:15000 and then acted at 37°C for 30min, 45min, and 60min, respectively. After the detection, the average P / N value was calculated, and the optimal enzyme-labeled secondary antibody dilution multiple and action time were determined according to the average P / N value.

[0148] Table 5. Determination of the 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 10,000 times and the action time was 30 minutes, the average P / N value was the highest (23.91). Therefore, the optimal dilution of the enzyme-labeled secondary antibody was determined to be 10,000 times and the action time was 30 minutes.

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

[0153] According to the optimal antigen coating concentration determined in the above test, the antigen plate was prepared, and 5 PEDV-positive sera and 3 PEDV-negative sera were selected for incubation according to the optimal serum dilution multiple and serum incubation time. The enzyme-labeled secondary antibody and the action time were carried out according to the determined optimal conditions. TMB (tetramethylbenzenediamine) was used as the substrate for 10 min, 15 min and 20 min respectively. After the reaction was terminated, the OD 450nm Read the values ​​at the wavelength, calculate the average P / N value, and determine the optimal substrate action time based on the average P / N value.

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

[0155]

[0156] The results showed (Table 6) that the average P / N value was the highest (19.59) when the substrate was exposed for 15 min, so the optimal substrate exposure 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 antibodies detected by the commercial IDEXX PEDVIgA antibody detection kit were selected, and positive and negative control wells were set. Then, according to the above optimal working conditions, ELISA detection was performed to calculate the average S / P value of the 49 negative sera (S / P value = (sample detection OD 450nm Value-negative control serum OD 450nm value) / (positive control serum OD 450nm - Negative control serum OD 450nm The critical value is calculated according to the formula critical value = (average S / P value of negative samples + 3 × s value) to determine the result judgment standard. The average S / P value of negative serum is calculated to be 0.0515 and the standard deviation is 0.0518. Therefore, the critical value of the test is set to 0.207. That is, when the S / P value of the sample to be tested is ≥0.207, it is judged as positive, and when the S / P value is <0.207, it is judged as negative.

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

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

[0161] (1) Antibody detection strips: Each kit contains 2 ELISA plates, each of which contains a removable ELISA strip coated with the detection antigen, with a specification of 8 wells x 12 strips.

[0162] Preparation of ELISA strips (antigen plates): The purified PEDV S1 recombinant protein obtained in Example 1 was diluted with 0.05M carbonate buffer (pH9.6), and the optimal coating concentration was determined to be 0.5ug / mL by the square array method. A detachable 96-well ELISA plate was taken, 100uL was added to each well, and after coating at 4°C for 24 hours, it was washed twice with PBS (pH7.4) washing solution (PBST) containing 0.05% Tween-20 (volume ratio), blocked with 5% skimmed milk powder (mass volume ratio) at 37°C for 1 hour, washed thoroughly with PBST, and air-dried at room temperature.

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

[0164] a. Negative control serum: serum from healthy piglets that have not been vaccinated and are PEDV negative in both fluorescent PCR and ELISA tests;

[0165] b. Positive control serum: Dilute the PEDV antibody positive serum (inactivated) to the working concentration in proportion with diluent.

[0166] Negative serum refers to the serum from healthy pigs that are not infected with PEDV, and positive serum refers to the serum from pigs collected after being inoculated with PEDV virus.

[0167] (3) Sample diluent (60 mL): general diluent of Changsha Jingyue Biotechnology Co., Ltd., catalog number: SEZH01.

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

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

[0170] (6) Substrate colorimetric solution (15 mL): Weigh 200 mg of tetramethylbenzidine (TMB), dissolve it in 100 mL of anhydrous ethanol or DMSO, and dilute to 1000 mL with double distilled water to prepare colorimetric solution A. Weigh 9.33 g of citric acid, 14.6 g of disodium hydrogen phosphate (Na2HPO4·12H2O), and 6.4 mL of 0.75% urea hydrogen peroxide, mix well, adjust the pH to 5.0-5.4, and dilute to 1000 mL with double distilled water to prepare colorimetric solution B. Mix equal volumes of colorimetric solutions A and B to obtain the substrate colorimetric solution.

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

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

[0173] (1) Specificity experiment

[0174] According to the optimal detection conditions determined in Example 2, the PEDV-S1-iELISA kit assembled in Example 3 was used to test 92 clinical samples that were negative for porcine epidemic diarrhea virus antibodies detected by the commercialized IDEXX PEDVIgA kit (wherein the porcine epidemic diarrhea virus negative samples include: swine fever positive serum, porcine reproductive and respiratory syndrome positive serum, porcine circovirus type 2 positive serum, pseudorabies positive serum) and 96 clinical samples that were positive for porcine epidemic diarrhea virus antibodies detected by the IDEXX PEDVIgA kit. The test results of the PEDV-S1-iELISA kit are as follows (Table 7): 89 of the 92 negative sera were tested negative and 3 were tested positive; 94 of the 96 porcine epidemic diarrhea virus positive sera were tested positive and 2 were tested negative. It can be seen that the specificity is 96.7% and the sensitivity is 97.9%. At the same time, the results of sera positive for classical swine fever virus (CSFV) antibody, porcine reproductive and respiratory syndrome virus (PRRSV) antibody, porcine circovirus 2 (PCV2) antibody, and pseudorabies virus (PRV) antibody were all negative, indicating that the method had good specificity (Table 8).

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

[0176]

[0177] Table 8. Serum cross-reaction test results

[0178]

[0179] (2) Sensitivity test

[0180] Using the optimal detection conditions determined by the PEDV-S1-iELISA kit assembled in Example 3, a pig milk sample that has been tested as positive for porcine epidemic diarrhea virus antibodies by the IDEXX PEDVIgA kit was diluted 2 times from 1:500 to measure the sample OD 450nm The S / P value was calculated, and the results are shown in Table 9: When the dilution of the positive milk sample was 512,000 times, the S / P value detected by the PEDV-S1-i ELISA kit was greater than 0.207, which was still positive, indicating that the detection method provided by the present disclosure has high sensitivity.

[0181] Table 9. Sensitivity test

[0182]

[0183] (3) Repeatability test

[0184] A total of 7 porcine clinical serum samples that were positive and negative for porcine epidemic diarrhea virus antibodies detected by the IDEXX PEDVIgA kit were selected and tested three times on the antigen-coated plates of the same batch of PEDV-S1-iELISA kits to evaluate intra-batch repeatability; the tests were performed on three antigen-coated plates from different batches to evaluate inter-batch repeatability. The average OD of each serum sample was calculated separately. 450nm value and standard deviation (SD), coefficient of variation

[0185] The test results are shown in Table 10. Intra-batch difference analysis showed that the coefficient of variation of the 7 sera was 0.16% to 6.88%, with an average of 2.87%. Inter-batch difference analysis showed that the coefficient of variation was 0.78% to 11.36%, with an average of 4.83%. The results show that the coefficient of variation is <15% for both intra-batch and inter-batch repeated tests, indicating that this kit has good intra-batch repeatability and inter-batch repeatability.

[0186] Table 10. PEDV-S1-ELISA repeatability test results

[0187]

[0188] (4) Stability test

[0189] Different batches of PEDV-S1-iELISA kits were prepared, and one batch of PEDV-S1-iELISA kits was randomly selected and placed in a 37°C incubator for a 15-day thermal stability experiment. The results were compared with the kits stored at 4°C, and the degradation rates of the OD values ​​of the positive control and clinical samples on the 4th and 15th days were calculated, as shown in Tables 11 and 12.

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

[0191] Sample No. 4℃ 37℃ Degradation rate Positive Control 1.302 1.158 11.060％ Negative samples 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 on the 15th day of the stability test of PEDV-S1-iELISA kit

[0193] Sample No. 4℃ 37℃ Degradation rate Positive Control 1.283 1.078 15.978％ Negative samples 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 test of PEDV-S1-iELISA kit on day 4 and day 15, OD of positive control and clinical samples 450nm The degradation rate of the value is less than 30%. According to the thermal stability experiment, the detection kit can be stored at 4°C with a shelf life of more than 1 year.

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

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

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

[0198]

[0199] Example 6. Indirect ELISA kit for porcine epidemic diarrhea virus S1 protein IgA antibody detection in anal swab samples

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

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

[0205] Table 15. IgA antibody levels in sera of pigs with different immune backgrounds

[0206]

[0207]

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

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

[0210] Table 16. IgA antibody levels in colostrum from pigs with different immune backgrounds

[0211]

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

[0213] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that The recombinant protein comprises any one or more of the following fragments: A first fragment having an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 60% sequence identity thereto; A second fragment having an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 60% sequence identity thereto; A third fragment having an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 60% sequence identity thereto; A fourth fragment having the amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 60% sequence identity thereto; and / or The fifth fragment has the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 60% sequence identity thereto.

2. The recombinant protein according to claim 1, characterized in that The first fragment, the second fragment, the third fragment, the fourth fragment and / or the fifth fragment are each independently connected by a connecting peptide, Preferably, the connecting peptide comprises a flexible connecting peptide, Preferably, the flexible connecting peptide comprises (G m S t G w ) n , (G n S) m 、(G) n , (EA3K) n or (XP) n , wherein n, m, t, and w are each independently selected from integers of 0 to 5, Preferably, the connecting peptides between the first fragment, the second fragment, the third fragment, the fourth fragment and / or the fifth fragment are the same or different.

3. The recombinant protein according to claim 1, characterized in that The recombinant protein includes 1 to 10 copies of any of the fragments; Preferably, the recombinant protein comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 60% sequence identity thereto and retaining the immunogenicity of the PEDV S1 protein.

4. A nucleic acid molecule, characterized in that The nucleotide sequence encodes the recombinant protein according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that: It comprises the nucleic acid molecule as claimed in claim 4.

6. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4, or the recombinant vector according to claim 5; Preferably, the cell comprises a eukaryotic or prokaryotic cell; Preferably, the prokaryotic cell comprises a bacterium; Preferably, the bacteria comprises Escherichia coli; Preferably, the eukaryotic cell comprises one or more of yeast, insect, plant or mammalian cells; Preferably, the insect cell comprises a Lepidoptera insect cell.

7. A kit for detecting anti-PEDV immunoglobulin, characterized in that: The kit comprises the recombinant protein according to any one of claims 1 to 3.

8. The kit according to claim 7, characterized in that: The kit also includes a secondary antibody labeled with a marker, a substrate and / or a control substance; Preferably, the kit further comprises a sample diluent, a washing solution and / or a stop solution; Preferably, the label comprises an enzyme; Preferably, the enzyme comprises horseradish peroxidase or alkaline phosphatase; Preferably, the substrate comprises tetramethylbenzidine or 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid; Preferably, the control substance includes a positive control substance and / or a negative control substance. Preferably, the immunoglobulin includes one or more of IgA, IgD, IgE, IgG and IgM.

9. A method for detecting anti-PEDV immunoglobulin in a sample using the kit according to claim 7 or 8, Preferably, the sample to be tested comprises a body fluid sample of a pig; Preferably, the pig body fluid sample includes one or more of blood, plasma, serum, milk, nasopharyngeal swab, oral swab, feces, anal swab or processed products thereof; Preferably, the pig body fluid sample includes one or more of a pig's oral swab, anal swab, blood or milk, more preferably colostrum.

10. Use of the recombinant protein according to any one of claims 1 to 3 or the nucleic acid molecule according to claim 4 in any one or more of the following (1) to (3): (1) preparing PEDV vaccines; (2) preparing an anti-PEDV immunoglobulin detection kit; (3) Preparing drugs for preventing or treating PEDV infection.

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