CN112285347B - ELISA detection kit for pathogenic antibodies in pig serum sample - Google Patents
ELISA detection kit for pathogenic antibodies in pig serum sample Download PDFInfo
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- CN112285347B CN112285347B CN202011062115.5A CN202011062115A CN112285347B CN 112285347 B CN112285347 B CN 112285347B CN 202011062115 A CN202011062115 A CN 202011062115A CN 112285347 B CN112285347 B CN 112285347B
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Abstract
The invention relates to a method for capturing antigen II DR molecules (SLA-DR) of swine leukocyte antigen on APCs of swine antigen processing presenting cells by using specific antibodies, eluting antigen peptide presented on SLA-DR by trifluoroacetic acid, carrying out mass spectrometry analysis after desalting treatment and resuspension to determine the amino acid sequence of the antigen peptide, identifying the antigen peptide sequence which can be processed and presented by the antigen presenting cells of organisms in all proteins coded by swine specific pathogens, and carrying out genetic engineering on the CD4 obtained by the method + The T epitope antigen peptide is respectively fused with NanoLuc luciferase, and then is subjected to in vitro recombinant expression and purification by using escherichia coli, and is used as an artificial antigen to carry out ELISA detection on a pig serum sample infected by corresponding pathogen so as to search for CD4 which can be identified by the pig serum sample infected by the pathogen + And (3) the T epitope antigen peptide sequence, and further, the antigen peptide sequences which are positive in ELISA detection of all antibodies are connected in series and then are subjected to recombinant expression in escherichia coli, and the antigen peptide sequence is used as an artificial antigen for specific antibody detection of given pathogen.
Description
Technical field:
the invention belongs to the technical field of biology, and particularly relates to a pathogenic antibody ELISA detection kit in pig serum samples, in particular to a method for separating and identifying PRRSV antigen peptide and application thereof, and particularly for detecting pathogenic antibody ELISA in pig serum samples.
The background technology is as follows:
in the process of reacting the immune system of the host organism to exogenous pathogenic infection, antigen stimulation or vaccine (hereinafter collectively referred to as exogenous immunogen) immunization, the exogenous antigen is captured mainly by Antigen Presenting Cells (APCs) in the immune system of the host organism, and processed and presented, and the complete exogenous immunogen protein is degraded and cut into antigen peptide fragments with the length of 12-24 amino acids, and the antigen peptide fragments can be assembled in an antigen peptide binding groove of a Major Histocompatibility Complex (MHC) class II molecule to form an MHC-II-antigen peptide complex. In pigs, the MHC-class II molecules responsible for this process are the porcine leukocyte antigen-II DR (Swine leukocyte antigen DR, SLA-DR) and are expressed on the surface of a variety of APCs, such as porcine alveolar macrophages, or macrophages and dendritic cells within lymphoid tissues. Antigenic peptide processed and presented by SLA-DR molecules on surface of pig APCs (APCs) can be used as pig specific CD4 + T cell epitope, corresponding CD4 in swine body + And the T cells are identified to finally activate the adaptive immune response of the pig organism, generate immune memory and protect the organism from pathogenic infection.
Thus, during activation of the porcine immune system, the only effective antigen component that ultimately elicits an immune response is the antigen peptide fragment of 12-24 amino acids in length (i.e., the porcine specific cd4+ T epitope) after the exogenous immunogen is presented by APCs processing, rather than the complete immunogen. Thus, CD4 derived from an immunogen is directly used + The T epitope fragment can activate the immune system of the organism by stimulating the organism, and can generate an immune response similar to the immune system of the organism stimulated by using the complete exogenous protein antigen. Furthermore, the use of one or more CD4 derived from an intact exogenous immunogen + The T epitope fragment can be expressed as a fragment containing only CD4 by a method of total artificial synthesis or genetic engineering recombinant expression + The recombinant protein of the T epitope can be used as a genetic engineering vaccine for preventing infectious diseases of pigs.
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) and characterized by sow abortion and piglet respiratory dysfunction, and can cause severe immunosuppression. The virus is widely spread in the global pig farm, causes huge economic loss to the pig industry in the world, becomes one of the main epidemic diseases of the global scale pig farm, and is also a great difficulty in controlling the global pig diseases. The existing PRRSV antibody ELISA detection kit in the market mainly uses the main nucleocapsid protein N of PRRSV virus as the wrapper antigen for detection, and has the detection omission phenomenon caused by virus antigen variation.
In the application, porcine and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is taken as an example, in vitro cultured porcine bone marrow induced dendritic cells (Bone Marrow derived dendritic cells, BM-DCs) are taken as porcine specific Antigen Presenting Cells (APCs), PRRSV is used for simulating the processing and presenting process of virus antigens coded by PRRSV infected hosts or immunized hosts in the porcine body, SLA-DR-antigen peptide complex on the BM-DCs is enriched through SLA-DR, the antigen peptide is eluted, the method of determining antigen peptide amino acid sequences of different virus proteins derived from PRRSV through mass spectrometry technology is carried out, then serum samples derived from porcine sources of the PRRSV virus are taken as positive control, the effective antigen peptide sequences which can be recognized by PRRSV infected porcine serum in the antigen peptide sequences obtained through all mass spectrometry analysis results are selected, and the effective antigen peptide combinations derived from different PRRSV proteins are prepared into artificial antigens to replace N antigens in the traditional PRRSV antibody ELISA detection kit, so that the accuracy of detection results is improved.
The invention comprises the following steps:
the first object of the present invention is to provide a method for screening a pathogenic or exogenous protein by combining mass spectrometry and antibody ELISA, which can effectively identify a specific antigen peptide (i.e., CD4 + T epitope) and will be described by way of example with respect to porcine reproductive respiratory syndrome.
The second object of the present invention is to replace the original pathogen or antigen by gene expression after combination and tandem connection with the antigen peptide which can be recognized by the corresponding pathogen or protein specific antibody and is obtained in the first object, and to use the antigen peptide for ELISA detection, so as to improve the detection accuracy.
The ELISA detection kit for pathogenic antibodies in pig serum samples is characterized by comprising a pig pathogen specific antigen peptide, wherein the antigen peptide is obtained by a method for screening and identifying pathogenic or exogenous proteins on pig species by utilizing pig-derived APCs (namely CD4+T epitopes), and the method comprises the following steps:
step one, preparing swine APCs cells;
step two, mass spectrum identification of antigen peptide;
step three, ELISA verification of antigen peptide based on NanoLuc fusion protein.
Preferably, the preparation of the swine APCs in the invention adopts the following method: (1) Pig bone marrow-derived dendritic cells are obtained by separating long leg bones of piglets, flushing bone marrow cavities by using a cell culture solution after opening holes, and culturing obtained bone marrow cells in vitro for 7 days after induction of pig granulocyte macrophage-colony stimulating factor (GM-CSF) (shown in figure 1); (2) Pig alveolar macrophages obtained by washing pig lungs and centrifuging the alveolar lavage fluid (as shown in fig. 2); (3) Collecting pig anticoagulation from pig peripheral blood mononuclear cells by using lymphocyte separation liquid, obtaining pig peripheral blood mononuclear cells from anticoagulation, adding pig granulocyte macrophage-colony stimulating factor (GM-CSF) and pig interleukin 4 (IL-4), and performing in vitro induction culture for 7 days to obtain the pig anticoagulation agent (shown in figure 3); or (4) immunizing pig individuals with an exogenous immunogen (including but not limited to pathogens, vaccines, protein antigens, etc.), thereafter isolating the spleen, lymph nodes of the pig, and grinding on an in vitro single cell filter to obtain an immune cell population comprising macrophages endogenous to the pig (as shown in fig. 4).
Preferably, the method for mass spectrometry identification of the antigen peptide comprises the following steps: (1) preparing an enriched "SLA-DR-antigenic peptide" complex; (2) obtaining an antigenic peptide amino acid sequence; (3) The molecular weight of the antigenic peptides was determined using a mass spectrometer and the amino acid sequence of the antigenic peptides was determined by means of searching a database of pathogenic protein amino acids by mass spectrometry software.
The preparation method of the composite enriched with the SLA-DR-antigenic peptide comprises the following steps: the APCs are subjected to immunogen stimulation (such as virus infection, antigen and APCs co-culture and the like) by using the APCs, then the APCs are lysed by using a cell lysis buffer to prepare a cell lysate, and an antibody for recognizing SLA-DR is used for enriching an SLA-DR-antigen peptide complex from the whole cell lysate.
The method for obtaining the antigenic peptide amino acid fragment comprises the following steps: and (3) treating the enriched 'SLA-DR-antigenic peptide' compound by using a trifluoroacetic acid buffer solution, separating and eluting the antigenic peptide from the SLA-DR, removing residual antibodies and SLA-DR molecular monomers by using a ultrafiltration tube with a cut-off molecular weight of 5kD, so that the filtered solution only contains the antigenic peptide, and desalting to obtain the antigenic peptide amino acid fragment.
Preferably, the ELISA verification method of the antigen peptide based on the NanoLuc fusion protein comprises the following steps: (1) Artificially synthesizing and cloning the obtained antigen peptide into a pET28-NanoLucC1 vector according to the cDNA sequence thereof, expressing the antigen peptide as NanoLuc fusion antigen peptide, and detecting fusion by using a NanoLuc antibody; (2) And respectively taking the nanoLuc fusion antigen peptide as a wrapper antigen, detecting a corresponding pathogenic infection pig serum sample by ELISA, wherein the peptide segment detected as positive by ELISA is the effective antigen peptide, otherwise, the peptide segment is not the effective antigen peptide.
The invention further connects in series a plurality of positive antigenic peptides which can be identified by the pig serum of the artificial infection, then recombining and expressing in colibacillus, and the obtained artificial recombinant protein can be used as the pig pathogen specific antigenic peptide for ELISA detection of the pig serum of the corresponding pathogen infection.
The preferred kit is an ELISA detection kit for PRRSV pathogenic antibodies in pig serum samples, and the porcine pathogenic specific antigen peptide is PRRSV antigen peptide.
Preferably, the PRRSV antigenic peptide is isolated and identified by a method comprising the steps of:
step one, preparing swine APCs cells;
step two, mass spectrum identification of antigen peptide;
step three, ELISA verification of antigen peptide based on NanoLuc fusion protein.
Preferably, the method for preparing the swine APCs comprises the following steps: (1) 4-6 weeks old piglet leg bones are used, after the middle section is sawed by a sterile saw blade, holes are drilled at the other end of the broken bones, and a serum-free RPMI 1640 culture medium containing EDTA is used for flushing a bone marrow cavity so as to obtain a pig bone marrow cell suspension; (2) Centrifuging the obtained pig bone marrow cell suspension at 300g centrifugal force for 10 min, discarding supernatant, adding erythrocyte lysate, culturing at 37deg.C for 20 min to completely lyse erythrocyte, adding 2 times volume of PBS to stop reaction, and centrifuging at 300g for 10 min to obtain purified bone marrow cells; (3) Bone marrow cells were counted at 1X 10 7 The individual bone marrow cells were placed in 10mL of RPMI 1640 medium containing 10% fetal bovine serum, and cultured by adding porcine GM-CSF at a concentration of 40ng per mL, with the medium being changed every 2 days, and after continuous culture for 7 days, the suspension cells were collected, which was bone marrow dendritic cells as porcine APCs.
Preferably, the method for mass spectrometry identification of the antigenic peptide comprises the following steps: (1) Infection with PRRSV-JXA1 strain at 1MOI dose of 1X 10 8 APCs cells were collected by centrifugation 24 hours after virus infection, APCs cell membrane proteins were extracted using a membrane protein extraction kit to avoid contamination with cytoplasmic proteins, and "SLA-DR antigen peptide" complexes were enriched from APCs cell membrane proteins by antibodies specifically recognizing SLA-DR (Purified Mouse Anti-Pic SLA-DR, BD Pharmingen ™, material Number: 553642); (2) Eluting the SLA-DR antigen peptide complex from the antibody using a 10% glycine solution, and eluting the antigen peptide from the SLA-DR antigen peptide complex using 10% trifluoroacetic acid (TFA); (3) Filtering 10% trifluoroacetic acid (TFA) eluent through an ultrafiltration tube with a molecular weight cutoff of 5kD to remove SLA-DR molecules, and collecting filtrate, namely the antigenic peptide eluted on SLA-DR; (4) Desalting and freeze-drying the antigen peptide filtrate,resuspension with molecular pure water, on-machine detection with Orbitrap Fusion Lumos Tribrid mass spectrometer, obtaining antigen peptide mass spectrum, setting all encoded protein amino acid sequences of PRRSV-JXA1 as PRRSV protein sequence library, using Proteome Discoverer or PEAKS ® And searching a library by using Studio X software to obtain the mass spectrum, and obtaining an antigen peptide sequence matched with PRRSV-JXA1 coding protein.
Preferably, the ELISA verification method of the antigen peptide based on the NanoLuc fusion protein comprises the following steps: (1) Artificially synthesizing and cloning the obtained PRRSV-JXA1 immune peptide into a pET28-NanoLucC1 vector according to the cDNA sequence, expressing the PRRSV-JXA1 immune peptide as a NanoLuc fusion antigen peptide, and detecting fusion by using a NanoLuc antibody; (2) The NanoLuc fusion antigen peptide was used as a wrapper antigen, and PRRSV-JXA1 infected pig serum samples were detected by ELISA.
The invention also claims recombinant proteins comprising PRRSV antigen peptides, which are PRRSV-fusion peptide segment A, respectively, having the amino acid sequence shown in SEQ ID NO:1 or PRRSV-fusion peptide segment B, the amino acid sequence of which is shown in SEQ ID NO: 2.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the invention screens the effective antigen peptide sequence on the immunogen (pathogen or protein antigen) to remove the useless peptide segment which does not have immune activity to the organism (namely can not activate the organism adaptive immune response), screens the antigen peptide with stronger immune activity and can efficiently stimulate the organism to generate immune response. Compared with the original immunogen, the antigen peptide has a short sequence (12-24 amino acid residues) and strong hydrophilicity (the antigen peptide needs to have the capability of combining with SLA-DR, and a hydrophobic peptide segment cannot combine with SLA-DR so as to be presented to activate CD4+ T cells), so that the antigen peptide is easy to express in vitro, and different antigen peptides can be randomly connected in series to prepare a composite antigen peptide, so that the problem of missed detection caused by using a single antigen as a wrapper antigen in a conventional ELISA experiment is avoided, and the sensitivity and accuracy of ELISA detection are improved.
Description of the drawings:
fig. 1: bone marrow dendritic cell morphology after 7 days of induced differentiation culture using GM-CSF stimulation: a: non-induced bone marrow cells; b: GM-CSF bone marrow dendritic cells BM-DCs.
Fig. 2: freshly isolated pig alveolar macrophage morphology photographs.
Fig. 3: morphology under pig peripheral blood mononuclear cell microscope.
Fig. 4: pig lymph nodes and spleen were ground to remove red blood cells and then lymphocytes.
Fig. 5: representative profile (nsp1β source) of antigenic peptide sequence mass spectrometry.
Fig. 6: examples of expression of luciferase NanoLuc fusion immune antigen peptide by escherichia coli.
Fig. 7: the antigen peptides are serially connected and then expressed in segments.
Fig. 8: the ELISA detection method provided by the invention is inconsistent with the detection of the Aide commercial monitoring kit, and 3 serum samples (sample numbers PC39, PC65 and PC 67) are verified to be positive to PRRSV antibodies by immunofluorescence detection of PRRSV infected MARC-145 cells.
The specific embodiment is as follows:
the present invention is further illustrated by the following specific embodiments, so that those skilled in the art can more clearly understand the technical scheme of the present invention, and the present invention is not limited thereto.
Example 1: preparation of swine APCs (examples are swine bone marrow dendritic cells)
1. The leg bones of 4-6 weeks old piglets are sawed off from the middle section by a sterile saw blade, holes are drilled at the other end of the broken bones, and the marrow cavity is flushed by using a serum-free RPMI 1640 culture medium containing EDTA, so as to obtain the pig marrow cell suspension.
The obtained swine bone marrow cell suspension was centrifuged at 300g for 10 minutes, the supernatant was discarded, red blood cell lysate was added to culture at 37℃for 20 minutes to completely lyse red blood cells, 2-fold volume of PBS was added to terminate the reaction, and again centrifuged at 300g for 10 minutes to obtain purified bone marrow cells.
Bone marrow cells were counted at 1X 10 7 The individual bone marrow cells were placed in 10mL of RPMI 1640 medium containing 10% fetal bovine serum at 40ng per mLPig GM-CSF was added at a concentration to culture, the culture medium was changed every 2 days, and after continuous culture for 7 days, suspension cells were collected, which were bone marrow dendritic cells as swine APCs, the morphology of which was shown in FIG. 1, and the next experiment was carried out.
Example 2 Mass spectrometric identification of antigenic peptides
1. Infection with PRRSV-JXA1 strain at 1MOI dose of 1X 10 8 APCs cells were collected by centrifugation 24 hours after virus infection, APCs cell membrane proteins were extracted using a membrane protein extraction kit to avoid contamination with cytoplasmic proteins, and "SLA-DR antigen peptide" complexes were enriched from APCs cell membrane proteins by antibodies specifically recognizing SLA-DR (Purified Mouse Anti-Pic SLA-DR, BD Pharmingen ™, material Number: 553642).
The SLA-DR antigen peptide complex was eluted from the antibody using a 10% glycine solution, and the antigen peptide was eluted from the SLA-DR antigen peptide complex using 10% trifluoroacetic acid (TFA).
Filtering 10% trifluoroacetic acid (TFA) eluent through an ultrafiltration tube with a molecular weight cutoff of 5kD to remove SLA-DR molecules, and collecting filtrate, which is the antigenic peptide eluted on SLA-DR.
4. After desalting and freeze-drying the antigen peptide filtrate, the filtrate was resuspended with molecular pure water and detected mechanically on a Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain a mass spectrum of the antigen peptide (representative spectrum is shown in fig. 5). All the coded protein amino acid sequences of PRRSV-JXA1 are set as PRRSV protein sequence library, and the obtained mass spectrum is searched by using Proteome Discoverer or PEAKS-Studio X software to obtain antigen peptide sequence matched with PRRSV-JXA1 coded protein, and the result is shown in Table 1.
Example 3: ELISA verification of antigenic peptides based on nanoLuc fusion proteins
1. The obtained PRRSV-JXA1 immunopeptides were artificially cloned into pET28-NanoLucC1 vector according to the cDNA sequence thereof, expressed as NanoLuc fusion antigen peptides, and the fusion was detected using NanoLuc antibodies, as shown in FIG. 6.
The NanoLuc fusion antigen peptide was used as a wrapper antigen, and PRRSV-JXA1 infected pig serum samples were detected by ELISA. The results are shown in Table 2.
TABLE 1 PRRSV-JXA1 Virus whole genome immune peptide sequence obtained by analytical Mass Spectrometry techniques
TABLE 2 detection of luciferase NanoLuc fusion immune antigen peptide Using 5 parts of PRRSV antibody positive serum and PRRSV antibody negative serum for searching for positive peptide fragment
Example 4: the recombinant expression protein of the composite PRRSV antigen peptide is used as a detection antigen for detecting serum samples of PRRSV infected individuals.
1. Selecting selected antigen peptide sequences, designing the antigen peptide sequences into recombinant PRRSV antigens in a G4S flexible connecting peptide interval mode, and naming the antigen peptide sequences as fusion peptide fragments A and B, wherein the sequences are SEQ ID NO:1 and SEQ ID NO:2, and carrying out recombinant expression, wherein the sequence and the expression result are shown in figure 7
2. 100 split pig serum samples were collected, ELISA was performed using recombinant PRRSV antigen as a wrapper antigen, the detection results are shown in Table 3, and detection was performed using the PRRSV X3 Herd Check kit of Aldes company as a control, the results are shown in Table 4
3. The results of the three serum samples are inconsistent with those of the ELISA kit of Edison company, wherein the detection by the method established by the invention is positive, and the ELISA kit of Edison company is negative, and the results are shown in Table 4. The PRRSV infected Marc-145 cells were further detected by immunofluorescence using diluted serum samples, and triplicate samples were determined to be PRRSV antibody positive samples, consistent with the established methods of the present invention. Immunofluorescence results are shown in FIG. 8.
TABLE 3 Table 3
Table 4: ELISA test results on 100 pig serum samples from the field using commercial kit from Aides company
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Sequence listing
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His His His
Claims (5)
1. A kit for ELISA detection of pathogenic antibodies in pig serum samples is characterized by comprising a pig pathogen specific antigen peptide, wherein the pig pathogen specific antigen peptide is used for carrying out CD4 on pathogenic or exogenous proteins on pig species by utilizing pig-derived APCs + The T epitope specific antigen peptide is obtained by a screening and identifying method, which comprises the following steps:
step one, preparation of swine APCs cells: 4-6 weeks old piglet leg bones are used, after the middle section is sawed by a sterile saw blade, holes are drilled at the other end of the broken bones, and a serum-free RPMI 1640 culture medium containing EDTA is used for flushing a bone marrow cavity so as to obtain a pig bone marrow cell suspension; centrifuging the obtained pig bone marrow cell suspension at 300g centrifugal force for 10 min, discarding supernatant, adding erythrocyte lysate, culturing at 37deg.C for 20 min to completely lyse erythrocyte, adding 2 times volume of PBS to stop reaction, and centrifuging at 300g for 10 min to obtain purified bone marrow cells; bone marrow cells were counted at 1X 10 7 Placing bone marrow cells in 10mL RPMI 1640 culture medium containing 10% foetal calf serum, adding pig granulocyte macrophage-colony stimulating factor GM-CSF according to the concentration of 40ng per mL, culturing, changing culture solution every 2 days, continuously culturing for 7 days, collecting suspension cells to obtain the invented pig source APCsBone marrow dendritic cells;
step two, mass spectrum identification of antigen peptide:
(1) Preparation of an enriched "SLA-DR-antigenic peptide" complex: co-culturing the APCs with viruses to enable the APCs to be infected by the viruses, then lysing the APCs with a cell lysis buffer to prepare a cell lysate, and enriching an SLA-DR-antigenic peptide complex from the whole cell lysate by using an antibody for recognizing SLA-DR;
(2) Obtaining an antigenic peptide amino acid sequence: treating the enriched SLA-DR-antigenic peptide complex with trifluoroacetic acid buffer solution, separating and eluting antigenic peptide from SLA-DR, removing residual antibody and SLA-DR molecular monomer by using a ultrafiltration tube with a cut-off molecular weight of 5kD, so that the filtered solution only contains antigenic peptide, and desalting to obtain the antigenic peptide amino acid fragment;
(3) Determining the molecular weight of the antigenic peptide using a mass spectrometer and searching a pathogen-derived protein amino acid database by mass spectrometry software to determine the antigenic peptide amino acid sequence;
step three, ELISA verification of antigen peptide based on NanoLuc fusion protein.
2. The kit for ELISA detection of pathogenic antibodies in porcine serum samples according to claim 1, wherein the porcine APCs cells are further prepared by: (1) Pig alveolar macrophages, obtained by washing pig lungs and centrifuging alveolar lavage fluid; (2) Collecting pig anticoagulation from pig peripheral blood mononuclear cells by using lymphocyte separation liquid, obtaining pig peripheral blood mononuclear cells from anticoagulation, adding pig granulocyte macrophage-colony stimulating factor GM-CSF and pig interleukin 4, and performing in vitro induction culture for 7 days to obtain the pig anticoagulation agent; or (3) immunizing individual pigs with an exogenous immunogen, then isolating spleen and lymph nodes of the pigs, and grinding on an in vitro single cell filter to obtain an immune cell population comprising endogenous macrophages of the pigs.
3. The kit for ELISA detection of pathogenic antibodies in porcine serum samples according to claim 1 or 2, wherein the kit is a kit for ELISA detection of PRRSV pathogenic antibodies in porcine serum samples, and the porcine pathogen specific antigenic peptide is a PRRSV antigenic peptide.
4. A kit for ELISA detection of pathogenic antibodies in porcine serum according to claim 3 wherein the PRRSV antigen peptide is isolated and identified by a method comprising the steps of:
step one, preparation of swine APCs cells: preparation of swine APCs cells: 4-6 weeks old piglet leg bones are used, after the middle section is sawed by a sterile saw blade, holes are drilled at the other end of the broken bones, and a serum-free RPMI 1640 culture medium containing EDTA is used for flushing a bone marrow cavity so as to obtain a pig bone marrow cell suspension; centrifuging the obtained pig bone marrow cell suspension at 300g centrifugal force for 10 min, discarding supernatant, adding erythrocyte lysate, culturing at 37deg.C for 20 min to completely lyse erythrocyte, adding 2 times volume of PBS to stop reaction, and centrifuging at 300g for 10 min to obtain purified bone marrow cells; bone marrow cells were counted at 1X 10 7 Placing the individual bone marrow cells in 10mL of RPMI 1640 culture medium containing 10% fetal bovine serum, adding pig GM-CSF according to the concentration of 40ng per milliliter for culture, changing the culture solution every 2 days, continuously culturing for 7 days, and collecting suspension cells, namely bone marrow dendritic cells serving as pig-derived APCs;
step two, mass spectrum identification of antigen peptide: infection with PRRSV-JXA1 strain at 1MOI dose of 1X 10 8 Centrifuging and collecting cells after virus infection for 24 hours, extracting APCs cell membrane proteins by using a membrane protein extraction kit to avoid pollution of cytoplasmic proteins, and enriching an 'SLA-DR antigen peptide' complex from the APCs cell membrane proteins by an antibody specifically recognizing SLA-DR; eluting the SLA-DR antigen peptide complex from the antibody using a 10% glycine solution, and eluting the antigen peptide from the SLA-DR antigen peptide complex using 10% trifluoroacetic acid; filtering 10% trifluoroacetic acid eluent through an ultrafiltration tube with a molecular weight cutoff of 5kD, removing SLA-DR molecules, and collecting filtrate, namely the antigenic peptide eluted on SLA-DR; desalting and freeze-drying the antigen peptide filtrate, re-suspending with molecular pure water at Orbitrap Fusion LuDetecting on-machine by a mos Tribrid mass spectrometer to obtain a mass spectrum of the antigen peptide; setting all coded protein amino acid sequences of PRRSV-JXA1 as PRRSV protein sequence library, searching the obtained mass spectrum by using Proteome Discoverer or PEAKS Studio X software to obtain antigen peptide sequence matched with PRRSV-JXA1 coded protein;
step three, ELISA verification of antigen peptide based on NanoLuc fusion protein.
5. The ELISA kit of claim 4, wherein the PRRSV antigen peptide comprises recombinant proteins of PRRSV antigen peptide, which are PRRSV-fusion peptide segment A, respectively, and the amino acid sequence of the PRRSV-fusion peptide segment A is shown in SEQ ID NO:1 or PRRSV-fusion peptide segment B, the amino acid sequence of which is shown in SEQ ID NO: 2.
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