CN112285347A - ELISA detection kit for pathogen antibody in porcine serum sample - Google Patents

ELISA detection kit for pathogen antibody in porcine serum sample Download PDF

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CN112285347A
CN112285347A CN202011062115.5A CN202011062115A CN112285347A CN 112285347 A CN112285347 A CN 112285347A CN 202011062115 A CN202011062115 A CN 202011062115A CN 112285347 A CN112285347 A CN 112285347A
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武春燕
南雨辰
周恩民
郑旭
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Abstract

The invention relates to a method for capturing porcine leukocyte antigen class II DR molecules (SLA-DR) responsible for processing and presenting antigens on porcine-derived antigen processing and presenting cells APCs by using specific antibodies, after eluting the antigenic peptides presented on SLA-DR by trifluoroacetic acid, carrying out mass spectrometry after desalting and resuspending to determine the amino acid sequence of the antigenic peptide, identifying the antigenic peptide sequence which can be processed and presented by organism antigen presenting cells in all proteins coded by porcine specific pathogens, and carrying out gene engineering on the CD4 obtained by the method+Respectively fusing the T epitope antigen peptide with NanoLuc luciferase, performing in-vitro recombinant expression and purification by using escherichia coli, performing ELISA detection on the T epitope antigen peptide serving as an artificial antigen and a pig serum sample infected by corresponding pathogen, and searching CD4 capable of being recognized by the pig serum sample infected by the pathogen+T epitope antigensAnd the peptide sequences are used for further carrying out recombinant expression in escherichia coli after all antigen peptide sequences which are used for detecting the antibodies ELISA to be positive are connected in series, and the antigen peptide sequences are used as artificial antigens for detecting specific antibodies of given pathogens.

Description

ELISA detection kit for pathogen antibody in porcine serum sample
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to an ELISA detection kit for a pathogenic antibody in a porcine serum sample, in particular to a method for separating and identifying PRRSV antigen peptide and application thereof, and particularly relates to an ELISA detection method for the pathogenic antibody in the porcine serum sample.
Background art:
in the process of responding to exogenous pathogenic infection, antigen stimulation or vaccine (hereinafter referred to as exogenous immunogen) immunization, the immune system of a host organism captures the exogenous antigen mainly through Antigen Presenting Cells (APCs) in the host immune system, processes and presents the antigen, degrades and cuts the complete exogenous immunogen protein into antigen peptide fragments with the length of 12-24 amino acids, and the antigen peptide fragments can be assembled in an antigen peptide combining groove of Major Histocompatibility Complex (MHC) II molecules to form an MHC-II-antigen peptide complex. In pigs, the MHC class II molecule responsible for this process is porcine leukocyte antigen-II DR (SLA-DR) and is expressed on the surface of a variety of APCs, such as porcine alveolar macrophages, or macrophages and dendritic cells within lymphoid tissues. The antigen peptide which is processed and presented by SLA-DR molecules on the surface of the porcine APCs can be used as porcine specific CD4+T cell epitope, corresponding CD4 in pig body+The T cells recognize the pig cell, and finally activate the adaptive immune response of the pig body, so that the immune memory is generated, and the body is protected from being infected by pathogen.
Therefore, during the activation of the pig immune system, the effective antigen component which finally triggers the immune response is only an antigenic peptide fragment (namely the pig specific CD4+ T epitope) with the length of 12-24 amino acids after the exogenous immunogen is processed and presented by APCs, but not the complete immunogen. Thus, CD4 derived from the immunogen was used directly+The T epitope fragment can activate the immune system of the organism by stimulating the organism, and generate an immune response similar to that of the complete exogenous protein antigen. In addition, one or more CD4 derived from intact exogenous immunogen(s) were used+The T epitope fragment can be obtained by full-artificial synthesis or recombinant expression method of genetic engineeringExpressed as a CD 4-only gene+T epitope recombinant protein, the recombinant protein obtained can be used as a genetic engineering vaccine for preventing swine infectious diseases.
Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease mainly characterized by sow abortion and piglet respiratory disorder caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and can cause severe immunosuppression. The virus is widely spread in global swinery, causes huge economic loss to the world pig industry, becomes one of the main epidemic diseases of global large-scale pig farms, and is also a big problem in global pig disease control. The existing PRRSV antibody ELISA detection kit in the market mainly uses the main nucleocapsid protein N of PRRSV virus as a plate antigen for detection, and has the phenomenon of missed detection caused by virus antigen variation.
The application takes porcine and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) as an example, takes in vitro cultured porcine Bone Marrow induced dendritic cells (BM-DCs) as porcine specific Antigen Presenting Cells (APCs), uses PRRSV infected BM-DCs to simulate the process of processing and presenting the virus antigen coded by the PRRSV infected host or immunized host in a pig body, enriches SLA-DR-antigenic peptide complexes on the BM-DCs through SLA-DR, measures the method of amino acid sequences of different virus protein antigenic peptides derived from the PRRSV by mass spectrometry after eluting the antigenic peptide, then takes an artificial infected PRRSV virus-derived serum sample as a positive control, detects the effective antigenic peptide sequences which can be identified by PRRSV infected pig serum in all the antigenic peptide sequences obtained by mass spectrometry analysis results, selects effective peptide combinations derived from different PRRSV proteins to prepare artificial antigens, the kit replaces the N antigen in the traditional PRRSV antibody ELISA detection kit to improve the accuracy of the detection result.
The invention content is as follows:
the first purpose of the invention is to provide a method for screening a pathogen or an exogenous protein in a porcine species by combining a mass spectrometry technique and an antibody ELISA screening techniqueOr a specific antigenic peptide recognized by a protein-specific antibody (i.e., CD 4)+T epitope) and the porcine reproductive respiratory syndrome is taken as an example for illustration.
The second purpose of the invention is to combine and connect the antigen peptide obtained in the first purpose and recognized by the corresponding pathogen or protein specific antibody in series, then carry out gene expression, replace the original pathogen or antigen, and use the original pathogen or antigen for ELISA detection, so as to improve the detection accuracy.
The kit for detecting the pathogen antibody ELISA in the porcine serum sample is characterized by comprising porcine pathogen specific antigenic peptide, wherein the antigenic peptide is obtained by a method for screening and identifying the specific antigenic peptide (namely CD4+ T epitope) of pathogen or exogenous protein on porcine species by utilizing porcine APCs, and the method comprises the following steps:
step one, preparing porcine APCs;
step two, mass spectrum identification of the antigen peptide;
and step three, performing ELISA verification on the antigen peptide based on the NanoLuc fusion protein.
Preferably, the preparation of the porcine APCs cells of the present invention adopts the following method: (1) porcine bone marrow-derived dendritic cells are obtained by separating long leg bones of piglets, opening holes, flushing marrow cavities by using cell culture solution, inducing the obtained bone marrow cells by porcine granulocyte macrophage-colony stimulating factor (GM-CSF), and culturing in vitro for 7 days (shown in figure 1); (2) porcine alveolar macrophages obtained by washing porcine lungs and then centrifuging alveolar lavage fluid (shown in figure 2); (3) collecting porcine peripheral blood mononuclear cell derived macrophages, collecting porcine anticoagulated blood, obtaining porcine peripheral blood mononuclear cells from the anticoagulated blood by using lymphocyte separating medium, adding porcine granulocyte macrophage-colony stimulating factor (GM-CSF) and porcine interleukin 4 (IL-4) for in vitro induction culture for 7 days (as shown in figure 3); or (4) immunizing individual pig with foreign immunogen (including but not limited to pathogen, vaccine, protein antigen, etc.), separating spleen and lymph node of pig, and grinding on unicellular filter net in vitro to obtain immune cell population containing pig endogenous macrophage (as shown in FIG. 4).
Preferably, the method for mass spectrometric identification of antigenic peptides according to the invention comprises the following steps: (1) preparing an enriched SLA-DR-antigenic peptide compound; (2) obtaining an antigenic peptide amino acid sequence; (3) the molecular weight of the antigen peptide is determined by using a mass spectrometer, and the amino acid sequence of the antigen peptide is determined by searching a pathogen-derived protein amino acid database through mass spectrometry software.
The method for preparing the enriched SLA-DR-antigenic peptide compound comprises the following steps: APCs are stimulated by immunogen (such as virus infection, antigen and APCs coculture and the like), then cell lysis buffer is used for lysing the APCs to prepare cell lysate, and SLA-DR-antigen peptide complex is enriched from the whole cell lysate by using SLA-DR recognizing antibody.
The method for obtaining the antigenic peptide amino acid fragment comprises the following steps: treating the enriched SLA-DR-antigen peptide compound with trifluoroacetic acid buffer solution, separating and eluting the antigen peptide from SLA-DR, removing residual antibody and SLA-DR molecular monomer by using an ultrafiltration tube with the molecular weight cutoff of 5kD, enabling the filtrate to only contain the antigen peptide, and desalting to obtain the antigen peptide amino acid fragment.
Preferably, the method for ELISA verification 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 of the antigen peptide, expressing the antigen peptide as NanoLuc fusion antigen peptide, and detecting the fusion by using a NanoLuc antibody; (2) the NanoLuc fusion antigen peptides are respectively used as envelope antigens, corresponding pathogen infection pig serum samples are detected through ELISA, peptide segments which are detected to be positive through ELISA are effective antigen peptides, and otherwise, the peptide segments are not effective antigen peptides.
The invention further connects a plurality of positive antigen peptides which can be identified by the serum of the artificially infected pig in series, and then the positive antigen peptides are recombined and expressed in colon bacillus, and the obtained artificially recombined protein can be used as the specific antigen peptide of the pig pathogen to be used for ELISA detection of the serum of the pig infected by the corresponding pathogen.
Preferably, the kit is an ELISA detection kit for PRRSV pathogenic antibodies in a porcine serum sample, and the porcine pathogenic specific antigen peptide is a PRRSV antigen peptide.
Preferably, the PRRSV antigen peptide is isolated and identified using a method comprising the steps of:
step one, preparing porcine APCs;
step two, mass spectrum identification of the antigen peptide;
and step three, performing ELISA verification on the antigen peptide based on the NanoLuc fusion protein.
Preferably, the method for preparing the porcine APCs cells comprises the following steps: (1) using leg bones of 4-6 weeks old piglets, sawing off from the middle section by using a sterile saw blade, drilling holes at the other end of the broken bones, and flushing marrow cavities by using a serum-free RPMI 1640 medium containing EDTA to obtain a pig bone marrow cell suspension; (2) centrifuging the obtained pig bone marrow cell suspension for 10 minutes by a centrifugal force of 300g, then discarding the supernatant, adding erythrocyte lysate, culturing for 20 minutes at 37 ℃ to fully lyse erythrocytes, adding PBS with 2 times volume to stop the reaction, and centrifuging for 10 minutes again by 300g to obtain purified bone marrow cells; (3) bone marrow cells were counted at 1X 107And (3) putting the bone marrow cells into 10mL of RPMI 1640 culture medium containing 10% fetal calf serum, adding pig GM-CSF at the concentration of 40ng per milliliter for culture, replacing the culture solution every 2 days, continuously culturing for 7 days, and collecting suspension cells, namely the bone marrow dendritic cells serving as the pig-derived APCs.
Preferably, the method for mass spectrometric identification of the antigenic peptide comprises the steps of: (1) 1X 10 infection with PRRSV-JXA1 Strain at 1MOI dose8APCs cells, centrifugally collecting the cells after the virus infection for 24 hours, extracting APCs cell membrane proteins by using a membrane protein extraction kit to avoid the pollution of cytoplasmic proteins, and enriching an SLA-DR antigenic peptide compound from the APCs cell membrane proteins by using an antibody (Purified Mouse Anti-Pig SLA-DR, BD Pharmingen, Material Number: 553642) for specifically recognizing the SLA-DR; (2) elution of SLA-DR antigenic peptide complexes from antibodies Using 10% Glycine solution followed by elution of antigen from SLA-DR antigenic peptide complexes Using 10% trifluoroacetic acid (TFA)A peptide; (3) filtering 10% trifluoroacetic acid (TFA) eluent through an ultrafiltration tube with the molecular weight cutoff of 5kD, removing SLA-DR molecules, and collecting filtrate, namely the antigen peptide eluted on SLA-DR; (4) desalting and freeze-drying antigen peptide filtrate, re-suspending with molecular pure water, detecting on Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain mass spectrum of antigen peptide, setting amino acid sequence of all encoded proteins of PRRSV-JXA1 as PRRSV protein sequence library, and using protome resolver or PEAKS®And (3) carrying out library searching on the obtained mass spectrum by the Studio X software to obtain an antigen peptide sequence matched with the PRRSV-JXA1 coding protein.
Preferably, the method for ELISA validation of the NanoLuc fusion protein-based antigenic peptide 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 of the immune peptide, expressing the immune peptide into NanoLuc fusion antigen peptide, and detecting fusion by using a NanoLuc antibody; (2) the NanoLuc fusion antigen peptide is respectively used as a plate-coated antigen, and PRRSV-JXA1 infected pig serum samples are detected by ELISA.
The invention also claims and protects recombinant proteins comprising PRRSV antigen peptides, which are PRRSV-fusion peptide segment A respectively, and the amino acid sequences of the recombinant proteins are shown as SEQ ID NO: 1 or PRRSV-fusion peptide segment B, the amino acid sequence of which is shown as SEQ ID NO: 2, respectively.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the invention screens effective antigen peptide sequence on immunogen (pathogen or protein antigen), removes useless peptide segment which has no immunocompetence (namely can not activate adaptive immune response) to organism in the immunogen, screens antigen peptide which has stronger immunocompetence and can efficiently stimulate the organism to generate immune response. Compared with the original immunogen, the antigen peptide has shorter sequence (12-24 amino acid residues) and stronger hydrophilicity (the antigen peptide needs to have the capability of combining with SLA-DR, and the hydrophobic peptide segment cannot be combined with SLA-DR so as to be presented and activate CD4+ T cells), so that the antigen peptide is easy to express in vitro, different antigen peptides can be randomly connected in series to prepare the composite antigen peptide, the problem of omission caused by using a single antigen as a coating antigen in the conventional ELISA experiment is avoided, and the sensitivity and the 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 myeloid dendritic cells BM-DCs.
FIG. 2: photographs of freshly isolated porcine alveolar macrophage morphology.
FIG. 3: porcine peripheral blood mononuclear cell microscopic morphology.
FIG. 4: the lymph cells are removed from the lymph nodes and spleen of pig after grinding.
FIG. 5: antigen peptide sequence mass spectrometry representative profiles (NSP 1 β source).
FIG. 6: an example of the expression of luciferase NanoLuc fusion immunogenic antigen peptide in escherichia coli.
FIG. 7: and (3) carrying out segmented expression on the antigen peptides after the antigen peptides are connected in series.
FIG. 8: the ELISA detection method obtained by the invention and the commercial Edison monitoring kit detect inconsistent 3 serum samples (sample numbers PC39, PC65 and PC 67) and the serum samples are proved to be positive by detecting the PRRSV infected MARC-145 cells through immunofluorescence.
The specific implementation mode is as follows:
the following specific embodiments are set forth to further illustrate the present invention so that those skilled in the art can more clearly understand the technical solutions of the present invention, and the present invention is not limited thereto.
Example 1: preparation of porcine APCs (taking porcine bone marrow dendritic cells as an example)
1. Leg bones of 4-6-week-old piglets are used, after being sawn off from the middle section by using a sterile saw blade, holes are drilled at the other end of the broken bones, and the marrow cavity is flushed by using serum-free RPMI 1640 medium containing EDTA to obtain a pig bone marrow cell suspension.
The obtained suspension of pig bone marrow cells was centrifuged at 300g for 10 minutes and then the supernatant was discarded, and erythrocyte lysate was added thereto and cultured at 37 ℃ for 20 minutes to sufficiently lyse erythrocytes, 2-fold volume of PBS was added thereto to terminate the reaction, and centrifuged again at 300g for 10 minutes to obtain purified bone marrow cells.
Bone marrow cells were counted at 1X 107The bone marrow cells are placed in 10mL RPMI 1640 medium containing 10% fetal calf serum, pig GM-CSF is added according to the concentration of 40ng per milliliter for culture, the culture solution is replaced every 2 days, after continuous culture is carried out for 7 days, suspension cells are collected, and the bone marrow dendritic cells serving as pig-derived APCs are obtained, the shape of the bone marrow dendritic cells is shown in figure 1, and the next experiment can be carried out.
Example 2 Mass Spectrometry identification of antigenic peptides
1. 1X 10 infection with PRRSV-JXA1 Strain at 1MOI dose8APCs cells, which are collected by centrifugation 24 hours after virus infection, are extracted by using a membrane protein extraction kit to avoid contamination of cytoplasmic proteins, and the "SLA-DR antigenic peptide" complex is enriched from the APCs cell membrane proteins by antibodies (Purified Mouse Anti-Pig SLA-DR, BD Pharmingen;, Material Number: 553642) that specifically recognize SLA-DR.
The SLA-DR antigenic peptide complex was eluted from the antibody using a 10% glycine solution, and the antigenic peptide was eluted from the SLA-DR antigenic peptide complex using 10% trifluoroacetic acid (TFA).
Filtering 10% trifluoroacetic acid (TFA) eluent through an ultrafiltration tube with the molecular weight cutoff of 5kD, removing SLA-DR molecules, and collecting filtrate, namely the antigen peptide eluted on SLA-DR.
4. Desalting and freeze-drying the antigen peptide filtrate, re-suspending with molecular pure water, and detecting on an Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain a mass spectrum of the antigen peptide (a representative spectrum is shown in FIG. 5). Setting amino acid sequences of all the encoding proteins of PRRSV-JXA1 as a PRRSV protein sequence library, and searching the library by using protein discover or PEAKS Studio X software to obtain a mass spectrum map, so as to obtain an antigenic peptide sequence matched with the PRRSV-JXA1 encoding protein, wherein the result is shown in Table 1.
Example 3: ELISA verification of antigen peptide based on NanoLuc fusion protein
1. The obtained PRRSV-JXA1 immune peptide was artificially synthesized and cloned according to its cDNA sequence into pET28-NanoLuc c1 vector, expressed as NanoLuc fusion antigen peptide, and fusion was detected using NanoLuc antibody, as shown in fig. 6.
The NanoLuc fusion antigen peptide is respectively used as a plate-coated antigen, and PRRSV-JXA1 infected pig serum samples are 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 technique
Figure DEST_PATH_IMAGE001
Table 2 detection of luciferase NanoLuc fusion immune antigen peptide by using 5 parts of PRRSV antibody positive serum and PRRSV antibody negative serum for searching positive peptide segment
Figure 408834DEST_PATH_IMAGE002
Figure DEST_PATH_IMAGE003
Example 4: the recombinant expression protein of the composite PRRSV antigen peptide is used as a detection antigen for detecting a PRRSV infected individual serum sample.
1. Selecting a selected antigen peptide sequence, designing the antigen into recombinant PRRSV antigens in a mode of G4S flexible connecting peptide spacing, and naming the antigens as fusion peptide segments A and B, wherein the sequences are respectively SEQ ID NO: 1 and SEQ ID NO: 2, and recombinant expression, the sequence and expression results are shown in FIG. 7
2. Collecting 100-field pig serum samples, performing ELISA detection by using recombinant PRRSV antigen as a plate-coated antigen, wherein the detection result is shown in Table 3, performing detection by using PRRSV X3 Herd Check kit of Edison corporation as a contrast detection coincidence rate, and the result is shown in Table 4
3. The control result of the ELISA kit of Edison shows that the results of three serum samples are inconsistent, wherein the detection result by the method established by the invention is positive, and the result of the ELISA kit of Edison is negative is shown in Table 4. Further detecting the PRRSV infected Marc-145 cells by immunofluorescence and diluted serum samples, and determining three samples as PRRSV antibody positive samples, which is consistent with the method established by the invention. The immunofluorescence results are shown in FIG. 8.
TABLE 3
Figure 331660DEST_PATH_IMAGE004
Figure DEST_PATH_IMAGE005
Table 4: ELISA test results of 100 swine serum samples from the field using a commercial kit from Edes
Figure 327298DEST_PATH_IMAGE006
Figure DEST_PATH_IMAGE007
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340 345 350
Phe Pro Leu Ala Thr Glu Gly Gly Gly Ser Thr Val Glu Phe Ser Leu
355 360 365
Pro Thr Gln His Thr Val Arg Leu Ile Arg Ala Thr Ala Ser Pro Ser
370 375 380
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu His His His
385 390 395 400
His His His

Claims (8)

1. The kit for detecting the pathogen antibody ELISA in the porcine serum sample is characterized by comprising porcine pathogen specific antigenic peptide, wherein the antigenic peptide is obtained by a method for screening and identifying the specific antigenic peptide (namely CD4+ T epitope) of pathogen or exogenous protein on porcine species by utilizing porcine APCs, and the method comprises the following steps:
step one, preparing porcine APCs;
step two, mass spectrum identification of the antigen peptide;
and step three, performing ELISA verification on the antigen peptide based on the NanoLuc fusion protein.
2. The ELISA test kit of claim 1, wherein the porcine APCs are prepared by the following method: (1) pig bone marrow-derived dendritic cells are obtained by separating long leg bones of piglets, flushing marrow cavities by using cell culture solution after opening holes, and culturing in vitro for 7 days after the obtained bone marrow cells are induced by porcine granulocyte macrophage-colony stimulating factor (GM-CSF); (2) pig alveolar macrophages are obtained by washing pig lungs and then centrifuging alveolar lavage fluid; (3) collecting porcine peripheral blood monocyte-derived macrophages, obtaining porcine peripheral blood monocytes from the anticoagulation by using lymphocyte separation fluid, adding porcine granulocyte macrophage-colony stimulating factor (GM-CSF) and porcine interleukin 4 (IL-4) for in vitro induction culture for 7 days to obtain porcine anticoagulation blood mononuclear cells; or (4) immunizing individual pig with foreign immunogen, separating spleen and lymph node of pig, and grinding in vitro on single cell filter net to obtain immune cell population containing pig endogenous macrophage.
3. The ELISA test kit of claim 1, wherein the mass spectrometric identification of the antigenic peptide comprises the steps of: (1) preparing an enriched SLA-DR-antigenic peptide compound; (2) obtaining an antigenic peptide amino acid sequence; (3) the molecular weight of the antigen peptide is determined by using a mass spectrometer, and the amino acid sequence of the antigen peptide is determined by searching a pathogen-derived protein amino acid database through mass spectrometry software.
4. The ELISA test kit according to claim 1, wherein the enriched SLA-DR-antigenic peptide complex is prepared by the following steps: APCs are stimulated by immunogen (such as virus infection, antigen and APCs coculture and the like), then cell lysis buffer is used for lysing the APCs to prepare cell lysate, and SLA-DR-antigen peptide complex is enriched from the whole cell lysate by using SLA-DR recognizing antibody.
5. The ELISA test kit of claim 1, wherein the method for obtaining the antigenic peptide amino acid fragment comprises: treating the enriched SLA-DR-antigen peptide compound with trifluoroacetic acid buffer solution, separating and eluting the antigen peptide from SLA-DR, removing residual antibody and SLA-DR molecular monomer by using an ultrafiltration tube with the molecular weight cutoff of 5kD, enabling the filtrate to only contain the antigen peptide, and desalting to obtain the antigen peptide amino acid fragment.
6. The ELISA detection kit of claim 1, wherein the kit is an ELISA detection kit for PRRSV pathogenic antibodies in a porcine serum sample, and the porcine pathogenic specific antigenic peptide is a PRRSV antigenic peptide.
7. The ELISA test kit of claim 6, wherein the PRRSV antigenic peptide is isolated and identified by a method comprising the steps of:
step one, preparing porcine APCs;
step two, mass spectrum identification of the antigen peptide;
step three, ELISA verification of antigen peptide based on NanoLuc fusion protein;
the method for mass spectrometric identification of the antigenic peptide comprises the following steps: (1) 1X 10 infection with PRRSV-JXA1 Strain at 1MOI dose8APCs cells, centrifugally collecting the cells after the virus infection for 24 hours, extracting APCs cell membrane proteins by using a membrane protein extraction kit to avoid the pollution of cytoplasmic proteins, and enriching an SLA-DR antigenic peptide compound from the APCs cell membrane proteins by using an antibody (Purified Mouse Anti-Pig SLA-DR, BD Pharmingen, Material Number: 553642) for specifically recognizing the SLA-DR; (2) eluting the SLA-DR antigenic peptide complex from the antibody using a 10% glycine solution, followed by eluting the antigenic peptide from the SLA-DR antigenic peptide complex using 10% trifluoroacetic acid (TFA); (3) filtering 10% trifluoroacetic acid (TFA) eluate through ultrafiltration tube with molecular weight cut-off of 5kD to remove SLA-DR molecule,collecting the filtrate, namely the antigen peptide eluted from the SLA-DR; (4) desalting and freeze-drying antigen peptide filtrate, re-suspending with molecular pure water, detecting on Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain mass spectrum of antigen peptide, setting amino acid sequence of all encoded proteins of PRRSV-JXA1 as PRRSV protein sequence library, and using protome resolver or PEAKS®And (3) carrying out library searching on the obtained mass spectrum by the Studio X software to obtain an antigen peptide sequence matched with the PRRSV-JXA1 coding protein.
8. The ELISA detection kit of claim 7, wherein the PRRSV antigen peptide comprises a recombinant protein of PRRSV antigen peptide, which is PRRSV-fusion peptide segment A, and the amino acid sequence thereof is shown as SEQ ID NO: 1 or PRRSV-fusion peptide segment B, the amino acid sequence of which is shown as SEQ ID NO: 2, respectively.
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