B cell epitope peptide based on gE protein conservation and its application in antibody diagnosis of porcine pseudorabies virus

Abstract

The application discloses a conserved B cell epitope peptide on a surface envelope glycoprotein of a porcine pseudorabies virus (PRV) and application of the B cell epitope peptide in PRV antibody diagnosis. The linear B cell epitope of the PRV gE is conserved, the sequence of the linear B cell epitope is GPGGGD, and the linear B cell epitope is named as GD-GG. The B cell epitope can be expressed on the surface of an inert carrier S9H bacteroid in a functional manner, and a GD-GG-antibody direct mediation agglutination test detection method established based on the surface expression of the S9H bacteroid on the GD-GG can specifically detect PRV infection antibodies, and does not cross-react with other porcine infectious disease antibodies. When two amino acids GG in the GD-GG sequence are mutated into RR, i.e., GPGGGD is mutated into GPRRGD, the specific combination reaction with the PRV infection antibodies does not occur. The diagnosis method has the advantages of high specificity, convenient operation and intuitive result determination, and is expected to become a technical means for precise visual diagnosis of PRV infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical and immunodiagnostic detection, and particularly relates to a B cell epitope peptide based on gE protein conservation and application thereof in diagnosis of porcine pseudorabies virus antibody, and especially to a characteristic sequence GPGGGD (GD-GG) of a B cell epitope peptide on a surface envelope glycoprotein (gE) of a porcine pseudorabies virus and a GD-GG-antibody direct-mediated agglutination test for detecting porcine pseudorabies virus infection antibody in a pig infected with a porcine pseudorabies virus, and accurate diagnosis of porcine pseudorabies virus infection and potential application. BACKGROUND

[0002] Porcine pseudorabies is an acute infectious disease caused by porcine pseudorabies virus (PRV) infection. Clinically, pigs of different ages exhibit different clinical symptoms, sows are characterized by reproductive disorders, newborn piglets exhibit neurological symptoms, and the mortality rate is as high as 100%, and pigs in the nursery and fattening stages exhibit respiratory distress and growth inhibition. Pigs are the natural host and reservoir of PRV, and pigs that have recovered carry the virus for a long time, which seriously hinders the development of the pig industry. Surface envelope glycoprotein gE is an important virulence factor of porcine pseudorabies virus. Since the successful construction of the gE gene deletion attenuated live vaccine strain Bartha-K61 in 1961, the world has effectively controlled the prevalence of porcine pseudorabies. However, after 2011, PRV infection was gradually reported in many domestic pig farms immunized with Bartha-K61, indicating that the traditional Bartha-K61 vaccine cannot effectively prevent infection with new strains. At present, the prevention and control of pseudorabies is becoming increasingly severe. The development of effective new vaccines is needed for the prevention and control of pseudorabies, and suitable detection methods are also needed for the monitoring of disease occurrence. The ultimate goal of epidemic prevention and control is the complete elimination of pathogens, and the elimination of wild virus-infected individuals from immunized pig populations is an effective measure for PRV purification. New Zealand and the United States successfully eliminated PRV by using a gE gene deletion vaccine in combination with a DIVA (distinguishing infected from vaccinated animals) detection method targeting the gE gene, gE protein, or anti-gE protein antibody. In the process of screening a large number of infected pigs, serological detection methods are the most economical and efficient, and are commonly used for PRV serum antibody detection. The enzyme-linked immunosorbent assay (ELISA), gel agglutination test (LAT), and serum virus neutralization test (SVNT) have limited specificity and sensitivity, and are time-consuming in laboratory detection, which is not convenient for clinical promotion. Therefore, it is urgent to develop a specific and convenient serological detection method to identify and detect PRV wild virus infection.

[0003] Among the existing PRV detection methods, the common detection targets are glycosylated proteins gB, gC, gD, gE and gI on the surface of virions. The structural proteins of the above viruses have good immunogenicity and high homology (>95%). After investigation, it is found that gE gene is the main missing target of attenuated vaccine, and the most commonly used pseudorabies virus vaccine in China is missing gE gene, so the gE protein antibody in pigs is specific to PRV wild virus infection, and the gE protein antibody cannot be produced in pigs immunized by gE gene deletion attenuated vaccine. gE antibody is a marker of wild virus infection, and gE antibody is also called infection antibody below to distinguish the immune antibody (not containing anti-gE antibody) induced by gE gene deletion attenuated vaccine. Therefore, a convenient agglutination test detection method targeting gE antibody can detect wild virus infection and help to distinguish infected pig population and vaccinated pig population. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0005] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0006] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0007] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0008] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0009] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0010] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0011] The technical problem to be solved by the present application is to provide a conserved B cell epitope peptide on the surface envelope glycoprotein of porcine pseudorabies virus.

[0012] Technical solution: In order to solve the above technical problems, the present application provides a pig pseudorabies virus surface envelope glycoprotein (gE) on the conservation of B cell epitope peptide, the amino acid sequence of the conservation of B cell epitope peptide is GPGGGD, named GD-GG.

[0013] The second aspect of the present application provides a nucleic acid molecule encoding the conservation of B cell epitope peptide, the nucleotide sequence of the conservation of B cell epitope peptide is GGCCCCGGCGGCGGCGAC, named AG1.

[0014] The third aspect of the present application provides a recombinant gene DNA fragment, which is obtained by introducing the nucleic acid molecule into the Peg pilus operon coding gene sequence, and the sequence of the recombinant gene DNA fragment is SEQ ID NO. 1.

[0015] The fourth aspect of the present application provides an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain, which contains the nucleic acid molecule of the conservation of B cell epitope peptide or the recombinant gene DNA fragment.

[0016] The fifth aspect of the present application provides a pig pseudorabies virus infection antibody specific detection system, which comprises the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain.

[0017] The sixth aspect of the present application provides a construction method of the recombinant vector, comprising the following steps:

[0018] (1) obtaining the DNA sequence of the nucleic acid molecule of the pig pseudorabies virus surface envelope protein conservative B cell epitope, and the DNA sequence is GGCCCCGGCGGCGGCGAC;

[0019] (2) introducing the DNA sequence obtained in step (1) into the Peg pilus operon coding gene sequence to construct a recombinant gene DNA fragment;

[0020] (3) connecting the recombinant gene DNA fragment obtained in step (2) to the vector to construct a recombinant vector.

[0021] The seventh aspect of the present application provides a construction method of the recombinant strain, which is obtained by introducing the recombinant vector into the vector bacteria by electroporation.

[0022] The eighth aspect of the present application provides the application of the conservation of B cell epitope peptide, the nucleic acid molecule, the recombinant gene DNA fragment, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain, and the detection system in the preparation of pig pseudorabies virus infection antibody specific detection reagent or kit.

[0023] The ninth aspect of the present application provides a reagent or kit for detecting porcine pseudorabies virus infection antibody, which comprises the conserved B cell epitope peptide, the nucleic acid molecule, the recombinant gene DNA fragment, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain, and the detection system.

[0024] The porcine pseudorabies virus gE conserved linear B cell epitope peptide of the present application has a sequence of GPGGGD, which is named as GD-GG. The B cell epitope can be functionally expressed on the surface of the inert carrier S9H bacterium, and the GD-GG-antibody direct mediation agglutination test detection method established based on the surface expression of GD-GG on the S9H bacterium can specifically detect only the porcine pseudorabies virus infection antibody, and has no cross reaction and non-specific binding reaction with other porcine infectious disease antibodies. However, if only two amino acids GG in the sequence of GD-GG are mutated to RR, i.e., GPGGGD is mutated to GPRRGD (GD-RR), no specific binding reaction with the porcine pseudorabies virus infection antibody occurs.

[0025] Advantages: Compared with the existing detection technology, the present application has the following significant advantages: the B cell epitope GD-GG of PRVgE of the present application is from the conserved antigen epitope determinant domain of gE, the functional B cell epitope GD-GG is expressed on the Peg pilus, and the GD-GG-antibody direct mediation agglutination test is established to detect the PRV infection antibody in the PRV infected pig. The functional B cell epitope GD-GG expressed on the surface of the bacterium specifically recognizes and binds the PRV infection antibody, thereby realizing specific, convenient and rapid detection of the PRV infection antibody. The specific detection system of the PRV infection antibody only has specific binding reaction with the PRV infection antibody, and has no cross reaction and non-specific reaction with the PRV vaccine immune antibody (not containing gE antibody) of the gE gene deletion PRV and other infectious disease pathogens, and can be used to identify the wild PRV infected pig from the immune pig group. The PRV infection in the pig group can be detected by the PRV infection antibody by the technical method of the present application. Since the present application has strong specificity and simple operation, it is convenient for clinical popularization and application, and is expected to become an important technical means for detecting PRV infection and a technical and platform for PRV pathogen purification. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a diagram of the bioinformatics software analysis results of the PRV gE B cell epitope. A: 56 DDLNGDLDGDDR 67 Conservative analysis comparison results; B: 157 PEVPR 161 Conservative analysis comparison results; C: 396 GPGGGD401 Conservative analysis alignment results.

[0027] Figure 2 Electrophoresis map for PCR amplification identification of recombinant gene DNA fragment peg-gE-AG1 for introduction. Lane M is Trans 2K Plus II DNA Marker, lane 1 is the genome of avian pathogenic E. coli isolate APEC-XM as negative control, lane 2 is the PCR amplification product of peg pilus operon gene, the template DNA is from chicken white dysentery Salmonella standard strain CVCC 526, as positive control, lane 3 is the PCR amplification product of peg-gE-AG1 (4847bp), lane 4 is the PCR amplification product of peg-CU2 (4847bp).

[0028] Figure 3 Electrophoresis map for enzyme digestion identification of recombinant vector pBR-Peg-gE-AG1 containing peg-gE-AG1. Lane M is Trans 2K Plus II DNA Marker; lane 1 is pBR322 circular plasmid; lane 2 is pBR-Peg-gE-AG1 recombinant vector (circular recombinant plasmid); lane 3 is the double enzyme digestion product of pBR-Peg-gE-AG1 with Nhe I and BamH I. Lane 4 is pBR-Peg-CU2 recombinant vector (circular recombinant plasmid); lane 5 is the double enzyme digestion product of pBR-Peg-CU2 with Nhe I and BamH I.

[0029] Figure 4 Schematic diagram of recombinant vector pBR-Peg-gE-AG1 containing gE-AG1.

[0030] Figure 5 Schematic diagram of recombinant vector pBR-Peg-CU2 containing CU2.

[0031] Figure 6Agglutination reaction results of S9H-pBR-Peg-gE-AG1 and control systems S9H-pBR-Peg, S9H-pBR-Peg-CU2 with PRV gE antibody positive serum, other pathogen antibody positive serum or negative serum. A: Agglutination reaction results of S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) with PRV gE antibody positive serum, white arrow indicates agglutination particles; B: S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) do not agglutinate with porcine reproductive and respiratory syndrome virus immune antibody positive serum; C: S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) do not agglutinate with Mycoplasma hyopneumoniae immune antibody positive serum; D: S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) do not agglutinate with Mycoplasma hyosynoviae immune antibody positive serum; E: S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) do not agglutinate with SPF pig serum; F: S9H-pBR-Peg (left), S9H-pBR-Peg-CU2 (middle), S9H-pBR-Peg-gE-AG1 (right) do not agglutinate with E. coli F4 fimbriae monoclonal antibody. DETAILED DESCRIPTION

[0032] Before further description of the specific embodiments of the application, it is to be understood that the application is not limited to the particular specific embodiments described as such and as such, the terminology used in the description is for the purpose of describing the particular specific embodiments only and is not intended to limit the scope of the present application. The test methods used in the following examples, unless otherwise indicated, were carried out according to the conventional conditions or according to the conditions recommended by the respective manufacturers.

[0033] When the examples give numerical ranges, it is to be understood that, unless the application indicates otherwise, every numerical range is to be understood to include both the two endpoints and any number within the range. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to specific methods, devices, materials, etc. described in the examples, any methods, devices, and materials similar or equivalent to those described in the examples can be used in the practice of the present application, according to the knowledge of those skilled in the art and the teachings of the present application.

[0034] Example 1 PRV gE protein B cell epitope prediction and dominant B cell epitope selection

[0035] The PRV gE protein information was retrieved through the NCBI (https: / / www.ncbi.nlm.nih.gov / ) Protein database, and the amino acid sequences of the gE proteins of 15 representative PRV isolates at home and abroad were selected, including classic strains and new variant strains. The names and detailed information of the gE protein amino acid sequences of each PRV isolate are as follows: Becker: NCBI accession number AEM64110.1, Ea: NCBI accession number AAD51327.1, Fa: NCBI accession number AAK95639.1, HB: NCBI accession number UBF23510.1, HeN1: NCBI accession number AJW72299.1, HLJ8: NCBI accession number AML81247.1, HN1201: NCBI accession number ALT14286.1, HNB: NCBI accession number AKG94161.1, HNX: NCBI accession number AKG94030.1, JS-2012: NCBI accession number AKP23873.1, LC: NCBI accession number AWM99391.1, LY: NCBI accession number QFR99244.1, SC: NCBI accession number AJE29736.1, TJ: NCBI accession number AIT55805.1, ZJ01: NCBI accession number AJD79548.1. The amino acid sequences of the 15 PRV gE proteins were aligned and analyzed for identity using the (DNAStar MegAlign) software. The results showed that the PRV gE protein amino acid sequence identity was 97.65% ± 2.35%, indicating that the PRV gE protein amino acid sequences of various strains had a high degree of conservation.

[0036] Based on the conservation of the PRV gE protein amino acid sequence, one of the gE protein amino acid sequences, Ea: NCBI accession number AAD51327.1, was further selected. First, the transmembrane domain was analyzed (protein transmembrane structure online analysis program TMHMM-2.0: https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). The results showed that the 1-430 amino acids of the PRV Ea strain gE protein were located outside the viral membrane, indicating that this region may contain B cell epitope peptides directly recognized by B cell receptors (BCR).

[0037] B-cell epitope prediction was performed on the amino acid sequence using B-cell epitope prediction software BepiPred-3.0 (https: / / services.healthtech.dtu.dk / services / BepiPred-3.0 / ), which scores each amino acid residue, and amino acid residues with scores higher than the default threshold (0.1512) are defined as part of a B-cell epitope, and polypeptides composed of consecutive amino acids with scores higher than the threshold (0.1512) are defined as B-cell epitope peptides. The results showed that the PRV Ea strain gE protein 1-430 amino acids contained 3 epitope peptides, respectively 56 DDLNGDLDGDDR 67 、 157 PEVPR 161 、 396 GPGGGD 401 Conservative analysis of the above 3 B-cell epitope peptides was performed using SnapGene 6.0.2 software. Among the gE protein amino acid sequences of 15 different PRV strains, the epitope peptide 56 DDLNGDLDGDDR 67 had 2 amino acid differences with the gE protein amino acid sequence of the Becker strain (NCBI accession number AEM64110.1); the epitope peptide 157 PEVPR 161 、 396 GPGGGD 401 was completely consistent among the gE protein amino acid sequences of 15 different PRV strains, and was highly conserved.

[0038] To ensure that the selected B-cell epitope peptides are highly specific in their herpes viruses and do not cross-react with other herpes virus antibodies, the above epitope peptides were aligned with the human herpes virus type 1 gE protein amino acid sequence (HSV-1 gE protein NCBI accession number CAF24756.1), the human herpes virus type 2 gE protein amino acid sequence (HSV-2 gE protein NCBI accession number ABU45436.1), the varicella-zoster virus gE protein amino acid sequence (VZV gE protein NCBI accession number NP_040190.1), and the Marek's disease virus gE protein amino acid sequence (MDV gE protein NCBI accession number AAA64969.1). Considering the complexity and strict specificity of the epitope peptides, according to the alignment results, the longer, conserved and specific epitope peptide 157 PEVPR 161 、 396 GPGGGD 401 was preferred 396 GPGGGD 401(named as GD-GG) for constructing a specific detection method for antibodies suitable for infection of various different PRV strains, and the above alignment results are shown in Figure 1 .

[0039] Example 2, Construction of a recombinant strain expressing the gE-conserved B cell epitope peptide GD-GG and verification of its functional expression

[0040] The nucleic acid molecule AG1 (GGCCCCGGCGGCGGCGAC) of the determined PRV gE-conserved B cell epitope peptide GD-GG and the nucleic acid molecule CU2 in which two amino acids GG of the GD-GG sequence are mutated to RR, i.e., GPGGGD is mutated to GPRRGD (GGCCCCCGTCGTGGCGAC) are respectively introduced into the Salmonella Peg pilus operon encoding gene sequence, and then ligated to the pBR322 plasmid to construct recombinant vectors pBR-Peg-gE-AG1 and pBR-Peg-CU2, and then the recombinant vectors are transformed into the carrier bacteria, and the functional GD-GG expressed on the surface of the carrier bacteria is verified.

[0041] The specific implementation procedure is as follows: Peg-gE-AG1 (4829 bp) and Peg-CU2 (4829 bp) chimeric genes are synthesized by Nanjing Qikexing Biotechnology Co., Ltd. (The reference sequence of the Peg pilus operon gene sequence is Salmonella enterica CYX strain, GenBank accession number CP113540.1, 1702724-1707552), and the recombinant gene DNA fragment obtained by replacing the site GTGAAACCGGATGAGGCG of the peg pilus operon gene sequence with gE-AG1 or CU2 is named peg-gE-AG1, peg-CU2.

[0042] The nucleotide sequence of the peg-gE-AG1 recombinant gene DNA fragment is shown in SEQ ID NO. 1, and the nucleotide sequence of the peg-CU2 recombinant gene DNA fragment is shown in SEQ ID NO. 2.

[0043] SEQ ID NO. 1:

[0044]

[0045]

[0046]

[0047]

[0048] SEQ ID NO. 2:

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Nanjing Qingke Biotechnology Co., Ltd. synthesized primers for amplifying the PEG fimbriae operon. The upstream primer is Peg-F: 5'-CGC. GCTAGC ATGAAACGTTCACTTATTGCTGCT-3'; Downstream primer Peg-R: 5'-CGT GGATCC TTAATTATAAGATACCACGATTAATGC-3', where the underlined sequences represent the Nhe I and BamHI restriction sites, respectively. Using the recombinant gene DNA fragments mentioned above as templates (SEQ ID NO.1 or SEQ ID NO.2, 1 μL, containing 1 ng of chimeric gene), the genome of the standard strain of Salmonella pullorum CVCC 526 (purchased from the China Institute of Veterinary Drug Control and the China Veterinary Microbial Culture Collection Center) was used as a positive control, and the genome of the avian pathogenic Escherichia coli isolate APEC-XM (Liu Jiaqi, Wu Hucong, Yin Yi, Zhang Dong, Xia Pengpeng, Ren Wenkai, Zhu Guoqiang. Research on the construction of a mouse model of neonatal Escherichia coli-induced meningitis by avian Escherichia coli [J]. Chinese Poultry, 2019, 41(10):26-30.) was used as a negative control. PCR amplification was performed using upstream and downstream primers. The amplification system was: 2 μL of pfu high-fidelity DNA polymerase (Beijing TransGen Biotech Co., Ltd., 2.5 U / μL), 10 μL of 5×pfu DNA polymerase as e buffer, and dNTPs. 5 μL of each of forward and reverse primers (10 mM), 2 μL of recombinant gene DNA fragment (1 ng / μL) or APEC-XM genome (250 ng / μL) or CVCC 526 genome (250 ng / μL), and 27 μL of ultrapure water were mixed thoroughly. PCR amplification was then performed using a Bio-Red thermal cycler with the following program: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 52℃ for 30 s, and extension at 72℃ for 5 min, for a total of 30 cycles. Further amplification was performed at 72℃ for 10 min. After amplification, the temperature was lowered to 12℃. A 1.5% agarose gel was prepared, electrophoresed at 100V for 50 min, stained with ethidium bromide, and imaged under UV light. The PCR results are shown below. Figure 2The PCR product of peg-gE-AG1 is 4847 bp, the PCR product of peg-CU2 is 4847 bp, the amplification size of the positive control is 4850 bp, and the negative control has no amplification band. The PCR amplification products (4847 bp) of peg-gE-AG1 and peg-CU2 were recovered using a universal DNA purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0055] The PCR amplification products of Peg-gE-AG1 and peg-CU2 were double digested with Nhe I and BamH I restriction enzymes (NEB), and then the linear DNA fragments were ligated using T4 DNA ligase (NEB) at 16°C metal bath overnight. The ligation system was as follows: pBR322 plasmid 2 μL (40.4 ng / μL), Peg-gE-AG1 fragment 6 μL (69.5 ng / μL) or peg-CU2 fragment 6 μL (63.7 ng / μL), T4 DNA ligase (NEB, 400 U / μL) 1 μL, 10×T4 DNA ligase reaction buffer 2 μL, ddH2O 9 μL, a total of 20 μL ligation system. The next day, the ligation product was transformed into S9H competent cells (S9H inert vector bacteria were derived from Chinese invention patent ZL202010427735.8 A universal inert vector Salmonella and its potential applications), and resistance screening was performed by coating with 100 μg / mL ampicillin solid medium. A single colony on the plate was inoculated into 100 μg / mL ampicillin LB liquid medium and grown to the platform phase, and the plasmid was recovered and double digested with Nhe I and BamH I restriction enzymes. A 1.0% agarose gel was prepared, electrophoresed at 100 V for 50 min, stained with ethidium bromide, and imaged under a UV imager. The results are shown in Figure 3 As shown, the Nhe I and BamH I double digestion products of the recombinant vectors pBR-Peg-gE-AG1 and pBR-Peg-CU2 contain a 4215 bp linear vector and a 4835 bp Peg-gE-AG1 or Peg-CU2 linear DNA fragment, which is consistent with the expected size. The schematic diagram of the recombinant vector pBR-Peg-gE-AG1 is shown in Figure 4 As shown, the schematic diagram of the recombinant vector pBR-Peg-CU2 is shown in Figure 5 As shown, the recombinant bacteria S9H-pBR-Peg-gE-AG1 and S9H-pBR-Peg-CU2 were screened.

[0056] The recombinant bacteria S9H-pBR-Peg-gE-AG1 and S9H-pBR-Peg-CU2 were grown in LB liquid medium containing 100 μg / mL ampicillin to the stationary phase, centrifuged at 4000 rpm for 5 min, and resuspended with an equal volume of sterile saline. This process was repeated twice to prepare the bacterial suspension (final concentration of 1 × 10 10 The prepared bacterial suspensions of the recombinant bacteria S9H-pBR-Peg-gE-AG1 and S9H-pBR-Peg-CU2 were subjected to agglutination test with PRV gE antibody-negative serum (10 samples of SPF pig serum, provided by the Animal Hospital of Zhejiang Agriculture and Forestry University), Salmonella Peg antibody-positive serum (10 samples, prepared and preserved by the experiment), PRV gE antibody-positive serum (10 samples of clinical samples, provided by Zhejiang Meibaolong Biotechnology Co., Ltd.), and PRV gE gene deletion attenuated live vaccine immunized pig serum (10 samples, provided by Zhejiang Meibaolong Biotechnology Co., Ltd.), respectively. The bacterial suspension of S9H engineering bacteria containing only pBR-Peg (final concentration of 1 × 10 10 The results are shown in Table 2. The bacterial suspensions of the three strains did not react with PRV gE antibody-negative serum. The bacterial suspensions of the three strains agglutinated with Peg antibody-positive serum, indicating that the functional Peg was successfully expressed on the surface of the carrier bacteria. The bacterial suspension of the recombinant S9H engineering bacteria S9H-pBR-Peg-gE-AG1 carrying the recombinant vector pBR-Peg-gE-AG1 showed obvious agglutination with PRV gE antibody-positive serum, with large agglutination particles and clear background. The bacterial suspension of S9H-pBR-Peg and the bacterial suspension of S9H-pBR-Peg-CU2 did not agglutinate with PRV gE antibody-positive serum, with no agglutination particles and a uniform turbid background. This result indicates that the PRV gE protein B cell epitope GD-GG can be expressed on the surface of the bacteria through the Peg pilus, and can specifically recognize and detect PRV gE antibody. However, when two amino acids GG in the GD-GG sequence are mutated to RR, i.e., GPGGGD is mutated to GPRRGD (GD-RR), no specific binding reaction with PRV gE antibody occurs.

[0057] In the antibody detection system, S9H-pBR-Peg bacterial suspension is used as a control system, and the antibody detection system only adds the B cell epitope GD-GG of PRV gE. The difference between S9H-pBR-Peg-CU2 and S9H-pBR-Peg-gE-AG1 is two amino acids, which excludes non-specific reactions. S9H-pBR-Peg-gE-AG1 specifically recognizes and binds to specific antibodies, thereby ensuring accurate diagnosis of individuals.

[0058] Table 2, verification of the function of the carrier bacteria S9H surface presentation expression Peg-gE-AG1

[0059]

[0060] Example 3, PRV infection antibody specificity diagnosis and cross-reaction test

[0061] Based on the functional verification of S9H-pBR-Peg-gE-AG1 expressed by the carrier bacteria S9H surface presentation, the PRV infection antibody specificity diagnosis and cross-reaction were tested, and the specific implementation procedures were as follows:

[0062] The control system S9H-pBR-Peg bacterial suspension, S9H-pBR-Peg-CU2 bacterial suspension and detection system S9H-pBR-Peg-gE-AG1 bacterial suspension (all with a final concentration of 1×10 10 CFU / mL) were prepared according to the method described in Example 2. Agglutination test was performed with different pathogen antibody positive serum, including 10 PRV gE antibody positive pig serum (provided by Zhejiang Meibaolong Biotechnology Co., Ltd.), 5 porcine reproductive and respiratory syndrome virus (PRRSV) immune antibody positive serum (provided by Zhejiang Meibaolong Biotechnology Co., Ltd.), 20 Mycoplasma hyopneumoniae immune antibody positive serum (provided by Taizhou Huaweite Biopharmaceutical Co., Ltd.), 6 Mycoplasma hyosynoviae immune antibody positive serum (provided by Jiangsu Academy of Agricultural Sciences), 25 SPF pig serum (provided by Zhejiang Agricultural University Animal Hospital), and 1 Escherichia coli F4 pilus monoclonal antibody (prepared and preserved by the laboratory). The detection results are shown in Figure 6 As shown in Table 3, in the PRV infection antibody specificity diagnosis, only specific agglutination reaction occurred with PRV gE antibody positive serum, and no cross agglutination reaction occurred with other infectious disease pathogen immune antibody positive serum, indicating that the detection system has good specificity.

[0063] Table 3, verification results of PRV infection antibody specificity diagnosis and cross-reaction

[0064]

[0065] Note: “-” indicates negative agglutination reaction; “+” indicates positive agglutination reaction.

[0066] Example 4, Test application of PRV infection antibody specificity detection system to clinical samples

[0067] The control system S9H-pBR-Peg bacterial suspension, the S9H-pBR-Peg-CU2 bacterial suspension and the detection system S9H-pBR-Peg-gE-AG1 bacterial suspension were prepared according to the method described in Example 2 (all with a final concentration of 1 x 10 10 The above three bacterial suspensions were respectively subjected to agglutination test with PRV gE antibody positive serum and PRV gE gene deletion live attenuated vaccine immunized pig serum. The serum and source involved in the detection included 100 PRV gE antibody positive pig serum (clinical infection pig serum, provided by Zhejiang Meibaolong Biotechnology Co., Ltd.) and 100 gE gene deletion live attenuated vaccine immunized serum (provided by Zhejiang Meibaolong Biotechnology Co., Ltd.). The detection results are shown in Table 4. The PRV infection antibody specificity detection system only agglutinates with the pig PRV gE antibody positive serum, and does not agglutinate with the PRV gE gene deletion live attenuated vaccine immunized pig serum (gE antibody negative), indicating that the present application can differentially diagnose PRV wild virus infection individuals and gE gene deletion live attenuated vaccine immunized individuals.

[0068] Table 4, Clinical sample detection results of pig PRV infection antibody specificity detection system

[0069]

[0070] Note: "-" indicates agglutination negative; "+" indicates agglutination positive.

Claims

1. A conserved B-cell epitope peptide on the surface glycoprotein of porcine pseudorabies virus, characterized in that, The amino acid sequence of the conservative B cell epitope peptide is GPGGGD.

2. A nucleic acid molecule encoding the conserved B cell epitope peptide of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule of the conservative B cell epitope peptide is GGCCCCGGCGGCGGCGAC.

3. A recombinant genetic DNA segment, comprising, The recombinant gene DNA fragment is obtained by introducing the nucleic acid molecule of claim 2 into the gene sequence of the Peg pilus operon, and the sequence of the recombinant gene DNA fragment is shown as SEQ ID NO.

1.

4. An expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the nucleic acid molecule of the conservative B cell epitope peptide of claim 2 or the recombinant gene DNA fragment of claim 3.

5. A detection system for antibodies specific to porcine pseudorabies virus infection, characterized by, The specific detection system for the antibody against the porcine pseudorabies virus infection comprises the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain of claim 4.

6. The method of constructing a recombinant vector of claim 4, wherein, The method comprises the following steps: (1) obtaining the DNA sequence of the nucleic acid molecule of the conservative B cell epitope of the surface membrane protein of the porcine pseudorabies virus, wherein the DNA sequence is GGCCCCGGCGGCGGCGAC; (2) introducing the DNA sequence obtained in step (1) into the gene sequence of the Peg pilus operon to construct a recombinant gene DNA fragment; (3) connecting the recombinant gene DNA fragment obtained in step (2) to a vector to construct a recombinant vector.

7. The method of constructing a recombinant strain of claim 4, wherein, The recombinant vector of claim 4 is introduced into a vector bacterium by means of electrotransformation.

8. The use of the conservative B cell epitope peptide of claim 1, the nucleic acid molecule of claim 2, the recombinant gene DNA fragment of claim 3, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain of claim 4, or the detection system of claim 5 in the preparation of a specific detection reagent or kit for the antibody against the porcine pseudorabies virus infection.

9. A reagent or kit for detecting antibodies to porcine pseudorabies virus infection, characterized by, The reagent or kit comprises the conservative B cell epitope peptide of claim 1, the nucleic acid molecule of claim 2, the recombinant gene DNA fragment of claim 3, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain of claim 4, or the detection system of claim 5.

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