Mouse anti-porcine herpesvirus type 1 gE monoclonal antibody, immunogen and application thereof

Abstract

The invention relates to the technical field related to immunological detection, in particular to a mouse anti-porcine herpesvirus type 1 gE monoclonal antibody as well as an immunogen and application of the mouse anti-porcine herpesvirus type 1 gE monoclonal antibody. The amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively as follows: GFSLSTSGMG, IVWGSETGRVTISRDNSK and VRYYDGDDD, and the amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a light chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively as follows: KSSQSLLYSDGKTFLN, LGSNRAS and SSLPHED. The monoclonal antibody can be specifically combined with gE proteins of all subtypes of the porcine herpesvirus type 1, and a porcine herpesvirus type 1 detection kit prepared from the monoclonal antibody has the advantages of high sensitivity, strong specificity, wide detection range, short detection time and the like.

Description

Technical Field

[0001] This application relates to the technical field of livestock antibody detection, specifically to a mouse anti-swine herpesvirus type 1 gE monoclonal antibody, its immunogen, and its application. Background Technology

[0002] Suid Herpesvirus 1 (SuHV-1), also known as Pseudorabies Virus (PRV), belongs to the Varicellavirus genus of the Alphaherpesvirinae subfamily of the Herpesviridae family. It is a double-stranded DNA virus with a typical herpesvirus structure. It is sensitive to lipid solvents, acids, alkalis, and high temperatures, but has a strong survival ability at low temperatures. This virus has a wide host range, high pathogenicity, and diverse transmission routes. It is the pathogen that causes pseudorabies (PR) in pigs and is also lethal to various livestock and wild animals such as cattle, sheep, dogs, and cats. It is one of the key animal disease pathogens that my country focuses on controlling. Pigs are the only natural reservoir host and asymptomatic carrier. Symptoms vary significantly among pigs of different ages (acute mortality in newborn piglets, reduced mortality in weaned piglets, and often latent infection in adult pigs or reproductive disorders in sows). In other mammals, infection is almost 100% fatal, and humans are not easily infected. Transmission routes include direct contact, indirect contact, vertical transmission, airborne transmission, and transmission by wild animals (such as wild boars), and it also has latent infection characteristics. Diagnosis requires a combination of clinical and laboratory tests. Real-time fluorescent PCR is rapid and sensitive in etiological testing, while gE-ELISA can differentiate between vaccine immunization and wild-type virus infection in serological testing. Prevention and control are centered on "vaccine immunization + biosafety + purification." There are no specific treatment drugs. Gene-deleted vaccines are the mainstream immunization method, which must be combined with strict isolation, introduction quarantine, environmental disinfection, and other biosafety measures. Large-scale pig farms can achieve virus purification by regularly culling positive pigs through gE-ELISA testing to reduce its harm to the livestock industry.

[0003] The detection of pseudorabies gE antibodies is the core means of distinguishing between wild-type virus infection and vaccine immunization. The commonly used methods are mainly enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and indirect immunofluorescence assay (IFA).

[0004] ELISA testing carries risks of cross-reactivity, has limited sensitivity, and takes a long time (typically 1-2 hours). Western blotting (WB) is cumbersome, time-consuming (3-4 hours or even longer), has low throughput (only a small number of samples can be tested at a time), and requires highly skilled operators. IFA testing relies on fluorescence microscopy, which is expensive and limits its application at the grassroots level. Interpretation is subjective and prone to human error. Furthermore, all of these methods rely on manual operation and cannot achieve fully automated testing. Summary of the Invention

[0005] To address the above issues, this application provides a mouse anti-porcine herpesvirus type 1 gE monoclonal antibody, its immunogen, and its applications. This monoclonal antibody, as a detection antibody, can specifically bind to the porcine herpesvirus type 1 antigen and can be applied to ELISA and CLIA assays, improving detection sensitivity and specificity while shortening detection time.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment, wherein the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment are amino acid sequences at positions 26-35, 50-57, and 88-96 of SEQ ID NO. 6, respectively; and the complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain variable region are amino acid sequences at positions 24-39, 55-61, and 95-102 of SEQ ID NO. 7, respectively.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the mouse anti-swine herpesvirus type 1 gE monoclonal antibody is as shown in SEQ ID NO. 6, or has 80% or more sequence similarity to SEQ ID NO. 6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 7, or has 80% or more sequence similarity to SEQ ID NO. 7.

[0009] Secondly, the present invention provides a nucleic acid molecule that encodes the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment.

[0010] Preferably, the nucleotide sequence encoding the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment heavy chain variable region is as shown in SEQ ID NO. 3, or has 80% or more sequence similarity to SEQ ID NO. 3; the amino acid sequence encoding the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment light chain variable region is as shown in SEQ ID NO. 4, or has 80% or more sequence similarity to SEQ ID NO. 4.

[0011] Thirdly, the present invention provides a biomaterial containing the aforementioned nucleic acid molecule; the biomaterial is an expression cassette, a vector, or a host cell.

[0012] Fourthly, the present invention provides a murine antibody conjugate, which is obtained by conjugating the murine antibody or its antigen-binding fragment or a combination thereof with a label, wherein the label is selected from one or more of horseradish peroxidase label, biotin label, chemiluminescent material label, nanomaterial label, and radioactive label.

[0013] Preferably, the marker is selected from horseradish peroxidase markers;

[0014] Preferably, the marker is selected from biotin.

[0015] Fifthly, the present invention provides a kit comprising the mouse antibody or its antigen-binding fragment, or the antibody conjugate, or the composition thereof.

[0016] Preferably, the kit is an ELISA antibody detection kit or a magnetic microparticle CLIA antibody immunoassay kit.

[0017] In a sixth aspect, the present invention provides a porcine herpesvirus type 1 gE multi-epitope fusion protein, the amino acid sequence of which is shown in SEQ ID NO. 5.

[0018] Beneficial effects:

[0019] (1) The murine antibody and its conjugate or composition provided by the present invention can specifically bind to the gE protein of any subtype of porcine herpesvirus type 1, and have no cross-reaction with other similar proteins, thus exhibiting high specificity.

[0020] (2) Based on this monoclonal antibody, a competitive ELISA method for detecting porcine herpesvirus type 1 gE antibody was established. It has high sensitivity, specificity and universality, and can accurately and quickly detect the level of porcine herpesvirus type 1 gE antibody in serum.

[0021] (3) Based on this monoclonal antibody, the competitive magnetic microparticle CLIA method for detecting porcine herpesvirus type 1 gE antibody has higher sensitivity, specificity and broad-spectrum adaptability. It can achieve accurate, high-throughput, rapid and fully automated detection of porcine herpesvirus type 1 gE antibody in serum and has good application prospects in the detection of porcine herpesvirus type 1 gE antibody. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0023] Figure 1 The image of the recombinant plasmid pET28a-SuHV-1-gE in Example 1 of this invention is shown.

[0024] Figure 2 This is the PCR identification result of the recombinant plasmid pET28a-SuHV-1-gE in Example 1 of the present invention;

[0025] Figure 3 This is a diagram showing the SDS-PAGE electrophoresis identification results of Example 1 of the present invention.

[0026] Among them, M1: Trans2K ® DNA Marker; 1: PCR product of empty vector pET28a; 2: PCR product of recombinant plasmid pET28a-SuHV-1-gE; M2: Protein molecular weight marker; 3: Protein yield after 8 hours of induction; 4: Protein yield after 12 hours of induction. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0028] Based on the embodiments of this invention, all other implementations obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The examples given are for illustrative purposes only and are not intended to limit the scope of this invention.

[0029] The above technical solution will be described in detail below with reference to specific embodiments.

[0030] Example 1: Expression of multi-epitope fusion proteins

[0031] (1) Preparation of the coding gene: 56 SuHV-1 strains published by NCBI were analyzed, and the highly conserved gE protein (Genbank accession number: MN443975.1) of the domestically prevalent representative strain JX18-2 was selected as the target protein region. B-cell epitopes were predicted from the nucleotide sequence of the gE protein using bioinformatics, and linear dominant epitopes were selected as the target gene epitopes for the multi-epitope fusion protein. The nucleotide sequences encoding the dominant epitope and the flexible linker peptide were ligated at the DNA level according to the correct reading frame, as shown in SEQ ID NO.1. The optimized coding gene sequence was obtained according to the codon preference of *E. coli*, as shown in SEQ ID NO.2.

[0032] (2) Construction of recombinant plasmid: The optimized coding gene sequence was ligated into the prokaryotic expression vector pET-28a using DNA splicing technology to obtain the recombinant plasmid pET28a-SuHV-1-gE. The recombinant plasmid map is shown below. Figure 1As shown. PCR identification was performed using the universal primers T7 / T7ter for the pET-28a vector, and the results are as follows. Figure 2 As shown.

[0033] (3) Expression of multi-epitope fusion proteins: The successfully ligated recombinant plasmid pET28a-SuHV-1-gE was transformed into BL21(DE3) competent cells. The transformed bacteria were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm for about 4 hours until OD500 was reached. 600 The concentration was 0.6. IPTG was added to a final concentration of 0.2 mmol / L, and the cells were collected after induction at 28°C and 100 rpm for 12 hours.

[0034] (4) Purification of the multi-epitope fusion protein: Crude purification was performed using a nickel ion affinity chromatography column, followed by further purification using a protein G purification column. The purified multi-epitope fusion protein is the porcine herpesvirus type 1 gE protein multi-epitope fusion protein. The amino acid sequence is shown in SEQ ID NO. 5, and the SDS-PAGE results are as follows: Figure 3 As shown. By Figure 1 It is known that the size of the multi-epitope fusion protein SEQ ID NO.5, which encodes gene SEQ ID NO.2, is 18.0 kDa.

[0035] (5) The purity of the multi-epitope fusion protein was determined by micro-spectrophotometer. The A280 / A260 ratio was 1.8 to 2.0, indicating high protein purity. It can be used for coating, labeling, detection, etc.

[0036] Example 2: Preparation of Monoclonal Antibodies

[0037] (1) Immunization of animals: Three BALB / c mice were immunized with purified SuHV-1 gE multi-epitope fusion protein, for a total of 4 immunizations. The immunization dose was 100 μg / mouse, and the immunization route was intraperitoneal immunization. For the first immunization, the multi-epitope fusion protein was emulsified with an equal volume of Freund's complete adjuvant. For the second and third immunizations, Freund's incomplete adjuvant was used for emulsification. The fourth immunization was a booster immunization 3 days before fusion, with an immunization dose of 50 μg / mouse, without the addition of adjuvant, and was administered intraperitoneally. After immunization, the spleen of the mice was harvested and ground to obtain splenic lymphocytes.

[0038] (2) Cell fusion: Splenic lymphocytes and myeloma cells (SP2 / 0) were mixed at a ratio of 1:10, and polyethylene glycol (PEG) was added as a fusion agent.

[0039] (3) Screening with HAT selective medium: After cell fusion, the mixed cell suspension was seeded into a 96-well plate containing HAT medium and incubated at 37°C in a 5% CO2 incubator.

[0040] (4) Obtaining cell lines: Recombinant gE protein was used as the coating antigen and indirect ELISA was performed to screen out positive hybridoma cell lines. After being subcloned into single cells by limiting dilution, the cells were expanded and cultured to finally obtain a stable monoclonal antibody cell line, named 2C4.

[0041] (5) Preparation of SuHV-1 gE monoclonal antibody: BALB / c mice were pretreated with liquid paraffin via intraperitoneal injection, and then inoculated with hybridoma cells in the logarithmic growth phase 1-2 weeks later. 5-10 days after inoculation, abdominal distension of the mice was observed, and ascites fluid was collected using a 16-gauge needle. Each mouse could be collected 2-3 times consecutively, yielding a total of 5-10 mL of ascites fluid. The ascites fluid was centrifuged (2000 r / min, 5 minutes) to remove cell debris, preliminarily purified by precipitation with saturated ammonium sulfate, and further purified by ion exchange chromatography to obtain SuHV-1 gE monoclonal antibody with a purity >95%.

[0042] (6) Sequence determination of heavy and light chain variable regions: Hybridoma cells in the logarithmic growth phase were collected and sent to Universal Biotech for sequencing. The amino acid sequences of the heavy chain variable region and the light chain variable region of the SuHV-1 gE monoclonal antibody are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively, and the encoding gene sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0043] Unless otherwise stated, the CDR sequence described in this invention is defined according to the Kabat numbering system.

[0044] Example 3: Labeling of Monoclonal Antibodies

[0045] (1) HRP labeling of monoclonal antibodies

[0046] Weigh 2 mg of HRP and dissolve it in 0.5 mL of distilled water. Add 0.5 mL of freshly prepared 0.06 mol / L NaIO4 solution to the above solution and incubate at 4°C in the dark for 30 min. Add 0.5 mL of 160 mmol / L ethylene glycol to the supernatant and incubate at room temperature for 30 min. Then add 2 mg of the monoclonal antibody to be labeled and mix well.

[0047] The above solution was placed in a dialysis bag and dialyzed in 2000 mL of 0.05 mmol / L CB Buffer (0.05 mol / L CB Buffer: Na2CO3 3.18 g + NaHCO3 5.88 g, with distilled water added to bring the volume to 2 L). The solution was stirred overnight at 4 °C.

[0048] Aspirate the dialysate into a 15 mL centrifuge tube, add 0.2 mL of freshly prepared 5 mg / mL NaBH4 solution, mix well, and incubate at 4 ℃ for 2 h. Remove the dialyzed labeled solution, add an equal volume of glycerol, and store at -20 ℃.

[0049] (2) Biotin labeling of monoclonal antibodies

[0050] Take 1 mg of biotin, bring it to room temperature, add it to 180 μL of ultrapure water, and dissolve and mix well.

[0051] Add 13.5 mg of the prepared SuHV-1 gE monoclonal antibody, mix thoroughly, and let stand at room temperature for 60 min. Add coupling buffer to the ultrafiltration tube, add PBS to the white line at the top of the tube, and centrifuge at 12000 rpm, 4 ℃ for 10 min.

[0052] Discard the waste liquid, wash three times with PBS, and recover the coupling solution.

[0053] Transfer the coupling solution to a clean centrifuge tube, then add an equal volume of glycerol, record the final volume and final concentration, and store at -20 ℃ for later use.

[0054] Example 4: ELISA-based kit for detecting porcine herpesvirus type 1 gE antibodies

[0055] In this embodiment, the main components of the ELISA kit for detecting porcine herpesvirus type 1 gE antibody are: coated microplate, horseradish peroxidase-labeled monoclonal antibody (HRP-labeled antibody), positive control, negative control, diluent, washing solution, chromogenic solution A, chromogenic solution B, and stop solution.

[0056] The preparation method of the coated ELISA plate specifically includes the following steps:

[0057] S401. Antigen Coating: Dilute the multi-epitope fusion protein to a final concentration of 2 μg / mL using CBS buffer (pH=9.6). Add 100 μL of the coating solution to each well of the ELISA plate and coat at 4°C for 12–18 h. Discard the solution in the wells of the ELISA plate. Wash with 300 μL of PBST buffer (0.05% Tween-20, pH=7.4) 5 times, 3 min each time.

[0058] S402. Blocking: Add blocking solution to the microplate obtained in step S401, 200 μL / well, block at 37°C for 2 h, and discard the solution in the wells of the microplate; add PBST buffer (0.05% Tween-20, pH=7.4) to wash, 300 μL / well, wash 5 times, 3 min / time; to obtain the coated microplate.

[0059] The blocking solution was prepared by diluting ovalbumin (OVA) with PBS buffer (pH=7.4), and the concentration of the OVA blocking solution was 5% (w / v).

[0060] Colorimetric solution A: prepared from 3,3',5,5'-tetramethylbenzidine (200 mg) and anhydrous ethanol (100 mL).

[0061] Colorimetric solution B: Add water for injection to 900 mL of Na2HPO4 (14.6 g) and citric acid (9.33 g), adjust the pH to 5.0-5.4, and then add water for injection to bring the volume to 1000 mL.

[0062] In this embodiment, the working principle of the ELISA kit for detecting porcine herpesvirus type 1 (SuHV-1) gE antibodies is as follows: a competitive method is used to determine whether the sample contains porcine herpesvirus type 1 (SuHV-1) gE antibodies. An ELISA plate is coated with SuHV-1 gE antigen (i.e., the multi-epitope fusion protein of this application) to prepare a stationary phase. The sample is added to the wells of the coated ELISA plate, followed by HRP-labeled SuHV-1 gE monoclonal antibody. The gE antibody in the sample and the HRP-labeled gE monoclonal antibody competitively bind to the SuHV-1 gE antigen, forming an antigen-antibody / ELISA antibody complex. After washing, a two-component 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution is added. TMB is converted to blue under the catalysis of HRP, and finally to yellow under acidic conditions. The intensity of the color is negatively correlated with the content of SuHV-1 gE antibody in the sample; the absorbance (OD value) is measured at a wavelength of 450 nm using an ELISA reader, and the presence of SuHV-1 gE antibody in the sample is calculated based on the OD value.

[0063] The working process of the kit for detecting porcine herpesvirus type 1 gE antibody based on ELISA includes the following steps:

[0064] S403. Experimental Preparation: Remove the coated plate and record the sample location. If only some strips are needed, remove the required strips for the experiment and store the remaining strips in a dry place at 2–8℃.

[0065] S404, Sample Addition / Control: Add 20µL of the test sample, negative control, and positive control to the wells of the antigen-coated plate, respectively, with 2 wells for each of the negative and positive controls; note that a different pipette tip should be used for each sample.

[0066] S405, Add enzyme: Add 80µL of enzyme-labeled antibody to each well, gently shake to mix, seal the plate and incubate at 37℃ for 30 minutes;

[0067] The enzyme-labeled antibody was an HRP-labeled SuHV-1 gE monoclonal antibody solution, diluted with PBST buffer (0.05 v / v Tween-20, pH=7.4) at a dilution ratio of 1:10000 (w / v).

[0068] S406 Washing: Discard the liquid in the wells, add 300µL of PBST buffer (0.05% Tween-20, pH=7.4) to each well, wash 4 times, 3min / wash, and after the last PBST buffer is discarded, pat dry the remaining PBST buffer in the wells.

[0069] S407. Color development: Add 100 μL of two-component TMB color development solution (color development solution A and color development solution B are prepared in a 1:1 volume ratio) to the microplate, gently shake to mix, seal the plate and place it at 37°C in the dark for 10 minutes.

[0070] S408, Termination: Add 50µL of stop solution to each well and gently shake the microplate until the color development is uniform;

[0071] S409. Reading: Place the microplate into the microplate reader and measure and read the absorbance OD value under dual wavelength conditions of 450nm test wavelength and 630nm reference wavelength.

[0072] Result determination:

[0073] The OD value of the negative control is denoted as OD. N The OD value of a positive control sample is recorded as OD. P The OD value of the sample to be tested is denoted as OD. S .

[0074] Experimental conditions: OD N Average value - OD P If the average value is ≥0.3, the test result is valid; otherwise, the test should be repeated.

[0075] S / N value = OD S / OD N value

[0076] Result interpretation: S / N value ≤ 0.3, judged as positive; S / N value > 0.3, judged as negative.

[0077] Example 5: Kit for detecting porcine herpesvirus type 1 gE antibody based on CLIA (computed molecular magnetic resonance imaging).

[0078] In this embodiment, the working principle of the kit for detecting porcine herpesvirus type 1 gE antibody based on CLIA (component-based magnetic microparticle immunoassay) is as follows: The test sample is mixed with biotin-labeled monoclonal antibody, acrid ester-labeled multi-epitope fusion protein, and streptavidin magnetic beads and incubated to form an immune complex. After removing unbound impurities by washing with solid-phase carrier magnetic microparticles, a luminescent substrate is added to induce luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is inversely proportional to the porcine herpesvirus type 1 gE antibody titer. The instrument's built-in standard curve outputs the RLU = corresponding porcine herpesvirus type 1 gE antibody titer value.

[0079] In this embodiment, the main components of the kit for detecting porcine herpesvirus type 1 gE antibody based on CLIA magnetic microparticles are: magnetic bead working solution, biotin-labeled antibody working solution, acrid ester-labeled working solution, pre-activation solution, activation solution, calibrator, positive control, and negative control.

[0080] Magnetic bead working solution: prepared by 225 μL of streptomycin affinity magnetic beads and 4275 μL of PBS-BSA solution (pH=7.4).

[0081] Antibody working solution: prepared by 5.5 μg of biotin-labeled monoclonal antibody and 11000 μL of PBS-BSA solution (pH=7.4).

[0082] Acridinium ester labeled working solution: prepared from 1.1 μg of acridinium ester labeled multi-epitope fusion protein and 11000 μL of PBS-BSA solution (pH=7.4).

[0083] Pre-activation solution: 0.1 mol / L hydrochloric acid solution containing 0.1% hydrogen peroxide

[0084] Activation solution: 0.25 M sodium hydroxide solution containing 2% Triton X-100

[0085] The working process of the kit for detecting porcine herpesvirus type 1 gE antibody based on CLIA (computed molecular microparticle) includes the following steps:

[0086] S501, sample (20 μL) + magnetic bead working solution (20 μL) + acrid ester labeled working solution (50 μL) and antibody working solution (50 μL), react at 37°C for 15 min, wash with 0.1 mol / L PBS buffer, add activation solution (100 μL) and activation solution (100 μL) and react at 37°C for 5 min, and detect the luminescence value.

[0087] Result determination: S / N value = luminescence value of the test sample / average luminescence value of the negative control

[0088] Validity condition: (average value of negative control luminescence - average value of positive control luminescence) / average value of negative control luminescence ≥ 0.3, otherwise the test should be repeated.

[0089] Result interpretation: S / N value > 0.25, judged as negative; S / N value ≤ 0.25, judged as positive.

[0090] Example 6: Reagent Kit Performance Testing

[0091] S601, Sensitivity Test

[0092] First, the positive control material for porcine herpesvirus type 1 gE antibody was serially diluted 2-fold using PBS buffer (0.05 v / v Tween-20, pH=7.4) to obtain 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, 512-fold, 1024-fold, 2048-fold, and 4096-fold sensitivity control materials, respectively.

[0093] Then, using sensitivity control samples as samples, the sensitivity of a domestic brand (Wuhan Keqian) porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, the SuHV-1 gE antibody competitive ELISA detection kit of this application, and the SuHV-1 gE antibody competitive magnetic particle CLIA detection kit of this application were tested, and the test results were compared and analyzed. The test results are shown in Table 1. The domestic brand could detect 512 times the sensitivity of the control sample, the SuHV-1 competitive ELISA antibody detection kit of this application could detect 1024 times the sensitivity of the control sample, and the competitive magnetic particle CLIA antibody detection kit of this application could detect 2048 times the sensitivity of the control sample. The two detection methods of this application are more sensitive than the domestic brand.

[0094] Table 1. Sensitivity test comparison results

[0095]

[0096] S602, Specificity Test

[0097] The SuHV-1 gE antibody competitive ELISA detection method and the SuHV-1 gE antibody competitive magnetic particle CLIA detection method constructed in this application were used to simultaneously detect positive sera for SuHV-1 gE, SuHV-1 gB, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and porcine parvovirus (PPV). The results are shown in Table 2. This method showed no cross-reactivity with other susceptible animal viruses and had good specificity.

[0098] Table 2. Specificity test results

[0099]

[0100] S603, Repeatability Test

[0101] Four swine serum samples with known background (one strongly positive SuHV-1 gE antibody sample, one positive SuHV-1 gE antibody sample, one weakly positive SuHV-1 gE antibody sample, and one negative SuHV-1 gE antibody sample) were selected and subjected to 20 replicate tests each using the competitive ELISA antibody detection method and the competitive magnetic microparticle CLIA antibody detection method established in this application. The coefficient of variation was calculated based on the absorbance or luminescence values ​​of the serum samples. The results are shown in Table 3. The coefficient of variation (COP) for strongly positive samples of the SuHV-1 gE antibody competitive ELISA method established in this application was 1.92%, for positive samples it was 2.52%, for weakly positive samples it was 2.91%, and for negative serum samples it was 2.14%. The COP for strongly positive serum samples of the SuHV-1 gE competitive magnetic microparticle CLIA antibody method established in this application was 1.44%, for positive serum samples it was 1.85%, for weakly positive serum samples it was 1.30%, and for negative serum samples it was 1.73%. This indicates that the SuHV-1 gE antibody detection method established in this application has high reproducibility.

[0102] Table 3. Repeatability Test Results

[0103]

[0104] S604, Clinical Sample Compliance Test

[0105] The SuHV-1 gE antibody competitive ELISA detection method and the SuHV-1 gE antibody competitive magnetic microparticle CLIA detection method of this application were used to simultaneously detect 112 clinical samples with known backgrounds. The detection results of the two methods were compared, and the concordance rate of the two methods was calculated. The results are shown in Table 4. The Kappa value of the two methods was 0.93, and the overall concordance rate was 97.32%, indicating that the concordance rate of the two methods was high.

[0106] Table 4.1 Results of the comparative test of 112 serum clinical samples

[0107]

[0108] Table 5. Sequence information of nucleotide sequences:

[0109]

[0110] Table 6. Sequence information of amino acid sequences:

[0111]

[0112] The foregoing provides a detailed description of a porcine herpesvirus 1 gE multi-epitope fusion protein, its encoding gene, and its use in detecting porcine herpesvirus 1 gE antibodies. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this application. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A mouse anti-porcine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment, characterized in that, The complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment are positions 26–35, 50–67, and 88–96 of SEQ ID NO. 6, respectively; the complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain variable region are positions 24–39, 55–61, and 95–102 of SEQ ID NO. 7, respectively.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment includes four heavy chain variable region framework regions: HFR1, HFR2, HFR3 and HFR4 and four light chain variable region framework regions: LFR1, LFR2 and LFR3 and LFR4; The amino acid sequence of HFR1 includes amino acid sequences selected from the following: A1) The first to 25th bits of SEQ ID NO. 6; A2) is a sequence that has more than 80% similarity to A1); The amino acid sequence of HFR2 includes amino acid sequences selected from the following: A3) Positions 36 to 49 of SEQ ID NO. 6; A4) and A3) have more than 80% identity sequences; The amino acid sequence of HFR3 includes amino acid sequences selected from the following: A5) Positions 68 to 87 of SEQ ID NO. 6; A6) and A5) have more than 80% identity sequences; The amino acid sequence of HFR4 includes amino acid sequences selected from the following: A7) Positions 97 to 107 of SEQ ID NO. 6; A8) and A7) have more than 80% identity sequences; The amino acid sequence of the LFR1 includes amino acid sequences selected from the following: A9) The first to 23rd bits of SEQ ID NO.7; Sequences A10 and A9 have more than 80% identity; The amino acid sequence of the LFR2 includes amino acid sequences selected from the following: A11) Positions 40 to 54 of SEQ ID NO. 7; A12) and A11) have more than 80% identity sequences; The amino acid sequence of the LFR3 includes amino acid sequences selected from the following: A13) Positions 62 to 94 of SEQ ID NO. 7; A14) and A13) have more than 80% identity sequences; The amino acid sequence of LFR4 includes amino acid sequences selected from the following: A15) Positions 103 to 112 of SEQ ID NO. 7; A16) is a sequence that has more than 80% similarity to A15).

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region of the mouse anti-swine herpesvirus type 1 gE monoclonal antibody is shown in SEQ ID NO. 6, or has 80% or more sequence similarity to SEQ ID NO. 6; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7, or has 80% or more sequence similarity to SEQ ID NO.

7.

4. A nucleic acid molecule, characterized in that, It encodes the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.3, or has 80% or more sequence similarity to SEQ ID NO.3; the nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.4, or has 80% or more sequence similarity to SEQ ID NO.

4.

6. A biomaterial, characterized in that, It contains the nucleic acid molecule as described in claim 4 or 5; the biological material is an expression cassette, a recombinant vector, or a host cell.

7. A mouse anti-porcine herpesvirus type 1 gE monoclonal antibody conjugate, characterized in that, It is obtained by conjugating the mouse anti-swine herpesvirus type 1 gE monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 with a marker, wherein the marker is selected from one or more of horseradish peroxidase markers, biotin markers, fluorescent dye markers, and chemiluminescent dye markers.

8. A porcine herpesvirus type 1 ELISA antibody detection kit, characterized in that, The kit includes the monoclonal antibody conjugate of claim 7.

9. A detection kit for porcine herpesvirus type 1 magnetic microparticle CLIA antibody, characterized in that, The kit includes the monoclonal antibody conjugate of claim 7.

10. The antibody detection kit according to claim 8 or claim 9, characterized in that, The monoclonal antibody was obtained by immunizing porcine herpesvirus type 1 gE multiepitope fusion protein.

11. The antibody detection kit according to claim 10, characterized in that, The amino acid sequence of the porcine herpesvirus type 1 gE multiepitope fusion protein is shown in SEQ ID NO.

5.

12. The antibody detection kit according to claim 11, characterized in that, The nucleotide sequence encoding the porcine herpesvirus type 1 gE multiepitope fusion protein is shown in SEQ ID NO.

2.

13. The antibody detection kit according to claim 11, characterized in that, The nucleotide sequence encoding the porcine herpesvirus type 1 gE multiepitope fusion protein is shown in SEQ ID NO. 1 before codon optimization.

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