Specific monoclonal antibody for resisting pseudorabies virus IE180 protein as well as preparation method and application of specific monoclonal antibody

Abstract

The invention provides a specific monoclonal antibody for resisting pseudorabies virus IE180 protein as well as a preparation method and application of the specific monoclonal antibody, and belongs to the technical field of biological medicines. The invention provides a hybridoma cell strain pIE180 mAb for producing a specific monoclonal antibody against the pseudorabies virus IE180 protein, and successfully prepares the monoclonal antibody against the PRV IE180 protein. An overlapping peptide library technology shows that the monoclonal antibody can recognize a new B cell epitope, and the epitope is highly conservative in various classic and mutant isolates, can be used for specific detection of PRV infection and development of diagnostic reagents, provides an important molecular basis for design of subunit vaccines, and has broad application prospects. Important clinical application and market prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a specific monoclonal antibody against pseudorabies virus IE180 protein, its preparation method, and its application. Background Technology

[0002] Pseudorabies virus (PRV) is a linear double-stranded DNA virus belonging to the Alphaerpesvirinae subfamily of the Herpesviridae family. The PRV genome is approximately 150 kb in length, with a GC content as high as 74%. Based on the strict temporal sequence of DNA replication and transcription after viral infection, PRV coding genes can be divided into early genes, early genes, and late genes, which are regulated sequentially in a cascade manner. The genome consists of unique long and short regions, as well as internal and end repetitive sequences, and contains at least 70 ORFs, encoding 70-100 proteins, while mature viral particles contain only about 50 proteins. Studies have shown that nearly 50% of mature viral particles consist of four parts: the genome, nucleocapsid, envelope, and envelope.

[0003] The PRV Immediate Early Gene 180 (IE180) is its only immediate early protein, activating the transcription of downstream early and late genes, and is crucial for viral replication. This gene begins transcription immediately upon the viral genome entering the cell nucleus, with a transcribed size of 4.4 kb and a translated size of 1462 aa. The IE180 gene is the first gene transcribed and translated after viral infection. The IE180 protein activates the transcription of downstream genes and acts as a trans-acting factor in overall gene expression. Studies have shown that viruses lacking IE180 cannot replicate effectively. After PRV infection, downstream genes (early and late genes) can only begin transcription under its activation if the IE180 gene begins transcription and is successfully translated into the IE180 protein; otherwise, viral replication is blocked. In other words, the transcription and translation of the IE180 gene are prerequisites for PRV replication. The expression of the IE180 protein is considered a marker of early PRV infection and latency-reactivation, and is also a key target protein in antiviral drug screening. However, there are currently no commercially available PRV IE180 monoclonal antibodies, nor are there any literature reports on PRV IE180 monoclonal antibodies. Summary of the Invention

[0004] This invention provides a specific monoclonal antibody against the pseudorabies virus IE180 protein, its preparation method, and its application. The monoclonal antibody can specifically bind to the PRV IE180 protein and can be used to establish ELISA, IFA, WB, and IHC detection methods for detecting early PRV infection and latent-reactivation status, providing an important research tool for studying early PRV infection and latent-reactivation.

[0005] This invention provides a hybridoma cell line pIE180 mAb that produces a specific monoclonal antibody against the pseudorabies virus IE180 protein. The hybridoma cell line pIE180 mAb has been deposited with the accession number CCTCC NO: C202638.

[0006] The present invention also provides a monoclonal antibody specific to the IE180 protein of pseudorabies virus produced using the above-mentioned hybridoma cell line pIE180 mAb.

[0007] In one specific embodiment of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4.

[0008] In one specific embodiment of the present invention, the nucleotide sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the nucleotide sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 2.

[0009] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, comprising the following steps: amplifying the IE180-S2 gene using the genomic DNA of pseudorabies virus as a template, and expressing and purifying the amplified IE180-S2 gene to obtain recombinant IE180-S2 protein. Animals were immunized with the recombinant IE180-S2 protein, and spleen cells were isolated and fused with myeloma cells. The hybridoma cell line pIE180 mAb was obtained by screening. The hybridoma cell line pIE180 mAb was inoculated into the peritoneum of an animal, and the monoclonal antibody was isolated and purified from the ascites fluid of the animal.

[0010] In one specific embodiment of the present invention, the primer pair for amplifying the IE180-S2 gene includes an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

[0011] In one specific embodiment of the present invention, the biological expression includes expression using prokaryotic bacteria.

[0012] The present invention also provides the application of the above-mentioned monoclonal antibody or the monoclonal antibody prepared by the above-mentioned preparation method in the preparation of a drug for treating pseudorabies virus.

[0013] The present invention also provides the application of the above-mentioned monoclonal antibody or the monoclonal antibody prepared by the above preparation method in the preparation of reagents for detecting antigens of pseudorabies virus.

[0014] In one specific embodiment of the present invention, the reagent for detecting the antigen of pseudorabies virus includes an immunoassay reagent.

[0015] Beneficial effects: This invention successfully prepared a method targeting Escherichia coli (E. coli). E. coli A monoclonal antibody (mAb) expressing the PRV IE180 protein was developed. Overlapping peptide library analysis revealed that this monoclonal antibody recognizes a novel B-cell epitope (amino acid positions 824-829 of the IE180 protein sequence). 824 WPEQPG 829 Furthermore, this epitope is highly conserved in a variety of classical and mutant isolates, and can be used for specific detection of PRV infection, development of diagnostic reagents, and to provide an important molecular basis for the design of subunit vaccines, with significant clinical applications and market prospects. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of SDS-PAGE Coomassie staining identification of the recombinant IE180-S2 purified protein in this invention; from left to right in the figure, the lanes represent the marker and the IE180 truncated protein S2, respectively. Figure 2 This is a schematic diagram of the Western blot analysis of the reactivity of purified protein with the HIS-tagged antibody in this invention; from left to right in the figure, the lanes represent the marker and the IE180 truncated protein S2, respectively. Figure 3 This is a schematic diagram illustrating the subclass identification of the three anti-PRV IE180 protein monoclonal antibodies in this invention. Figure 4 This is a schematic diagram showing the location of the antigen recognition region of the monoclonal antibody 4E1 in this invention; Figure 5 This is a schematic diagram of Western blot analysis for identifying the epitope binding to the 4E1 binding antigen of the IE180 monoclonal antibody in this invention; the left side of the diagram, from top to bottom, shows the epitope identification diagrams for the first, second, and third rounds, while the right side, from top to bottom, shows the epitope identification diagrams for the fourth and fifth rounds. Figure 6 This is a schematic diagram illustrating the conservation of antigen recognition epitopes by comparing the amino acid sequences of IE180 of 20 PRV virus strains based on the NCBI database in this invention. Figure 7 This is a schematic diagram of the Western blot analysis of the reactivity of VERO CCL-81 cells with 4E1 monoclonal antibody after PRV infection in this invention; Figure 8 This is a schematic diagram of the Western blot analysis of the reactivity of 4E1 monoclonal antibody after 239T cells were transfected with the IE180-S2 eukaryotic expression vector in this invention. Figure 9 This is a schematic diagram illustrating the indirect immunofluorescence detection of PRV IE180 protein expression and localization in cells using the 4E1 monoclonal antibody in this invention. Figure 10 This is a schematic diagram of the immunohistochemical detection of IE180 protein distribution and expression in PRV-infected pig tissues using the 4E1 monoclonal antibody in this invention. The diagram shows immunohistochemical staining analysis of lung, spleen, lymph nodes, trigeminal nerve, and brain sections of PRV-infected pigs, with the staining results observed under a 20X microscope.

[0017] Biological Preservation Information The hybridoma cell line pIE180 mAb was deposited at the China Center for Type Culture Collection (CCTCC) on February 27, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202638. Detailed Implementation

[0018] This invention provides a hybridoma cell line pIE180 mAb that produces a specific monoclonal antibody against the pseudorabies virus IE180 protein. The hybridoma cell line pIE180 mAb has been deposited with the accession number CCTCC NO: C202638.

[0019] The present invention also provides a monoclonal antibody specific to the IE180 protein of pseudorabies virus produced using the above-mentioned hybridoma cell line pIE180 mAb.

[0020] The amino acid sequence of the light chain variable region of the monoclonal antibody of the present invention is shown in SEQ ID No. 3, and the nucleotide sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4, and the nucleotide sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 2.

[0021] SEQ ID No.1:GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCGGTCAGAGCCTTTTATATAGTAACAATCAAAAGAATTATTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGGTATTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA; SEQ ID No.2:CAGATTCAGCTTAAGGAGTCTGGACCTGCTGTCATCAAGCCATCACAGTCACTGTCTCTCACCTGCATAGTCTCTGGATTCTCCATCACAAGTAGTAGTTATTGCTGGCACTGGATCCGCCAGCCCCCAGGAAAGGGGTTAGAGTGGATGGGGCGCATATGTTATGAAAATTCAATATACTATAGTCCATCCATCAAAAGCCGCAGCACCATCTCCAGAGACACATCTCTGAACAAATTCTTTATCCAGCTGAGCTCTGTGACAAATGAGGACACAGCCATGTACTACTGTTCCAGGGAAAACGTTAACTACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; SEQ ID No.3:DIVMSQSPSSLAVSVGEKVTMSCKSGQSLLYSNNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYRYWTFGGGTKLEIK; SEQ ID No. 4: QIQLKESGPAVIKPSQSLSLTCIVSGFSITSSSYCWHWIRQPPGKGLEWMGRICYENSIYYSPSIKSRSTISRDTSLNKFFIQLSSVTNEDTAMYYCSRENVNYAMDYWGQGTSVTVSS.

[0022] The monoclonal antibody described in this invention is an IgG1 / κ isotype immunoglobulin that specifically recognizes the C-terminal 824-829aa fragment of the IE180 protein. The amino acid sequence of the recognizable B-cell epitope, as demonstrated in the examples, is shown in SEQ ID No. 7. 824 WPEQPG 829 .

[0023] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, comprising the following steps: amplifying the IE180-S2 gene using the genomic DNA of pseudorabies virus as a template, and expressing and purifying the amplified IE180-S2 gene to obtain recombinant IE180-S2 protein. Animals were immunized with the recombinant IE180-S2 protein, and spleen cells were isolated and fused with myeloma cells. The hybridoma cell line pIE180 mAb was obtained by screening. The hybridoma cell line pIE180 mAb was inoculated into the peritoneum of an animal, and the monoclonal antibody was isolated and purified from the ascites fluid of the animal.

[0024] In this invention, the gene ID of the IE180 gene is MZ063026. In one embodiment of this invention, a primer pair is designed with the 610-1465 aa fragment of the protein encoded by the IE180 gene as the target sequence, including an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

[0025] Upstream primer (SEQ ID No. 5): 5'-AAATGGGTCGCGGATCCGAATTCCCGCACATCGGGGACG-3'; Downstream primer (SEQ ID No. 6): 5'-TGCTCGAGTGCGGCCGCAAGCTTTCAGCGGAGCAGCAGG-3'.

[0026] This invention utilizes the fragment amplified by the above primer pairs, inserts it into the EcoRI and HindIII restriction endonuclease sites of a prokaryotic expression vector such as pET-28a, to construct a recombinant expression vector expressing the recombinant IE180-S2 protein. The recombinant expression vector is then transformed into prokaryotic expression cells such as Escherichia coli, and the recombinant IE180-S2 protein is obtained after induction of expression, separation, and purification.

[0027] In one embodiment of the present invention, mice were immunized with the recombinant IE180-S2 protein, and spleen cells were isolated and fused with myeloma cells to screen for positive hybridoma cell lines IE180 mAb. The hybridoma cell line IE180 mAb was inoculated into the peritoneal cavity of the animals, and the ascites fluid was separated and purified to prepare IE180-S2 protein-specific monoclonal antibodies.

[0028] The present invention also provides the application of the above-mentioned monoclonal antibody or the monoclonal antibody prepared by the above-mentioned preparation method in the preparation of a drug for treating pseudorabies virus.

[0029] The present invention also provides the application of the above-mentioned monoclonal antibody or the monoclonal antibody prepared by the above preparation method in the preparation of reagents for detecting pseudorabies virus.

[0030] The monoclonal antibody described in this invention can be used for immunodetection of PRV, including Western blotting (WB), immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC), and can be used for early detection of PRVIE180 protein and transcriptional function studies of the viral IE180 gene.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a specific monoclonal antibody against pseudorabies virus IE180 protein, its preparation method, and its application, is provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are conventional commercially available materials in the art, and "%" in the embodiments refers to volume percentage. The sources of some reagents and materials are disclosed as follows: VeroCCL-81, HEK 293T, and SP2 / 0 myeloma cells (purchased from the American Technology and Research Center for Biological Standards, ATCC). VeroCCL-81 and HEK 293T cells were cultured in DMEM medium (purchased from Gibco) containing 10% fetal bovine serum (purchased from Invitrogen), SP2 / 0 myeloma cells were cultured in DMEM medium containing 20% ​​fetal bovine serum, and fusion cells were cultured in 1640 medium (purchased from Gibco). PRV Bartha-K61 (GenBank: JF797217.1) was donated by Professor Gao Song of Yangzhou University. PRV DX strain (GenBank: MZ063026.1) was isolated and stored by the Key Laboratory of Animal Virology, Ministry of Agriculture, College of Animal Science, Zhejiang University, and has been published in the paper (Front. Microbiol. 13:943707. doi: 10.3389 / fmicb.2022.943707).

[0033] The following plasmids were used: pET-28a(+), pCAGGS, and pEGFP (purchased from Miaoling Biotechnology); Phanta Super-Fidelity DNA Polymerase (purchased from Vazyme); 2×Taq Plus Master Mix enzyme (purchased from Vazyme); GeneRuler 100bp DNA Ladder (purchased from Thermo); PCR purification and recovery kit (purchased from Vazyme); gel DNA recovery kit (purchased from Vazyme); plasmid mini-extraction kit (purchased from TIANGEN); restriction endonucleases EcoRI, XhoI, BamHI, SacI, kpnI, and HindIII (purchased from Takara); kanamycin and ampicillin (purchased from Biosharp); agarose and tryptone (purchased from Solarbio); Protein Marker (purchased from Thermo); donkey anti-mouse IgG (H+L) Alexa Fluor 488 (purchased from Thermo); horseradish peroxidase HRP-labeled goat anti-mouse IgG and goat anti-rabbit IgG (purchased from Sangon). Biotech), Lipomaster 3000 Transfection Reagent (purchased from Vazyme), PRV gC monoclonal antibody (prepared by Huang Wenxiang of the Key Laboratory of Animal Virology, Ministry of Agriculture, College of Animal Sciences, Zhejiang University, by immunizing mice with purified gC protein; the applicant hereby promises that this material will be released to the public within twenty years from the date of application), HIS-tagged monoclonal antibody, FLAG-tagged monoclonal antibody, β-ACTIN-tagged polyclonal antibody, EGFP monoclonal antibody, Freund's adjuvant (purchased from Sigma), PEG4000 (purchased from Sigma), 50×HAT selective medium (purchased from Biodragon).

[0034] Example 1: Process for PRV IE180-S2 gene expression and monoclonal antibody preparation (1) Cloning and expression of the IE180-S2 gene, protein purification and preparation of monoclonal antibodies Two high-scoring truncated segments of IE180 were predicted using BEPIPRED-3. The 610-1465aa fragment of the IE180 gene was selected and named IE180-S2. Primers targeting the N-terminus were designed based on the truncated IE180 segments: a forward primer (SEQ ID No. 5) and a reverse primer (SEQ ID No. 6). The amplicon, along with the EcoRI and HindIII restriction endonuclease sites, was cloned into pET-28a to obtain the prokaryotic expression system pET-28a-IE180-S2, with an expressed protein size of 107 kDa. Subsequently, the 610-1465aa fragment of the IE180-S2 gene was inserted into the FLAG-tagged mammalian expression vector pCAGGS to obtain the eukaryotic target system vector pCAGGS-FLAG-IE180-S2.

[0035] Plasmid pET-28a-IE180-S2 was transformed into *E. coli* BL21(DE3) to express the histidine (His)-labeled IE180-S2 protein. Single colonies of each transformant were expanded in Luria Bertani (LB) medium containing 30 μg / mL kanamycin. Optimal induction conditions were achieved when the culture density at 600 nm reached 0.6–0.8. Protein expression was induced for 12 h at 16°C with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation at 4000 rpm for 15 min, resuspended in pre-chilled PBS, and lysed by sonication at 300 W for 6 s followed by a 6 s pause on ice for 30 min. The supernatant was then collected from the cell lysate after centrifugation at 12,000 rpm for 30 min.

[0036] For IE180-S2 protein purification, the supernatant was filtered using a 0.22 μm filter before purifying the IE180-S2 protein through a Ni-NTA resin column. The nickel column was then activated, washed with 5 mM NaOH, and then soaked in 2 mL of NaOH for 10 min. Next, the column was washed with 20% ethanol, and then soaked in 2 mL of NaOH for 10 min. Finally, the column was washed thoroughly with the mother liquor. For purification, the conjugate was transferred to an elution column, followed by washing with the mother liquor. Elution was then performed with 10 mM imidazole solution to below 0.1 ng / μL, followed by 20 mM imidazole solution to below 0.1 ng / μL, and so on, until elution with 200 mM imidazole solution (50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole, pH 8.0) to below 0.1 ng / μL. The protein eluent from each imidazole concentration was collected, and the protein concentration was measured using a UV spectrophotometer. The eluent was aliquoted into 1 mL tubes and stored at -80°C. Subsequent SDS-PAGE analysis was used to detect the expression of IE180-S2 protein and cell lysates. Western blot analysis using SDS-PAGE and anti-His antibody was used to validate the purified IE180-S2 protein.

[0037] The results are as follows Figure 1 As shown, the soluble IE180-S2 protein purified by nickel column was identified by SDS-PAGE, and the purified IE180-S2 protein could be recognized by the HIS-tagged antibody in Western blot. Figure 2 ).

[0038] Female BALB / c mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Experimental Animal License No.: ZJU20240878). Immunization experiments were conducted using these BALB / c mice within the Biosafety Level 2 (B2C) animal experimental platform of the College of Animal Sciences, Zhejiang University. Six- to eight-week-old BALB / c mice were subcutaneously immunized with purified pIE180-S2 protein, with immunizations performed every 14 days for a total of three immunizations. Five days after each immunization, blood was collected from the tail vein of the mice to separate serum. Mice with the highest antibody ELISA titers in their serum were given intraperitoneal booster immunizations. For the first immunization, 0.2 mg of protein was mixed with Freund's complete adjuvant at a 1:1 (v / v) ratio; for subsequent immunizations, 0.15 mg of protein was mixed with Freund's incomplete adjuvant at a 1:1 (v / v) ratio before injection.

[0039] Peritoneal feeder cells were prepared using healthy female BALB / c mice aged 6-8 weeks, 1 day before cell fusion. Before the experiment, the paraffin plates and worktables were irradiated with UV light for 30 min, and the culture medium and serum were preheated to 37°C. Blood was collected from the eyes of selected mice using forceps into 1.5 mL EP tubes. Mice were euthanized by cervical dislocation and immersed in alcohol for 10-15 min. The mice were then fixed with paraffin plates and pins to expose the abdominal skin. The abdominal skin was carefully cut open with scissors, taking care not to cut into the peritoneum. 25 mL of 1640 culture medium was added to a 10 cm petri dish. 10 mL of 1640 culture medium was quickly drawn up using a 10 mL syringe, and the needle was replaced with a 5 mL syringe needle. The needle was carefully inserted into the peritoneum, avoiding other tissues, and the culture medium was slowly injected into the peritoneal cavity. During injection, the bulging peritoneal cavity was gently moved with fingers to aspirate the peritoneal fluid, avoiding other tissues. The needle was removed, and the peritoneal fluid was injected into an aspiration tank. Repeat the operation 2-3 times, add 1640 medium to make up to 50 mL of feeder cell solution, and spread 80 μL of each cell solution into 5 96-well plates. After 12 h, perform the fusion experiment.

[0040] Mice exhibiting good reactivity to serum ELISA, IFA, and Western blot were selected and boosted with 200 μg of antigen via intraperitoneal injection. Three days after the booster immunization, blood was collected after enucleation, and serum was separated as a negative control. Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 10 min. Spleen cells were collected from the euthanized mice for a fusion experiment with spleen cells and feeder cells. The main steps of the cell fusion procedure were enucleation, blood collection, and euthanasia. The cadavers were then immersed in alcohol for 10–15 min and pre-washed with 5 mL of 1640 medium in petri dishes. Subsequently, the mice were restrained, and the skin was cut open with scissors. When cutting the peritoneum, the scissors were replaced, and the spleen was removed. The spleen was rinsed with 5 mL of 1640 medium to remove surface blood. The spleen was then placed in a glass mortar and pestle, and 1 mL of 1640 medium was added for grinding. The grinding solution was tilted and transferred to a clean 50 mL centrifuge tube. This step was repeated until the medium was no longer red. Add liquid to a 50 mL centrifuge tube containing spleen cells to bring the volume to 10 mL using 1640 medium. Centrifuge at 1000 rpm for 3 min. Remove connective tissue using a 1 mL pipette tip. Repeat 3 times. After slightly digesting two prepared T75 sp2 / 0 cells, pipette each with 5 mL of 1640 medium (total 10 mL). Transfer these cells to a new 50 mL centrifuge tube and centrifuge together with the spleen cells. Repeat 3 times. Discard the supernatant from all cells. Gently tap the cells on a table to loosen and evenly distribute them. In a 37°C water bath, slowly add 1 mL of PEG4000 fusion agent dropwise over 2 min while shaking. Then, slowly add 5 mL of 1640 medium containing 20% ​​fetal bovine serum (FBS) over 2 min, followed by 10 mL of 1640 medium containing 20% ​​FBS. Centrifuge at 1000 rpm for 5 min. Mix thoroughly with 10% FBS + 2% HAT + 2‰ penicillin-dextrose antibody, depending on the plating volume. The centrifuged fusion cells were gently resuspended in prepared culture medium, and the resuspended cell solution was then mixed into the culture medium. 120 μL of the resuspended cells were seeded into each well and incubated for 7 days. The fusion cells were then continuously cultured in DMEM supplemented with 2% HAT and 20% fetal bovine serum. After cell fusion culture, positive cell lines were screened using indirect ELISA, and the supernatant was used as the primary antibody. Cells from wells that tested positive by ELISA were first evenly seeded into new wells. Wells that remained positive after a second ELISA test were then subcloned using a combination of limiting dilution and serial dilution to identify single positive clones. This subcloning process was repeated continuously. After three rounds of subcloning of hybridoma cells, three PRV IE180 protein monoclonal antibody-positive cell lines (2H9, 4E1, and 4G12) were obtained. Verification revealed that they recognized the same antigenic epitope, and these were subsequently collectively referred to as monoclonal antibody line 4E1.

[0041] 6-8 week old BALB / c mice were intraperitoneally injected with 0.2-0.3 mL of incomplete Freund's adjuvant per mouse. Three days later, they were intraperitoneally injected with 1-3 × 10⁻⁶ mouse hybridoma cell lines. 6 Five days after collection of cells, the abdominal distension of mice was observed to determine the production of ascites. When the mice showed obvious abdominal swelling and tension, ascites was collected using a syringe. The ascites was collected by centrifugation at 2000 rpm for 5 min 2-3 times, removing the upper layer of adipose tissue and the lower layer of red blood cells. The supernatant was collected and the IE180 protein-specific monoclonal antibody was purified, aliquoted into 1 mL tubes, and stored at -80℃.

[0042] Example 2 Identification of anti-PRV IE180 protein monoclonal antibody (1) Identification of eukaryotic and viral cell-expressed proteins by detection of anti-PRV IE180 protein monoclonal antibody To detect the reactivity of monoclonal antibodies to the IE180-S2 recombinant protein, Western blotting was used to perform antibody reactivity experiments under cell infection and transfection states. Vero CCL-81 cells were infected with PRV DX strain at MOI=0.1, and cell samples were collected 24 h post-infection, with uninfected PRV serving as the control group. Simultaneously, HEK293T cells were transfected with the pCAGGS-FLAG-IE180-S2 plasmid constructed in Example 1 at a dose of 0.5 μg, with the original pCAGGS plasmid serving as the control group. Cells were collected after 48 h of culture. Cells were lysed on ice for 15 min with cell lysis buffer containing PMSF, centrifuged at 13000 r / min for 15 min at 4°C, and the supernatant was transferred to a new centrifuge tube. 5× loading buffer was added, and the cells were incubated at 100°C for 10 min for SDS-PAGE analysis. The pIE180-S2 protein was then separated by electrophoresis on a 12% SDS-PAGE gel and transferred to a 0.22 μm reinforced nitrocellulose membrane (NC membrane) and incubated at 100 V for 90 min. The membrane surface was then blocked with 5% skim milk and incubated at room temperature for 1 h. Primary antibody incubation was performed using monoclonal antibody (1:500 dilution) against IE180 protein hybridoma cell supernatant and polyclonal antibody (1:3000 dilution). After washing five times with PBST (80 mM Na2HPO4, 20 mM NaH2PO4·2H2O, and 100 mM NaCl, 0.05% Tween-20), horseradish peroxidase-labeled goat anti-mouse IgG (1:3000 dilution) was added, and the mixture was incubated at room temperature for 1 h. Finally, the target bands were exposed under a developer.

[0043] The results are as follows Figure 7 As shown, the 4E1 strain monoclonal antibody was positive for PRV-infected cell samples. (See figure) Figure 8 As shown, the monoclonal antibody against the PRV IE180 protein strain 4E1 reacted with the IE180-S2 protein under eukaryotic expression but did not react with the empty plasmid transfected sample, indicating that the epitope recognized by 4E1 is linear.

[0044] (2) Identification of the protein domain recognized by anti-PRV IE180 protein monoclonal antibody To identify the epitopes recognized by the anti-PRV IE180 protein monoclonal antibody 4E1, a series of pCAGGS eukaryotic expression vectors were designed, with FLAG tags fused to SacⅠ and KpnⅠ sites and expressed in HEK293T cells, and pEGFP eukaryotic expression vectors fused to XHOⅠ and HindⅢ sites. The truncated construct sites are as follows: Figure 4 As shown.

[0045] First, the truncated IE180 protein was divided into nine fragments: E1 (600-710 aa), E2 (700-810 aa), E3 (800-900 aa), E4 (866-966 aa), E5 (951-1074 aa), E6 (1054-1174 aa), E7 (1154-1274 aa), E8 (1254-1374 aa), and E9 (1361-1465 aa), resulting in nine eukaryotic expression plasmids: pCAGGS-IE180-E1-E9. The monoclonal antibody 4E1 recognized E3 but not other peptides, indicating that the 4E1 epitope is within the 800-900 amino acid range. A truncated peptide library was further constructed using the pEGFP vector, and the recombinant vector was expressed in HEK293T cells. The results were then analyzed by Western blotting. The second round of design included nine eukaryotic expression plasmids: E10 (800-835 aa), E11 (830-865 aa), E12 (860-895 aa), E13 (896-925 aa), E14 (920-955 aa), E15 (950-985 aa), E16 (980-1015 aa), E17 (1010-1045 aa), and E18 (1040-1074 aa). After Western blot validation, the epitope map range was narrowed down to 36 amino acids in E10 (800-835). The third round of design involved three eukaryotic expression plasmids: E19 (800-814 aa), E20 (812-825 aa), and E21 (823-835 aa), further targeting 18 amino acids in E21 (823-835 aa). Finally, 16 eukaryotic expression plasmids, E22-E37, were designed using a truncated approach, identifying WPEQPG (824-829 aa) as the minimal B-cell epitope recognized by 4E1. Figure 5 ).

[0046] The conservation of monoclonal antibody recognition epitopes was assessed by amino acid sequence alignment of IE180 sequences from 20 PRV strains obtained from GenBank. Epitopes targeting monoclonal antibody recognition were identified. 824 WPEQPG 829 The conservation of the IE180 protein recognition epitope sequence among different PRV genotypes was analyzed using MEGA12 software and ClustalW multiple sequence alignment. All reference sequences were obtained from the GenBank database. Results are shown below. Figure 6 The plants showed high conservation, with sequence similarity reaching 100%.

[0047] Cells with three positive clones underwent nucleotide sequencing, which was performed by Nanjing Detai Biotechnology Co., Ltd. The nucleotide sequence of the variable region of the light chain of mouse monoclonal antibody 4E1 is shown in SEQ ID No. 1, and the nucleotide sequence of the variable region of the heavy chain of mouse monoclonal antibody 4E1 is shown in SEQ ID No. 2. The amino acid sequences were deduced from the nucleotide sequences. The amino acid sequence of the light chain of mouse monoclonal antibody 4E1 is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain of monoclonal antibody 4E1 is shown in SEQ ID No. 4.

[0048] Example 3: Identification of Anti-IE180 Monoclonal Antibody Subclasses The subtypes of monoclonal antibodies from three ascites strains of IE180 were detected using the mouse monoclonal antibody subtype identification kit from Proteintech Group (Wuhan Sanying Biotechnology Co., Ltd.). The number of ELISA strips was selected based on the sample quantity. Subclonal cell supernatant was diluted 1×PBST 1:100 and added to the wells of the strips (50 μL / well). Goat anti-mouse ELISA-labeled secondary antibody was then added, and the plates were incubated for 1 h. The liquid in the wells was discarded, the plates were washed three times with 1×PBST, blotted dry on absorbent paper, and 100 μL of chromogenic reagent was added. The OD values ​​were read using an ELISA reader. 450 The result is as follows Figure 3 As shown, the subclass of the three monoclonal antibodies against PRV IE180 protein is IgG1 / κ.

[0049] Example 4: Indirect immunofluorescence identification of IE180 protein expression and localization in cells using anti-PRV IE180 protein monoclonal antibody. In the IFA experiment, the digested Vero CCL-81 cells were divided into groups of 3 × 10⁻⁶ cells. 5The cells were seeded into 12-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until they formed a monolayer. After infection with PRV DX and Bartha-K61 strains (MOI=0.1) for 48 h, the cells were fixed with frozen methanol solution stored at -20°C for 20 min. The methanol was then discarded, and the cells were either air-dried in a biosafety cabinet or stored in PBS at 4°C. The fixed cells were incubated with anti-PRV IE180 protein monoclonal antibody (1:500 dilution) at 37°C for 1 h, washed three times with PBS, and then mixed with 488-labeled goat anti-mouse IgG secondary antibody (1:3000 dilution) and washed three times with PBS. 4',6-diamidinyl-2-phenylindole (DAPI) dye (1:10000 dilution) was added, and the cells were incubated at room temperature for 10 min, followed by washing with PBS. Finally, the cells were observed under a microscope.

[0050] Immunofluorescence assay results as follows Figure 9 As shown, the PRV 4E1 antibody responded well to VEROCCL-81 cells infected with PRV DX and Bartha-K61 strains.

[0051] Example 5 Immunohistochemical detection of the distribution and expression of IE180 protein in PRV-infected pig tissues using anti-PRV IE180 protein monoclonal antibody Ten 28-day-old SPF pigs (purchased from Jiangsu Qianyue Biotechnology Co., Ltd.) were selected and divided into two groups of five: an infection group and a control group. The infection group consisted of 10 pigs. 5 TCID 50 Piglets in the PRV DX strain were infected with 2 mL of DMEM culture medium via intranasal inoculation. The control group received 2 mL of DMEM culture medium via intranasal inoculation. On the third day post-challenge, infected piglets developed fever and neurological symptoms, and were then dissected. Brain, trigeminal, lung, spleen, and inguinal lymph nodes were collected. 1 cm tissue samples were harvested. 3 The sections were fixed in a solution containing 4% paraformaldehyde for 24 h. After dewaxing with xylene, they were rehydrated with 95%, 85%, and 75% ethanol solutions, decreasing the concentration to 70% ethanol, for 5–10 min each time. Antigen retrieval was performed using sodium citrate buffer (pH 6.0). After blocking with bovine serum albumin (BSA) at 37°C for 2 h, the sections were incubated overnight at 4°C with a monoclonal antibody against IE180 protein (1:500 dilution). The stained sections were hybridized for 1 h using horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:500 dilution). Finally, the immune complexes were detected using a 3,3'-diaminobenzidine liquid substrate system.

[0052] Experimental results are as follows Figure 10As shown, after PRV infection, IE180 protein is mainly concentrated in the inguinal lymph nodes and spleen, followed by the brain, trigeminal nerve, and lungs. No positive staining was observed in the control group tissues, thus establishing a negative control. Lymph node staining was most prominent, with strongly positive cells appearing yellowish-brown.

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hybridoma cell line pIE180 mAb producing a specific monoclonal antibody against the pseudorabies virus IE180 protein, characterized in that, The hybridoma cell line pIE180 mAb has been deposited with accession number CCTCC NO: C202638.

2. A monoclonal antibody specific to the IE180 protein of pseudorabies virus produced using the hybridoma cell line pIE180 mAb as described in claim 1.

3. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

4.

4. The monoclonal antibody according to claim 2 or 3, characterized in that, The nucleotide sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the nucleotide sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.

2.

5. The method for preparing the monoclonal antibody according to any one of claims 2 to 4, characterized in that, The steps include: amplifying the IE180-S2 gene using the genomic DNA of pseudorabies virus as a template, and then expressing and purifying the amplified IE180-S2 gene to obtain recombinant IE180-S2 protein. Animals were immunized with the recombinant IE180-S2 protein, and spleen cells were isolated and fused with myeloma cells. The hybridoma cell line pIE180 mAb described in claim 1 was then screened to obtain the hybridoma cell line pIE180 mAb described in claim 1. The hybridoma cell line pIE180 mAb was inoculated into the peritoneum of an animal, and the monoclonal antibody was isolated and purified from the ascites fluid of the animal.

6. The preparation method according to claim 5, characterized in that, The primer pair for amplifying the IE180-S2 gene includes an upstream primer with nucleotide sequences as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No.

6.

7. The preparation method according to claim 5, characterized in that, The biological expression includes expression using prokaryotic bacteria.

8. The use of the monoclonal antibody according to any one of claims 2 to 4 or the monoclonal antibody prepared by the preparation method according to any one of claims 5 to 7 in the preparation of anti-pseudorabies virus drugs.

9. The use of the monoclonal antibody according to any one of claims 2 to 4 or the monoclonal antibody prepared by the preparation method according to any one of claims 5 to 7 in the preparation of reagents for detecting pseudorabies virus antigen.

10. The application according to claim 9, characterized in that, The reagents for detecting pseudorabies virus antigen include immunoassay reagents.

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