Neutralizing monoclonal antibody of pseudorabies virus gB protein as well as antigen epitope peptide and application of neutralizing monoclonal antibody

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a neutralizing monoclonal antibody of a pseudorabies virus gB protein and an antigen epitope peptide and application thereof. An escherichia coli expression system capable of stably expressing the PRV gB protein is designed and constructed, a mouse is immunized by taking the purified gB protein as an immunogen, and the monoclonal cell strain resisting the PRV gB protein is screened through an immunoperoxidase monolayer cell test (IPMA) by utilizing a cell fusion technology and taking the stably expressed gB protein as a detection antigen. The titer of the monoclonal antibody IPMA generated by the cell strain is 1: 32000, the neutralizing titer is 1: 24, the monoclonal antibody can specifically recognize and combine with a 615RVPGERGTC623 sequence region on the gB protein, the interaction between an antigen and the antibody can be known, the monoclonal antibody has important guiding significance on development of vaccines and detection reagents based on the gB protein, and a technical support is provided for prevention, control and treatment of PRV.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to a monoclonal antibody against the gB protein of pseudorabies virus. Background Technology

[0002] Pseudorabies (PR) is caused by the porcine pseudorabies virus (PRV). Pesudorabies virus Pigs infected with Propionibacterium avium (PRV) are an acute and highly contagious disease that can infect various poultry, including pigs, cattle, and sheep, as well as wild animals. Pigs are the natural host and primary source of infection. PRV infection has caused devastating economic losses to the global pig industry and severely hinders its healthy development. Newborn piglets infected with PRV often exhibit acute encephalitis, with a mortality rate as high as 100%. Adult pigs infected with PRV frequently show symptoms such as respiratory distress and growth retardation. Pregnant sows are prone to reproductive disorders such as abortion, stillbirths, or weak piglets. Furthermore, infected sows carry the virus for life, becoming a continuously spreading "mobile virus reservoir," posing a significant obstacle to disease eradication efforts on pig farms.

[0003] In recent years, the emergence of PRV variants has further increased the difficulty of prevention and control. Traditional vaccine strains (such as Bartha-K61 strain) have significantly reduced cross-protection against variants due to antigenic differences, leading to frequent outbreaks in some immunized pig farms. Therefore, establishing accurate and efficient PRV detection methods and developing highly specific and active antibody reagents have become core components of early PRV diagnosis, dynamic monitoring of the epidemic, and evaluation of immunization efficacy. They are also key supports for overcoming current prevention and control bottlenecks and building a precise prevention and control system.

[0004] The PRV genome encodes 11 glycoproteins, among which the gB protein, as a core structural protein of the viral envelope, is highly conserved and possesses strong immunogenicity, making it a key molecule in PRV infection and the immune response. On one hand, the gB protein participates in key infection processes throughout the entire process, including viral adsorption and fusion of the envelope with the host cell membrane. On the other hand, this protein exhibits high sequence conservedness and extremely strong immunogenicity, containing multiple immunodominant epitopes in its molecular structure, which can efficiently induce the body to produce neutralizing antibodies. These antibodies can interfere with viral invasion by binding to the gB protein, while simultaneously mediating complement-dependent cytotoxicity and opsonization of phagocytes, thereby enhancing the body's ability to clear the virus. Based on these characteristics, the gB protein has become an ideal molecular target for PRV antibody detection, vaccine development, and targeted prevention and treatment technologies.

[0005] Monoclonal antibodies, due to their advantages such as high specificity, high purity, good batch stability, and scalability, have demonstrated application value in PRV clinical detection and disease control. They can be used to construct various detection methods such as ELISA, immunofluorescence assay (IFA), and immunoperoxidase monolayer assay (IPMA), achieving accurate qualitative and quantitative detection of PRV and providing a potential technical tool for passive immunotherapy of PRV. Existing patent 202211426483.2 discloses an anti-porcine pseudorabies virus gB monoclonal antibody; patent 201710671265.8 discloses a PRV gB monoclonal antibody and its application. By screening hybridoma cells that stably secrete PRV gB protein monoclonal antibodies, high-titer monoclonal antibodies can be prepared, laying the foundation for the prevention and control of porcine pseudorabies. However, most publicly available monoclonal antibodies focus on binding activity to conserved regions of the gB protein, without clearly identifying the specific immunodominant epitopes they recognize. Furthermore, their specific binding ability and neutralizing activity against the gB protein of PRV variants need further improvement. Simultaneously, the development of highly specific antibody reagents based on clearly defined epitopes remains scarce, making it difficult to meet the needs of precise detection and control in the current context of variant epidemics. Therefore, developing highly specific and highly active monoclonal antibodies against the PRV gB protein, and further precisely screening for its specific immunodominant epitopes, is not only of significant theoretical importance for elucidating the immune mechanism and biological function of the gB protein, but also provides core technical support for developing novel precise detection reagents and targeted prevention and treatment drugs against PRV, and has important practical value for overcoming current bottlenecks in PRV prevention and control. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a neutralizing monoclonal antibody against pseudorabies virus gB protein, its antigenic epitope peptide, and its applications.

[0007] The technical solution of this invention is implemented as follows:

[0008] This application constructs a PRV gB prokaryotic expression vector to achieve the expression of recombinant gB protein, the amino acid sequence of which is shown in SEQ ID NO.12 and the encoded nucleotide sequence is shown in SEQ ID NO.13.

[0009] The preparation method of recombinant gB protein is as follows: The codon-optimized gB protein gene was constructed into the pET-32a vector, transformed into Rosetta competent cells, and gB protein was expressed. The gB protein was then purified through denaturation and renaturation. Subsequently, animals were immunized with PRV gB recombinant protein and attenuated live vaccine as immunogens to prepare a monoclonal antibody against PRV gB. After IPMA screening, a monoclonal antibody with neutralizing activity, 4A10, was identified. IFA and WB experiments verified that it specifically recognizes the anti-pseudorabies virus gB protein with a titer as high as 1:2.56×10⁻⁶.5 The IPMA titer is 1:3200, and the neutralizing titer is 1:2. 4 .

[0010] On one hand, the present invention provides a neutralizing monoclonal antibody against pseudorabies virus gB protein, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising the amino acid sequences CDRH1 as shown in SEQ ID NO.1, CDR H2 as shown in SEQ ID NO.2, and CDR H3 as shown in SEQ ID NO.3; the light chain variable region comprising the amino acid sequences CDR L1 as shown in SEQ ID NO.4, CDR L2 as shown in SEQ ID NO.5, and CDR L3 as shown in SEQ ID NO.6.

[0011] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0012] The aforementioned monoclonal antibodies exhibit high specificity and do not react with glycoproteins of other herpesviruses. They provide important guidance for the development of vaccines and diagnostic reagents based on PRV gB protein and offer technical support for the prevention and treatment of PRV.

[0013] Secondly, the antigenic epitope peptide specifically recognized by the aforementioned neutralizing monoclonal antibody is located in the 615-623 peptide segment of the pseudorabies virus gB protein, and its amino acid sequence is shown in SEQ ID NO.11.

[0014] Thirdly, a gene encoding the above-mentioned neutralizing monoclonal antibody, wherein the DNA sequence encoding the heavy chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO.9, and the DNA sequence encoding the light chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO.10.

[0015] Fourthly, expression vectors containing the aforementioned genes.

[0016] Fifthly, the host cell containing the aforementioned expression vector is a prokaryotic cell, preferably Escherichia coli.

[0017] The sixth aspect concerns the use of the aforementioned neutralizing monoclonal antibody, the aforementioned epitope peptide, the aforementioned gene, the aforementioned expression vector, or the aforementioned host cell in the preparation of reagents or kits for detecting pseudorabies virus.

[0018] Seventhly, the use of the aforementioned neutralizing monoclonal antibody, the aforementioned antigenic epitope peptide, the aforementioned gene, the aforementioned expression vector, or the aforementioned host cell in the preparation of drugs for the prevention or treatment of pseudorabies virus infection.

[0019] Eighthly, a kit for detecting pseudorabies virus, the kit comprising the aforementioned neutralizing monoclonal antibody.

[0020] Ninthly, a drug for treating pseudorabies virus, wherein the active ingredient of the drug is the aforementioned neutralizing monoclonal antibody.

[0021] The present invention has the following beneficial effects:

[0022] This invention discloses a neutralizing monoclonal antibody against pseudorabies virus gB protein and its antigenic epitope peptide. The invention first designs and constructs an *E. coli* expression system capable of stably expressing PRV gB protein. Purified gB protein is used as an immunogen to immunize mice. Cell fusion technology is employed, with stably expressed gB protein as a detection antigen. An immunoperoxidase monolayer assay (IPMA) is used to screen for monoclonal cell lines anti-PRV gB protein. The monoclonal antibody produced by this cell line has an IPMA titer of 1:3200 and a neutralizing titer of 1:2. 4 It can specifically recognize and bind to gB proteins. 615 RVPGERGTC 623 The sequence region does not react with glycoproteins of other herpesviruses, which helps to understand the interaction between antigens and antibodies. It has important guiding significance for the development of vaccines and detection reagents based on PRV gB protein, and provides technical support for the prevention and treatment of PRV. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The SDS-PAGE identification in Example 1 of this application is as follows: M is the marker, and bands 1-3 are, in order, post-induction sonication precipitate, post-induction sonication supernatant and pre-induction whole bacteria.

[0025] Figure 2 This is the Western Blot identification in Example 1 of this application; where Figure A shows the refolding of the target protein after purification, Figure B shows the reaction of the target protein with His monoclonal antibody, Figure C shows the reaction of the target protein with PRV positive serum; M is the marker.

[0026] Figure 3 This is the indirect ELISA screening of positive hybridoma cell lines after fusion in Example 2 of this application.

[0027] Figure 4 This refers to the positive hybridoma cell line selected by IPMA fusion in Example 2 of this application.

[0028] Figure 5 The SDS-PAGE analysis of the purified ascites fluid from the monoclonal antibody in Example 3 of this application is shown; where M is the marker, and bands 1, 3, and 5 are the purification results of ascites fluid from 4A10, 1D12, and 5E7, respectively, while bands 2, 4, and 6 are the purification results of ascites fluid from 4A10, 1D12, and 5E7, respectively.

[0029] Figure 6 This study aims to determine the affinity of the 4A10 monoclonal antibody prepared in Example 3 of this application.

[0030] Figure 7 The neutralizing titer of the 4A10 monoclonal antibody prepared in Example 3 of this application.

[0031] Figure 8 This is for the identification of the recognition antigenic epitope of the 4A10 monoclonal antibody prepared in Example 3 of this application. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1: Preparation of PRV gB recombinant protein

[0035] 1. Construction of pET-32a-gB recombinant plasmid

[0036] Based on the PRV gene sequence in the GenBank database (GenBank accession No. QQL12166.1), primers were designed using SnapGene software (Table 1). EcoR1 and Xho 1 were selected as restriction enzyme sites. The codon-optimized gB gene (sequence shown in SEQ ID NO.14) was amplified by PCR and ligated into the pET-32a vector. The plasmid was then transformed into Rosetta competent cells to construct the pET-32a-gB-Rosetta expression bacterium.

[0037] Table 1 Primer sequence listing

[0038]

[0039] The results showed that using the gB target gene as a template, PCR amplification with synthesized gB-specific primers yielded a 2186 bp band, the molecular weight of which matched the expected size of the target gene. Based on the bacterial PCR results, the pET-32a-gB vector was preliminarily identified as successfully constructed.

[0040] 2. Expression of PRV gB protein

[0041] After initial induction of expression in the constructed pET-32a-gB-Rosetta expression bacterium, it was sonicated and centrifuged. The supernatant was collected, and the precipitate was resuspended in cell disruption buffer. SDS-PAGE was used to analyze the expression of gB protein. The results showed that the pET-32a-gB-Rosetta expression bacterium was in the form of inclusion bodies, with a molecular weight of approximately 100 kDa. Figure 1 ).

[0042] 3. Purification and identification of PRV gB protein

[0043] Orthogonal experiments were used to optimize the induction time, IPTG concentration, and temperature of PRV gB protein expression. The optimized conditions were then used to induce large-scale expression of gB protein, which was purified using nickel affinity chromatography. Western blotting was used to identify the specificity and reactivity of the refolded protein. Results showed that the refolded gB recombinant protein reacted with positive sera containing both anti-His monoclonal antibody and PRV antibody, indicating that the refolded gB recombinant protein possessed good specificity and reactivity. Figure 2 ).

[0044] Example 2: Preparation of PRV gB recombinant protein monoclonal antibody

[0045] 1. Animal immunization and preparation of polyclonal antibodies

[0046] The gB recombinant protein was mixed with an adjuvant in equal volumes, emulsified, and then used to immunize mice. The mice were immunized once every 14 days for a total of 3 times. The serum titer of the immunized mice was determined by indirect ELISA.

[0047] (1) Indirect ELISA determination of serum titer in mice

[0048] ① Dilute the purified gB protein to 2 μg / mL, add 100 μL / well to the microplate, and incubate at 37℃ for 2 h.

[0049] ② Discard the coating solution, wash 3 times with PBST, discard the washing solution, add 300 μL of 5% skim milk per well, and block at 37℃ for 2 h.

[0050] ③ Discard the blocking solution, wash 3 times with PBST, discard the washing solution, add 1:200 diluted mouse polyclonal antibody serum to the first well, and serially dilute it 2 times with PBS. Add 100 μL / well to the microplate, incubate at 37℃ for 1 h, wash 6 times with PBST, and discard the washing solution.

[0051] ④ Dilute PCoat Anti-Mouse IgG / HRP (1:5000) with 5% skim milk and add 100 μL / well to the microplate. Incubate at 37°C for 1 h, wash 6 times with PBST, and discard the washing solution.

[0052] ⑤ Add TMB colorimetric solution at a rate of 100 μL / well and react in the dark for 8-10 min.

[0053] ⑥ Add 100 μL / well of 2% H2SO4 as a stop solution to terminate the reaction, and read the OD. 450 nm value.

[0054] Serum from immunized mice was serially diluted as the primary antibody, and Goat Anti-Mouse IgG / HRP (1:5000) was used as the secondary antibody. Serum titers were detected by indirect ELISA; the titers of both mice were 1:1.024×10⁻⁶. 5 .

[0055] 2. Cell fusion

[0056] Three days before cell fusion, mice with the highest titer were selected and injected intraperitoneally with 100 µg of recombinant gB protein to induce hyperimmunity. Cell fusion was performed three days after hyperimmunity. First, a healthy Kunming mouse was euthanized by cervical insemination and immersed in 75% alcohol. The mouse was fixed on a dissecting board, and its skin was cut open with sterile scissors and forceps in a laminar flow hood. 20 mL of RPMI-1640 medium containing HAT and 10% fetal bovine serum was injected into the peritoneal cavity of the mouse using a sterile syringe. The medium was then aspirated, mixed with 80 mL of medium, and spread into 96-well cell culture plates, 100 μL per well. The next day, ocular blood was collected from the hyperimmune mice. After incubating the blood at 37°C for 2 hours, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected as a positive control, aliquoted, and stored at -20°C for later use. Next, the superimmune mice were euthanized by cervical indwelling and immersed in 75% alcohol for 5 minutes. Afterward, they were fixed on a dissecting board, and their fur was cut open using sterile scissors and forceps in a laminar flow hood. A second set of sterile scissors and forceps was used to cut open the mouse's peritoneum. The spleen was then removed and placed on a 200-mesh sterile nylon mesh, and ground with scissors while continuously adding GNK to rinse the cells, allowing them to filter individually into a sterile beaker below. The spleen cells were transferred to a 50 mL sterile centrifuge tube and centrifuged together with cultured SP2 / 0 cells at 1000 rpm for 10 minutes. After centrifugation, the supernatant was discarded, and the cells were gently ejected. Each cell was resuspended in 20 mL of GNK and counted. The spleen cells and SP2 / 0 cells were then mixed together at an 8:1 ratio and centrifuged at 1000 rpm for 10 minutes. After centrifugation, discard the supernatant, gently disperse the cells at the bottom, and place the centrifuge tube in a 37°C water bath. Add 1 mL of PEG1500 to the bottom of the centrifuge tube over 90 seconds, and let it stand for 90 seconds. Then, slowly add 15 mL of GNK to stop the process, and let it stand for another 5 minutes. Next, add 25 mL of GNK and centrifuge at 1000 rpm for 10 minutes. After centrifugation, discard the supernatant, gently disperse the cells at the bottom, add 100 mL of culture medium and mix gently. Spread 100 μL of the mixture into each well of a 96-well cell culture plate containing feeder cells, for a final volume of 200 μL per well. Incubate in a cell culture incubator for 7 days.

[0057] 3. Screening of hybridoma cell positive wells

[0058] Mice with the highest serum antibody titers were boosted with immunization before fusion. Spleen cells from these immunized mice were fused with pre-prepared SP2 / 0 cells, and positive hybridoma cell wells were screened using indirect ELISA. The gB recombinant protein and the vector-expressed protein were used as coating agents, and cell supernatant was used as the primary antibody. Hybridoma cell lines that reacted with the gB recombinant protein but not with the vector-expressed protein were selected. The initially screened positive hybridoma cell lines were then expanded and subcloned, and identified using IPMA.

[0059] (1) Indirect ELISA screening

[0060] First, indirect ELISA was used to screen for monoclonal hybridoma cell lines specifically targeting the PRV gB protein. Recombinant PRV gB protein was used as the coating agent for preliminary screening of the hybridoma cell supernatant. The initially screened positive hybridoma cell lines were validated three times using indirect ELISA. After excluding false-positive monoclonal cell lines, the positive cell lines were transferred to 48-well plates for further culture. Detailed procedures were followed as per the indirect ELISA titer determination for mouse serum.

[0061] (2) IPMA screening

[0062] ①Put PK 15 according to 1.5×10 4 Cells / wells were seeded in 96-well cell culture plates. When the cell confluence reached approximately 80%, the PRV HeNLH / 2017 virus was diluted with DMEM maintenance medium containing 2% FBS at a concentration of 200 TCID⁻¹. 50 100 μL was seeded into the cell plate.

[0063] ② When obvious lesions appear in the cells at the bottom of the culture plate, discard the cell supernatant and wash with PBS 3 times.

[0064] ③ Add 50 μL / well of pre-cooled 4% cell fixative to fix at room temperature for 20 min, discard the fixative, wash 3 times with PBS, discard the washing solution, add 300 μL / well of 5% skim milk, and block at 37℃ for 2 h.

[0065] ④ Add the hybridoma cell secretion supernatant directly to the sealed cell plate at a rate of 50 μL / well and incubate at 37°C for 1 h.

[0066] ⑤ Wash 3 times with PBS, then add 100 μL of Goat Anti Mouse IgG / HRP diluted 1:5000 with 5% skim milk to each well and incubate at 37°C for 1 h.

[0067] ⑥ Wash 3 times with PBS, then wash twice with double-distilled water (DDW).

[0068] ⑦ Prepare AEC colorimetric solution according to the ratio of double distilled water (DDW): solution A: solution B: solution C = 850 μL: 50 μL: 50 μL: 50 μL. After developing the color for 15 min at a volume of 50 μL / well, stop the reaction with an equal volume of DDW. Observe the results under a microscope and take pictures.

[0069] Identification of preliminarily screened OD using indirect ELISA450 Positive hybridoma cell lines >1.0

[0070] 4. Subcloning and further identification of positive cells

[0071] Subcloning was performed using HT / 1640 10% FBS medium. The selected positive hybridoma cells were diluted to 1 cell / well and added to 96-well plates pre-coated with 100 μL of feeder cells. The subcloned cells were incubated statically for 5–7 days, and the number of cell clusters in each well was observed and recorded. Positive monoclonal cell lines were screened using indirect ELISA. If necessary, 2–3 subcloning cycles could be performed until a neutralizing monoclonal cell line secreting specific PRV gB protein was obtained. The obtained neutralizing monoclonal cell line was then progressively expanded in T25 cell culture flasks at a growth rate of 1.5–2 × 10⁻⁶ cells / well. 6 Cells / vials are frozen for later use.

[0072] Six positive hybridoma cell lines were obtained and named 5E7, 2G5, 4A10, 5E11, 4F3, and 1D12, respectively. The supernatant titers of the six hybridoma cell lines ranged from 1:800 to 1:3200. Figure 3 All six positive hybridoma cell lines reacted with the gB recombinant protein but not with the carrier protein, and all were positive according to IPMA results. Figure 4 ).

[0073] Example 3: Identification of PRV gB recombinant protein monoclonal antibody

[0074] 1. Preparation, purification, and identification of monoclonal antibodies

[0075] Multiparous BALB / c female mice were intraperitoneally injected with 500 μL of Freund's incomplete adjuvant. The monoclonal cell lines 4A10, 1D12, and 5E7 from Example 2 were expanded and cultured. Seven days after the mice were injected with Freund's incomplete adjuvant, they were further cultured at a rate of 1×10⁻⁶. 6 Cells were injected into the peritoneal cavity of mice at a ratio of 500 μL / cell. After approximately 7 days, when the mice exhibited noticeable abdominal distension, slowed activity, and decreased appetite, ascites fluid was collected. The ascites fluid was centrifuged at 6000 r / min for 15 min, and the clear, pale yellow intermediate layer was collected. The monoclonal antibody was purified using the ammonium octanoate-sulfate method, and the purification results were analyzed by SDS-PAGE. Figure 5As shown, the purified monoclonal antibody exhibited two expected target bands, with sizes of 55 kDa and 25 kDa, indicating that the study successfully obtained monoclonal antibodies with a purity greater than 90%. The antibody concentrations of 4A10, 1D12, and 5E7 after purification were determined by UV spectrophotometry to be 3.1 mg / mL, 2.32 mg / mL, and 1.4 mg / mL, respectively. The titers of the purified monoclonal antibodies (4A10, 1D12, and 5E7) were determined by indirect ELISA, showing titers of 1:2.56 × 10⁻⁶. 5 1:1.28×10 5 1:6.4×10 4 .

[0076] 2. Determination of the affinity and neutralizing activity of monoclonal antibodies.

[0077] The affinity of the 4A10 monoclonal antibody, which had the highest titer in ascites fluid, was determined. The equilibrium dissociation constant (KD) of the 4A10 monoclonal antibody was determined using an indirect non-competitive enzyme-linked immunosorbent assay (ELISA) to assess its affinity. Purified gB recombinant antigen was diluted to suitable concentrations of 2 μg / mL and 1 μg / mL with coating buffer, and coated overnight at 4°C. After washing with PBST, the antibody was blocked with 5% skim milk at 37°C for 2 h. After washing, serially diluted 4A10 monoclonal antibody was added, and incubated at 37°C for 1 h. After washing, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody was added, and incubated at 37°C for 1 h. After thorough washing with PBST, TMB chromogenic solution was added for color development in the dark. The reaction was terminated with 2 mol / L H2SO4, and the absorbance (OD) was measured at 450 nm. 450 (Plotting antibody concentration on the x-axis, the corresponding OD...) 450 The saturation binding curve was plotted with values ​​on the ordinate, and the half-maximal effective concentration (EC50) was calculated using nonlinear regression fitting. 50 The equilibrium dissociation constant (KD) of the antibody was calculated using a formula, and the KD value was used to evaluate the binding affinity between the 4A10 monoclonal antibody and the antigen. Results are shown below. Figure 6 Its affinity constant is 3.4 × 10⁻⁶. 8 L / moL.

[0078] 3. Neutralization activity assay of monoclonal antibodies

[0079] The IPMA titer of the purified 4A10 monoclonal antibody with the highest titer was determined, showing an IPMA titer of 1:3200. PK-15 cells in good growth condition were seeded in 96-well plates one day in advance. The following day, when the cell density reached approximately 80%, the PRV HeNLH / 2017 wild-type strain was diluted to 200 TCID in 2% DMEM medium.50 / 100 μL, the purified monoclonal antibody ascites fluid was diluted 0, 2, 4, 8, 16, 32, and 64 times with 2% DMEM medium. 100 μL of antibody from each dilution was then mixed with 100 μL of 200 TCID45 molecules. 50 Mix 100 μL of virus solution thoroughly and incubate at 37°C in a CO2 incubator for 1 h. Wash cells twice with sterile PBS, add the virus and antibody mixture, and perform 5 replicates for each dilution. Include positive and blank controls; the positive control contains only 100 TCID45 molecules. 50 / 100 μL of PRV HeNLH / 2017 epidemic strain, with a blank control containing only DMEM medium, and antibody neutralization titer determined by IPMA after 24 h. Results are as follows. Figure 7 As shown, the neutralizing valence is 1:2. 4 .

[0080] 4. Identification of antigenic epitopes recognized by monoclonal antibodies

[0081] The antigenic epitopes recognized by the monoclonal antibody were preliminarily identified using peptide scanning technology. The peptide was synthesized by Jier Biochemical (Shanghai, China) based on the PRV gB protein sequence (GenBank accession No. QQL12166.1) from the GenBank database. This peptide was conjugated to bovine serum albumin (BSA) carrier protein via Sulfo-SMCC, and the conjugated peptide was spotted onto nitrocellulose membranes at 1 μL per well. gB-positive serum was used as a positive control, and BSA as a negative control, with 1 μL spotted onto each well of the nitrocellulose membrane. After drying, the supernatant of hybridoma cells from strain 4A10 was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The reactivity of the hybridoma cell supernatant with the peptide was detected by Dot-blot.

[0082] Preliminary reaction results of hybridoma cell supernatant and peptide showed that the 4A10 monoclonal antibody recognized peptide p7, the antigenic site of which is located at peptide 615-623 of the pseudorabies virus gB protein. 615 RVPGERGTC 623 ), the results are shown Figure 8 .

[0083] Example 4: Cloning of the variable region gene of PRV gB recombinant protein monoclonal antibody

[0084] 4A10 hybridoma cells in the logarithmic growth phase were collected by centrifugation, and total RNA was extracted according to the RNA extraction kit instructions. RNA concentration and purity were determined using a nucleic acid micro-detector, expressed as OD0.05. 260 / OD 280A value between 1.8 and 2.0 is considered acceptable; RNA integrity is also verified by 1% agarose gel electrophoresis.

[0085] Using the extracted total RNA as a template, the first strand of cDNA was synthesized using a reverse transcription kit:

[0086] The reaction system was as follows: 5 μL total RNA, 1 μL Oligo (dT) primer, 1 μL dNTP Mix, and RNase-free water to a total volume of 10 μL; after mixing, incubate at 70°C for 5 min, then immediately on ice for 2 min; subsequently, add 4 μL 5×RT buffer, 1 μL RNase inhibitor, 1 μL M-MLV reverse transcriptase, and RNase-free water to a total volume of 20 μL.

[0087] Reaction conditions: 42℃ for 60 min, 70℃ for 10 min to inactivate reverse transcriptase, and store the reaction product at -20℃ for later use.

[0088] Based on the conserved sequences of murine immunoglobulin genes, the heavy chain variable region (VH) and light chain variable region (VL) were amplified using universal degenerate primers, respectively:

[0089] PCR reaction system (25 μL): 2 μL cDNA template, 2.5 μL 10×PCR buffer, 2 μL dNTP, 1 μL each of forward and reverse primers, 0.3 μL high-fidelity DNA polymerase, and ddH2O to a final volume of 25 μL.

[0090] PCR amplification program: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 35 cycles; 72℃ for 10 min, incubate at 4℃.

[0091] Five μL of PCR product was analyzed by 1.5% agarose gel electrophoresis. The expected band size was: VH: approximately 350-400 bp; VL: approximately 320-380 bp. The target band was purified by gel extraction using a gel extraction kit. The purified product was ligated to the TA cloning vector overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar containing ampicillin. The cells were incubated upside down at 37°C for 12-16 h. Single colonies were picked for colony PCR identification. Positive colonies were inoculated into LB liquid medium for expansion and plasmid extraction. At least three independent positive clones were selected for each target fragment, and bidirectional sequencing was performed using universal primers (M13F / M13R). The sequencing results were analyzed by sequence splicing, alignment, and open reading frame analysis to determine the correct nucleotide and amino acid sequences of the 4A10 antibody VH and VL.

[0092] Table 2. Amino acid sequences of the variable regions FR and CDR in the heavy chain of monoclonal antibodies.

[0093]

[0094] Table 3. Amino acid sequences of the variable regions FR and CDR of the light chain of monoclonal antibodies.

[0095]

[0096] The variable region sequence of the screened neutralizing monoclonal antibody 4A10 was amplified using the molecular cloning techniques described above, and then sequenced. The nucleotide and amino acid sequences of the heavy chain variable region of the obtained neutralizing monoclonal antibody are shown in SEQ ID NO.9 and SEQ ID NO.7, respectively; the nucleotide and amino acid sequences of the light chain variable region are shown in SEQ ID NO.10 and SEQ ID NO.8, respectively; the amino acid sequences of the heavy chain variable regions CDR H1, CDR H2, and CDR H3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively (Table 2), and the amino acid sequences of the light chain variable regions CDR L1, CDR L2, and CDR L3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively (Table 3).

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A neutralizing monoclonal antibody against pseudorabies virus gB protein, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes CDR H1 with the amino acid sequence shown in SEQ ID NO.1, CDR H2 with the amino acid sequence shown in SEQ ID NO.2, and CDR H3 with the amino acid sequence shown in SEQ ID NO.3; the light chain variable region includes CDR L1 with the amino acid sequence shown in SEQ ID NO.4, CDR L2 with the amino acid sequence shown in SEQ ID NO.5, and CDR L3 with the amino acid sequence shown in SEQ ID NO.

6.

2. The neutralizing monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The antigenic epitope peptide specifically recognized by the neutralizing monoclonal antibody according to claim 2, characterized in that: The antigenic epitope peptide is located in the 615-623 peptide segment of the pseudorabies virus gB protein, and its amino acid sequence is shown in SEQ ID NO.

11.

4. A gene encoding the neutralizing monoclonal antibody of claim 2, characterized in that: The DNA sequence encoding the heavy chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO.9, and the DNA sequence encoding the light chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO.

10.

5. An expression vector comprising the gene of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. The use of the neutralizing monoclonal antibody of claim 1 or 2, the antigenic epitope peptide of claim 3, the gene of claim 4, the expression vector of claim 5, or the host cell of claim 6 in the preparation of reagents or kits for detecting pseudorabies virus.

8. The use of the neutralizing monoclonal antibody of claim 1 or 2, the antigenic epitope peptide of claim 3, the gene of claim 4, the expression vector of claim 5, or the host cell of claim 6 in the preparation of a drug or vaccine for the prevention or treatment of pseudorabies virus infection.

9. A kit for detecting pseudorabies virus, characterized in that: The kit contains the neutralizing monoclonal antibody as described in claim 1 or 2.

10. A drug for treating pseudorabies virus, characterized in that, The active ingredient of the drug is the neutralizing monoclonal antibody as described in claim 1 or 2.

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