Anti-pseudorabies virus gD protein neutralizing monoclonal antibodies and uses thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an anti-pseudorabies virus (PRV) gD protein neutralizing monoclonal antibody and application thereof. The heavy chain variable region of the monoclonal antibody comprises CDR1 with an amino acid sequence of GYTFTDYW, CDR2 with an amino acid sequence of IDTSDSYT and CDR3 with an amino acid sequence of ARRTVPFDY; the light chain variable region of the monoclonal antibody comprises CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS and CDR3 with an amino acid sequence of QHIRELTR. The antibody provides a new raw material for research on a pathogenic mechanism of the pseudorabies virus, development of an early detection kit and a therapeutic antibody or vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a neutralizing monoclonal antibody against pseudorabies virus (PRV) gD protein and its application. Background Technology

[0002] Pseudorabies virus (PRV), also known as Olsonsky virus or suid herpesvirus 1, is the pathogen that causes pseudorabies (PR). New variants of pseudorabies virus with even stronger virulence can overcome the immune protection of traditional vaccines, significantly increasing the difficulty of prevention and control. Therefore, in addition to expanding our understanding of the pseudorabies virus proteome and individual protein functions, it is also necessary to find an appropriate balance between antibody and cell-mediated pseudorabies virus immune responses. These are of great significance for the rational design of targeted vaccines.

[0003] Pseudorabies virus encodes 70 to 100 different proteins. The gD protein, composed of 402 amino acids, has a highly conserved nucleotide sequence and is located in the US region of the pseudorabies virus genome. As an important immunogenic protein, the gD protein effectively activates the host's humoral and cellular immune responses, inducing high levels of neutralizing antibodies. This characteristic, coupled with its irreplaceable role in viral invasion, makes the gD protein an ideal target for developing novel pseudorabies virus subunit vaccines and genetically engineered vaccines. The mechanism of action of neutralizing antibodies mainly involves altering the conformation of the viral surface, preventing viral adsorption to susceptible cells, and thus preventing the virus from penetrating and proliferating within susceptible cells. Screening for neutralizing antibodies against the gD protein will enhance our understanding of the humoral immune response in pigs, providing a theoretical basis for designing novel pseudorabies virus vaccines and effectively controlling pseudorabies. Summary of the Invention

[0004] This invention screened neutralizing antibodies against the gD protein, providing a theoretical basis for designing novel vaccines against pseudorabies virus and effectively controlling pseudorabies.

[0005] The present invention specifically adopts the following technical solution:

[0006] In a first aspect, the present invention provides a neutralizing monoclonal antibody against pseudorabies virus gD protein, wherein the heavy chain variable region of the monoclonal antibody comprises CDR1 with the amino acid sequence GYTFTDYW (as shown in SEQ ID NO:7), CDR2 with the amino acid sequence IDTSDSYT (as shown in SEQ ID NO:8), and CDR3 with the amino acid sequence ARRTVPFDY (as shown in SEQ ID NO:9); and the light chain variable region of the monoclonal antibody comprises CDR1 with the amino acid sequence KSVSTSGYSY (as shown in SEQ ID NO:10), CDR2 with the amino acid sequence LVS, and CDR3 with the amino acid sequence QHIRELTR (as shown in SEQ ID NO:11).

[0007] In a further embodiment, the monoclonal antibody is an antigen-binding fragment, which is a Fab fragment, an F(ab')2 fragment, or a single-chain antibody.

[0008] In a further embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0009] Secondly, the present invention also provides a nucleic acid encoding the neutralizing monoclonal antibody against the pseudorabies virus gD protein. The sequences of the nucleic acid encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0010] The monoclonal antibody is prepared based on the nucleic acid using conventional genetic engineering or protein engineering methods. Therefore, the present invention also claims protection for biological materials used in genetic engineering or protein engineering to prepare the monoclonal antibody. Thus, in a third aspect, the present invention provides biological materials comprising the nucleic acid, said biological material being an expression cassette, transposon, plasmid vector, viral vector, or host cell.

[0011] Furthermore, this invention has discovered that the monoclonal antibody can effectively inhibit the replication of pseudorabies virus, meaning that the monoclonal antibody of this invention has neutralizing activity. Therefore, in a fourth aspect, this invention provides the use of the monoclonal antibody, the nucleic acid, and the biological material in any of the following aspects:

[0012] A: Used to prepare drugs for the prevention or treatment of diseases caused by pseudorabies virus infection;

[0013] B: Used to prepare pseudorabies virus detection reagents or kits.

[0014] Fifthly, the present invention provides a drug, a pseudorabies virus detection reagent or kit containing the aforementioned monoclonal antibody.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention utilizes cell fusion and monoclonal screening techniques to successfully obtain a monoclonal antibody with neutralizing activity specifically targeting the gD protein of pseudorabies virus. This monoclonal antibody exhibits neutralizing activity in vitro, preventing pseudorabies virus infection of susceptible cells, and demonstrates a strong affinity for the gD protein, providing a reliable research tool for further investigation of the gD protein's function.

[0017] The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term passage or cryopreservation of hybridoma cells. It also facilitates the optimization of the antibody at the gene and protein levels, thereby improving the antibody's specificity and affinity.

[0018] The monoclonal antibody prepared by this invention can specifically recognize and neutralize pseudorabies virus. Therefore, this antibody provides new raw materials for research on the pathogenic mechanism of pseudorabies virus, early detection kits, and the development of therapeutic antibodies or vaccines. Attached Figure Description

[0019] Figure 1 Preparation and identification results of pseudorabies virus gD recombinant protein; A. Coomassie brilliant blue staining results, B. Western blot results.

[0020] Figure 2 : Results of polyclonal antibody titer detection; 1#, 2#, 3#, 4#, and 5# in the figure represent mice immunized with pseudorabies virus gD recombinant protein.

[0021] Figure 3 IFA was used to identify the specificity of monoclonal antibodies; A. SP2 / 0 cell supernatant was used on pseudorabies virus-infected cells; B. 3A10 cell supernatant was used on pseudorabies virus-infected PK15 cells.

[0022] Figure 4 : Results of monoclonal antibody subclass identification.

[0023] Figure 5 SDS-PAGE electrophoresis identification of monoclonal antibodies obtained after purification of ascites fluid.

[0024] Figure 6 The titer of the monoclonal antibody obtained after purification of ascites fluid was measured.

[0025] Figure 7 Results of the identification of the neutralizing activity of monoclonal antibody 3A10.

[0026] Figure 8Agarose gel electrophoresis results of gene amplification products of monoclonal antibody 3A10 heavy chain variable region and κ light chain variable region. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Experimental materials used in the examples:

[0029] SP2 / 0 and PK15 cells were preserved in the inventor's laboratory and can also be obtained by those skilled in the art through purchase. The purified gD recombinant protein and recombinant fluorescent virus rPRV-EGFP-Fluc were constructed and preserved by the inventor (reference: Yu Zhang, Xiangtong Li, Juan Zhang, Yueyue Duan, Peibin Chen, Lei Shi, CongYuan, Liyan Cao, Maowen Sun, Yating Wang, Xiangyu Kong, Haixue Zheng, QiWang. (2024). A rapid and versatile reverse genetic approach and visualization animal models for emerging zoonotic pseudorabies virus. Antiviral Res. Dec:232:106036.). Freund's complete and incomplete adjuvants and polyethylene glycol (PEG) were purchased from Sigma-Aldrich. Horseradish peroxidase-labeled goat anti-mouse IgG antibody (HRP-IgG) and fluorescein isothiocyanate-labeled goat anti-mouse antibody (FITC-IgG) were purchased from Abcam. Prime STAR® Max DNA Polymerase (Premix) and PrimeScript II 1 st Strand cDNA Synthesis Kit purchased from Takara, SBAClonotyping TMThe System / HRP antibody subclass identification kit was purchased from Southern Biotech, the mouse IgG control was purchased from Solarbio, and the BALB / c female mice were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute (Lanzhou Branch of China Animal Health and Epidemiology Center), Chinese Academy of Agricultural Sciences.

[0030] Example 1: Preparation of recombinant pseudorabies virus gD protein

[0031] (1) Construction of a stable gD expression cell line

[0032] Using the genome of pseudorabies virus PRV CD22 strain (GenBank accession number: OR666765) as a template, the extracellular region sequence of the gD protein (corresponding to the US6 gene) was amplified. This extracellular region sequence is Ala. 18 -Arg 353 The encoding nucleotide sequence of the gD protein extracellular region was obtained; the encoding nucleotide sequence of the signal peptide was fused to the N-terminus of the gD protein extracellular region sequence, and the encoding nucleotide sequence of the 6×His tag was fused to the C-terminus. The above fusion sequence (as shown in SEQ ID NO:1) was inserted between the EcoRI and BamHI restriction sites of the pLV-IRES-puro lentiviral vector using molecular cloning technology to construct the recombinant lentiviral vector pLVS-IRES-puro-gD.

[0033] Next, 10 μg of pLVS-IRES-puro-gD, 6.6 μg of psPAX2 packaging plasmid, and 3.3 μg of pMD2.G envelope plasmid were co-transfected into HEK-293T cells. Forty-eight hours after transfection, the cell culture supernatant was collected, which was the packaged lentiviral particle. The packaged lentiviral particle was transduced into fresh monolayer HEK-293T cells, with uninoculated HEK-293T cells serving as a blank control. Forty-eight hours after transduction, 4 μg / mL of puromycin was added to the cell culture system for pressure selection. Selection continued until the blank control cells were completely inactivated. The surviving cells were then expanded to obtain a stable HEK-293T cell line expressing the gD recombinant protein.

[0034] The amino acid sequence of the gD recombinant protein is shown in SEQ ID NO:2.

[0035] (2) Expression and purification of gD recombinant protein

[0036] HEK-293T cell lines stably expressing recombinant gD protein were expanded and cultured. 1 L of cell culture supernatant was collected and centrifuged at 12000 rpm for 10 min. The supernatant was then collected, and the recombinant gD protein was purified using Ni column affinity chromatography. The eluted protein was replaced with PBS buffer to obtain a purified recombinant pseudorabies virus gD protein solution.

[0037] (3) Identification of gD recombinant protein

[0038] Two gels were prepared for SDS-PAGE electrophoresis, one for Coomassie brilliant blue staining and the other for Western blotting (WB) detection of proteins. The specific procedures are as follows:

[0039] SDS-PAGE electrophoresis: Take 80 μL of gD protein solution, add 20 μL of 5× protein loading buffer, and boil in a metal bath at 100℃ for 10 min, followed by centrifugation at 12000 g for 5 min. Perform electrophoresis using a 10% separating gel, setting the voltage to 150 V and the electrophoresis time to 60 min.

[0040] Coomassie Brilliant Blue staining: The gel after electrophoresis was completely immersed in Coomassie Brilliant Blue staining solution and stained by gentle shaking at room temperature for 2 h, followed by destaining for 2 h. Finally, the gel was imaged and analyzed using an imaging system.

[0041] Western blot (WB) detection: The electrophoresed gel was transferred to a nitrocellulose membrane (NC membrane) under wet transfer conditions for 90 min. After transfer, the NC membrane was placed in 5% bovine serum albumin (BSA) diluted with TBST buffer and gently shaken at room temperature for 1 h. After blocking, His mouse primary antibody was diluted to 1:5000 with TBST buffer and added to the NC membrane for incubation at room temperature with gentle shaking for 1 h. After incubation, the NC membrane was washed 4 times with TBST buffer for 10 min each time. Then, goat anti-mouse HRP-IgG secondary antibody was diluted to 1:10000 with TBST buffer and added to the NC membrane for incubation at room temperature with gentle shaking in the dark for 1 h. After incubation, the NC membrane was washed 4 times with TBST buffer for 10 min each time in the dark. Finally, imaging analysis was performed using an imaging system.

[0042] The results are as follows Figure 1 As shown, Coomassie brilliant blue staining ( Figure 1 A) and WB ( Figure 1 B) A single band of approximately 55 kDa appears in both bands.

[0043] Example 2: Screening and identification of monoclonal antibodies against pseudorabies virus gD protein

[0044] 1. Animal immunization

[0045] Five 6-week-old female BALB / c mice (labeled 1#, 2#, 3#, 4#, and 5#) were selected, and purified gD recombinant protein was immunized at a dose of 30 μg / mouse. For the initial immunization, the gD recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites (one on the back and two on the abdomen). Booster immunizations were administered every two weeks for a total of four immunizations. For the booster immunizations, an equal volume of gD recombinant protein and Freund's incomplete adjuvant were emulsified using the same method as the initial immunization. Seven days after the fourth immunization, tail blood was collected to determine the polyclonal antibody titer.

[0046] 2. ELISA method for detecting polyclonal antibody titers

[0047] The purified gD recombinant protein was diluted to 1 μg / mL with coating buffer and added to ELISA plates at 50 μL / well. The plates were incubated at 37°C for 1 h. After washing four times with PBST (K2HPO4 0.26 g, Na2HPO4·12H2O 2.89 g, NaCl 8.50 g, Tween-20 0.5 mL, diluted to 1 L with water), 5% skim milk powder was added, and the plates were blocked at 37°C for 1 h. Positive sera from five immunized mice and negative sera from one non-immunized mouse (NC) were serially diluted with PBST at dilutions ranging from 1:1000 to 1:2048000 (12 dilutions). The plates were incubated at 37°C for 30 min. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. After washing four times with PBST, TMB was developed, and the OD was measured using a microplate reader. 450 The P / N ratio is calculated based on the OD values ​​of positive and negative sera for determination. A P / N ratio ≥ 2.0 is considered a positive reaction. The highest serum dilution factor that achieves a positive result is defined as the antibody titer of the sample.

[0048] The results are as follows Figure 2 As shown, the serum antibody titers of mice #2 and #5, which were immunized with gD protein, were both greater than 1:64000, meeting the requirements for cell fusion.

[0049] 3. Preparation of monoclonal antibodies

[0050] Spleen cells from a gD recombinant protein-immunized mouse were mixed with SP2 / 0 cells at a ratio of 5:1 and fused using PEG fusion agent. The fused cells were seeded into 96-well cell culture plates and cultured at 37°C in a CO2 incubator. Once the hybridoma cells covered the bottom 1 / 10 of the cell plate, the supernatant was collected for ELISA detection (ELISA coating method as described in "ELISA Method for Detecting Polyclonal Antibody Titer"). Wells identified as positive by ELISA were then validated by indirect immunofluorescence assay (IFA). Selected ELISA and IFA positive wells were subcloned using a limiting dilution method. Wells showing only a single clone were observed under an inverted microscope; the supernatant was collected and antibody detected using the ELISA and IFA methods described above. Positive cells were then subjected to the next round of subcloning, for a total of three cycles.

[0051] IFA test: 1×10 4 PK15 cells were seeded in 96-well cell culture plates. After the cells formed a monolayer, they were inoculated with 0.01 MOI of pseudorabies virus (PRV) CD22. Forty-eight hours after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with 0.01 mol / L PBS (K₂HPO₄ 0.26 g, Na₂HPO₄·12H₂O 2.89 g, NaCl 8.50 g, diluted to 1 L with water, pH 7.4). The cells were then permeabilized with 0.1% Triton X-100 at room temperature for 10 minutes, followed by three washes with PBS. The hybridoma cell supernatant and anti-mouse FITC-labeled goat anti-mouse IgG were then incubated. After the reaction was complete, the results were observed under a fluorescence microscope.

[0052] 3. Monoclonal antibody specificity identification

[0053] After three subclonings, the cell supernatant secreted by the obtained single cell line (3A10) was subjected to IFA analysis. IFA results showed that the 3A10 cell supernatant, when used to treat PK15 cells infected with pseudorabies virus, produced a specific green fluorescent signal. Figure 3 B), while no fluorescent signal was observed in pseudorabies virus-infected cells treated with SP2 / 0 cell supernatant (B). Figure 3 A).

[0054] 4. Identification of monoclonal antibody subclasses

[0055] According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Instructions: This kit identifies the antibody subclass of monoclonal antibodies obtained from the supernatant of 3A10 cells. The monoclonal antibody's heavy chain constant region is IgG1 type, and its light chain constant region is Kappa type. Figure 4As shown.

[0056] 5. Preparation, purification, and potency testing of ascites fluid

[0057] 5.1 Preparation of Ascites

[0058] Female Balb / c mice aged 10–12 weeks were administered 0.5 mL of Freund's incomplete adjuvant intraperitoneally. One week later, each mouse was administered 5 × 10⁻⁶ mL of the adjuvant intraperitoneally. 5 In the preparation of monoclonal antibodies, positive hybridoma cells (0.2 mL) obtained after three subclonings were collected. After 7-10 days, the abdominal cavity of the mice became significantly distended. Ascites fluid was collected, aliquoted, and stored at -80℃.

[0059] 5.2 Purification of Ascites Fluid

[0060] According to PIERCE company NAB TM The protein G was purified by affinity chromatography using the Protein G Spin Purification Kit, and the purity was then identified by SDS-PAGE electrophoresis.

[0061] Depend on Figure 5 It can be concluded that the heavy chain of the monoclonal antibody obtained after purification of ascites fluid is about 55 kDa and the light chain is about 25 kDa. The monoclonal antibody is named 3A10.

[0062] 5.3 Monoclonal Antibody Titer Detection

[0063] The monoclonal antibody 3A10 obtained from purified ascites fluid was used to determine the antibody concentration by the BCA method. It was then diluted with PBST to 1 mg / mL and serially diluted with PBS (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000). 50 μL of the diluted monoclonal antibody solution was added to each well, and its titer was measured. The specific experimental steps were the same as in Case 2, "Detection of Polyclonal Antibody Titer by ELISA".

[0064] The results are as follows Figure 6 As shown, the monoclonal antibody 3A10 maintained a significantly higher absorbance value than the negative control well at a dilution of 1:128000, and the P / N ratios all met the positive criteria.

[0065] Example 3: Identification of the neutralizing activity of monoclonal antibody 3A10 against pseudorabies virus gD protein.

[0066] 1. Neutralization experiment

[0067] To verify whether monoclonal antibody 3A10 has neutralizing activity, a neutralization experiment was conducted. The specific steps are as follows:

[0068] (1) Take one vial of PK15 cells frozen in liquid nitrogen, thaw rapidly in a 37°C water bath, centrifuge at 500 g for 5 min, discard the supernatant, resuspend the cells in 1 mL of DMEM medium containing 10% FBS and 2% antibiotics, passage 3 times, and then use 1×10⁻⁶ cells per cell. 5 Cells / well were seeded into 96-well cell culture plates and incubated at 37°C for 24 h.

[0069] (2) The fluorescent virus rPRV-EGFP-Fluc (7.5×10⁻⁶) constructed in the inventor's laboratory 7 TCID 50 ( / mL) Dilute to 2000 TCID with DMEM 50 / mL, the virus was mixed with DMEM, mouse IgG and 3A10 antibody (1 mg / mL) in equal volumes, and incubated at 37℃ for 1 h. Then, the cells in (1) were inoculated and cultured for another 24 h. At the same time, a blank cell control group (Mock) was set up without virus inoculation.

[0070] (3) After culturing at 37℃ for 2-3 days, observe the fluorescence of cells infected with the fluorescent virus rPRV-EGFP-Fluc using an inverted fluorescence microscope, take pictures, record and save them.

[0071] Experimental results are as follows Figure 7 As shown, compared with the untreated group (DMEM) and the unrelated antibody treatment group (mouse IgG), the fluorescence of rPRV-EGFP-Fluc infected cells was significantly reduced in the monoclonal antibody 3A10 treatment group, indicating that antibody 3A10 can effectively inhibit the replication of pseudorabies virus.

[0072] Example 4: PCR amplification and sequencing of the variable region gene of the anti-pseudorabies virus gD protein monoclonal antibody 3A10

[0073] RNA was extracted from 3A10 hybridoma cells and analyzed using PrimeScript II 1. st The Strand cDNA Synthesis Kit reverse transcribes it to synthesize cDNA.

[0074] The antibody variable region gene was amplified using nested PCR. First, using the aforementioned cDNA as a template, the antibody variable region gene was amplified using a first-round primer sequence of murine IgG and κ light chain antibodies. Then, using the first-round product as a template, the antibody variable region gene was amplified using a second-round primer sequence of murine IgG and κ light chain antibodies. PCR amplification was performed using Prime STAR® Max DNA Polymerase (Premix), with the following reaction program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min. Primers for antibody variable region gene amplification are referenced in the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., & Nussenzweig, MC(2016). Sequencing and cloning of antigen-specific antibodies from mousememory B cells. Nature protocols, 11(10), 1908–1923.).

[0075] After amplification, 1% agarose gel electrophoresis was performed. The gene size of the variable regions of the heavy chain and κ light chain of monoclonal antibody 3A10 was approximately 400 bp. Figure 8 The target fragment was then excised and recovered. This recovered fragment was inserted into the pMD-18T vector and sequenced. The sequencing results were compared with the antibody gene library (IMGT), confirming that the amplified sequence was DNA from the heavy and light chain variable regions of the monoclonal antibody.

[0076] Specifically, the DNA sequence encoding the heavy chain variable region of mouse anti-pseudorabies virus gD protein monoclonal antibody 3A10 is shown in SEQ ID NO:3; the DNA sequence encoding the light chain variable region of mouse anti-pseudorabies virus gD protein monoclonal antibody 3A10 is shown in SEQ ID NO:4.

[0077] The amino acid sequence of the heavy chain variable region of mouse anti-pseudorabies virus gD protein monoclonal antibody 3A10 is shown in SEQ ID NO:5; the amino acid sequence of the light chain variable region of mouse anti-pseudorabies virus gD protein monoclonal antibody 3A10 is shown in SEQ ID NO:6. The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of the 3A10 monoclonal antibody are shown in Table 1 below.

[0078] Table 1. Amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 3A10.

[0079]

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A neutralizing monoclonal antibody against pseudorabies virus gD protein, characterized in that, The heavy chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence GYTFTDYW, CDR2 with the amino acid sequence IDTSDSYT, and CDR3 with the amino acid sequence ARRTVPFDY; the light chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence KSVSTSGYSY, CDR2 with the amino acid sequence LVS, and CDR3 with the amino acid sequence QHIRELTR.

2. The neutralizing monoclonal antibody against pseudorabies virus gD protein according to claim 1, characterized in that, The monoclonal antibody is an antigen-binding fragment, which may be a Fab fragment, an F(ab')2 fragment, or a single-chain antibody.

3. The neutralizing monoclonal antibody against pseudorabies virus gD protein according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

4. The nucleic acid encoding the neutralizing monoclonal antibody against pseudorabies virus gD protein as described in claim 1.

5. The nucleic acid according to claim 4, characterized in that, The sequences of the nucleic acids encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

6. A biological material comprising the nucleic acid of claim 4 or 5, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

7. The use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, or the biological material according to claim 6 in any of the following aspects: A: Used to prepare drugs for the prevention or treatment of diseases caused by pseudorabies virus infection; B: Used to prepare pseudorabies virus detection reagents or kits.

8. A drug comprising the monoclonal antibody according to any one of claims 1 to 3.

9. A pseudorabies virus detection reagent or kit containing the monoclonal antibody according to any one of claims 1 to 3.

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