Anti-pseudorabies virus gD protein neutralizing monoclonal antibody, its preparation method and application

Abstract

This invention discloses a neutralizing monoclonal antibody against pseudorabies virus (PRV) gD protein, its preparation method, and its application. It belongs to the field of immunoglobulin technology. The neutralizing monoclonal antibody against PRV gD protein includes a heavy chain variable region and a light chain variable region. The complementarity-determining regions (CDR1, CDR2, and CDR3) in the heavy chain variable region include amino acid sequences as shown in positions 31-35, 50-66, and 99-110 of SEQ ID NO:1, respectively. The complementarity-determining regions (CDR1, CDR2, and CDR3) in the light chain variable region include amino acid sequences as shown in positions 24-34, 50-56, and 89-97 of SEQ ID NO:3, respectively. This neutralizing monoclonal antibody can specifically react with the gD protein, effectively recognize PRV, and efficiently neutralize PRV infection.

Description

Technical Field

[0001] This invention relates to the field of immunoglobulin technology, specifically to a neutralizing monoclonal antibody against pseudorabies virus gD protein, its preparation method, and its application. Background Technology

[0002] Pseudorabies (PR), also known as Aujeszky disease, is a highly contagious disease caused by the pseudorabies virus (PRV), affecting a variety of domestic and wild mammals. Domestic pigs and wild boars are the primary natural hosts of PRV. PRV can infect pigs of all ages. Adult domestic pigs are usually asymptomatic carriers, and pregnant sows are prone to abortion or giving birth to mummified fetuses, and may carry and shed the virus for a long period. Young piglets infected with PRV exhibit central nervous system disorders and high fever as the main clinical symptoms, with a mortality rate as high as 100%. The prevalence of PRV has caused enormous economic losses to the livestock industry.

[0003] Varicellovirus (PRV) belongs to the genus Varicellovirus of the subfamily Alphaherpesvirinae within the family Orthoherpesviridae, and is also known as suid herpesvirus I (SuHV1). The glycoprotein gD on the surface of the PRV viral envelope is an essential structural protein for PRV to invade host cells and is also an important immunogenic protein. Antibodies produced by gD provide high protection for animals. Although the gD gene varies among different genotypes, it is relatively conserved, making it a good target protein for subunit vaccines.

[0004] Vaccination is crucial for eradicating PRV. Although the introduction of the live attenuated vaccine Bartha-K61 has effectively controlled the spread of PRV in my country, its protective effect against novel variants is very limited. Therefore, the development of new vaccines requires precise tools for evaluating immunization efficacy. Currently, there are no mature antibodies for evaluating the immunization efficacy of vaccines targeting the gD protein subunit. Therefore, obtaining neutralizing antibodies is of great application value for evaluating vaccine immunization efficacy and developing new diagnostic technologies for PRV gD protein antibodies. Summary of the Invention

[0005] The purpose of this invention is to provide a neutralizing monoclonal antibody against pseudorabies virus gD protein, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A neutralizing monoclonal antibody against pseudorabies virus gD protein or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region comprise amino acid sequences as shown in positions 31-35, 50-66, and 99-110 of SEQ ID NO:1, respectively; and the complementarity-determining regions CDR1, CDR2, and CDR3 in the light chain variable region comprise amino acid sequences as shown in positions 24-34, 50-56, and 89-97 of SEQ ID NO:3, respectively.

[0007] Furthermore, the neutralizing monoclonal antibody or its antigen-binding fragment against the pseudorabies virus gD protein also includes four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, and four light chain variable region framework regions named LFR1, LFR2 and LFR3 and LFR4, respectively.

[0008] The HFR1 comprises the amino acid sequence shown as positions 1 to 30 of SEQ ID NO:1; The HFR2 comprises the amino acid sequence shown as positions 36 to 49 of SEQ ID NO:1; The HFR3 comprises the amino acid sequence shown at positions 67 to 98 of SEQ ID NO:1; The HFR4 comprises the amino acid sequence shown in positions 111 to 121 of the sequence listing SEQ ID NO:1.

[0009] Furthermore, the LFR1 comprises the amino acid sequence shown in positions 1 to 23 of SEQ ID NO:3; The LFR2 comprises the amino acid sequence shown as positions 35 to 49 of SEQ ID NO:3; The LFR3 comprises the amino acid sequence shown at positions 57 to 88 of SEQ ID NO:3; The LFR4 comprises the amino acid sequence shown in positions 98 to 107 of the sequence listing SEQ ID NO:3.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned neutralizing monoclonal antibody against pseudorabies virus gD protein or its antigen-binding fragment, comprising the following steps: Nucleic acid molecules encoding the heavy chain variable region and the light chain variable region are introduced into host cells for expression. The neutralizing monoclonal antibody against the anti-pseudorabies virus gD protein or its antigen-binding fragment was isolated and purified from the host cell culture.

[0011] Another object of the present invention is to provide biological materials related to the above-mentioned anti-pseudorabies virus gD protein neutralizing monoclonal antibodies or their antigen-binding fragments, wherein the biological materials are any one of the following: A nucleic acid molecule encoding the neutralizing monoclonal antibody or its antigen-binding fragment against the pseudorabies virus gD protein; An expression cassette containing the nucleic acid molecule; A recombinant vector containing the nucleic acid molecule or expression cassette; Recombinant microorganisms containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors; Recombinant cells containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors.

[0012] Another object of the present invention is to provide the use of the above-mentioned anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment or the above-mentioned biological material in at least one of the following: Prepare products for the detection or auxiliary detection of pseudorabies virus; To prepare products for the detection or auxiliary detection of diseases caused by pseudorabies virus infection; Prepare products for screening or assisting in the screening of pseudorabies virus; To prepare products for screening or assisting in the screening of diseases caused by pseudorabies virus infection; Prepare products for the detection or auxiliary detection of pseudorabies virus gD protein; Preparation of a product that binds to the pseudorabies virus gD protein; To prepare products that neutralize pseudorabies virus infection; To prepare products for the prevention and treatment of diseases caused by pseudorabies virus infection.

[0013] The anti-pseudorabies virus gD protein neutralizing monoclonal antibody provided by this invention can specifically react with the gD protein, effectively recognize PRV, and efficiently neutralize PRV infection. The monoclonal antibody provided by this invention can be used in PRV immunological and serological diagnosis, evaluation of immunogenicity, and related experiments. It is also a good antiviral drug candidate, providing antibody resources for the effective detection and purification of PRV. Attached Figure Description

[0014] Figure 1 The results are from an indirect immunofluorescence assay of monoclonal antibody TPH027 and pseudorabies virus genotype II strain.

[0015] Figure 2 The results are from a Western blot experiment of monoclonal antibody TPH027 and pseudorabies virus gD protein.

[0016] Figure 3The reaction results of monoclonal antibody TPH027 with PRV genotype I strain (Bartha-K61) were verified by indirect immunofluorescence assay.

[0017] Figure 4 The results were obtained to verify the neutralizing ability of the monoclonal antibody TPH027 prepared from ascites fluid against the PRV-YY strain using indirect immunofluorescence assays.

[0018] Figure 5 The SDS-PAGE purification results of the monoclonal antibody TPH027 were used to express and purify it.

[0019] Figure 6 Results of the reaction between monoclonal antibody TPH027 prepared by genetic engineering methods and PRV genotype II strain (YY). Detailed Implementation

[0020] The technical solutions 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.

[0021] In one embodiment of the present invention, a neutralizing monoclonal antibody against pseudorabies virus gD protein or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region, wherein the complementarity-determining regions CDR1, CDR2 and CDR3 in the heavy chain variable region respectively comprise amino acid sequences as shown in positions 31-35, 50-66 and 99-110 of SEQ ID NO:1; and the complementarity-determining regions CDR1, CDR2 and CDR3 in the light chain variable region respectively comprise amino acid sequences as shown in positions 24-34, 50-56 and 89-97 of SEQ ID NO:3.

[0022] In a preferred embodiment of the present invention, the anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment further includes four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, and four light chain variable region framework regions named LFR1, LFR2 and LFR3 and LFR4, respectively.

[0023] Specifically, HFR1 includes the amino acid sequence shown in positions 1 to 30 of SEQ ID NO:1; HFR2 includes the amino acid sequence shown in positions 36 to 49 of SEQ ID NO:1; HFR3 includes the amino acid sequence shown in positions 67 to 98 of SEQ ID NO:1; and HFR4 includes the amino acid sequence shown in positions 111 to 121 of SEQ ID NO:1.

[0024] The LFR1 described above includes the amino acid sequence shown in positions 1 to 23 of SEQ ID NO:3; the LFR2 described above includes the amino acid sequence shown in positions 35 to 49 of SEQ ID NO:3; the LFR3 described above includes the amino acid sequence shown in positions 57 to 88 of SEQ ID NO:3; and the LFR4 described above includes the amino acid sequence shown in positions 98 to 107 of SEQ ID NO:3.

[0025] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:1 or a sequence having at least 99%, 95%, 90%, 85%, 80% or 75% identity with it and retaining antigen-binding activity; the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:3 or a sequence having at least 99%, 95%, 90%, 85%, 80% or 75% identity with it and retaining antigen-binding activity.

[0026] As used in this invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding moiety" or "antigen-binding domain." In some cases, the antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0027] The antibody or its antigen-binding fragment described in this invention includes a heavy chain variable region and a light chain variable region, both of which are composed of a complementarity-determining region and a framework region; the complementarity-determining regions of the heavy chain variable region and the light chain variable region are each composed of their respective CDR1, CDR2 and CDR3. The CDR described in this invention is a "complementarity-determining region," which is a region in the antibody variable domain that is highly variable in sequence and forms a structurally defined "hypervariant loop" and / or contains antigen contact residues, or "antigen contact sites." The CDR is primarily responsible for binding to antigen epitopes. A variable region typically contains three CDR regions, named CDR1, CDR2, and CDR3 sequentially from the N-terminus.

[0028] The framework region of the heavy chain variable region consists of four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4 respectively; the framework region of the light chain variable region consists of four light chain variable region framework regions named LFR1, LFR2 and LFR3 and LFR4 respectively.

[0029] This invention can achieve the determination of sequence identity percentages through various known methods, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for sequence alignment can be determined, including the algorithms required to achieve maximum alignment across the entire length of the compared sequences. However, for the purposes of this invention, the sequence comparison computer program ALIGN-2 is used to generate the sequence identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code has been submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559, under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0030] Specifically, the at least 75% identity includes at least 80% identity. Specifically, the at least 75% identity includes at least 85% identity. Specifically, the at least 75% identity includes at least 90% identity. Specifically, the at least 75% identity includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. More specifically, the at least 75% identity can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0031] The term "antibody" as used in the broadest sense in this invention includes polyclonal and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments, including antigen-binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, and multispecific antibodies, such as bispecific antibodies, tri- and tetra-antibodies, tandem double scFvs, and tandem triple scFvs. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. The term also covers intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may contain a human IgG1 constant region. The antibody may contain a human IgG4 constant region.

[0032] The antibody or its antigen-binding fragment described above may further include a constant region.

[0033] In the antibody or its antigen-binding fragment, the constant region may be a heavy chain constant region and a light chain constant region.

[0034] In the antibody or its antigen-binding fragment, the heavy chain constant region may be IgG, IgM, or IgA.

[0035] In the antibody or its antigen-binding fragment, the IgG may be IgG1, IgG2, IgG3 or IgG4.

[0036] In the antibody or its antigen-binding fragment, the light chain constant region may be a lambda(λ) constant region or a kappa(κ) constant region.

[0037] The antibody or its antigen-binding fragment may be mouse, human, chimeric, or humanized.

[0038] The term "monoclonal antibody" as used in this invention, unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are generally classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of this invention, the heavy chain may be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0039] In this invention, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0040] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0041] In another embodiment of the present invention, a method for preparing the above-mentioned anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment is also provided, specifically including the following steps: Nucleic acid molecules encoding the aforementioned heavy chain variable regions and light chain variable regions were introduced into host cells for expression. The above-mentioned anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment was isolated and purified from the host cell culture.

[0042] In another embodiment of the present invention, a biological material related to the above-mentioned anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment is also provided, wherein the above-mentioned biological material is any one of the following materials: Nucleic acid molecules encoding the above-mentioned neutralizing monoclonal antibody or its antigen-binding fragment against the pseudorabies virus gD protein; Expression cassettes containing the aforementioned nucleic acid molecules; Recombinant vectors containing the aforementioned nucleic acid molecules or expression cassettes; Recombinant microorganisms containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors; Recombinant cells containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors.

[0043] In the aforementioned biological materials, the nucleic acid molecule can be either a DNA molecule or an RNA molecule. The DNA molecule can be genomic DNA or a cDNA molecule. The nucleic acid molecule can be the genomic gene or cDNA gene of the protein.

[0044] In the aforementioned biological materials, the expression cassette refers to DNA capable of expressing the protein in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all regulatory sequences necessary for the expression of the nucleic acid molecule containing the protein. The regulatory sequences, under compatible conditions, guide the expression of the protein in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0045] In the aforementioned biological materials, the recombinant vector can be a cloning vector or an expression vector. As used in this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0046] When preparing an expression vector, the nucleic acid molecule encoding the aforementioned protein can be located within the vector so that it can be operatively linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, integrates into the chromosome and replicates along with the integrated chromosome. The vector contains one or more selection markers that facilitate the selection of transformed cells. A selection marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers protrophic anatrophy, etc. Examples of bacterial selection markers include the dal gene of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable stable integration of the vector into the host cell genome or ensure autonomous replication of the vector within the cell, independent of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling autonomous replication within the target host cell. The origin of replication may carry a mutation that makes it temperature-sensitive in the host cell (see, for example, f. Ehrlich, 1978, Proceedings of the National Academy of Sciences 75: 1433). The yield of the gene product can be increased by inserting more than one copy of the nucleic acid molecule encoding the aforementioned protein into the host cell. This copy number increase can be achieved by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selection marker along with the nucleic acid molecule, and by culturing cells in the presence of a suitable selection reagent to select cells containing the amplified copy of the selection marker gene, thereby containing the additional copy of the nucleic acid molecule. The operations for connecting the above elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0047] In the above-mentioned biological materials, the recombinant microorganisms may specifically be bacteria, yeast, algae, and fungi. The bacteria may be any of the following: 1) prokaryotic microorganisms; 2) Gram-negative bacteria; 3) Escherichia coli; 4) Escherichia coli.

[0048] In the aforementioned biological materials, the recombinant cells can be animal cells, and the animal cell lines mentioned above can be non-reproductive materials. The animal cells can be isolated mammalian cells. The mammals include humans or mice. The mammalian cells may exclude animal germ cells, animal fertilized eggs, and animal embryonic stem cells; they can be somatic cells or cell lines. The animal cells can be cell lines or somatic cells derived from mice.

[0049] In another embodiment of the present invention, the use of the above-described anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment or the above-described biological material in at least one of the following: Prepare products for the detection or auxiliary detection of pseudorabies virus; To prepare products for the detection or auxiliary detection of diseases caused by pseudorabies virus infection; Prepare products for screening or assisting in the screening of pseudorabies virus; To prepare products for screening or assisting in the screening of diseases caused by pseudorabies virus infection; Prepare products for the detection or auxiliary detection of pseudorabies virus gD protein; Preparation of a product that binds to the pseudorabies virus gD protein; To prepare products that neutralize pseudorabies virus infection; To prepare products for the prevention and treatment of diseases caused by pseudorabies virus infection.

[0050] In this invention, the pseudorabies virus includes circulating strains of pseudorabies virus.

[0051] The aforementioned products include the aforementioned antibodies or their antigen-binding fragments, or the aforementioned biological materials.

[0052] The products described in this invention are drugs, compositions, health products, functional foods, foods for special medical purposes, or other biological products.

[0053] In this invention, the dosage form of the drug is an injection, a lyophilized powder for injection, an aerosol, a large-volume infusion, a drop, a pill, a powder, a granule, a tablet, a capsule, an oral liquid, or an emulsion.

[0054] In this invention, the product also includes a pharmaceutically acceptable carrier, which may be an excipient, stabilizer, suspending agent or diluent, etc., as is well known to those skilled in the art.

[0055] In this invention, the pharmaceutically acceptable excipients, diluents, or carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Additionally, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation.

[0056] The following embodiments are examples of practical applications of the technical solution of the present invention, but are not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0057] The insect baculovirus expression vector pFastBac 1 in the following examples is described in the following literature: Mi Shijiang. Identification of monoclonal antibodies and broad-spectrum monoclonal antibodies for differentiating between epidemic strains and vaccine strains of classical swine fever virus and their antigenic epitope analysis [D]. Jilin University, 2022.

[0058] The prevalent PRV strains of genotype II in the following examples are described in the following literature: Yang Taotao, Zhao Dun, Liu Chongling, et al. Isolation and identification of four pseudorabies virus strains in Hunan Province and sequence analysis of their immune and virulence-related genes [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2016, 43(01):50-57. The public may obtain this biological material from the applicant (Jilin University) in accordance with the relevant national biosafety regulations. This biological material is only for repeating the relevant experiments of this invention and shall not be used for other purposes.

[0059] The protein-tagged antibody anti-His Tag mAb used in the following examples was purchased from Solarbio, catalog number K200060M.

[0060] Example 1: Preparation of monoclonal antibody against pseudorabies virus gD protein: In this embodiment, the immune antigen is the gD protein of pseudorabies virus (PRV) genotype II strain YY (PRV-YY) (named PRV-YY gD protein). PRV-YY gD protein was prepared using an insect baculovirus expression system. The steps were as follows: The transmembrane and intramembrane regions of the gD protein encoding gene (GenBank accession number: KP259814.1, positions 1-1209) containing the signal peptide were removed. A His tag (His tag nucleotide sequence 5'-CATCATCACCATCACCAT-3', as shown in SEQ ID NO:5) was added to the 3' end and cloned into the insect baculovirus expression vector pFastBac1 to obtain a recombinant expression vector. This recombinant expression vector was transformed into DH10Bac competent cells to obtain recombinant baculovirus particles containing the target gene fragment. The baculovirus particles were transfected into Sf9 cells for protein expression. The purified PRV-YY gD protein was collected using fixed metal ion affinity chromatography, and the purified protein was used to obtain an immunogenic antigen.

[0061] I. Preparation of hybridoma cell lines and monoclonal antibodies: (1) Immunization of mice: SPF-grade female BALB / c mice aged 6-8 weeks were immunized. PRV-YY gD purified protein was emulsified with an equal volume of 206 adjuvant (Seppic). The mice were immunized by subcutaneous injection at multiple points. Each mouse was immunized with 50 μg of emulsified protein. The mice were immunized once every two weeks for a total of 3 times. One week after the third immunization, 100 μg of PRV-YY gD purified protein without adjuvant was injected intraperitoneally for shock immunization. The mice were then fed for another 3 days.

[0062] (2) Cell fusion: After euthanizing the mice by dislocation, the spleen was removed, gently crushed in a nylon sieve, washed with sterile PBS, and the washing fluid was collected. Red blood cells were removed using red blood cell lysis buffer, and the spleen cells were counted. SP2 / 0 cells and spleen cells were mixed at a ratio of 1:5, centrifuged at 1200 r / min for 3 min, and the supernatant was discarded. The pellet was washed twice with 10 mL of electrofusion buffer (BTX), and then resuspended in 9 mL of electrofusion buffer. The cells were transferred to an electrofusion instrument for fusion, and the fusion parameters were set to 800 V, 40 μs, and 1 time. After fusion, the cells were activated for 10 h in Advanced RPMI 1640 medium containing 5% FBS (Thermo Fisher Scientific, catalog number 12633012), and then transferred to semi-solid medium (Bio-Long Biotech, catalog number BTYA0607) for 10 days.

[0063] (3) Hybridoma cell screening: Pick the monoclonal cell clusters in the semi-solid culture medium in step (2) and transfer them to Advanced RPMI 1640 medium containing HAT (purchased from Thermo Fisher, catalog number 21060017). After culturing for 3 days, use indirect immunofluorescence assay to screen antibody-positive cell wells. After subcloning, identify a hybridoma cell line (named hybridoma cell TPH027) and name the monoclonal antibody secreted by it as monoclonal antibody TPH027.

[0064] (4) Identification of monoclonal antibody type: The Ig class and light chain type of monoclonal antibody were identified by indirect ELISA. The secondary antibodies used for different types of antibodies were all from the Enzyme-Label Secondary Antibody Kit for Ig Class Identification of Mouse Monoclonal Antibody (purchased from Bio-Long Company, catalog number BF16002X). The results showed that the antibody type of monoclonal antibody TPH027 was IgG1, κ light chain.

[0065] (5) Identification of the type of reaction between monoclonal antibody and virus: A. Reaction of monoclonal antibody with PRV virus: The monoclonal antibody TPH027 was reacted with the PRV genotype II strain (YY) using an indirect immunofluorescence assay (IFA). The steps are as follows: 1. Cell inoculation: PRV virus was inoculated at 200 TCID50 / well. PK-15 cells were added to 96-well plates and incubated at 37°C and 5% CO2 for 18 h.

[0066] 2. Cell fixation: Discard the cell culture supernatant, add 200 μL PBS to each well of a 96-well plate and wash three times, then add 100 μL of 80% cold acetone stored at -20℃ and fix in a -20℃ freezer for 1 h.

[0067] 3. Primary antibody incubation: Discard the cold acetone fixative, add 200 μL of PBS to each well and wash 3 times, add 100 μL of hybridoma cell culture supernatant to each well, and incubate at 37℃ for 1 h.

[0068] 4. Secondary antibody incubation: Discard the primary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times. Dilute the Alexa Fluor 488 fluorescent secondary antibody 1:500 with PBS, and add 0.01% Evans Blue and 5% FBS. Mix thoroughly and add 100 μL to each well of the cell plate. Incubate at 37°C for 1 h.

[0069] 5. Fluorescence observation: Discard the secondary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times, then observe the reaction between the antibody and infected cells under a fluorescence microscope.

[0070] The results are as follows Figure 1 The results showed that the monoclonal antibody TPH027 reacted with the PRV-YY strain with significant fluorescence.

[0071] B. Reaction of monoclonal antibody with PRV viral gD protein: Western blot experiments were performed on the monoclonal antibody TPH027 and the PRV-YY gD protein expressed via an insect baculovirus expression system, with the protein tag antibody anti-His Tag mAb used as a control. The steps are as follows: 1. Protein treatment: Add gD protein to 4× loading buffer in proportion and boil for 10 min.

[0072] 2. Protein electrophoresis: Load the processed protein (10 μL per lane) into a 10% density SDS-PAGE gel for protein electrophoresis. The program is 55 V for 50 min and 110 V for 80 min.

[0073] 3. Transfer: Use a semi-dry transfer method to transfer the proteins after electrophoresis to an NC membrane. The program is 23 V for 25 min.

[0074] 4. Blocking: Add 5 g of skim milk powder to every 100 mL of PBS and shake thoroughly to prepare the blocking solution. Add 5 mL of the blocking solution to a resealable bag and place it on a shaker at room temperature to block the NC membrane for 1 h.

[0075] 5. Incubation of primary antibody: Use blocking buffer to dilute hybridoma cell culture supernatant 1:100, and Anti-His Tag antibody diluted 1:3000 as a positive control. Incubate overnight at 4°C on a shaker.

[0076] 6. Incubation with secondary antibody: Wash the membrane 3 times with PBS, add Alexa Fluor 680-labeled fluorescent secondary antibody diluted 1:5000 with PBS, and incubate on a shaker at room temperature in the dark for 1 h.

[0077] 7. Scan the NC membrane: Wash the membrane three times with PBS, place it in a two-color infrared laser imaging system for scanning, and save the image.

[0078] The results are as follows Figure 2 As shown, the monoclonal antibody TPH027 reacts with the gD protein of the PRV-YY strain.

[0079] Identification of the reaction profile between monoclonal antibody and PRV virus: The monoclonal antibody TPH027 was subjected to IFA testing with PRV genotype I strain (Bartha-K61) to verify the antibody's reactivity with different genotype strains. The procedure was the same as step (5) of Example 1, identification of the reaction between monoclonal antibody and PRV virus. The results are as follows: Figure 3As shown, the monoclonal antibody TPH027 showed significant fluorescence in response to the PRV genotype I strain (Bartha-K61).

[0080] II. Preparation of Monoclonal Antibody Ascites Fluid from Hybridoma Cell Lines and Identification of Neutralizing Capacity: Six- to eight-week-old female Balb / C mice were intraperitoneally injected with 300 μL of mouse ascites adjuvant. Twelve days later, 1 × 10⁶ hybridoma cells (TPH027) prepared in step 1 above were injected intraperitoneally. When the mice's abdominal circumference was significantly enlarged, the ascites was aspirated with a syringe, centrifuged at 12,000 r / min for 10 min, and the supernatant was collected and stored to obtain the monoclonal antibody TPH027 prepared from the ascites.

[0081] The ability of the monoclonal antibody TPH027 prepared from ascites fluid to neutralize the PRV-YY strain was identified using an indirect immunofluorescence assay.

[0082] The identification method is as follows: The PRV-YY strain was diluted to 4000 TCID50 / mL using MEM, and 100 μL of the virus dilution was added to a 96-well plate. The monoclonal antibody TPH027 prepared from ascites fluid was serially diluted 2-fold starting at 1:100 using MEM, with 50 μL added to each concentration gradient to the corresponding virus solution. An antibody-free MEM control group (without monoclonal antibody) was set up, and the cells were incubated at 37°C for 1 h. 100 μL of PK-15 cells passaged 1:4 were added to the virus / antibody neutralization solution, and the cells were cultured at 37°C and 5% CO2 for 18 h. IFA (using the same method as steps 2-5 in the monoclonal antibody-virus reaction section above) was used to determine whether the neutralizing monoclonal antibody could neutralize PRV.

[0083] The results are as follows Figure 4 As shown, when a higher concentration of purified monoclonal antibody was added, no obvious green fluorescence was observed in cells infected with PRV. As the antibody dilution factor increased, green fluorescence appeared in cells infected with different strains, indicating that monoclonal antibody TPH027 has good neutralizing ability.

[0084] Example 2: Sequence determination of monoclonal antibody TPH027: Sequence determination of monoclonal antibody TPH027: Nucleic acid was extracted from hybridoma cells TPH027 prepared in Example 1 using a nucleic acid extraction kit (Magen, R4410-250). Reverse transcription was performed using M-MLV reverse transcriptase (TaKaRa, 2641B) to obtain hybridoma cell cDNA. PCR was used to amplify the variable regions of the heavy and light chains. The primers for amplifying the heavy chain variable region were F1: 5'-CAGGTTCAGCTGCAGCAGTC-3' (as shown in SEQ ID NO:8); R1: 5'-TGAGGAGACGGTGACTGAG-3' (as shown in SEQ ID NO:9). The primers for amplifying the light chain variable region were F2: 5'-GATGTTGTGATGACCCAA-3' (as shown in SEQ ID NO:10); R2: 5'-ACTGAGGCACCTCCAGATG-3' (as shown in SEQ ID NO:11). The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence obtained is as follows: The amino acid sequence of the heavy chain variable region of monoclonal antibody TPH027 is shown in SEQ ID NO:1 of the sequence listing, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:2 of the sequence listing; the amino acid sequence of the light chain variable region of monoclonal antibody TPH027 is shown in SEQ ID NO:3 of the sequence listing, and the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:4 of the sequence listing.

[0085] The amino acid sequences of CDR1 in the heavy chain variable region are shown in positions 31-35 of SEQ ID NO:1; the amino acid sequences of CDR2 in the heavy chain variable region are shown in positions 50-66 of SEQ ID NO:1; the amino acid sequences of CDR3 in the heavy chain variable region are shown in positions 99-110 of SEQ ID NO:1; the amino acid sequences of CDR1 in the light chain variable region are shown in positions 24-34 of SEQ ID NO:3; the amino acid sequences of CDR2 in the light chain variable region are shown in positions 50-56 of SEQ ID NO:3; and the amino acid sequences of CDR3 in the light chain variable region are shown in positions 89-97 of SEQ ID NO:3. The CDRs are determined using the Kabat coding system. The nucleotide sequences described above can be synthesized artificially.

[0086] Example 3: Preparation of monoclonal antibody TPH027 using genetic engineering methods: I. Construction of recombinant expression plasmids: To express the monoclonal antibody TPH027, heavy chain expression vectors and light chain expression vectors were prepared separately: The nucleotide sequence of the TPH027 heavy chain gene consisted of the nucleotide sequence of the heavy chain variable region coding gene (SEQ ID NO:2) and the mouse-IgG2a template sequence (heavy chain constant region sequence, as shown in SEQ ID NO:6 in the sequence listing), with the last base of SEQ ID NO:2 linked to the first base of SEQ ID NO:6. The nucleotide sequence of the TPH027 light chain gene consisted of the nucleotide sequence of the light chain variable region coding gene (SEQ ID NO:4) and the mouse-kappa template sequence (light chain constant region sequence, as shown in SEQ ID NO:7 in the sequence listing), with the last base of SEQ ID NO:4 linked to the first base of SEQ ID NO:7. The heavy chain gene and light chain gene of monoclonal antibody TPH027 were cloned into the Xba I and EcoR V cleavage sites of vector pcDNA3.4 (HonorGene, catalog number HG-VPH1386) to obtain heavy chain expression vector and light chain expression vector, respectively.

[0087] II. Antibody Expression: (1) Freshly digested 293T cells were seeded into 175 cm⁻¹ soil. 2 Add 35 mL of DMEM medium containing 8% FBS (purchased from Corning, catalog number 10-013-CVRC) to the culture flasks and culture the cells to a density of 90%. A total of 4 flasks were cultured.

[0088] (2) Dilute 200 μg of the heavy chain expression vector and light chain expression vector (100 μg each) and 200 μL of QuickShuttle-293 cell transfection reagent (Biolong, KX0110044) into 1 mL of physiological saline.

[0089] (3) Combine the two solutions from step (2) above and mix them well. This is the complex required for one bottle of cell transfection.

[0090] (4) Add the above complex directly to the cell culture medium in step (1) and mix it with a pipette.

[0091] (5) Transfer the cell plate to a 37℃ / 5% CO2 incubator for culture, and take the supernatant for purification after 3 days.

[0092] III. Antibody purification: (1) Buffer preparation: Add Na2HPO4·12H2O to sterile ddH2O to make the final concentration 0.2 M, and shake well to mix.

[0093] (2) Preparation of pre-elution buffer: Add 0.1 M citric acid to the buffer solution to make the volume ratio of citric acid 20%.

[0094] (3) Preparation of elution buffer: Add 0.1 M citric acid to the buffer solution to make the volume ratio of citric acid 60%.

[0095] (4) Sample processing: Take 30 mL of cell expression supernatant from step (2), add the buffer solution prepared in step (1) at a volume ratio of 1:1, filter with a pore size of 0.22 μm and prepare for column loading.

[0096] (5) Equilibrate the column: Use a constant flow pump to slowly pass 10 mL of buffer solution through a pre-packed Protein A / G 4FF column (Sangon Biotech, C600983) at a flow rate of 1 mL / min.

[0097] (6) Sample loading: Use a constant flow pump to slowly pass the solution from step (4) through the Protein A / G column at a flow rate of 1 mL / min.

[0098] (7) Washing: Use a constant flow pump to slowly pass 10 mL of washing buffer through the Protein A / G column at a flow rate of 1 mL / min.

[0099] (8) Pre-elution: Use a constant flow pump to slowly pass 10 mL of pre-elution solution through the Protein A / G column at a flow rate of 1 mL / min.

[0100] (9) Elution: Using a constant flow pump, 15 mL of elution buffer was slowly passed through the Protein A / G column at a flow rate of 1 mL / min. The elution product was aliquoted into 1.5 mL centrifuge tubes to obtain the purified monoclonal antibody TPH027 solution.

[0101] The concentration of the purified monoclonal antibody TPH027 solution was 0.7 mg / mL.

[0102] IV. Validation of antibody expression: Take the purified monoclonal antibody TPH027 solution obtained above, add non-reducing buffer (lane 1) without DTT and reducing buffer (lane 2) containing DTT in proportion, and load the sample for SDS-PAGE experiment.

[0103] The results are as follows Figure 5 As shown in the figure, lane 1 has a clear single band at a position greater than 180 kDa. After treatment with reducing buffer, the monoclonal antibody TPH027, as shown in lane 2, has two clear bands at approximately 25 kDa and 50 kDa, representing the light and heavy chains, indicating that the antibody has been well purified.

[0104] Example 4: Identification of antibodies prepared by genetic engineering methods: The purified monoclonal antibody TPH027 solution obtained in Example 3 was subjected to an IFA experiment with the PRV genotype II strain (YY) to verify antibody activity. The purified monoclonal antibody TPH027 solution diluted 1:800 with PBS was used as the primary antibody, and the remaining steps were the same as in step (5) of Example 1 for the identification of the reaction between the monoclonal antibody and the PRV virus. The results are as follows: Figure 6 As shown, the monoclonal antibody TPH027 reacts with the PRV genotype II strain (YY).

[0105] The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. Anti-pseudorabies virus gD protein neutralizing monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that, The complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region are composed of amino acid sequences as shown in positions 31-35, 50-66, and 99-110 of SEQ ID NO:1, respectively; the complementarity-determining regions CDR1, CDR2, and CDR3 in the light chain variable region are composed of amino acid sequences as shown in positions 24-34, 50-56, and 89-97 of SEQ ID NO:3, respectively.

2. The anti-pseudorabies virus gD protein neutralizing monoclonal antibody or antigen binding fragment thereof according to claim 1, characterized in that, It also includes four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, and four light chain variable region framework regions named LFR1, LFR2, LFR3 and LFR4. The HFR1 consists of an amino acid sequence as shown in positions 1 to 30 of SEQ ID NO:1; The HFR2 consists of an amino acid sequence as shown in positions 36 to 49 of SEQ ID NO:1; The HFR3 consists of an amino acid sequence as shown in positions 67 to 98 of SEQ ID NO:1; The HFR4 consists of an amino acid sequence as shown in positions 111 to 121 of the sequence listing SEQ ID NO:

1.

3. The anti-pseudorabies virus gD protein neutralizing monoclonal antibody or antigen binding fragment thereof according to claim 2, characterized in that, The LFR1 consists of an amino acid sequence as shown in positions 1 to 23 of SEQ ID NO:3; The LFR2 consists of an amino acid sequence as shown in positions 35 to 49 of SEQ ID NO:3; The LFR3 consists of an amino acid sequence as shown in positions 57 to 88 of SEQ ID NO:3; The LFR4 consists of an amino acid sequence as shown in positions 98 to 107 of SEQ ID NO:

3.

4. The method of producing an anti-pseudorabies virus gD protein neutralizing monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, Includes the following steps: Nucleic acid molecules encoding the heavy chain variable region and the light chain variable region are introduced into host cells for expression. The neutralizing monoclonal antibody against the anti-pseudorabies virus gD protein or its antigen-binding fragment was isolated and purified from the host cell culture.

5. Biological material associated with the neutralizing monoclonal antibody or antigen-binding fragment thereof against the anti-pseudorabies virus gD protein according to any one of claims 1 to 3, characterized in that, The biomaterial is any one of the following materials: A nucleic acid molecule encoding the neutralizing monoclonal antibody or its antigen-binding fragment against the pseudorabies virus gD protein; An expression cassette containing the nucleic acid molecule; A recombinant vector containing the nucleic acid molecule or expression cassette; Recombinant microorganisms containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors; Recombinant cells containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors.

6. The use of the anti-pseudorabies virus gD protein neutralizing monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, or the biological material as described in claim 5, in at least one of the following: Prepare products for detecting or assisting in the detection of pseudorabies virus; To prepare products for the detection or auxiliary detection of diseases caused by pseudorabies virus infection; Prepare products for screening or assisting in the screening of pseudorabies virus; To prepare products for screening or assisting in the screening of diseases caused by pseudorabies virus infection; Prepare products for the detection or auxiliary detection of pseudorabies virus gD protein; To prepare products that neutralize pseudorabies virus infection; To prepare products for the prevention and treatment of diseases caused by pseudorabies virus infection.

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