Neutralizing monoclonal antibody of anti-pseudorabies virus gD protein, hybridoma cell and application thereof

Abstract

The invention relates to an anti-PRV gD (pseudorabies virus gD) protein neutralizing monoclonal antibody, a hybridoma cell and application of the anti-PRV gD protein neutralizing monoclonal antibody. The anti-PRV gD neutralizing monoclonal antibody is prepared by constructing a PRV gD eukaryotic expression vector, transduction the PRV gD eukaryotic expression vector into a CHO (Chinese hamster ovary) cell for expression and then carrying out animal immunization by taking a PRV gD recombinant protein and an attenuated live vaccine as immunogens. The neutralizing monoclonal antibody or hybridoma cell provided by the invention lays a foundation for the development of a rapid detection technology of the pseudorabies virus and the gD protein thereof, has wide research and application values and commercial use values in immunodetection related to the pseudorabies virus, and has important guiding significance for vaccine development based on the pseudorabies virus.

Description

Technical Field

[0001] The present invention relates to the field of biological immunology technology, and specifically to neutralizing monoclonal antibodies against pseudorabies virus gD protein, hybridoma cells and applications thereof. Background Art

[0002] Porcine pseudorabies (PR), also known as Aujeszky's disease (AD), is a type of infectious disease caused by pseudorabies virus (PRV). Pigs are the main natural host of PRV, but many mammals including rodents, cats, dogs and cattle may be infected with PRV. After infection, the main clinical symptoms are fever, encephalomyelitis and neurological diseases. It has a high mortality rate and has caused great losses to the breeding industry worldwide. In recent years, there have been articles reporting cases of human infection with PRV. PRV infection often causes clinical symptoms such as visual impairment and headaches. In severe cases, it can cause blindness or even death in patients. However, there is currently no specific antiviral drug that can effectively control the diseases caused by PRV infection in humans.

[0003] Pseudorabies virus (PRV) gD protein is a glycoprotein necessary for the virus to penetrate the membrane and is also a key cell surface receptor binding protein. It is mainly responsible for binding to host cell surface receptors and promoting membrane fusion. The gD protein is also the main target of neutralizing monoclonal antibodies, inducing the body to produce high-titer neutralizing monoclonal antibodies, thereby protecting animals from pseudorabies virus (PRV). It has high homology in different pseudorabies virus (PRV) mutant strains. In the process of pseudorabies virus (PRV) invading cells, gD protein binds to receptor proteins such as HVEM (Herpesvirus entry mediator receptor), HS (Heparan Sulfate) and Nectin-1 on the cell surface, captures virus particles on the host cell surface, and mediates the cascade reaction of virus membrane fusion to help the virus invade the host cell. When pseudorabies virus (PRV) invades the host cell, gC protein first binds to HS, and then gD protein recognizes and binds to the receptor protein on the host cell surface, and finally stimulates gB, gH and gL proteins to form a complex to mediate the viral envelope and host cell membrane to complete cell fusion. This process is completely irreversible. Therefore, the development of neutralizing monoclonal antibodies and hybridoma cells against gD protein is of great significance for the detection technology and vaccine development of pseudorabies virus (PRV), which is helpful for the prevention, control and purification of pseudorabies.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] Studies have found that gD protein does not participate in virus replication and reproduction, and does not participate in the transmission of pseudorabies virus (PRV) between cells. Virus particles lacking the gD protein gene do not have the ability to invade cells, which can greatly reduce the virus's infection efficiency. gD protein is the main immunogenic glycoprotein of pseudorabies virus (PRV), the most important protective antigen of pseudorabies virus (PRV), and the most effective virus-neutralizing monoclonal antibody target in pseudorabies virus (PRV).

[0006] Based on this, the present application screened and obtained a neutralizing monoclonal antibody and hybridoma cell against pseudorabies virus gD protein.

[0007] In the first aspect disclosed in the present application, a neutralizing monoclonal antibody against pseudorabies virus gD protein is provided, wherein the heavy chain variable region includes CDR H1, CDR H2, and CDRH3, and the amino acid sequences thereof are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; and the light chain variable region includes CDR L1, CDRL2, and CDR L3, and the amino acid sequences thereof are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0008] In some embodiments of the present disclosure, the amino acid sequence of the heavy chain variable region of the neutralizing monoclonal antibody is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region thereof is shown as SEQ ID NO.8.

[0009] The second aspect disclosed in the present application provides a gene encoding the 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 is shown in SEQ ID NO.10.

[0010] The third aspect disclosed in the present application provides a hybridoma cell, the monoclonal antibody secreted by the hybridoma cell contains the heavy chain variable region and the light chain variable region mentioned above.

[0011] In a fourth aspect disclosed in the present application, the neutralizing monoclonal antibody, the gene or the hybridoma cell is used in the preparation of a pseudorabies virus detection reagent.

[0012] In a fifth aspect disclosed in the present application, the neutralizing monoclonal antibody, the gene or the hybridoma cell is used in the preparation of an immunological detection reagent.

[0013] In a sixth aspect disclosed in the present application, the monoclonal antibody or the hybridoma cell is used in the preparation of an antibody or vaccine against pseudorabies virus.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. The present application constructs a PRV gD eukaryotic expression vector and transduces it into CHO cells for mass expression. Recombinant PRV gD soluble protein is purified by Ni column affinity chromatography. Subsequently, animals are immunized with PRV gD recombinant protein and attenuated live vaccine as immunogens to prepare neutralizing monoclonal antibodies against PRV gD. The light chain of the neutralizing monoclonal antibody is Lambda, and the heavy chain subtype is IgG1. It has high neutralization, and its neutralization activity IC 50 318.5 ng / ml, with a titer of no less than 1:5.12×10 5 , the affinity constant is 7.01×10 8 L / mol, and has high specificity, does not react with the glycoproteins of other herpes viruses, has good protection and treatment capabilities for animals, has important guiding significance for the development of PRV-based vaccines, and provides technical support for the prevention, control and treatment of PRV.

[0015] 2. The neutralizing monoclonal antibody in this application can specifically recognize gD protein, laying the foundation for the solution of PRV gD protein rapid detection technology, and has extensive research application value and commercial use value in PRV-related immune detection, and has important guiding significance for the development of PRV-based vaccines.

[0016] 3. Based on the heavy chain variable region sequence and light chain variable region sequence of the neutralizing monoclonal antibody disclosed in the present application, one or more amino acids can be added, deleted, replaced, etc. through conventional genetic engineering and protein engineering to obtain its active fragments or conservative variants, but still able to specifically bind to the pseudorabies virus gD protein, laying the foundation for further modifying its antibody variable region sequence to prepare genetically engineered antibodies in different combinations, so as to further enhance the specificity of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the screening results of a CHO cell line that stably expresses PRV gD recombinant protein in an embodiment of the present application.

[0018] Figure 2 This is a diagram showing the results of PRV gD protein purification in one embodiment of the present application.

[0019] Figure 3This is a diagram showing the Western Blot identification results of the reaction between PRV gD protein and positive serum in one embodiment of the present application.

[0020] Figure 4 This is an IPMA chart of mouse serum titer after the third immunization of mice in an embodiment of the present application.

[0021] Figure 5 This is a graph showing the IPMA results of the neutralizing monoclonal antibody 3F7 in one embodiment of the present application.

[0022] Figure 6 This is the Biacore affinity kinetics determination of the antibody in one embodiment of the present application.

[0023] Figure 7 This is a diagram showing the specificity of the neutralizing monoclonal antibody in one embodiment of the present application.

[0024] Figure 8 This is a diagram for identifying the neutralizing activity of the neutralizing monoclonal antibody 3F7 in one embodiment of the present application. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described below in conjunction with examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present application in any way.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and materials involved are all conventional commercial products unless otherwise specified; the tests and detection methods involved are all conventional methods unless otherwise specified.

[0027] Example 1. Preparation of recombinant PRV gD protein After the gene sequence encoding the PRV gD protein was codon-optimized according to the CHO expression system, it was synthesized by Shanghai Shenggong Biotechnology Co., Ltd. to obtain a plasmid named pCGS3-gD. Subsequently, the Ig Kappa signal peptide and Kozaka sequence were added to the C-terminus of the protein, and the His tag was added to the N-terminus, and it was constructed into the pLVX-IRES-ZsGreen1 vector. The recombinant plasmid pLVX-IRES-ZsGreen1-gD was finally successfully obtained through molecular cloning.

[0028] The lentivirus was successfully obtained through the lentivirus packaging system and transduced into CHO cells. A stable cell line that can stably express PRV gD protein was obtained through subclone screening and named CHO / gD ( Figure 1 ). CHO / gD was adjusted to 0.5×10 6cells / mL and cultured for 168 h. The supernatant was then filtered using a 0.22 μm filter membrane and purified using Ni affinity chromatography. The eluted target protein was dialyzed against PB buffer and concentrated using an ultrafiltration tube to obtain the PRVgD recombinant protein.

[0029] The recombinant PRV gD protein sample was taken for SDS-PAGE identification ( Figure 2 ) with a purity of 90% and a protein concentration of 0.7 mg / mL. It was divided into aliquots as needed and stored at -80°C for subsequent experiments as an immunogen.

[0030] Western Blot test was performed using PRV positive pig serum as primary antibody and Goat Anti-pig IgG / HRP as secondary antibody. The results are as follows: Figure 3 As shown, the recombinant PRV gD protein reacted well with positive pig serum, further confirming the successful preparation of PRVgD protein.

[0031] Example 2. Preparation and identification of neutralizing monoclonal antibodies against PRV gD protein Main reagents and materials: Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, PEG-1500, 1640 culture medium, Goat Anti-pig IgG / HRP, Goat Anti-Mouse IgG / HRP, pseudorabies live attenuated vaccine (Bartha-K61 strain) were purchased from Zhejiang Shihua Nobiwei Biotechnology Co., Ltd., ultrasensitive ECL luminescence kit, antibody subtype identification kit, SMM CHO-SI culture medium, BALB / c mice were purchased from the Animal Experiment Center of Zhengzhou University.

[0032] 1. Animal immunization (1) Immunization of BALB / c mice with recombinant PRV gD protein ① The immunization dose was 50 μg of recombinant PRV gD protein. The protein was mixed with an equal volume of Freund's complete adjuvant, and the mixture was emulsified to an oil-in-water state using an emulsifier and transferred to a 1 mL syringe. BALB / c mice were immunized by multiple subcutaneous injections on the back.

[0033] ② After 14 days, the mice were given a second booster immunization. The immunization dose was the same as the first immunization, but the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.

[0034] ③ After 28 days, the mice were immunized for the third time, and the tail blood was collected on the 35th day. The serum was collected after centrifugation at 3000 r / min for 10 min, and the titer of mouse polyclonal antibody serum was determined by i-ELISA.

[0035] (2) Immunization regimen with live attenuated vaccines ① Resuspend the vaccine powder in sterile PBS in a biosafety cabinet and immunize mice.

[0036] ② Select 14 days as an immunization cycle and use subcutaneous multiple injections for immunization. The rest of the experimental operations are the same as the method of immunizing mice with recombinant PRV gD protein in step (1).

[0037] ③ Select mice with high antibody titers and good growth status and give them shock immunization by intraperitoneal injection at a dose of 100 μg / mouse on day 42.

[0038] 2. ELISA to determine the titer of mouse serum (1) i-ELISA titer measurement ① Dilute the purified gD protein to 2 μg / mL using CBS buffer, add it to the enzyme label strip at a volume of 100 μL / well, and coat at 37℃ for 2 h.

[0039] ② Discard the coating solution, wash with PBST three times, pat dry, and block with 5% skim milk 200 μL / well at 37℃ for 2 h.

[0040] ③ Discard the blocking solution, wash three times with PBST and pat dry, add 1:200 diluted mouse polyclonal serum to the first well, and dilute it 2-fold with PBS, add 100 μL / well to the ELISA plate, incubate at 37℃ for 1 h, wash five times with PBST and pat dry.

[0041] ④ Dilute Goat Anti Mouse IgG / HRP with 5% skim milk at 1:5000, add 100 μL / well to the ELISA plate, incubate at 37°C for 1 h, wash 5 times with PBST and pat dry.

[0042] ⑤ Add TMB colorimetric solution at 100 μL / well and react for 10 min in the dark.

[0043] ⑥ 2% H2SO4 was used as the stop solution to terminate the reaction and the OD was read using a microplate reader. 450nm The numerical value of .

[0044] (2) IPMA potency test ① Increase PK 15 by 1.5×104 cells / well were inoculated in a 96-well cell culture plate. When the cell confluence reached about 80%, the PRV HeNLH / 2017 virus was diluted in DMEM maintenance medium containing 2% FBS to 200 TCID 50 / 100 μL was inoculated into the cell plate.

[0045] ② When the cells at the bottom of the culture plate show obvious lesions, discard the cell supernatant and wash three times with PBS.

[0046] ③ Add 4% pre-cooled cell tissue fixative at 50 μL / well and fix at room temperature for 20 min. Discard the fixative and wash three times with PBS. Then add 5% skim milk at a volume of 200 μL / well and block at 37°C for 2 h.

[0047] ④ Dilute the serum of the immunized mice at a ratio of 1:100 with PBS, dilute it 2-fold in a dilution cup, and add it to the sealed cell plate at a volume of 100 μL / well, then incubate at 37°C for 1 h.

[0048] ⑤ Wash three times with PBS, dilute Goat Anti Mouse IgG / HRP with 5% skim milk at a ratio of 1:2000, add at a volume of 100 μL / well, and incubate at 37°C for 1 h.

[0049] ⑥ Wash three times with PBS and then wash twice with DDW.

[0050] ⑦ Prepare AEC colorimetric solution at a ratio of DDW: A solution: B solution: C solution = 850 μL: 50 μL: 50 μL: 50 μL, add to the plate at a volume of 50 μL / well, develop for 15 min, then terminate with an equal volume of DDW, observe the results under a microscope and take photos.

[0051] The antibody titer test results are as follows Figure 4 , Figure 5 As shown, the ELISA results showed that the polyclonal antibody titers of mice No. 1 and No. 4 were ≥6400, and the IPMA titers were ≥800. Two mice were used to prepare and screen neutralizing monoclonal antibodies.

[0052] 3. Cell Fusion (1) Preparation of spleen cell suspension: The mice were euthanized by cervical dislocation and then immersed in 75% alcohol for 5 min. The spleen was removed with sterile scissors and forceps. After the spleen was rinsed with GNK solution, it was placed on a sterile 200-mesh nylon mesh and ground into spleen cell homogenate with scissors; centrifuged at 1200 r / min for 10 min, SP2 / 0 was taken at a ratio of 8:1 for spleen cells: SP2 / 0 cells, a certain amount of SP2 / 0 suspension was added to the spleen cell suspension, and centrifuged at 1200 r / min for 10 min.

[0053] (2) Cell fusion: PEG 1500 was used as a fusion agent for cell fusion. The fused cells were resuspended in HAT medium and added to a 96-well plate at 100 μL / well. The plates were cultured in a 37°C 5% CO2 cell culture incubator. Obvious cell clusters could be observed under a microscope about 5 days after fusion. On the 10th day, 50 μL of cell supernatant was aspirated and primary screening was performed using i-ELISA, secondary screening was performed using IPMA, and neutralizing monoclonal antibodies were screened using a virus neutralization experiment.

[0054] Example 3: Identification of hybridoma cell lines secreting neutralizing monoclonal antibodies against PRV gD protein 1. Screening and subcloning of hybridoma cells (1) i-ELISA screening: First, i-ELISA was used to screen the monoclonal hybridoma cell lines specific for PRV gD protein. Recombinant PRV gD protein was used as the coating source to perform preliminary screening on the hybridoma cell supernatant. The positive hybridoma cell lines obtained in the preliminary screening were verified three times using i-ELISA. After excluding false positive monoclonal cell lines, the positive cell lines were transferred to 48-well plates for further culture.

[0055] (2) IPMA screening: The specific steps are the same as step 3 (2) in Example 2, except that the primary antibody is replaced with the culture supernatant of the positive hybridoma cell line.

[0056] (3) Virus neutralization test ① One day in advance, culture PK15 cells at a rate of 1.5×10 4 cells / well were seeded in 96-well cell culture plates.

[0057] ② Collect the supernatant of positive monoclonal cells and centrifuge at 12,000 r / min and 4°C for 10 min.

[0058] ③ When the cell confluence reaches 70-80% the next day, dilute the sample to be tested 2-fold in the maintenance medium in a 96-well plate, set aside the cell control and negative control, and add 50 μL of 200TCID 50 The virus was diluted and incubated in a 37°C incubator for 1 h.

[0059] ④ Discard the supernatant from the cell culture plate with PK 15 cells, wash it three times with sterile PBS, and then quickly and gently add the sample to be tested, virus and maintenance culture medium.

[0060] ⑤ Incubate at 37℃ 5% CO2 for 48 h. When obvious lesions appear in the negative control wells, record the number of lesions in the sample wells to be tested and fix the cell plate with 4% cell tissue fixative.

[0061] ⑥ Discard the cell supernatant in the culture plate, wash three times with 0.01 M sterile PBS, add 4% cell tissue fixative at 50 μL / well and let stand at room temperature for 20-30 min, discard the tissue fixative and wash three times with 0.01 M PBS, then block with 5% skim milk 200 μL / well at 37℃ for 2 h, or block overnight at 4℃, and wash three times with 0.01 M PBS after blocking.

[0062] ⑦ The primary antibody was diluted with the eyeball blood of the fused mouse at a ratio of 1:800 with antibody diluent, added to a 96-well plate at 100 μL / well, incubated in a 37°C incubator for 1 h, and then washed three times with 0.01 M PBS.

[0063] ⑧ Secondary antibody: Dilute Goat Anti Mouse IgG / HRP 1: 2000 and add 100 μL / well to a 96-well plate. Incubate at 37°C for 1 h, then wash three times with 0.01 M PBS and twice with DDW.

[0064] ⑨ Color development: AEC color development solution was prepared according to the volume ratio of A:B:C:DDW of 50 μL:50 μL:50 μL:850 μL, and then the color development solution was added according to the volume of 50 μL / well, and the reaction was carried out at room temperature in the dark for 15 minutes. 100 μL / wellDDW was added to terminate the color development, and the samples were observed under a microscope and photographed.

[0065] (4) Subcloning and further identification of positive cells: HT / 1640 10% FBS medium was used for subcloning. The screened positive hybridoma cells were diluted to 1 cell / well and added to a 96-well plate pre-coated with 100 μL of feeder cells. The subcloned cell plate was cultured for 5-7 days. The number of cell clusters in each well was observed and recorded. Positive monoclonal cell lines were screened by i-ELISA. If necessary, 2-3 subclones were performed until a neutralizing monoclonal cell line that could secrete specific PRVgD protein was finally obtained. The neutralizing monoclonal cell line was then gradually expanded and cultured in a T25 cell culture flask at a rate of 1.5-2×10 6 The cells / branch were frozen for future use.

[0066] Example 4. Preparation of ascites of neutralizing monoclonal antibodies against PRV gD protein 1. Determination of the neutralizing titer of PRV gD protein in monoclonal cell supernatants (1) Dilute the gD protein to 2 μg / mL using CBS buffer and add it to the ELISA strip at a volume of 100 μL / well. After coating at 37°C for 2 h, continue to block with 5% skim milk (200 μL / well) at 37°C for 2 h.

[0067] (2) Discard the blocking solution, wash three times with PBST and tap dry, dilute the supernatant of monoclonal cells with PBS 2-fold, 100 μL / well, incubate at 37°C for 1 h, wash five times with PBST and tap dry.

[0068] (3) Dilute Goat Anti Mouse IgG / HRP at 1:5000 with PBS solution and add 100 μL / well to the ELISA plate. Incubate at 37°C for 1 h, wash 5 times with PBST solution and pat dry.

[0069] (4) Add TMB colorimetric solution at 100 μL / well and react for 10 min in the dark.

[0070] (5) 2% H2SO4 was used as the stop solution to terminate the reaction. The OD was read using an ELISA reader. 450nm The numerical value of .

[0071] 2. Preparation of Monoclonal Antibody Ascites (1) Multiparous BALB / c female mice were selected and intraperitoneally injected with 500 μL of Freund's incomplete adjuvant.

[0072] (2) The secretory neutralizing monoclonal cell line in Example 2 was expanded and cultured. Seven days after the mice were injected with Freund's incomplete adjuvant, 1×10 6The cells were injected into the peritoneal cavity of mice at a concentration of 500 μL.

[0073] (3) After about 7 days, when the mouse abdomen feels noticeably swollen, its activity slows down, and its appetite decreases, the ascites can be collected.

[0074] (4) After centrifuging the ascites at 6000 r / min for 15 min, collect the clear and transparent light yellow liquid in the middle layer and store it at -20°C for later use.

[0075] Example 5. Determination of properties of neutralizing monoclonal antibodies against PRV gD protein 1. Biacore determination of antibody-protein affinity / kinetics (1) After the gD protein was diluted with buffer (concentration of about 100 ng / mL), 20 μL was added to the CM5 chip, and the coupling amount was set to 100 RU. The software automatically controlled the injection time and selected the Fc1 channel as the reference channel.

[0076] (2) Antibodies 3F7, 2F4G10, 10F5, 11H7D4, and 7H3B9 were prepared in a concentration gradient of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, and 0 nM, respectively, with a volume of 110 μL.

[0077] (3) Prepare running buffer: HBS-P + , injection: 30 μL / min, association 120 s, dissociation 300 s.

[0078] (4) Using the default method of Biacore software, the results were fitted by kinetics, and the affinity constant of the neutralizing monoclonal antibody 3F7 was 1.71E-12 M and the dissociation constant was 2.14E-07 S -1 ( Figure 6 ).

[0079] 2. Specificity identification of neutralizing monoclonal antibodies (1) The PRV-gB, PRV-gC, VZV-gE, VZV-gB proteins expressed in the laboratory earlier and the PRVgD expressed in this example were coated on the enzyme label strip at 200 ng / well.

[0080] (2) The cell culture supernatant of the monoclonal hybridoma cell line was added to the ELISA plate as the primary antibody. The specific experimental steps were the same as step 3 (2) in Example 2. The results were as follows: Figure 7 shown.

[0081] 3. Identification of neutralizing activity of neutralizing antibodies (1) PK15 cells were cultured at 1.5×10 4 cells / well were evenly spread into a 96-well plate. When the cell confluence at the bottom of the plate reached about 80%, the cell culture supernatant was discarded, the cells were washed three times with sterile PBS, and maintenance solution was added for later use.

[0082] (2) The antibody 3F7 to be tested was serially diluted to 25 μg / mL in the first well, and then added to a 96-well cell culture plate at a volume of 50 μL / well. Eight replicates were made for each dilution. 50 μL of 200TCID 50 PRV HeNLH / 2017 was incubated in a 37℃ 5% CO2 incubator for 1 h.

[0083] (3) The virus-antibody mixture after co-incubation was added to a 96-well cell culture plate with PK 15 cells. Each 96-well plate tested 11 samples with gradient dilutions. The first 4 wells in the 12th column were added with maintenance culture medium as a blank control, and the last 4 wells were added with 200 TCID 50 PRV was used as negative control, and the cells were cultured in a 37°C cell culture incubator for 48 h. When obvious CPE appeared in the negative control group, and no CPE appeared in the blank control group and the experimental group, the number of wells with CPE in each dilution gradient was recorded. The experiment was repeated three times to obtain three independent lesion rates and protection rates for each gradient. The experimental results were then analyzed using GraphPad Prism 9.0 software and the IC 50 , the results are as follows Figure 8 shown.

[0084] 4. Amplification of variable region sequences of monoclonal antibodies The variable region sequence of the screened neutralizing monoclonal antibody 3F7 was amplified by molecular cloning technology and sent to Anhui General Biotechnology Co., Ltd. for sequencing.

[0085] The amino acid sequences of the heavy chain variable regions CDR H1, CDR H2, and CDR H3 of the neutralizing monoclonal antibody obtained are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the light chain variable regions CDR L1, CDRL2, and CDR L3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The amino acid sequence of the heavy chain variable region of the neutralizing monoclonal antibody 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.

[0086] 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 is shown in SEQ ID NO.10.

[0087] Although some preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations to the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A neutralizing monoclonal antibody against pseudorabies virus gD protein, characterized in that: Its heavy chain variable region includes CDRH1, CDR H2, and CDR H3, and its amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; its light chain variable region includes CDR L1, CDR L2, and CDR L3, and its amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

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

8.

3. A gene encoding the neutralizing monoclonal antibody according to claim 1, 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 is shown in SEQ ID NO.

10.

4. A hybridoma cell, which secretes a monoclonal antibody comprising the heavy chain variable region and light chain variable region as claimed in claim 1.

5. Use of the neutralizing monoclonal antibody according to claim 1, the gene according to claim 3 or the hybridoma cell according to claim 4 in the preparation of a pseudorabies virus detection reagent.

6. Use of the neutralizing monoclonal antibody according to claim 1, the gene according to claim 3 or the hybridoma cell according to claim 4 in the preparation of an immunological detection reagent.

7. Use of the neutralizing monoclonal antibody according to claim 1 or the hybridoma cell according to claim 4 in the preparation of antibodies or vaccines against pseudorabies virus.

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