Neutralizing monoclonal antibody for resisting feline panleucopenia virus and application of neutralizing monoclonal antibody

Abstract

The invention discloses a neutralizing monoclonal antibody for resisting feline panleucopenia virus and application of the neutralizing monoclonal antibody, and belongs to the technical field of bioengineering. The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2; the antibody is an IgG2a subtype and kappa light chain, can specifically bind to FPV VP2 protein, and effectively neutralizes infection of an F81 cell by an FPV-ZZ20230 strain under the dilution ratio of 1: 64. The invention also provides nucleic acid molecules (SEQ ID NO: 8-9) for coding the antibody, an expression vector, a host cell and a preparation method. The antibody can be used for preparing medicines for preventing or treating feline panleucopenia, and can also be used as a detection antibody for ELISA (enzyme-linked immuno sorbent assay), Western blot or indirect immunofluorescence (IFA) diagnostic kits. The antibody is clear in structure and high in neutralizing activity, does not need to be preserved by hybridoma, and has good clinical application and industrialization prospects.

Description

A neutralizing monoclonal antibody against feline panleukopenia virus and its uses Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a neutralizing monoclonal antibody against feline panleukopenia virus and its uses. Background Technology

[0002] Feline panleukopenia (FP), also known as feline distemper or feline parvovirus disease, is a highly contagious, acute, and fatal infectious disease caused by the feline panleukopenia virus (FPV). FPV belongs to the family Parvoviridae and the genus Protoparvovirus, and is a non-enveloped, single-stranded DNA virus. The disease primarily infects felines but can also spread to wild animals such as mustelids and raccoons. Typical clinical manifestations of FPV infection include high fever, severe vomiting, bloody diarrhea, severe dehydration, and significant leukopenia. It is particularly dangerous for unvaccinated kittens aged 3-6 months, with an infection rate as high as 70% and a mortality rate of 50%-90%, exceeding 80% in severe cases. Because the virus is extremely stable in the environment (surviving for months or even years) and can be transmitted through direct contact, contaminated objects, or arthropods, it easily breaks out in catteries, shelters, and multi-cat households, causing significant economic losses and animal welfare problems.

[0003] Currently, clinical treatment for cats already infected with the virus primarily relies on symptomatic and supportive therapies, including intravenous fluid replacement to correct dehydration and electrolyte imbalances, antiemetics and antidiarrheals, and broad-spectrum antibiotics to prevent secondary infections. However, this strategy has fundamental limitations: the lack of specific antiviral drugs or neutralizing agents that directly target FPV makes it impossible to effectively inhibit viral replication and spread in the body, resulting in long treatment cycles, low cure rates, high medical costs, and difficulty in significantly reducing the mortality rate of severe cases.

[0004] In terms of prevention, inactivated vaccines or live attenuated vaccines are currently the main means of control. The principle is to stimulate the body to produce an immune response mainly composed of neutralizing antibodies by inoculating attenuated or inactivated intact virus particles, thereby obtaining resistance to subsequent infections. Although vaccines play an important role in group control, there are still obvious shortcomings: (1) Incomplete immune protection: kittens often fail to receive immunization due to interference from maternal antibodies, and older cats or immunosuppressed individuals often fail to receive immunization due to low immune response; (2) No therapeutic effect: vaccines are only used for prevention and are ineffective against animals that have already been infected and developed the disease; (3) Safety risks: live attenuated vaccines have the potential risk of virulence reversion or causing disease in immunodeficient cats.

[0005] Studies have shown that the VP2 protein of FPV is the main structural protein of its capsid, accounting for more than 90% of the total protein in the viral particle. VP2 not only determines the virus's host range, tissue tropism, hemagglutination activity, and pathogenicity, but is also a key protective antigen that induces the body to produce neutralizing antibodies. Therefore, VP2 is widely used in subunit vaccine design and immunogen development. However, despite the continuous progress in VP2-based vaccine research, highly efficient, high-affinity monoclonal antibodies with definite neutralizing activity have not yet been reported, severely restricting the development of rapid diagnostic reagents for FPV, passive immunotherapy agents, and novel vaccine evaluation systems.

[0006] Therefore, there is an urgent need to develop a monoclonal antibody that can specifically recognize the FPV VP2 protein, has a strong virus neutralizing ability, and has a clear and reproducible molecular structure, in order to fill this key technological gap and provide core support for the accurate diagnosis of FPV, passive immunotherapy, and the development of new vaccines. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a neutralizing monoclonal antibody against feline panleukopenia virus and its uses, which solves the problem of the lack of efficient and specific neutralizing monoclonal antibodies against FPV in the prior art, and provides a key tool for the rapid diagnosis of FPV, passive immunization therapy and vaccine efficacy evaluation.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a neutralizing monoclonal antibody against feline panleukopenia virus (FPV), wherein the monoclonal antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); the amino acid sequence of the VH is shown in SEQ ID NO:1 in the sequence listing, and the amino acid sequence of the VL is shown in SEQ ID NO:2 in the sequence listing; wherein, the monoclonal antibody can specifically bind to the FPV VP2 protein and neutralize the infection of F81 cells by the FPV-ZZ20230 strain at a dilution of 1:64.

[0009] Furthermore, the monoclonal antibody is the IgG2a subtype, and the light chain is the κ chain.

[0010] Furthermore, the VH of the monoclonal antibody comprises the following complementarity-determining regions: CDR-H1, CDR-H2, and CDR-H3; the amino acid sequence of CDR-H1 of the VH is GYTFTDYY, as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence of CDR-H2 of the VH is INPNGDT, as shown in SEQ ID NO: 4 in the sequence listing; and the amino acid sequence of CDR-H3 is ARNYGSSYWYFDV, as shown in SEQ ID NO: 5 in the sequence listing.

[0011] Furthermore, the VL of the monoclonal antibody comprises the following complementarity-determining regions: CDR-L1, CDR-L2, and CDR-L3; the amino acid sequence of CDR-L1 of the VL is QSIVHSDGNTY, as shown in SEQ ID NO: 6 in the sequence listing; the amino acid sequence of CDR-L2 of the VL is KLS; and the amino acid sequence of CDR-L3 of the VL is FQGSHVPFT, as shown in SEQ ID NO: 7 in the sequence listing.

[0012] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody, the nucleic acid molecule comprising a nucleotide sequence encoding the VH and / or a nucleotide sequence encoding the VL; the coding sequence of the VH is shown in SEQ ID NO:8, and the coding sequence of the VL is shown in SEQ ID NO:9.

[0013] This invention provides an expression vector containing the aforementioned nucleic acid molecules and a host cell. By cloning the nucleic acid sequences encoding the variable regions of the heavy and light chains into expression vectors (such as pcDNA3.1, pCHO1.0, etc.) and co-transfecting them into mammalian cells (such as HEK293F, CHO-K1) or prokaryotic expression systems (such as Escherichia coli), the monoclonal antibody can be efficiently expressed and purified.

[0014] The preparation method of the above monoclonal antibody is as follows: (1) Design and expression of VP2 truncated protein: Using the VP2 gene of FPV China-HN1 strain as a template, specific primers were designed, and an Nde I restriction site was introduced at the 5' end of the upstream primer and an Xho I restriction site was introduced at the 5' end of the downstream primer; the target fragment with a length of 960 bp was obtained by PCR amplification and cloned into the prokaryotic expression vector pET-22b to construct the recombinant plasmid pET-22b-tVP2; the verified pET-22b-tVP2 plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the VP2 truncated protein in the form of inclusion bodies was obtained by IPTG induction expression; the VP2 truncated protein was purified by Ni-Sepharose 6 Fast Flow affinity chromatography column, and obtained high-purity, immunogenic and reactive soluble VP2 truncated protein by gradient imidazole elution (optimal elution concentration is 250 mM) and dialysis refolding.

[0015] (2) Establishment of hybridoma cell line: BALB / c mice were immunized with purified VP2 truncated protein and the individuals with the highest antibody titer were screened; their spleens were ground to prepare single cell suspensions, which were mixed with SP2 / 0 myeloma cells at a ratio of 10:1 and PEG1500 was added for fusion; the fused cells were subjected to HAT selection culture and three limiting dilution subclonings to finally obtain a hybridoma cell line that stably secretes anti-FPV VP2 monoclonal antibody, named 9H12D6D6E6.

[0016] (3) Purification and characterization of monoclonal antibodies: High-purity monoclonal antibodies were obtained by ascites induction method and Protein G affinity chromatography. Gene sequencing showed that the amino acid sequence of the heavy chain variable region of the monoclonal antibody was SEQ ID NO:1 and the amino acid sequence of the light chain variable region was SEQ ID NO:2. The antibody subtype identification results showed that the antibody was of type IgG2a and the light chain was κ chain.

[0017] The monoclonal antibody described above in this invention can be used to prepare drugs for the prevention or treatment of feline panleukopenia. This antibody exerts a neutralizing effect by blocking FPV virus invasion of host cells and / or inhibiting viral replication.

[0018] The present invention also provides a kit for detecting feline panleukopenia virus, comprising the monoclonal antibody as a detection antibody, which can be used for ELISA, Western blot or indirect immunofluorescence (IFA) detection of FPV VP2 antigen.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) potent neutralizing activity: verified by indirect immunofluorescence (IFA) neutralization assay, the monoclonal antibody (mAbs-9H12D6D6E6) of the present invention can effectively block the infection of FPV-ZZ20230 strain on F81 cells at a dilution of 1:64, significantly inhibiting the virus from entering the host cell and replicating, demonstrating excellent in vitro neutralizing ability. This activity level far exceeds that of conventional polyclonal antibodies or non-neutralizing monoclonal antibodies, providing a feasible molecular tool for clinical passive immunotherapy.

[0020] (2) High specificity and high affinity: This antibody can specifically recognize FPV VP2 protein (including the truncated protein expressed in prokaryotes and the natural conformation of VP2 in virus-infected cells). The ELISA detection titer is as high as 1:500,000 and the indirect immunofluorescence (IFA) titer is 1:64,000, indicating that it has extremely high binding affinity and antigen recognition specificity, which can effectively avoid cross-reaction and improve diagnostic accuracy.

[0021] (3) Clear structure: The present invention has fully disclosed the amino acid sequence and encoding gene (SEQ ID NO:8-9) of the heavy chain variable region (VH, SEQ ID NO:1) and light chain variable region (VL, SEQ ID NO:2) of the antibody, which enables those skilled in the art to stably and reproducibly produce the antibody through conventional recombinant expression systems (such as CHO or HEK293 cells), which is beneficial to industrialization.

[0022] (4) Wide range of applications and high safety: The antibody of this invention can be used as a multifunctional biological agent in multiple fields. In terms of treatment: it can be used to prepare passive immunization agents against FPV, which are particularly suitable for emergency prevention or early treatment of kittens, senior cats or individuals with weakened immune function after viral exposure, effectively filling the protection gap during the vaccine immunization period; in terms of diagnosis: it can be used as a highly specific recognition element to develop ELISA kits, indirect immunofluorescence (IFA) detection reagents or colloidal gold rapid detection cards to achieve sensitive, accurate and convenient diagnosis of FPV infection; in terms of scientific research and quality control: it can be used as a standard neutralizing antibody for FPV vaccine efficacy evaluation, standardized quality control of virus neutralization tests and research on the structure and function of VP2 protein. Moreover, this antibody is a single-component recombinant or purified monoclonal antibody, which does not contain live virus, inactivated virus or attenuated components, and has higher biosafety and clinical reliability.

[0023] In summary, this invention, through a technical pathway of "advantageous epitope screening—truncated antigen expression—high-titer immunization—monoclonal antibody screening—functional verification," successfully obtained an anti-FPV neutralizing antibody with clinical application potential. It provides a more effective tool for the prevention and control of feline panleukopenia, significantly improving the cure rate and reducing mortality. It also solves key bottlenecks in existing FPV prevention and treatment methods, such as the "lack of direct antiviral drugs" and "limitations in vaccine protection," and has significant veterinary clinical value and socio-economic benefits in improving the prevention and control of feline panleukopenia. Attached Figure Description

[0024] Figure 1 shows the results of double enzyme digestion identification of recombinant plasmid pET-22b-tVP2; Figure 2 shows the optimized results of imidazole elution concentration of recombinant protein; Figure 3 shows the results of recombinant protein activity identification; Figure 4 shows the serum titer of mice after immunization; Figure 5 shows the FPV VP2-9H12 monoclonal cell clusters; Figure 6 shows the purification of mAbs-9H12D6D6E6 ascites fluid (M: trichrome pre-stained protein marker; lane 1: unpurified ascites fluid; lane 2: purified ascites fluid); Figure 7 shows the antibody titer of mAbs-9H12D6D6E6; Figure 8 shows the specificity and titer identification of mAbs-9H12D6D6E6 (A: IFA identification of monoclonal antibody recognizing eukaryotic protein FPV-VP2 and antibody titer detection; B: IFA identification of monoclonal antibody quantitative analysis; C: Western spectroscopy). Figure 9 shows the viral neutralizing activity and neutralizing titer of the monoclonal antibody as detected by IFA; Figure 10 shows the results of monoclonal antibody subtype identification. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 11: Bioinformatics analysis and recombinant expression vector construction of FPV VP2 truncated protein Using the VP2 gene of feline panleukopenia virus (FPV) China-HN1 strain (GenBank accession number: PP738170) as a reference sequence, SnapGene 6.0 software was used to predict antigenic epitopes and perform structural analysis to screen out advantageous antigenic regions with high hydrophilicity, high flexibility and surface accessibility. Based on this, amplification primers were designed, with an Nde I restriction site (containing a protective base) introduced at the 5' end of the upstream primer and an Xho I restriction site (containing a protective base) introduced at the 5' end of the downstream primer. The primer sequences are as follows: Forward primer (SEQ ID NO:10): 5′-GGAATTCCATATGGCTAAGAGTGCAAAATCTTGTAAAGC-3′; Reverse primer (SEQ ID NO:11): 5′-CCGCTCGAGTTACTTATCGTCGTCATCCTTGTAATC-3′; The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and PCR amplification was performed using FPV China-HN1 strain cDNA as a template to obtain a target fragment of 960 bp in length. The PCR product and pET-22b(+) vector were digested with Nde I and Xho I, respectively, and then ligated with T4 DNA ligase and transformed into E. coli DH5α competent cells. Positive clones were selected for initial screening by bacterial PCR and sent for sequencing verification. The recombinant plasmid with correct sequencing was named pET-22b-tVP2.

[0029] pET-22b-tVP2 was identified by double digestion with Nde I / Xho I. Agarose gel electrophoresis showed two bands with sizes of 960 bp and 5493 bp (as shown in Figure 1), which were consistent with the theoretical values, proving that the recombinant expression vector was successfully constructed.

[0030] Example 2: Prokaryotic expression, purification and activity verification of VP2 truncated protein.

[0031] 1. Induction and Purification of Recombinant Protein: The pET-22b-VP2 recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. Overnight culture was performed using ampicillin-resistant solid LB medium for screening. Single colonies with good growth were selected, expanded, and activated. 8 ml of LB medium was added to each shaker tube (bacterial culture to medium ratio 1:100, kanamycin to bacterial culture ratio 1:1000). The tubes were placed in a shaker at 37℃ and 220 rpm for colony activation. The activated bacterial culture was inoculated at a 1:100 ratio into 200 mL of LB medium containing ampicillin. The culture was shaken at 37℃ and 200 rpm for 3-4 h. When the OD600 reached 0.4-0.6, 10 mL of the culture was stored at 4℃ for later use. IPTG was added to the remaining culture to a final concentration of 1 Mm, and the culture was induced at 37℃ for 8 h. The culture was then collected. Resuspend the precipitate in 15 mL of 1×PBS and sonicate on ice for 30 min. Centrifuge and discard the supernatant. Resuspend the precipitate in 15 mL of 8M urea lysis buffer and shake in a rotary mixer at 4 °C for 12 h. Centrifuge and collect the supernatant.

[0032] Centrifuge and collect the supernatant, then load it onto a Ni Sepharose 6 Fast Flow affinity chromatography column (GE Healthcare). Elute sequentially with elution buffers containing 10, 20, 50, 100, 250, and 500 mM imidazole. SDS-PAGE analysis showed that the target protein had the highest purity in the 250 mM imidazole elution fraction (as shown in Figure 2). This fraction was collected, dialyzed to remove urea, and then refolded.

[0033] 2. Western blot validation of recombinant proteins: After electrophoresis, cut a suitable size of the lower layer of the SDS-PAGE gel according to the marker position and place the cut gel in transfer buffer. Cut a PVDF membrane of the same size as the SDS-PAGE gel and activate it in anhydrous ethanol for 1 min. Arrange the membrane in the transfer tank in the following order from negative to positive: sponge pad, filter paper, protein gel, PVDF membrane, filter paper, and sponge pad. Add rapid transfer buffer and wet transfer at a constant current of 0.4 A for 25 min. Blocking: After transfer, remove the PVDF membrane with tweezers and place it in an incubation box. Wash 2-3 times with PBST. Add PBST containing 5% skim milk powder to the incubation box for blocking. Incubate the box on a horizontal shaker at room temperature for 2 minutes. h, discard the blocking solution, wash the PVDF membrane three times with PBST for 10 min each time; Primary antibody incubation: Dilute the anti-His-tagged mouse monoclonal antibody 1:2000 with universal antibody diluent, add the diluted primary antibody to the incubation chamber, incubate overnight at 4°C, recover the primary antibody, wash the PVDF membrane three times with PBST solution for 10 min each time; Secondary antibody incubation: Dilute the goat anti-mouse IgG antibody 1:2000 with PBST containing 5% skim milk powder, add the diluted secondary antibody to the incubation chamber, incubate at room temperature for 2 h, wash three times with PBST for 10 min each time; Chromogenic imaging: Add an appropriate amount of chromogenic solution to the PVDF membrane using a pipette, and take pictures using a chemiluminescence imaging system. After immunizing animals with tVP2 recombinant protein, collect immune serum as primary antibody for Western blot identification.

[0034] Western blot results (as shown in Figure 3) revealed a specific band at approximately 36 kDa, indicating successful expression of the recombinant tVP2 protein. Furthermore, Western blot analysis using serum obtained from mice immunized with this protein as the primary antibody also showed a positive signal at the same location, demonstrating that the protein effectively induces the production of specific antibodies and can neutralize these antibodies. This indicates that the recombinant tVP2 protein possesses good immunogenicity and reactivity.

[0035] Example 3: Preparation of anti-FPV monoclonal antibody and establishment of hybridoma cell line.

[0036] 1. Immunization of mice: Three 6-8 week old female BALB / c mice (purchased from the Experimental Animal Center of Zhengzhou University) were used for the primary immunization by intraperitoneal injection of 50 μg of refolded tVP2 recombinant protein and an equal volume of Freund's complete adjuvant emulsion. Two weeks later, a second immunization was performed using Freund's incomplete adjuvant emulsion with the same dose of recombinant protein. A third immunization was performed two weeks later, again using Freund's incomplete adjuvant.

[0037] Seven days after the last immunization, blood was collected from the tail vein of mice, and serum was separated. The serum was serially diluted (1:100 to 1:2,500,000), and ELISA plates were coated with 4 μg / mL tVP2 protein as the antigen. Antibody titers were detected using an indirect ELISA method. The primary antibody was the diluted immune serum, and the secondary antibody was HRP-labeled goat anti-mouse IgG (H+L). Odulocyte count (OD) was measured after colorimetric development. 450 value.

[0038] As shown in Figure 4, the antibody titers of the three mice were: Mouse 1:1:100000, Mouse 2:1:500000, and Mouse 3:1:100000. Among them, the antibody titer of Mouse 2 was significantly higher than that of the other individuals (P / N>2.1), indicating that it had the strongest immune response. Therefore, this mouse was selected for hyperimmunization (without adjuvant, 50 μg protein) for subsequent hybridoma cell fusion.

[0039] 2. Three days after fusion of spleen cells and myeloma cells with hyperimmune immunotherapy, Mouse 2 cells were sacrificed, and the spleen was aseptically removed. The spleen was resuspended in PBS and ground to prepare a single-cell suspension. Spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1, and PEG1500 (molecular weight 1500) was added for cell fusion. After the fusion reaction was complete, the reaction was terminated with RPMI-1640 complete medium, and the fused cells were evenly seeded into 96-well plates, added to HAT-containing selective medium, and cultured in a 37°C, 5% CO2 incubator.

[0040] On day 4, half of the preheated HAT medium was replaced, and culture continued until day 7, at which point initial screening of positive wells began. The cell supernatant from each well was used as the primary antibody, and the binding ability to tVP2 protein was detected by indirect ELISA. The criterion was OD (octane rating). 450 A ≥2.1-fold negative control was considered positive. Multiple positive clones were obtained through initial screening, and further subcloning was performed using a three-stage limiting dilution method (each dilution to single cell / well). Finally, a stable monoclonal cell line secreting high-affinity antibodies was selected from 1000 hybridoma cell wells and named 9H12D6D6E6.

[0041] The cell line exhibits a typical circular colony morphology under a microscope and is in good growth condition (as shown in Figure 5). After multiple passages, it still maintains stable antibody secretion capacity and has good genetic stability.

[0042] Example 4 This example describes the preparation of monoclonal antibodies using the ascites induction method. (1) Preparation and selection of monoclonal cell subclones and ascites fluid. Indirect ELISA detection of D 450Wells with nm values ​​2.1 times higher than the negative control were transferred to 96-well plates and serially diluted. Observation was performed under a microscope until 80-90 cells were found in each well. The cells were then thoroughly mixed with RPMI-1640 medium and seeded onto 96-well plates for expansion culture. Once the subcloned cells reached confluence, ELISA was performed. Positive clones were selected for a second subcloning. After the second subcloning, ELISA was performed again until the positive rate of cell supernatant in single-cloned wells was 100%.

[0043] Freund's incomplete adjuvant was injected intraperitoneally into BALB / c mice at a dose of 500 μL per mouse. Seven days later, hybridoma cells were injected intraperitoneally at a dose of 1 × 10⁶. 6 / 200μL. After the mouse abdomen is significantly distended (about 7-14 days), the ascites fluid is extracted, centrifuged at 12000rpm for 10min, and the supernatant is collected and stored at -80℃ for later use.

[0044] (2) Monoclonal antibody purification: The collected ascites fluid samples were purified. First, the ascites fluid was diluted with an equal volume of PBS, and saturated ammonium sulfate solution was added dropwise while stirring until the final concentration reached 50%. The solution was then allowed to stand at 4°C for 2-4 hours. Subsequently, the solution was centrifuged at 12,000 rpm for 10 min, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in an equal volume of PBS as the original ascites fluid and placed in a dialysis bag. Dialysis was performed overnight at 4°C to remove residual ammonium sulfate.

[0045] The ascites fluid after dialysis was loaded onto a Protein G affinity chromatography column (Cytiva) as follows: (i) Column pretreatment: Equilibrate the column with 10 column volumes of PBS; (ii) Sample loading: Slowly add the filtered ascites fluid to the column at a flow rate of 1 mL / min; (iii) Washing: Wash with Wash Buffer until no protein is detected in the eluent; (iv) Antibody elution: Elute the antibody with 0.1 M glycine (pH 2.8) and collect the eluent until no protein is detected; (v) pH adjustment: Adjust the pH of the elution product to neutral with saturated sodium carbonate.

[0046] The purified antibody was analyzed by SDS-PAGE, and the results are shown in Figure 6: Lane 1 is the unpurified ascites fluid, where multiple non-specific protein bands are visible; Lane 2 is the purified ascites fluid, showing two main bands located at approximately 50 kDa (heavy chain) and 25 kDa (light chain), respectively, indicating that the antibody was successfully purified and has high purity.

[0047] Example 5: This example demonstrates the determination of the working concentration of purified monoclonal antibody in an indirect ELISA.

[0048] The titer of purified monoclonal antibodies was determined using an indirect ELISA method. The prokaryotic recombinant protein pET-22b-tVP2 (i.e., a truncated FPV VP2 protein) was diluted to 5 μg / mL with 1×PBS and coated onto ELISA plates, 100 μL per well, and incubated at 37°C for 2 h. The plates were then blocked with 5% skim milk for 2 h, followed by sequential addition of serially diluted mAbs-9H12D6D6E6 (dilutions: 1:100, 1:1000, 1:10000, 1:100000, 1:500000, 1:2500000, 1:3000000, 1:5000000) as primary antibody, and incubated at 37°C for 1 h. HRP-labeled goat anti-mouse IgG (H+L) was used as secondary antibody, and the plates were incubated at 37°C for 1 h. After each incubation, the plates were washed three times with PBST. Finally, add TMB substrate and develop color for 15 min. After adding stop solution, measure OD value at 450 nm wavelength.

[0049] The results are shown in Figure 7. As the antibody dilution factor increases, OD 450 The value gradually decreases, but a significant signal (OD) is still maintained even at a dilution factor of 1:500,000. 450 >0.2), while the negative control remained at a low level (OD). 450 <0.1). Therefore, the ELISA titer of this monoclonal antibody is 1:500000.

[0050] Example 6: This example describes the determination of the working concentration of the purified monoclonal antibody IFA.

[0051] (1) IFA detection specificity and titer: F81 cells in good growth condition were prepared into single-cell suspension and seeded into 24-well plates. When the cells adhered to the plate to 70% confluence, FPV-ZZ20230 virus was diluted to 200 TCID with DMEM. 50 Infecting F81 cells with the appropriate infectious dose, while simultaneously setting up a blank control group uninfected with the virus, and incubating at 37℃ and 5% CO2 for 36 h. Infrared alpha (IFA) assays were performed using monoclonal antibodies diluted at different ratios as primary antibodies to determine the IFA availability and titer of the monoclonal antibodies.

[0052] Discard the cell culture medium. Fix with 300 μL / well of 4% paraformaldehyde at room temperature for 30 min, then wash three times with PBS. Permeate with 300 μL / well of 0.1% Triton X-100 at room temperature for 10 min, then wash three times with PBS. Block with PBS containing 10% fetal bovine serum at room temperature for 1 h, then add 300 μL / well and wash three times with PBS. Apply primary antibody: Incubate with 300 μL / well of purified monoclonal antibody at room temperature for 1 h, then wash three times with PBS. Subsequent operations were performed sequentially under light-protected conditions. FITC-labeled goat anti-mouse IgG was diluted 1:1000, 300 μL / well, and incubated at room temperature for 1 h. After washing 3 times with PBS, the results of the inverted fluorescence microscope images showed (see Figures 8A-B): obvious green fluorescence signals could be observed at dilutions from 1:1000 to 1:64000, indicating that the antibody could effectively recognize VP2 protein in virus-infected cells; when diluted to 1:256000, the fluorescence almost disappeared, indicating that its IFA titer was 1:64000; the positive control (PC) was strongly positive, and the negative control (NC) showed no fluorescence.

[0053] Total protein was extracted from FPV-ZZ20230-infected F81 cells, separated by SDS-PAGE, and transferred to a membrane. The primary antibody was mAbs-9H12D6D6E6, and the secondary antibody was HRP-labeled for color development. Western blot results are shown in Figure 8C. The antibody detected a specific band in the FPV-infected cell sample, with an apparent molecular weight of approximately 55-60 kDa, consistent with the typical migration characteristics of FPV VP2 protein in SDS-PAGE. This confirms that the antibody can recognize native eukaryotic VP2 protein expression and exhibits high specificity.

[0054] Example 7: This example describes the assay of the neutralizing activity of a monoclonal antibody. FPV-ZZ20230 was diluted to 200 TCID50 using DMEM. Ascites fluid collected was serially diluted 2-fold. The diluted ascites fluid and virus solution were mixed and incubated at 37°C with 5% CO2 for 1 h. Healthy F81 cells were mixed with the virus-ascites fluid mixture and seeded into 96-well plates. Control groups without ascites fluid and without virus were included. Cells were cultured at 37°C with 5% CO2 for approximately 30 h (no cytopathic effect was observed). The cell culture medium was discarded, and the cells were fixed with 300 μL / well of 4% paraformaldehyde at room temperature for 30 min, followed by washing three times with PBS. The cells were permeabilized with 300 μL / well of 0.1% Triton X-100 at room temperature for 10 min, followed by washing three times with PBS. The cells were then blocked with PBS containing 10% fetal bovine serum at room temperature for 1 h, followed by washing three times with PBS (300 μL / well). Primary antibody application: 300 μL of purified monoclonal antibody was incubated at room temperature for 1 h, followed by washing three times with PBS. Subsequent operations were performed under light-protected conditions. FITC-labeled goat anti-mouse IgG was diluted 1:1000, 300 μL / well, incubated at room temperature for 1 h, followed by washing three times with PBS. The images were taken using an inverted fluorescence microscope, and the results were analyzed.

[0055] The results are shown in Figure 9: the virus control group (PC) without antibodies showed widespread green fluorescence, indicating successful viral invasion and replication; the negative control group (NC) showed no fluorescence; at dilutions of 1:4 and 1:8, a small number of fluorescent spots remained; at a dilution of 1:64, the fluorescence signal almost disappeared, indicating that the virus was effectively blocked. This demonstrates that mAbs-9H12D6D6E6, at a dilution of 1:64, can effectively neutralize the infection of FPV-ZZ20230 strain on F81 cells, exhibiting strong neutralizing ability.

[0056] Example 8: This example describes the identification of monoclonal antibody subtypes. To determine the subtype of the monoclonal antibody, the commercially available Mouse Monoclonal Antibody Isotyping ELISA Kit (Thermo Fisher) was used to identify the subtype of mAbs-9H12D6D6E6. Hybridoma cell culture supernatant was taken and reacted with antibodies against IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, κ chain, and λ chain, respectively, and the OD was measured. 450 value.

[0057] The results are shown in Figure 10: the IgG2a subtype had the highest OD value, significantly higher than other IgG subtypes; the κ chain (Kappa) OD value was significantly higher than the λ chain; other subtypes showed very weak or no signal. Therefore, this monoclonal antibody is the IgG2a subtype with the κ chain as its light chain, consistent with typical murine monoclonal antibody characteristics.

[0058] Example 9: Monoclonal Antibody Variable Region Gene Sequencing and Sequence Disclosure. Total RNA was extracted from 9H12D6D6E6 cells, reverse transcribed, and the VH / VL gene was amplified and sequenced to obtain the following sequence: Heavy Chain Variable Region Nucleotide Sequence (SEQ ID NO: 1) NO:8): GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACCTTCACTGACTACTACATGAAGTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGATACTTTC TACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCCGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAACTACGGTAGTAGTTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0059] Heavy chain variable region amino acid sequence (SEQ ID NO:1): EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNNGDTFYNQKFKGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCARNYGSSYWYFDVWGAGTTVTVSS.

[0060] Light chain variable region nucleotide sequence (SEQ ID NO:9):GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGTCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTGCATAGTGATGGAAACACCTATTTAGACTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAA CTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAAATAAAA.

[0061] The amino acid sequence of the light chain variable region (SEQ ID NO:2): DVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSDGNTYLDWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK.

[0062] Complementarity-determining region (CDR) sequence analysis: According to the Kabat numbering system, the heavy chain variable region of the monoclonal antibody contains the following complementarity-determining regions (CDRs): CDR-H1: GYTFTDYY (SEQ ID NO:3); CDR-H2: INPNGDT (SEQ ID NO:4); CDR-H3: ARNYGSSYWYFDV (SEQ ID NO:5).

[0063] The light chain variable region of the monoclonal antibody includes the following complementarity-determining regions (CDRs): CDR-L1: QSIVHSDGNTY (SEQ ID NO:6); CDR-L2: KLS; CDR-L3: FQGSHVPFT (SEQ ID NO:7).

[0064] The above examples fully demonstrate that the monoclonal antibody provided by this invention has a well-defined molecular structure, excellent specificity, high titer, and strong neutralizing activity, and can be used for the diagnosis, treatment, and basic research of FPV. All experiments were conducted with the approval of the Experimental Animal Ethics Committee of Henan Agricultural University (Approval No.: HNND20211921901) and comply with animal welfare regulations.

[0065] The foregoing provides a detailed description of a neutralizing monoclonal antibody against feline panleukopenia virus and its uses. Specific examples have been used to illustrate the principles and implementation methods of the invention. These examples are merely illustrative and are intended to aid in understanding the method and core concepts of the invention. It should be noted that those skilled in the art can make various modifications and improvements to the invention without departing from its principles, and these modifications and improvements also fall within the scope of protection of the claims.

Claims

1. A neutralizing monoclonal antibody against feline panleukopenia virus (FPV), characterized in that, The monoclonal antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); the amino acid sequence of the VH is shown in SEQ ID NO:1 in the sequence listing, and the amino acid sequence of the VL is shown in SEQ ID NO:2 in the sequence listing; wherein, the monoclonal antibody can specifically bind to the FPV VP2 protein and neutralize the infection of F81 cells by the FPV-ZZ20230 strain at a dilution of 1:

64.

2. The neutralizing monoclonal antibody against feline panleukopenia virus according to claim 1, characterized in that, The monoclonal antibody is the IgG2a subtype, and the light chain is the κ chain.

3. The neutralizing monoclonal antibody against feline panleukopenia virus according to claim 1 or 2, characterized in that, The VH of the monoclonal antibody comprises the following complementarity-determining regions: CDR-H1, CDR-H2, and CDR-H3; the amino acid sequence of CDR-H1 of the VH is GYTFTDYY, as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence of CDR-H2 of the VH is INPNGDT, as shown in SEQ ID NO: 4 in the sequence listing; and the amino acid sequence of CDR-H3 is ARNYGSSYWYFDV, as shown in SEQ ID NO: 5 in the sequence listing.

4. The neutralizing monoclonal antibody against feline panleukopenia virus according to claim 1 or 2, characterized in that, The VL of the monoclonal antibody comprises the following complementarity-determining regions: CDR-L1, CDR-L2, and CDR-L3; the amino acid sequence of CDR-L1 of the VL is QSIVHSDGNTYLD, as shown in SEQ ID NO: 6 in the sequence listing; the amino acid sequence of CDR-L2 of the VL is KLS; and the amino acid sequence of CDR-L3 of the VL is FQGSHVPFT, as shown in SEQ ID NO: 7 in the sequence listing.

5. A nucleic acid molecule encoding the monoclonal antibody as described in claim 1, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the VH and / or a nucleotide sequence encoding the VL; the coding sequence of the VH is shown in SEQ ID NO:8, and the coding sequence of the VL is shown in SEQ ID NO:

9.

6. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, It includes the expression vector as described in claim 6.

8. Use of the monoclonal antibody as described in claim 1 or 2 in the preparation of a medicament for the prevention or treatment of feline panleukopenia.

9. The use according to claim 8, characterized in that, The monoclonal antibody exerts a neutralizing effect by blocking FPV virus invasion of host cells and / or inhibiting viral replication.

10. A kit for detecting feline panleukopenia virus, characterized in that, Containing the monoclonal antibody as described in claim 1 as a detection antibody, for use in ELISA, Western blot or indirect immunofluorescence (IFA) detection of FPV VP2 antigen.

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