Monoclonal antibody of feline parvovirus VP2 protein, antigen epitope peptide recognized by monoclonal antibody and application of monoclonal antibody

Abstract

The invention discloses a monoclonal antibody of feline parvovirus VP2 protein, an antigen epitope peptide recognized by the monoclonal antibody and application of the monoclonal antibody. A variable region of an antibody heavy chain of the monoclonal antibody comprises CDR1-3 consisting of amino acid sequences shown as SEQ ID NO.1-3; a variable region of a light chain of the antibody comprises CDR1-3 consisting of amino acid sequences as shown in SEQ ID NO.4-6. The invention further discloses a preparation method of the antibody. The obtained non-neutralizing monoclonal antibody with high specificity and high conservative property aiming at the FPV VP2 protein P1 region can specifically recognize and combine with a plurality of FPV strains, can be used as a candidate antibody for FPV universal antigen detection, provides an effective tool for analyzing the immunological characteristics of FPV, and has a wide application prospect. The method also has potential application value for subsequent diagnostic reagent development and vaccine strategy research. The further obtained antigen epitopes are extremely conservative and appear for the first time in China, and all the cat parvoviruses can be detected, so that the possibility of missing detection is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical antibody technology, specifically to a monoclonal antibody against feline parvovirus VP2 protein, the antigenic epitope peptide it recognizes, and its applications. Background Technology

[0002] Feline panleukopenia virus (FPV), also known as feline infectious enteritis virus or feline distemper virus, is a highly fatal pathogen in felines, especially posing a significant threat to pet health management, particularly to kittens. Therefore, early diagnosis of FPV and the development of effective prevention and control measures are of great importance. Although some vaccines and treatments exist, issues such as unstable vaccine efficacy and insufficient diagnostic sensitivity still hinder effective control.

[0003] The VP2 protein of FPV, as a major structural protein of the virus, not only participates in the assembly of viral particles but is also a key target for host immune recognition. Its high conservation and surface exposure characteristics make it an ideal antigen for the preparation of monoclonal antibodies. Monoclonal antibodies possess high specificity and affinity, playing a crucial role in virus detection, vaccine design, and virus neutralization, helping to block viral invasion and enhance the immune response. Therefore, developing monoclonal antibodies targeting the VP2 protein holds promise for improving FPV detection capabilities and expanding prevention and control strategies. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody against feline parvovirus VP2 protein, its recognized antigenic epitope peptide, and its applications; specifically, it provides a monoclonal antibody (mAbs) against FPV VP2 protein and identifies its antigenic epitope. This invention uses FPV strain SH0918 VP2 protein as an immunogen, immunizing BALB / c mice via intraperitoneal injection. Hybridoma technology is used for cell fusion and screening to obtain the specific monoclonal antibody. Subsequently, ascites titer is measured using an indirect enzyme-linked immunosorbent assay (ELISA), and its neutralizing activity and reactivity are evaluated using a hemagglutination inhibition assay (HI), Western blot, and indirect immunofluorescence assay (IFA).

[0005] The objective of this invention is achieved through the following technical solution: <First Aspect> This invention provides a monoclonal antibody against the feline parvovirus VP2 protein, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain. The variable region of the antibody heavy chain includes CDR1, composed of the amino acid sequence shown in SEQ ID NO.1 (GYTFTDYS), CDR2, composed of the amino acid sequence shown in SEQ ID NO.2 (INTETGEP), and CDR3, composed of the amino acid sequence shown in SEQ ID NO.3 (AMLR). The variable region of the antibody light chain includes CDR1 composed of the amino acid sequence shown in SEQ ID NO.4 (GNIHNY), CDR1 composed of the amino acid sequence shown in SEQ ID NO.5 (NAK), and CDR3 composed of the amino acid sequence shown in SEQ ID NO.6 (QHFRGTPFT).

[0006] In this invention, the feline parvovirus monoclonal antibody comprises a heavy chain and a light chain. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain includes a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into complementarity-determining regions (CDRs), with a frame region (FR) inserted between each pair of complementarity-determining regions. Each VH and VL includes three CDRs and four FRs, connected in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0007] As one embodiment of the present invention, the monoclonal antibody comprises the heavy chain variable region amino acid sequence as shown in SEQ ID NO. 7 or its homologous sequence; and / or, It contains the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 8 or its homologous sequence.

[0008] The homologous sequence is a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity.

[0009] In one embodiment of the present invention, the monoclonal antibody is prepared by animal immunization using FPV VP2 protein as an immunogen.

[0010] <Second aspect> The present invention provides a nucleic acid encoding a heavy chain of a monoclonal antibody as described in any of the preceding claims, and / or encoding a light chain of a monoclonal antibody as described in any of the preceding claims.

[0011] As one embodiment of the present invention, the nucleic acid includes nucleotide sequences encoding CDR1, CDR2, and CDR3 for the heavy chain variable region, and / or, Nucleotide sequences used to encode the CDR1, CDR2, and CDR3 regions of the light chain variable region.

[0012] As one embodiment of the present invention, the nucleic acid further includes nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region; and / or, Nucleotide sequences used to encode the FR1, FR2, FR3, and FR4 regions of the light chain variable region.

[0013] <Third aspect> This invention provides an antigenic epitope peptide of feline parvovirus VP2 protein, which specifically binds to the aforementioned monoclonal antibody.

[0014] As one embodiment of the present invention, the amino acid sequence of the antigenic epitope peptide is as follows: 50 EFKFLENGWVEITANS 65 As shown in SEQ ID NO.25.

[0015] <Fourth Aspect> The present invention provides a biological material containing the above-mentioned nucleic acid, wherein the biological material is one or more of an expression vector, a eukaryotic or prokaryotic host cell.

[0016] <Fifth Aspect> The present invention also provides the application of the monoclonal antibody described in any of the above claims in FPV detection.

[0017] As one embodiment of the present invention, the monoclonal antibody is used in the preparation of an FPV detection kit.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention successfully prepared and identified a highly specific and conserved non-neutralizing monoclonal antibody targeting the P1 region of the FPV VP2 protein, with the subclass identified as IgG1 / κ. This monoclonal antibody specifically recognizes and binds to multiple FPV strains, but lacks neutralizing activity. The targeted linear antigenic epitope is located in the P1-1 region of the VP2 protein. Comparison with the corresponding amino acid sequences of 61 FPV strains from both domestic and international sources showed that this epitope is highly conserved. This monoclonal antibody can serve as a candidate antibody for the detection of universal FPV antigens.

[0019] 2) The antigenic epitopes obtained by this invention are extremely conserved, which is the first time they have appeared in China. They can detect all feline parvoviruses, greatly reducing the possibility of missed detection.

[0020] 3) This invention successfully prepared a highly specific monoclonal antibody against the FPV VP2 protein. This antibody not only provides an effective tool for analyzing the immunological characteristics of FPV, but also has potential application value for the development of subsequent diagnostic reagents and vaccine strategy research. Attached Figure Description

[0021] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a Western blot image of the reaction between the hybridoma cell supernatant and the VP2 protein of FPV strains SH0918 and BJ19 in Example 1. Figure 2 This is a Western blot diagram of the reaction between mAb ascites fluid and VP2 protein of different FCV strains in Example 1. Figure 3 The results of Western blot analysis for the identification of monoclonal antibody antigenic epitopes in Example 2 are shown; wherein, Figure 3 A represents the reaction result of the monoclonal antibody with the VP2-1 fragment; Figure 3 B represents the reaction results of the monoclonal antibody with fragments P1-1, P1-2, P1-3, and P1-4; Figure 4 This refers to the results of using ChimeraX software in step 2 to locate the antigenic epitope on the VP2 protein trimer; among which, Figure 4 A is a plan view of the VP2 protein monomer structure, with the location of the monoclonal antibody epitope in the VP2 protein monomer marked. Figure 4 B is a plan view of the FPV viral capsid structure composed of VP2 protein polymers. Figure 4 C represents the location of the monoclonal antibody epitope in the FPV viral capsid structure composed of VP2 protein multimers. Figure 5 To implement the sequence identifier map generated by Weblogo for the 50-65aa antigenic epitopes of VP2 protein in 61 FPV strains in step 2; Figure 6 The gel electrophoresis results are for the light and heavy chain variable region genes amplified in Example 3; where M band is the marker; 1 band is the light chain variable region; 2 bands are the heavy chain variable region; and - band is the negative control.

[0022] Figure 7 This is a schematic diagram of the amino acid sequences of the light and heavy chain variable regions of the monoclonal antibody obtained in Example 3. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0024] The cell lines and virus strains used in this invention include the feline kidney passaged cell line (F81), the human embryonic kidney cell line (293T), and the FPV strains SH0918, BJ19, ZZ4, and SH202401, all of which were preserved by the Companion Animal Biosafety Risk Early Warning and Control Technology Team of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Among them, the FPV strain BJ19 has been published in reference 1 (Tang Aoxing, Liu Chuncao, Wang Zhenzhen, et al. Isolation, identification and genetic evolution analysis of three feline parvovirus strains [J]. Chinese Journal of Animal Infectious Diseases, 2023, 31(02):86-92. DOI:10.19958 / j.cnki.cn31-2031 / s.20210629.002.); ZZ4 is a strain isolated in our laboratory, Genbank accession number: PX804535; SH0918 is a strain isolated in our laboratory, Genbank accession number: PX804536; SH202401 is a strain isolated in our laboratory, which has been sequenced in full length, Genbank accession number: PV402123. The expression plasmids of each strain were obtained using conventional methods.

[0025] The plasmid pCMV-3 used in this invention Flag-14, pCMV-3×Flag-14-eGFP, and pCold Ⅰ were purchased from Thermo Fisher Scientific, USA.

[0026] The competent cells DH5α, BL21 (DE3) and Rosetta (DE3) used in this invention were purchased from Shanghai Sangon Biotech Co., Ltd.

[0027] The reagents and materials used in this invention include BamH I and Hind III restriction endonucleases, plasmid extraction kit, 2×Es Taq MasterMix (Dye) kit, gel extraction kit, complete Freund's adjuvant, incomplete Freund's adjuvant, PEG SOLUTION 50%, horseradish peroxidase-labeled goat anti-mouse IgG (IgG-HRP), goat anti-mouse IgG FITC, anti-His tag mAb, infrared fluorescently labeled goat anti-mouse IgG (IgG-RBITC), goat anti-cat IgG (H+L)-HRP, Alexa Flour 488-Conjugated Goat anti-Rabbit IgG (H+L), His-tagged protein purification kit (denaturing formulation), affinity chromatography column, protein marker, and IgG antibody subclass identification kit, which were purchased from TAKARA, Tiangen Biotech (Beijing) Co., Ltd., Novizan Biotech Co., Ltd., Sigma-Aldrich, QIAGEN, Beyotime, and Jackson Immuno, respectively. The experimental animals were female SPF BALB / c mice, aged 6-8 weeks, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd., Shanghai Shenger Biotechnology Co., Ltd., Shanghai Beyotime Biotechnology Co., Ltd., and Southern Biotech Co., Ltd.

[0028] Example 1: Preparation of hybridoma cells and identification of monoclonal antibodies 1.1 Preparation of Immunogen Using the successfully isolated and identified FPV SH0918 strain sequence as a reference sequence, DNA was extracted and used as a template for PCR amplification with designed specific primers. The correctly identified PCR product was purified using a gel extraction kit to obtain the VP2 target gene. The purified VP2 target gene DNA fragment was homologously recombinated with the pCold I vector, which had been linearized by double digestion with BamH I and Hind III. The recombinant product was transformed with DH5α cloned bacteria, and positive recombinant bacteria were selected and sent for sequencing. The recombinant bacteria with correct sequencing results were named FPV-VP2-pCold I, and the recombinant plasmid was extracted and identified by double enzyme digestion.

[0029] The correctly identified recombinant plasmid FPV-VP2-pCold Ⅰ was transformed into Rosetta (DE3) competent cells for inclusion body expression. After denaturation with 8M urea, the protein was purified by affinity chromatography using a denaturing-resistant nickel column. The eluted target protein was renatured by dialyzing and then concentrated by ultrafiltration. Finally, the concentration was determined using the Beyotime BCA Protein Quantitative Kit (Enhanced Version). After aliquoting, the protein was stored at -80℃ for later use.

[0030] The recombinant VP2 protein (gene sequence shown in SEQ ID NO.9) was mixed with an equal volume of complete Freund's adjuvant and thoroughly emulsified. The emulsified antigen was then incubated at 4 °C for at least 8 h in preparation for mouse immunization.

[0031] 1.2 Mouse Immunization Female BALB / c mice aged 6-8 weeks were used for immunization. The initial immunization was performed via subcutaneous injection, with each mouse receiving 200 μL of the emulsified antigen (containing 50 μg VP2 protein). Booster immunizations were administered at weeks 2 and 4 following the initial immunization, respectively, using an equal volume mixture of VP2 protein and incomplete Freund's adjuvant. One week after the final immunization, blood was collected from the eye to obtain serum, and the antibody titer against FPV in the immunized mouse serum was determined using an indirect ELISA method. One to two mice with a titer higher than 1:20000 were selected for subsequent cell fusion experiments.

[0032] 1.3 Cell Fusion Mice with the highest serum titers were selected, and spleens were harvested after booster immunization. Spleen cells were isolated, and PEG 1000 was used as a fusion agent to mix spleen cells with SP2 / 0 myeloma cells at an appropriate ratio (1:1) for cell fusion experiments.

[0033] 1.4 Screening and Cloning Purification of Positive Hybridoma Cells Indirect ELISA was used to screen for positive hybridoma cell clones that could continuously secrete anti-VP2 protein-specific antibodies. The initially screened positive wells were then subjected to limiting dilution subcloning until hybridoma cell lines capable of stably secreting the target monoclonal antibody were obtained.

[0034] Through three cloning and purification processes, a hybridoma cell line capable of stably secreting mAbs targeting the FPV VP2 protein was obtained and named SH2501A.

[0035] 1.5 Ascites preparation and purification Sensitization was achieved by intraperitoneal injection of sterile paraffin oil (0.5 mL / mouse) into 6-8 week old BALB / c mice. One week later, approximately 5 × 10⁵ g of paraffin oil was injected intraperitoneally into each mouse. 6 Hybridoma cell lines in the logarithmic growth phase. After the mouse abdomen became significantly distended, ascites fluid was aspirated, centrifuged at 12,000 r / min for 10 min, and the supernatant was collected. Monoclonal antibodies were purified using the ammonium sulfate precipitation method.

[0036] 2. Identification of monoclonal antibodies 2.1 Western blot identification of hybridoma supernatant FPV strains SH0918 and BJ19 were inoculated into F81 cells. After obvious cytopathic effect (CPE) was observed, cells were collected and total protein was extracted. Protein samples from the diseased cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. Using the supernatant of the positive hybridoma cells (SH2501A) obtained in step 1.4 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, ×Western blot analysis was performed to verify the binding specificity of the monoclonal antibody to the viral protein.

[0037] Western blot results are as follows Figure 1 As shown in the figure. The results showed that the hybridoma cell supernatant could specifically recognize the VP2 protein of SH0918 and BJ19, demonstrating good reaction specificity.

[0038] 2.2 Hemagglutination Inhibition Assay (HI) The neutralizing activity of monoclonal antibodies was assessed using a viral hemagglutination inhibition assay. 25 μL of pH 6.4 PBS was added to each well of a 96-well hemagglutination plate. 25 μL of hybridoma cell supernatant obtained in step 1.4 was added to well 1, and the plate was serially diluted 2-fold up to well 10, with the final 25 μL discarded. 25 μL of 4 HAU virus solution was added to wells 1 through 11, with well 12 serving as a blank control. After mixing, the plate was incubated at 4 °C for 1 h. Then, 25 μL of 1% porcine erythrocyte suspension was added to each well, and the plate was incubated at 4 °C for 2 h. The hemagglutination inhibition titer was observed and determined. The results showed that no HI titer was detected in the hybridoma cell supernatant, indicating that the antibody did not possess FPV neutralizing ability.

[0039] 2.3 Subtype Identification Using a commercially available mouse immunoglobulin subtype identification kit, the monoclonal antibody purified from the ascites fluid in step 1.5 was analyzed for subtypes via indirect ELISA. Following the standard operating procedure recommended in the manufacturer's instructions, the immunoglobulin subtype was determined based on the binding reaction of the monoclonal antibody with different types of capture antibodies. The results showed that the heavy chain of this monoclonal antibody was IgG1, and the light chain was κ.

[0040] 2.4 Indirect ELISA Ascites Titer Determination The reaction conditions were optimized using a checkerboard titration method. VP2 recombinant protein was coated onto ELISA plates overnight at 4 °C with concentration gradients of 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL. Negative and positive sera diluted 1:100, 1:200, 1:400, and 1:800 were used for detection, and the optical density (OD) values ​​at 450 nm were read. The optimal reaction conditions were determined based on the highest P / N ratio, and the ascites titer was measured accordingly.

[0041] The ascites titer of the purified monoclonal antibody from step 1.5 was determined by indirect ELISA using a commercially available kit, following the standard operating procedure recommended in the instructions. The results showed that the ascites titer of the monoclonal antibody was ≥1:64000.

[0042] 2.5 Western blot analysis F81 cells were infected with FPV strains SH0918, BJ19, and ZZ4 as antigens. After significant cell erosion (CPE) was observed, cells were collected, and total protein was extracted using RIPA lysis buffer. Protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. Using the monoclonal antibody purified from the ascites fluid in step 1.5 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, ×Western blot experiments were performed to detect the reactivity of the antibodies with the VP2 protein of different FPV strains.

[0043] Western blot results are as follows Figure 2 As shown in the figure. The results show that the monoclonal antibody can specifically bind to the VP2 protein of three FPV strains: SH0918, BJ19, and ZZ4.

[0044] In summary, the monoclonal antibody prepared by this invention can specifically recognize cell samples infected with multiple genotypes of FPV strains, and is suitable for FPV detection.

[0045] Example 2: Identification of antigenic epitopes 1. Eukaryotic expression Based on the spatial structure of the VP2 protein and the major antigenic region obtained from preliminary analysis, a eukaryotic expression plasmid containing the antigenic segment (VP2-1, amino acid sequence as shown in SEQ ID NO. 10) was constructed using conventional methods: pCMV-3×Flag-14-VP2-1. The plasmid was transfected into 293T cells, and cells were harvested 48 hours later. Total protein was extracted using RIPA lysis buffer, and ×Western blot analysis was performed to determine whether the monoclonal antibody obtained in Example 1 recognized the target region. Figure 3 The results from A showed that the monoclonal antibody could specifically bind to the VP2-1 fragment.

[0046] 2. Refinement of antigen regions and eukaryotic expression Based on the preliminary localization results, a pair of specific primers (shown as SEQ ID NO. 10 and 11 in Table 1) were designed to target fragment P1 (50aa-100aa) in the positive target region VP2-1. Fragment P1 was cloned into the pCMV-3×Flag-14-eGFP eukaryotic expression vector to construct the recombinant plasmid pCMV-3×Flag-14-eGFP-P1. The plasmid was transfected into 293T cells, and after 48 hours, cells were collected and total protein was extracted. Western blot analysis was used to further identify whether the monoclonal antibody obtained in Example 1 recognized the target region. The results showed that the monoclonal antibody specifically reacted with the P1 fragment.

[0047] 3. Precise identification of antigenic epitopes Based on the Western blot results in section 3.2, four pairs of primers (as shown in SEQ ID NO.12-19 in Table 1) were designed to divide the P1 positive region into four fragments: P1-1 (50aa-65aa), P1-2 (61aa-77aa), P1-3 (73aa-88aa), and P1-4 (84aa-100aa). These fragments were cloned into the pCMV-3×Flag-14-eGFP eukaryotic expression vector to construct four eukaryotic expression plasmids: pCMV-3×Flag-14-eGFP-P1-1, pCMV-3×Flag-14-eGFP-P1-2, pCMV-3×Flag-14-eGFP-P1-3, and pCMV-3×Flag-14-eGFP-P1-4. These plasmids were then transfected into 293T cells. Cells were lysed using Ripa, protein samples were extracted, and Western blot analysis ultimately limited the antibody epitopes to within 15 amino acids. Results are as follows: Figure 3 As shown in Figure B, this monoclonal antibody reacts only with the P1-1 fragment, indicating that the monoclonal antibody recognizes the VP2 protein sequence. 50 EFKFLENGWVEITANS 65 .

[0048] Further analysis using ChimeraX software to locate the epitope on the VP2 protein trimer revealed that the antigenic epitope is located at the apex of the FPV viral capsid structure composed of the VP2 protein multimer, i.e., on the surface of the viral particle (e.g., ...). Figure 4(As shown). The amino acid sequences corresponding to the VP2 position of 61 FPV strains from both domestic and international sources were compared. Weblogos (50-65 aa) were created using the Weblogo3 website. The illustrations show that this epitope is highly conserved (e.g., ...). Figure 5 (As shown).

[0049] Table 1 Primer Design Example 3: Acquisition of the variable region sequence of a monoclonal antibody 1. Total RNA extraction and cDNA synthesis from hybridoma cells Select target hybridoma cells in good growth condition and collect approximately 1×10⁶ cells. 6 Total RNA was extracted from cells using the TRIzol reagent method, and RNA integrity was verified by agarose gel electrophoresis. Using 1 μg of total RNA as a template, cDNA was synthesized using the HanHai New Enzyme 1st Stand cDNA Synthesis Kit, strictly following the instructions. The resulting product was stored at -20 ℃ for later use.

[0050] 2. Amplification and purification of the variable regions of the light and heavy chains of monoclonal antibodies. Using the cDNA synthesized in step 3.1 as a template, PCR amplification was performed using mouse antibody variable region-specific primers (primer sequence information is shown in Table 2) with a 2×Es Taq MasterMix (Dye) kit to amplify the light and heavy chain variable region genes of the monoclonal antibody, respectively. After amplification, 5 μL of the PCR product was subjected to 1.5% agarose gel electrophoresis for preliminary verification. The electrophoresis results are shown below. Figure 6 As shown, band M is the marker; band 1 is the light chain variable region; band 2 is the heavy chain variable region; and band - is the negative control. The target band was clearly detected by gel electrophoresis, and its size was within the expected range according to DNA molecular weight standards.

[0051] For target bands that are single and of the correct size, gel extraction and purification were performed using an agarose gel DNA recovery kit, and the samples were sent to Suzhou Renduan Biomedical Technology Co., Ltd. for sequencing analysis. The sequencing results are as follows: Figure 7 As shown, the results indicate that the full-length heavy chain variable region sequence of the monoclonal antibody is 333 bp, encoding a peptide containing 111 amino acids (the amino acid sequence is shown in SEQ ID NO.7, and its CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO.1–3); the full-length light chain variable region sequence is 321 bp, encoding a peptide containing 107 amino acids (shown in SEQ ID NO.8, and its CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO.4–6).

[0052] Table 2 Primer names and sequences In summary, the development of the FPV monoclonal antibody in this invention is of great significance for establishing highly sensitive antibody detection kits, evaluating vaccine immunization efficacy, and developing novel and effective therapeutic drugs. This invention successfully prepared and identified a hybridoma cell line that stably secretes anti-VP2 protein monoclonal antibodies by using recombinant FPV VP2 protein as an immunogen. This antibody exhibits high specificity, recognizing different FPV strains and showing no cross-reactivity with negative controls, demonstrating good broad-spectrum and specificity.

[0053] The results of this invention echo existing research and provide a new perspective. Multiple reports have shown that monoclonal antibodies targeting specific epitopes of VP2 can effectively neutralize FPV, inhibit viral replication in vitro, and alleviate clinical symptoms and enteroviral load in infection models. One research team identified a conserved linear epitope located at 300-315 aa using phage display technology, providing a potential target for vaccine design. However, most of these studies have focused on antibodies with neutralizing activity. This invention, however, reveals a class of antibodies targeting a highly conserved non-neutralizing epitope (P1-1), which, while lacking therapeutic potential, have significant value in the diagnostic field. The hybridoma cell line monoclonal antibody screened in this invention recognizes an antigenic epitope located on the VP2 protein. 50 EFKFLENGWVEITANS 65 The region (SEQ ID NO. 25) has been confirmed to have extremely high conservation through sequence alignment with 61 FPV strains from both domestic and international sources. This makes this antibody a promising core ingredient for developing universal FPV antigen detection reagents, avoiding detection failures caused by epitope mutations between viral strains.

[0054] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A monoclonal antibody against feline parvovirus VP2 protein, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region of the antibody heavy chain includes CDR1, composed of the amino acid sequence shown in SEQ ID NO.1, CDR2, composed of the amino acid sequence shown in SEQ ID NO.2, and CDR3, composed of the amino acid sequence shown in SEQ ID NO.3; The variable region of the antibody light chain includes CDR1, composed of the amino acid sequence shown in SEQ ID NO.4, CDR2, composed of the amino acid sequence DTS, and CDR3, composed of the amino acid sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody against feline parvovirus VP2 protein according to claim 1, characterized in that, The monoclonal antibody comprises the heavy chain variable region amino acid sequence as shown in SEQ ID NO.7 or its homologous sequence; and / or, It contains the amino acid sequence of the light chain variable region as shown in SEQ ID NO.8 or its homologous sequence.

3. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain of a monoclonal antibody against the feline parvovirus VP2 protein as described in any one of claims 1-2; and / or, the nucleic acid encodes the light chain of a monoclonal antibody against the feline parvovirus VP2 protein as described in any one of claims 1-2.

4. The nucleic acid according to claim 3, characterized in that, Including the nucleotide sequences of CDR1, CDR2, and CDR3 used to encode the variable regions of the heavy chain, and / or, Nucleotide sequences used to encode the CDR1, CDR2, and CDR3 regions of the light chain variable region.

5. An antigenic epitope peptide of feline parvovirus VP2 protein, characterized in that, The antigenic epitope peptide specifically binds to the monoclonal antibody according to any one of claims 1-2.

6. The antigenic epitope peptide according to claim 5, characterized in that, Its amino acid sequence is 50 EFKFLENGWVEITANS 65 .

7. A biomaterial containing the nucleic acid of claim 3 or 4, characterized in that, The biological material is one or more of an expression vector, eukaryotic or prokaryotic host cells.

8. The use of a monoclonal antibody as described in any one of claims 1-2 in FPV detection.

9. The application according to claim 8, characterized in that, This includes the application of monoclonal antibodies in the preparation of FPV detection kits.

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