Anti-African swine fever virus P15 protein antibody and application thereof

Abstract

The invention discloses a hybridoma cell. The microbial preservation number of the hybridoma cell is CCTCC (China Center For Type Culture Collection) NO: C202557. The antibody secreted by the hybridoma cell can be combined with African swine fever virus P15 protein, can inhibit proliferation of the African swine fever virus, and has a medical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products and relates to an anti-African swine fever virus P15 protein antibody and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and wild boars. Sequence analysis confirmed that the strain initially introduced into my country was a highly virulent genotype II strain. As the ASF virus became prevalent in my country, field detection and isolation of moderately virulent genotype II natural variants and low-virulence genotype I strains were detected. These strains have a long incubation period, mild symptoms, and irregular toxin excretion, but they still possess a strong ability to spread, making them more susceptible to widespread spread and more difficult to eradicate.

[0003] African swine fever virus (ASFV) is the only member of the genus Asfenovirus in the family Assurviridae and the only arbovirus-borne DNA virus. The virion has a complex icosahedral symmetric structure composed of five layers: the genome, nucleocapsid, inner membrane, capsid, and outer membrane. ASFV encodes 150–167 open reading frames (ORFs), encoding 68 structural proteins and over 100 nonstructural proteins.

[0004] The ASFV core capsid protein P15, a key component in viral assembly and replication, has garnered significant attention in recent years. Research has shown that the P15 protein is composed of approximately 100 amino acids, and its high-resolution three-dimensional structure, determined through methods such as X-ray crystallography, has revealed unique structural features. The P15 protein exhibits dual functions: First, a specific structural domain confers high affinity for DNA, enabling it to specifically bind to the viral genome and regulate packaging and replication. Second, P15 interacts with host cell membranes, recognizing and binding to membrane lipids to promote virion assembly and stabilization. This dual function not only provides new molecular insights into the ASFV assembly mechanism but also provides a theoretical basis for the development of future P15-based antiviral strategies. These findings suggest that the P15 protein is a crucial regulator of the ASFV life cycle and a potential drug target and biomarker. Therefore, the development of antibodies against the P15 protein has potential applications, despite the limited availability of such antibodies. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a hybridoma cell, which is a hybridoma cell with a microbial deposition number of CCTCC NO: C202557 or a subculture cell of a hybridoma cell with a microbial deposition number of CCTCC NO: C202557; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial deposition number of CCTCC NO: C202557 maintains specific binding activity to the P15 protein of African swine fever virus.

[0006] In some embodiments, the amino acid sequence of the African swine fever virus P15 protein is shown as SEQ ID NO.2.

[0007] A second aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0008] P1: monoclonal antibody

[0009] The monoclonal antibody maintains specific binding activity to the African swine fever virus P15 protein;

[0010] The monoclonal antibody comprises a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0011] The monoclonal antibody heavy chain includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3;

[0012] The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3;

[0013] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52;

[0014] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7, positions 70-77;

[0015] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128;

[0016] The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52;

[0017] The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72;

[0018] The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117;

[0019] P2: combination of monoclonal antibody heavy chain and monoclonal antibody light chain

[0020] The monoclonal antibody heavy chain and the monoclonal antibody light chain combination maintain specific binding activity to the African swine fever virus P15 protein;

[0021] The monoclonal antibody heavy chain includes the amino acid sequence of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and a tag peptide and / or a signal peptide for isolating and purifying the protein;

[0022] The monoclonal antibody light chain includes the amino acid sequence of light chain CDR1, light chain CDR2, light chain CDR3 and a tag peptide and / or a signal peptide for isolating and purifying the protein;

[0023] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52;

[0024] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7, positions 70-77;

[0025] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128;

[0026] The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52;

[0027] The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72;

[0028] The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117;

[0029] P3: Antibody derivatives

[0030] The antibody derivative maintains specific binding activity to the African swine fever virus P15 protein;

[0031] The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3;

[0032] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52;

[0033] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7, positions 70-77;

[0034] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128;

[0035] The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52;

[0036] The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72;

[0037] The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117;

[0038] The form of the antibody derivative is selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0039] P4: RNA combination

[0040] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0041] The monoclonal antibody heavy chain RNA can be translated to produce the monoclonal antibody heavy chain described in P1 or P2;

[0042] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2;

[0043] P5: Genetic combination

[0044] The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2;

[0045] P6: Gene expression cassette combination

[0046] The gene expression product in the gene expression cassette combination is the RNA combination described in P4;

[0047] P7: Genetic Engineering Vector

[0048] The genetic engineering vector contains the gene expression cassette described in P6;

[0049] The genetic engineering vector encodes the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA; or

[0050] The genetic engineering vector is a combination of a vector encoding the monoclonal antibody heavy chain RNA and a vector encoding the monoclonal antibody light chain RNA;

[0051] P8: Cells

[0052] The cells contain the genetic engineering vector described in P7;

[0053] The encoded protein in the gene expression cassette of the genetic engineering vector is constitutively expressed or artificially induced;

[0054] When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the cell contains a vector encoding the monoclonal antibody heavy chain RNA and a vector encoding the monoclonal antibody light chain RNA; or

[0055] The cell is a combination of a cell containing a vector encoding the monoclonal antibody heavy chain RNA and a cell containing a vector encoding the monoclonal antibody light chain RNA;

[0056] P9: Composition

[0057] The composition contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; and

[0058] P10: Test kit

[0059] The kit contains the monoclonal antibody described in P1, the monoclonal antibody heavy chain and monoclonal antibody light chain combination described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

[0060] In some embodiments, in P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention.

[0061] In some embodiments, in P1, the amino acid sequence of the monoclonal antibody heavy chain is shown in SEQ ID NO.7;

[0062] The amino acid sequence of the light chain of the single monoclonal antibody is shown in SEQ ID NO.9.

[0063] In some embodiments, the amino acid sequence of the African swine fever virus P15 protein is shown as SEQ ID NO.2.

[0064] Use of the hybridoma cell described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in the preparation of a preparation for inhibiting the proliferation of African swine fever virus.

[0065] Use of the hybridoma cell described in the first aspect of the invention or the biomaterial described in the second aspect of the invention in the preparation of a preparation for treating, preventing or alleviating African swine fever.

[0066] The use of the hybridoma cells described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in preparing a preparation for identifying African swine fever virus P15 protein, African swine fever virus subviral particles containing African swine fever virus P15 protein, or African swine fever virus particles containing African swine fever virus P15 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The results of the detection of ASFV neutralization by P15 monoclonal antibody cell supernatant are shown.

[0068] Figure 2 The results of the detection of p15-4E2 monoclonal antibody neutralizing ASFV are shown.

[0069] Figure 3 This is the SPR detection result. DETAILED DESCRIPTION

[0070] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0071] Materials and instruments not described in the present invention are conventional materials and instruments in the field. Operation details not described in the present invention are conventional operations in the field. The software used in the present invention is operated by conventional methods with reference to the instructions of the software provider. The kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0072] The nucleic acid sequences shown in the present invention are written from left to right in the direction of 5' to 3', and the protein sequences are written from left to right in the direction of N-terminus to C-terminus.

[0073] Strains, cells and biomaterials

[0074] (1) Primary porcine alveolar macrophages

[0075] Primary porcine alveolar macrophages (PAMs) were obtained from 30-50 day old healthy SPF pigs and cultured in 1640 medium containing 10% (v / v) FBS in a 37°C, 5% CO2 incubator. They were prepared and used by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0076] (5)ASFV HLJ / 18-6GD strain

[0077] The ASFV gene-deleted virus strain (ASFV HLJ / 18-6GD strain, referred to as HLJ / 18-6GD strain), which lacks six genes in the MGF360 / 505 region, was prepared and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The preparation method is described in ASFV-△6GD strain in Aseven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs, Sci China Life Sci, Weiye Chen, et al., 2020; 63(5): 623-634. The HLJ / 18-6GD strain is a recombinant strain obtained by replacing six different genes with the expression of enhanced green fluorescent protein eGFP based on the HLJ / 18 (GenBank No. MK333180.1, full name Pig / HLJ / 2018) as the backbone.

[0078] Example 1: Preparation and identification of monoclonal antibodies

[0079] (1) Preparation of ASFV P15 protein

[0080] The present invention uses the gene of the HLJ / 18 strain (GenBank No. MK333180.1, full name Pig / HLJ / 2018). The P15 protein is processed into a mature virion structural protein by pS273R protease. In the pp62 polyprotein precursor (encoded by gene CP530R), the coding sequence of the P15 protein after codon optimization is as follows (SEQ ID NO.1):

[0081] ATGCCCTCAAATATGAAGCAATTTTGTAAAATATCTGTTTTGGCTGCAGCAACATGATCCGGACCTGCTGGAGATCATCAACAACCTGTGCATGCTGGGTAATCTGAGCGCAGCCAAATACAAGCACGGCGTCACCTTTATCTACCCGAAACAGGCTAAGATCCGCGATGAAATCAAGAAACACGCGTATAGCAACGACCCGTCGCAAGCGATCAAGACCTTGGAGTCCCTGATTTTACCGTT CTACATTCCGACTCCGGCTGAATTCACCGGTGAAATTGGCAGCTATACCGGCGTGAAACTGGAGGTTGAAAAAACGGAGGCCAATAAGGTGATTCTTAAGAACGGCGAGGCGGTGTTGGTTCCGGCGGCGGATTTTAAACCGTTCCCGGACCGTCGTCTGGCAGTTTGGATTATGGAAAGCGGTTCCATGCCACTGGAGGGTCCGCCTTATAAG

[0082] The P15 protein sequence encoded by the aforementioned CP530R gene is as follows (SEQ ID NO.2):

[0083] MPSNMKQFCKISVWLQQHDPDLLEIINNLCMLGNLSAAKYKHGVTFIYPKQAKIRDEIKKHAYSNDPSQAIKTLESLILPFYIPTPAEFTGEIGSYTGVKLEVEKTEANKVILKNGEAVLVPAADFKPFPDRRLAVWIMESGSMPLEGPPYK

[0084] The sequence encoding the P15 protein containing the above-mentioned CP530R gene was recombined into the pET-42b plasmid and transformed into Escherichia coli (E. coli BL21) to induce the expression of exogenous P15 protein. A His-tagged protein was introduced through primer design, and the resulting protein was purified using a His purification system to obtain a purified P15 recombinant protein.

[0085] (2) Preparation of monoclonal antibodies against ASFV P15 protein

[0086] The purified P15 protein solution (1 mg / ml, the solvent is sterile PBS (0.01 mol / L, pH 7.4)) was mixed and emulsified with equal volumes of complete Freund's adjuvant, and 6-8 week old female BALB / c mice were subcutaneously immunized, a total of 6 mice, with an immunization dose of 50 μg P15 protein / mouse. Then, the same dose of P15 protein solution was mixed and emulsified with equal volumes of incomplete Freund's adjuvant to prepare the immunogen, and two booster immunizations were performed, with an interval of 2 weeks between each immunization. The dose was the same each time. Ten days after the second booster immunization, blood was collected from each mouse, serum was separated, and antibody titer was determined using the indirect P15-ELISA method. The mouse with the highest antibody titer was selected and the purified P15 protein solution was used for the final booster immunization without adjuvant, with an immunization dose of 50 μg P15 protein / mouse. After 3 days, the mouse was euthanized, and spleen cells were taken and fused with SP2 / 0 myeloma cells. The fused hybridoma cells were cultured in HAT selection culture for 10 days. The purified P15 protein prepared in step (1) was used as the coating antigen, and the P15 antibody in the cell supernatant 10 days after fusion was detected by indirect ELISA. The antibody-positive wells were subcloned three times by limiting dilution, and finally three positive hybridoma cells with a single genetic background that could secrete antibodies against the P15 protein were obtained. They were named cell lines 1G9, 4E2, and 4C7, and the monoclonal antibodies secreted by these hybridoma cells were called P15-1G9, P15-4E2, and P15-4C7.

[0087] Example 2: Virus inhibition experiment (neutralization test) of monoclonal antibodies

[0088] (I) Effect of P15 monoclonal antibody cell supernatant on ASFV replication

[0089] ASFV HLJ / 18-6GD strains diluted in 1640 medium supplemented with 10% FBS were incubated with supernatants from P15 monoclonal antibody P15-1G9, P15-4E2, and P15-4C7 cells (cell supernatants were collected when the monoclonal antibody cells reached 90% viability) at a final virus dose of 0.1. The mixtures (a 1:1 ratio of diluted virus volume to monoclonal cell supernatant volume) were incubated at 37°C for 2 hours. After incubation, the supernatant in the PAM was discarded, and the mixtures were added to the respective PAMs. A negative control group was not supplemented with monoclonal antibody cell supernatant and was not infected with ASFV. A positive control group was not supplemented with monoclonal cell supernatant but was infected with ASFV. All other procedures and dosages were the same as those in the previous experiment.

[0090] 48 hours after infection, cells were photographed using a fluorescence microscope and then the cell supernatant was collected for nucleic acid extraction. ASFV was detected using the WOAH-recommended qPCR method. Primers F1 (SEQ ID NO. 3) and R1 (SEQ ID NO. 4) were used with a probe (partial nucleic acid sequence: SEQ ID NO. 5) to detect the viral B646L gene fragment and calculate the ASFV genome copy number.

[0091] F1: 5'-3'CTGCTCATGGTATCAATCTTATCGA.

[0092] R1: 5'-3'GATACCACAAGATCAGCCGT.

[0093] Probe: 5'-3'(FAM)CCACGGGAGGAATACCAACCCAGTG(TAMRA).

[0094] The results are as follows Figure 1 As shown in the figure, a is the statistical result of the p72 gene CT value of each group, and b is the photographic result of each group (the upper figure is fluorescence mode, the lower figure is bright field). The qPCR test results show that the cell supernatant of the P15-4E2 monoclonal antibody has a significant difference in ASFV, indicating that the cell supernatant of this monoclonal antibody can inhibit ASFV replication. There is no significant difference between the cell supernatant of the P15-1G9 and P15-4C7 monoclonal antibodies and the positive control, indicating that the cell supernatant of these two monoclonal antibodies has no effect on viral replication. The above qPCR test results echo the fluorescence microscopy results, indicating that the cell supernatant of the P15-4E2 monoclonal antibody can inhibit ASFV replication.

[0095] (II) Effect of P15-4E2 monoclonal antibody on ASFV replication

[0096] ASFV HLJ / 18-6GD strain diluted in 1640 medium containing 10% FBS was incubated with the P15-4E2 monoclonal antibody (the monoclonal antibody was diluted to 1000 μg / mL, 800 μg / mL, 500 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL in 1640 medium containing 10% FBS, respectively). The virus dose was adjusted to a final MOI of 0.1. The mixture (1:1 volume of diluted virus to diluted antibody) was incubated at 37°C for 2 hours. After incubation, the supernatant in the PAM was discarded, and the mixture was added to the PAM. A negative control group was not treated with the monoclonal antibody and was not infected with ASFV. A positive control group was not treated with the monoclonal antibody but was infected with ASFV. All other procedures and dosages were the same as those in the previous experiment.

[0097] After 24 and 48 hours of infection, the cells were photographed using a fluorescence microscope and then the number of fluorescent cells in the images was calculated using Image J software. Finally, the concentration of monoclonal antibodies that inhibited 50% ASFV replication at different time points was calculated using GraphPad Prism software.

[0098] The results are as follows Figure 2 As shown, images were taken using a fluorescence microscope, and the number of cells in each fluorescence image was counted using Image J software, and then calculated using GraphPad Prism software. The experimental results showed that the concentrations of P15-4E2 monoclonal antibody that could inhibit ASFV replication by 50% at 24h and 48h, respectively, were 257μg / mL and 366μg / mL.

[0099] Example 3: Characterization of Monoclonal Antibodies

[0100] (I) Typing of P15 protein monoclonal antibodies

[0101] The monoclonal antibody P15-4E2 was typed and identified using a mouse immunoglobulin typing kit (supplier: SouthernBiotech, catalog number 5300-05). The results showed that the monoclonal antibody P15-4E2 was a kappa light chain IgG1 type.

[0102] (II) Gene sequence determination of P15 protein monoclonal antibody

[0103] Total RNA from hybridoma 4E2 was obtained using the Trizol method and reverse transcribed into cDNA. Multiple primer pairs were designed based on the conserved sequences of mouse antibody genes. The cDNA was amplified by PCR. The amplified products were sequenced and, using a combination of NCBI NucleotideBLAST, IMGT / V Quest program, and NCBI IgBLAST tools, the full-length coding sequences of the antibody heavy and light chains were assembled. The sequences are as follows:

[0104] 1) The heavy chain coding sequence is (SEQ ID NO.6):

[0105]

[0106] Heavy chain CDR1 coding sequence: positions 133-156.

[0107] Heavy chain CDR2 coding sequence: positions 208-231.

[0108] Heavy chain CDR3 coding sequence: positions 346-384.

[0109] (2) The heavy chain protein sequence is (SEQ ID NO.7):

[0110] MMGSSIILFLVATATGVHSQVQLHQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGEIHPKSGNTNYNENFKGKATLTVDTSSSTAYVDLSSLASEDSAVYYC ARSGGTYYYAMDSWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTK VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAP IEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0111] Heavy chain CDR1 protein sequence: positions 45-52.

[0112] Heavy chain CDR2 protein sequence: positions 70-77.

[0113] Heavy chain CDR3 protein sequence: positions 116-128.

[0114] (3) The light chain coding sequence is (SEQ ID NO.8):

[0115] ATGATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACACTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAACACCCTGGAGCAAGAAGATTTTGCCACTTACTTTTGTCAACAGGGTAATACGTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATT GTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0116] Light chain CDR1 coding sequence: positions 139 - 156.

[0117] Light chain CDR2 coding sequence: positions 208 - 216.

[0118] Light chain CDR3 coding sequence: positions 325 - 351.

[0119] (4) The light chain protein sequence is (SEQ ID NO.9):

[0120] MMMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDTSNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTINTLEQEDFATYFCQQGNTSPY TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0121] Light chain CDR1 protein sequence: positions 47-52.

[0122] Light chain CDR2 protein sequence: positions 70-72.

[0123] Light chain CDR3 protein sequence: positions 109-117.

[0124] (3) Hybridoma Deposit

[0125] The hybridoma cells (secreting monoclonal antibody p15-4E2) prepared by the present invention were deposited with a patent procedure-approved depository institution. The depository institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The microbial accession number is CCTCC NO: C202557. The culture is named "Hybridoma cell line p15-4E2"; the Chinese classification name is "Hybridoma Cell"; the English classification name is "Hybridoma Cell"; the deposit date is February 28, 2025, and the identified survival date is March 6, 2025.

[0126] (4) Surface plasmon resonance detection experiment

[0127] P15 protein was immobilized on a CM5 sensor chip using Biacore® 8k (GE Healthcare) and PBS (containing 0.05% Tween-20) buffer via NHS / EDC. Multi-cycle kinetic analysis of the Fab was performed to determine its affinity. Data were analyzed using Biacore® 8k evaluation software (GE Healthcare).

[0128] The results are as follows Figure 3 As shown in the figure, the P15-4E2 monoclonal antibody tightly binds to P15 (KD=6.39 nM) as detected by surface plasmon resonance, wherein the black line represents the actual data curve and the colored line represents the fitted data curve, and the difference between the two is not significant.

[0129] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A hybridoma cell, wherein the hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C202557 or a subculture cell of a hybridoma cell with a microbial deposit number of CCTCC NO: C202557; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial deposit number of CCTCC NO: C202557 maintains specific binding activity to the P15 protein of African swine fever virus.

2. The hybridoma cell according to claim 1, wherein The amino acid sequence of the African swine fever virus P15 protein is shown in SEQ ID NO.

2.

3. A biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody retains specific binding activity to the African swine fever virus P15 protein; The monoclonal antibody comprises a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52; The heavy chain CDR2 protein sequence is shown in positions 70-77 of SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128; The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52; The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72; The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117; P2: Combination of monoclonal antibody heavy chain and monoclonal antibody light chain The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to the African swine fever virus P15 protein; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and an amino acid sequence of a tag peptide and / or a signal peptide for separating and purifying the protein; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2, a light chain CDR3 and an amino acid sequence of a tag peptide and / or a signal peptide for separating and purifying the protein; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52; The heavy chain CDR2 protein sequence is shown in positions 70-77 of SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128; The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52; The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72; The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117; P3: Antibody derivatives The antibody derivative retains specific binding activity to the African swine fever virus P15 protein; The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.7, positions 45-52; The heavy chain CDR2 protein sequence is shown in positions 70-77 of SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.7, positions 116-128; The light chain CDR1 protein sequence is shown in SEQ ID NO.9, positions 47-52; The light chain CDR2 protein sequence is shown in SEQ ID NO.9, positions 70-72; The light chain CDR3 protein sequence is shown in SEQ ID NO.9, positions 109-117; The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA combination The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or P2; The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2; P5: Genetic combination The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2; P6: Gene expression cassette combination The gene expression product in the gene expression cassette combination is the RNA combination described in P4; P7: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in P6; The genetic engineering vector encodes the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA; or The genetic engineering vector is a combination of a vector encoding the monoclonal antibody heavy chain RNA and a vector encoding the monoclonal antibody light chain RNA; P8: Cells The cell contains the genetic engineering vector described in P7; The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively or artificially induced; When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the cell contains a vector encoding the monoclonal antibody heavy chain RNA and a vector encoding the monoclonal antibody light chain RNA; or The cell is a combination of a cell containing a vector encoding the monoclonal antibody heavy chain RNA and a cell containing a vector encoding the monoclonal antibody light chain RNA; P9: Composition The composition contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; and P10: Test kit The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

4. The biomaterial according to claim 3, characterized in that In P1, the monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2.

5. The biomaterial according to claim 3 or 4, characterized in that In P1, the amino acid sequence of the monoclonal antibody heavy chain is shown in SEQ ID NO.7; The amino acid sequence of the light chain of the single monoclonal antibody is shown in SEQ ID NO.

9.

6. The biomaterial according to any one of claims 3 to 5, characterized in that The amino acid sequence of the African swine fever virus P15 protein is shown in SEQ ID NO.

2.

7. Use of the hybridoma cell according to claim 1 or 2 or the biomaterial according to any one of claims 2 to 6 in the preparation of a preparation for inhibiting the proliferation of African swine fever virus.

8. Use of the hybridoma cell according to claim 1 or 2 or the biomaterial according to any one of claims 2 to 6 in the preparation of a preparation for treating, preventing or alleviating African swine fever.

9. Use of the hybridoma cell according to claim 1 or 2 or the biological material according to any one of claims 2 to 6 in preparing a preparation for identifying African swine fever virus P15 protein, African swine fever virus subviral particles containing African swine fever virus P15 protein, or African swine fever virus particles containing African swine fever virus P15 protein.

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