Antibody specifically binding to African swine fever virus E165R protein or antigen binding fragment thereof and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an antibody specifically bound with African swine fever virus E165R protein or an antigen binding fragment thereof and application of the antibody or the antigen binding fragment. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 3. High blocking activity and excellent specificity are realized. The invention also provides nucleic acid molecules encoding the antibody or the antigen binding fragment thereof, expression cassettes, recombinant vectors and host cells. A blocking ELISA detection kit established on the basis of the antibody or the antigen binding fragment thereof can realize high-sensitivity detection of the E165R antibody in porcine serum through a competitive binding principle of coated E165R protein and an enzyme-labeled antibody. The invention provides a key tool for structural analysis, serological diagnosis and genetic engineering antibody development of the African swine fever virus E165R protein.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to an antibody that specifically binds to the E165R protein of African swine fever virus or its antigen-binding fragment and its applications. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and highly contagious disease caused by the African swine fever virus (ASFV). Clinically, it is characterized by its acute onset, fever, hemorrhagic nature, high morbidity, and high mortality. Based on the virulence of the infecting strain, ASFV infection can be classified as peracute, acute, subacute, and chronic. The main clinical symptoms are high fever, tissue edema, and hemorrhagic lesions; the mortality rate from the peracute stage can reach 100%.

[0003] ASFV is a large double-stranded DNA virus encoding over 200 proteins. Among them, the E165R protein, an ASFV-encoded deoxyuridine triphosphate pyrophosphatase (dUTPase), plays a crucial role in viral DNA synthesis, maintaining genome replication fidelity by hydrolyzing dUTP. Studies have shown that knocking out or inhibiting the E165R gene significantly reduces viral replication levels in macrophages, confirming its important value as an antiviral target. Simultaneously, the E165R protein is also a key target protein for ASFV serological diagnostics and immunological research. Monoclonal antibody studies targeting E165R have revealed a specific antibody-binding epitope in the motif V region of this protein. This epitope exhibits viral species specificity—monoclonal antibodies targeting this epitope recognize only ASFV dUTPase and not host porcine dUTPase, providing a theoretical basis for establishing specific serological differential diagnostic methods.

[0004] Although the E165R protein plays a dual role in the pathogenesis and diagnostic techniques of ASFV, its three-dimensional molecular structure remains unresolved, hindering structure-based drug design and in-depth epitope discovery. At the detection level, while various serological detection methods for ASFV exist, high-specificity, high-blocking-activity monoclonal antibody tools targeting the E165R protein are still lacking. Current antibody development largely focuses on viral structural proteins (such as p72 and p54), and the lack of high-quality monoclonal antibodies targeting key functional proteins like E165R leads to the following technical challenges that urgently need to be addressed:

[0005] Lack of structural biology research tools: The lack of highly specific and high-affinity monoclonal antibodies as "molecular probes" hinders the crystal structure analysis and functional domain study of the E165R protein.

[0006] Serological testing lacks specificity: Existing testing systems cannot accurately target the E165R protein, making it impossible to quantify antibody levels against this functional protein and accurately assess vaccine efficacy.

[0007] Therefore, there is an urgent need in this field to develop monoclonal antibodies and their derivatives that specifically bind to the ASFV E165R protein and have high blocking activity, in order to meet the pressing needs of viral structure analysis, specific serological detection, and immunological research. Summary of the Invention

[0008] One of the purposes of this application is to provide an antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus E165R protein and its application, so as to provide a monoclonal antibody with high blocking activity that specifically binds to the ASFV E165R protein and its derivative tools.

[0009] To achieve the above objectives, this application adopts the following technical solution:

[0010] An antibody or antigen-binding fragment thereof that specifically binds to the E165R protein of African swine fever virus, said antibody or antigen-binding fragment comprising: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3.

[0011] Furthermore, the antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment.

[0012] Furthermore, the antibody is a monoclonal antibody 1C10, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

[0013] Furthermore, the antibody is a single-chain antibody, wherein the heavy chain variable region and the light chain variable region are linked by a linker peptide.

[0014] Biological materials related to the antibodies or antigen-binding fragments of this application, wherein the biological materials are any of the following:

[0015] (a) A nucleic acid molecule containing a sequence encoding a heavy chain variable region and / or a light chain variable region encoding the antibody or an antigen-binding fragment thereof;

[0016] (b) Expression cassette containing the nucleic acid molecules in (a);

[0017] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0018] (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c);

[0019] (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0020] Furthermore, the nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO:2; and / or

[0021] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO:4.

[0022] The application of antibodies or their antigen-binding fragments or biological materials in the preparation of African swine fever virus detection products;

[0023] Optionally, the product is a blocking ELISA antibody detection kit.

[0024] An African swine fever virus blocking ELISA antibody detection kit, the kit comprising: a support medium coated with ASFVE165R protein, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody of this application or its antigen-binding fragment.

[0025] Furthermore, the coating concentration of ASFV E165R protein was 0.5-1.0 μg / mL;

[0026] Optionally, the supporting medium is a microtiter plate;

[0027] Optionally, the enzyme labeled with the enzyme may be horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0028] Furthermore, the test reagent includes a colorimetric solution and a stop solution. The colorimetric solution includes colorimetric solution A and colorimetric solution B. Colorimetric solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and colorimetric solution B contains 0.02% w / v tetramethylbenzidine and 1% v / v anhydrous ethanol.

[0029] Optionally, the stop solution is a 10% hydrochloric acid solution;

[0030] Optionally, the kit may also include at least one of a positive control, a negative control, a washing solution, and a sample diluent;

[0031] Optionally, positive controls are positive serum from pigs immunized with 5% v / v E165R protein, PBS solution containing 20% ​​v / v bovine serum and 0.1% v / v Proclin 300;

[0032] Optionally, the negative control is a PBS solution containing 5% v / v porcine negative serum, 20% v / v bovine serum and 0.1% v / v Proclin 300;

[0033] Optionally, the sample diluent is a PBS solution containing 20% ​​v / v newborn calf serum and 0.1% v / v Proclin 300.

[0034] The technical effects of this application are as follows:

[0035] This application utilizes hybridoma technology to screen and obtain a monoclonal antibody targeting the E165R protein of African swine fever virus (ASFV). This antibody contains unique and well-defined heavy and light chain variable regions, enabling precise recognition of the functional epitopes of the E165R protein and achieving highly specific binding to this key viral protein. The antibody exhibits significant blocking activity, effectively interfering with the interaction between the viral protein and ligand. Furthermore, it shows no cross-reactivity with various common porcine pathogens, including ASFV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), ensuring specificity and accuracy of detection. A blocking ELISA detection system based on this core antibody, through systematic optimization of key reaction parameters, has formed a stable and reliable reagent combination, achieving highly sensitive detection and accurate quantification of E165R protein antibodies in porcine serum. In clinical background serological validation, it demonstrates excellent positive and negative concordance rates and good reproducibility. This detection system is not only suitable for rapid screening and diagnosis of clinical samples but also effectively used for dynamic antibody monitoring and immunization efficacy evaluation after vaccination, capturing the temporal patterns and intensity changes of the immune response.

[0036] Furthermore, this application provides a complete antibody-encoding gene sequence, supporting the preparation of single-chain antibodies and other derivative forms through genetic engineering methods. The resulting derivative molecules maintain high blocking activity while exhibiting excellent expression efficiency and detection efficacy, providing key technical tools for structural biology research of E165R protein, serological differential diagnosis of African swine fever virus, and development of genetically engineered antibody drugs. It also provides a novel detection tool for E165R protein, improving the technical level of disease prevention and control. Detailed Implementation

[0037] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0038] Words such as “further,” “even further,” and “especially” are used to describe the purpose and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0039] "Optional," "optional," and "optional" mean that something is optional, that is, it means that it can be selected from either "with" or "without." If there are multiple "optional" options in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, then each "optional" option is independent.

[0040] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] Unless otherwise specified, all percentage concentrations mentioned in this application are mass / volume percentages (w / v) or volume / volume percentages (v / v), and the concentration ranges include endpoint values ​​and all subranges within that range.

[0042] "Antibody" refers to an immunoglobulin molecule that can specifically bind to antigens. Its basic structural unit consists of two heavy chains and two light chains linked by disulfide bonds, and each chain contains a variable region and a constant region. This term encompasses complete antibody molecules, their functional fragments (such as antigen-binding fragments), derivatives (such as chimeric and humanized antibodies), and polypeptides containing antigen-binding domains.

[0043] "Monoclonal antibody" refers to a group of antibodies that are substantially homologous and whose amino acid sequences are essentially identical, produced by a single B cell clone. The monoclonal antibody of this application specifically refers to a murine antibody against the E165R protein of African swine fever virus, and more particularly to an antibody secreted by the hybridoma cell line 1C10 that has a specific variable region sequence (SEQ ID NO: 1 and 3).

[0044] "Heavy chain variable region (VH)" and "light chain variable region (VL)" refer to the N-terminal domains responsible for antigen recognition in the antibody heavy and light chains, respectively. VH and VL together constitute the antigen binding site through their respective complementarity-determining regions. In this application, the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:3 are specifically referred to.

[0045] "Antigen-binding fragments" refer to antibody fragments that contain the complete variable region (VH and / or VL) of the antibody, capable of specifically binding to antigens but lacking some or all of the constant region. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies. These fragments can be obtained by enzymatic digestion of complete antibodies or recombinant DNA technology.

[0046] Fab fragment: A monovalent antigen-binding fragment consisting of a light chain (containing a variable region and a constant region) and the N-terminal portion of a heavy chain (containing the heavy chain variable region and the first constant region CH1).

[0047] Fab' fragment: A monovalent antigen-binding fragment that includes a portion of the heavy chain hinge region sequence on the Fab basis, containing one or more cysteine ​​residues from the hinge region.

[0048] F(ab')2 fragment: A bivalent antigen-binding fragment formed by two Fab' fragments linked by disulfide bonds in the hinge region, containing two antigen-binding sites.

[0049] scFv fragment: Single-chain variable region fragment, a single polypeptide chain formed by directly linking the heavy chain variable region and the light chain variable region through an artificially designed linker peptide, retaining the antigen-binding specificity of the complete antibody.

[0050] Fv fragment: The smallest antigen-binding unit formed by the non-covalent binding of the heavy chain variable region and the light chain variable region.

[0051] "Linker peptides" are short peptide sequences used to link the heavy chain variable region and the light chain variable region in single-chain antibody forms such as scFv. They are usually composed of repeating units of flexible amino acid residues (such as glycine and serine) to provide sufficient degrees of freedom of movement for the two domains.

[0052] "Nucleic acid molecule" refers to deoxyribonucleic acid or ribonucleic acid encoding the antibody or its antigen-binding fragment (especially its heavy chain variable region and / or light chain variable region) described in this application, including but not limited to cDNA, synthetic DNA or optimized gene sequences.

[0053] The “expression cassette” comprises the nucleic acid molecule described in this application and a DNA construct operatively linked thereto with regulatory sequences (such as promoters, terminators, enhancers, signal peptide sequences, polyadenylation signals) necessary to control its transcription and / or translation.

[0054] "Recombinant vector" refers to a recombinant nucleic acid molecule obtained by inserting the nucleic acid molecule or expression cassette of this application into a suitable vector backbone (such as a plasmid, bacteriophage, or viral vector) for delivery and expression of the nucleic acid sequence.

[0055] "Recombinant host cells" are eukaryotic or prokaryotic cells introduced into the nucleic acid molecules, expression cassettes, or recombinant vectors of this application through transfection, transformation, or transduction, and can be used to produce the antibodies or their antigen-binding fragments.

[0056] "Blocking ELISA Antibody Detection Kit" refers to an enzyme-linked immunosorbent assay (ELISA) system based on the principle of antigen-antibody competitive binding. It contains essential components such as solid-phase coated antigen and enzyme-labeled antibody. In this method, the target antibody in the test sample competes with the enzyme-labeled antibody to bind to the solid-phase coated antigen. The degree of signal inhibition is positively correlated with the content or activity of the target antibody in the test sample; that is, the higher the content of the target antibody, the lower the colorimetric signal.

[0057] The antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus E165R protein provided in this application contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0058] SDVQVQESGPGLVKPSQSLSLTCTVTGYSISSDYAWNWIRQFPGNRLEWMAYISYSGDTRYNPSLKSRISITRDTSKNQVFLQLNSVTSEDTATYYCAGGGGYWGQGTTLTVSS (SEQ ID NO: 1).

[0059] NIVMTQTPKFLLASAGDRVTITCKASQSVSNDVAWYQQKSGQSPKLLIYHASTRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPYTFGGGTKLEIKR (SEQ ID NO: 3).

[0060] In some embodiments, the antibody or its antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment. In some embodiments, the antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody.

[0061] In a preferred embodiment, the antibody is monoclonal antibody 1C10 obtained in the embodiments of this application. The heavy chain subclass of monoclonal antibody 1C10 is IgG1, and the light chain subclass is kappa.

[0062] Antibodies are immunoglobulins (Igs) used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. Immunoglobulins are mainly classified into five types: IgA, IgD, IgE, IgG, and IgM. IgG antibodies have four subtypes: IgG1, IgG2, IgG3, and IgG4, with differences in the position and number of disulfide bonds between different subtypes. The light chain of immunoglobulin (Ig) is divided into two subtypes: κ (kappa) and λ (lambda). Experimental testing and identification have confirmed that the monoclonal antibody 1C10 protected in this application has the heavy chain subtype IgG1 and the light chain subtype kappa.

[0063] In a preferred embodiment, the antibody is the single-chain antibody 1C10 obtained in the embodiments of this application, and the heavy chain variable region and the light chain variable region of the single-chain antibody 1C10 are linked by a linker peptide.

[0064] Here, the heavy chain variable region and light chain variable region of the single-chain antibody 1C10 can be linked using linker peptides commonly used in the art.

[0065] This application also provides biological materials related to the antibody or its antigen-binding fragment of this application, wherein the biological material is any one of the following:

[0066] (a) A nucleic acid molecule containing a sequence encoding a heavy chain variable region and / or a light chain variable region encoding the antibody or an antigen-binding fragment thereof.

[0067] (b) Expression cassette containing nucleic acid molecules from (a).

[0068] (c) A recombinant vector containing either the nucleic acid molecule in (a) or the expression cassette in (b).

[0069] (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0070] (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0071] In a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 2; preferably, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 4.

[0072] TCTGATGTGCAGGTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCAGCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGCCTACATAAGTTACAGTGGTGA CACTCGCTACAACCCGTCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGGTCTTCTTGCAGTTGAATTCTGTGACTTCTGAGGACACAGCCACATATTACTGTGCGGGGGGGGGGGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 2).

[0073] AATATTGTGATGACCCAGACTCCCAAATTCCTGCTTGCATCAGCCGGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGTCAGGGCAGTCTCCGAAATTGCTGATATATCATGCATCCACTCGCTA CACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTTATTTCTGTCAGCAGGATTATAACTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG (SEQ ID NO: 4).

[0074] This application also provides the use of the above-mentioned antibody or its antigen-binding fragment or biological material in the preparation of products for detecting African swine fever virus.

[0075] In some embodiments, the product is a reagent or kit. The heavy chain variable region and light chain variable region provided in this application can specifically bind to African swine fever virus. Therefore, this property and commonly used detection methods in the art can be used to detect African swine fever virus and prepare a product. In a preferred embodiment, the kit is an African swine fever virus blocking ELISA antibody detection kit.

[0076] This application also provides an African swine fever virus blocking ELISA antibody detection kit, which includes: a support medium coated with E165R protein, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody or its antigen-binding fragment provided in this application.

[0077] In a preferred embodiment, the enzyme-labeled reagent is an enzyme-labeled monoclonal antibody 1C10.

[0078] In a preferred embodiment, the coating concentration of E165R protein is 0.5-1.0 μg / mL, preferably 0.6 μg / mL. Preferably, the supporting medium is a microtiter plate; preferably, the enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0079] It should be noted that the coating concentration of E165R protein can be, but is not limited to, 0.50 μg / mL, 0.51 μg / mL, 0.52 μg / mL, 0.53 μg / mL, 0.54 μg / mL, 0.55 μg / mL, 0.56 μg / mL, 0.57 μg / mL, 0.58 μg / mL, 0.59 μg / mL, 0.60 μg / mL, 0.61 μg / mL, 0.62 μg / mL, 0.63 μg / mL, 0.64 μg / mL, 0.65 μg / mL, 0.66 μg / mL, 0.67 μg / mL, 0.68 μg / mL, 0.69 μg / mL, 0.70 μg / mL, 0.71 μg / mL, 0.72 μg / mL, 0.73 μg / mL, 0.7... 4μg / mL, 0.75μg / mL, 0.76μg / mL, 0.77μg / mL, 0.78μg / mL, 0.79μg / mL, 0.80μg / mL ,0.81μg / mL, 0.82μg / mL, 0.83μg / mL, 0.84μg / mL, 0.85μg / mL, 0.86μg / mL, 0.87μg / mL, 0.88μg / mL, 0.89μg / mL, 0.90μg / mL, 0.91μg / mL, 0.92μg / mL, 0.93μg / mL, 0.9 4μg / mL, 0.95μg / mL, 0.96μg / mL, 0.97μg / mL, 0.98μg / mL, 0.99μg / mL or 1.00μg / mL.

[0080] In a preferred embodiment, the detection reagent includes a colorimetric solution and a stop solution. Preferably, the colorimetric solution includes colorimetric solution A and colorimetric solution B, wherein colorimetric solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid, and 0.03% w / v urea peroxide, and colorimetric solution B contains 0.02% w / v tetramethylbenzidine and 1% v / v anhydrous ethanol. Preferably, the stop solution is a 10% hydrochloric acid solution.

[0081] In a preferred embodiment, the kit further includes at least one of a positive control, a negative control, a washing buffer, and a sample diluent. Preferably, the positive control is a PBS solution containing 5% v / v E165R protein-immunized pig positive serum, 20% v / v bovine serum, and 0.1% v / v Proclin 300; preferably, the negative control is a PBS solution containing 5% v / v porcine negative serum, 20% v / v bovine serum, and 0.1% v / v Proclin 300; preferably, the washing buffer is a 20× concentrated washing buffer, prepared by dissolving 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, and 10ml Tween 20 in purified water and bringing the volume to 1000ml, then diluting 20 times with purified water before use; preferably, the sample diluent is a PBS solution containing 20% ​​v / v newborn calf serum and 0.1% v / v Proclin 300.

[0082] The present application is further described below with reference to specific embodiments. The advantages and features of the present application will become clear from the description. The embodiments described are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0083] To make this application easier to understand, specific embodiments are described below to further illustrate this application. Unless otherwise specified, the experimental methods described in this application are conventional methods; and unless otherwise specified, the biological materials described are all commercially available.

[0084] Example 1: Preparation and Identification of ASFV E165R Protein

[0085] African swine fever virus E165R protein was prepared according to the method of Chinese patent application CN116019905A; the concentration of E165R protein was determined to be 1.2 mg / ml according to the instructions of the BCA protein concentration assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.).

[0086] Example 2: Preparation and Identification of ASFV E165R Protein Monoclonal Antibody

[0087] 2.1 Screening of hybridoma cells

[0088] Five female BALB / c mice aged 4–6 weeks were immunized with ASFV E165R protein every 2 weeks using a subcutaneous multi-site immunization method at a dose of 100 μg / mouse (400 μl volume). For the first immunization, 100 μg of protein was emulsified with Freund's complete adjuvant and then immunized. Subsequent immunizations were performed using 100 μg of protein emulsified with Freund's incomplete adjuvant and then immunized. A total of 3 immunizations were performed.

[0089] After triple immunization, mouse serum was collected and serum titer was determined using an indirect ELISA method with ASFV E165R protein: The protein was diluted to 0.5 μg / ml and coated onto an ELISA plate (100 μl / well), incubated at 2–8°C for 16–24 hours; the liquid in the plate was discarded, blocking buffer was added (200 μl / well), and the plate was blocked at 2–8°C for 16–24 hours, followed by washing; the test sample (hybridoma cell supernatant diluted 1:100 and then serially diluted, mouse serum diluted 1:1000 and then serially diluted) was added (100 μl / well), with a negative control containing PBS (0.01 mol / L, pH 7.4) also included. The plates were incubated at 37°C. Incubate at ℃ for 60 minutes, wash the plate; add 100 μl of enzyme-labeled secondary antibody diluted to the working concentration per well, incubate at 37℃ for 30 minutes, wash the plate; add 50 μl of chromogenic reagent A and B per well sequentially, vortex to mix, incubate at 37℃ in the dark for 15 minutes, then add 50 μl of stop solution per well; set the microplate reader wavelength to 450 nm and detect the OD value of each well; the test is valid when the negative control OD value is <0.2; a S / N (sample OD value / negative control OD value) ≥2.1 is considered positive; a S / N (sample OD value / negative control OD value) <2.1 is considered negative. The highest sample dilution corresponding to the positive well is taken as the titer of that sample. Using this method to test the serum of 5 mice, the results showed that the serum titer of one mouse (2#) was the highest, reaching 1:512000. ASFVE165R protein was injected intraperitoneally at a dose of 100 μg into mice to induce shock immunization. Cell fusion was performed 3 days after immunization. The fused cells were subjected to multiple subclonal screenings to obtain 10 positive hybridoma cell lines.

[0090] 2.2 Selection of Monoclonal Antibodies with Blocking Activity for ELISA

[0091] A positive serum blocking assay was performed on the supernatant of 10 hybridoma cell lines. The procedure was as follows: 100 µl of cell supernatant was added for detection; wells containing sample dilution buffer were designated as negative controls. The plates were incubated at 37°C for 60 minutes and washed. 100 µl of 1:100 diluted African swine fever positive serum was added to each well, and the plates were incubated at 37°C for 60 minutes and washed. 100 µl of diluted HRP-labeled anti-swine IgG was added to each well, and the plates were incubated at 37°C for 30 minutes and washed. 50 µl of chromogenic reagents A and B were added to each well, and the mixture was vortexed and incubated at 37°C in the dark for 15 minutes. 50 µl of stop solution was added to each well. The OD value of each well was measured at 450 nm using a microplate reader. The sample blocking rate was calculated as: (OD value of negative control wells - OD value of sample wells) / OD value of negative control wells. Three hybridoma cell lines (1C10, 4G3, and 5F8) with high blocking rates were selected for ascites preparation. The ascites was diluted 100-fold, and the blocking rate was evaluated using a positive serum blocking test. The results showed that the blocking rates of monoclonal antibodies 4G3 and 5F8 were 42% and 35%, respectively, both not exceeding 50%, and were therefore discarded. The blocking rate of monoclonal antibody 1C10 was 88%, which was a more ideal result. The ascites was purified by Protein G affinity chromatography and used for subsequent studies.

[0092] 2.3 Identification of Monoclonal Antibodies

[0093] 2.3.1 Subclass Identification

[0094] The subclasses of monoclonal antibody 1C10 were identified using a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclass of 1C10 was IgG1, and the light chain subclass was kappa.

[0095] 2.3.2 Western blot identification

[0096] First, ASFV E165R protein was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a membrane, Western blot was performed using a dilution of monoclonal antibody 1C10 as the primary antibody and a dilution of HRP-labeled goat anti-mouse IgG as the secondary antibody. The results showed that monoclonal antibody 1C10 reacted with ASFV E165R protein to produce a specific band, indicating that monoclonal antibody 1C10 can recognize ASFV E165R protein.

[0097] 2.3.3 Specificity Identification

[0098] IFA antigen plates for classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus were prepared separately. After fixation with 80% cold acetone, the plates were air-dried and stored at -20℃ for later use. Before use, each antigen plate was warmed to room temperature, washed once with PBS, and then diluted with monoclonal antibody 1C10. The plates were then detected using the standard IFA method. Results showed that no specific fluorescence was observed in the cell wells inoculated with different viruses containing monoclonal antibody 1C10, indicating that monoclonal antibody 1C10 does not react with common porcine viruses and exhibits good specificity.

[0099] Example 3: Establishment of the ASFV E165R protein blocking ELISA antibody detection kit

[0100] 3.1 Preparation and Identification of Enzyme-Labeled Antibodies

[0101] 3.1.1 Preparation

[0102] Horseradish peroxidase (HRP) labeling of monoclonal antibody 1C10 was performed using a modified sodium periodate method. 20 mg of horseradish peroxidase (HRP) was dissolved in 1 ml of ultrapure water, and 1 ml of freshly prepared NaIO4 solution (30 mg NaIO4 dissolved in 1 ml of ultrapure water, prepared fresh before use) was added. The mixture was incubated at 2–8°C in the dark for 30 minutes. 40 μl of ethylene glycol was added to the above solution, and the mixture was incubated at 2–8°C in the dark for 30 minutes. Following this, 2 mg of purified monoclonal antibody was added to 100 μl of the above mixture, and the mixture was added to a dialysis bag. After mixing, the mixture was dialyzed against CB buffer for 6 hours. The entire procedure must be performed in the dark. Transfer the dialyzed mixture to a 1.5 ml EP tube, add 10 μl of freshly prepared NaBH4 solution (20 mg NaBH4 dissolved in 1 ml ultrapure water, prepared fresh before use), and incubate at room temperature for 2 hours, mixing every 30 minutes. Add an equal volume of saturated ammonium sulfate, mix well, and incubate at 2–8 °C for 15 minutes. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant. Resuspend the precipitate in a mixture of PBS and glycerol (V:V = 1:1) equal to the volume of purified antibody.

[0103] 3.1.2 Identification

[0104] Appearance: At room temperature, it is a reddish-brown liquid with no flocculent precipitate observed.

[0105] Quality assessment: The enzyme-labeled antibody was diluted 10-fold and its absorbance (A) at 403 nm and 280 nm was measured using a UV spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:

[0106] Enzyme amount (mg / ml) = A 403nm ×0.4×Dilution factor.

[0107] IgG level (mg / ml) = (A 280nm -A 403nm ×0.3) ×0.62 × dilution factor.

[0108] Molecular ratio (E / P) = Enzyme amount × 4 / IgG amount.

[0109] Labeling rate = A 403nm / A 280nm .

[0110] The specific results after absorbance detection and calculation are shown in Table 1:

[0111] Table 1. Quality evaluation results of enzyme-labeled antibodies

[0112] 3.2 Preparation of ASFV E165R protein blocking ELISA antibody detection kit

[0113] Antigen coating plate: The E165R protein prepared in Example 1 was diluted to 0.6 μg / ml with carbonate buffer (0.05 mol / L, pH 9.6) and coated at 100 μl / well. The plate was incubated at 2–8°C for 16–24 hours. After washing with washing buffer, blocking buffer (50 g sucrose, 10 g BSA, 200 ml bovine serum, 1.0 ml Proclin 300, and PBS buffer (0.01 mol / L, pH 7.4) was added to make up to 1000 ml) at 2–8°C for 16–24 hours. After discarding the blocking buffer, the plate was dried, sealed, and stored at 2–8°C for later use.

[0114] Enzyme-labeled reagent: The enzyme-labeled monoclonal antibody 1C10 prepared in Example 3.1.1 was diluted with enzyme-labeled dilution buffer (30g trehalose, 200ml bovine serum, 1.0ml Proclin 300, 10ml Tween 20, 0.04g AM dye, and PBS buffer (0.01mol / L, pH 7.4) to a final volume of 1000ml) and stored at 2–8℃.

[0115] Positive control: Take 50ml of porcine E165R protein-positive serum, 200ml of bovine serum, and 1.0ml of Proclin 300. Add PBS buffer (0.01mol / L, pH 7.4) to a final volume of 1000ml. Mix well and filter through a 0.22μm filter. Aseptically aliquot the mixture into a single volume as a positive control and store at 2–8℃.

[0116] Negative control: Take 50 ml of porcine negative serum, 200 ml of bovine serum, and 1.0 ml of Proclin 300. Add PBS buffer (0.01 mol / L, pH 7.4) to a final volume of 1000 ml. Mix well and filter through a 0.22 μm filter. Aseptically aliquot the mixture into a negative control and store at 2–8 °C.

[0117] Sample dilution solution: Take 8g sodium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 0.2g potassium chloride, 600ml purified water, 1ml Proclin 300, and 200ml bovine serum. Dissolve and bring the volume to 1000ml with purified water. Mix well and filter through a 0.22μm filter. Aseptically dispense and store at 2–8℃.

[0118] 20× Concentrated Washing Solution: Dissolve 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, and 10ml Tween 20 in purified water and bring the volume to 1000ml. Mix well and filter through a 0.22μm filter. Aseptically dispense. Dilute 20 times with purified water before use.

[0119] Colorimetric reagent A: Dissolve 14.7g of disodium hydrogen phosphate, 9.3g of citric acid, and 0.3g of urea peroxide in purified water, bring the volume to 1000ml, mix well, and aseptically dispense. Dissolve 0.2g of tetramethylbenzidine (TMB) and 10ml of anhydrous ethanol in purified water, bring the volume to 1000ml, mix well, and aseptically dispense.

[0120] Termination solution: 10% hydrochloric acid.

[0121] Assemble the above components into a kit.

[0122] 3.3 Establishment of Detection Methods

[0123] The testing steps are as follows:

[0124] (1) Numbering: Number the microplates corresponding to the samples in sequence. Each plate should have 2 negative control (NC) wells, 2 positive control (PC) wells and 1 blank control well (blank control wells are not required when using dual-wavelength detection).

[0125] (2) Add sample diluent: Add 50µl of sample diluent to each well, except for negative control wells, positive control wells and blank control wells.

[0126] (3) Sample addition: Add 50µl of the sample to be tested and 100µl of the negative and positive controls to the corresponding wells, except for the blank control wells. Gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 45 minutes.

[0127] (4) Washing: Wash 3 times with detergent, and dry after the last wash.

[0128] (5) Add enzyme-labeled reagent: Add 100 μl of enzyme-labeled reagent to each well except for the blank control well; seal the plate with sealing film and incubate at 37°C for 30 minutes.

[0129] (6) Washing: Wash 3 times with detergent, and dry after the last wash.

[0130] (7) Color development: Add 50µl of color developer A and B solution to each well in sequence, mix well, and incubate at 37℃ in the dark for 15 minutes.

[0131] (8) Termination: Add 50µl of stop solution to each well, gently shake to mix, and measure the results with an enzyme-linked immunosorbent assay (ELISA) reader within 10 minutes.

[0132] (9) Measurement:

[0133] Dual-wavelength measurement (recommended): Set dual wavelengths to 450nm / 600~650nm and measure the A value of each well. OD 450nm Subtract OD 600～650nm The A value is for the sample or negative / positive control.

[0134] For single-wavelength assays, the OD value of each well was measured by setting the microplate reader to 450 nm. The A value of the sample or negative / positive control was obtained by subtracting the OD value of the blank control well.

[0135] (10) Result determination: Calculate the S / N of the sample = sample A value / mean A value of negative control. When S / N≤0.5, it is judged as positive for ASFV E165R protein antibody; when S / N>0.5, it is judged as negative for ASFV E165R protein antibody.

[0136] 3.4 Evaluation of the ASFV E165R protein blocking ELISA antibody detection kit

[0137] 3.4.1 Sensitivity

[0138] The kit prepared in Example 3.2 was used to detect serially diluted porcine E165R protein-positive serum. The results showed that the kit was positive for porcine E165R protein-positive serum at dilutions from 1 to 16 times, and negative for dilutions from 32 to 256 times. The detection limit was 16 times dilution.

[0139] 3.4.2 Specificity

[0140] The kit prepared in Example 3.2 was used to test 8 positive serum samples of common porcine viruses (including positive serum for porcine pseudorabies, porcine reproductive and respiratory syndrome, classical swine fever, porcine circovirus type 2, porcine epidemic diarrhea, porcine transmissible gastroenteritis, porcine rotavirus disease, and foot-and-mouth disease type O), 10 SPF porcine serum samples, 10 newborn swine serum samples that had not consumed colostrum, and 50 serum samples of conventionally vaccinated swine that were negative for ASFV antigen. The results showed that the S / N values ​​of all tested samples were >0.5, indicating that all samples were negative, demonstrating that the kit had good specificity.

[0141] 3.4.3 Repeatability

[0142] Three batches of reagent kits were prepared according to the preparation method in Example 3.2. Each batch was tested five times with a 10-fold dilution of porcine E165R protein-positive serum. Intra-batch and inter-batch repeatability were calculated. The results showed that the intra-batch and inter-batch repeatability were both no higher than 8%, indicating good repeatability.

[0143] 3.4.4 Fluctuation of Serum Detection

[0144] The kit prepared in Example 3.2 was used to detect porcine E165R protein immune serum. All serum samples turned positive one week after the second immunization, indicating good sensitivity.

[0145] 3.4.5 Clinical Application

[0146] Based on the above results, the kit prepared in Example 3.2 was used for clinical application to detect 20 ASF-positive swine serum samples and 1000 negative swine serum samples collected before 2018. The results showed that the S / N values ​​of the 20 ASF-positive swine serum samples were all ≤0.5, indicating that they were all positive; the S / N values ​​of the 1000 negative swine serum samples collected before 2018 were >0.5, indicating that they were all negative.

[0147] Example 4: Sequencing of the 1C10 variable region of the ASFV E165R protein monoclonal antibody.

[0148] Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed:

[0149] F: 5'-ACTAGTCGACATGAGAGTGCTGATT-3' (SEQ ID NO: 5).

[0150] R: 5'- CCAGGGRCCARKGGATARACN-3' (SEQ ID NO: 6).

[0151] Design primer sequences for the light chain variable region:

[0152] F: 5'-ACTAGTCGACATGGGCWTCAAGAT-3' (SEQ ID NO: 7).

[0153] R: 5'- CCCAAGCTTACTGGATGGTG-3' (SEQ ID NO: 8).

[0154] In the primer sequences above, "R" represents the base "A or G", "K" represents the base "G or T", "N" represents the base "A or T or G or C", and "W" represents the base "A or T".

[0155] Hybridoma cells were collected, RNA was extracted and reverse transcribed to serve as a template, and the variable region sequence was amplified using the primers described above. The amplified product was cloned, and positive clones were screened and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The results showed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1C10 were as shown in SEQ ID No: 1 and SEQ ID No: 3, respectively, and the gene sequences were as shown in SEQ ID No: 2 and SEQ ID No: 4, respectively.

[0156] Example 5: Preparation and Identification of Single-Chain Antibody 1C10

[0157] The heavy chain variable region (VH) gene and light chain variable region (VL) gene of the monoclonal antibody were amplified, and after being transferred into a linker peptide, they were ligated into the prokaryotic expression vector pET-32a(+) to construct a recombinant plasmid. This plasmid was then transformed into BL21 competent cells for expression to obtain the fusion protein. The corresponding single-chain antibody 1C10 was prepared from the variable region sequence of the monoclonal antibody 1C10 obtained according to the method described in Example 4. A positive serum blocking assay was performed on the single-chain antibody 1C10 according to the method in Example 2. The results showed that the blocking rate of the single-chain antibody 1C10 was >80%, which was satisfactory, indicating that the prepared single-chain antibody had good blocking activity. The ELISA titer of 1C10 was determined according to the method in Example 2. The results showed that the ELISA titer of the single-chain antibody 1C10 was 1:512000, indicating good reactivity. IFA testing was performed using an ASFV antigen plate purchased from the European African Swine Fever Reference Laboratory (Centro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain). The result was positive, indicating that the single-chain antibody 1C10 can recognize ASFV.

[0158] The results above show that the variable region sequences shown in SEQ ID No: 1, SEQ ID No: 3 or SEQ ID No: 2, SEQ ID No: 4 can be used for the preparation of genetically engineered antibodies against African swine fever virus.

[0159] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0160] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus E165R protein, characterized in that, The antibody or its antigen-binding fragment comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

3.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is Fab, Fab', F(ab')2, scFv, or Fv.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody is a monoclonal antibody 1C10, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a single-chain antibody, wherein the heavy chain variable region and the light chain variable region are linked by a linker peptide.

5. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, characterized in that, The biomaterial is any one of the following: (a) A nucleic acid molecule containing a sequence encoding a heavy chain variable region and / or a light chain variable region encoding the antibody or an antigen-binding fragment thereof; (b) Expression cassette containing the nucleic acid molecules in (a); (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c); (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

6. The biomaterial according to claim 5, characterized in that, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 2; and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

4.

7. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, or the biological material as described in claim 5 or 6, in the preparation of African swine fever virus detection products; Optionally, the product is a blocking ELISA antibody detection kit.

8. An African swine fever virus blocking ELISA antibody detection kit, characterized in that, The kit comprises: a support medium coated with ASFV E165R protein, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

9. The reagent kit according to claim 8, characterized in that, The coating concentration of ASFV E165R protein was 0.5-1.0 μg / mL; Optionally, the supporting medium is a microtiter plate; Optionally, the enzyme labeled with the enzyme may be horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

10. The kit according to claim 8 or 9, characterized in that, The test reagents include a colorimetric solution and a stop solution. The colorimetric solution includes colorimetric solution A and colorimetric solution B. Colorimetric solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and colorimetric solution B contains 0.02% w / v tetramethylbenzidine and 1% v / v anhydrous ethanol. Optionally, the stop solution is a 10% hydrochloric acid solution; Optionally, the kit may also include at least one of a positive control, a negative control, a washing solution, and a sample diluent; Optionally, positive controls are positive serum from pigs immunized with 5% v / v E165R protein, PBS solution containing 20% ​​v / v bovine serum and 0.1% v / v Proclin 300; Optionally, the negative control is a PBS solution containing 5% v / v porcine negative serum, 20% v / v bovine serum and 0.1% v / v Proclin 300; Optionally, the sample diluent is a PBS solution containing 20% ​​v / v newborn calf serum and 0.1% v / v Proclin 300.

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