African swine fever virus CD2v protein-specific antibody and its application

Abstract

The present invention discloses an antibody specific for African swine fever virus CD2v protein and an application thereof. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown as SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively. The antibody can specifically bind to the African swine fever virus CD2v protein and has good binding activity, and can be used for the early diagnosis and screening of African swine fever virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to an antibody specific to the CD2v protein of African swine fever virus and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute viral hemorrhagic disease caused by African swine fever virus (ASFV) infecting pigs. It is the only known double-stranded DNA arbovirus. Persistently infected wild boars and soft ticks are important sources of infection for African swine fever. Clinically, it is characterized by high fever, systemic hemorrhage and high mortality. Domestic pigs and wild boars of all ages are susceptible, and the mortality rate is as high as 100% in the case of infection with strong strains. African swine fever is listed as a notifiable animal disease by the World Organization for Animal Health and is a Class I animal disease in my country. There is currently no recognized effective vaccine or treatment. Therefore, detecting the presence of ASFV is crucial to the prevention and control of African swine fever.

[0003] The CD2v protein encoded by the ASFV EP204R gene is a late-expressed protein and an important viral glycosylated protein, consisting of an N-terminal signal peptide, an extracellular region, a transmembrane region, and a C-terminal cytoplasmic tail. It has been found to exist in the viral envelope and is involved in cell-to-cell adhesion, virulence enhancement, and immune response regulation. The two immunoglobulin-like domains in the extracellular region have a high homology with mammalian CD2, which is involved in cell-to-cell adhesion and T-cell-mediated immune responses, while the intracellular region has no similarity. In addition, CD2v is also involved in ASFV immune evasion and adsorption of red blood cells after infection, which can destroy lymphocyte function and play an important role in ASFV virus invasion and spread.

[0004] ASFV is a complex DNA virus that has a significant impact on the global breeding industry. Due to the lack of safe and effective vaccines, the only way to prevent the spread of the disease and cause significant economic losses is to cull the pig herd. Therefore, the development of a sensitive, rapid, simple to operate, highly specific and sensitive early ASFV diagnostic method is of great significance for early detection of infected pigs and monitoring of African swine fever disease to reduce economic losses in the breeding industry. In view of this, the purpose of the present invention is to provide an African swine fever virus CD2v protein-specific antibody, which has good binding activity to the African swine fever virus CD2v protein and can be used for early detection and screening of African swine fever virus infection. Summary of the invention

[0005] The object of the present invention is to provide an antibody specific for the CD2v protein of African swine fever virus, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively. The antibody can specifically bind to the CD2v protein of African swine fever virus. The present invention provides a tool for early detection and screening of African swine fever virus infection in the art.

[0006] The present invention adopts the following technical solutions to achieve the above purpose:

[0007] In a first aspect, the present invention provides an antibody specific for the CD2v protein of African swine fever virus.

[0008] Further, the antibody comprises complementary determining regions CDR1, CDR2, CDR3 of the heavy chain variable region and complementary determining regions CDR1, CDR2, CDR3 of the light chain variable region;

[0009] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0010] The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9;

[0012] The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:17.

[0013] Further, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR4, and the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4;

[0014] The amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively;

[0015] The amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0016] In the present invention, the antibody also includes an antibody corresponding to an amino acid sequence having at least 90% homology with the amino acid sequence as described above. In a specific embodiment of the present invention, the antibody comprises:

[0017] (1) a heavy chain variable region sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO:9;

[0018] (2) a light chain variable region sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 17; or

[0019] (3) The heavy chain variable region sequence described in (1) and the light chain variable region sequence described in (2).

[0020] In the present invention, the antibodies include not only the antibodies as described above, but also the functional variants of the antibodies as described above. If the variant can compete with the parent antibody for specific binding to the CD2v protein, the variant molecule is considered to be a functional variant of the antibody of the present invention. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence but contain in vitro or in vivo chemical and / or biochemical modifications that are not found in the parent antibody, for example. Such modifications include acetylation, phthalidation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation, and the like. In other words, the modification in the amino acid and / or nucleotide sequence of the parent antibody does not significantly affect or change the binding properties of the antibody encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the antibody can still recognize and bind to its target. The functional variants of the antibody are also included in the protection scope of the present invention.

[0021] Further, the functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications may be introduced by standard techniques known in the art, such as directed mutagenesis and random PCR-mediated mutagenesis, and may include natural and non-natural nucleotides and amino acids. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue with similar structure or chemical properties. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chain amino acids (e.g., aspartic acid, glutamic acid), uncharged polar side chain amino acids (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chain amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chain amino acids (e.g., threonine, valine, isoleucine) and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan). Those skilled in the art will appreciate that other amino acid residue family classifications other than the above families may also be used. In addition, variants may have non-conservative amino acid substitutions, such as amino acids replaced by another amino acid residue having a different structure or chemical property. Similar minor variations may also include amino acid deletions and / or insertions. Computer programs well known in the art may be used to find and determine which amino acid residues may be substituted, inserted or deleted without eliminating immunologically active guidance.

[0022] In the present invention, the term "homology" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions that can be compared in each sequence. When the position in the compared sequence is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 90% homology" is about 90% homology, about 91% homology, about 92% homology, about 93% homology, about 94% homology, about 95% homology, about 96% homology, about 97% homology, about 98% homology, about 99% homology, about 99.9% homology, or a range (including endpoints) between any two of these values ​​or any value therein.

[0023] In a second aspect, the present invention provides a nucleic acid molecule.

[0024] Furthermore, the nucleic acid molecule encodes the antibody described in the first aspect of the present invention.

[0025] In the present invention, the nucleic acid molecule can be DNA or RNA. The DNA comprising the nucleotide sequence encoding the antibody described herein generally comprises a promoter operably connected to the nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in the expression cell of interest. The nucleic acid can also include a selective marker that can be used to select cells containing the nucleic acid of interest. Useful selective markers are well known to those skilled in the art. The precise nucleotide sequence of the nucleic acid is not particularly limited, as long as the nucleotide sequence encodes the antibody described herein. Codons can be selected, for example, to match the codon preference of the expression cell of interest (e.g., mammalian cells, such as human cells) and / or for convenience during cloning. The DNA can be a plasmid, for example, and the plasmid can include a replication origin (e.g., for replication of the plasmid in prokaryotic cells).

[0026] In a third aspect, the present invention provides an expression vector.

[0027] Further, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention;

[0028] Preferably, the nucleic acid molecule in the expression vector is operably linked to a promoter;

[0029] More preferably, the promoter includes tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, SV40 promoter, CMV promoter, MMTV promoter;

[0030] More preferably, the vector comprises a plasmid vector, a cosmid vector, or a viral vector;

[0031] Most preferably, the viral vector comprises a bacteriophage vector, an adenoviral vector, a retroviral vector, or an adeno-associated viral vector.

[0032] In a fourth aspect, the present invention provides a host cell.

[0033] Further, the host cell comprises the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention;

[0034] Preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0035] More preferably, the host cell is a eukaryotic cell;

[0036] Most preferably, the eukaryotic cells include mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algal host cells, nematode host cells, protozoan host cells, and fish host cells.

[0037] In some embodiments, the host cell includes but is not limited to: prokaryotic cells, fungal cells, yeast cells or higher eukaryotic cells, such as mammalian cells. In other embodiments, the host cell can be a prokaryotic cell (e.g., Escherichia coli). In other embodiments, the host cell can also be a eukaryotic cell (e.g., yeast cell, e.g., COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell or myeloma cell). In other embodiments, the host cell can be a yeast cell. In other embodiments, the host cell can be a mammalian cell, for example, the mammalian cell can be a CHO-K1 cell.

[0038] In a fifth aspect, the present invention provides a reagent for detecting African swine fever virus.

[0039] Furthermore, the reagent comprises the antibody described in the first aspect of the present invention.

[0040] Furthermore, the antibody is an antibody labeled with a detectable marker.

[0041] Furthermore, the detectable label includes an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label or a magnetic label.

[0042] Enzyme labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and luciferase. Substrates for these enzymes are well known in the art. Suitable substrates for detection include diaminobenzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art. Fluorescent labels, for example, include 5-carboxyfluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, and Cy5, fluorescent proteins such as GFP (green fluorescent protein), Texas Red, and Alexa dyes. Radioactive labels, for example, include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus. Radioactive labels can be detected by any known and appropriate method, such as photographic film or phosphorimager. Magnetic labels, for example, include paramagnetic and superparamagnetic labels. Chemiluminescent labels used may include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts or oxalate esters.

[0043] In a sixth aspect, the present invention provides a product for detecting African swine fever virus.

[0044] Further, the product comprises the antibody described in the first aspect of the present invention;

[0045] Preferably, the product comprises a kit;

[0046] More preferably, the kit includes an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit, a colloidal gold immunoassay kit, a chemiluminescent immunoassay kit, and a radioimmunoassay kit.

[0047] In a seventh aspect, the present invention provides a pharmaceutical composition for treating African swine fever virus infectious diseases.

[0048] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the antibody described in the first aspect of the present invention.

[0049] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0050] Further, the one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients include, but are not limited to: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); preservatives; and any other pharmaceutically or physiologically acceptable carriers, diluents or excipients reported in any prior art that can be used in pharmaceutical compositions.

[0051] In the present invention, the term "effective amount" refers to the amount required to produce a physiological change in the subject to which it is administered. The "therapeutically effective amount" of the antibody described in the first aspect of the present invention refers to the amount that effectively achieves the desired treatment or prevention result at the necessary dosage and time period. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0052] In the present invention, the term "treatment" refers to a clinical intervention that attempts to change the natural course of the treated individual, and can be performed for prevention or in the course of clinical pathology. The desired effects of treatment include, but are not limited to: preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the pharmaceutical composition of the present invention is used to delay the development of African swine fever virus infection or to slow the progression of African swine fever virus infection.

[0053] In some embodiments, the pharmaceutical composition comprising the antibody described in the first aspect of the present invention can be produced by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. The pharmaceutical composition can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants, which help process the protein into a preparation that can be used pharmaceutically. Suitable formulations depend on the selected route of administration.

[0054] In an eighth aspect, the present invention provides any of the following methods:

[0055] (1) A method for preparing the antibody according to the first aspect of the present invention, the method comprising the following steps: culturing the host cell according to the fourth aspect of the present invention, and isolating the antibody according to the first aspect of the present invention from the culture;

[0056] In some embodiments, the antibodies described in the first aspect of the present invention can be prepared and produced by conventional methods known in the art, such as phage display technology well known in the art. Alternatively, the antibodies described in the first aspect of the present invention can be expressed in other cell lines, and the sequences encoding the antibodies described in the first aspect of the present invention can be used to transform suitable host cells, and then the host cells are cultured and the antibodies are purified. The transformation can be carried out by any method known in the art, for example, the polynucleotides are packaged in viruses (or viral vectors) and the host cells are transduced with viruses (or vectors). The transformation method used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes, and direct microinjection of DNA into the nucleus.

[0057] (2) A method for detecting African swine fever virus in a sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample with the antibody described in the first aspect of the present invention, and detecting the formation of a complex between the antibody and the African swine fever virus CD2v protein;

[0058] (3) A method for specifically inhibiting the activity of CD2v protein for non-diagnostic and non-therapeutic purposes in vitro, the method comprising the following steps: introducing the nucleic acid molecule described in the second aspect of the present invention into a cell of an organism, and inhibiting the activity of CD2v protein by expressing the antibody described in the first aspect of the present invention.

[0059] In the present invention, the term "subject" or "individual" is preferably various domestic pigs and wild boars, and the term "sample" or "sample to be tested" includes tissues, cells and biological fluids separated from the subject, as well as tissues, cells and fluids present in the subject. Biological samples disclosed herein include, for example but not limited to: whole blood, plasma, semen, saliva, tears, urine, feces, sweat, oral cavity, skin, cerebrospinal fluid and hair. Biological samples can also be obtained from the internal organs of the subject.

[0060] In a ninth aspect, the present invention provides any of the following applications:

[0061] (1) Use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the reagent described in the fifth aspect of the present invention in the preparation of a product for detecting or assisting in the detection of African swine fever virus;

[0062] (2) Use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the reagent described in the fifth aspect of the present invention in the preparation of a product for diagnosing or assisting in the diagnosis of African swine fever virus infectious diseases;

[0063] (3) Use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the reagent described in the fifth aspect of the present invention in the preparation of a product for detecting African swine fever virus CD2v protein;

[0064] (4) Use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the pharmaceutical composition described in the seventh aspect of the present invention in the preparation of a drug for treating African swine fever virus infectious diseases.

[0065] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0066] The present invention provides an antibody specific for the CD2v protein of the African swine fever virus. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown as SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively. The antibody can specifically bind to the CD2v protein of the African swine fever virus and has good binding activity. The antibody can be used in the early diagnosis and screening of the African swine fever virus, and plays an important role in the prevention and control of African swine fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the electrophoresis result diagram for detecting monoclonal antibody 4H8;

[0068] Figure 2 This is a graph showing the HPLC results for detecting monoclonal antibody 4H8;

[0069] Figure 3 This is the result of ELISA testing the binding activity of monoclonal antibody 4H8. DETAILED DESCRIPTION

[0070] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0071] Example 1 Screening of monoclonal antibodies

[0072] 1. Recombinant expression of immunogens

[0073] The African swine fever virus CD2v protein sequence was synthesized and constructed into the pEM5.1 vector; the transfection plasmid was extracted; the HEK293 cells were transfected and the cells were cultured for 7 days; the supernatant was harvested, purified by Ni column, and concentrated and replaced with buffer to obtain the recombinant African swine fever virus CD2v protein. The recombinant African swine fever virus CD2v protein sequence was derived from Uniprot. The sequence is shown in SEQ ID NO: 1, see Table 1 below.

[0074] Table 1 Sequence of recombinant African swine fever virus CD2v protein

[0075]

[0076] 2. Immunity

[0077] The first immunization was performed with Freund's complete adjuvant, 100 μg per mouse, intraperitoneally, with a total dose of 0.5 mL / mouse. The second immunization was performed 3 weeks later. The second immunization was performed with Freund's incomplete adjuvant, with a dose of 50 μg / 0.5 mL / mouse. The third immunization was performed 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0078] Take feeder cells and press 10 5 Use 100 μl / well and plate 10 μl of the solution one day before fusion. 5 Mouse immune spleen cells were taken and fused with the prepared myeloma cells using the fusion agent PEG, and then plated into a 96-cell culture plate that had been added with feeder cells, 100 μL / well.

[0079] 3. Screening and cloning of hybridoma cells

[0080] Positive wells were screened by ELISA detection method, and African swine fever virus CD2v protein was plated overnight; the plate was washed, and skimmed milk powder was added for blocking at 37°C for 1 hour; the plate was washed, and 100 μL of 96-well culture supernatant was added, and the culture was incubated at 37°C for 1 hour; the plate was washed, and HRP-labeled goat anti-mouse secondary antibody was added, and the culture was incubated at 37°C for 30 minutes; the plate was washed, and the color developing solution was added, and the color was developed for 10 minutes, and the stop solution was added to read the OD 450 value; the cell lines with high expression were screened for subclone culture.

[0081] 4. Sequencing

[0082] Collect cells, extract total RNA with Trizol, and use oligo(dT)20 as primer to reverse transcribe and generate cDNA. Then use specific primers to PCR amplify the heavy and light chain variable region genes respectively. After electrophoresis purification, the PCR products are inserted into the vector through TA cloning, transformed, and positive clones are selected for sequencing.

[0083] 5. Experimental results

[0084] The anti-CD2v monoclonal antibody 4H8 targeting the CD2v protein of African swine fever virus was screened out, and the sequence is shown in Table 2.

[0085] Table 2 Sequence of monoclonal antibody 4H8

[0086]

[0087]

[0088] Example 2 Functional study of monoclonal antibody 4H8

[0089] 1. Expression and purification of monoclonal antibodies

[0090] 1) Chemically synthesize the selected sequences and clone them into eukaryotic expression vectors.

[0091] 2) Amplify and extract the plasmid.

[0092] 3) The antibody encoding plasmid was transiently transfected into mammalian HEK293 cells.

[0093] 4) Collect the supernatant and purify the monoclonal antibody using affinity chromatography.

[0094] 5) The results showed that the expression level of the purified antibody was 235 mg / L.

[0095] 2. Physical and chemical properties of monoclonal antibodies

[0096] 2.1 Gel electrophoresis to detect the purity of monoclonal antibodies

[0097] 1) Instruments and Equipment

[0098] The instruments and equipment used in the experiment are shown in Table 3.

[0099] Table 3 Instruments and equipment

[0100] name Manufacturer model Chemiluminescence imager Tanon Tanon-5200 Electrophoresis Apparatus BIO-RAD PowerPac Basic Electrophoresis tank BIO-RAD DYC-Mini4

[0101] 2) Main reagents

[0102] The main reagents used in the experiment are shown in Table 4.

[0103] Table 4 Main reagents

[0104]

[0105]

[0106] 3) Sample preparation

[0107] Take 20 μL of sample and mix with 5 μL of 5× reduction buffer, heat at 95°C for 5 min, and cool;

[0108] Take 20 μL of sample and mix with 5 μL of 5× non-reducing buffer.

[0109] 4) Electrophoresis

[0110] Prepare gel, add appropriate amount of electrophoresis buffer, add sample, and perform electrophoresis.

[0111] 5) Dyeing and bleaching

[0112] After the electrophoresis is finished, take the gel and put it into an appropriate amount of Coomassie Brilliant Blue staining solution, and stain it at room temperature for 1 hour or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining decolorizing solution, and decolorize it at room temperature for 4-24 hours. After the decolorization is completed, soak it in ddH2O, refer to the marker protein, compare it with the unstained gel, cut out the gel of the required protein component, and collect it. Then separate the protein to be purified from the gel.

[0113] 6) Experimental results

[0114] The results are as follows Figure 1 As shown, the bands from left to right are marker and reduction bands respectively; the electrophoresis result graph shows that the detection purity of the monoclonal antibody is greater than 95%.

[0115] 2.2 HPLC detection of monoclonal antibody purity

[0116] 1) Instruments and Equipment

[0117] The instruments and equipment used in the experiment are shown in Table 5.

[0118] Table 5 Instruments and equipment

[0119]

[0120] 2) Main reagents

[0121] The main reagents used in the experiment are shown in Table 6.

[0122] Table 6 Main reagents

[0123] name Manufacturer Specification Part Number Potassium Hydrogen Phosphate Trihydrate Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10017592 Potassium dihydrogen phosphate Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10017692 Potassium chloride Sinopharm Chemical Reagent Co., Ltd. 500g / bottle 10016392

[0124] 3) Mobile phase preparation

[0125] Add potassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride to about 900 mL of purified water, stir to dissolve, dilute to 1 L, measure with a pH meter to determine that the pH is between 6.2±0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0126] 4) Sample preparation

[0127] System suitability sample: MIL62 standard diluted to 2 mg / mL with mobile phase;

[0128] Test sample: Dilute the sample to be tested to 2 mg / mL with mobile phase.

[0129] 5) Chromatographic conditions

[0130] The specific chromatographic conditions are shown in Table 7.

[0131] Table 7 Chromatographic conditions

[0132]

[0133] 6) Experimental results

[0134] The results are as follows Figure 2 As shown, the liquid phase detection results showed that the detection purity of the monoclonal antibodies was greater than 95%.

[0135] 3. Binding activity detection of monoclonal antibodies

[0136] 1) Coating: Dilute the antigen CD2v protein to 2 μg / mL with coating solution, mix well, add to 96-well coating plate, 100 μL / well, seal the plate, and incubate at 4°C overnight.

[0137] 2) Wash the plate 3 times in a plate washer. No liquid should remain on the plate during the last wash. Pat the liquid on the surface of the plate dry with absorbent paper.

[0138] 3) Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37° C. for 1 h, and wash the plate 3 times according to step 2).

[0139] 4) Dilute the antibody in a gradient manner, 100 μL / well, react at 37°C for 1 h, and wash the plate 3 times according to step 2).

[0140] 5) Add secondary antibody: dilute with DPBS at 1:2000, add to 96-well plate, 100 μL / well, react at 37°C for 1 hour, and wash the plate 3 times according to step 2).

[0141] 6) Color development: Add TMB, 100 μL / well, and develop color for 10 min at room temperature in the dark.

[0142] 7) Stop: Add 2N H2SO4, 100 μL / well.

[0143] 8) Measure OD450 with an enzyme-labeled instrument within 10 minutes.

[0144] 9) Experimental results

[0145] The results are as follows Figure 3 As shown, the results showed that monoclonal antibody 4H8 could specifically bind to CD2v protein in a concentration-dependent manner, with an EC50 of 0.02916 μg / mL.

[0146] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody specific for the CD2v protein of African swine fever virus, characterized in that: The antibody comprises complementary determining regions CDR1, CDR2, CDR3 of the heavy chain variable region and complementary determining regions CDR1, CDR2, CDR3 of the light chain variable region; The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9; The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

17.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to claim 1 or 2.

4. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 3.

5. The expression vector according to claim 4, characterized in that The nucleic acid molecule in the expression vector is operably linked to a promoter.

6. The expression vector according to claim 5, characterized in that The promoter includes tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, SV40 promoter, CMV promoter or MMTV promoter.

7. The expression vector according to claim 4, characterized in that The vector includes a plasmid vector, a cosmid vector or a viral vector.

8. The expression vector according to claim 7, characterized in that The viral vector includes a phage vector, an adenoviral vector, a retroviral vector or an adeno-associated viral vector.

9. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 3 or the expression vector of any one of claims 4-8.

10. The host cell according to claim 9, characterized in that The host cell includes a eukaryotic cell or a prokaryotic cell.

11. The host cell according to claim 10, characterized in that The host cell is a eukaryotic cell.

12. The host cell according to claim 11, characterized in that The eukaryotic cell includes a mammalian host cell, an insect host cell, a fungal host cell, a eukaryotic algae host cell, a nematode host cell, a protozoan host cell or a fish host cell.

13. A reagent for detecting African swine fever virus, characterized in that: The reagent comprises the antibody according to claim 1 or 2.

14. A product for detecting African swine fever virus, characterized in that: The product comprises the antibody according to claim 1 or 2.

15. The product according to claim 14, characterized in that The products include kits.

16. The product according to claim 15, characterized in that The kit includes an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit, a colloidal gold immunoassay kit, a chemiluminescent immunoassay kit or a radioimmunoassay kit.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to claim 1 or 2.

18. A method for preparing the antibody according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: culturing the host cell according to any one of claims 9 to 12, and isolating the antibody according to claim 1 or 2 from the culture.

19. A method for detecting African swine fever virus in a sample for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises the following steps: contacting the sample to be tested with the antibody according to claim 1 or 2, and detecting the formation of a complex between the antibody and the African swine fever virus CD2v protein.

20. A method for specifically inhibiting CD2v protein activity for non-diagnostic and non-therapeutic purposes in vitro, characterized in that: The method comprises the following steps: introducing the nucleic acid molecule of claim 3 into a cell of an organism, and inhibiting the activity of the CD2v protein by expressing the antibody of claim 1 or 2.

21. Use of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the expression vector of any one of claims 4 to 8, the host cell of any one of claims 9 to 12, or the reagent of claim 13 in the preparation of a product for detecting or assisting in the detection of African swine fever virus.

22. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to any one of claims 4 to 8, the host cell according to any one of claims 9 to 12, or the reagent according to claim 13 in the preparation of a product for diagnosing or assisting in the diagnosis of African swine fever virus infectious diseases.

23. Use of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the expression vector of any one of claims 4 to 8, the host cell of any one of claims 9 to 12, or the reagent of claim 13 in the preparation of a product for detecting African swine fever virus CD2v protein.

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