Seneca virus type A 3A protein antigenic epitope peptide, monoclonal antibody and its application

Abstract

The present invention belongs to the field of biomedicine technology and specifically discloses a type A Senecavirus 3A protein antigenic epitope peptide or its encoding nucleic acid, a monoclonal antibody that specifically binds to the type A Senecavirus 3A protein antigenic epitope, a hybridoma cell line that secretes the monoclonal antibody, and its use in the preparation of a medicament for preventing and / or treating type A Senecavirus infection, or a reagent for detecting and / or diagnosing type A Senecavirus infection. The monoclonal antibody provided by the present invention is secreted by the hybridoma cell line SVA-3A-5A7, deposited with CCTCC NO: C202466. The monoclonal antibody can specifically bind to the type A Senecavirus 3A protein (the antigenic epitope is located at amino acids 5-36 of the 3A protein extracellular domain) and can be used to prepare a medicament for preventing and / or treating type A Senecavirus infection, or a reagent for detecting and / or diagnosing type A Senecavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a type A Seneca virus 3A protein antigenic epitope peptide or its encoded nucleic acid, a monoclonal antibody that specifically binds to the type A Seneca virus 3A protein antigenic epitope, a hybridoma cell line that secretes the monoclonal antibody, and its application in the preparation of drugs for the prevention and / or treatment of type A Seneca virus infection, or reagents for the detection and / or diagnosis of type A Seneca virus infection. Background Technology

[0002] Senecavirus A (SVA) is a non-enveloped, single-stranded RNA virus belonging to the family Picornaviridae and the sole member of the genus Senecavirus. Pigs are the primary susceptible animals. The clinical symptoms of SVA infection are remarkably similar to those of other swine infectious vesicular diseases such as foot-and-mouth disease (FMD), vesicular stomatitis (SVD), and vesicular stomatitis (VS). Infected pigs often exhibit anorexia, lethargy, lameness, and blisters on the muzzle, snout, and coronary band. SVA also possesses oncolytic properties, selectively infecting neuroendocrine tumor cells, showing promise for cancer treatment in early clinical trials. Due to its widespread prevalence and unique natural oncolytic activity, SVA has become increasingly well-known.

[0003] SVA virus particles have an icosahedral structure, are spherical, and have a diameter of 17-25 nm. The SVA genome is approximately 7300 nt in length, consisting of a 6543 nt open reading frame, a 668 nt 5' untranslated region, and a 68 nt 3' untranslated region with a poly(A) tail. The SVA open reading frame encodes approximately 2180 amino acids and has a typical L-4-3-4 genome layout of other piconelasviruses, namely, a leader protein L, intermediate protein P1 (which cleaves into four structural proteins: VP1, VP2, VP3, and VP4), P2 (which cleaves into three non-structural proteins: 2A, 2B, and 2C), and P3 (which cleaves into four non-structural proteins: 3A, 3B, 3C, and 3D). Prediction revealed that protein 3A is a transmembrane protein composed of an extracellular domain, a transmembrane domain, and an intracellular domain. Sequence comparison revealed that the extracellular and transmembrane domain sequences of the 3A protein are relatively conserved, while the intracellular domain sequences show greater variation, which may be related to the interaction between host proteins.

[0004] As one of the non-structural proteins of SVA, research on the 3A protein is currently very limited. However, studies have found that the 3A proteins of other viruses within the Picornaviridae family play important roles. For example, the differences in the intracellular domain sequence of the 3A protein in foot-and-mouth disease may be related to cross-species transmission mechanisms. The membrane anchoring region of the hepatitis A virus 3A protein can bind to the transduction protein MAVS on the outer mitochondrial membrane, leading to the degradation of MAVS by the 3C protein and inhibiting the host's innate immune response. The 3A protein of enterovirus 71 can inhibit the expression of host antiviral response-related proteins by preventing the formation of endoplasmic reticulum transport vesicles. Simultaneously, the 3A protein can bind to and protect long-chain dsRNA from Dicer cleavage, effectively inhibiting the RNAi pathway and thus promoting viral replication. However, the role of the SVA 3A protein in evading the host cell's antiviral innate immune response remains unreported. Therefore, screening for 3A monoclonal antibodies is helpful in studying the biological function of the 3A protein.

[0005] Meanwhile, studies analyzing antibody kinetics in experimental pigs after viral challenge revealed significantly higher antibody levels against the non-structural protein 3AB compared to other non-structural proteins and the structural protein VP1. Furthermore, protein 3A exists not only in its mature form but also as a component of the precursor protein 3AB, and is expressed in both early and late stages of viral infection, exhibiting good immunogenicity. Therefore, SVA antibody detection methods based on protein 3A may have higher sensitivity and potential as a diagnostic target. Summary of the Invention

[0006] The main technical problem solved by this invention is to provide an antigenic epitope peptide of Seneca virus type A 3A protein, which can be used to stimulate and induce the body to produce effector molecules (antibodies) and effector cells, exert an immune effect, and thus kill Seneca virus type A.

[0007] Secondly, this invention provides an application of the type A Seneca virus 3A protein antigenic epitope peptide or its encoded nucleic acid.

[0008] Furthermore, the present invention provides a monoclonal antibody that specifically binds to the antigenic epitope of the Seneca virus type A 3A protein, and a hybridoma cell line that secretes the monoclonal antibody.

[0009] Furthermore, the present invention provides the application of a monoclonal antibody or hybridoma cell line against type A Seneca virus 3A protein.

[0010] Finally, the present invention provides a medicament for preventing and / or treating Seneca virus type A infection, or a reagent for detecting and / or diagnosing Seneca virus type A infection.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0012] An antigenic epitope peptide of type A Seneca virus 3A protein, the antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 1.

[0013] The antigenic epitope provided by this invention is located at amino acids 5-36 of the extracellular domain of the Seneca virus 3A protein (GenBank: QOJ79820.1, amino acid sequence region 1397-1486, 90aa, as shown in SEQ ID NO: 2).

[0014] The nucleic acid encoding an epitope peptide of the Seneca virus type A 3A protein antigen.

[0015] Specifically, the nucleic acid encoded by the antigenic epitope peptide is DNA or RNA. Sequence optimization can be performed based on codon bias without altering the encoded amino acid sequence.

[0016] A biological material containing an antigenic epitope peptide of type A Seneca virus 3A protein or its encoded nucleic acid.

[0017] Specifically, the biological materials include, but are not limited to, recombinant expression vectors (such as plasmid vectors and viral vectors), gene expression cassettes, recombinant bacteria, and host cells (such as prokaryotic cells and eukaryotic cells).

[0018] Application of an antigenic epitope peptide of type A Seneca virus 3A protein or its encoded nucleic acid and biological materials.

[0019] As a preferred embodiment of the present invention, the application includes any one or more of the following:

[0020] (1) Use in the preparation of medicines for the prevention and / or treatment of Seneca virus type A infection;

[0021] (2) Application in the preparation of reagents for the detection and / or diagnosis of Seneca virus type A infection.

[0022] A medicine for the prevention and / or treatment of Seneca virus type A infection, said medicine comprising one or more of the following pharmacologically active ingredients:

[0023] (1) Seneca virus type A 3A protein antigenic epitope peptide;

[0024] (2) The nucleic acid encoding the antigenic epitope peptide of the Seneca virus type A 3A protein;

[0025] (3) Biological materials containing the antigenic epitope peptide of type A Seneca virus 3A protein or its encoded nucleic acid.

[0026] Specifically, the drug is a preventive and / or therapeutic vaccine. The vaccine can be a nucleic acid vaccine, containing the nucleic acid encoding the antigenic epitope peptide of the Seneca A virus 3A protein, such as a DNA vaccine or an RNA vaccine. The vaccine can also be a peptide vaccine, a recombinant protein vaccine, or a synthetic long peptide vaccine, containing the antigenic epitope peptide of the Seneca A virus 3A protein.

[0027] In a preferred embodiment of the present invention, the vaccine further comprises an immunomodulator or adjuvant selected from poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, Montanide ISA. TM One or more of the following: (such as Montanide ISA51).

[0028] A reagent for detecting and / or diagnosing Seneca virus type A infection, said reagent comprising one or more of the following components:

[0029] (1) Seneca virus type A 3A protein antigenic epitope peptide;

[0030] (2) Primers and / or probes for detecting the nucleic acid encoding the antigenic epitope peptide of the Seneca virus type A 3A protein.

[0031] Specifically, the reagent is an immunoassay and / or diagnostic reagent containing an epitope peptide of the Seneca A virus 3A protein, which can be used to detect the presence of antibodies produced after Seneca A virus infection in porcine body fluids.

[0032] Specifically, the reagent is a molecular detection and / or diagnostic reagent, which contains primers, probes, etc. that encode nucleic acids for detecting the antigenic epitope peptide of Seneca virus type A 3A protein, and can be used to detect the presence of Seneca virus type A (i.e., Seneca virus type A 3A protein) in pig body fluids.

[0033] In a preferred embodiment of the present invention, the reagent further comprises pharmaceutically acceptable carriers, excipients, etc.

[0034] Specifically, for immunoassay and / or diagnostic reagents, the reagents may also include one or more of the following: sample diluent, ELISA plate, blocking solution, washing solution, substrate, stop solution, negative control, positive control, etc.

[0035] Specifically, for molecular detection and / or diagnostic reagents, the reagents may also contain one or more of the following: polymerase (RNA- and / or DNA-dependent polymerase), amplification reaction buffer, positive control, negative control, etc.

[0036] A monoclonal antibody against Seneca virus type A 3A protein, wherein the monoclonal antibody is secreted by the hybridoma cell line SVA-3A-5A7, which is deposited at the China Center for Type Culture Collection with accession number CCTCC NO: C202466.

[0037] Specifically, the monoclonal antibody specifically recognizes and binds to the Seneca virus type A 3A protein, particularly amino acids 5-36 of the extracellular domain of the 3A protein.

[0038] Specifically, the monoclonal antibody is of type IgG2b and the light chain type is Kappa.

[0039] A hybridoma cell line for preparing monoclonal antibodies against Seneca virus type A 3A protein. The hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C202466.

[0040] Specifically, the hybridoma cell line was obtained by immunizing mice with Seneca virus type A 3A protein as an immunogen.

[0041] Application of a monoclonal antibody against type A Seneca virus 3A protein and a hybridoma cell line.

[0042] As a preferred embodiment of the present invention, the application includes any one or more of the following:

[0043] (1) Use in the preparation of medicines for the prevention and / or treatment of Seneca virus type A infection;

[0044] (2) Application in the preparation of reagents for the detection and / or diagnosis of Seneca virus type A infection.

[0045] A medicine for the prevention and / or treatment of Seneca virus type A infection, said medicine comprising one or more of the following pharmacologically active ingredients:

[0046] (1) Monoclonal antibody against Seneca virus type A 3A protein;

[0047] (2) Hybridoma cell lines used to prepare monoclonal antibodies against type A Seneca virus 3A protein.

[0048] In a preferred embodiment of the present invention, the dosage form of the drug is pharmaceutically acceptable and includes a pharmaceutically acceptable carrier, excipients and / or adjuvants.

[0049] In a preferred embodiment of the present invention, the drug can be administered topically at a pharmaceutically acceptable dose.

[0050] A reagent for detecting and / or diagnosing Seneca virus type A infection, said reagent comprising one or more of the following components:

[0051] (1) Monoclonal antibody against Seneca virus type A 3A protein;

[0052] (2) Hybridoma cell lines used to prepare monoclonal antibodies against type A Seneca virus 3A protein.

[0053] Specifically, the reagent can be used to detect the presence of type A Seneca virus (i.e., type A Seneca virus 3A protein) in porcine body fluids.

[0054] As a preferred embodiment of the present invention, the reagent further comprises pharmaceutically acceptable carriers, excipients, etc., as described above.

[0055] The beneficial effects of this invention are:

[0056] Based on the sequence of Seneca virus type A 3A protein (GenBank: QOJ79820.1, amino acid sequence region 1397-1486, 90aa) published by NCBI, this invention constructs a prokaryotic expression system to express and purify the 3A protein, and uses it as an immunogen to immunize BALB / c mice to prepare hybridoma cell lines.

[0057] The monoclonal antibody provided by this invention is secreted by the hybridoma cell line SVA-3A-5A7, which is deposited at the China Center for Type Culture Collection (CCTCCNO: C202466). It can specifically bind to the Seneca virus type A 3A protein and can be used to prepare drugs for the prevention and / or treatment of Seneca virus type A infection, or reagents for the detection and / or diagnosis of Seneca virus type A infection.

[0058] This invention, through analysis of the epitope region of the 3A protein, reveals that most regions of the 3A protein possess good antigenicity. The antigenic epitope recognized by the monoclonal antibody 5A7 is located at amino acids 5-36 of the extracellular domain of the 3A protein. The synthesized antigenic epitope peptide can also be used to prepare drugs for the prevention and / or treatment of Seneca virus A infection, or reagents for the detection and / or diagnosis of Seneca virus A infection, demonstrating promising clinical application prospects and high economic value. Attached Figure Description

[0059] Figure 1 The optimal IPTG induction concentration of protein 3A was verified by SDS-PAGE in the experimental example.

[0060] Figure 2 This experiment uses SDS-PAGE to verify the optimal expression mode of the 3A protein.

[0061] Figure 3 The SDS-PAGE test in the experimental example was used to verify the expression level of 3A protein after induction at 37℃ for 6 hours.

[0062] Figure 4 The SDS-PAGE test in the experimental example was used to verify the expression level of 3A protein after induction at 25℃ for 12 h.

[0063] Figure 5 The SDS-PAGE test in the experimental example was used to verify the expression level of 3A protein after induction at 16℃ for 20 h.

[0064] Figure 6 To verify the difference in protein concentration of protein 3A after imidazole elution in the experimental example using SDS-PAGE;

[0065] In the figure, 1: 3A protein before passing through the nickel column; 2: 3A protein flow-through buffer; 3: 20 mM imidazole elution buffer; 4: 40 mM imidazole elution buffer; 5: First ml of elution buffer after 250 mM imidazole elution; 6: Third ml of elution buffer after 250 mM imidazole elution; 7: Fifth ml of elution buffer after 250 mM imidazole elution; 8: Seventh ml of elution buffer after 250 mM imidazole elution; 9: Ninth ml of elution buffer after 250 mM imidazole elution; 10: Eleventh ml of elution buffer after 250 mM imidazole elution; 11: Thirteenth ml of elution buffer after 250 mM imidazole elution; 12: Fifteenth ml of elution buffer after 250 mM imidazole elution.

[0066] Figure 7 In this experimental case, Western blot was used to identify the reactivity of the monoclonal antibody SVA-3A-5A7 with the 3A protein.

[0067] Figure 8 The immunofluorescence assay was used to identify the reactivity of the monoclonal antibody with SVA in the experimental example.

[0068] Figure 9 For the identification of the 5A7 subtype of monoclonal antibody in the experimental example;

[0069] Figure 10 Prediction of the 3A protein antigenic epitope in the experimental example;

[0070] Figure 11 This refers to the reaction between the his antibody and amino acid regions 5-36 and 67-87 of 3A in the experimental example.

[0071] Figure 12 This refers to the reaction between the 5A7 antibody and the amino acid regions at positions 5-36 and 67-87 of 3A in the experimental example.

[0072] To more clearly illustrate the technical solution protected by this invention, the accompanying drawings in the embodiments or experimental examples have been briefly described above. It should be understood that the above drawings should not be considered as any limitation on the scope of protection of this invention. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0073] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples.

[0074] Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solutions and effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Other technical solutions obtained by those skilled in the art based on the following embodiments without creative effort, such as those obtained through modifications, variations, or simple substitutions, are all within the scope of protection of the present invention.

[0075] Unless otherwise specified, the methods used in the examples and experimental cases are conventional methods.

[0076] Unless otherwise specified, the raw materials, reagents, equipment, etc. used in the examples and experimental cases are all commercially available products.

[0077] The preservation information involved in this invention is as follows:

[0078] Preservation Information

[0079] Preservation name: Hybridoma cell line SVA-3A-5A7.

[0080] Accession number: CCTCC NO: C202466.

[0081] Depository: China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China.

[0082] Date of preservation: August 17, 2024.

[0083] Example 1

[0084] This embodiment provides an antigenic epitope peptide of type A Seneca virus 3A protein and its encoded nucleic acid, wherein the antigenic epitope peptide contains the amino acid sequence shown in SEQ ID NO: 1.

[0085] This embodiment also provides a recombinant bacterium or host cell containing the antigenic epitope peptide of type A Seneca virus 3A protein.

[0086] This embodiment also provides a recombinant expression vector (such as a plasmid vector or viral vector) or gene expression cassette that encodes a nucleic acid containing an epitope peptide of the type A Seneca virus 3A protein antigen.

[0087] This embodiment also provides the use of Seneca virus type A 3A protein antigenic epitope peptide, recombinant bacteria or host cells in the preparation of medicaments for the prevention and / or treatment of Seneca virus type A infection.

[0088] This embodiment also provides the application of the nucleic acid encoding the Seneca virus type A 3A protein antigenic epitope peptide, recombinant expression vector, or gene expression cassette in the preparation of reagents for detecting and / or diagnosing Seneca virus type A infection.

[0089] Example 2

[0090] This embodiment provides a drug for preventing Seneca virus type A infection. The drug is an mRNA vaccine containing a pharmaceutically effective amount of mRNA delivered by lipid nanoparticles (LNPs) and an appropriate amount of adjuvant. The mRNA is codon-optimized and encodes an antigenic epitope peptide of the Seneca virus type A 3A protein (amino acid sequence shown in SEQ ID NO: 1).

[0091] This embodiment also provides a drug for treating Seneca virus type A infection, the drug being a synthetic polypeptide vaccine containing a pharmaceutically effective amount of Seneca virus type A 3A protein antigenic epitope peptide (as above) and an appropriate amount of adjuvant.

[0092] This embodiment also provides a reagent for detecting and / or diagnosing Seneca virus type A infection. The reagent is an enzyme-linked immunosorbent assay (ELISA) reagent, containing an epitope peptide of the Seneca virus type A 3A protein antigen, as well as sample diluent, ELISA plate, blocking buffer, washing buffer, substrate, stop solution, negative control, and positive control. This reagent is mainly used to detect the presence of antibodies produced after Seneca virus type A infection in porcine body fluids.

[0093] This embodiment also provides a reagent for detecting and / or diagnosing Seneca virus type A infection. The reagent is a molecular diagnostic reagent containing primers and probes encoding the nucleic acid of the antigenic epitope peptide of the Seneca virus type A 3A protein, as well as a polymerase (RNA- and / or DNA-dependent polymerase), amplification reaction buffer, a positive control, and a negative control. This reagent is mainly used to detect the presence of Seneca virus type A (i.e., Seneca virus type A 3A protein) in porcine body fluids.

[0094] Example 3

[0095] This embodiment provides a monoclonal antibody against Seneca virus type A 3A protein. The monoclonal antibody is secreted by the hybridoma cell line SVA-3A-5A7, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202466. This monoclonal antibody is of the IgG2b type, with a Kappa light chain, and can specifically recognize and bind to the Seneca virus type A 3A protein, particularly amino acids 5-36 of the extracellular domain of the 3A protein.

[0096] This embodiment also provides a hybridoma cell line for preparing monoclonal antibodies against Seneca virus type A 3A protein. The hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C202466. This hybridoma cell line was obtained by immunizing mice with Seneca virus type A 3A protein as an immunogen.

[0097] This embodiment also provides the application of an anti-Seneca virus type A 3A protein monoclonal antibody or hybridoma cell line in the preparation of a drug for the prevention and / or treatment of Seneca virus type A infection.

[0098] This embodiment also provides the application of a monoclonal antibody against Seneca virus type A 3A protein or a hybridoma cell line in the preparation of reagents for detecting and / or diagnosing Seneca virus type A infection.

[0099] Example 4

[0100] This embodiment provides a drug for the prevention and / or treatment of Seneca virus type A infection, the drug comprising a pharmaceutically effective amount of anti-Seneca virus type A 3A protein monoclonal antibody (same as in Embodiment 3), and an appropriate amount of adjuvant.

[0101] This embodiment also provides a reagent for detecting and / or diagnosing Seneca virus type A infection. The reagent is an enzyme-linked immunosorbent assay (ELISA) reagent containing a monoclonal antibody against Seneca virus type A 3A protein, as well as sample diluent, ELISA plate, blocking buffer, washing buffer, substrate, stop solution, negative control, and positive control. This reagent is mainly used to detect the presence of Seneca virus type A (i.e., Seneca virus type A 3A protein) in porcine body fluids.

[0102] In other embodiments of the present invention, the medicament further comprises other pharmacologically active ingredients for combined or adjunctive prevention and / or treatment of Seneca virus type A infection.

[0103] In other embodiments of the present invention, the dosage form of the drug is other pharmaceutically acceptable dosage forms, such as capsules, tablets, etc., which contain other pharmaceutically acceptable carriers and excipients.

[0104] In other embodiments of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage, and the dosage of the drug is determined according to the condition and can be followed as prescribed by a doctor.

[0105] Experimental Example

[0106] I. Experimental Materials

[0107] The expression strain containing the recombinant plasmid PET-28a-SVA-3A was preserved in the laboratory of Henan Agricultural University.

[0108] BALB / c mice were purchased from the Henan Experimental Animal Center.

[0109] 0.01M PBS: Weigh 8g sodium chloride, 0.2g potassium chloride, 3.63g disodium hydrogen phosphate dodecahydrate, and 0.24g potassium dihydrogen phosphate, dissolve them in deionized water, bring the volume to 1L, and autoclave for later use.

[0110] 8M urea: Weigh 480g of urea and dissolve it in 0.01M PBS, then bring the volume to 1L.

[0111] LB liquid medium: Weigh 2g Tryptone, 1g Yeast Extract, and 2g sodium chloride, place them in a 500mL Erlenmeyer flask, add 200mL of single-distilled water, and after complete dissolution, adjust the pH to 7.0. After autoclaving, store at 4℃ for later use.

[0112] II. Experimental Methods

[0113] 1. Preparation of SAV 3A protein

[0114] 1.1 Screening of IPTG Concentration as an Inducer

[0115] Take one frozen pET-28a-3A expression strain and inoculate it into 5 mL of K. + The culture was incubated in 50 mg / mL liquid LB medium at 37°C on an air shaker. When the OD600nm value of the bacterial culture was between 0.6 and 0.8, it was added to 5 groups of 5 mL of K-containing medium at a volume ratio of 1:100. + The mother bacteria were stored at 4°C for a short period in 50 mg / mL liquid LB medium for subsequent experiments. Five groups of bacterial suspensions were then cultured on an air shaker. When the OD600nm value of the bacterial suspensions was between 0.6 and 0.8, IPTG was added to the five groups of bacterial suspensions at final concentrations of 0, 0.3, 0.5, 0.8, and 1 mM, respectively, and expression was induced for another 6 h. After induction, the bacterial cells were enriched by centrifugation at 12000 rpm for 2 min. The supernatant was discarded, and the cells were resuspended in 250 μL of PBS. 10 μL of the resuspended cells were then used to prepare an SDS-PAGE sample for analysis. The results are as follows: Figure 1 As shown.

[0116] 1.2 Determination of protein expression in supernatant and precipitate

[0117] The preserved bacterial culture was added to 5 mL of antibiotic-free LB medium at a volume ratio of 1:100 and activated for 1 h. After activation, kanamycin was added to a final concentration of 50 g / mL, and the culture was then incubated on an air shaker until the OD600nm value was between 0.6 and 0.8. IPTG, the inducer, was added to a final concentration of 0.5 mM, and expression was induced for another 6 h. After induction, the bacterial culture was centrifuged at 12000 rpm for 2 min to enrich the bacterial cells. The supernatant was discarded, and the cells were resuspended in 250 μL of PBS. The ultrasonic homogenizer was set to the following parameters: at 0℃, ultrasonication was performed for 1 second on, 1 second off, for a total of 15 min. After ultrasonication, the cells were centrifuged at 9000 rpm for 15 min, and the inclusion bodies were dissolved in 250 μL of 8M urea. 10 μL of each solution was taken from the supernatant and inclusion body solution to prepare SDS-PAGE samples for analysis. The results are as follows: Figure 2 As shown.

[0118] 1.3 Screening for optimal temperature and time for induction expression

[0119] The preserved bacterial culture was added to 5 mL of antibiotic-free LB medium in three groups at a volume ratio of 1:100 and activated for 1 h. After activation, kanamycin was added to a final concentration of 50 μg / mL, and the cultures were then incubated on an air shaker until the OD600nm value was between 0.6 and 0.8. IPTG was added to each group to a final concentration of 0.5 mM, and expression was induced at 37℃ for 6 h, 25℃ for 12 h, and 16℃ for 20 h, respectively. After induction, the bacterial culture was centrifuged at 12000 rpm for 2 min, the supernatant was discarded, and the enriched bacterial cells were resuspended in 250 μL of PBS. The ultrasonic homogenizer was set to the following parameters: at 0℃, ultrasonication was performed for 1 second on, 1 second off, for a total of 15 min. After ultrasonication, the culture was centrifuged at 9000 rpm for 15 min, and the inclusion bodies were dissolved in 250 μL of 8M urea. 10 μL of each solution was taken from the supernatant and inclusion body solution to prepare SDS-PAGE samples for analysis. The results are as follows: Figure 3-5 As shown.

[0120] 1.4 Ni-Agarose Resin Chromatographic Purification

[0121] Ni-Agarose Resin packing material was mixed and added to the chromatography column. The packing material was washed three times with PBS before use. The nickel column was equilibrated with TBS containing 10 mM imidazole. The supernatant from the disrupted bacteria was then mixed with the packing material by pipetting, transferred to a centrifuge tube, and inverted for 3 hours at 4°C to ensure complete protein binding. The mixed protein was added to the chromatography column, and 10 mL of 20 mM and 40 mM imidazole were added to wash away impurities. The target protein was then eluted with 15 mL of 250 mM imidazole, and the eluent was collected in 1 mL increments. The nickel column was rinsed with distilled water and stored in 20% anhydrous ethanol. The eluent was placed in a dialysis bag with an 8000D pore size and dialyzed overnight at 4°C in 0.01 M PBS. Protein concentration was determined, aliquoted, and stored at -80°C. SDS-PAGE samples were prepared from the 10 mM, 20 mM, and 250 mM imidazole eluents and analyzed by SDS-PAGE. The results are as follows: Figure 6 As shown.

[0122] 2. Preparation of monoclonal antibodies

[0123] 2.1 Screening of positive hybridoma cell lines

[0124] Purified SVA 3A protein was injected subcutaneously into BALB / c mice at a dose of 50 μg / mouse via multiple injection sites. After multiple immunizations, the serum titer of the immunized mice was measured using an indirect ELISA method with SVA. When the titer reached the cell fusion threshold, mouse spleen cells were fused with mouse myeloma cells (SP2 / 0). Cells that did not fuse successfully were screened using HAT selective medium. After 5-7 days, the cell clusters grew to a suitable size, and the medium was changed. Two to three days later, the cell culture medium was extracted, and indirect ELISA was performed using an ELISA plate coated with 3A protein. Preliminary results showed that the cells in two wells of the cell culture plate could produce antibodies that could react with the 3A prokaryotic expression protein. Subsequently, specific cell lines were screened using an ELISA plate coated with 3A protein, SVA virus solution, and pET-28a irrelevant protein expressed by the same E. coli expression system. Cells were counted using a limiting dilution method, and 200 cells were used for subcloning. The presence of target cells in the cell wells was determined using an indirect ELISA method. After repeating this process multiple times, the selected cell lines were finally amplified and cryopreserved. Cell supernatant was used to verify the interaction between the cell lines and the 3A protein and SVA virus. The results are as follows: Figure 7-8 As shown.

[0125] 2.2 Hybridoma cell culture and preparation of monoclonal antibody ascites

[0126] SVA-5A7 hybridoma cells were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cells were washed with serum-free hybridoma culture medium and cultured in a T75 culture flask with hybridoma culture medium containing 1% HT. The cells were maintained in good condition until they reached a plump and translucent state (i.e., the optimal state).

[0127] Healthy BALB / c mice were selected and injected intraperitoneally with 500 μL of liquid paraffin one week prior to cell injection. Hybridoma cells were washed with serum-free DMEM and then resuspended in 500 μL of serum-free DMEM at a concentration of 1 × 10⁶ cells / mL. 6 Hybridoma cells were collected, and ascites was extracted from mice when their abdomens began to swell. The ascites was centrifuged at 12000 rpm for 5 min, discarding the uppermost layer of oil and the bottom precipitate. The fluid was then filtered through a 0.22 μm filter before use. Antibody subtype identification was performed using an antibody subtype identification kit, and the results are as follows: Figure 9 As shown.

[0128] 2.3 Prediction and Identification of SVA 3A Protein Epitopes

[0129] The 3A protein was analyzed using an antigenic epitope prediction website. The cutoff position for the 3A protein was selected based on the distribution of antigenic epitope regions, such as... Figure 10 As shown. The truncated protein base sequence was ligated into the pET-28a plasmid, and the truncated protein was obtained by expression in E. coli BL21(DE3) expression bacteria. The reactivity between the monoclonal antibody SVA-3A-5A7 and the 3A truncated protein was then identified by Western blot. The results are shown below. Figure 11-12 As shown.

[0130] III. Experimental Results

[0131] 1. Expression and purification of 3A protein

[0132] 1.1 Determination of the optimal concentration of the inducer IPTG

[0133] like Figure 1 As shown, when the IPTG concentration is in the range of 0.3-0.8 mM, the expression level of protein 3A in the supernatant is relatively high, and the optimal concentration of IPTG was finally determined to be 0.5 mM.

[0134] 1.2 Determination of protein supernatant expression and precipitation expression mode

[0135] like Figure 2 As shown, the expression level of protein 3A in the supernatant was high after SDS-PAGE Coomassie Brilliant Blue staining.

[0136] 1.3 Screening for optimal temperature and time for induction expression

[0137] like Figure 3-5As shown, the expression level of 3A protein was highest after 6 hours of induction at 37℃.

[0138] 1.4 Ni-Agarose Resin Chromatographic Purification

[0139] like Figure 6 As shown, SDS-PAGE with Coomassie Brilliant Blue staining revealed that proteins bound to the nickel column were competitively eluted with 250 mM imidazole, and high concentrations of the target protein were found in the first to fifteenth milliliters of elution buffer.

[0140] 2. Preparation of 3A monoclonal antibodies

[0141] 2.1 Screening of positive hybridoma cell lines

[0142] like Figure 7 As shown, Western blot analysis confirmed that the antibody produced by the selected cell line specifically reacted with the purified 3A protein, resulting in a clear band. However, no band was observed when incubated with the pET-28a blank vector expressing the protein in prokaryotes. This indicates that the monoclonal antibody produced by the selected cell line has good specificity, can recognize the 3A protein, and exhibits good reactivity with it.

[0143] like Figure 8 As shown, the antibodies produced by the screened cell lines were verified by immunofluorescence assay (IFA) to react with SVA-infected IBRS-2 cells, producing fluorescence in the cells, while uninfected IBRS-2 cells did not show fluorescence. This indicates that the monoclonal antibodies produced by the screened cell lines have good reactivity with the SVA virus.

[0144] The final selected cell line was named SVA-3A-5A7. A portion of the cell line was sent to the China Center for Type Culture Collection (CCTCC) at Wuhan University for preservation using patent procedures. The preservation name was Hybridoma cell line SVA-3A-5A7, and the preservation number was CCTCC NO: C202466.

[0145] 2.2 Monoclonal antibodies produced by hybridoma cell line SVA-3A-5A7

[0146] like Figure 9 As shown, the results of monoclonal antibody subtype identification indicate that monoclonal antibody 5A7 is of type IgG2b, and the light chain type is Kappa.

[0147] 2.3 Prediction and Identification of 3A Protein Epitopes

[0148] The 3A protein is composed of an extracellular domain, a transmembrane domain, and an intracellular domain, such as... Figure 10As shown, analysis using an antigen epitope prediction website suggests that monoclonal antibody 5A7 may primarily recognize amino acid regions at positions 5-36 and 67-87 of the 3A protein.

[0149] like Figure 11 As shown, amino acid regions 5-36 and 67-87 of protein 3A were normally expressed using a prokaryotic expression system.

[0150] like Figure 12 As shown, the identification results indicate that the 5A7 monoclonal antibody reacts with the amino acid region from position 5 to 36 of the 3A protein.

[0151] IV. Conclusion

[0152] Since its discovery in 2002, SVA has gradually shown a global prevalence. The clinical symptoms of this disease are extremely similar to those of foot-and-mouth disease (FMD), a Category A highly contagious animal disease classified by the World Organisation for Animal Health (OIE), impacting the global swine trade. Simultaneously, due to its oncolytic properties, it is well-known in cancer treatment. However, the pathogenic mechanism of SVA remains poorly understood, severely hindering the development of highly effective SVA control products and oncolytic agents. The SVA 3A protein plays a crucial role in resisting host innate immunity and viral replication. Research on the 3A protein will contribute to a deeper understanding of the biological characteristics of SVA.

[0153] This invention obtained a large quantity and purification of type A Seneca virus 3A protein via prokaryotic expression and prepared a monoclonal antibody against the type A Seneca virus 3A protein. This provides a material basis for future research on the mechanism of the 3A protein, the establishment of clinical serological detection methods for Seneca virus, and the screening of clinical therapeutic drugs.

[0154] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A type A Seneca virus 3A protein antigenic epitope peptide, characterized in that: The amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the antigenic epitope peptide of the type A Seneca virus 3A protein as described in claim 1.

3. The application of the type A Seneca virus 3A protein antigenic epitope peptide as described in claim 1 or the nucleic acid encoded as described in claim 2, characterized in that: The applications include any one or more of the following: (1) Use in the preparation of medicaments for the prevention and / or treatment of Seneca virus type A infection; (2) Application in the preparation of reagents for the detection and / or diagnosis of Seneca virus type A infection.

4. A drug for preventing and / or treating Seneca virus type A infection or a reagent for detecting and / or diagnosing Seneca virus type A infection, characterized in that: The drug or reagent contains one or more of the following components: (1) The type A Seneca virus 3A protein antigenic epitope peptide as described in claim 1; (2) The nucleic acid encoded by the antigenic epitope peptide of type A Seneca virus 3A protein as described in claim 2.

5. A monoclonal antibody against Seneca virus type A 3A protein, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line SVA-3A-5A7, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202466; the monoclonal antibody specifically recognizes and binds to the type A Seneca virus 3A protein antigenic epitope peptide as described in claim 1.

6. A hybridoma cell line for preparing a monoclonal antibody against Seneca virus type A 3A protein as described in claim 5, characterized in that: The hybridoma cell line has the accession number CCTCC NO: C202466 and is deposited at the China Center for Type Culture Collection.

7. The application of a monoclonal antibody against Seneca virus type A 3A protein as described in claim 5 or a hybridoma cell line as described in claim 6, characterized in that: The applications include any one or more of the following: (1) Use in the preparation of medicaments for the prevention and / or treatment of Seneca virus type A infection; (2) Application in the preparation of reagents for the detection and / or diagnosis of Seneca virus type A infection.

8. A drug for the prevention and / or treatment of Seneca virus type A infection, characterized in that: The drug contains one or more of the following active ingredients: (1) The monoclonal antibody against type A Seneca virus 3A protein as described in claim 5; (2) The hybridoma cell line as described in claim 6.

9. A reagent for detecting and / or diagnosing Seneca virus type A infection, characterized in that: The reagent contains one or more of the following components: (1) The monoclonal antibody against type A Seneca virus 3A protein as described in claim 5; (2) The hybridoma cell line as described in claim 6.

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