Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application of Senecavirus type A 3A protein antigen epitope peptide and monoclonal antibody

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a Seneca virus type A 3A protein antigen epitope peptide or a coding nucleic acid thereof, and a monoclonal antibody specifically bound with a Seneca virus type A 3A protein antigen epitope. The invention also discloses a hybridoma cell strain secreting the monoclonal antibody and application of the hybridoma cell strain in preparation of drugs for preventing and / or treating Senecavirus A infection or reagents for detecting and / or diagnosing Senecavirus A infection. The monoclonal antibody provided by the invention is secreted by a hybridoma cell strain SVA-3A-5A7 with the preservation number of CCTCC NO: C202466, the monoclonal antibody can be specifically combined with the Senecavirus A 3A protein (the antigen epitope is located at the 5th-36th amino acid of the extracellular domain of the 3A protein), and the monoclonal antibody can be used for preparing medicines for preventing and / or treating Senecavirus A infection. Or a reagent for detecting and / or diagnosing Senecavirus type A infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a type A Senecavirus 3A protein antigen epitope peptide or its encoding nucleic acid, a monoclonal antibody that specifically binds to the type A Senecavirus 3A protein antigen epitope, a hybridoma cell line that secretes the monoclonal antibody, and the use of the monoclonal antibody in preparing a drug for preventing and / or treating type A Senecavirus infection, or a reagent for detecting and / or diagnosing type A Senecavirus infection. Background Art

[0002] Senecavirus A (SVA) is a non-enveloped single-stranded RNA virus belonging to the Picornaviridae family and the only member of the Senecavirus genus. The main susceptible animals to this virus are pigs. The clinical symptoms caused by SVA infection are extremely similar to those of infectious vesicular diseases of pigs, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS). Infected pigs often show clinical symptoms such as anorexia, lethargy, lameness, and blisters on the nose, mouth, and hoof crown. At the same time, SVA has oncolytic properties and can selectively infect neuroendocrine tumor cells, showing prospects for cancer treatment in early clinical trials. Due to the widespread prevalence of SVA and its unique natural oncolytic effect, it has gradually become well-known.

[0003] The SVA virus particle is an icosahedral structure, spherical, and 17-25nm in diameter. The total length of the SVA genome is about 7300nt, consisting of an open reading frame of 6543nt, a 5' non-coding region of 668nt, and a 3' non-coding region of 68nt with a poly(A) tail. The open reading frame of SVA can encode approximately 2180 amino acids, and has the typical L-4-3-4 genome layout of other picornaviruses, namely the leader protein L, the intermediate protein P1 (cleaved into four structural proteins VP1, VP2, VP3 and VP4), P2 (cleaved into three non-structural proteins 2A, 2B and 2C), and P3 (cleaved into four non-structural proteins 3A, 3B, 3C and 3D). It was predicted that the 3A protein is a transmembrane protein, which is composed of an extracellular domain, a transmembrane domain, and an intracellular domain. Through sequence comparison, it was found that the extracellular structure sequence and transmembrane domain sequence of the 3A protein are relatively conserved, while the intracellular domain sequence is more diverse. This diversity may be related to the interaction between host proteins.

[0004] 3A is one of the non-structural proteins of SVA, and there are very few studies on 3A protein. However, studies have found that the 3A proteins of other viruses in the same Picornaviridae family play an important role. For example, in the 3A protein of foot-and-mouth disease, the difference in the sequence of its intracellular domain may be related to the cross-species transmission mechanism. The membrane anchor region of the 3A protein of hepatitis A virus can bind to the transduction protein MAVS on the outer membrane of mitochondria, resulting in MAVS being degraded by the 3C protein and inhibiting the host's natural immune response. The 3A protein of enterovirus type 71 can inhibit the expression of host antiviral response-related proteins by preventing the formation of endoplasmic reticulum transport vesicles. At the same time, the 3A protein can bind to and protect long-chain ds RNA from Dicer cleavage, which can effectively inhibit the RNAi pathway and promote viral replication. However, there are still no reports on the ability of SVA 3A protein to evade the host cell's antiviral natural immune response. Therefore, the screening of 3A monoclonal antibodies is helpful for the study of the biological functions of 3A protein.

[0005] At the same time, a study found that when analyzing the antibody dynamics in pigs after virus infection, the level of antibodies produced against the non-structural protein 3AB was significantly higher than that of other non-structural proteins and the structural protein VP1. The 3A protein not only exists in a mature form, but can also be used as a component of the precursor protein 3AB. It is expressed in both the early and late stages of viral infection and has good immunogenicity. Therefore, the SVA antibody detection method based on the 3A protein may have higher sensitivity and has the potential to be used as a diagnostic target. Summary of the invention

[0006] The main technical problem solved by the present invention is to provide an A-type Seneca virus 3A protein antigen epitope peptide for stimulating and inducing the body to produce effector molecules (antibodies) and effector cells, exerting immune effects and thereby killing the A-type Seneca virus.

[0007] Secondly, the present invention provides an application of a type A Senecavirus 3A protein antigen epitope peptide or a nucleic acid encoding the same.

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

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

[0010] Finally, the present invention provides a drug for preventing and / or treating type A Senecavirus infection, or an agent for detecting and / or diagnosing type A Senecavirus infection.

[0011] In order to solve the above technical problems, the present invention provides the following technical solutions:

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

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

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

[0015] Specifically, the nucleic acid encoding the antigen epitope peptide is DNA or RNA. Without changing the encoded amino acid sequence, the sequence can be optimized according to codon preference.

[0016] A biological material comprising an antigenic epitope peptide of type A Seneca virus 3A protein or a nucleic acid encoding the same.

[0017] Specifically, the biological material includes but is not limited to recombinant expression vectors (such as plasmid vectors, viral vectors), gene expression cassettes, recombinant bacteria, host cells (such as prokaryotic cells, eukaryotic cells), etc.

[0018] Application of a type A Seneca virus 3A protein antigen epitope peptide or its encoding nucleic acid and biological material.

[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 a drug for preventing and / or treating type A Seneca virus infection;

[0021] (2) Use in the preparation of reagents for detecting and / or diagnosing type A Seneca virus infection.

[0022] A drug for preventing and / or treating type A Seneca virus infection, comprising one or more of the following active ingredients:

[0023] (1) Senecavirus type A 3A protein antigen epitope peptide;

[0024] (2) nucleic acid encoding an antigenic epitope peptide of the 3A protein of Senecavirus type A;

[0025] (3) A biological material comprising an antigenic epitope peptide of the 3A protein of Seneca virus type A or a nucleic acid encoding the same.

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

[0027] As 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 (such as Montanide ISA51) and the like.

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

[0029] (1) Senecavirus type A 3A protein antigen epitope peptide;

[0030] (2) Primers and / or probes for detecting nucleic acids encoding antigenic epitope peptides of type A Senecavirus 3A protein.

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

[0032] Specifically, the reagent is a molecular detection and / or diagnostic reagent, which contains primers, probes, etc. for detecting nucleic acid encoding antigenic epitope peptides of type A Senecavirus 3A protein, and can be used to detect whether type A Senecavirus (i.e., type A Senecavirus 3A protein) is present in pig body fluids.

[0033] As 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 further include one or more of a sample diluent, an ELISA plate, a blocking solution, a washing solution, a substrate, a stop solution, a negative control, a positive control, and the like.

[0035] Specifically, for molecular detection and / or diagnostic reagents, the reagents further include one or more of a polymerase (RNA and / or DNA-dependent polymerase), an amplification reaction buffer, a positive control, a negative control, and the like.

[0036] The invention discloses an anti-type A Seneca virus 3A protein monoclonal antibody, which is secreted and produced by a hybridoma cell line SVA-3A-5A7 which is deposited in the China Center for Type Culture Collection and has a deposit number of CCTCC NO: C202466.

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

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

[0039] A hybridoma cell line for preparing monoclonal antibodies against type A Seneca virus 3A protein. The hybridoma cell line is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C202466.

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

[0041] Application of an anti-type A Seneca virus 3A protein monoclonal antibody 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 a drug for preventing and / or treating type A Seneca virus infection;

[0044] (2) Use in the preparation of reagents for detecting and / or diagnosing type A Seneca virus infection.

[0045] A drug for preventing and / or treating type A Seneca virus infection, comprising one or more of the following active ingredients:

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

[0047] (2) A hybridoma cell line for preparing monoclonal antibodies against type A Seneca virus 3A protein.

[0048] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, comprising a pharmaceutically acceptable carrier, auxiliary material and / or adjuvant.

[0049] As a preferred embodiment of the present invention, the drug can be used by local administration, and the dosage is a pharmaceutically acceptable dosage.

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

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

[0052] (2) A hybridoma cell line for preparing monoclonal antibodies against type A Seneca virus 3A protein.

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

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

[0055] Beneficial effects of the present invention:

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

[0057] The monoclonal antibody provided by the present invention is secreted and produced by the hybridoma cell line SVA-3A-5A7 (Hybridoma cell line SVA-3A-5A7) deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: C202466, can specifically bind to the type A Seneca virus 3A protein, and can be used to prepare a drug for preventing and / or treating type A Seneca virus infection, or a reagent for detecting and / or diagnosing type A Seneca virus infection.

[0058] The present invention analyzes the epitope region of the 3A protein and finds that most regions of the 3A protein have good antigenicity, and the antigen epitope recognized by the monoclonal antibody 5A7 is located at amino acids 5-36 of the extracellular domain of the 3A protein. The antigen epitope peptide synthesized in this way can also be used to prepare drugs for preventing and / or treating type A Seneca virus infection, or reagents for detecting and / or diagnosing type A Seneca virus infection, and has good clinical application prospects and high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0060] Figure 2 The best expression method of 3A protein was verified by SDS-PAGE in the experimental example;

[0061] Figure 3 The SDS-PAGE in the experiment verifies the expression of 3A protein when induced at 37°C for 6 hours;

[0062] Figure 4 The SDS-PAGE in the experiment verifies the expression of 3A protein when induced at 25℃ for 12h;

[0063] Figure 5 The SDS-PAGE in the experiment verified the expression of 3A protein when induced at 16℃ for 20h;

[0064] Figure 6 This is the difference in protein concentration after SDS-PAGE verification of 3A protein imidazole elution in the experimental example;

[0065] In the figure, 1: 3A protein before passing through the nickel column; 2: 3A protein flow-through; 3: 20mM imidazole eluent; 4: 40mM imidazole eluent; 5: the first milliliter of eluent after 250mM imidazole elution; 6: the third milliliter of eluent after 250mM imidazole elution; 7: the fifth milliliter of eluent after 250mM imidazole elution; 8: the seventh milliliter of eluent after 250mM imidazole elution; 9: the ninth milliliter of eluent after 250mM imidazole elution; 10: the eleventh milliliter of eluent after 250mM imidazole elution; 11: the thirteenth milliliter of eluent after 250mM imidazole elution; 12: the fifteenth milliliter of eluent after 250mM imidazole elution;

[0066] Figure 7 Western blot was used to identify the reactivity of monoclonal antibody SVA-3A-5A7 with 3A protein in the experimental example;

[0067] Figure 8 For the experimental example, immunofluorescence was used to identify the reactivity of monoclonal antibodies with SVA;

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

[0069] Fig.10 Prediction of 3A protein antigen epitopes in the experimental examples;

[0070] Fig.11 It is the reaction of his antibody with the amino acid regions of positions 5-36 and 67-87 of 3A in the experimental example;

[0071] Fig.12 It is the reaction of 5A7 antibody with the amino acid regions of 5-36 and 67-87 of 3A in the experimental example;

[0072] In order to more clearly illustrate the technical solution to be protected by the present invention, the drawings in the embodiments or experimental examples are briefly introduced above. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0073] The technical solution of the present invention will be clearly and completely explained below in conjunction with 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 technical effects of the present invention and should not be regarded as any limitation on the protection scope of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the protection scope of the present invention.

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

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

[0077] The deposit information involved in the present invention is as follows:

[0078] Collection Information

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

[0080] Deposit 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 a nucleic acid encoding the same. The antigenic epitope peptide comprises an amino acid sequence as shown in SEQ ID NO: 1.

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

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

[0087] This embodiment also provides a use of a type A Senecavirus 3A protein antigen epitope peptide, a recombinant bacterium or a host cell in the preparation of a drug for preventing and / or treating type A Senecavirus infection.

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

[0089] Example 2

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

[0091] This embodiment also provides a drug for treating type A Seneca virus infection, which is a synthetic polypeptide vaccine. The vaccine contains a pharmaceutically effective amount of type A Seneca virus 3A protein antigen epitope peptide (same as above) and an appropriate amount of adjuvant.

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

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

[0094] Example 3

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

[0096] This embodiment also provides a hybridoma cell line for preparing anti-type A Seneca virus 3A protein monoclonal antibody, the hybridoma cell line is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: C202466. The hybridoma cell line is prepared by immunizing mice with type A Seneca virus 3A protein as an immunogen.

[0097] This embodiment also provides a use of an anti-type A Senecavirus 3A protein monoclonal antibody or hybridoma cell line in the preparation of a drug for preventing and / or treating type A Senecavirus infection.

[0098] This embodiment also provides a use of an anti-type A Senecavirus 3A protein monoclonal antibody or hybridoma cell line in the preparation of a reagent for detecting and / or diagnosing type A Senecavirus infection.

[0099] Example 4

[0100] This embodiment provides a drug for preventing and / or treating type A Senecavirus infection, wherein the drug comprises a pharmaceutically effective amount of an anti-type A Senecavirus 3A protein monoclonal antibody (same as in Example 3) and an appropriate amount of an adjuvant.

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

[0102] In other embodiments of the present invention, the medicine further comprises other medicinal ingredients for combined or auxiliary prevention and / or treatment of type A Seneca virus 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 of the disease and can be followed according to the doctor's advice.

[0105] Experimental example

[0106] 1. Experimental Materials

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

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

[0109] 0.01M PBS: weigh 8 g sodium chloride, 0.2 g potassium chloride, 3.63 g disodium hydrogen phosphate dodecahydrate, and 0.24 g potassium dihydrogen phosphate, dissolve in deionized water, make up to 1 L, and sterilize by high pressure for later use.

[0110] 8M urea: Weigh 480 g urea and dissolve it in 0.01 M PBS and make up to 1 L.

[0111] LB liquid medium: weigh 2 g Tryptone, 1 g Yeast Extract, and 2 g sodium chloride, place in a 500 mL conical flask, add 200 mL single distilled water, adjust the pH to 7.0 after complete dissolution, sterilize under high pressure and store at 4°C for later use.

[0112] 2. Experimental Methods

[0113] 1. Preparation of SAV 3A protein

[0114] 1.1 Screening of inducer IPTG concentration

[0115] Take a frozen pET-28a-3A expression strain and inoculate it into 5 mL of K + (50 mg / mL) in liquid LB medium and cultured on an air shaker at 37°C. When the OD600nm value of the bacterial solution was between 0.6 and 0.8, it was added to 5 groups of 5 mL of K-containing + (50mg / mL) of liquid LB culture medium, the mother bacteria were placed at 4℃ for short-term storage for subsequent experiments. Then, the 5 groups of bacterial solutions were placed on an air shaker for culture. When the OD600nm value of the bacterial solution was between 0.6-0.8, IPTG with a final concentration of 0, 0.3, 0.5, 0.8, and 1mM was added to the 5 groups of bacterial solutions, respectively, and the induction expression was continued for 6h. After induced expression, the 5 groups of bacterial solutions were centrifuged at 12000rpm for 2min to enrich the bacteria, the supernatant was discarded, 250μL PBS was added for resuspending, and 10μL was taken out after resuspending to make SDS-PAGE samples for SDS-PAGE analysis. The results are shown in Figure 1 shown.

[0116] 1.2 Determination of protein supernatant expression and precipitation expression

[0117] The preserved bacterial solution was added to 5 mL of antibiotic-free LB medium at a volume ratio of 1:100 for activation for 1 hour. After activation, kanamycin was added at a final concentration of 50 g / mL, and then cultured on an air shaker until the OD600nm value was between 0.6-0.8. The inducer IPTG was added at a final concentration of 0.5 mM, and the induced expression was continued for 6 hours. After induced expression, the bacterial solution was centrifuged at 12000 rpm for 2 minutes to enrich the bacteria, the supernatant was discarded, and 250 μL PBS was added for resuspending. The working parameters of the ultrasonic disruptor were set as follows: at 0°C, ultrasound was turned on for 1 second, stopped for 1 second, and ultrasound was performed for a total of 15 minutes. After the ultrasound was completed, centrifuged at 9000 rpm for 15 minutes, the inclusion bodies were dissolved with 250 μL 8M urea, and 10 μL was taken out from the supernatant and inclusion body solution to make SDS-PAGE samples for SDS-PAGE analysis. The results are as follows Figure 2 shown.

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

[0119] The preserved bacterial solution was added to 3 groups of 5 mL of anti-antibody LB culture medium at a volume ratio of 1:100 for activation for 1 hour. After activation, kanamycin was added at a final concentration of 50 μg / mL, and then cultured on an air shaker until the OD600nm value was between 0.6-0.8. The inducer IPTG was added to each group at a final concentration of 0.5 mM, and induced expression was carried out at 37°C for 6 hours, 25°C for 12 hours, and 16°C for 20 hours. After induced expression, the bacterial solution was centrifuged at 12000 rpm for 2 minutes, the supernatant was discarded, and 250 μL PBS was added to the enriched bacteria for resuspending. The working parameters of the ultrasonic disruptor were set as follows: at 0°C, ultrasound was turned on for 1 second, stopped for 1 second, and ultrasound was performed for a total of 15 minutes. After the ultrasound, centrifuged at 9000 rpm for 15 minutes, the inclusion bodies were dissolved with 250 μL 8M urea, and 10 μL was taken out from the supernatant and inclusion body solution to make SDS-PAGE samples for SDS-PAGE analysis. The results are as follows Figure 3-5 shown.

[0120] 1.4Ni-Agarose Resin Chromatography Purification

[0121] Mix the Ni-Agarose Resin filler and add it to the chromatography column. Wash the filler three times with PBS before use. Equilibrate the nickel column with TBS containing 10mM imidazole. Then, mix the supernatant of the broken bacteria and the filler by blowing, transfer to a centrifuge tube and mix by inversion at 4°C for 3 hours to allow the protein to fully combine with the filler. Add the mixed protein to the chromatography column, add 10mL of 20mM and 40mM imidazole respectively to wash away the impurities, and then elute the target protein with 15mL of 250mM imidazole, and collect the eluate per 1mL. After rinsing the nickel column with distilled water, it is stored in 20% anhydrous ethanol. The eluate is loaded into a dialysis bag with a pore size of 8000D, dialyzed in 0.01M PBS at 4°C overnight, the protein concentration is determined, and it is divided and stored at -80°C. Take out the 10mM, 20mM, and 250mM imidazole eluates to make SDS-PAGE samples for SDS-PAGE analysis. The results are as follows Figure 6 shown.

[0122] 2. Preparation of monoclonal antibodies

[0123] 2.1 Screening of positive hybridoma cell lines

[0124] Purified SVA 3A protein was inoculated into BALB / c mice at a dose of 50 μg / mouse by subcutaneous multi-point injection. After multiple immunizations, the titer of the immunized mouse serum was detected by the 3A indirect ELISA method. When the titer reached the cell fusion condition, the mouse spleen cells were taken for cell fusion with mouse myeloma cells (SP2 / 0). The cells that failed to fuse were screened and excluded using HAT selective culture medium. After 5-7 days, the cell clusters grew to a suitable size and the medium was changed. After 2-3 days, the cell culture medium was extracted and indirect ELISA was performed using an ELISA plate coated with 3A protein. It was preliminarily determined that the cells in the two cell culture plate wells could produce antibodies that could react with the 3A prokaryotic expression protein. Subsequently, the specific cell strain was screened using an ELISA plate coated with 3A protein, SVA virus liquid, and an unrelated protein of the vector pET-28a expressed by the same Escherichia coli expression system. The cells were counted by limiting dilution method, 200 cells were taken for subcloning, and the indirect ELISA method was used to determine whether the target cells existed in the cell wells. After repeated several times, the screened cell lines were finally amplified and frozen in time. The cell supernatant was taken to verify the interaction with 3A protein and SVA virus. Figure 7-8 shown.

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

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

[0127] Healthy BALB / c mice were selected and injected with 500 μL of liquid paraffin via intraperitoneal injection one week before cell injection. Hybridoma cells were washed with serum-free DMEM and resuspended in 500 μL of serum-free DMEM at 1×10 6 When the mouse abdomen begins to swell, extract the ascites. Centrifuge the ascites at 12,000 rpm for 5 minutes, discard the top layer of fat and the bottom sediment, and then filter it through a 0.22 μm filter before use. Use the antibody subtype identification kit to identify the subtype. The results are as follows: Fig. 9 shown.

[0128] 2.3 Prediction and identification of SVA 3A protein antigenic epitopes

[0129] The 3A protein was analyzed using the antigen epitope prediction website, and the truncation position of the 3A protein was selected according to the distribution of the antigen epitope region, such as Fig.10 The truncated protein base sequence was connected to 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 in Figure 11-12 shown.

[0130] 3. Experimental Results

[0131] 1. 3A protein expression and purification

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

[0133] like Figure 1 As shown, when the IPTG concentration was in the range of 0.3-0.8 mM, the expression level of 3A protein supernatant was 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

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

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

[0137] like Figure 3-5As shown, the expression level of 3A protein was the highest when induced at 37°C for 6 h.

[0138] 1.4Ni-Agarose Resin Chromatography Purification

[0139] like Figure 6 As shown, by SDS-PAGE Coomassie Brilliant Blue staining, it was found that after competitive elution of the protein bound to the nickel column with 250 mM imidazole, it was found that the first to fifteenth milliliters of the eluate contained a higher concentration of the target protein.

[0140] 2. Preparation of 3A monoclonal antibody

[0141] 2.1 Screening of positive hybridoma cell lines

[0142] like Figure 7 As shown in the figure, the antibody produced by the screened cell line was verified by Western blot to react specifically with the purified 3A protein, and an obvious band appeared, while no band appeared when incubated with the pET-28a blank vector prokaryotic expression protein. This shows that the monoclonal antibody produced by the screened cell line has good specificity, can recognize 3A protein, and has good reactivity with 3A protein.

[0143] like Figure 8 As shown, the antibodies produced by the screened cell lines were verified by immunofluorescence assay (IFA) to react with IBRS-2 cells infected with SVA, and fluorescence appeared in the cells, while IBRS-2 cells not infected with SVA 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 cell line finally screened was named SVA-3A-5A7, and a portion of it was sent to the China Type Culture Collection of Wuhan University for patent deposit. The deposited name was Hybridoma cell line SVA-3A-5A7 (Hybridoma cellline SVA-3A-5A7), and the deposit number was CCTCC NO: C202466.

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

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

[0147] 2.3 Prediction and identification of 3A protein antigenic epitopes

[0148] The 3A protein consists of an extracytoplasmic domain, a transmembrane domain, and an intracytoplasmic domain, such as Fig.10As shown, analysis of the antigen epitope prediction website showed that monoclonal antibody 5A7 may mainly recognize the amino acid regions at positions 5-36 and 67-87 of the 3A protein.

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

[0150] like Fig.12 As shown, the identification results showed that the 5A7 monoclonal antibody reacted with the 5-36 amino acid region of the 3A protein.

[0151] IV. Conclusion

[0152] Since its discovery in 2002, SVA has gradually become a global epidemic. The clinical symptoms of the disease are extremely similar to foot-and-mouth disease, a Class A infectious disease in animals published by the World Organization for Animal Health, which affects the global pig trade. At the same time, due to its oncolytic properties, it is well known in tumor treatment. However, the pathogenic mechanism of SVA is still poorly understood, which seriously restricts the research and development progress of SVA efficient prevention and control products and oncolytic preparations. SVA 3A protein plays an important role in anti-host natural immunity and viral replication. Research on 3A protein will help to further understand the biological characteristics of SVA.

[0153] The present invention obtains a large amount of purified A-type Seneca virus 3A protein by prokaryotic expression, and prepares monoclonal antibodies against A-type Seneca virus 3A protein, providing a material basis for future research on the mechanism of 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 implementation methods 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 protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Senecavirus type A 3A protein antigen epitope peptide, characterized in that: The antigen epitope peptide comprises the amino acid sequence shown in SEQ ID NO:

1.

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

3. A biological material comprising the antigenic epitope peptide of the type A Seneca virus 3A protein as claimed in claim 1, or the encoding nucleic acid thereof as claimed in claim 2.

4. A use of the A-type Senecavirus 3A protein antigen epitope peptide as claimed in claim 1, or the encoding nucleic acid as claimed in claim 2, or the biomaterial as claimed in claim 3, characterized in that: The application includes any one or more of the following: (1) Use in the preparation of a drug for preventing and / or treating type A Seneca virus infection; (2) Use in the preparation of reagents for detecting and / or diagnosing type A Seneca virus infection.

5. A drug for preventing and / or treating type A Senecavirus infection, or a reagent for detecting and / or diagnosing type A Senecavirus infection, characterized in that: The drug or agent comprises one or more of the following components: (1) The antigenic epitope peptide of Seneca virus type A 3A protein as described in claim 1; (2) A nucleic acid encoding the antigenic epitope peptide of the Senecavirus type A 3A protein as described in claim 2; (3) The biological material comprising the Senecavirus type A 3A protein antigen epitope peptide or its encoding nucleic acid as described in claim 3.

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

7. A hybridoma cell line for preparing the anti-type A Seneca virus 3A protein monoclonal antibody according to claim 6, characterized in that: The hybridoma cell line has a deposit number of CCTCC NO: C202466 and is deposited in China Center for Type Culture Collection.

8. A use of the anti-Senecavirus type A 3A protein monoclonal antibody according to claim 6 or the hybridoma cell line according to claim 7, characterized in that: The application includes any one or more of the following: (1) Use in the preparation of a drug for preventing and / or treating type A Seneca virus infection; (2) Use in the preparation of reagents for detecting and / or diagnosing type A Seneca virus infection.

9. A drug for preventing and / or treating Senecavirus type A infection, characterized in that: The medicine contains one or more of the following active ingredients: (1) The anti-type A Seneca virus 3A protein monoclonal antibody according to claim 6; (2) The hybridoma cell line for preparing anti-type A Seneca virus 3A protein monoclonal antibody as described in claim 7.

10. A reagent for detecting and / or diagnosing Seneca virus type A infection, characterized in that: The reagent comprises one or more of the following components: (1) The anti-type A Seneca virus 3A protein monoclonal antibody according to claim 6; (2) The hybridoma cell line for preparing anti-type A Seneca virus 3A protein monoclonal antibody as described in claim 7.

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