Truncated foot-and-mouth disease virus-like particle antigen A and application thereof

Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to an A-type truncated foot-and-mouth disease virus-like particle antigen and application thereof. Compared with a wild type foot-and-mouth disease virus structural protein precursor protein P1, the N end of the A type truncated foot-and-mouth disease virus structural protein precursor protein P1 is truncated by 10-40 amino acids. While the relatively strong capability of inducing an organism to generate an antibody of the full-length wild capsid protein is reserved, the protein has the characteristics of high expression quantity, easiness in purification, high uniformity, high stability and the like.

Description

Technical Field

[0001] The present application belongs to the technical field of veterinary biological products, and specifically relates to a type A truncated foot-and-mouth disease virus-like particle antigen and its application. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease that can spread rapidly over long distances. It is the most contagious disease in mammals, and even-toed ungulates, in particular, are particularly susceptible to the disease, causing significant economic losses worldwide. Animals affected by FMD include cattle, sheep, goats, and pigs. The causative agent, foot-and-mouth disease virus (FMDV), belongs to the genus FMDV of the family Picornaviridae. It is a non-enveloped virus with a single, linear, positive-strand RNA genome consisting of approximately 8,500 bp, which is the basis for FMDV host infection and inheritance. The virus is divided into seven serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3), of which types O and A are the most widespread. Vaccine immunization is an effective measure to control the disease and protect livestock from harm.

[0003] Currently, research on expression, purification, and assembly of FMDV-like particles based on the E. coli expression system mainly includes two forms: soluble expression, purification, and assembly and inclusion body expression, purification, and assembly. Soluble expression, purification, and assembly have obvious advantages.

[0004] Although soluble expression, purification, and assembly can maintain the relatively native conformation and immune activity of VLPs, the low soluble expression level, purification yield, and assembly efficiency, as well as the presence of multimers, pose significant challenges to the large-scale industrial production of FMDV VLPs.

[0005] Therefore, how to obtain target proteins with high soluble expression, high purification yield, stable VLPs after assembly, and at the same time maintain their native conformation has become the research focus of researchers. Summary of the Invention

[0006] The present application provides a type A truncated foot-and-mouth disease virus-like particle antigen and its application, in order to provide a target protein with high solubility, high expression, high purification yield, stable VLPs after assembly while maintaining its natural conformation.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] The invention relates to a type A truncated foot-and-mouth disease virus structural protein precursor protein P1. Compared with the wild-type foot-and-mouth disease virus structural protein precursor protein P1, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 has its N-terminus truncated by 10 to 40 amino acids.

[0009] Furthermore, the N-terminus is truncated by 10, 15, 20 or 40 amino acids.

[0010] Furthermore, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 includes VP0 protein, VP3 protein and VP1 protein in sequence, the VP0 protein has the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9, the VP3 protein has the amino acid sequence shown in SEQ ID NO.11, and the VP1 protein has the amino acid sequence shown in SEQID NO.13.

[0011] Furthermore, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is expressed by a SUMO expression system, and the protein expressed in series in the form of SUMOVP0, SUMOVP3 and SUMOVP1 can bind to a Ni affinity chromatography medium in a solution with a pH of 7.5 to 8.5;

[0012] Preferably, the proteins expressed in series as SUMOVP0, SUMOVP3 and SUMOVP1 can assemble to form stable VLPs in a solution with a pH of 7.5 to 8.5 and a salt concentration of 0 mM to 500 mM.

[0013] A type A truncated foot-and-mouth disease virus-like particle antigen is obtained by self-assembly of VP0 protein, VP3 protein and VP1 protein of the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus.

[0014] The biomaterial related to the protein P1 is characterized in that the biomaterial comprises any one of the following:

[0015] (1) a nucleic acid fragment encoding the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus;

[0016] (2) an expression cassette containing the nucleic acid fragment of (1);

[0017] (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2);

[0018] (4) A recombinant cell containing the nucleic acid in (1), the expression cassette in (2), or the vector in (3);

[0019] Preferably, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 includes VP0 protein, VP3 protein and VP1 protein in sequence, the nucleic acid fragment encoding the VP0 protein has the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO8 or SEQ ID NO.10, the nucleic acid fragment encoding the VP3 protein has the nucleotide sequence shown in SEQ ID NO.12, and the nucleic acid fragment encoding the VP1 protein has the nucleotide sequence shown in SEQ ID NO.14.

[0020] Application of the protein P1 or antigen or biological material in the preparation of medicine for preventing type A foot-and-mouth disease virus infection.

[0021] A type A foot-and-mouth disease subunit vaccine comprises a pharmaceutically acceptable carrier and an immunizing amount of the above-mentioned type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or type A truncated foot-and-mouth disease virus-like particle antigen.

[0022] Furthermore, the content of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or the type A truncated foot-and-mouth disease virus-like particle antigen is ≥160 μg / ml, preferably 160-240 μg / ml.

[0023] Furthermore, the pharmaceutically acceptable carrier includes at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative;

[0024] Preferably, the adjuvant comprises one or more of aluminum gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, and muramyl dipeptide;

[0025] Preferably, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, more preferably 50% V / V;

[0026] Preferably, the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids;

[0027] Preferably, the immunostimulant comprises α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin-2.

[0028] Compared with the prior art, the technical effects of this application are:

[0029] The type A truncated foot-and-mouth disease virus structural protein precursor protein P1 of the present application can be soluble expressed in Escherichia coli at a high expression level, achieving a high yield. The purification method is relatively simple and easy to operate. In particular, after soluble expression in Escherichia coli, the Escherichia coli can be lysed, and then the lysate can be subjected to chromatographic treatment, such as chromatography treatment, thereby obtaining high-purity type A truncated foot-and-mouth disease virus structural protein precursor protein P1 (purity can reach at least 90% or higher, such as 95%, 96%, 97%, 98%, 99%). The type A truncated foot-and-mouth disease virus structural protein precursor protein P1 has good uniformity and stability, and is not easy to aggregate after assembly. At the same time, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or its variants can induce the body to produce high-titer antibodies.

[0030] It can be seen that the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 of the present application retains the strong ability of the full-length wild-type capsid protein to induce the body to produce antibodies, while also having the characteristics of high expression level, easy purification, high uniformity and stability. DETAILED DESCRIPTION

[0031] Explanation of relevant terms in this application:

[0032] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and the Orthomavirus genus. It has seven serotypes: O, A, C, SAT1, SAT2, SAT3 (South African FMDV types 1, 2, and 3), and Asia1 (Asia1). There is no cross-protection between the types, and each type contains multiple subtypes. At the center of the virus lies a single-stranded, positive-sense RNA, consisting of approximately 8,000 bases, which serves as the basis for infection and inheritance. The surrounding proteins determine the virus's antigenicity, immunogenicity, and serological reactivity. The viral capsid is a symmetrical icosahedron. FMDV is the causative agent of foot-and-mouth disease, a highly contagious disease of even-toed ungulates. The Office International des Epizooties (OIE) lists FMD as the first on its "List of Category A Animal Infectious Diseases." China classifies it as a "Class I Infectious Disease for Animal Quarantine in Entry." Prevention and control of FMD in my country primarily involves vaccination, and animals infected with FMD are culled.

[0033] In this application, a protein "truncated at the N-terminus by X amino acids" refers to a protein obtained by replacing amino acid residues 1 to X at the N-terminus of the protein with a methionine residue encoded by the start codon. For example, a truncated foot-and-mouth disease virus structural protein precursor protein P1 type A with an N-terminal truncation of 10 amino acids refers to a protein obtained by replacing amino acid residues 1 to 10 at the N-terminus of the foot-and-mouth disease virus structural protein precursor protein P1 with a methionine residue encoded by the start codon.

[0034] "Antigen" refers to a substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0035] Virus-like particles (VLPs) are particles assembled from one or more viral structural proteins. They have an external structure and antigenicity similar to viral particles, but do not contain viral genes.

[0036] Foot-and-mouth disease virus VP0, VP3, and VP1 proteins: The FMDV structural protein precursor protein P1 is processed by protease 3C into VP0, VP3, and VP1 proteins. These three proteins can self-assemble into the icosahedral viral capsid. VP0 is an intermediate formed after protease 3C cleavage of P1. In the final stage of virion formation, VP0 matures and cleaves into VP4 and VP2.

[0037] The terms "vaccine" and "vaccine composition" refer to a pharmaceutical composition containing the structural protein precursor protein P1 of type A truncated foot-and-mouth disease virus or a type A truncated foot-and-mouth disease virus-like particle antigen, which can induce, stimulate or enhance the immune response of pigs against foot-and-mouth disease.

[0038] The term "immunizing amount" should be understood as an "immunologically effective amount", also known as an immunoprotective amount or an effective amount to produce an immune response, which is the amount of antigen that can effectively induce an immune response in the recipient, and this amount is sufficient to prevent or improve the signs or symptoms of the disease, including adverse health effects or its complications. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of the disease, including adverse health consequences or complications caused by infection caused by pathogens. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be indirectly assessed by, for example, measuring antibody titers, lymphocyte proliferation assays, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain, and the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs of the subject such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, inducing cellular and / or humoral immunity.

[0039] The term "pharmaceutically acceptable carrier" refers to all other components of the vaccine composition of the present application except for the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus or the type A truncated foot-and-mouth disease virus-like particle antigen, and is a carrier or diluent that does not stimulate the body and does not hinder the biological activity and properties of the compound used, preferably an adjuvant. The term "adjuvant" may include alumina adjuvants; saponins, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oil, in particular isobutene or deuterene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / deuterate), glycerol tri-(caprylate / deuterate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, esters of mannide (e.g. anhydrous mannitol oleate), esters of fatty glycols, esters of polyglycerols, esters of propylene glycol, and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which may be ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, in particular L 121. See Hunter et al., The theory and practical application of adjuvants (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al., Vaccine (1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adiuvant approach" edited by Powell M and Newman M (Plenum Press, 1995) can be used.The term "polymer of acrylic acid or methacrylic acid" is preferably a cross-linked polymer of acrylic acid or methacrylic acid, in particular cross-linked with a polyalkenyl ether of sugar or a polyol, such compounds being known as carbomers (trade name Carbopol) (Phameuropa, 1996, 8(2)). Those skilled in the art may also refer to U.S. Pat. No. 2,909,462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. These products are sold under the name Carbopol, (BF Goodrich, Ohio, USA) being particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P may be mentioned, with Carbopol 971P being most preferred. The term "copolymers of maleic anhydride and alkenyl derivatives" also includes copolymers of maleic anhydride and ethylene such as EMA (Monsanto). These polymers dissolve in water to produce an acidic solution, which is then neutralized, preferably to physiological pH, to produce an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin (recombinant or other), cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant, etc. Preferably, the adjuvant comprises one or more of alumina adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide or Gel adjuvant.

[0040] "Gene recombination" refers to the recombining of genes that control different traits. Modern genetic engineering techniques, also known as recombinant DNA, implement genetic recombination in vitro according to artificial design. The goal is to transfer the genetic genes in one individual's cells to the DNA molecules in another individual's cells with different traits, thereby causing genetic variation. After the target gene from the donor is transferred into the recipient bacteria, the gene product can be expressed, resulting in products that are difficult to obtain using conventional methods.

[0041] "Transformation" refers to the acquisition of a new genetic phenotype by cells or cultured recipient cells through the automatic or artificial introduction of exogenous DNA.

[0042] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, enabling expression of the genetic material they carry. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, and cosmids.

[0043] The term "assembly" refers to the process by which regular granular structures are formed through various interactions between viral structural proteins (such as capsid proteins) or between structural proteins and nucleic acids, which includes the assembly of natural viral particles and virus-like particles.

[0044] In the present application, host cell disruption can be achieved by various methods well known to those skilled in the art, including but not limited to homogenizer disruption, homogenizer disruption, ultrasonic treatment, grinding, high-pressure extrusion, lysozyme treatment, and the like.

[0045] The term "SUMO (small ubiquitin-related modifier)" tag protein is a small molecule ubiquitin-related modifier protein, a class of large proteins that are highly conserved in eukaryotes and participate in protein ubiquitination-related modifications. Compared with GST, MBP or NusA, SUMO can not only be used as a fusion tag for recombinant protein expression, but also has the function of a molecular chaperone, can promote the correct folding of proteins, is resistant to heat and proteases, and is more helpful in maintaining the stability of the target protein. In addition, the SUMO tag has a matching protease (high specificity) that recognizes the tertiary structure of SUMO and has extremely high cleavage specificity without any amino acid residues, making it suitable for recombinant protein expression.

[0046] The term "high-performance liquid gel filtration chromatography" refers to a key branch of chromatography. Using a liquid as the mobile phase, a high-pressure infusion system pumps a single solvent of varying polarity, a mixed solvent of varying proportions, a buffer, or other mobile phases through a chromatographic column containing a stationary phase. After the components are separated within the column, they are detected by a detector, enabling analysis of the sample. During HPLC, proteins are separated based on their size, as they follow different paths through the column.

[0047] The term "Ulp1" refers to Ubl-specific protease 1 (Ulp1), which can specifically recognize the spatial structure of SUMO and cleave it to remove the SUMO tag in the fusion protein, and is dependent on the spatial structure of SUMO.

[0048] The term "biphasic adjuvant" refers to a water-in-oil-in-water adjuvant.

[0049] The term "prevent" when referring to FMDV infection means inhibiting the replication of FMDV, inhibiting the spread of FMDV or preventing FMDV from establishing in its host, as well as alleviating the symptoms of a disease or condition caused by FMDV infection.

[0050] Below, the specific implementation methods of this application are described in detail.

[0051] This application has screened out a series of type A truncated foot-and-mouth disease virus structural protein precursor proteins P1 through a large number of experiments. While retaining immunogenicity, this protein P1 can be expressed in high-expression soluble tandem in the form of VP0-VP3-VP1 in Escherichia coli, and purified by chromatography. The high-purity truncated proteins thus obtained have good uniformity and stability, and can induce the body to produce high-titer antibodies.

[0052] The present application provides a type A truncated foot-and-mouth disease virus structural protein precursor protein P1, which has an N-terminal truncation of 10 to 40 amino acids compared to the wild-type foot-and-mouth disease virus structural protein precursor protein P1. For example, but not limited to, the type A foot-and-mouth disease virus structural protein precursor protein P1 may be truncated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 N-terminal amino acids.

[0053] In some embodiments, the N-terminus of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is truncated by 10-40 amino acids, such as 10, 15, 20 or 40 amino acids, compared with the wild-type foot-and-mouth disease virus structural protein precursor protein P1.

[0054] In some embodiments, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 includes VP0 protein, VP3 protein and VP1 protein in sequence, and the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is expressed in series in the form of VP0, VP3, and VP1. The VP0 protein has the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, the VP3 protein has the amino acid sequence shown in SEQ ID NO.11, and the VP1 protein has the amino acid sequence shown in SEQ ID NO.13.

[0055] Amino acid sequence of VP0 of type A foot-and-mouth disease virus:

[0056] GAGQSSPATGSQNQSGNTGSIINNYYMQQYQNSMDTQL GDNAISGGSNEGSTDTTSTHTTNTQNNDWFSKLASSAFTGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTCGYSTGEDHVSGPNTSGLETRVVQAERFFKKHLFDWTTDKPFGHTEKLELPTEH KGVYGQLVESFAYMRNGWDVEVSAVGNQFNGGCLLVAMVPEFKEFTQREKYQLTLFPHQFISPRTNMTAHITVPYLGVNRYDQYKKHKPWTLVVMVVSPLTTSSIGATQIKVYANIAPTHVHVAGELPSKE(SEQ ID NO.1).

[0057] Nucleotide sequence of VP0 of type A foot-and-mouth disease virus:

[0058] GGTGCTGGTCAGTCTTCTCCGGCTACCGGTTCTCAGA ACCAGTCTGGTAACACCGGTTCTATCATCAACAACTACTACATGCAGCAGTACCAGAACTCTATGGACACCCAGCTGGGTGACAACGCTATCTCTGGTGGTTCTAACGAAGGTTCTACCGACACCACCTCTACCCACACCACCAACACCCAGAACAACGACTGGTTCTCTAAACTGGCTTCTTCTGCTTTCACCGGTCTGTTCGGTGCTCTGCTGGCTGACAAAAAAACCGAAGAAACCACCCTGCTGGAAGACCGTATCCTGACCACCCGTAACGGTCACACCACCTCTACCACCCAGTCTTCTGTTGGTGTTACCTGCGGTTACTCTACCGGTGAAGACCACGTTTCTGGTCCGAACACCTCTGGTCTGGAAACCCGTGTTGTTCAGGCTGAACGTTTCTTCAAAAAACACCTGTTCGACTGGACCACCGACAAACCGTTCGGTCACACCGAAAAACTGGAACTGCCGACCGAACACAAAGGTGTTTACGGTCAGCTGGTTGAATCTTTCGCTTACATGCGTAACGGTTGGGACGTTGAAGTTTCTGCTGTTGGTAACCAGTTCAACGGTGGTTGCCTGCTGGTTGCTATGGTTCCGGAATTCAAAGAATTCACCCAGCGTGAAAAATACCAGCTGACCCTGTTCCCGCACCAGTTCATCTCTCCGCGTACCAACATGACCGCTCACATCACCGTTCCGTACCTGGGTGTTAACCGTTACGACCAGTACAAAAAACACAAACCGTGGACCCTGGTTGTTATGGTTGTTTCTCCGCTGACCACCTCTTCTATCGGTGCTACCCAGATCAAAGTTTACGCTAACATCGCTCCGACCCACGTTCACGTTGCTGGTGAACTGCCGTCTAAAGAA(SEQ ID NO.2).

[0059] Amino acid sequence of VP0 with 10 amino acids truncated at the N-terminus of foot-and-mouth disease virus type A:

[0060] SQNQSGNTGSIINNYYMQQYQNSMDTQLGDNAISGGSN EGSTDTTSTHTTNTQNNDWFSKLASSAFTGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTCGYSTGEDHVSGPNTSGLETRVVQAERFFKKHLFDWTTDKPFGHTEKLELPTEHKGVYG QLVESFAYMRNGWDVEVSAVGNQFNGGCLLVAMVPEFKEFTQREKYQLTLFPHQFISPRTNMTAHITVPYLGVNRYDQYKKHKPWTLVVMVVSPLTTSSIGATQIKVYANIAPTHVHVAGELPSKE(SEQ ID NO.3).

[0061] The nucleotide sequence of VP0 of foot-and-mouth disease virus type A with 10 amino acids truncated at the N-terminus:

[0062] TCTCAGAACCAGTCTGGTAACACCGGTTCTATCATCA ACAACTACTACATGCAGCAGTACCAGAACTCTATGGACACCCAGCTGGGTGACAACGCTATCTCTGGTGGTTCTAACGAAGGTTCTACCGACACCACCTCTACCCACACCACCAACACCCAGAACAACGACTGGTTCTCTAAACTGGCTTCTTCTGCTTTCACCGGTCTGTTCGGTGCTCTGCTGGCTGACAAAAAAACCGAAGAAACCACCCTGCTGGAAGACCGTATCCTGACCACCCGTAACGGTCACACCACCTCTACCACCCAGTCTTCTGTTGGTGTTACCTGCGGTTACTCTACCGGTGAAGACCACGTTTCTGGTCCGAACACCTCTGGTCTGGAAACCCGTGTTGTTCAGGCTGAACGTTTCTTCAAAAAACACCTGTTCGACTGGACCACCGACAAACCGTTCGGTCACACCGAAAAACTGGAACTGCCGACCGAACACAAAGGTGTTTACGGTCAGCTGGTTGAATCTTTCGCTTACATGCGTAACGGTTGGGACGTTGAAGTTTCTGCTGTTGGTAACCAGTTCAACGGTGGTTGCCTGCTGGTTGCTATGGTTCCGGAATTCAAAGAATTCACCCAGCGTGAAAAATACCAGCTGACCCTGTTCCCGCACCAGTTCATCTCTCCGCGTACCAACATGACCGCTCACATCACCGTTCCGTACCTGGGTGTTAACCGTTACGACCAGTACAAAAAACACAAACCGTGGACCCTGGTTGTTATGGTTGTTTCTCCGCTGACCACCTCTTCTATCGGTGCTACCCAGATCAAAGTTTACGCTAACATCGCTCCGACCCACGTTCACGTTGCTGGTGAACTGCCGTCTAAAGAA(SEQ ID NO.4).​​​​​GNTGSIINNYYMQQYQNSMDTQLGDNAISGGSNEGSTD TTSTHTTNTQNNDWFSKLASSAFTGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTCGYSTGEDHVSGPNTSGLETRVVQAERFFKKHLFDWTTDKPFGHTEKLELPTEHKGVYGQLVESFAYMRNGWDVEVSAVGNQFNGGCLLVAMVPEFKEFTQREKYQLTLFPHQFISPRTNMTAHITVPYLGVNRYDQYKKHKPWTLVVMVVSPLTTSSIGATQIKVYANIAPTHVHVAGELPSKE(SEQ IDNO.5).

[0065] N-terminal truncated VP0 nucleotide sequence of Foot-and-Mouth Disease Virus serotype A with 15 amino acids deleted

[0066] GGTAACACCGGTTCTATCATCAACAACTACTACATGC AGCAGTACCAGAACTCTATGGACACCCAGCTGGGTGACAACGCTATCTCTGGTGGTTCTAACGAAGGTTCTACCGACACCACCTCTACCCACACCACCAACACCCAGAACAACGACTGGTTCTCTAAACTGGCTTCTTCTGCTTTCACCGGTCTGTTCGGTGCTCTGCTGGCTGACAAAAAAACCGAAGAAACCACCCTGCTGGAAGACCGTATCCTGACCACCCGTAACGGTCACACCACCTCTACCACCCAGTCTTCTGTTGGTGTTACCTGCGGTTACTCTACCGGTGAAGACCACGTTTCTGGTCCGAACACCTCTGGTCTGGAAACCCGTGTTGTTCAGGCTGAACGTTTCTTCAAAAAACACCTGTTCGACTGGACCACCGACAAACCGTTCGGTCACACCGAAAAACTGGAACTGCCGACCGAACACAAAGGTGTTTACGGTCAGCTGGTTGAATCTTTCGCTTACATGCGTAACGGTTGGGACGTTGAAGTTTCTGCTGTTGGTAACCAGTTCAACGGTGGTTGCCTGCTGGTTGCTATGGTTCCGGAATTCAAAGAATTCACCCAGCGTGAAAAATACCAGCTGACCCTGTTCCCGCACCAGTTCATCTCTCCGCGTACCAACATGACCGCTCACATCACCGTTCCGTACCTGGGTGTTAACCGTTACGACCAGTACAAAAAACACAAACCGTGGACCCTGGTTGTTATGGTTGTTTCTCCGCTGACCACCTCTTCTATCGGTGCTACCCAGATCAAAGTTTACGCTAACATCGCTCCGACCCACGTTCACGTTGCTGGTGAACTGCCGTCTAAAGAA(SEQ ID NO.6).

[0067] Amino acid sequence of VP0 with the N-terminus truncated by 20 amino acids of foot-and-mouth disease virus type A:

[0068] IINNYYMQQYQNSMDTQLGDNAISGGSNEGSTDTTSTH TTNTQNNDWFSKLASSAFTGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTCGYSTGEDHVSGPNTSGLETRVVQAERFFKKKHLFDWTTDKPFGHTEKLEPTEHKGVYGQLVESFAYMRNGWDVEVSAVGNQFNGGCLLVAMVPEFKEFTQREKYQLTLFPHQFISPRTNMTAHITVPYLGVNRYDQYKKHKPWTLVVMVVSPLTTSsIGATQIKVYANIAPTHVHVAGELPSKE(SEQ IDNO.7)。

[0069] VP0

[0070] ATCATCAACAACTACTACATGCAGCAGTACCAGAACT CTATGGACACCCAGCTGGGTGACAACGCTATCTCTGGTGGTTCTAACGAAGGTTCTACCGACACCACCTCTACCCACACCACCAACACCCAGAACAACGACTGGTTCTCTAAACTGGCTTCTTCTGCTTTCACCGGTCTGTTCGGTGCTCTGCTGGCTGACAAAAAAACCGAAGAAACCACCCTGCTGGAAGACCGTATCCTGACCACCCGTAACGGTCACACCACCTCTACCACCCAGTCTTCTGTTGGTGTTACCTGCGGTTACTCTACCGGTGAAGACCACGTTTCTGGTCCGAACACCTCTGGTCTGGAAACCCGTGTTGTTCAGGCTGAACGTTTCTTCAAAAAACACCTGTTCGACTGGACCACCGACAAACCGTTCGGTCACACCGAAAAACTGGAACTGCCGACCGAACACAAAGGTGTTTACGGTCAGCTGGTTGAATCTTTCGCTTACATGCGTAACGGTTGGGACGTTGAAGTTTCTGCTGTTGGTAACCAGTTCAACGGTGGTTGCCTGCTGGTTGCTATGGTTCCGGAATTCAAAGAATTCACCCAGCGTGAAAAATACCAGCTGACCCTGTTCCCGCACCAGTTCATCTCTCCGCGTACCAACATGACCGCTCACATCACCGTTCCGTACCTGGGTGTTAACCGTTACGACCAGTACAAAAAACACAAACCGTGGACCCTGGTTGTTATGGTTGTTTCTCCGCTGACCACCTCTTCTATCGGTGCTACCCAGATCAAAGTTTACGCTAACATCGCTCCGACCCACGTTCACGTTGCTGGTGAACTGCCGTCTAAAGAA(SEQ ID NO.8).

[0071] Amino acid sequence of VP0 with 40 amino acids truncated at the N-terminus of foot-and-mouth disease virus type A:

[0072] NAISGGSNEGSTDTTSTHTTNTQNNDWFSKLASSAFTGL FGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTCGYSTGEDHVSGPNTSGLETRVVQAERFFKKHLFDWTTDKPFGHTEKLELPTEHKGVYGQLVESFAYMRNGWDVE VSAVGNQFNGGCLLVAMVPEFKEFTQREKYQLTLFPHQFISPRTNMTAHITVPYLGVNRYDQYKKHKPWTLVVMVVSPLTTSSIGATQIKVYANIAPTHVHVAGELPSKE(SEQ ID NO.9).

[0073] The nucleotide sequence of VP0 of foot-and-mouth disease virus type A with 40 amino acids truncated at the N-terminus:

[0074] AACGCTATCTCTGGTGGTTCTAACGAAGGTTCTACCG ACACCACCTCTACCCACACCACCAACACCCAGAACAACGACTGGTTCTCTAAACTGGCTTCTTCTGCTTTCACCGGTCTGTTCGGTGCTCTGCTGGCTGACAAAAAAACCGAAGAAACCACCCTGCTGGAAGACCGTATCCTGACCACCCGTAACGGTCACACCACCTCTACCACCCAGTCTTCTGTTGGTGTTACCTGCGGTTACTCTACCGGTGAAGACCACGTTTCTGGTCCGAACACCTCTGGTCTGGAAACCCGTGTTGTTCAGGCTGAACGTTTCTTCAAAAAACACCTGTTCGACTGGACCACCGACAAACCGTTCGGTCACACCGAAAAACTGGAACTGCCGACCGAACACAAAGGTGTTTACGGTCAGCTGGTTGAATCTTTCGCTTACATGCGTAACGGTTGGGACGTTGAAGTTTCTGCTGTTGGTAACCAGTTCAACGGTGGTTGCCTGCTGGTTGCTATGGTTCCGGAATTCAAAGAATTCACCCAGCGTGAAAAATACCAGCTGACCCTGTTCCCGCACCAGTTCATCTCTCCGCGTACCAACATGACCGCTCACATCACCGTTCCGTACCTGGGTGTTAACCGTTACGACCAGTACAAAAAACACAAACCGTGGACCCTGGTTGTTATGGTTGTTTCTCCGCTGACCACCTCTTCTATCGGTGCTACCCAGATCAAAGTTTACGCTAACATCGCTCCGACCCACGTTCACGTTGCTGGTGAACTGCCGTCTAAAGAA(SEQ ID NO.10).

[0075] Amino acid sequence of foot-and-mouth disease virus VP3

[0076] GIVPVACSDGYGGLVTTDPKTADPAYGMVYNPPRTNYP GRFTNLLDVAEACPTFLCFDDGKPYIVTRTDEQRLLAKFDLSLAAKHMSNTYLSGIAQYYAQYSGTINLHFMFTGSTDSKARYMVAYVPPGAETPPDTPEKAAHCIHAEWDTGLNSKFTFSIPYVSAADYAYTASDEAETTNVQGWVCIYQITHGKAEQDTLVVSVSAGKDFELRLPIDPRAQ(SEQ ID NO.11).

[0077] Nucleotide sequence of Foot-and-Mouth Disease Virus serotype A VP3:

[0078] GGTATCGTTCCGGTTGCTTGCTCTGACGGTTACGGTGG TCTGGTTACCACCGACCCGAAAACCGCTGACCCGGCTTACGGTATGGTTTACAACCCGCCGCGTACCAACTACCCGGGTCGTTTCACCAACCTGCTGGACGTTGCTGAAGCTTGCCCGACCTTCCTGTGCTTCGACGACGGTAAACCGTACATCGTTACCCGTACCGACGAACAGCGTCTGCTGGCTAAATTCGACCTGTCTCTGGCTGCTAAACACATGTCTAACACCTACCTGTCTGGTATCGCTCAGTACTACGCTCAGTACTCTGGTACCATCAACCTGCACTTCATGTTCACCGGTTCTACCGACTCTAAAGCTCGTTACATGGTTGCTTACGTTCCGCCGGGTGCTGAAACCCCGCCGGACACCCCGGAAAAAGCTGCTCACTGCATCCACGCTGAATGGGACACCGGTCTGAACTCTAAATTCACCTTCTCTATCCCGTACGTTTCTGCTGCTGACTACGCTTACACCGCTTCTGACGAAGCTGAAACCACCAACGTTCAGGGTTGGGTTTGCATCTACCAGATCACCCACGGTAAAGCTGAACAGGACACCCTGGTTGTTTCTGTTTCTGCTGGTAAAGACTTCGAACTGCGTCTGCCGATCGACCCGCGTGCTCAG(SEQ IDNO.12).

[0079] A - type foot - and - mouth disease virus VP1 amino acid sequence:

[0080] TTATGESADPVTTTVENYGGETQVQRRYHTDVGFLMD RFVQIKPVGPTHVIDLMQTHQHGLVGAMLRAATYYFSDLEIVVNHTGNLTWVPNGAPEAALQNTSNPTAYHKAPFTRLALPYTAPHRVLATVYSGTSKYSAPQNRRGDSGPLAARLAAQLPASFNFGAIRATEIRELLVRMKRAELYCPRPLLAVEVSSQDRHKQKIIAPAKQLL(SEQ IDNO.13).

[0081] A - type foot - and - mouth disease virus VP1 nucleotide sequence:

[0082] ACCACCGCTACCGGTGAATCTGCTGACCCGGTTACCA CCACCGTTGAAAACTACGGTGGTGAAACCCAGGTTCAGCGTCGTTACCACACCGACGTTGGTTTCCTGATGGACCGTTTCGTTCAGATCAAACCGGTTGGTCCGACCCACGTTATCGACCTGATGCAGACCCACCAGCACGGTCTGGTTGGTGCTATGCTGCGTGCTGCTACCTACTACTTCTCTGACCTGGAAATCGTTGTTAACCACACCGGTAACCTGACCTGGGTTCCGAACGGTGCTCCGGAAGCTGCTCTGCAGAACACCTCTAACCCGACCGCTTACCACAAAGCTCCGTTCACCCGTCTGGCTCTGCCGTACACCGCTCCGCACCGTGTTCTGGCTACCGTTTACTCTGGTACCTCTAAATACTCTGCTCCGCAGAACCGTCGTGGTGACTCTGGTCCGCTGGCTGCTCGTCTGGCTGCTCAGCTGCCGGCTTCTTTCAACTTCGGTGCTATCCGTGCTACCGAAATCCGTGAACTGCTGGTTCGTATGAAACGTGCTGAACTGTACTGCCCGCGTCCGCTGCTGGCTGTTGAAGTTTCTTCTCAGGACCGTCACAAACAGAAAATCATCGCTCCGGCTAAACAGCTGCTG(SEQ ID NO.14).

[0083] In some embodiments, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is expressed by a SUMO expression system, and the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is expressed in series as SUMOVP0, SUMOVP3 and SUMOVP1. The protein expressed in series as SUMOVP0, SUMOVP3 and SUMOVP1 can bind to a Ni affinity chromatography medium (e.g., Ni Sepharose 6 Fast Flow) in a solution with a pH of 7.5 to 8.5 (e.g., pH 8.0).

[0084] In some embodiments, proteins expressed in tandem as SUMOVP0, SUMOVP3, and SUMOVP1 can assemble to form stable VLPs in a solution with a pH of 7.5 to 8.5 (eg, pH 8.0) and a salt concentration of 0 mM to 500 mM (eg, 300 mM).

[0085] The present application provides a type A truncated foot-and-mouth disease virus-like particle antigen, which is obtained by self-assembly of VP0 protein, VP3 protein and VP1 protein of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 of the present application.

[0086] A biological material related to the structural protein precursor protein P1 of type A foot-and-mouth disease virus, the biological material comprising:

[0087] (1) a nucleic acid fragment encoding the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus;

[0088] (2) an expression cassette containing the nucleic acid fragment of (1);

[0089] (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2);

[0090] (4) A recombinant cell containing the nucleic acid fragment in (1) or the expression cassette in (2) or the vector in (3).

[0091] In some embodiments, the nucleic acid fragment encoding the VP0 protein expressed in tandem has the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10, the nucleic acid fragment encoding the VP3 protein has the nucleotide sequence shown in SEQ ID NO.12, and the nucleic acid fragment encoding the VP1 protein has the nucleotide sequence shown in SEQ ID NO.14.

[0092] In some embodiments, the present application relates to polynucleotides encoding the type A truncated protein P1 of the present application or its variants or fusion proteins of the present application, and vectors containing the polynucleotides. Vectors that can be used to insert the polynucleotide of interest are well known in the art, including but not limited to cloning vectors and expression vectors. In one embodiment, for example, the vector is a plasmid, etc.

[0093] In some embodiments, the application also relates to host cells comprising the above-mentioned polynucleotides or vectors. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell of the present application can also be a cell line, such as 293T cells.

[0094] Application of the truncated type A foot-and-mouth disease virus structural protein precursor protein P1 or type A foot-and-mouth disease virus-like particle antigen or biological material in the preparation of drugs for preventing type A foot-and-mouth disease virus infection.

[0095] In some embodiments, the drug is a subunit vaccine for foot-and-mouth disease type A.

[0096] The present application provides a type A foot-and-mouth disease subunit vaccine, which comprises a pharmaceutically acceptable carrier and an immunizing amount of the above-mentioned type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or type A truncated foot-and-mouth disease virus-like particle antigen.

[0097] In some embodiments, the content of type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or type A truncated foot-and-mouth disease virus-like particle antigen is ≥100 μg / ml, preferably 100-200 μg / ml.

[0098] In some embodiments, the pharmaceutically acceptable carrier includes at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

[0099] Preferably, the adjuvant includes one or more of aluminum gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, and muramyl dipeptide.

[0100] In some embodiments, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, and more preferably 50% V / V.

[0101] In some embodiments, the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

[0102] Preferably, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin-2.

[0103] The present application will be further described below in conjunction with specific embodiments, and the advantages and features of the present application will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present application in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0104] Materials and Reagents: Gene synthesis in this application was performed entirely by Suzhou Jinweizhi Biotechnology Co., Ltd. Restriction enzymes such as Xba I, Nco I, BamH I, and Xho I were purchased from Takara. All other reagents and chemicals were of analytical grade. Escherichia coli BL21 (DE3) was purchased from New England Biolabs. The experimental methods described in this application are conventional unless otherwise noted. The biological materials described are commercially available unless otherwise noted.

[0105] Example 1: Construction of a tandem expression vector encoding truncated foot-and-mouth disease virus type A capsid proteins SUMO-VP0, SUMO-VP3 and SUMO-VP1

[0106] 1.1 Preparation and transformation of recombinant plasmids

[0107] Suzhou Jinweizhi Biotechnology Co., Ltd. synthesized the A type foot-and-mouth disease virus VP0 gene fragment shown in SEQ ID NO.2 of the sequence list, the A type foot-and-mouth disease virus N-terminal truncated 10 amino acids VP0 gene fragment (VP0△10) shown in SEQ ID NO.4, the A type foot-and-mouth disease virus N-terminal truncated 15 amino acids VP0 gene fragment (VP0△15) shown in SEQ ID NO.6, the A type foot-and-mouth disease virus N-terminal truncated 20 amino acids VP0 gene fragment (VP0△20) shown in SEQ ID NO.8, the A type foot-and-mouth disease virus N-terminal truncated 40 amino acids VP0 gene fragment (VP0△40) shown in SEQ ID NO.10, the A type foot-and-mouth disease virus VP3 gene fragment shown in SEQ ID NO.12 of the sequence list, and the A type foot-and-mouth disease virus VP1 gene fragment shown in SEQ ID NO.14 of the sequence list, and connected them to the pETSUMO vector respectively. Then, the fragments containing SUMO-VP0, SUMO-VP3 and SUMO-VP1 were amplified using the successfully connected recombinant plasmids as templates. The fragments obtained after digestion with the corresponding endonucleases were cloned step by step into the same pET28a expression vector to obtain the recombinant plasmid containing SUMO-VP0-SUMO-VP3-SUMO-VP1, named: pET28a-SUMOVP0-SUMOVP3-SUMOVP1. Similarly, recombinant plasmids of different truncation lengths were obtained: pET28a-SUMOVP0△10-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△15-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△20-SUMOVP3-SUMOVP1, and pET28a-SUMOVP0△40-SUMOVP3-SUMOVP1.

[0108] The above-mentioned pET28a-SUMOVP0-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△10-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△15-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△20-SUMOVP3-SUMOVP1, and pET28a-SUMOVP0△40-SUMOVP3-SUMOVP1 recombinant plasmids were respectively transformed into competent Escherichia coli BL21 (DE3), spread on kanamycin-resistant solid LB medium, and cultured at 37°C for 10-12 hours until single colonies were clearly visible. Single colonies were picked and transferred to test tubes of liquid LB medium containing kanamycin. The culture was shaken at 37°C and 230 rpm for 12 hours, and the supernatant was discarded by centrifugation. The cells were then resuspended in sterile LB liquid medium and glycerol was added to a final concentration of 15%. After shaking, the cells were stored at -80°C.

[0109] 1.2 Expression of recombinant proteins

[0110] BL21 (DE3) Escherichia coli strains carrying the recombinant plasmids pET28a-SUMOVP0-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△10-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△15-SUMOVP3-SUMOVP1, pET28a-SUMOVP0△20-SUMOVP3-SUMOVP1, and pET28a-SUMOVP0△40-SUMOVP3-SUMOVP1, respectively, were taken out from -80°C and inoculated into 50 ml of kanamycin-resistant LB liquid medium. After shaking culture at 37°C and 230 rpm for 12 hours, the culture was transferred to 1 L of LB liquid medium and cultured at 37°C to prepare seed liquid for fermentation.

[0111] The fermentation tank used was a 50L fermentation tank from Shanghai Baoxing Biological Company. 30L of culture medium was prepared and placed in the fermentation tank and sterilized at 121°C for 30 minutes. The next day, 3L of seed solution was added to the fermentation tank and the culture solution concentration reached OD 600 When the value is about 10, the culture temperature is lowered to 25℃, and IPTG is added to a final concentration of 0.5mM to induce the culture for 12 hours. The fermentation density is about 40 (OD 600 ) Stop the culture and collect the bacteria by centrifugation.

[0112] The cells were resuspended and disrupted four times using a homogenizer at 800 bar. The cells were centrifuged at 13,500 rpm for 40 minutes. The supernatant was collected and the protein was crudely purified using ammonium sulfate fractionation precipitation, followed by chromatographic purification. Protein concentration was measured using BCA assay, and the results are shown in Table 1. 031 represents the untruncated VP0 FMDV type A capsid protein. The results showed that the concentration of the purified truncated FMDV type A capsid protein increased with increasing amino acid truncation length, and the purity reached over 90%.

[0113] Table 1 Protein concentrations after purification of different truncated proteins Protein type Protein concentration (mg / mL) purity(%) 031 3.2 91 031-VP0△10 4.1 92 031-VP0△15 4.2 90.5 031-VP0△20 5.8 95 031-VP0△40 6.1 90

[0114] Example 2: Assembly and characterization of truncated foot-and-mouth disease virus type A capsid proteins VP0, VP3 and VP1

[0115] The purified protein solution (20 mM sodium hydrogen phosphate buffer) from the previous step was incubated in 5 U / 0.5 ml Ulp1 protease, 0.5 M (NH4)2SO4, pH 8.0 at 15°C for 24 hours. Nickel filler was then added to adsorb SUMO protein to obtain the final assembly state of FMDV VLPs. The assembled particles were subjected to liquid chromatography and dynamic light scattering detection to evaluate the assembly of the VLPs. The results are shown in Table 2.

[0116] High-performance liquid gel filtration chromatography (HPSEC) analysis was performed using an Agilent 1260 system. A TSK SWXL series column (300 × 7.8 mm, ID, 45 nm pore size) was used for VLP analysis. The mobile phase was PBS, the injection volume was 50 μL, the flow rate was 0.5 mL / min, and the UV detector was set to a wavelength of 280 nm.

[0117] Dynamic light scattering (DLS) detection was performed using a Zetasizer ZSE dynamic light scattering instrument produced by Malvern.

[0118] The results showed that after VLP assembly, the untruncated protein 031 formed aggregates in approximately 48% of the assembled protein. Truncation of the VP0 N-terminus improved protein aggregation. Truncation lengths of 10, 15, and 20 amino acids allowed VLP assembly and reduced aggregation. As the truncation length increased, the proportion of aggregates decreased, the number of VLPs increased, and the assembly efficiency increased. When the truncation length was 40 amino acids, aggregates were almost absent, but the VLP assembly efficiency decreased. These experimental results indicate that truncation of the VP0 N-terminus by 10-20 amino acids does not affect VLP assembly, and that the stability of the VLPs increases with increasing truncation length. However, the assembly efficiency of the VLPs decreased with increasing truncation length to 40 amino acids. The particle sizes of these assembled samples, as measured by DLS, ranged from 26.37 nm to 35.62 nm.

[0119] Table 2 VLPs assembly efficiency detected by HPSEC and diameter detected by DLS Protein type Aggregate ratio VLPs share Diameter (nm) 031 48％ 52％ 35.62 031-VP0△10 4％ 96％ 29.05 031-VP0△15 4％ 96％ 28.46 031-VP0△20 3％ 97％ 28.48 031-VP0△40 1％ 88％ 26.37

[0120] The VLPs 031-VP0△10, 031-VP0△15 and 031-VP0△20 with better VLP assembly efficiency were stored at 2-8℃ for 3 months. The VLPs were very stable and the proportion of VLPs did not change significantly (see Table 3 for the results).

[0121] Table 3 Shelf life test: HPSEC detection of VLPs assembly efficiency and DLS detection of diameter Protein type Aggregate ratio VLPs share Diameter (nm) 031-VP0△10 4％ 96％ 29.05 031-VP0△15 4％ 96％ 28.46 031-VP0△20 3％ 97％ 28.48

[0122] Example 3: Analysis of the immunogenicity of VLPs assembled from truncated foot-and-mouth disease virus type A capsid proteins VP0, VP3 and VP1

[0123] 1. Vaccine Preparation

[0124] Take the foot-and-mouth disease virus-like particles obtained in Example 2 with 10, 15 and 20 amino acids truncated and slowly add them to an equal amount of adjuvant. During the addition process, stir continuously with an emulsifier at a speed of 400 rpm for 10 minutes, mix well, and store at 4°C to obtain a vaccine composition containing type A foot-and-mouth disease virus-like particle antigen. The specific ratio is shown in Table 4. The adjuvants suitable for this application can be any adjuvant known to those skilled in the art. In this application, the adjuvant selected is a biphasic adjuvant (water-in-oil-in-water emulsion), for example, the adjuvant ISA 206 (Seppic, France).

[0125] Table 4 Composition ratio of the A-type foot-and-mouth disease virus-like particle vaccine composition Components Vaccine 1 Vaccine 2 Vaccine 3 031-VP0△10(μg / ml) 160 - - 031-VP0△15(μg / ml) - 200 - 031-VP0△20(μg / ml) - - 240 Biphasic adjuvant (V / V%) 50％ 50％ 50％

[0126] 2. Immunization Procedure

[0127] Twenty healthy, one-month-old pigs that were negative for foot-and-mouth disease type A antibodies were screened using an ELISA kit and randomly divided into four groups: a immunization group of five pigs and a control group of five pigs. Group 1 served as the PBS control group, while groups 2 to 4 received Vaccine 1, Vaccine 2, and Vaccine 3, as prepared above. The immunization group received 2 mL of PBS intramuscularly, while the PBS control group received the same amount of PBS. Blood was collected from each pig before immunization and weekly for three weeks after immunization, continuing for six weeks.

[0128] 3. Antibody level detection

[0129] The collected serum was tested for FMD A antibody using an ELISA kit. The specific results of FMD ELISA antibody levels in pigs after vaccination are shown in Table 5.

[0130] Table 5 ELISA antibody levels

[0131] The results showed that all pigs had negative ELISA antibody levels before vaccination. Two weeks after vaccination, all pigs had positive antibodies. Three weeks after vaccination, all pigs had antibodies above 1:180. No decrease in ELISA antibody levels was observed after six weeks. Antibody levels in pigs in the PBS control group remained negative and unchanged.

[0132] It is proved that the truncated type A foot-and-mouth disease virus-like particle vaccine provided in this application can quickly form high levels of specific antibodies and play a good immune protection role in pigs.

[0133] The above description is merely a preferred embodiment of the present application and does not constitute any form of limitation to the present application. Although the present application has been disclosed as a preferred embodiment, it is not intended to limit the present application. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present application. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present application that do not depart from the content of the technical solution of the present application are still within the scope of the technical solution of the present application.

Claims

1. A truncated foot-and-mouth disease virus structural protein precursor protein P1 type A, characterized in that: Compared with the wild-type foot-and-mouth disease virus structural protein precursor protein P1, the N-terminus of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is truncated by 10 to 40 amino acids.

2. The protein P1 according to claim 1, characterized in that The N-terminus is truncated by 10, 15, 20 or 40 amino acids.

3. The protein P1 according to claim 1, characterized in that The type A truncated foot-and-mouth disease virus structural protein precursor protein P1 includes VP0 protein, VP3 protein and VP1 protein in sequence, the VP0 protein has the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9, the VP3 protein has the amino acid sequence shown in SEQ ID NO.11, and the VP1 protein has the amino acid sequence shown in SEQ ID NO.

13.

4. The protein P1 according to claim 3, characterized in that The type A truncated foot-and-mouth disease virus structural protein precursor protein P1 is expressed by a SUMO expression system, and the protein expressed in series in the form of SUMOVP0, SUMOVP3 and SUMOVP1 can bind to the Ni affinity chromatography medium in a solution with a pH of 7.5 to 8.5; Preferably, the proteins expressed in series as SUMOVP0, SUMOVP3 and SUMOVP1 can assemble to form stable VLPs in a solution with a pH of 7.5 to 8.5 and a salt concentration of 0 mM to 500 mM.

5. A type A truncated foot-and-mouth disease virus-like particle antigen, characterized in that: The antigen is obtained by self-assembly of VP0 protein, VP3 protein and VP1 protein of the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus according to any one of claims 1 to 4.

6. A biomaterial related to the protein P1 according to any one of claims 1 to 4, characterized in that: The biological material includes any one of the following: (1) a nucleic acid fragment encoding the structural protein precursor protein P1 of the type A truncated foot-and-mouth disease virus; (2) an expression cassette containing the nucleic acid fragment of (1); (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2); (4) A recombinant cell containing the nucleic acid in (1), the expression cassette in (2), or the vector in (3); Preferably, the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 includes VP0 protein, VP3 protein and VP1 protein in sequence, the nucleic acid fragment encoding the VP0 protein has the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO8 or SEQID NO.10, the nucleic acid fragment encoding the VP3 protein has the nucleotide sequence shown in SEQ ID NO.12, and the nucleic acid fragment encoding the VP1 protein has the nucleotide sequence shown in SEQ ID NO.

14.

7. Use of the protein P1 according to any one of claims 1 to 4, the antigen according to claim 5, or the biomaterial according to claim 6 in the preparation of a medicament for preventing infection by type A foot-and-mouth disease virus.

8. A subunit vaccine for foot-and-mouth disease type A, characterized in that: The subunit vaccine comprises a pharmaceutically acceptable carrier and an immunizing amount of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 according to any one of claims 1 to 4 or the type A truncated foot-and-mouth disease virus-like particle antigen according to claim 5.

9. The subunit vaccine according to claim 8, characterized in that The content of the type A truncated foot-and-mouth disease virus structural protein precursor protein P1 or the type A truncated foot-and-mouth disease virus-like particle antigen is ≥160 μg / ml, preferably 160-240 μg / ml.

10. The subunit vaccine according to claim 8 or 9, characterized in that The pharmaceutically acceptable carrier includes at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; Preferably, the adjuvant comprises one or more of aluminum gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, and muramyl dipeptide; Preferably, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, more preferably 50% V / V; Preferably, the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids; Preferably, the immunostimulant comprises α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin-2.

Images