Immunogenic composition containing p30 protein of african swine fever virus, and use thereof

Abstract

The present invention relates to an immunogenic fragment of a p30 protein of an African swine fever virus, a recombinant protein, an immunogenic composition, and a use. The immunogenic fragment of the p30 protein of the African swine fever virus or a variant having immunogenicity can greatly improve the expression quantity, and can also reserve strong immunocompetence.

Description

Immunogenic composition of African swine fever virus p30 protein and its application Technical Field

[0001] The present disclosure relates to immunogenic compositions, and in particular to African swine fever virus p30 protein or its immunogenic fragments, corresponding encoding nucleotide sequences, immunogenic compositions and uses thereof. Background Art

[0002] African swine fever is a viral disease caused by infection with the African swine fever virus (ASFV). The acute form clinically manifests as high fever, depression, anorexia, cyanosis of the skin, and bleeding in various organs. The disease is highly contagious and fatal, with morbidity and mortality rates reaching 100%.

[0003] Due to the large genome structure and complex immune escape mechanisms of ASFV, developing an effective vaccine is extremely difficult. To date, no safe and effective vaccine exists for epidemic prevention and control. Previous research on African swine fever vaccines has shown that inactivated vaccines can induce a high level of humoral immune response but do not provide immune protection. Therefore, current approaches to African swine fever vaccine design primarily focus on attenuated and subunit vaccines.

[0004] Research on attenuated vaccines is progressing more rapidly across various countries, but their challenges are becoming increasingly apparent. Attenuated vaccines typically only protect against homologous strains of the same genotype and offer no protection against heterologous virus challenges. Attenuated vaccines are also often associated with adverse side effects, such as skin lesions and joint swelling. Furthermore, attenuated vaccines can lead to chronic or persistent infection and potentially revert to virulence.

[0005] Compared to attenuated vaccines, subunit vaccines offer a targeted approach with fewer side effects and greater safety. Previous research has shown that various ASFV antigens can induce neutralizing antibodies and provide partial immune protection, paving the way for the development of a safe and effective ASF vaccine. With in-depth research into the structure and immunology of ASFV, the design of effective subunit vaccines capable of generating protective antibodies and specific cellular immune responses has become a hot topic in the field.

[0006] Summary of the Invention

[0007] In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure targets the African swine fever virus (ASFV) p30 protein and finds that through reasonable truncation expression, the expression level of the p30 protein can be increased while retaining strong immune activity.

[0008] According to one aspect of the present disclosure, an immunogenic fragment or an immunogenic variant thereof is provided, wherein the immunogenic fragment comprises at least the amino acid fragment from positions 110 to 194 of the amino acid sequence shown in SEQ ID NO: 1.

[0009] In some embodiments, the immunogenic fragment includes at most amino acids 44 to 194 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes at most amino acids 57 to 194 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes at most amino acids 65 to 194 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes amino acids 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 to 194 of the amino acid sequence shown in SEQ ID NO: 1.

[0010] In a preferred embodiment, the immunogenic fragment may include amino acid fragments at positions 44 to 194, 57 to 194, 65 to 194, 92 to 194, and 110 to 194 of the amino acid sequence shown in SEQ ID NO: 1.

[0011] In some embodiments, the immunogenic fragment can have an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and / or SEQ ID NO: 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity thereto.

[0012] According to another aspect of the present disclosure, a recombinant protein is provided, comprising: a first domain comprising the above-mentioned immunogenic fragment or an immunogenic variant thereof; and a second domain comprising a lytic tag or a backbone polypeptide for forming nanoparticles.

[0013] In some embodiments, the second domain serves as a scaffold polypeptide and is capable of being assembled into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[0014] In some embodiments, the second domain can be selected from, for example, I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT2, I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1, I53-50B4 PostT1, sumo, Encapsulin, LS (dioxotetrahydropteridine synthase), E2P (dihydrosulfonyl acetyltransferase) and at least one backbone polypeptide among I3.

[0015] In a specific embodiment, the second domain can be selected from I52-32A, LS.

[0016] In some embodiments, each of the second domains (e.g., LS) can independently self-assemble into sixty-mer nanoparticles. In some embodiments, the second domains can be paired and assembled into sixty-mer nanoparticles, such as I52-32A paired with I52-32B. In some embodiments, the first domain and the second domain are connected by a linker to form a fusion protein.

[0017] In some embodiments, the second domain is used as a lytic tag and is fused with the immunogenic fragment or its immunogenic variant gene to express, which can significantly promote the correct folding of the expressed protein and improve the solubility of the expressed protein.

[0018] In some embodiments, the second domain is selected from at least one solubility-promoting tag selected from sumo (molecular ubiquitin-like modifier protein), GST (glutathione S-transferase), MBP (mannose binding protein), Trx (thioredoxin) and NusA (N utilization protein A).

[0019] In some embodiments, the first domain is directly connected to the second domain or is connected through a linker.

[0020] According to another aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein of the present disclosure.

[0021] According to another aspect of the present disclosure, an expression vector is provided, which includes the above-mentioned nucleic acid molecule of the present disclosure.

[0022] In some embodiments, the expression vector can be selected from a viral or bacterial vector, such as, but not limited to, an African swine fever virus vector, a lentiviral vector, an avian pox virus vector, a canine measles virus vector, a herpes virus vector, a varicella virus vector, an adenovirus vector, an adeno-associated virus vector, and the like.

[0023] According to another aspect of the present disclosure, a host cell is provided, which comprises the aforementioned nucleic acid molecule, or is capable of expressing the aforementioned immunogenic fragment or immunogenic variant thereof or the aforementioned recombinant protein of the present disclosure.

[0024] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0025] In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc.

[0026] In some embodiments, the eukaryotic cells can be selected from yeast cells, insect cells, plant cells, animal cells, etc., such as yeast cells, CHO cells, 293 cells, Vero cells, or NSO cells.

[0027] According to another aspect of the present disclosure, a nanoparticle is provided, comprising the immunogenic fragment or immunogenic variant thereof or the recombinant protein disclosed herein, wherein the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

[0028] In some embodiments, the nanoparticles include hexamers self-assembled from scaffold proteins, and the scaffold proteins can be selected from, for example, LS, E2P, and I3.

[0029] In some embodiments, the nanoparticles include a sixty-mer formed by pairing and assembling the backbone protein in the recombinant protein with another backbone protein, for example, I53-34A is paired with I53-34B, I53-40A is paired with I53-40B, I53-47A is paired with I53-47B, I53-50A is paired with I53-50B, I53-51A is paired with I53-51B, I52-03A is paired with I52-03B, I52-32A is paired with I52-32B, I52-33A is paired with I52-33B, I32-06A is paired with I32-06B, I32-19A is paired with I32-19B, and I32-28A is paired with I32-28B.

[0030] According to another aspect of the present disclosure, an immunogenic composition is provided, comprising: the above-mentioned immunogenic fragment or immunogenic variant thereof, or the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned nanoparticle; and a pharmaceutically acceptable carrier.

[0031] In some embodiments, the immunogenic composition may include: one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof, or the above-mentioned recombinant proteins, or the above-mentioned nanoparticles, or nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants or recombinant proteins disclosed herein.

[0032] In some embodiments, the immunogenic composition may further include additional African swine fever virus antigens.

[0033] In some embodiments, the pharmaceutically acceptable carrier includes an adjuvant, which includes: a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative polymer; an immunostimulatory sequence (ISS), such as an oligodeoxyribonucleotide sequence (CpG ODN) having one or more unmethylated CpG units; a water-in-oil (W / O) adjuvant, an oil-in-water (O / W) adjuvant or an oil-in-water-in-oil (W / O / W) adjuvant, such as Freund's adjuvant, SPT emulsion, MF59, ISA 206, ISA72, adjuvant-65, SAF, etc.; a cationic lipid containing a quaternary ammonium salt such as DDA; a cytokine; aluminum hydroxide or aluminum phosphate; a saponin (e.g., Quil A, QS-21, GPI-0100); or any combination or mixture thereof.

[0034] In a preferred embodiment, the saponin is Quil A, QS-21, or GPI-0100.

[0035] In a preferred embodiment, the adjuvant comprises an emulsion; the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed by combining an oil with an emulsifier, the emulsion being based on light liquid paraffin oil, isoprenoid oils produced by olefin oligomerization (such as squalane or squalene oil, olefins, especially oils produced by oligomerization of isobutylene or decene), linear alkyl esters of acids or alcohols (more especially vegetable oils, ethyl oleate, propylene glycol di-(octanoate / caprylate), glycerol tri-(octanoate / caprylate) or propylene glycol dioleate), branched fatty acids or esters of alcohols (especially isostearates); emulsifiers are nonionic surfactants (especially esters of polyoxyethylated fatty acids (for example oleic acid), esters of sorbitan, esters of mannide (such as anhydrous mannitol oleate), esters of aliphatic diols, esters of glycerol, esters of polyglycerols, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which esters may be ethoxylated, ethers of fatty alcohols and polyols (for example oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially L121).

[0036] In a preferred embodiment, the polymer of acrylic acid or methacrylic acid is a cross-linked acrylic acid or methacrylic acid polymer, in particular a compound cross-linked with a polyalkenyl ether of a sugar or a polyol, carbomer, preferably Carbopol 974P, 934P and 971P.

[0037] In a preferred embodiment, the copolymer of maleic anhydride and an alkenyl derivative is a copolymer of maleic anhydride and ethylene, EMA. In a preferred embodiment, the adjuvant is Gel 01 adjuvant.

[0038] In some embodiments, the immunogenic composition can be administered orally, intradermally, intramuscularly, or intranasally.

[0039] According to another aspect of the present disclosure, provided is the use of one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, the above-mentioned host cells, the above-mentioned nanoparticles or the above-mentioned immunogenic compositions in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

[0040] According to yet another aspect of the present disclosure, a method for preventing and / or treating African swine fever virus infection in a subject is provided.

[0041] In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal of the Suidae family, such as a pig. In some embodiments, the individual or subject can be a wild boar (Sus scrofa), a domestic pig (Sus scrofa domesticus), a warthog (Potamochoerus), a forest pig (Hylochoerus), a giant forest pig (Hylochoerus), an African wild boar (Potamochoerus), or a feral pig.

[0042] In some embodiments, the African swine fever virus infection can be a pathogenic African swine fever virus infection. In some embodiments, the symptoms or diseases of pathogenic African swine fever virus infection can be selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleed, abortion, leukopenia, and thrombocytopenia.

[0043] According to another aspect of the present disclosure, a kit for detecting African swine fever virus infection is provided, wherein the kit comprises the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein of the present disclosure.

[0044] According to another aspect of the present disclosure, provided is the use of the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein in the preparation of a kit for detecting African swine fever virus infection.

[0045] In some embodiments, the sample is selected from a body fluid or tissue sample from a subject. In some embodiments, the sample can be selected from a blood, saliva or serum sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1 shows the SDS-PAGE results of different p30 recombinant proteins.

[0047] FIG2 shows the results of the binding strength of different p30 recombinant proteins or their assembled particles to African swine fever positive serum.

[0048] FIG3 shows the results of immunogenicity assays of different p30 recombinant proteins or their assembled particles. DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below with reference to the following embodiments. The specific embodiments described herein are intended only to illustrate the present disclosure and are not intended to limit the present disclosure in any way. In addition, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0050] The African swine fever virus (ASFV) genome is over 170 kb and contains over 150 open reading frames (ORFs). Viral particles exceed 200 nm in diameter. The virus forms a viral factory around the cell nucleus, where viral replication and assembly occur. ASFV particles are icosahedral and possess a multilayered envelope structure, consisting of an internal core (also called a nucleoid or viral nucleoid), a core shell, an inner envelope, a capsid, and an outer envelope (primarily composed of lipids and a small amount of protein). ASFV-encoded proteins play important roles in viral assembly, DNA replication and repair, and gene expression. Furthermore, the ASFV genome encodes numerous proteins involved in immune evasion, including proteins that inhibit type I interferon and induce apoptosis, such as DP96R, MGF-505-7R, and pE199L.

[0051] The p30 protein is encoded by the CP204L gene of the African swine fever virus (ASFV). P30 is typically expressed early in ASFV infection and remains expressed throughout the infection period. The p30 protein triggers a strong immune response, and antibodies against p30 can be detected early in infection. Antibodies against p30 can inhibit ASFV replication, making it a promising protective antigen.

[0052] In some embodiments, the full-length p30 protein of African swine fever virus has the amino acid sequence shown in SEQ ID NO:1.

[0053] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0054] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0055] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0056] The ASFV immunogenic compositions described herein are preferably subunit vaccines. The "subunit vaccines" described herein comprise one or more polypeptides or proteins derived from ASFV, or immunogenic fragments thereof, or one or more nucleic acid molecules encoding immunogenic fragments thereof, and wherein the nucleic acid molecules are capable of being expressed in pigs. These polypeptides or proteins, immunogenic fragments thereof, or one or more nucleic acid molecules encoding immunogenic fragments thereof can be prepared using techniques known in the art.

[0057] As used herein, the term "immunogenic composition" refers to a composition comprising at least one antigen that induces an immunological response in a host or individual to which the immunogenic composition is administered. The immunological response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the present disclosure. Preferably, the immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more clinical signs of ASFV infection. Preferably, any host or individual referred to herein is an animal.

[0058] As used herein, numerical ranges are to be understood as including all numbers within the range. For example, a range of 1 to 20 is to be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0059] The term "connector" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. In some embodiments, the amino acid sequences of all the connectors present in the recombinant protein of the present invention are identical. In other embodiments, the amino acid sequences of at least two connectors present in the recombinant protein of the present invention are different. The connector should have a length suitable for connecting two or more monomer domains in this way, and the connector can ensure that the different domains it is connected to are correctly folded and appropriately presented, thereby exerting the function of its biological activity. In different embodiments, the connector has a flexible conformation. Suitable flexible connectors include, for example, having glycine, glutamine and / or serine residues.

[0060] In some embodiments, the linker of the present disclosure can be a flexible peptide linker. In some embodiments, the peptide linker is rich in glycine, serine, alanine, proline and / or glutamine residues. In some embodiments, the peptide linker can be selected from (G n S) m , wherein n and m are each independently selected from an integer of 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5.

[0061] "Percent (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the sequences and (as needed) introducing gaps to obtain maximum percent sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, alignment can be performed in various ways within the scope of this art, for example, using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves maximum alignment over the full length of the compared sequences.

[0062] The variants of the immunogenic fragments of the p30 protein disclosed herein can be obtained by substitution, addition or deletion of one or more amino acids, thereby having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the immunogenic fragments of the p30 protein in terms of amino acid sequence, while retaining comparable immunogenicity thereto.

[0063] The immunogenic variants of the p30 protein immunogenic fragments disclosed herein can be obtained by replacing one or more conservative amino acids in the p30 protein immunogenic fragments. In some embodiments, the replacement of conservative amino acids can mean that an amino acid residue is replaced with a residue with similar biological properties. Particularly preferred substitutions are generally conservative in nature, that is, those that occur within the amino acid family. For example, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative changes include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, or substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, or similar conservative substitutions of amino acids with structurally related amino acids that do not significantly affect biological activity. Thus, proteins having an amino acid sequence substantially identical to a reference molecule but with minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide.

[0064] The second domain described herein can self-assemble in vitro or be paired with another scaffold protein to form nanoparticles. The immunogenic fragment of the p30 protein or its variant used herein forms a fusion protein with the second domain, and the immunogenic fragment of the p30 protein or its variant is ultimately displayed on the surface of the nanoparticle.

[0065] In one embodiment, the second domain can be selected from some polypeptides synthesized in vitro that can self-assemble into nanoparticles, such as I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52 These polypeptides can self-assemble in pairs into nanostructures, such as icosahedral nanostructures, in vitro.

[0066] The small ubiquitin-like modifier (SUMO) is a molecular chaperone with a structure similar to ubiquitin (a globular fold of β-pleated sheets wrapped around an α-helix). It is conserved in eukaryotes and participates in various physiological processes, including protein post-translational modification. Because it has a highly hydrophobic core that provides a nucleation site for the folding of fusion proteins, it serves as a molecular chaperone and fusion tag to improve the stability and solubility of exogenous proteins. The tertiary structure of SUMO proteins is fully recognized by SUMO proteases, and after specific enzymatic cleavage, the target protein retains its native N-terminus, ensuring its activity. The SUMO expression system significantly outperforms traditional expression systems in terms of expression levels and product solubility.

[0067] As used herein, the term "immunogenic composition" generally refers to a composition comprising at least one antigen or an immunogenic portion thereof that elicits an immune response in a host, either a cellular immune response or an antibody-mediated immune response, against the composition. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more of the clinical symptoms of ASFV infection. In cases where the host exhibits a protective immune response resulting in increased resistance to the novel infection and / or reduced clinical severity of the disease, the immunogenic composition may also be referred to as a "vaccine."

[0068] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0069] As used herein, the term "prevent and / or treat" refers to reducing the incidence of a specific ASFV infection, or reducing the severity of clinical symptoms caused by or associated with a specific ASFV infection. Additionally, the term "prevent and / or treat" may also refer to reducing the number of animals infected with a specific ASFV (i.e., reducing the incidence of ASFV infection), or reducing the severity of clinical symptoms typically associated with or caused by an ASFV infection, in a group of animals that have received an effective amount of an immunogenic composition as provided herein, compared to a group of animals that have not received the immunogenic composition.

[0070] As used herein, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect.

[0071] The following examples and figures are provided to facilitate understanding of the present disclosure. However, it should be understood that these examples and figures are intended to illustrate the present disclosure only and are not intended to limit the present disclosure in any way. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present disclosure.

[0072] The p30 protein is an early-stage protein expressed by the African swine fever virus (ASFV). It has strong immunogenicity and can induce the production of neutralizing antibodies in infected hosts. The p30 protein is crucial for the establishment of effective ASFV infection, and therefore the development of a subunit vaccine containing the p30 protein is of great significance for the prevention and control of ASF. The present disclosure provides a simple, convenient, and low-cost recombinant protein capable of soluble expression of p30 and its truncations while maintaining excellent immunogenicity, which will facilitate the development of a subunit vaccine for ASF.

[0073] Example 1: Construction of plasmid

[0074] The gene encoding the full-length or truncated p30 protein is ligated to genes encoding various lytic tags (SUMO, GST, MBP, etc.) or multimeric scaffold proteins (I52-32A, LS, etc.) via a nucleic acid sequence encoding a "linker." Overlapping PCR is then used to generate the recombinant prokaryotic gene fragment. The recombinant gene fragment is double-digested with Nde I and Xho I restriction endonucleases. The digested fragment is then ligated with a linearized pET-28a vector using DNA ligase to generate the prokaryotic recombinant plasmid. The N-terminal or C-terminal His tag encoded in the pET-28a vector can be used for subsequent protein purification.

[0075] The sequence information involved in this example is shown in Table 1 below.

[0076] Table 1

[0077] Example 2: Prokaryotic expression, purification and detection of p30 protein

[0078] Taking His-SUMO fusion protein as an example, the specific steps are as follows:

[0079] (1) Protein expression: The constructed p30 expression plasmid was transformed into BL21 (DE3) and cultured in LB medium at 37°C overnight to serve as seed bacteria. The next day, the seed bacteria were inoculated into 1 L LB medium at a ratio of 1:100 and cultured at 37°C until the OD value reached 0.6-1.0. The culture was then cooled to 20°C and induced with 0.5 mM IPTG overnight.

[0080] (2) Protein purification: Collect the bacteria and add Tirs buffer (40mL / L bacterial solution) to the bacteria and resuspend them evenly. After the above bacterial solution is crushed with a high-pressure crusher, centrifuge at high speed and collect the supernatant. The supernatant slowly flows through a nickel affinity chromatography column equilibrated with Tris buffer. After the sample is loaded, the Ni column is rinsed with Tris buffer containing 0.5% Triton X-114 for 5 column volumes to remove endotoxins and foreign proteins. After rinsing with Tris buffer for 5 column volumes again to remove residual Triton X-114, the target protein is eluted with Tris buffer containing 500mM imidazole and collected. The collected target protein is mounted on an anion exchange chromatography column and eluted with a gradient salt concentration to collect the target protein. After adding Ulp1 enzyme digestion, the nickel affinity chromatography column is reversely mounted and the flow-through is collected as the final target protein.

[0081] (3) SDS-PAGE Identification of Target Protein: Take an appropriate amount of purified target protein, add the corresponding proportion of loading buffer, and treat in boiling water for 10 minutes. Perform SDS-PAGE and then stain with Coomassie Brilliant Blue. The SDS-PAGE results of exemplary recombinant proteins of SUMO and p30 protein and its truncated form are shown in Figure 1 below. The purified protein meets the expected molecular weight.

[0082] (4) Evaluation of target protein yield: The protein concentration of the purified p30 protein was determined using a NanoDrop instrument. Yield per liter (mg / L) = product volume (ml) × protein concentration (mg / ml) / fermentation volume (L). The results are shown in Table 2 below.

[0083] Table 2 Expression levels of p30 protein in each construct (mg / L)

[0084] Example 3: The purified recombinant protein was subjected to a sixty-mer assembly experiment

[0085] The purified P30 recombinant proteins from Example 2 were self-assembled or mixed with another scaffold protein paired with the scaffold protein for paired assembly. After mixing, the mixture was allowed to stand in a refrigerator at 4°C for 24 hours to complete the assembly. A negatively stained grid was prepared for the sample, and the assembly effect of the nanoparticles was detected using a 120 kV electron microscope. The results are summarized in Table 3 below.

[0086] Table 3. Statistics of the effect of African swine fever P30 protein assembly into sixty-mers. Note: In the assembly effect, + represents that the particles can be assembled into sixty-mer particles, and - represents that the particles cannot be assembled into sixty-mer particles.

[0087] Example 4 African swine fever positive serum binding experiment

[0088] The p30 recombinant protein purified in Example 2 and the sixty-mer particle antigen assembled in Example 3 were used as coating antigens, African swine fever positive serum (purchased from the China Veterinary Drug Administration) was used as the blocking solution, goat anti-swine IgG-HRP was used as a marker, and TMB was used as a detection substrate. An enzyme-linked immunosorbent assay (ELISA) experiment was performed to determine the binding ability of the recombinant protein to the positive serum.

[0089] The specific steps are as follows: Take each purified recombinant protein and dilute it with 1×PBS (pH 8.0) to a 1μg / mL coating working solution. Add 100μL to each well of a 96-well microtiter plate and place it at 4°C overnight (12-16 hours). Remove the microtiter plate and discard the liquid in the wells. Wash it three times with PBST (1×PBS plus 0.5‰ Tween 20) and pat it dry on absorbent paper. Add 200μL of blocking buffer (PBST plus 0.2% BSA) to each well and place it at room temperature for 1 hour. Discard the blocking solution, wash it three times with PBST, and pat it dry on absorbent paper. Dilute African swine fever-positive serum 1:5000 in blocking buffer and add 100 μL to an antigen-coated ELISA plate. Incubate at room temperature for 60 minutes. Wash the plate three times with PBST. Then, add a goat anti-swine IgG-HRP marker diluted 1:5000 in blocking buffer to each well. Incubate at room temperature for 60 minutes. Wash the plate five times with PBST. After patting dry, add 100 μL of the substrate TMB to each well. Incubate at room temperature in the dark for 2-10 minutes. Add 50 μL of stop solution (2 M sulfuric acid) to each well. Measure absorbance at 450 nm using 630 nm as the reference wavelength. A positive result is determined when the absorbance is 2.0 times greater than that of the negative control (cell culture medium).

[0090] The exemplary results of each P30 recombinant protein binding to African swine fever positive serum are shown in Figure 2. The results show that each p30 recombinant protein and the sixty-mer particles assembled therefrom have a high binding strength to African swine fever positive serum, indicating that the above-mentioned recombinant proteins expressed in the present invention and the sixty-mer particles assembled therefrom have the correct spatial conformation.

[0091] Example 5 Antigen immunogenicity determination experiment

[0092] Six-week-old BALB / c mice were immunized using the purified portion of the p30 recombinant protein described in Example 2 and the partially assembled hexameric particle antigen described in Example 3 as immunogens. The first immunization consisted of a subcutaneous injection of 100 μg of antigen in Freund's complete adjuvant. Four weeks later, a secondary immunization was performed with 100 μg of antigen per mouse in Freund's incomplete adjuvant. Blood samples were collected before the first immunization (day 0), two weeks after the first immunization (week 2), four weeks after the first immunization (week 4), and two weeks after the second immunization (week 6), and antibody levels were subsequently determined by ELISA.

[0093] The specific steps are as follows:

[0094] (1) Six-week-old BALB / c mice were immunized with different immunogens using conventional methods. The first primary immunization was subcutaneous injection of 10 μg of antigen using Freund's complete adjuvant. The second immunization was performed 4 weeks later with 10 μg / mouse using Freund's incomplete adjuvant.

[0095] (2) Blood samples were collected before the first basic immunization (week 0), two weeks after the first immunization (week 2), four weeks after the first immunization (week 4), and two weeks after the second immunization (week 6). The intracellular immunofluorescence staining method was used to analyze the strength of the antibody level in the serum.

[0096] (3) Take the purified p30 recombinant protein or its assembled particles, dilute it with 1×PBS (pH 8.0) to a 1μg / mL coating working solution, add 100μL to each well of a 96-well ELISA plate, and place it at 4°C overnight (12-16 hours). Remove the ELISA plate and discard the liquid in the wells, wash it three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat it dry on absorbent paper. Add 200μL of blocking buffer (PBST plus 0.2% BSA) to each well and place it at room temperature for 1 hour. Discard the blocking solution, wash it three times with PBST, and pat it dry on absorbent paper. Sera from immunized mice at each time point were diluted 1:5000 in blocking buffer, and 100 μL was added to an antigen-coated ELISA plate. The plate was incubated at room temperature for 60 minutes, and the plate was washed three times with PBST. Then, goat anti-swine IgG-HRP conjugate diluted 1:5000 in blocking buffer was added to each well. The plate was incubated at room temperature for 60 minutes, and the plate was washed five times with PBST. After patting dry, 100 μL of TMB substrate was added to each well. The plate was incubated at room temperature in the dark for 2-10 minutes, and 50 μL of stop solution (2 M sulfuric acid) was added to each well. The absorbance at 450 nm was measured with a reference wavelength of 630 nm. A positive result was determined if the absorbance was 2.0 times greater than that of the negative control (cell culture medium).

[0097] The results of antigen immunogenicity assays of exemplary P30 prokaryotic recombinant proteins are shown in Figure 3. The results showed that each p30 recombinant protein had high immunogenicity, and the immunogenicity of the particulate antigen was significantly higher than that of the non-particulate antigen.

[0098] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An immunogenic fragment or an immunogenic variant thereof, characterized in that: The immunogenic fragment at least includes the amino acid fragment from positions 110 to 194 of the amino acid sequence shown in SEQ ID NO:

1.

2. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that: The immunogenic fragment includes at most amino acids 44 to 194 of the amino acid sequence shown in SEQ ID NO:

1.

3. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that: The immunogenic fragment has an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and / or SEQ ID NO: 14, or an amino acid sequence having at least 85% sequence identity thereto.

4. A recombinant protein, characterized in that The recombinant protein comprises: A first domain comprising the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3; and The second domain includes a solubility-promoting tag or a backbone polypeptide for forming nanoparticles.

5. The recombinant protein according to claim 4, characterized in that The lytic tag is selected from at least one of sumo, GST, MBP, Trx and NusA, and / or The backbone polypeptide is selected from I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT2, I53-47B.1, I53-47B.1 At least one of NegT2, I53-50A.1, I53-50A.1 NegT2, I53-50A.1 PostT1, I53-50B4PostT1, LS, E2P and I3, preferably, the second domain can form a hexameric nanoparticle, Preferably, the first domain is directly connected to the second domain or connected via a linker.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or encodes the recombinant protein according to claim 4 or 5.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 6, or is capable of expressing the immunogenic fragment or immunogenic variant thereof of any one of claims 1 to 3, or the recombinant protein of claim 4 or 5; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

8. A nanoparticle, characterized in that: The nanoparticle comprises the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5, Preferably, the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

9. An immunogenic composition, characterized in that The immunogenic composition comprises: an immunogenic fragment or an immunogenic variant thereof according to any one of claims 1 to 3, a recombinant protein according to claim 4 or 5, a nucleic acid molecule according to claim 6, a host cell according to claim 7 or a nanoparticle according to claim 8; and a pharmaceutically acceptable carrier, Preferably, the immunogenic composition further comprises an additional African swine fever virus antigen.

10. Use of the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4 or 5, the nucleic acid molecule according to claim 6, the host cell according to claim 7, the nanoparticle according to claim 8 or the immunogenic composition according to claim 9 in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

11. The use according to claim 10, characterized in that: The subject includes a mammal, Preferably, the subject comprises an animal of the family Suidae or a pig, More preferably, the subject includes wild boar (Sus scrofa), domestic pig (Sus scrofa domesticus), warthog (Potamochoerus), forest pig (Hylochoerus), giant forest pig (Hylochoerus), African wild boar (Potamochoerus) and feral pig.

12. The use according to claim 10 or 11, characterized in that: The African swine fever virus infection is a pathogenic African swine fever virus infection, Preferably, the disease or symptom of African swine fever virus infection is selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin maculopathy, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia.

13. A kit for detecting African swine fever virus infection, the kit comprising the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5.

14. Use of the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5 in the preparation of a kit for detecting African swine fever virus infection, Preferably, the African swine fever virus is from a body fluid or tissue sample of a subject. More preferably, the body fluid sample is selected from a blood, saliva or serum sample.

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