African swine fever virus-like particle and application thereof in preparation of African swine fever virus vaccine

Abstract

The invention provides an African swine fever virus-like particle and application thereof in preparation of an African swine fever virus vaccine, and relates to the technical field of biology. The capsid protein, the protein A, the protein B and the protein C provided by the invention have good immune effects and can be used in African swine fever virus vaccines. Wherein the protein C is a four-layer nested particle and maintains a three-dimensional conformation of neutralizing epitopes on the surface of the particle, so that the structural rigidity of the particle is enhanced, and a better immune effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an African swine fever virus-like particle and its application in the preparation of an African swine fever virus vaccine. Background Technology

[0002] African swine fever (ASF) is a highly contagious and deadly disease in pigs, listed as a notifiable disease by the World Organisation for Animal Health (WOAH), causing enormous economic losses to the global pig farming industry. Vaccination is the best strategy for preventing and controlling ASF outbreaks, but all vaccine technologies have failed to achieve safety and efficacy, including naturally occurring attenuated strains, artificially passaged attenuated strains, artificially gene-deleted attenuated strains, whole-virus inactivated vaccines, genetically engineered subunit vaccines, viral vector vaccines, and DNA vaccines. To date, there is no commercially available vaccine approved by the WOAH. Newly developed mRNA vaccines and T / B cell epitope vaccines have not yet achieved the expected results.

[0003] The infection, pathogenesis, genetic variation, and immune escape mechanisms of African swine fever virus (ASFV) are complex, and insufficient basic research is a significant factor hindering vaccine development. Since its discovery in 1921, researchers have attempted whole-virus inactivated vaccines, attenuated vaccines, genetically engineered subunit vaccines, and viral vector vaccines, all of which have ended in failure.

[0004] Since the discovery of ASFV, traditional methods were initially used to research vaccines. After long-term, extensive trials, the generally accepted view is that whole-virus inactivated vaccines have weak immunogenicity and cannot protect immunized animals against infection and disease. Various types of attenuated strains, whether passaged attenuated, genetically engineered attenuated, or naturally occurring attenuated strains, result in immunized animals carrying the virus for a certain period, posing a significant risk of viral mutation and recombination, and raising widespread concerns about biosafety. Subunit vaccines often vary significantly due to differences in protein types and combinations, expression hosts, immunization doses, adjuvant types, challenge strains, and doses, making reproducibility difficult and providing only partial protection, insufficient for prevention and control needs. Live vectors, primarily poxviruses, herpesviruses, and adenoviruses, have antigens largely similar to subunit vaccines, also failing to provide complete protection. Currently, given the aforementioned biosafety issues and the limitation of only partial protection, no commercially available attenuated, subunit, or live vector vaccines are on the market.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide an antigen to solve the aforementioned technical problems.

[0007] A second objective of this invention is to provide biological materials.

[0008] A third objective of this invention is to provide the application of the above-mentioned antigen in the preparation of African swine fever virus vaccines.

[0009] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a capsid protein comprising pB646L protein, molecular chaperone proteins pB602L and pM1249L protein, and a fusion protein of pB438L protein, pH240R protein, and pE120R protein; wherein the fusion protein of pB438L protein, pH240R protein, and pE120R protein is composed of pB438L protein, pH240R protein, and pE120R protein in tandem from the N-terminus to the C-terminus; the amino acid sequence of pB646L protein is shown in SEQ ID NO.11; the amino acid sequence of molecular chaperone protein pB602L protein is shown in SEQ ID NO.12; the amino acid sequence of pM1249L protein is shown in SEQ ID NO.13; the amino acid sequence of pB438L protein is shown in SEQ ID NO.14; the amino acid sequence of pH240R protein is shown in SEQ ID NO.15; and the amino acid sequence of pE120R protein is shown in SEQ ID NO.16.

[0010] As a further technical solution, the amino acid sequence of the fusion protein of pB438L protein, pH240R protein and pE120R protein is shown in SEQ ID NO.21.

[0011] In a second aspect, the present invention provides a protein A, comprising, from the inside out, a capsid protein and an outer membrane protein; the outer membrane protein comprising a fusion protein of pEP402R protein and pEP153R protein; the amino acid sequence of the pEP402R protein is shown in SEQ ID NO.17; the amino acid sequence of the pEP153R protein is shown in SEQ ID NO.18.

[0012] As a further technical solution, the capsid protein includes pB646L protein, molecular chaperone proteins pB602L and pM1249L protein, and a fusion protein of pB438L protein, pH240R protein and pE120R protein; the fusion protein of pB438L protein, pH240R protein and pE120R protein is composed of pB438L protein, pH240R protein and pE120R protein in series from N-terminus to C-terminus; Preferably, the amino acid sequence of the pB646L protein is shown in SEQ ID NO.11; the amino acid sequence of the molecular chaperone protein pB602L is shown in SEQ ID NO.12; the amino acid sequence of the pM1249L protein is shown in SEQ ID NO.13; the amino acid sequence of the pB438L protein is shown in SEQ ID NO.14; the amino acid sequence of the pH240R protein is shown in SEQ ID NO.15; and the amino acid sequence of the pE120R protein is shown in SEQ ID NO.16.

[0013] Preferably, the amino acid sequence of the fusion protein of pB438L protein, pH240R protein and pE120R protein is shown in SEQ ID NO.21; Preferably, the fusion protein of pEP402R and pEP153R proteins is composed of pEP402R and pEP153R proteins linked together from the N-terminus to the C-terminus; Preferably, in the fusion protein, the pEP402R protein and the pEP153R protein are linked by a furin cleavage site; Preferably, the amino acid sequence of the fusion protein of pEP402R and pEP153R proteins is shown in SEQ ID NO. 22.

[0014] Thirdly, the present invention provides a protein B, which comprises, from the inside out, an inner membrane protein, the capsid protein and the outer membrane protein; The inner membrane proteins include fusion proteins of pD117L, pKP177R, and pO61R proteins, as well as fusion proteins of pE248R, pE199L, pE183L, pH108R, and pCP204L proteins.

[0015] As a further technical solution, the amino acid sequence of the pD117L protein is shown in SEQ ID NO.3; the amino acid sequence of the pKP177R protein is shown in SEQ ID NO.4; the amino acid sequence of the pO61R protein is shown in SEQ ID NO.5; the amino acid sequence of the pE248R protein is shown in SEQ ID NO.6; the amino acid sequence of the pE199L protein is shown in SEQ ID NO.7; the amino acid sequence of the pE183L protein is shown in SEQ ID NO.8; the amino acid sequence of the pH108R protein is shown in SEQ ID NO.9; and the amino acid sequence of the pCP204L protein is shown in SEQ ID NO.10. Preferably, the fusion protein of pD117L protein, pKP177R protein and pO61R protein is composed of pD117L protein, pKP177R protein and pO61R protein in series from N-terminus to C-terminus. Preferably, in the fusion protein, the pD117L protein, pKP177R protein, and pO61R protein are linked by a furin cleavage site. Preferably, the amino acid sequence of the fusion protein of pD117L protein, pKP177R protein and pO61R protein is shown in SEQ ID NO.19.

[0016] Preferably, the fusion protein of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein is composed of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein in series from N-terminus to C-terminus. Preferably, in the fusion protein, pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein are linked by furin cleavage sites. Preferably, the amino acid sequence of the fusion protein of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein is shown in SEQ ID NO.20.

[0017] Fourthly, the present invention provides a protein C, comprising, from the inside out, a core-shell protein, the inner membrane protein, the capsid protein, and the outer membrane protein; the core-shell protein includes pCP530R protein and pCP2475L protein.

[0018] As a further technical solution, the amino acid sequence of the pCP530R protein is shown in SEQ ID NO.1; the amino acid sequence of the pCP2475L protein is shown in SEQ ID NO.2.

[0019] Fifthly, the present invention provides a biomaterial selected from any one of the following: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding the capsid protein, a nucleotide sequence encoding protein A, a nucleotide sequence encoding protein B, or a nucleotide sequence encoding protein C; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid in a, or containing the vector in b, or expressing the capsid protein, protein A, protein B, or protein C.

[0020] In a sixth aspect, the present invention provides the use of the above-mentioned capsid protein, protein A, protein B or protein C in the preparation of African swine fever virus vaccines.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The capsid protein provided by this invention is obtained by mutation based on wild-type capsid protein. The inventors have found that the mutated capsid protein of this invention has a better immune effect than the unmutated one and can be used in African swine fever virus vaccines.

[0022] The protein A provided by this invention is derived from the wild-type outer membrane protein through mutation. Protein A composed of the mutated outer membrane protein has a better immune effect than the unmutated protein and can be used in African swine fever virus vaccines.

[0023] The protein B provided by this invention is based on protein A with the addition of inner membrane protein. The inventors have found that protein B has a better immune effect than protein A and can be used in African swine fever virus vaccines.

[0024] The protein C provided by this invention further adds nucleocapsid protein to protein B, forming a four-layered nested particle that maintains a three-dimensional conformation with neutralizing epitopes on the particle surface. This strengthens the rigidity of the particle structure and provides better immunogenicity than protein B, making it suitable for use in African swine fever virus vaccines. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 For electron microscopic observation of the experimental group in Example 4; Figure 2 The control group in Example 4 was observed using electron microscopy; Figure 3 Electron microscopy was used to observe the experimental group in Example 5; Figure 4 This serves as the control group for electron microscopy observation in Example 5; Figure 5 For electron microscopic observation of the experimental group in Example 6; Figure 6 The control group in Example 6 was observed using electron microscopy; Figure 7 For electron microscopic observation of the experimental group in Example 7; Figure 8 This serves as the control group for electron microscopy observation in Example 7; Figure 9 For electron microscopic observation of test group 1 in Example 8; Figure 10 For electron microscopy observation of nuclear components in test group 2 of Example 8; Figure 11 For electron microscopic observation of test group 3 in Example 8; Figure 12 For electron microscopic observation of test group 4 in Example 8; Figure 13 For electron microscopy observation of test group 5 in Example 8. Detailed Implementation

[0027] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] Materials and Methods: The gene synthesis in this embodiment was commissioned to General Biotech (Anhui) Co., Ltd.

[0029] PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier, and adjustments can be made through simple experiments if necessary.

[0030] The rod-particle extraction experiment was based on Thermo's PureLink. TM Follow the instructions in the HiPure Plasmid Filter Maxiprep Kit manual. If necessary, adjustments can be made through simple experiments.

[0031] The VLP electron microscopy observations were commissioned to the Peking University Medical and Health Analysis Center.

[0032] 2YT agar plates: Tryptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L, agar 15 g / L, kanamycin 50 μg / mL, gentamicin 7 μg / mL, tetracycline 10 μg / mL, Bluo-gal 100 μg / mL, IPTG 40 μg / mL.

[0033] 2YT liquid medium: Tryptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L, kanamycin 50 μg / mL, gentamicin 7 μg / mL, tetracycline 10 μg / mL.

[0034] Protein Maker: Thermo, Catalog No.: 26616.

[0035] 50% Glycerin: First, heat 100mL of 100% glycerin in a container. Then, add 100mL of water to the heated glycerin container and stir well. This will give you 50% glycerin. Sterilize it under high pressure before use.

[0036] Example 1: Optimization of African Swine Fever Virus Protein Structure and Construction of Plasmids 1. Construction of nucleoshell protein particles 1.1 The core protein pCP530R is expressed alone.

[0037] The amino acid sequence is shown in SEQ ID NO.1.

[0038] Plasmid construction: The pCP530R gene codon was optimized and inserted downstream of the PpH promoter in the pFastBac Dual vector between BamHⅠ (GGA TCC) and Hind Ⅲ (AAG CTT), plasmid number 7. The CP530R nucleotide sequence is identical to that of the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1), plasmid number 7 hlj.

[0039] 1.2 The nucleocapsid protein pCP2475L was expressed alone.

[0040] The amino acid sequence is shown in SEQ ID NO.2.

[0041] Plasmid construction: The pCP2475L gene codon was optimized and inserted into the MCS between BamHⅠ (GGATCC) and HindⅢ (AAGCTT) downstream of the PpH promoter in the pFastBac1 vector. The plasmid was numbered 9. Using the same method, a plasmid of the CP2475L gene from ASFV isolatePig / HLJ / 2018 (GenBank: MK333180.1) was constructed, numbered 9hlj, with the corresponding protein being pCP2475L.hlj.

[0042] 2. Construction of inner membrane protein particles 2.1 The inner membrane proteins pD117L, pKP177R and pO61R were fused and expressed; the proteins were linked together by furin cleavage sites.

[0043] The amino acid sequence is shown in SEQ ID NO.19.

[0044] Plasmid construction: pD117L, pKP177R, and pO61R were fused, and the proteins were linked by nucleotide sequences corresponding to furinase cleavage sites. The codons of the fusion gene D117L-KP177R-O61R were optimized and inserted into the XhoⅠ (CTC GAG) - KpnⅠ (GGT ACC) space downstream of the Pp10 promoter in the pFastBac Dual vector. The plasmid was numbered 6. Using the same method, the fusion gene plasmid of D117L, KP177R, and O61R from ASFVisolate Pig / HLJ / 2018 (GenBank: MK333180.1) was constructed, numbered 6hlj, and the corresponding protein was pD117L-KP177-RO61R.hlj. Specifically, the stop codon was deleted from the 3' end of the D117L gene, the start codon was deleted from the 5' end and the stop codon was deleted from the 3' end of the KP177R gene, and the start codon was deleted from the 5' end of the O61R gene.

[0045] 2.2 The inner membrane proteins pE248R, pE199L, pE183L, pH108R and pCP204L were fused and expressed, and the proteins were linked by furin cleavage sites; the fusion proteins were partially mutated at glycosylation sites to increase the protein surface area density and enhance immunogenicity.

[0046] The amino acid sequence is shown in SEQ ID NO.20.

[0047] Plasmid construction: pE248R, pE199L, pE183L, pH108R, and pCP204L were fused, with partial glycosylation site mutations, and the proteins were linked by furinase cleavage sites; the codons of the fusion gene E248R-E199L-E183LH108R-CP204L were optimized and inserted into the XhoⅠ (CTC GAG) - KpnⅠ (GGT ACC) space downstream of the Pp10 promoter in the pFastBac Dual vector, and the plasmid was numbered 8. Using the same method, fusion gene plasmids of E248R, E199L, E183L, H108R, and CP204L from the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1) were constructed, designated as 8hlj, with the corresponding protein being pE248R-E199L-E183L-H108R-CP204L.hlj. Specifically, the stop codon was deleted from the 3' end of the E248R gene; the start codon was deleted from the 5' end and the stop codon from the 3' end of the E199L, E183L, and H108R genes; and the start codon was deleted from the 5' end of the CP204L gene.

[0048] 3. Construction of capsid protein particles 3.1 The major capsid protein pB646L was expressed alone; The amino acid sequence is shown in SEQ ID NO.11.

[0049] Plasmid construction: The B646L gene codon was optimized and inserted downstream of the PpH promoter in the pFastBac Dual vector between BamHⅠ (GGA TCC) and HindⅢ (AAG CTT), and the plasmid was designated as plasmid 1. Using the same method, a plasmid of the B646L gene from the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1) was constructed, designated as 1hlj, with the corresponding protein being pB646L.hlj.

[0050] 3.2 Molecular chaperone protein pB602L expressed alone; The amino acid sequence is shown in SEQ ID NO.12.

[0051] Plasmid construction: The molecular chaperone protein B602L gene was inserted downstream of the Pp10 promoter in the pFastBac Dual-vector XhoⅠ (CTC GAG)-KpnⅠ (GGT ACC), and the plasmid was numbered 2. The nucleotide sequence of B602L is identical to that of the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1), and the plasmid was numbered 2 hlj.

[0052] 3.3 pM1249L expressed alone; The amino acid sequence is shown in SEQ ID NO.13.

[0053] Plasmid construction: The M1249L gene codon was optimized and inserted downstream of the PpH promoter in the pFastBac Dual vector between BamHⅠ (GGA TCC) and HindⅢ (AAG CTT), and the plasmid was designated as plasmid 3. Using the same method, a plasmid of the M1249L gene from the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1) was constructed, designated as 3hlj, with the corresponding protein being pM1249L.hlj.

[0054] 3.4 Fusion expression of pB438L, pH240R and pE120R, with each protein linked by a furinase cleavage site; codon optimization of the fusion gene B438L-H240R-E120R.

[0055] The amino acid sequence is shown in SEQ ID NO.21.

[0056] Plasmid construction: The fusion gene B438L-H240R-E120R was inserted downstream of the PpH promoter in the pFastBac Dual vector (pFastBac Dual vector #3) between BamHⅠ (GGA TCC) and Hind Ⅲ (AAG CTT), and the plasmid was numbered 5. Using the same method, a fusion gene plasmid of B438L, H240R, and E120R from ASFVisolate Pig / HLJ / 2018 (GenBank: MK333180.1) was constructed, numbered 5hlj, and the corresponding protein was pB438L-H240R-E120R.hlj. Specifically, the stop codon was deleted from the 3' end of the B438L gene, the start codon was deleted from the 5' end and the stop codon was deleted from the 3' end of the H240R gene, and the start codon was deleted from the 5' end of the E120R gene.

[0057] 4. Construction of outer membrane protein particles The pCD2v (pEP402R) and C-type lectin (pEP153R) were fused and expressed, with the two proteins linked by a furinase cleavage site. The fusion protein was partially mutated at its glycosylation sites to increase its surface area and enhance its immunogenicity. Two 6×His tag sequences were also embedded in the middle of the fusion protein to facilitate subsequent purification.

[0058] The amino acid sequence is shown in SEQ ID NO.22.

[0059] Plasmid construction: pEP402R and pEP153R were fused together using a furin restriction site. The fusion gene EP402R-EP153R was optimized and inserted downstream of the Pp10 promoter in the pFastBac Dual vector between XhoⅠ and KpnⅠ, and the plasmid was designated as plasmid 4. Using the same method, the fusion gene plasmid of EP402R and EP153R from the ASFV isolate Pig / HLJ / 2018 (GenBank: MK333180.1) was constructed, designated as 4hlj, and the corresponding protein was pEP402R-EP153R.hlj. Specifically, the stop codon was deleted from the 3' end of the EP402R gene, and the start codon was deleted from the 5' end of the EP153R gene.

[0060] The plasmid information is shown in Tables 1 and 2.

[0061] Table 1 VLP plasmid construction information

[0062] Note: The sequences in this table are mutated or optimized sequences.

[0063] Table 2. Construction information of the control ASFV isolate Pig / HLJ / 2018 plasmid (.hlj).

[0064] Note: The sequences in this table are the original sequences.

[0065] Example 2: Expression and detection method of African swine fever virus protein.

[0066] After diluting the synthesized plasmid, 5 ng was added to DH10Bac competent cells. The mixture was gently mixed, incubated on ice for 30 min, then heat-shocked at 42°C for 45 s, followed by an ice bath for 2 min. 700 μL of antibiotic-free 2YT medium was added, and the mixture was incubated at 37°C with shaking at 200 rpm for 4 h. The mixture was then plated onto 2YT agar plates and incubated at 37°C for 48 h. White colonies were selected and re-plated onto fresh 2YT agar plates. The plates were incubated at 37°C for 48 h. Single white colonies were picked and added to 500 μL of antibiotic-containing 2YT liquid medium. After incubation at 250 rpm for 4 h, the culture was inoculated into 250 mL of 2YT liquid medium using Thermo PureLink. TM The HiPure Plasmid Filter Maxiprep Kit was used for rod extraction. The experimental procedures were performed according to the kit's instructions, and the rod concentration was determined using a UV spectrophotometer.

[0067] ExpiFectamine TM After inverting the Sf Transfection Reagen transfection reagent 5-10 times, pipette 30µL into 1 mL of Opti-MEM. TM In the serum-reduced medium, invert the container 5-10 times to mix thoroughly, incubate at room temperature for 5 min, then add the 12.5 ng of rod particles mentioned above, invert the container 5-10 times to mix thoroughly, incubate at room temperature for 5 min, and then inoculate into 25 mL of Expi-sf9 cell culture medium with a culture density of 5E6 cells / mL. Label the container and incubate at 27°C. 3-4 days after transfection, count the cells. When the cell viability reaches about 80%, collect the cell supernatant (i.e., P0 generation virus) and determine the titer of the P0 generation virus.

[0068] Protein expression was performed using Gibco's ExpiSf™ Protein Production Kit (catalog number: A3767806). Cell density was adjusted to 5E6 cells / mL with a viability ≥90% using fresh ExpiSf CD medium, and ExpiSf was added immediately. TMThe enhancer (100 μL / 25 mL) was placed in a shaker at 27°C for 18-24 h. The cells were infected with P0 generation recombinant baculovirus at MOI=1 and cultured at 27°C and 125 rpm for 96 h. After centrifugation at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in 480 μL PBS. The cells were then mixed with 6X protein loading at a ratio of 1:5 and incubated in a metal bath at 100°C for 10 min to obtain the cell protein sample. Protein expression was identified by SDS-PAGE and WB assays.

[0069] Resuspend the cells harvested by centrifugation as described above in an equal volume of 50 mM Tris + 300 mM NaCl (with protease inhibitor added according to the protease inhibitor's instructions), pH 8.0 buffer, and sonicate under the following conditions: 650 W at 15% power, 8 min sonication time, 3 s working time, 10 s interval. Centrifuge the sonicated cell suspension at 12000 g for 20-30 min to separate the supernatant. Prepare protein samples. Both the working solution (50 mM Tris + 300 mM NaCl, pH=8.0) and the sample (the separated protein supernatant) must be filtered through a 0.45 µm filter membrane. Before use, degas the protein using low-frequency sonication for 10 min. Purify the protein according to the system and CaptoCore700 user manual. Identify the purified protein by SDS-PAGE and WB assays, and quantify the purified protein using the BCA method (average of three assays).

[0070] Example 3 VLP Immunotesting Protection Test 1. Test Protocol 1.1 Vaccine formulation: MONTANIDE ISA 206 adjuvant, virus-like particle protein 200 μg / mL, mixed in a 1:1 ratio, homogenized using a high-pressure homogenizer, to prepare a W / O / W type vaccine; 1.2 Immunization dose: 100 μg / mL / dose; 1.3 Immunization route: intramuscular injection in the neck; 1.4 Immunization schedule: First immunization on day 0, second immunization on day 14; 1.5 Animal grouping: 15~18Kg piglets were randomly divided into groups of 5; (1) Experimental group: each piglet was injected intramuscularly with 206 adjuvant + 1 mL of VLP protein emulsion vaccine; (2) Control group: each piglet was injected intramuscularly with 206 adjuvant + 1 mL of VLP protein emulsion vaccine; (3) Blank group: each piglet was injected intramuscularly with 206 adjuvant + 1 mL of PBS emulsion.

[0071] 2. Infection dosage On day 21 after the second immunization, administer 10 doses of African swine fever virus type I+II strain orally.2.0 HAU / head; 2. Post-challenge observation and testing 3.1 Clinical observation 3.2 After immunization, observe the health status daily, and after challenge, observe the clinical symptoms daily, record the onset and death times, until the trial endpoint on the 28th day after challenge.

[0072] 3.3 Viral load detection: After challenge, the Ct values ​​in oropharyngeal swabs, anal swabs and whole blood of animals were detected by real-time quantitative PCR.

[0073] 4. Result Determination For quantitative real-time PCR, a Ct value of 38-40 is considered suspicious, and ≤35 is considered positive. The endpoint of the experiment is 28 days after challenge, and the number of surviving animals in each group is counted.

[0074] Example 4 This example compares the capsid protein before and after mutation. The experimental groups are shown in the table below.

[0075]

[0076] 1. Plasmid construction, protein expression, and VLP assembly Refer to “Construction of Capsid Protein Granules” in Example 1 and Example 2.

[0077] VLP such as Figure 1 and Figure 2 (Scale bar is 100 nm) shows that Figure 1 The VLPs in the experimental group appeared as dispersed, non-uniform spherical particles in the field of view, with a particle size distribution ranging from 38 to 80 nm. The amount of purified protein was 205 μg / mL. Figure 2 The VLPs in the control group appeared as dispersed, non-uniform spherical particles in the field of view, with a particle size distribution ranging from 33 to 71 nm, and a purified protein content of 193 μg / mL.

[0078] 2. Immune protection Refer to Example 3: VLP immune challenge protection test.

[0079] Within 3 days of viral challenge, none of the groups showed clinical symptoms. From the 4th day onwards, all groups developed body temperatures ≥40℃. The control group began dying on the 5th day, with 5 / 5 dying by the 10th day. The experimental group began dying on the 5th day, with 5 / 5 dying by the 13th day, and no survivors. All pigs in the experimental groups began dying on the 6th day, with 5 / 5 dying by the 15th day, and no survivors. All three groups of pigs died before the end of the experiment, with only differences in the onset of illness and the time of death. Prior to death, all pigs had body temperatures ≥41℃, and all samples showed positive Ct values.

[0080] Example 5 This embodiment compares the outer membrane protein before and after mutation in the combination of capsid protein and outer membrane protein. The experimental groups are shown in the table below.

[0081]

[0082] 1. Plasmid construction, protein expression, and VLP assembly Refer to Example 1 for “Construction of capsid protein particles and outer membrane protein particles” and Example 2; VLP such as Figure 3 and Figure 4 Display (scale bar is 100 nm). Figure 3 The particles in the experimental group were observed to be dispersed, spherical or irregular particles of varying sizes in the field of view, with a particle size distribution range of 40~96 nm and a purified protein amount of 175 μg / mL. Figure 4 It can be observed that the particles in the control group are dispersed, spherical or irregular particles of varying sizes in the field of view, with a particle size distribution range of 40~73 nm and a purified protein amount of 151 μg / mL. The background of both VLP images is relatively clean, with only a small amount of protein impurities.

[0083] 1. Immune protection Refer to Example 3: VLP immune challenge protection test.

[0084] Within 3 days after the challenge, no clinical symptoms were observed in any group. On day 4, the control group showed elevated body temperature / positive Ct values ​​in oropharyngeal / anal swabs / whole blood. On day 6, 1 / 5 of the control group died, and 3 / 5 of the control group exhibited lethargy and body temperature ≥41℃. 2 / 5 of the control and experimental groups showed lethargy. On day 7, a total of 3 / 5 of the control group, 1 / 5 of the control group, and 1 / 5 of the experimental group died. By day 10, a total of 5 / 5 of the control group, 2 / 5 of the control group, and 2 / 5 of the experimental group died. By day 12, 4 / 5 of the control group and 3 / 5 of the experimental group died. By day 17, 4 / 5 of both the control and experimental groups died. By day 25, 5 / 5 of the control group died. By day 28, a total of 4 / 5 of the experimental group died, with 1 / 5 surviving. The Ct values ​​of tissue samples from the dead pigs were all positive. The Ct values ​​of oropharyngeal / anal swab samples from the surviving pigs changed from positive to negative, while the Ct values ​​of whole blood samples remained negative.

[0085] Example 6 This example compares the inner membrane protein before and after mutation in the combination of inner membrane protein, capsid protein, and outer membrane protein. The experimental groups are shown in the table below.

[0086]

[0087] 1. Plasmid construction, protein expression, and VLP assembly Refer to Example 1 for “Construction of inner membrane protein particles, construction of capsid protein particles, and construction of outer membrane protein particles” and Example 2; VLP such as Figure 5 and Figure 6 Display (scale bar is 200 nm). Figure 5 The particles in the experimental group were dispersed spherical or irregularly shaped particles of varying sizes in the field of view, with a particle size distribution range of 55~145 nm and a purified protein amount of 138 μg / mL. Figure 6 The control group consisted of dispersed spherical particles of varying sizes in the field of view, with a particle size distribution range of 51–136 nm and a purified protein content of 127 μg / mL.

[0088] 2. Immune protection Within 3 days after challenge, no clinical symptoms were observed in any group. On day 4, pigs in the control group developed fever and positive Ct values ​​in oropharyngeal swabs / anal swabs / whole blood. On day 7, 2 / 5 of the control group showed lethargy and 2 / 5 died. On day 8, 3 / 5 of the control group and 2 / 5 of the experimental group showed lethargy. On day 11, 5 / 5 of the control group died. On day 21, 3 / 5 of the control group and 2 / 5 of the experimental group died. On day 25, 4 / 5 of the control group and 3 / 5 of the experimental group died. On day 28, 4 / 5 of the control group died, with 1 / 5 surviving; and 3 / 5 of the experimental group died, with 2 / 5 surviving. Ct values ​​in tissue samples from the dead pigs were all positive; Ct values ​​in the surviving pigs' tissues changed from positive to negative, while Ct values ​​in whole blood samples remained negative.

[0089] Example 7 This embodiment compares the nucleocapsid protein before and after mutation in a combination of nucleocapsid protein, inner membrane protein, capsid protein, and outer membrane protein. The experimental groups are shown in the table below.

[0090]

[0091] 1. Plasmid construction, virus-like particle expression and detection Refer to Example 1 for “Construction of nucleoshell protein particles, construction of inner membrane protein particles, construction of capsid protein particles, and construction of outer membrane protein particles” and Example 2; Experimental group VLP such Figure 7 As shown (scale bar is 100 nm), the internal particles exhibit a pseudo-hexagonal shape, with an external membrane structure, resulting in an overall regular circular appearance; the overall particle size is approximately 230 nm, the internal particle size is approximately 188 nm, the thickness of the external membrane structure is approximately 60 nm, the pore diameter on the surface of the internal particles is 16 nm, and the purified protein content is 107 μg / mL; the control group VLP is as follows. Figure 8As shown (scale bar is 100 nm), the particles are regular circles with a diameter distribution range of 103~146 nm, and the amount of purified protein is 102 μg / mL.

[0092] 2. Immune protection Within 3 days after the challenge, no clinical symptoms were observed in any group. On day 4, pigs in the control group developed fever and positive Ct values ​​in oropharyngeal / anal swabs / whole blood. On day 6, 1 / 5 of the control group died. On day 7, 2 / 5 of the control group died. On day 9, 4 / 5 of the control group died. On day 12, 5 / 5 of the control group died, and 1 / 5 of the control group died. On day 17, 2 / 5 of the control group died. On day 24, 2 / 5 of the control group died. On day 26, 1 / 5 of the experimental group died. On day 28, the end of the experiment, 2 / 5 of the control group had died, and 3 / 5 survived. 1 / 5 of the experimental group had died, and 4 / 5 survived. The Ct values ​​of tissue samples from the dead pigs were all positive. The Ct values ​​of oropharyngeal / anal swab samples from the surviving pigs all changed from positive to negative, while the Ct values ​​of whole blood samples remained negative.

[0093] Example 8 This example compares various mutant combinations in combinations containing all four proteins: nucleocapsid protein, inner membrane protein, capsid protein, and outer membrane protein. The experimental groups are shown in the table below.

[0094]

[0095] 1. Plasmid construction, virus-like particle expression and detection Refer to Example 1 for “Construction of nucleoshell protein particles, construction of inner membrane protein particles, construction of capsid protein particles, and construction of outer membrane protein particles” and Example 2; Experimental group 1 as Figure 9 As shown (scale bar is 200 nm), most of the particles are approximately spherical, with a diameter ranging from 62 to 151 nm. The purified protein content is 61 μg / mL. Experimental group 2 is as follows... Figure 10 As shown (scale bar is 200 nm), most of the particles are approximately spherical, with a diameter ranging from 55 to 167 nm. The purified protein content is 70 μg / mL. Experimental group 3 is as follows... Figure 11 As shown (scale bar is 200 nm), most particles are approximately spherical, with a diameter distribution ranging from 50 to 180 nm, of which more than 50% of the particles have a diameter between 50 and 100 nm; purified protein content: 74 μg / mL. Experimental group 4 is as follows... Figure 12 As shown (scale bar is 200 nm), most of the particles are approximately spherical, with a diameter ranging from 75 to 180 nm. The purified protein content is 81 μg / mL. Experimental group 5 is as follows... Figure 13As shown (scale bar is 200 nm), most of the particles are approximately spherical, with a diameter distribution range of 90~205 nm, and the amount of purified protein is 89 μg / mL.

[0096] 2. Immune protection Experimental Group 1: Within 7 days after challenge, no clinical symptoms were observed; on day 8, 1 / 5 of the pigs showed fever, and 2 / 5 had positive Ct values ​​in oropharyngeal swabs / anal swabs / whole blood; on day 12, 1 / 5 showed lethargy and 1 / 5 died; on day 19, 1 / 5 showed lethargy, and a cumulative mortality of 2 / 5; on day 25, a cumulative mortality of 3 / 5; at the end of the experiment on day 28, a cumulative mortality of 3 / 5 and surviving pigs remained. The Ct values ​​in the oropharyngeal / anal swab samples of the surviving pigs all changed from positive to negative, while the Ct values ​​in the whole blood samples remained negative.

[0097] Experimental Group 2: Within 9 days after challenge, no clinical symptoms were observed; on day 10, 2 / 5 of the pigs showed elevated body temperature, and 2 / 5 had positive Ct values ​​in oropharyngeal / anal swabs / whole blood; on day 13, 1 / 5 died; on day 17, 2 / 5 showed lethargy; on day 21, 2 / 5 remained lethargic, with a cumulative mortality of 2 / 5; on day 26, a cumulative mortality of 3 / 5; at the end of the experiment on day 28, a cumulative mortality of 3 / 5 was observed, with 2 / 5 surviving. The Ct values ​​in the oropharyngeal / anal swab samples of the surviving pigs all changed from positive to negative, while the Ct values ​​in the whole blood samples remained negative.

[0098] Experimental Group 3: Within 8 days after challenge, no clinical symptoms were observed; on day 9, 1 / 5 of the pigs showed fever and 1 / 5 had positive Ct values ​​in oropharyngeal / anal swabs / whole blood; on day 15, 1 / 5 died; on day 17, 1 / 5 showed lethargy; on day 20, 2 / 5 showed lethargy, with a cumulative mortality of 2 / 5; on day 25, a cumulative mortality of 2 / 5; at the end of the experiment on day 28, a cumulative mortality of 3 / 5 was observed, with 2 / 5 surviving. The Ct values ​​in the oropharyngeal / anal swab samples of the surviving pigs all changed from positive to negative, while the Ct values ​​in the whole blood samples remained negative.

[0099] Experimental Group 4: Within 10 days after challenge, no clinical symptoms were observed; on day 11, 1 / 5 of the pigs showed fever and 1 / 5 had positive Ct values ​​in oropharyngeal / anal swabs / whole blood; on day 16, 1 / 5 showed lethargy and 1 / 5 died; on day 19, 2 / 5 showed lethargy; on day 25, 2 / 5 had died cumulatively; on day 28, the experimental endpoint, 2 / 5 had died cumulatively, and 3 / 5 survived. The Ct values ​​in the oropharyngeal / anal swab samples of the surviving pigs all changed from positive to negative, while the Ct values ​​in the whole blood samples remained negative.

[0100] Experimental Group 5: Within 12 days after challenge, no clinical symptoms were observed; on day 13, 1 / 5 of the pigs showed fever and 1 / 5 had positive Ct values ​​in oropharyngeal / anal swabs / whole blood; on day 15, 2 / 5 showed lethargy; on day 23, 1 / 5 showed lethargy and 1 / 5 died; on day 27, a total of 2 / 5 died; on day 28, the experimental endpoint, a total of 2 / 5 died and 3 / 5 survived. The Ct values ​​in the oropharyngeal / anal swab samples of the surviving pigs all changed from positive to negative, while the Ct values ​​in the whole blood samples remained negative.

[0101] Control group: Within 3 days after challenge, no clinical symptoms were observed; on day 4, 2 / 5 of the animals had a body temperature ≥40℃; on day 5, 2 / 5 showed lethargy and 2 / 5 died; on day 7, 2 / 5 showed listlessness and a cumulative death rate of 3 / 5; on day 9, 5 / 5 died. Prior to death, all animals had a body temperature ≥41℃, and all samples had positive Ct values.

[0102] The African swine fever nested virus-like particles provided by this invention can protect animals against recombinant African swine fever virus type I+II strain 10 2.0 HAU attack resulted in 4 / 5 of the pigs inoculated with the virus surviving and being able to clear the infected virus.

[0103] The WOAH-recommended evaluation standard for African swine fever vaccines is a challenge dose of 10 for virulent strains. 4.0 HAU intramuscular injection; this experiment used a recombinant virulent strain of African swine fever virus genotype I+II. 2.0 Oral infection with HAU resulted in 4 / 5 protection up to the trial endpoint of 28 days after challenge. Although the virulence and dosage were lower than the WOAH recommended standard, it still provided basic data for in-depth research and development of African swine fever VLP vaccines.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A capsid protein, characterized in that, This includes fusion proteins of pB646L, pB602L, and pM1249L, as well as pB438L, pH240R, and pE120R proteins. The fusion protein of pB438L protein, pH240R protein and pE120R protein is composed of pB438L protein, pH240R protein and pE120R protein in series from N-terminus to C-terminus. The amino acid sequence of the pB646L protein is shown in SEQ ID NO.11; The amino acid sequence of the molecular chaperone protein pB602L is shown in SEQ ID NO.12; The amino acid sequence of the pM1249L protein is shown in SEQ ID NO.13; The amino acid sequence of the pB438L protein is shown in SEQ ID NO.14; The amino acid sequence of the pH240R protein is shown in SEQ ID NO.15; The amino acid sequence of the pE120R protein is shown in SEQ ID NO.

16.

2. The capsid protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein of pB438L, pH240R and pE120R proteins is shown in SEQ ID NO.

21.

3. A protein A, characterized in that, From the inside out, they consist of capsid proteins and outer membrane proteins; The outer membrane protein includes a fusion protein of pEP402R and pEP153R proteins; the amino acid sequence of the pEP402R protein is shown in SEQ ID NO.17; and the amino acid sequence of the pEP153R protein is shown in SEQ ID NO.

18.

4. The protein A according to claim 3, characterized in that, The capsid proteins include pB646L protein, molecular chaperone proteins pB602L and pM1249L protein, and a fusion protein of pB438L protein, pH240R protein and pE120R protein. The fusion protein of pB438L protein, pH240R protein and pE120R protein is composed of pB438L protein, pH240R protein and pE120R protein in series from N-terminus to C-terminus. Preferably, the amino acid sequence of the pB646L protein is shown in SEQ ID NO.11; the amino acid sequence of the molecular chaperone protein pB602L is shown in SEQ ID NO.12; the amino acid sequence of the pM1249L protein is shown in SEQ ID NO.13; the amino acid sequence of the pB438L protein is shown in SEQ ID NO.14; the amino acid sequence of the pH240R protein is shown in SEQ ID NO.15; and the amino acid sequence of the pE120R protein is shown in SEQ ID NO.

16. Preferably, in the fusion protein of pB438L protein, pH240R protein and pE120R protein, pB438L protein, pH240R protein and pE120R protein are linked by furin cleavage sites. Preferably, the amino acid sequence of the fusion protein of pB438L protein, pH240R protein and pE120R protein is shown in SEQ ID NO.21; Preferably, the fusion protein of pEP402R and pEP153R proteins is composed of pEP402R and pEP153R proteins linked together from the N-terminus to the C-terminus; Preferably, in the fusion protein, the pEP402R protein and the pEP153R protein are linked by a furin cleavage site; Preferably, the amino acid sequence of the fusion protein of pEP402R and pEP153R proteins is shown in SEQ ID NO.

22.

5. A protein B, characterized in that, From the inside out, it includes an inner membrane protein, a capsid protein as described in claim 3 or 4, and an outer membrane protein as described in claim 3 or 4; The inner membrane proteins include fusion proteins of pD117L, pKP177R, and pO61R proteins, as well as fusion proteins of pE248R, pE199L, pE183L, pH108R, and pCP204L proteins.

6. Protein B according to claim 5, characterized in that, The amino acid sequence of the pD117L protein is shown in SEQ ID NO.3; the amino acid sequence of the pKP177R protein is shown in SEQ ID NO.4; the amino acid sequence of the pO61R protein is shown in SEQ ID NO.5; and the amino acid sequence of the pE248R protein is shown in SEQ ID NO.

6. The amino acid sequence of the pE199L protein is shown in SEQ ID NO.7; The amino acid sequence of the pE183L protein is shown in SEQ ID NO.8; the amino acid sequence of the pH108R protein is shown in SEQ ID NO.9; and the amino acid sequence of the pCP204L protein is shown in SEQ ID NO.

10. Preferably, the fusion protein of pD117L protein, pKP177R protein and pO61R protein is composed of pD117L protein, pKP177R protein and pO61R protein in series from N-terminus to C-terminus; Preferably, in the fusion protein, the pD117L protein, pKP177R protein, and pO61R protein are linked by a furin cleavage site. Preferably, the amino acid sequence of the fusion protein of pD117L protein, pKP177R protein and pO61R protein is shown in SEQ ID NO.19; Preferably, the fusion protein of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein is composed of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein in series from N-terminus to C-terminus. Preferably, in the fusion protein, pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein are linked by furin cleavage sites. Preferably, the amino acid sequence of the fusion protein of pE248R protein, pE199L protein, pE183L protein, pH108R protein and pCP204L protein is shown in SEQ ID NO.

20.

7. A protein C, characterized in that, From the inside out, it includes, in sequence, the nucleocapsid protein, the inner membrane protein as described in claim 5 or 6, the capsid protein as described in claim 3 or 4, and the outer membrane protein as described in claim 3 or 4; The nucleocapsid proteins include pCP530R and pCP2475L proteins.

8. The protein C according to claim 7, characterized in that, The amino acid sequence of the pCP530R protein is shown in SEQ ID NO.1; the amino acid sequence of the pCP2475L protein is shown in SEQ ID NO.

2.

9. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a capsid protein of claim 1 or 2, a nucleotide sequence encoding protein A of claim 3 or 4, a nucleotide sequence encoding protein B of claim 5 or 6, or a nucleotide sequence encoding protein C of claim 7 or 8; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing the capsid protein of claim 1 or 2, protein A of claim 3 or 4, protein B of claim 5 or 6, or protein C of claim 7 or 8.

10. The use of the capsid protein of claim 1 or 2, protein A of claim 3 or 4, protein B of claim 5 or 6, or protein C of claim 7 or 8 in the preparation of an African swine fever virus vaccine.

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