Encapsulin fusion protein for expressing Seneca virus epitope and application of Encapsulin fusion protein in preparation of subunit vaccine

Abstract

The invention relates to the technical field of genetic engineering, in particular to Encapsulin fusion protein for expressing Seneca virus epitope and application of Encapsulin fusion protein in preparation of subunit vaccines. The invention discovers that Seneca virus antigen epitopes are inserted among the 62nd to 63rd positions, the 124 to 125th positions, the 138 to 139th positions and the 239th to 240th positions of amino acids of an Encapsulin protein fragment subjected to amino acid sequence modification, and / or the 62nd to 63rd positions, the 124 to 125th positions, the 138 to 139th positions and the 239th to 240th positions of amino acids are replaced by the Seneca virus antigen epitopes, so that efficient and soluble expression of a target antigen in escherichia coli can be realized; the protein cage nano antigen particles are successfully self-assembled; the protein nano antigen particle can induce a widely neutralizing Seneca virus antibody, improves the immune efficacy, and has the potential of becoming a Seneca multi-epitope vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to an Encapsulin fusion protein expressing Senecavirus antigen epitopes and application thereof in preparing a subunit vaccine. BACKGROUND

[0002] The structural protein of Senecavirus (SVA) is a key functional protein for completing the assembly process of the virus, and determines the antigen specificity of the virus, and thus is a core antigen component of the virus. Based on this characteristic, preparing a subunit vaccine by expressing the highly immunogenic Senecavirus structural protein has become one of the key research directions of scholars in the field. At present, various protein expression systems have been applied to the preparation of Senecavirus VP2 protein, but these technical solutions generally have common defects: first, the solubility of the target protein is poor, which affects subsequent purification and application; second, the immunogenicity of the expression product is weak, and it is difficult to induce a strong immune response; third, the in vitro assembly efficiency of the virus coat protein is low, which restricts the preparation efficiency of the vaccine; and fourth, the overall preparation cost is high, which is not conducive to large-scale production and promotion.

[0003] Although the dominant antigen epitopes of Senecavirus have been clearly identified, and Senecavirus antigen peptides of different sources also have certain immunogenicity and can stimulate the body to produce a basic immune response, when animals are immunized with short peptides as antigens alone, the core problem of insufficient immunogenicity still exists. In the prior art, a complete antigen is usually prepared by coupling a short peptide with an exogenous carrier protein to improve the immunogenicity of the short peptide molecule; however, this method has obvious limitations: on the one hand, it needs to be immunized for multiple times to induce the body to produce high-level specific antibodies, and the operation process is complicated and time-consuming; on the other hand, the exogenous carrier protein may interfere with the specific immune response targeted by the short peptide, affecting the stability and specificity of the immune effect.

[0004] With the help of DNA recombination technology, fusion proteins can be efficiently expressed in prokaryotic expression systems and eukaryotic expression systems, and their products have been widely used in the fields of biology and medicine. In the application of vectors, protein cage nanoparticles are often used as carriers for targeted drug delivery. Ferritin nanocage has unique advantages. It not only can encapsulate exogenous small molecule proteins in its nanocage, effectively reducing the degradation of small molecule antigens and increasing the effective concentration of antigens, but also can assist target cells in uptaking and processing corresponding antigens, thereby significantly enhancing the stability and immunogenicity of antigen proteins. However, there are still technical challenges in introducing antigen epitope sequences into nanocage systems. On the one hand, the operation complexity of the introduction process is high, and if the antigen epitope sequence is not properly selected or the introduction strategy is not reasonably designed, it is easy to cause nanocage assembly failure. On the other hand, even if the nanocage is successfully assembled, the protein cage nanoparticles formed may interfere with the immunogenicity of the target antigen, and ultimately fail to induce the body to produce specific neutralizing antibodies, affecting the immune protection effect. SUMMARY

[0005] In view of the above technical problems, the present application has carried out a systematic study on the insertion site of the antigen epitope of the Encapsulin protein fragment, and found that the insertion of Senecavirus antigen epitope between the 62-63, 124-125, 138-139 and 239-240 amino acids of the Encapsulin protein fragment with the amino acid sequence of SEQ ID NO. 1, and / or the replacement of the 62-63, 124-125, 138-139 and 239-240 amino acids of the Encapsulin protein fragment with the amino acid sequence of SEQ ID NO. 1 with viral antigen epitopes can realize the efficient and soluble expression of the target protein in Escherichia coli, and can successfully self-assemble into protein cage nanoparticles. The self-assembled Encapsulin protein nanoantigen particles as a vaccine research platform can be used to prepare subunit vaccines of different viruses, and have a broad application prospect.

[0006] Specifically, the application provides the following technical solutions:

[0007] In a first aspect, the application provides an application of self-assembled Encapsulin protein nanoantigen particles prepared by inserting Senecavirus antigen epitopes between the 62-63, 124-125, 138-139 and 239-240 amino acids of the Encapsulin protein fragment with the amino acid sequence of SEQ ID NO. 1, and / or replacing the 62-63, 124-125, 138-139 and 239-240 amino acids of the Encapsulin protein fragment with the amino acid sequence of SEQ ID NO. 1 with Senecavirus antigen epitopes.

[0008] Preferably, the Senecavirus antigen epitope is a Senecavirus B cell antigen epitope.

[0009] Preferably, the Senecavirus B cell antigen epitope is selected from at least one of SEQ ID NO. 3-6.

[0010] In a second aspect, the present application provides a chimeric Encapsulin fusion protein expressing an antigen epitope, wherein the Encapsulin fusion protein is: inserting a Senecavirus antigen epitope between the 62-63th, 124-125th, 138-139th and 239-240th amino acids of the Encapsulin protein fragment with the amino acid sequence shown in SEQ ID NO. 1, and / or replacing the 62-63th, 124-125th, 138-139th and 239-240th amino acids of the Encapsulin protein fragment with the amino acid sequence shown in SEQ ID NO. 1 with a Senecavirus antigen epitope.

[0011] Preferably, the antigen epitope is a Senecavirus B cell antigen epitope.

[0012] Preferably, the Senecavirus B cell antigen epitope is selected from at least one of SEQ ID NO. 3-6.

[0013] Preferably, the amino acid sequence of the Encapsulin fusion protein is shown in SEQ ID NO. 11.

[0014] In a third aspect, the present application provides an Encapsulin protein nano-antigen particle self-assembled from the Encapsulin fusion protein of the second aspect.

[0015] In a fourth aspect, the present application provides a use of the Encapsulin fusion protein of the second aspect or the Encapsulin protein nano-antigen particle of the third aspect in the preparation of a Senecavirus subunit vaccine.

[0016] In a fifth aspect, the present application provides a preparation method of the Encapsulin protein nano-antigen particle of the third aspect, the method comprising the following steps:

[0017] (1) synthesizing a gene fragment encoding the Encapsulin fusion protein;

[0018] (2) connecting the gene fragment of step (1) to a prokaryotic expression vector to construct a recombinant expression plasmid;

[0019] (3) transforming Escherichia coli with the recombinant expression plasmid of step (3), and obtaining the Encapsulin protein fusion protein through induction expression and purification;

[0020] (4) the Encapsulin protein fusion protein self-assembles to form a protein cage nanoparticle.

[0021] Preferably, the gene fragment encoding the Encapsulin fusion protein is as shown in SEQ ID NO. 12.

[0022] In a sixth aspect, the present application provides a Senecavirus subunit vaccine, which comprises the Encapsulin fusion protein of the second aspect or the Encapsulin protein nanoparticle of the third aspect.

[0023] The beneficial effects of the present application are:

[0024] (1) The present application inserts the Senecavirus antigen epitope between the 62-63th, 124-125th, 138-139th and 239-240th amino acids of the Encapsulin protein fragment with the amino acid sequence as shown in SEQ ID NO. 1, and / or replaces the 62-63th, 124-125th, 138-139th and 239-240th amino acids of the Encapsulin protein fragment with the amino acid sequence as shown in SEQ ID NO. 1 with the antigen epitope to obtain an Encapsulin protein nanoparticle expressing the antigen epitope; the Encapsulin protein nanoparticle expressing the antigen epitope can display the fusion-expressed antigen epitope peptide segment in the form of multiple copies on the surface of the particle, improving the immunogenicity of the antigen epitope; the protein cage nanoparticle itself is a macromolecule as a virus-like particle, which has good ability to stimulate immune response of the body; and the purification of the fusion protein is simple and easy to operate, and the self-assembled protein cage nanoparticle has good thermal stability, which does not affect the assembly of the nanoparticle and the immunogenicity thereof.

[0025] (2) The present application takes Senecavirus as an example to prepare an Encapsulin protein nanoparticle expressing Senecavirus antigen; the Senecavirus subunit vaccine prepared by emulsifying the Encapsulin protein nanoparticle with an adjuvant has good immune protection effect, and can induce the body to produce virus-neutralizing antibodies after immunizing mice, and can be used for preparation of Senecavirus subunit vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Schematic diagram of construction of recombinant plasmid for inserting antigen epitope into new site of Encapsulin protein;

[0027] Figure 2SDS-PAGE identification results of recombinant protein expressing chimeric SVA antigen epitopes; wherein M is protein molecular weight marker, S1 is IPTG induced bacteria lysis supernatant, P1 is IPTG induced bacteria lysis precipitate;

[0028] Figure 3 SDS-PAGE identification results of purified recombinant protein of chimeric SVA antigen epitopes; wherein M is protein molecular weight marker, 1 is eluted purified recombinant protein;

[0029] Figure 4 Electron microscope analysis results of nanoparticle assembly;

[0030] Figure 5 Antibody level of mouse serum after immunization of chimeric antigen epitope nanoparticles;

[0031] Figure 6 Neutralizing antibody level of mouse serum after immunization of chimeric antigen epitope nanoparticles. DETAILED DESCRIPTION

[0032] The above scheme is further described in combination with specific examples. It should be understood that these examples are used to illustrate the present application and are not limited to limit the scope of the present application. The implementation conditions used in the examples can be further adjusted according to the specific conditions of the manufacturer, and the implementation conditions not mentioned are usually the conditions in the conventional experiments.

[0033] The experiments described in the following examples obtained the biosafety license and the Seneca laboratory activity license:

[0034] According to the relevant requirements of biosafety level 3 laboratory (BSL-3) related biosafety, the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, through the biosafety committee of Lanzhou Veterinary Research Institute, the experimental animal ethics committee, the biosafety committee of Chinese Academy of Agricultural Sciences, the experimental animal ethics committee of Lanzhou Veterinary Research Institute, the biosafety committee of Lanzhou Veterinary Research Institute, obtained the license for carrying out high pathogenicity foot-and-mouth disease virus and Seneca virus and other pathogens and animal research, and has been recorded in the Ministry of Agriculture and Rural Affairs, which meets the requirements of national biosafety level.

[0035] Explanation and explanation of related terms in the present application:

[0036] The term "E. coli expression system" refers to a system composed of E. coli (strains) and vectors, wherein the E. coli (strains) is derived from commercially available, herein exemplified but not limited to: BL21(DE3), BL21(DE3)pLysS, B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, GI698, preferably BL21(DE3).

[0037] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted so as to bring about the expression of the protein. A vector can transform a host cell with its genetic material elements to be expressed in the host cell by transformation, transduction or transfection. For example, vectors include: plasmids; bacteriophages; cosmids; and the like.

[0038] The term "vaccine" refers to a biological preparation that provides protective responses in an animal, wherein the vaccine has been delivered and cannot cause serious illness. The vaccine of the present application is a genetically engineered subunit vaccine composed of Seneca virus antigen epitopes.

[0039] The vaccine of the present application, further optionally comprises one or more adjuvants, excipients, carriers and diluents. Adjuvants can be any suitable adjuvants, chemical immunological adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, Span, etc.; microbial immunological adjuvants such as Mycobacterium, BCC, lipopolysaccharide, muramyl dipeptide, cytoplasmic peptide, liposoluble wax D, Corynebacterium parvum; plant immunological adjuvants are mostly polysaccharides extracted from plants or large fungi, such as Pachyman, Safflower polysaccharide, Chinese herbal medicine, etc. And biochemical immunological adjuvants such as thymus peptide, transfer factor, interleukin, etc. The preferred adjuvant can be nano-adjuvant biological adjuvant, interleukin, interferon, etc.

[0040] The administration of the vaccine of the present application can be by a convenient route, for example, intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal, etc. The attenuated vaccine of the present application is preferably administered by intramuscular injection. The vaccine can be administered after a prime-boost regimen. For example, after the first vaccination, the subject can receive a second boost administration after a period of time (e.g., about 7, 14, 21 or 28 days). Typically, the dose of the boost administration is the same or lower than the dose of the prime administration. In addition, a third boost immunization can also be performed, for example, 2-3 months, 6 months or one year after immunization.

[0041] The materials and methods used in the following examples are shown below:

[0042] Construction of vectors: Ministry of Agriculture

[0043] The Seneca virus B cell antigen epitopes are derived from Seneca virus strain SVA / FJ2017, and the amino acid sequences of the B antigen epitopes are shown in SEQ ID NO. 3-6, respectively; the nucleotide sequences for expressing the B cell antigen epitopes are shown in SEQ ID NO. 7-10.

[0044] The Encapsulin protein fragment is a protein gene fragment isolated from a thermophilic bacteria (Thermotoga maritima), and the gene fragment is shown as SEQ ID NO. 2, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

[0045] The amino acid sequence of the constructed insertion plug of the capsid virus antigen epitope fusion protein is shown as SEQ ID NO. 11; and the nucleotide sequence encoding the insertion plug of the capsid virus antigen epitope fusion protein is shown as SEQ ID NO. 12.

[0046] The T4 ligase, the restriction endonuclease BamH I and Xba I are all purchased from the UK NEB company; the DNA molecular weight standard DL10000 is purchased from TaKaRa company; the Prestained Protein Marker I is purchased from Thermo company; the plasmid small amount rapid extraction kit and the DNA gel recovery kit are purchased from OMEGA.Bio-tek company.

[0047] Example 1 Preparation of chimeric capsid virus antigen epitope Encapsulin protein cage nanoantigen particles

[0048] 1.1 Construction and identification of recombinant plasmid

[0049] The construction schematic diagram of the recombinant protein expression plasmid of the chimeric capsid virus antigen epitope is shown in Figure 1 , and the construction process of each plasmid is as follows:

[0050] 1) The vectors pMV-62H and pMV-SVA-4B (D62B-D124B-D138B-D239B) are synthesized by Beijing Lihe Huada Gene Technology Co., Ltd., wherein pMV-62H is a control vector with Encapsulin gene; pMV-SVA-4B is a transition vector in which the Encapsulin fusion protein expression fragment (shown as SEQ ID NO. 11) of the SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 shown capsid virus B cell epitope is sequentially inserted or replaced between the 62-63th, 124-125th, 138-39th and 239-240th positions of the Encapsulin protein (shown as SEQ ID NO. 1).

[0051] 2) The transition vector pMV-SVA-4B of the expression fragment of the Encapsulin fusion protein with the B cell epitope of Senecavirus and the expression vector pMV-62H of only the inserted Encapsulin protein were respectively subjected to Xba I and BamH I double enzyme digestion and DNA fragment recovery and then inserted into the prokaryotic expression vector pET28 (insertion site between Xba I and BamH I) to obtain recombinant expression plasmids pET28-62H and PET28-SVA-4B respectively.

[0052] 3) The recombinant vectors were transformed into E. coli DH5a, coated on Luria-Bertanil (LB) solid medium containing kanamycin (Kna 50 mg / L), cultured at 37°C, and positive clones were selected and sent to Yangling Okeye Biotechnology Co., Ltd. for sequencing identification after Nde I and Xho I double enzyme digestion verification.

[0053] 1.2 Induced expression and solubility identification of recombinant proteins

[0054] The correctly identified recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells. Single colonies containing recombinant plasmids were picked on LB solid medium containing kanamycin 50 mg / L to obtain positive clones.

[0055] The above positive clones were respectively inoculated in LB liquid medium containing 50 mg / L of kanamycin and cultured at 37°C overnight. The obtained bacterial solution was inoculated in 100 mL of LB liquid medium containing 50 mg / L of kanamycin at a volume ratio of 1:100 and cultured at 37°C, 220 r / min until the logarithmic phase (OD 600 0.6-0.8) for induced expression, and the expression of proteins was detected by SDS-PAGE. The induced concentration of the inducer IPTG was 0.5 mmol / L, and the optimal induced expression conditions were 16°C, 220 r / min for 12 h. The next day after induction, 1 mL of bacterial solution was centrifuged to remove the supernatant, resuspended with PBS, added with an equal volume of 2x SDS-PAGE loading buffer, mixed well, and boiled in water for 10 min. The remaining bacterial solution was centrifuged to collect the precipitate, resuspended with 10 mL of Binding Buffer (pH 8.0), and added with lysozyme (1 g / L) for ice bath for 30 min, then the bacterial body was broken by low-temperature ultrasonic wave (ultrasonic time 5 s, intermittent 5 s, working time 10 min).

[0056] The supernatant and precipitate were respectively collected for SDS-PAGE analysis to detect the solubility expression of the fusion protein.

[0057] The SDS-PAGE identification results are as follows Figure 2As shown, obvious target bands can be seen in the supernatant (S1) of the recombinant protein, indicating that the Encapsulin fusion protein with inserted Cocal virus epitope after induction expression has a protein band at about 42 kDa consistent with the expected size, indicating that the recombinant plasmid can efficiently and solubly express the Encapsulin fusion protein with insertion or substitution of B cell epitopes at amino acid positions 62-63, 124-125, 138-139 and 239-240.

[0058] 1.3 Purification of recombinant protein

[0059] The protein-expressing E. coli was induced for expression at 16°C, 220 r / min, with IPTG induction at a final concentration of 0.5 mmol / L. After 12 h of induction, the bacterial cells were collected by centrifugation, resuspended in binding Buffer (pH 8.0) at a volume of 10 mL / g, and broken by a high-pressure homogenizer with lysozyme (1 g / L). After breaking, the bacterial cells were centrifuged at 18000 rpm for 1 h at low temperature. The supernatant was heated at 65°C for 30 min, and then centrifuged at 10000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter, and then purified by molecular sieving. The sample purified by molecular sieving was collected.

[0060] 100 μL of the supernatant was taken and added to the Loading buffer for boiling for 10 min to prepare the SDS-PAGE electrophoresis sample, which was stored at -20°C or subjected to electrophoresis identification.

[0061] The results of purification of the recombinant protein are shown in Figure 3 The results show that most of the impurities can be removed by breaking the bacteria and high-temperature treatment of the Encapsulin fusion protein containing the Cocal virus B cell epitope. Further purification results show that obvious target protein bands can be seen at the target protein position, indicating that this method can effectively and rapidly obtain purified Encapsulin fusion protein containing the B cell epitope.

[0062] 1.4 Formation and physical characterization (electron microscopy detection) of protein cage nanoparticles

[0063] To further analyze whether the recombinant protein forms protein cage nanoparticles and whether the formed particles are uniform, the Encapsulin fusion protein containing the Cocal virus B cell epitope was subjected to transmission electron microscopy (TEM) physical characterization.

[0064] Specific steps: take the purified recombinant protein, after different gradient dilution, take 10 μL protein drop on the copper net, stand for 10 min, use filter paper to suck the liquid from one side of the copper net; then add 10 μL 1% phosphotungstic acid staining solution, stand for 2 min, then use filter paper to suck the staining solution from one side of the copper net; use forceps to clamp the copper net into a glass dish, let the liquid on the copper net dry naturally; fix the prepared copper net on the sample stage of the sample holding rod, insert into the sample chamber, after vacuumizing, find the appropriate field of view in the observation window, observe and analyze whether the purified recombinant protein self-assembles to form protein cage nanoparticles.

[0065] The results are shown in Figure 4 After 5-fold dilution of the purified recombinant protein, complete nanoparticles can be observed under electron microscope, indicating that the recombinant protein has good self-assembly ability and forms 25-30 nm protein cage nanoparticles.

[0066] The expression vector of the recombinant protein is successfully constructed, and the preparation method and efficient purification method of the recombinant protein are established. The electron microscope results show that the recombinant protein forms protein cage nanoparticles.

[0067] In this example, the Senecavirus B B cell epitopes represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 are inserted between the 62-63th, 124-125th, 138-139th and 239-240th positions of the Encapsulin protein represented by SEQ ID NO. 1, respectively, to obtain a fusion protein, which indicates that the insertion or replacement of antigen epitopes at the 62-63th, 124-125th, 138-139th and 239-240th positions of the Encapsulin protein represented by SEQ ID NO. 1 can express and self-assemble into protein cage nanoparticles containing antigen epitopes, proving that the 62-63th, 124-125th, 138-139th and 239-240th positions of the Encapsulin protein represented by SEQ ID NO. 1 are new insertion sites of antigen epitopes. However, the order and type of antigen epitopes are not limited by the present application, and those skilled in the art can reasonably expect that the insertion or replacement of other same or different Senecavirus antigen epitopes at the 62-63th, 124-125th, 138-139th and 239-240th positions of the Encapsulin protein represented by SEQ ID NO. 1 can also successfully prepare protein cage nanoparticles containing corresponding antigen epitopes.

[0068] Example 2 Detection of neutralizing antibody response in Senecavirus subunit vaccine immunized animals

[0069] 2.1 Immunization of mice with Senecavirus Unit vaccine

[0070] The protein cage nanoparticles obtained in Example 2 above were emulsified with 61VG adjuvant to prepare Senecavirus Unit vaccines, respectively. Four to six-week-old BALB / C mice (from the Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were subcutaneously injected with the Senecavirus Unit vaccines, 5 mice per group. Seneca SVA / FJ2017 virus inactivated vaccine was used as a positive control group (IV), and the PBS immunization group was used as a negative control group. The first immunization was followed by a booster immunization 21 days later. The mice were euthanized 14 days after the second immunization. Blood was collected every week after immunization to separate serum for detecting serum antibody and neutralizing antibody levels.

[0071] The method for detecting serum antibody levels is as follows: First, coat the ELISA plate with SVA VP2 protein at a final concentration of 1 ng / μL, 50 μL / well, 4°C, coat the 96-well enzyme-labeled plate overnight; wash the plate 5 times with 1×PBST, pat dry, add 5% bovine serum albumin (BSA), 120 μL / well, 4°C, block for 10 h; wash the plate 5 times with 1×PBST, add the serum to be tested diluted with 1:1000, 50 μL / well, 37°C, incubate for 30 min; wash the plate 5 times with 1×PBST, pat dry, add HRP-labeled secondary antibody diluted with secondary antibody diluent at 1:10000, 50 μL / well, 37°C, incubate for 30 min; wash the plate 5 times with 1×PBST, pat dry, add TMB substrate developing solution, 50 μL / well, 37°C, avoid light, incubate for 12 min; add 2 mol / L sulfuric acid, 50 μL / well, stop the reaction, read the OD 450 value; when [OD value of the serum to be tested (S-OD value of the blank hole (B)] / [OD value of the negative serum hole (N-OD value of the blank hole (B)] ≥ 2.1, it is determined that the serum antibody is positive.

[0072] The specific method for detecting serum neutralizing antibody levels is as follows: The prepared BHK-21 cells were plated at a cell density of 2.5-3×10 5 / mL in a 96-well cell culture plate. When the cell confluence reached 80-90%, the separated serum was diluted in a serum dilution plate at a 2-fold dilution ratio, with each serum repeated 8 times. Then the detection virus was diluted to 200 TCID 50 preparation, and added to the serum diluted with a 2-fold dilution ratio. The mixture was incubated at 37°C for 1 h, and then added to the cell culture plate. The plate was further incubated at 37°C and 5% CO2 for 2-3 days. When the positive control cells showed obvious CPE, the CPE (cytopathic effect) of each serum was observed, and the neutralizing titer was calculated according to the Reed-Muench method.

[0073] The results of detecting the antibody level in the serum of mice are shown in Table 1. Figure 5 As shown in Table 1, the antibody level in the serum of mice immunized with the SVA4B vaccine prepared by emulsifying the protein cage nanoparticles prepared in Example 1 with an adjuvant showed a growth trend.

[0074] The results of detecting the neutralizing antibody in the serum of mice are shown in Table 2. Figure 6 As shown in Table 2, the neutralizing antibody level in the serum of mice immunized with the SVA4B vaccine prepared by emulsifying the protein cage nanoparticles prepared in Example 1 with an adjuvant was improved, indicating that the recombinant protein embedding multiple antigen epitopes of Senecavirus was able to produce a high level of virus neutralizing antibody in the immunized mice, which was equivalent to that of the inactivated vaccine.

[0075] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent transformation or modification according to the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. Use of an Encapsulin fusion protein for the preparation of self-assembling protein nano-antigenic particles by inserting a Senecavirus antigenic epitope between amino acids 62-63, 124-125, 138-139, 239-240 of an Encapsulin protein fragment having an amino acid sequence as set forth in SEQ ID NO. 1 and / or replacing amino acids 62-63, 124-125, 138-139 and 239-240 of an Encapsulin protein fragment having an amino acid sequence as set forth in SEQ ID NO. 1 with a Senecavirus antigenic epitope.

2. Use according to claim 1, wherein The Senecavirus antigenic epitope is selected from a Senecavirus B cell antigenic epitope.

3. A chimeric Encapsulin fusion protein expressing an epitope of an antigen, characterized in that, The Encapsulin fusion protein is an Encapsulin fusion protein having an amino acid sequence as set forth in SEQ ID NO.

11.

4. The Encapsulin fusion protein of claim 3, wherein, The Senecavirus antigenic epitope is selected from a Senecavirus B cell antigenic epitope.

5. The Encapsulin fusion protein of claim 4, wherein, The Senecavirus B cell antigenic epitope is selected from at least one of SEQ ID NO. 3-6.

6. The Encapsulin fusion protein of claim 5, wherein, The Encapsulin fusion protein has an amino acid sequence as set forth in SEQ ID NO.

11.

7. An Encapsulin protein nano-antigenic particle self-assembled from the Encapsulin fusion protein of any one of claims 3-6.

8. Use of the Encapsulin fusion protein of any one of claims 3-6 or the Encapsulin protein nano-antigenic particle of claim 6 for the preparation of a Senecavirus subunit vaccine.

9. A Senecavirus subunit vaccine, characterized in that, The Senecavirus subunit vaccine comprises the Encapsulin fusion protein of any one of claims 3-6 or the Encapsulin protein nano-antigenic particle of claim 7.

Images