Insertion site of antigen epitope of self-assembled protein cage nanoparticle and application of insertion site in preparation of subunit vaccine

Abstract

The invention relates to the technical field of genetic engineering, in particular to Encapsulin fusion protein for expressing foot and mouth disease virus epitopes and application of the Encapsulin fusion protein in preparation of subunit vaccines. The invention discovers that different foot-and-mouth disease 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 different foot-and-mouth disease virus antigen epitopes; efficient and soluble expression of a target antigen in escherichia coli can be realized, and protein cage nano antigen particles are successfully self-assembled; the protein nano antigen particle can induce a widely neutralized foot-and-mouth disease virus antibody, improves the immune efficacy, and has the potential of becoming a broad-spectrum foot-and-mouth disease vaccine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the insertion site of antigenic epitopes in self-assembled protein cage nanoparticles and their application in the preparation of subunit vaccines. Background Technology

[0002] Foot-and-mouth disease virus (FMDV) structural proteins have two core functions: mediating viral assembly and determining viral antigenic specificity, thus serving as crucial antigenic components of the virus. Based on this characteristic, preparing subunit vaccines by expressing highly immunogenic FMDV structural proteins has become a key research focus in the field. Currently, various protein expression systems have been applied to the preparation of FMDV VP1 protein, but these techniques generally suffer from common drawbacks: firstly, the target protein has poor solubility, affecting subsequent purification efficiency; secondly, the expressed product has weak immunogenicity, making it difficult to induce a strong immune response; thirdly, the in vitro assembly efficiency of the viral coat protein is low, hindering vaccine preparation; and fourthly, the overall preparation cost is high, hindering large-scale production and promotion. Although the dominant antigenic epitopes of FMDV have been clearly identified, and FMDV antigenic peptides from different sources possess basic immunogenicity and can stimulate a preliminary immune response, the core problem of insufficient immunogenicity remains when using short peptides alone as antigens to immunize animals. In existing solutions, complete antigens are typically prepared by conjugating short peptides with exogenous carrier proteins to enhance the immunogenicity of short peptide molecules. However, this method has significant limitations: on the one hand, multiple immunizations are required to induce the body to produce high levels of specific antibodies, making the process cumbersome and time-consuming; on the other hand, exogenous carrier proteins may interfere with the specific immune response targeted by the short peptides, affecting the stability and specificity of the immunization effect.

[0003] With the help of DNA recombination technology, fusion proteins can be efficiently expressed in both prokaryotic and eukaryotic expression systems, and their products have been widely used in the fields of biology and medicine. In vector applications, protein cage nanoparticles are often used as targeted drug delivery carriers. For example, ferritin nanocages have unique functional advantages. They can not only encapsulate exogenous small molecule proteins within the cage, effectively reducing the degradation of small molecule antigens and increasing their effective concentration, but also assist target cells in the uptake and processing of corresponding antigens, thereby significantly enhancing the stability and immunogenicity of antigen proteins. However, introducing antigenic epitope sequences into nanocage systems still faces significant technical challenges: on the one hand, the introduction process is highly complex, and if the antigenic epitope sequence is not properly selected or the introduction strategy is not designed reasonably, it is very easy to cause nanocage assembly failure, directly affecting the realization of the carrier function; on the other hand, even if the nanocage is successfully assembled, the resulting protein cage nanoparticles may interfere with the immunogenicity of the target antigen, ultimately failing to induce the body to produce specific neutralizing antibodies and losing the key immune protection effect. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention systematically studied the insertion sites of antigenic epitopes in Encapsulin protein fragments. It was found that inserting foot-and-mouth disease virus antigenic epitopes between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment shown in SEQ ID NO.1, and / or replacing amino acids 62-64, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment shown in SEQ ID NO.1 with viral antigenic epitopes, can achieve efficient and soluble expression of the target protein in *E. coli*, and successfully self-assemble into protein cage nanoparticles. These self-assembled Encapsulin protein nanoparticles, as a vaccine research platform, can be used to prepare subunit vaccines for different viruses, and have broad application prospects.

[0005] The specific content of the invention is as follows: In a first aspect, the present invention provides an application for preparing self-assembled Encapsulin protein nanoparticles by inserting viral antigenic epitopes between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1, and / or by replacing amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1 with viral antigenic epitopes.

[0006] Preferably, the viral antigenic epitope is selected from foot-and-mouth disease virus antigenic epitopes.

[0007] Preferably, the foot-and-mouth disease virus antigenic epitope is a foot-and-mouth disease virus B-cell antigenic epitope.

[0008] Preferably, the foot-and-mouth disease virus is type O foot-and-mouth disease virus.

[0009] Preferably, the foot-and-mouth disease virus B-cell antigen epitope is selected from at least one of the epitopes shown in SEQ ID NO. 3-6.

[0010] Secondly, the present invention provides an Encapsulin fusion protein that chimericly expresses an antigenic epitope. The Encapsulin fusion protein is characterized by: inserting a viral antigenic epitope between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1, and / or replacing amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1 with a viral antigenic epitope.

[0011] Preferably, the viral antigenic epitope is selected from foot-and-mouth disease virus antigenic epitopes.

[0012] Preferably, the antigenic epitope is a B-cell antigenic epitope of the same serotype of foot-and-mouth disease virus.

[0013] Preferably, the foot-and-mouth disease virus is type O foot-and-mouth disease virus.

[0014] Preferably, the foot-and-mouth disease virus B-cell antigenic epitope is selected from at least one type O foot-and-mouth disease virus B-cell antigenic epitope shown in SEQ ID NO.3-6.

[0015] Preferably, the amino acid sequence of the Encapsulin fusion protein is shown in SEQ ID NO.11.

[0016] Thirdly, the present invention provides Encapsulin protein nanoantigen particles formed by the self-assembly of the Encapsulin fusion protein described in the second aspect above.

[0017] Fourthly, the present invention provides the application of the Encapsulin fusion protein described in the second aspect or the Encapsulin protein nanoantigen particles described in the third aspect in the preparation of subunit vaccines.

[0018] Fifthly, the present invention provides a method for preparing the Encapsulin protein nanoparticles described in the third aspect above, the method comprising the following steps: (1) Synthesize the gene fragment encoding the Encapsulin fusion protein; (2) The gene fragment described in step (1) is ligated into a prokaryotic expression vector to construct a recombinant expression plasmid; (3) Transform the recombinant expression plasmid described in step (3) into Escherichia coli, and obtain the Encapsulin protein fusion protein after induction expression and purification; (4) The Encapsulin protein fusion protein self-assembles to form protein cage nanoparticles.

[0019] Preferably, the gene fragment encoding the Encapsulin fusion protein is shown in SEQ ID NO.12.

[0020] In a sixth aspect, the present invention provides a subunit vaccine comprising the Encapsulin fusion protein described in the second aspect or the Encapsulin protein nanoantigen particles described in the third aspect.

[0021] The beneficial effects of this invention are: (1) This invention first discovered potential sites for inserting antigenic epitopes in the Encapsulin protein fragment with an amino acid sequence as shown in SEQ ID NO.1 (amino acids 62-63, 124-125, 138-139 and 239-240 of the Encapsulin protein fragment shown in SEQ ID NO.1). The discovery of these antigenic epitope insertion sites expands the antigenic epitope insertion space of self-assembled Encapsulin protein nanoparticles, laying the foundation for further designing self-assembled Encapsulin protein nanoparticle vaccines with multiple antigenic epitopes. Using self-assembled Encapsulin protein nanoparticles as a vaccine research platform, it can be used to prepare subunit vaccines of different viruses, which has broad application prospects.

[0022] (2) In this invention, foot-and-mouth disease virus antigenic epitopes are inserted between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1, and / or amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1 are replaced with antigenic epitopes to prepare Encapsulin protein nanoparticles expressing antigenic epitopes; the Encapsulin protein nanoparticles expressing antigenic epitopes can display the fused expressed antigenic epitope peptide in multiple copies on the particle surface, thereby improving the immunogenicity of the antigenic epitope; the protein cage nanoparticles themselves, as a large molecule of virus-like particles, have a good ability to stimulate the body's immune response; moreover, this fusion protein purification method is simple and easy to implement, and the self-assembled protein cage nanoparticles have good thermal stability and do not affect the assembly of nanoparticles or their own immunogenicity.

[0023] (3) Taking foot-and-mouth disease virus as an example, the present invention prepared Encapsulin protein nanoantigen particles expressing foot-and-mouth disease virus antigen; the foot-and-mouth disease subunit vaccine prepared by emulsifying the Encapsulin protein nanoantigen particles with adjuvant has a good immune protection effect, and can induce the body to produce virus neutralizing antibodies after immunizing guinea pigs, and can be used for the preparation of foot-and-mouth disease subunit vaccine. Attached Figure Description

[0024] Figure 1 A schematic diagram of recombinant plasmid construction with a new site for inserting a Flag tag into the Encapsulin protein; Figure 2 SDS-PAGE identification results of recombinant protein expression; where M is the protein molecular weight marker, S is the supernatant of induced cell lysis, and P is the precipitate of induced cell lysis. Figure 3 SDS-PAGE identification results of purified recombinant protein; where M is the protein molecular weight marker, and 1 is the purified protein eluted from the elution. Figure 4 Identification results of novel sites on nanoparticles; where M is the protein molecular weight marker, Inp is the sample incubated with anti-flag immunomagnetic beads, E1, E2, and E3 are the magnetic bead elution solutions, and Mb is the protein-magnetic bead complex eluted sample. Figure 5 SDS-PAGE identification results of recombinant protein expression with chimeric FMDV antigenic epitopes; where M is the protein molecular weight marker, S is the supernatant of IPTG-induced bacterial lysis, P is the precipitate of IPTG-induced bacterial lysis, and H is the purification effect of bacterial lysis supernatant after high-temperature treatment. Figure 6 SDS-PAGE identification results of purified recombinant protein with chimeric FMDV antigenic epitopes; where M is the protein molecular weight marker, and 1 is the purified recombinant protein eluted. Figure 7 Electron microscopy analysis results of nanoparticle assembly; Figure 8 Serum antibody levels in guinea pigs immunized with nanoparticles containing chimeric antigenic epitopes. Figure 9 Neutralizing antibody levels in guinea pig serum after immunization with nanoparticles containing chimeric antigenic epitopes. Detailed Implementation

[0025] The above-described solution will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The implementation conditions used in the embodiments may be further adjusted according to the conditions of specific manufacturers, and the implementation conditions not specified are generally those in routine experiments.

[0026] The experiments described in the following examples obtained biosafety clearance and foot-and-mouth disease laboratory activity permits: In accordance with the requirements for a Biosafety Level 3 (BSL-3) laboratory and foot-and-mouth disease-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, through a hierarchical reporting process involving the Lanzhou Veterinary Research Institute's Biosafety Committee, Laboratory Animal Ethics Committee, the Chinese Academy of Agricultural Sciences' Biosafety Committee, the Lanzhou Veterinary Research Institute's Laboratory Animal Ethics Committee, and the Lanzhou Veterinary Research Institute's Biosafety Committee, obtained permission from the Ministry of Agriculture and Rural Affairs to conduct research on highly pathogenic FMDV and other pathogens and related animals. This permission has been registered with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0027] Explanation and interpretation of relevant terms in this invention: The term "Escherichia coli expression system" refers to a system consisting of Escherichia coli (strain) and a vector, wherein the Escherichia coli (strain) is derived from commercially available sources, including, but not limited to, BL21(DE3), BL21(DE3)pLysS, B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, GI698, with BL21(DE3) being preferred.

[0028] The term "vector" refers to a nucleic acid delivery system that allows the insertion of a polynucleotide encoding a protein, thereby enabling the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed in the host cells. Examples of vectors include plasmids, bacteriophages, and cosmids.

[0029] The term "vaccine" refers to a biological agent capable of providing a protective response in animals, wherein the vaccine has been delivered and does not cause serious disease. The vaccine of this invention is a genetically engineered subunit vaccine composed of foot-and-mouth disease virus antigenic epitopes.

[0030] The vaccine of the present invention optionally further comprises one or more adjuvants, excipients, carriers, and diluents. The adjuvant can be any suitable adjuvant, including chemical adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, Span, etc.; microbial adjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, cytosine, lipid-soluble wax D, and short rod-shaped bacteria; and plant-based adjuvants, which are mostly polysaccharides extracted from plants or macrofungi, such as Poria cocos polysaccharide, safflower polysaccharide, and traditional Chinese medicine. Biochemical adjuvants include thymosin, transfer factor, and interleukins. Preferred adjuvants may be nano-adjuvants, biological adjuvants, interleukins, interferon, etc.

[0031] The vaccine of the present invention can be administered via convenient routes, such as intramuscular injection, intranasal administration, oral administration, subcutaneous administration, transdermal administration, and vaginal administration. The attenuated vaccine of the present invention is preferably administered via intramuscular injection. The vaccine can be administered after a primary immunization-boost regimen. For example, after the first vaccination, the subject can receive a second booster dose after a period of time (e.g., approximately 7, 14, 21, or 28 days). Typically, the booster dose is the same as or lower than the primary immunization dose. Furthermore, a third booster immunization can also be performed, for example, 2-3 months, 6 months, or one year after immunization.

[0032] The materials and methods used in the following embodiments are shown below: Carrier construction: The FMDV B-cell antigenic epitope is derived from type O foot-and-mouth disease virus strain O / Mya / BY / 2010, and the amino acid sequences of the B-cell antigenic epitope are shown in SEQ ID NO. 3-6, respectively; the nucleotide sequences expressing the B-cell antigenic epitope are shown in SEQ ID NO. 7-9.

[0033] The Encapsulin protein fragment was obtained from a thermophilic bacterium ( Thermotoga maritima The protein gene fragment isolated from the sample is shown in SEQ ID NO.2, and the amino acid sequence of the protein is shown in SEQ ID NO.1. The amino acid sequence of the constructed FMDV epitope fusion protein is shown in SEQ ID NO.11; the nucleotide sequence encoding the FMDV epitope fusion protein is shown in SEQ ID NO.12.

[0034] T4 ligase, restriction endonuclease BamH I and Xba All reagents were purchased from NEB (UK); DNA molecular weight standard DL10000 was purchased from TaKaRa; Prestained Protein Marker I was purchased from Thermo; Plasmid small-volume rapid extraction kit and DNA gel recovery kit were purchased from OMEGA.Bio-tek; Flag-tagged magnetic bead immunoprecipitation kit was purchased from Shanghai Yamei Biomedical Technology Co., Ltd.

[0035] Example 1: Selection, Display, and Identification of Novel Sites in Encapsulin Protein 1.1 Selection of novel sites for Encapsulin protein Based on the amino acid sequence and 3D structure analysis of Encapsulin protein, loop structures on the surface of assembled nanoparticles were selected for amino acid substitution and insertion prediction. Finally, suitable amino acid sites were selected for recombinant protein expression verification.

[0036] 1.2 Construction and Identification of Recombinant Plasmids A schematic diagram of the construction of a recombinant protein expression plasmid with a Flag tag inserted at a novel site in Encapsulin protein nanocage particles is shown below. Figure 1 As shown, the plasmid construction process is as follows: 1) Carrier pMV 42H and pMV 5tag-2 was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., in which pMV 42H is a control vector carrying the Encapsulin gene; pMV 5tag-2 is a transition vector for expressing the Encapsulin fusion protein fragment with a Flag tag inserted at position 62, an HA tag inserted at position 124, a Myc tag inserted at position 138, and a V5 tag inserted at position 239, respectively. The pET28 vector plasmid was preserved by the Foot-and-Mouth Disease and Emerging Disease Epidemiology Innovation Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0037] 2) Transition vector pMV for expressing Encapsulin fusion protein fragment with insert tag 5tag-2 and the expression vector pMV containing only the Encapsulin protein 42H respectively Xba I and Bam After double digestion with HI, the recovered DNA fragment was inserted into the pET28 prokaryotic expression vector (insertion site: Xba I and Bam Between HI, the recombinant expression plasmid pET28 was constructed and obtained respectively. 42H and pET28 5tag-2.

[0038] 3) The recombinant vector was converted to... E.coli DH5α, applied to Luria containing kanamycin (Kan 50 mg / L) Bertanil (LB) solid medium, cultured at 37°C, positive clones selected, and then... Nde I and Xho After double enzyme digestion verification, the recombinant plasmid was sent to Yangling Aoke Biotechnology Co., Ltd. for sequencing identification.

[0039] 1.3 Induction of recombinant protein expression and identification of solubility The correctly identified recombinant plasmids were transformed into [the appropriate transfection sites]. E.coli BL21(DE3) competent cells. Single colonies containing recombinant plasmids were picked from LB solid medium containing 50 mg / L kanamycin to obtain positive clones.

[0040] The above positive clones were inoculated into LB liquid medium containing 50 mg / L kanamycin and incubated overnight at 37°C. The resulting bacterial suspension was then inoculated into 100 mL of LB liquid medium containing 50 mg / L kanamycin at a volume ratio of 1:100 and cultured at 37°C with shaking at 220 r / min until mid-log phase (P<0.05). OD 600 Protein expression was induced by an IPTG concentration of 0.6-0.8, and SDS-PAGE was used to detect protein expression. The final concentration of IPTG was 0.5 mmol / L, and the optimal induction conditions were 16℃ and 220 r / min for 12 h. After induction the next day, 1 mL of bacterial culture was taken, centrifuged to remove the supernatant, resuspended in PBS, and mixed with an equal volume of 2×SDS-PAGE loading buffer. The mixture was then boiled in water for 10 min. The remaining bacterial culture was centrifuged to collect the precipitate, and the cells were resuspended in 10 mL of Binding Buffer (pH 8.0). Lysozyme (1 g / L) was added, and the cells were incubated on ice for 30 min. The cells were then lysed by low-temperature sonication (5 s sonication time, 5 s interval, working time 10 min).

[0041] The supernatant and precipitate were collected separately for SDS-PAGE analysis to detect the soluble expression of the fusion protein.

[0042] SDS-PAGE identification results are as follows: Figure 2 As shown, a clear target band was visible in the supernatant (S2) of lysed E. coli cells, indicating that the Encapsulin fusion protein with inserted Flag, HA, Myc, and V5 tags after induction expression had a protein band of the expected size at around 50 kDa. This indicates that the recombinant plasmid can efficiently and solublely express the Encapsulin fusion protein with inserted Flag, HA, Myc, and V5 tags at amino acid sites 62-63, 124-125, 138-139, and 239-240.

[0043] 1.4 Purification of recombinant proteins Positive clones were induced to express protein by using IPTG at a final concentration of 0.5 mmol / L, at 16℃ and 220 r / min. After 12 h of induction, the bacterial cells were collected by centrifugation. The cells were resuspended in binding buffer (pH 8.0) at a volume of 10 mL / g, and then homogenized with lysozyme (1 g / L) using a high-pressure homogenizer. After homogenization, the cells were centrifuged at 18000 rpm for 1 h. The precipitate was discarded, and the supernatant was heated at 65℃ for 30 min, followed by centrifugation at 10000 rpm for 30 min. The precipitate was discarded, and the supernatant was filtered through a 0.45 μm filter and purified by molecular sieve. The purified sample was collected.

[0044] Take 100 μL of supernatant, add loading buffer, and boil for 10 min to prepare SDS-PAGE electrophoresis samples. Store at -20℃ or identify by electrophoresis. The recombinant protein purification results are as follows: Figure 3 As shown, the results indicate that most of the impurities in the Encapsulin fusion protein with Flag, HA, Myc, and V5 tags inserted at amino acid sites 62-63, 124-125, 138-139, and 239-240 can be removed by high-temperature treatment after lysis. Further purification results show that a clear target protein band is visible at the target protein position, indicating that this method can effectively and rapidly obtain purified Encapsulin fusion proteins containing exogenous amino acid sequences or epitopes.

[0045] 1.5 Validation of the display of exogenous sequences on the surface of nanoparticles 1.5.1 Pretreatment of magnetic beads Gently pipette the anti-flag, anti-HA, anti-Myc, and anti-V5 immunomagnetic beads to fully suspend them. Take 25 µL of the magnetic bead suspension and place it in a 1.5 mL centrifuge tube. Add 500 µL of lysis / wash buffer to each tube and gently pipette to resuspend the magnetic beads. Then, let the tube stand on a magnetic rack for 1 min until the magnetic beads adhere to the side wall of the centrifuge tube. Discard the supernatant. Repeat the above steps twice.

[0046] 1.5.2 Sample binding, washing, and elution Add 500 µL of the protein sample prepared in step 1.4.1 to the pretreated magnetic beads and incubate on a reverse mixer (room temperature for 1 h, 4℃ for 4~6 h or overnight); place the mixture on a magnetic rack and let it stand for 1 min, then transfer the supernatant to a new centrifuge tube for later use (the supernatant can be used to detect whether there is any residue of the tagged protein), and the remaining contents in the original centrifuge tube are the protein-magnetic bead complex. Add 500 µL of lysis / wash buffer to the obtained protein-magnetic bead complex, gently resuspend by pipetting, then incubate on a magnetic rack for 1 min and discard the supernatant; repeat approximately three times until the supernatant is obtained after washing. OD 280 Up to less than 0.05; Add 80–100 µL of 1× SDS-PAGE loading buffer (diluted from 5× to 1×) to the washed protein-magnetic bead complex and mix well. Heat at 100 °C for 10 min. After cooling, place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to adhere to the sidewall of the centrifuge tube. Collect the supernatant and perform SDS-PAGE and Western blot analysis.

[0047] The results of the identification are as follows Figure 4 As shown, the results indicate that after incubation with immunomagnetic beads, Encapsulin fusion proteins with Flag, HA, Myc, and V5 tags inserted at amino acid sites 62-63, 124-125, 138-139, and 239-240 showed obvious target protein bands in the eluted protein-magnetic bead complex, while no target protein was detected in the intermediate magnetic bead wash solution. This method demonstrates that purified Encapsulin fusion proteins containing exogenous amino acid sequences or epitopes can effectively display short peptides with different tags inserted at amino acid sites 62-63, 124-125, 138-139, and 239-240 on the surface of nanoparticles and be captured by the tag antibodies labeled on the magnetic beads.

[0048] Example 2: Preparation of Encapsulin protein cage nanoparticles containing chimeric foot-and-mouth disease virus antigenic epitopes 2.1 Construction and Identification of Recombinant Plasmids A schematic diagram of the construction of a recombinant protein expression plasmid containing chimeric foot-and-mouth disease virus antigenic epitopes is shown below. Figure 1 As shown, the construction process of each plasmid is as follows: 1) Carrier pMV 62H and pMV-5tag-4B (D62B-D124B-D138B-D239B) were synthesized by Beijing Liuhe BGI Genomics Co., Ltd., among which pMV 62H is a control vector carrying the Encapsulin gene; pMV-5tag-4B is a transition vector in which the Encapsulin fusion protein expression fragments of the foot-and-mouth disease B cell epitopes shown in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 are inserted sequentially between positions 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein (shown in SEQ ID NO. 1).

[0049] 2) The transition vector pMV-5tag-4B containing an Encapsulin fusion protein expression fragment with foot-and-mouth disease virus B-cell epitopes and the expression vector pMV containing only the Encapsulin protein. 62H respectively Xba I and BamH After double enzyme digestion and DNA fragment recovery, the fragment was inserted into the pET28 prokaryotic expression vector (insertion site: Xba I and BamH Between I, the recombinant expression plasmid pET28 was constructed to obtain I. 62H and PET28-5tag-4B.

[0050] 3) The recombinant vector was converted to... E.coli DH5α, applied to Luria containing kanamycin (Kna 50 mg / L) Bertanil (LB) solid medium, cultured at 37°C, positive clones selected, and then... Nde I and Xho After double enzyme digestion verification, the sample was sent to Yangling Aoke Biotechnology Co., Ltd. for sequencing identification.

[0051] 2.2 Induction of recombinant protein expression and identification of solubility The specific experimental method is the same as in 1.3.

[0052] The supernatant and precipitate were collected separately for SDS-PAGE analysis to detect the soluble expression of the fusion protein.

[0053] SDS-PAGE identification results are as follows: Figure 5 As shown, the target bands were clearly visible in the supernatant (S) of the recombinant protein, indicating that the Encapsulin fusion protein with inserted FMDV epitopes after induction expression had protein bands of the same size as expected at around 50 kDa. This indicates that the recombinant plasmid can efficiently and solublely express the Encapsulin fusion protein with inserted B cell epitopes at amino acid sites 62-63, 124-125, 138-139, and 239-240.

[0054] 2.3 Purification of recombinant proteins The specific experimental method is the same as in 1.4.

[0055] The results of recombinant protein purification are as follows Figure 6 As shown, the results indicate that most of the impurities in the Encapsulin fusion protein containing foot-and-mouth disease virus B-cell epitopes can be removed by high-temperature treatment after lysis. Figure 5 (H) Further purification results showed that a clear target protein band was visible at the target protein location, indicating that this method can effectively and rapidly obtain purified Encapsulin fusion protein containing B cell epitopes.

[0056] 2.4 Formation and physical characterization of protein cage nanoparticles (electron microscopy) To further analyze whether the recombinant protein formed protein cage nanoparticles and whether the formed particles were uniform, the Encapsulin fusion protein containing foot-and-mouth disease virus B cell epitopes was characterized by transmission electron microscopy (TEM).

[0057] Specific steps: Take the purified recombinant protein and dilute it in different gradients. Take 10 µL of the protein and drop it onto a copper grid. Let it stand for 10 min and use filter paper to absorb the liquid from one side of the copper grid. Then add 10 µL of 1% phosphotungstic acid staining solution and let it stand for 2 min. Use filter paper to absorb the staining solution from one side of the copper grid. Use tweezers to pick up the copper grid and place it in a glass petri dish, allowing the liquid on the copper grid to air dry naturally. Fix the prepared copper grid on the sample stage of the sample holder and insert it into the sample chamber. After vacuuming, find a suitable field of view in the observation window to observe and analyze whether the purified recombinant protein self-assembles into protein cage nanoparticles.

[0058] The results are as follows Figure 7 As shown, after the purified recombinant protein was diluted 5-fold, fully assembled nanoparticles could be seen under an electron microscope, indicating that the recombinant protein has good self-assembly ability and assembled into protein cage nanoparticles of 25-30 nm.

[0059] This embodiment successfully constructed an expression vector for recombinant proteins and established a method for preparing and efficiently purifying recombinant proteins. Electron microscopy results showed that the recombinant proteins formed protein cage nanoparticles.

[0060] In this embodiment, fusion proteins were obtained by inserting antigenic epitopes at positions 62-63, 124-125, SEQ ID NO.5, and SEQ ID NO.6 of the Encapsulin protein shown in SEQ ID NO. 1. This indicates that inserting antigenic epitopes between positions 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein shown in SEQ ID NO. 1 can express, purify, and self-assemble into protein cage nanoparticles containing antigenic epitopes. This demonstrates that positions 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein shown in SEQ ID NO. 1 are the insertion sites for new antigenic epitopes. However, this invention does not limit the order and type of antigenic epitopes. Those skilled in the art can reasonably expect that inserting the same or different antigenic epitopes between positions 62-63, 124-125, 138-139 and 239-240 of the Encapsulin protein shown in SEQ ID NO.1 can eventually successfully prepare protein cage nanoparticles containing the corresponding antigenic epitopes.

[0061] Example 3: Detection of neutralizing antibody response in animals immunized with foot-and-mouth disease subunit vaccine 1. Foot-and-mouth disease subunit vaccine immunizes guinea pigs.

[0062] The protein cage nanoparticles obtained in Example 2 were emulsified with 61VG adjuvant to obtain foot-and-mouth disease (FMD) subunit vaccines (5tag2-4B). Five guinea pigs (approximately 200g each, sourced from the Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were immunized intramuscularly with this FMD subunit vaccine. An inactivated FMD O / Mya / BY / 2010 strain vaccine was used as the positive control (IV), and a PBS immunization group served as the negative control. A booster immunization was given 21 days after the first immunization, and the guinea pigs were euthanized 14 days after the second immunization. Blood samples were collected weekly after immunization to separate serum for detecting serum antibody and neutralizing antibody levels.

[0063] The serum antibody level detection method is as follows: First, coat the ELISA plate with 50 μL / well of FMDV VP1 protein (final concentration 1 ng / μL), at 4℃, and incubate overnight in a 96-well microplate; wash 5 times with 1×PBST, blot dry, add 120 μL / well of 5% bovine serum albumin (BSA), and block at 4℃ for 10 h; wash 5 times with 1×PBST, add 50 μL / well of serum diluted 1:1000, and incubate at 37℃ for 30 min; wash 5 times with 1×PBST, blot dry, add 50 μL / well of HRP-labeled secondary antibody diluted 1:10000 with secondary antibody dilution buffer, and incubate at 37℃ for 30 min; wash 5 times with 1×PBST, blot dry, add 50 μL / well of TMB substrate chromogenic solution, and incubate at 37℃ in the dark for 12 min; add 2 mol / L sulfuric acid, 50 μL / well of... μL / well, terminate the reaction, and read the reading. OD 450 Value; when [serum to be tested / detected well] OD Value - Blank Hole OD Value] / [Negative serum well] OD Value - Blank Hole OD If the value is ≥ 2.1, it is considered as a positive serum antibody.

[0064] The specific method for detecting serum neutralizing antibody levels is as follows: Prepared BHK-21 cells are packed at a rate of 2.5-3 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of / mL in 96-well cell culture plates. When the cell confluence reached 80-90%, the separated serum was serially diluted 2-fold in serum dilution plates, with each serum sample diluted 8 times. Then, the virus for detection was prepared at 200 TCID50. 50 Prepare the virus dilution solution and add it to the serially diluted serum. Incubate at 37°C for 1 hour. Then, add the neutralized serum and virus mixture to a cell culture plate and continue culturing at 37°C and 5% CO2 for 2-3 days. Once the positive control cells show obvious CPE, observe the CPE (cytopathic effect) of each serum group and calculate the neutralization titer according to the Reed-Muench method.

[0065] The results of guinea pig serum antibody level detection are shown in the figure. Figure 8 As shown, PBS was used as the immunization control group, and IV was used as the inactivated vaccine control group. The results showed that serum antibody levels in guinea pigs immunized with the foot-and-mouth disease subunit vaccine obtained by adjuvant emulsification of the protein cage nanoparticles prepared in Example 2 of this invention exhibited an increasing trend.

[0066] Detection of neutralizing antibodies in guinea pig serum, such as Figure 9As shown, the serum neutralizing antibody level detection results of guinea pigs immunized with the foot-and-mouth disease subunit vaccine obtained by emulsifying the protein cage nanoparticles prepared by Example 2 of the present invention with adjuvant showed that the recombinant protein with multiple antigenic epitopes of chimeric foot-and-mouth disease virus described in the present invention produced a high level of virus neutralizing antibodies equivalent to or higher than that of the inactivated vaccine after immunizing guinea pigs.

[0067] The above examples are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of preparing self-assembled Encapsulin protein nanoparticles by inserting viral antigenic epitopes between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment with the amino acid sequence as shown in SEQ ID NO.1, and / or by replacing amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment with viral antigenic epitopes.

2. The application as described in claim 1, characterized in that, The viral antigenic epitopes are selected from foot-and-mouth disease virus antigenic epitopes.

3. A chimeric encapsulin fusion protein expressing an antigenic epitope, characterized in that, The Encapsulin fusion protein is characterized by inserting viral antigenic epitopes between amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1, and / or replacing amino acids 62-63, 124-125, 138-139, and 239-240 of the Encapsulin protein fragment as shown in SEQ ID NO.1 with viral antigenic epitopes.

4. The Encapsulin fusion protein as described in claim 3, characterized in that, The viral antigenic epitopes are selected from foot-and-mouth disease virus antigenic epitopes.

5. The Encapsulin fusion protein as described in claim 4, characterized in that, The foot-and-mouth disease virus antigenic epitopes are B-cell antigenic epitopes of the same serotype of foot-and-mouth disease virus.

6. The Encapsulin fusion protein as described in claim 5, characterized in that, The foot-and-mouth disease virus B-cell antigenic epitope is selected from at least one type O foot-and-mouth disease virus B-cell antigenic epitope shown in SEQ ID NO.3-6.

7. The Encapsulin fusion protein as described in claim 6, characterized in that, The amino acid sequence of the Encapsulin fusion protein is shown in SEQ ID NO.

11.

8. Encapsulin protein nanoantigen particles formed by the self-assembly of the Encapsulin fusion protein as described in any one of claims 3-7.

9. The use of the Encapsulin fusion protein as described in any one of claims 3-7 or the Encapsulin protein nanoantigen particles as described in claim 8 in the preparation of subunit vaccines.

10. A subunit vaccine, characterized in that, The subunit vaccine comprises the Encapsulin fusion protein of any one of claims 3-7 or the Encapsulin protein nanoantigen particles of claim 8.

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