Porcine encephalitis myocarditis virus-like particles, their preparation methods and applications

Abstract

This invention discloses porcine encephalomyocarditis virus-like particles (EMCVs), their preparation method, and applications. The invention involves amplifying the P1 and 3CD genes of EMCV and then ligating them to the PpH and Pp10 promoters of the pFastBacDual vector, respectively, to construct the recombinant transfer vector pFBD-P1-3CD. pFBD-P1-3CD was transformed into E. coli DH10Bac competent cells, and recombinant rod-like particles rBacmid-P1-3CD were obtained through blue-white screening. rBacmid-P1-3CD was transfected into sf9 insect cells, and finally purified using a sucrose density gradient to obtain virus-like particles EMCV-VLPs. Experiments demonstrate that the obtained EMCV-VLPs possess good immunogenicity, can induce a favorable immune response, and can be used as a strategic reserve vaccine for the prevention and control of EMCV.

Description

Technical Field

[0001] This invention relates to a virus-like particle, its preparation method, and its application, particularly to a porcine encephalitis / myocarditis virus-like particle prepared using an insect cell-baculovirus expression system, its preparation method, and its application. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Encephalomyocarditis virus (EMCV) was first isolated in 1945 by Helwig and Schmidt from a captive male gibbon in Miami, Florida, that suddenly died of pulmonary edema and myocarditis. EMCV is not only a cause of myocarditis and encephalitis, but also a cause of neurological diseases, reproductive diseases, and diabetes in many mammals, and is commonly used as a model for diabetes and viral myocarditis. Although the incidence of EMCV infection in humans is low, the development and prevalence of xenotransplantation, such as human-pig heart transplantation, makes the prevention and control of EMCV increasingly important.

[0003] EMCV is the sole member of the genus Cardivir in the family Picornaviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus with an icosahedral capsid approximately 27 nm in diameter. Its genome is about 7.8 kb long. The 3' end of the RNA is a polyA, and the 5' end lacks a cap structure, directly covalently linked to the VPg sequence. The EMCV genome open reading frame (ORF) encodes a large polyprotein (L-1ABCD-2ABC-3ABCD), which can cleave into precursor proteins P1, P2, and P3. Precursor protein P1 cleaves into viral capsid proteins VP0 (35.9 kD), VP3 (25.1 kD), and VP1 (30.5 kD), while VP0 can further cleave into VP4 (7.3 kD) and VP2 (28.6 kD). Precursor proteins P2 and P3 are precursors of non-structural proteins. P3 is cleaved into 3A, 3B, 3C, and 3D, which is related to the cleavage of the P1 protein. Like other viruses in the Picornaviridae family, EMCV has an icosahedral morphology composed of a viral capsid. Each structural protein has 60 copies. VP1, VP2, and VP3 are located on the surface of the viral particle, while the VP4 protein is located on the inner surface of the capsid and interacts closely with the viral RNA.

[0004] Virus-like particles (VLPs) are nanoscale empty capsid structures composed of one or more viral structural proteins. They have a structure similar to natural viruses, do not contain viral inherited material, and therefore exhibit good immunogenicity and safety. VLPs can be generated in a variety of different expression host systems, including mammalian cell expression systems, baculovirus-insect cell expression systems, yeast expression systems, E. coli expression systems, and plant expression systems. The choice of expression system depends on several factors, including the structure of the VLPs, production costs, and application.

[0005] Baculovirus-insect cell expression systems are widely used in the production of recombinant proteins. This system achieves gene expression by infecting insect cells with baculoviruses. Currently, the two main insect cell lines used for producing recombinant proteins using this system are sf9 cells and High Five cells. As a relatively mature eukaryotic expression system, it has advantages that prokaryotic expression systems lack. Insect cells can recognize and process signal peptides, support oligomerization, and perform post-translational modifications such as glycosylation, phosphorylation, acylation, and disulfide bond formation, thereby obtaining correctly folded target proteins, which is beneficial for the correct assembly of VLPs. Insect cells can also be grown in serum-free media and can be easily scaled up in shaker culture or bioreactors at 27-28°C. This expression system also has other advantages, such as inherent safety, scalability, and speed. Furthermore, baculovirus design is a rapid and simple procedure, making it suitable for the production of viral vaccines. This has led to the system's widespread use in the production of human and veterinary vaccines. The baculovirus-insect cell expression system operates in two phases: infection and production. This system produces protein quantities comparable to those of *E. coli* and yeast, and exhibits enhanced capabilities in performing complex post-translational modifications. Therefore, this system has been used to produce numerous VLP types, including chikungunya, HIV, and porcine parvovirus-like particles. Summary of the Invention

[0006] The purpose of this invention is to provide a porcine encephalitis myocarditis virus-like particle, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] This invention proposes a method for preparing porcine encephalomyocarditis virus-like particles. The method includes transfecting recombinant rod particles expressing encephalomyocarditis virus P1 and 3CD proteins into insect cells, and after cytopathic effects, isolating and purifying porcine encephalomyocarditis virus-like particles from the cell culture medium.

[0009] Preferably, the carrier used to construct the recombinant rod particles is pFastBacDual.

[0010] Preferably, the insect cells are selected from Sf9, Sf21 or High Five.

[0011] Preferably, the method involves amplifying the P1 and 3CD genes of EMCV and then ligating them to the PpH and Pp10 promoters of the pFastBacDual vector, respectively, to construct the recombinant transfer vector pFBD-P1-3CD. The identified recombinant transfer vector pFBD-P1-3CD is then transformed into E. coli DH10Bac competent cells. Recombinant rod midparticles rBacmid-P1-3CD are obtained by blue-white screening. The identified recombinant rod midparticles rBacmid-P1-3CD are then transfected into sf9 insect cells. Finally, encephalocarditis virus-like particles are obtained by purification using a sucrose density gradient.

[0012] Preferably, the method includes the following steps:

[0013] (1) RNA extraction and reverse transcription

[0014] Total RNA was extracted from EMCV viral fluid, and then cDNA was obtained by reverse transcription. After the concentration was measured, the cDNA was stored at -20°C for later use.

[0015] (2) Amplification of P1 and 3CD fragments

[0016] Based on the gene sequences of EMCV-P1 and EMCV-3CD published in GenBack by NCBI, specific primers for amplifying their full-length sequences were designed using primer design software. Primers P1-FP / P1-RP were designed to amplify P1 using the recognition sites of BamHI and HindIII restriction enzymes, respectively. Primers 3CD-FP / 3CD-RP were designed to amplify 3CD using the recognition sites of NheI and KpnI restriction enzymes, respectively. Using EMCV cDNA as a template, the target fragments of P1 and 3CD were amplified, respectively.

[0017] P1-FP: 5'-GGATCCATGTCCTCAGACAAGAATAACT-3'

[0018] P1-RP: 5'-AAGCTTTTATAGCATCAAGACTCCAGCT-3'

[0019] 3CD-FP: 5'-GCTAGCATGCCGAACCCTGTGATGGACTT-3'

[0020] 3CD-RP: 5'-GGTACCTTAGAACAGACTCCTCCATCTGT-3'

[0021] (3) Construction of pFBD-P1

[0022] The P1 target gene purified by gel recovery and pFastBacDual vector was double-digested with BamHI and HindIII, respectively. The digested P1 target fragment was then ligated with the pFastBacDual vector to obtain the recombinant plasmid pFBD-P1.

[0023] (4) Construction of pFBD-P1-3CD

[0024] The 3CD target gene purified by gel recovery and recombinant plasmid pFBD-P1 was double-digested with NheI and KpnI, respectively. The digested 3CD target fragment was ligated into the pFBD-P1 vector to obtain the recombinant transfer plasmid pFBD-P1-3CD.

[0025] (4) The recombinant transfer plasmid pFBD-P1-3CD was transformed into E. coli DH10Bac competent cells.

[0026] The constructed pFBD-P1-3CD was transformed into E.coli DH10Bac competent cells, and positive colonies were obtained by blue-white screening. The colonies were cultured and recombinant rod mid-p1-3CD was extracted from them.

[0027] (5) Expression of recombinant baculovirus

[0028] Recombinant rod-like particle rBacmid-P1-3CD was transfected into sf9 insect cells to obtain the P1 generation strain. The P1 generation strain was then used to infect sf9 insect cells to obtain the P2 generation strain with a higher titer. Similarly, the P2 generation virus solution was used to infect sf9 insect cells to obtain the P3 generation strain.

[0029] (6) Purification and identification of EMCV-VLPs

[0030] sf9 insect cells were infected with P3 generation virus solution, and the cell culture medium was harvested. After repeated freeze-thaw cycles, centrifugation, concentration, emulsification, and sucrose density gradient purification, purified porcine encephalomyocarditis virus-like particles were obtained.

[0031] Preferably, the nucleotide sequence encoding the P1 protein of encephalomyocarditis virus is shown in SEQ ID NO.1, and the nucleotide sequence encoding the 3CD protein of encephalomyocarditis virus is shown in SEQ ID NO.2.

[0032] Porcine encephalitis myocarditis virus-like particles prepared according to the method are also within the scope of protection of this invention.

[0033] Furthermore, the present invention also proposes the use of the aforementioned porcine encephalitis myocarditis virus-like particles in any of the following:

[0034] 1) Used to prepare polyclonal antibodies or monoclonal antibodies against porcine encephalitis myocarditis virus;

[0035] 2) Used to prepare a reagent for detecting porcine encephalitis myocarditis virus antibodies;

[0036] 3) Used to prepare a genetically engineered subunit vaccine against porcine encephalomyocarditis virus.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] This invention involves sequentially ligating the P1 and 3CD genes of porcine encephalomyocarditis virus (EMCV) into two expression elements of the pFastBacDual (pFBD) vector to construct the recombinant transfer vector pFBD-P1-3CD. The identified recombinant transfer vector pFBD-P1-3CD was transformed into E. coli DH10Bac competent cells, and the recombinant rod midline rBacmid-P1-3CD was obtained through blue-white screening. The identified recombinant rod midline rBacmid-P1-3CD was then transfected into sf9 insect cells, and finally, virus-like particles were obtained through purification. This invention is the first to utilize the P1 and 3CD protein strategy to achieve the expression of EMCV viral structural proteins and the assembly of virus-like particles in insect cells. The obtained virus-like particles can increase the antibody and neutralizing antibody levels in immunized animals, thereby enhancing their resistance to porcine encephalomyocarditis virus infection. Attached Figure Description

[0039] Figure 1 The results are the PCR and enzyme digestion identification of P1;

[0040] Figure 2 The results are PCR and enzyme digestion identification of 3CD.

[0041] Figure 3 The PCR identification results for rBacmid-P1-3CD;

[0042] Figure 4 The result of baculovirus transfection into sf9 cells;

[0043] Figure 5 Western blotting results for EMCV-VLPs;

[0044] Figure 6 Transmission electron microscopy results for EMCV-VLPs;

[0045] Figure 7 Immunoelectron microscopy results of EMCV-VLPs;

[0046] Figure 8 The mouse immune antibody level of EMCV-VLPs. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1

[0049] 1. RNA extraction and reverse transcription

[0050] Total RNA was extracted from EMCV viral fluid using the Trizol method, and then GoScript was used. TM For the reverse transcription system, add 3 μL of RNA template and 2 μL of Random Primer to a PCR tube and mix well. Incubate the mixture in a 70°C metal bath for 5 min, then remove it and place it on ice for 5 min. Centrifuge for 10 s and then place it on ice again. Then, add 1 μL of GoScript™ Reverse Transcripase, 4 μL of GoScript™ 5×Reaction Buffer, 5 μL of MgCl2, 1 μL of PCR Nucleotide Mix, 0.5 μL of Recombinant RNasin Ribonuclease Inhibitor, and 3.5 μL of Nuclease-Free Water to a new PCR tube and mix well to prepare the RT-mix. Add the mixture from the two PCR tubes to a single PCR tube. Set the PCR instrument to the reverse transcription program, including annealing at 25°C for 5 min, extension at 42°C for 60 min, and reverse transcriptase inactivation at 70°C for 15 min. After the program is complete, obtain cDNA, measure the concentration, and store at -20°C for later use.

[0051] 2. Amplification of P1 and 3CD fragments

[0052] Based on the gene sequences of EMCV-P1 and EMCV-3CD published in NCBI GenBack, specific primers for amplifying their full-length sequences were designed using primer design software. Primers P1-FP / P1-RP were designed to amplify P1 using the recognition sites of BamHI and HindIII restriction enzymes, respectively. Primers 3CD-FP / 3CD-RP were designed to amplify 3CD using the recognition sites of NheI and KpnI restriction enzymes, respectively. Using EMCV cDNA as a template, the target fragments of P1 and 3CD were amplified, respectively. After the PCR program was completed, the PCR products were subjected to 1% agarose gel electrophoresis and observed under 365nm UV light in a laboratory gel imaging system for the presence of specific bands. The gel containing the target band was excised and purified according to the instructions of the gel extraction kit, and the concentration was determined for later use.

[0053] P1-FP: 5'-GGATCCATGTCCTCAGACAAGAATAACT-3'

[0054] P1-RP: 5'-AAGCTTTTATAGCATCAAGACTCCAGCT-3'

[0055] 3CD-FP: 5'-GCTAGCATGCCGAACCCTGTGATGGACTT-3'

[0056] 3CD-RP: 5'-GGTACCTTAGAACAGACTCCTCCATCTGT-3'

[0057] 3. Construction of the recombinant transfer vector pFBD-P1-3CD

[0058] 3.1 Construction of pFBD-P1

[0059] 1) The connection reaction of pFBD-P1

[0060] The purified P1 target gene and vector were double-digested with BamHI and HindIII, respectively. The digested products were subjected to 1% agarose gel electrophoresis, and the presence of specific bands was observed under 365 nm UV light in a laboratory gel imaging system. After excising the gel containing the target band, purification was performed according to the gel extraction kit instructions, and the concentration was determined for later use. The digested P1 target fragment was mixed with the pFastBacDual (pFBD) vector at a concentration ratio of 1:3, and after adding an equal volume of Solution I, the mixture was incubated overnight at 16°C in a metal bath. After thawing DH5α competent cells on ice, 10 μL of ligation product was added, and the mixture was kept on ice for 30 min. The mixture was then heat-shocked in a pre-prepared 42℃ metal bath for 90 s, followed by an ice bath for 3 min. In a clean bench, 800 μL of preheated antibiotic-free LB liquid medium was added to the mixture, and the mixture was incubated at 37℃ on a shaker at 200 rpm for 1 h. After centrifugation at 4500 rpm at room temperature for 2 min, 700 μL of the supernatant was discarded in a clean bench, leaving approximately 200 μL to suspend the precipitate. This precipitate was then added to LB agar plates containing kanamycin (50 μg / mL), spread evenly with a spreader, and incubated at 37℃ for 12–16 h until colonies appeared. Plasmids were extracted after positive colonies were identified by PCR.

[0061] 2) Double enzyme digestion identification of recombinant plasmid pFBD-P1

[0062] The extracted plasmids were identified by double digestion with BamHI and HindIII. Positive plasmids were sent for sequencing, and those correctly sequenced were named pFBD-P1 (see...). Figure 1 ).

[0063] Construction of 3.2pFBD-P1-3CD

[0064] The purified 3CD target gene and recombinant plasmid pFBD-P1 were double-digested with NheI and KpnI, respectively. The digestion products were subjected to 1% agarose gel electrophoresis. The gel containing the specific band was excised and recovered according to the gel extraction kit instructions. After determining the concentration of the recovered product, ligation and transformation were performed. Then, bacterial PCR was used for identification. Positive plasmids were extracted and further analyzed by double digestion with NheI and KpnI. Correctly identified plasmids were sent for sequencing and named pFBD-P1-3CD. The procedure was the same as in 3.1 (see...). Figure 2 The nucleotide sequence encoding the P1 protein of encephalomyocarditis virus is shown in SEQ ID NO.1, and the nucleotide sequence encoding the 3CD protein of encephalomyocarditis virus is shown in SEQ ID NO.2.

[0065] 4. Construction of recombinant rod-like particles rBacmid-P1-3CD

[0066] 4.1 Transformation of recombinant transfer plasmid into E. coli DH10Bac competent cells

[0067] The constructed pFBD-P1-3CD was transformed into competent cells. The detailed steps are as follows:

[0068] 1) After thawing E. coli DH10Bac competent cells on ice, add 2 μL of pFBD-P1-3CD plasmid and place on ice for 30 min;

[0069] 2) Place the mixture in a metal bath preheated to 42°C for 90 seconds for heat shock, and then quickly place it in an ice bath for 3 minutes;

[0070] 3) Add 800 μL of preheated LB liquid medium containing kanamycin and tetracycline resistance to the mixture in a clean bench, and incubate at 37℃ and 200 r / min for 1 h on a shaker.

[0071] 4) Centrifuge at 4500 r / min at room temperature for 2 min. Discard 700 μL of supernatant in a clean bench, and suspend the precipitate in about 200 μL of liquid. Add the precipitate to an LB agar plate containing X-Gal and IPTG and kanamycin (50 μg / mL). Spread the agar evenly with a spreader and incubate at 37℃ for 12-16 h until colonies grow on the plate.

[0072] 5) Blue-white screening: Select a single white colony, inoculate it into 5 mL of LB liquid medium containing triple antibodies, and incubate it on a shaker at 37℃ and 200 r / min for about 12 h.

[0073] 4.2 Extraction of recombinant rod-like particles rBacmid-P1-3CD

[0074] Because the recombinant baculovirus plasmid is large, it cannot be extracted using a kit. Detailed steps are as follows:

[0075] 1) Collect the bacterial culture in a 1.5 mL centrifuge tube, centrifuge at 10000 r / min for 1 min at room temperature, discard the supernatant, repeat the operation to collect all the bacterial cells, and aspirate to completely remove the residual liquid;

[0076] 2) Add 250 μL of Solution I containing RNase A, which was pre-cooled and stored at 4°C, to a centrifuge tube, suspend the bacterial cells, and let stand at room temperature for 10 min to allow for full reaction.

[0077] 3) Add 250μL SolutionⅡ, gently invert 5-10 times, and let stand at room temperature for 2-4 minutes. At this time, the liquid will become clear and stringy when the cap is opened.

[0078] 4) Add 350 μL Solution Ⅲ, gently invert and mix 5-10 times, let stand for 2 min, then centrifuge at 12000 r / min for 10 min to precipitate white flocculent matter;

[0079] 5) Transfer the supernatant to another centrifuge tube containing 500 μL of isopropanol, let it stand for 10 min, then centrifuge at 12000 r / min for 15 min, remove the supernatant with a pipette and mark the location of the precipitate.

[0080] 6) Add 500 μL of 75% ethanol, gently invert several times to wash the precipitate, centrifuge at 12000 r / min for 5 min, remove the supernatant with a pipette, open the lid and dry at room temperature for 10 min until the ethanol has completely evaporated;

[0081] 7) Add 30 μL of preheated ultrapure water (65°C) to the centrifuge tube to dissolve the precipitate. After measuring the concentration, store at -20°C for later use.

[0082] 4.3 PCR identification of recombinant rod-like particle rBacmid-P1-3CD

[0083] The extracted recombinant rod-like particle rBacmid-P1-3CD was amplified and identified by PCR using universal primer M13F as the upstream primer and 3CDR-KpnI as the downstream primer (see [link to relevant documentation]). Figure 3 ).

[0084] 3CD-RP-KpnI:5'-GGTACCTTAGAACAGACTCCTCCATCTGT-3'

[0085] 5. Expression of recombinant baculovirus

[0086] 5.1 Obtaining and amplifying the P1 generation strain (IFA identification)

[0087] The recombinant rod granule rBacmid-P1-3CD was transfected into sf9 insect cells, and the specific steps are as follows:

[0088] 1) Dilute sf9 cells with a viability greater than 90% to 5.5 × 10⁻⁶. 5 / mL, seeded into 6-well plates, adding 2mL to each well to achieve a total cell count of 1.1×10⁶ cells per well. 6 Place the cells in a 27°C incubator for 1 hour to allow them to adhere to the cell wall.

[0089] 2) Take 1 μg of purified rod DNA and 6 μL of inverted mixed Cellfectin reagent and dilute them into 100 μL of unsupplemented Grace medium. Then mix the two and incubate at room temperature for 30 min.

[0090] 3) Add 800 μL of culture medium to the mixture, mix well, and then add it to a six-well plate from which the cell culture medium has been discarded. Incubate at 27°C for 5 hours.

[0091] 4) Discard the mixture in the six-well plate, add 2 mL of complete culture medium, and incubate at 27°C for 72 h;

[0092] 5) After 72 hours, obvious lesions appeared in the cells. The culture medium was collected, and cell debris was removed by centrifugation. The supernatant obtained was the P1 generation strain and stored at -80℃ for later use.

[0093] 6) The transfection efficiency of recombinant rod granules was detected using indirect immunofluorescence technique. As shown in the figure, compared with the control, the recombinant rod granules have been successfully transfected into insect cells. (See...) Figure 4 ).

[0094] Because the P1 generation virus strain has a relatively low viral titer, it is necessary to continue infecting sf9 insect cells with the P1 generation virus strain to obtain a higher-titer virus strain. Add the P1 generation virus solution to a cell culture flask filled with sf9 insect cells at a ratio of 1:10, and incubate at 27°C for 72 hours. Once obvious cell pathogenesis occurs, collect the culture medium, centrifuge to remove cell debris, and the resulting supernatant is the P2 generation virus strain, stored at -80°C for later use. Similarly, infect sf9 insect cells with the P2 generation virus solution to obtain the P3 generation virus strain.

[0095] 6. Purification and identification of EMCV-VLPs

[0096] 6.1 Purification of EMCV-VLPs

[0097] 1) Expression of VLPs: SF9 cells were revived and cultured in SF9 insect cell culture medium at 27°C. When the cell density reached approximately 80%, the cells were infected with P3 generation virus solution at a ratio of 1:10. After 72 hours, the cell culture medium was harvested and subjected to three freeze-thaw cycles at -80°C. Then, the cells were centrifuged at 8000 rpm for 30 minutes at 4°C to remove cell debris. This process was repeated once.

[0098] 2) Sample concentration: The VLPs culture medium was concentrated to about 80 mL by filtration through a membrane bag; it was then dispensed into 4 ultrafiltration tubes and centrifuged at 40,000 r / min for 2 h at 4 °C, and the supernatant was discarded.

[0099] 3) Sample emulsification: The precipitate obtained after centrifugation was blown off with 1 mL of PBS and collected into an emulsification tube. The emulsification rod was pulled to spread it fully. The tube was centrifuged at 10000 r / min for 30 min at 4℃ and the supernatant was collected.

[0100] 4) Purification of VLPs: The final supernatant was added to a sucrose supernatant with a density gradient of 15%-45%, and centrifuged at 36,000 r / min for 2.5 h at 4 °C. The white virus band between the sucrose surface was collected, which is the highly purified VLPs.

[0101] 6.2 Identification of EMCV-VLPs

[0102] 6.2.1 Western blot identification of EMCV-VLPs

[0103] Take 80 μL of concentrated and purified VLPs sample, add 20 μL of 5× loading buffer, and boil in a 100℃ metal bath for 10 min. After SDS-PAGE electrophoresis, transfer the protein to an NC membrane using a wet transfer method. Remove the NC membrane containing the protein, wash once with TBST, and cover the NC membrane with 5% skim milk prepared with TBST solution. Block at room temperature for 1 h. Discard the blocking solution, and incubate the prepared polyclonal antibody 1000-fold diluted with 1% skim milk prepared with TBST solution overnight at 4℃. Then place on a circular shaker and wash 5 times with TBST for 8 min each time. Dilute HRP-labeled rabbit anti-IgG 1:3000 with 1% skim milk prepared with TBST solution and apply to the NC membrane. Incubate slowly on a shaker at room temperature for 1 h, and wash 5 times with TBST for 8 min each time. Prepare the ECL chemiluminescence solution according to the instructions: add 3 μL of solution B to 1 mL of solution A, mix well, and evenly apply to the NC membrane. Incubate in the dark for 1 min. Display the results using a Tanon 5200 fully automated chemiluminescence imaging analysis system. The results showed that the expressed EMCV-VLPs had three bands, the same as the viral control, and the size was consistent with the structural proteins of the EMCV virus. Figure 5 ).

[0104] 6.2.2 Transmission electron microscopy identification of EMCV-VLPs

[0105] 10 μL of sample was dropped onto the front side of a copper plate and allowed to adsorb for 2 minutes. Residual sample was then blotted dry with filter paper. The plate was then washed twice with 20 μL of PBS. Finally, it was negatively stained with 3% phosphotungstic acid for 1 minute. Residual staining solution was blotted dry with filter paper, and the plate was air-dried at room temperature before observation, imaging, and analysis using a transmission electron microscope (TEM). The results showed particles with a diameter similar to that of virus particles (approximately 20 nm) in the TEM, confirming the successful acquisition of EMCV-VLPs in this study. Figure 6 ).

[0106] 6.2.3 Immunoelectron microscopy identification of EMCV-VLPs

[0107] The purified VLPs sample was mixed with 4% paraformaldehyde (PFA) at a 1:1 ratio (total volume 50 μL). 20 μL of the mixture was dropped onto the front side of an electron microscope copper grid and incubated at room temperature for 20 min. The copper grid was washed twice with PBS for 3 min each time, followed by three washes with 50 mM glycine for 3 min each time. 20 μL of 5% BSA blocking solution was dropped onto the front side of the copper grid and incubated for 10 min. The polyclonal antibody serum was diluted 10-fold with primary antibody dilution buffer, and 20 μL was dropped onto the copper grid and incubated for 1 h. 20 μL of wash buffer was dropped onto the copper grid and washed six times for 3 min each time. Colloidal gold particles were diluted 10-fold with secondary antibody dilution buffer, and 20 μL was dropped onto the copper grid and incubated for 1 h. 20 μL of wash buffer was dropped onto the copper grid and incubated for 1 h. The buffer was dropped onto a copper grid and washed six times for 2 minutes each time, followed by six washes with PBS for 2 minutes each time. 20 μL of 1% glutaraldehyde was then dropped onto the copper grid and allowed to stand for 2 minutes. Next, 20 μL of deionized water was used to wash six times for 2 minutes each time. Finally, the grid was negatively stained with 3% phosphotungstic acid for 1 minute. Residual staining was blotted dry with filter paper, and the grid was air-dried at room temperature before observation, photography, and analysis using a transmission electron microscope. The results showed that EMCV-VLPs bound well to the secondary antibody containing colloidal gold, confirming that the VLPs obtained in this study were indeed EMCV-VLPs. Figure 7 ).

[0108] 6.2.4 Mouse Immunization Experiment with EMCV-VLPs

[0109] This experiment used purified EMCV-VLPs to immunize mice. Mice were divided into four groups of five each. Group 1 was the PBS control group, with each mouse receiving 200 μL intramuscularly. Group 2 was the VLPs + 206 group, with each mouse receiving 50 μg intramuscularly. Group 3 was the 1 / 2 VLPs + 206 group, with each mouse receiving 25 μg intramuscularly. Group 4 was the VLPs group, with each mouse receiving 50 μg intramuscularly. The immunization protocol was as follows: initial immunization, a second booster immunization two weeks later, and blood samples were collected from mice four weeks later for immune-related marker detection. The animal immunization results showed that the EMCV-VLPs obtained in this study could stimulate the body to produce good neutralizing and specific antibodies. Figure 8 ).

Claims

1. A method for preparing porcine encephalomyocarditis virus-like particles, characterized in that, The method includes the following steps: (1) RNA extraction and reverse transcription Total RNA was extracted from EMCV viral fluid, and then cDNA was obtained by reverse transcription. After the concentration was measured, the cDNA was stored at -20°C for later use. (2) Amplification of P1 and 3CD fragments Based on the gene sequences of EMCV-P1 and EMCV-3CD published in GenBack by NCBI, specific primers for amplifying their full-length sequences were designed using primer design software. Primers P1-FP / P1-RP were designed to amplify P1 using the recognition sites of BamHI and HindIII restriction enzymes, respectively. Primers 3CD-FP / 3CD-RP were designed to amplify 3CD using the recognition sites of NheI and KpnI restriction enzymes, respectively. Using EMCV cDNA as a template, the target fragments of P1 and 3CD were amplified, respectively. P1-FP: 5'-GGATCCATGTCCTCAGACAAGAATAACT-3' P1-RP: 5'-AAGCTTTTATAGCATCAAGACTCCAGCT-3' 3CD-FP: 5'-GCTAGCATGCCGAACCCTGTGATGGACTT-3' 3CD-RP: 5'-GGTACCTTAGAACAGACTCCTCCATCTGT-3' The nucleotide sequence encoding the P1 protein of encephalomyocarditis virus is shown in SEQ ID NO.1, and the nucleotide sequence encoding the 3CD protein of encephalomyocarditis virus is shown in SEQ ID NO.2; (3) Construction of pFBD-P1 The purified P1 target gene and the pFastBacDual vector were respectively used... Bam HI and Hind III. The target fragment of P1 was digested with enzymes and then ligated into the pFastBacDual vector to obtain the recombinant plasmid pFBD-P1. (4) Construction of pFBD-P1-3CD The purified 3CD target gene and recombinant plasmid pFBD-P1 were respectively used... Nhe I and Kpn I was double-digested with enzymes, and the digested 3CD target fragment was ligated into the pFBD-P1 vector to obtain the recombinant transfer plasmid pFBD-P1-3CD; (5) The recombinant transfer plasmid pFBD-P1-3CD was transformed into E. coli DH10Bac competent cells Convert the constructed pFBD-P1-3CD to E. coli DH10Bac competent cells were screened by blue-white screening to obtain positive colonies. The colonies were cultured and recombinant rod mid-P1-3CD was extracted from them. (6) Expression of recombinant baculovirus Recombinant rod-like particle rBacmid-P1-3CD was transfected into sf9 insect cells to obtain the P1 generation strain. The P1 generation strain was then used to infect sf9 insect cells to obtain the P2 generation strain with a higher titer. Similarly, the P2 generation virus solution was used to infect sf9 insect cells to obtain the P3 generation strain. (7) Purification and identification of EMCV-VLPs sf9 insect cells were infected with P3 generation virus solution, and the cell culture medium was harvested. After repeated freeze-thaw cycles, centrifugation, concentration, emulsification, and sucrose density gradient purification, purified porcine encephalomyocarditis virus-like particles were obtained.

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