Fusion protein and nanoparticle of porcine reproductive and respiratory syndrome virus recombinant epitope and application of fusion protein and nanoparticle

Abstract

The invention belongs to the technical field of veterinary vaccines, and particularly relates to fusion protein and nanoparticles of porcine reproductive and respiratory syndrome virus recombinant epitopes and application of the fusion protein and the nanoparticles. A GP3 protein antigen epitope, a GP4 protein antigen epitope, a GP5 protein antigen epitope and an M protein antigen epitope are recombined, the obtained recombinant protein is closely related to protective immunity of the PRRSV, a strong humoral immune response aiming at the PRRSV can be generated after piglets are immunized, and a good protection effect is generated on infection of the PRRSV. The fusion protein is connected with the beta-cyclopeptide, and the beta-cyclopeptide serving as a nano skeleton can spontaneously form 24-surface nanoparticles, so that the antigen can be effectively presented, and the immune effect of the vaccine is promoted. Furthermore, the N terminal of the beta cyclopeptide is connected with an OX40L protein, and the OX40L protein belongs to a tumor necrosis factor ligand superfamily, so that the cellular immune effect can be further improved.

Description

Technical Field

[0001] This invention belongs to the field of veterinary vaccine technology, specifically relating to a fusion protein and nanoparticles of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus and their applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-sense RNA virus belonging to the genus Betaarterivirus, family Arteriviridae, and order Nidovirales. The PRRSV genome is approximately 15.4 kb in length and encodes eight structural proteins, including four membrane-associated glycoproteins (GP2a, GP3, GP4, GP5), three unglycosylated membrane proteins (E, ORF5a, M), and one nucleocapsid protein (N). Since its emergence, PRRSV has been a significant viral disease affecting the swine industry, characterized by respiratory symptoms in pigs of all ages and reproductive disorders in sows.

[0003] Although some drugs targeting PRRSV infection have been developed and genetically edited pigs targeting PRRSV infection have been proposed, vaccination still plays a crucial role in controlling the spread of PRRSV. Currently, there are two main types of commercial vaccines against PRRSV: modified live attenuated vaccines (MLV) and inactivated vaccines. MLV vaccines are generally effective against homologous strains but have limited effectiveness against heterologous strains and carry the risk of virulence reversion, while inactivated vaccines often have limited efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a fusion protein and nanoparticles of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) and their applications. Based on the fusion protein and nanoparticles, a PRRSV nanoparticle vaccine is prepared, which can stimulate a strong immune response in the body and provide effective protection against PRRSV infection.

[0005] This invention provides a fusion protein of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus, the fusion protein comprising GP3 protein antigenic epitope, GP4 protein antigenic epitope, GP5 protein antigenic epitope-1, GP5 protein antigenic epitope-2 and M protein antigenic epitope sequentially linked by a flexible peptide. The amino acid sequence of the GP3 protein antigenic epitope is shown in SEQ ID NO:1; The amino acid sequence of the GP4 protein antigenic epitope is shown in SEQ ID NO:2; The amino acid sequence of the GP5 protein antigen epitope-1 is shown in SEQ ID NO:3; The amino acid sequence of the GP5 protein antigenic epitope-2 is shown in SEQ ID NO:4; The amino acid sequence of the M protein antigenic epitope is shown in SEQ ID NO:5.

[0006] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:6.

[0007] This invention provides a DNA fragment encoding the fusion protein described in the above-mentioned technical solution.

[0008] This invention provides a porcine reproductive and respiratory syndrome virus nanoparticle, the nanoparticle comprising a fusion protein and a β-cyclic peptide linked to the N-terminal flexible peptide of the fusion protein; The amino acid sequence of the β-cyclic peptide is shown in SEQ ID NO:8; The fusion protein is the fusion protein described in the above technical solution or the fusion protein encoded by the DNA fragment described in the above technical solution.

[0009] Preferably, the nanoparticles further include an OX40L protein linked to the N-terminal flexible peptide of the β-cyclic peptide, the amino acid sequence of which is shown in SEQ ID NO:10.

[0010] This invention provides a DNA fragment encoding the porcine reproductive and respiratory syndrome virus nanoparticles described in the above-mentioned technical solution.

[0011] This invention provides a method for preparing porcine reproductive and respiratory syndrome virus (PRRSV) nanoparticles as described in the above technical solution, comprising the following steps: cloning the DNA fragment of the PRSV nanoparticles as described in the above technical solution into a vector to obtain a recombinant vector; transforming the recombinant vector into a host bacterium to obtain an engineered bacterium; and culturing, expressing, and purifying the engineered bacterium to obtain the PRSV nanoparticles.

[0012] This invention provides a biological material comprising a recombinant vector or engineered bacteria, wherein the recombinant vector comprises a base vector and a target gene inserted into the base vector; The engineered bacteria include a basic bacterium and a target gene or recombinant vector introduced into the basic bacterium; The target gene is the DNA fragment described in the above technical solution.

[0013] This invention provides the application of the fusion protein described in the above-described technical solution, or the fusion protein encoded by the DNA fragment described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles encoded by the DNA fragment described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles prepared by the preparation method described in the above-described technical solution, or the biomaterials described in the above-described technical solution, in the preparation of porcine reproductive and respiratory syndrome vaccines.

[0014] This invention provides a vaccine for porcine reproductive and respiratory syndrome, comprising an antigen and an adjuvant; The antigen is the fusion protein described in the above technical solution, or the fusion protein encoded by the DNA fragment described in the above technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticle described in the above technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticle encoded by the DNA fragment described in the above technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticle prepared by the preparation method described in the above technical solution.

[0015] Beneficial effects: This invention provides a fusion protein of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus (PRRSV). The fusion protein comprises GP3, GP4, GP5-1, GP5-2, and M protein epitopes linked sequentially by flexible peptides, with amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:5. The structural proteins GP3, GP4, GP5, and M of PRRSV are important immunogenic proteins. This invention selects GP3, GP4, GP5, and M protein epitopes and recombines them. The resulting recombinant protein is closely related to protective immunity against PRRSV, and immunizing piglets with it induces a strong humoral immune response against PRRSV, providing excellent protection against PRRSV infection.

[0016] This invention links a fusion protein to a β-cyclic peptide. The β-cyclic peptide, acting as a nanoframework, can spontaneously form 24-sided nanoparticles, effectively presenting antigens and enhancing the immune response of vaccines. Furthermore, this invention links the OX40L protein, belonging to the tumor necrosis factor ligand superfamily, to the N-terminus of the β-cyclic peptide, which can further improve cellular immunity. Results from the examples show that the specific antibody levels in the OX40L-β-MEs and β-MEs vaccine groups were higher than those in the monomeric fusion protein (MEs) group, and there was no significant difference between the OX40L-β-MEs and β-MEs vaccine groups. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Schematic diagram of the recombinant protein structures of MEs, β-MEs, and OX40L-β-MEs; Figure 2PCR identification diagrams of recombinant plasmids pColdⅡ-MEs, pColdⅡ-β-MEs, and pColdⅡ-OX40L-β-MEs; Figure 3 SDS-PAGE and Western blotting images of purified fusion proteins MEs; Figure 4 SDS-PAGE and Western blotting images of purified fusion protein β-MEs; Figure 5 SDS-PAGE and Western blotting images of the purified fusion protein OX40L-β-MEs; Figure 6 Protein particle size distributions determined by dynamic laser scattering for fusion proteins β-MEs and OX40L-β-MEs; Figure 7 These are the results of transmission electron microscopy observations of the fusion protein β-MEs; the red arrows indicate nanoparticles. Figure 8 The results of transmission electron microscopy observation of the fusion protein OX40L-β-MEs are shown; the red arrows indicate nanoparticles. Figure 9 The results of detecting PRRSV-specific antibody levels in serum at different time points after immunization of piglets; ns indicates no significant difference. express P <0.05, express P <0.01, express P <0.001, express P <0.0001. Detailed Implementation

[0019] This invention provides a fusion protein of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus, the fusion protein comprising GP3 protein antigenic epitope, GP4 protein antigenic epitope, GP5 protein antigenic epitope-1, GP5 protein antigenic epitope-2 and M protein antigenic epitope sequentially linked by a flexible peptide. The amino acid sequence of the GP3 protein antigenic epitope is shown in SEQ ID NO:1; The amino acid sequence of the GP4 protein antigenic epitope is shown in SEQ ID NO:2; The amino acid sequence of the GP5 protein antigen epitope-1 is shown in SEQ ID NO:3; The amino acid sequence of the GP5 protein antigenic epitope-2 is shown in SEQ ID NO:4; The amino acid sequence of the M protein antigenic epitope is shown in SEQ ID NO:5.

[0020] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:6.

[0021] In one embodiment, the amino acid sequence of the flexible peptide connecting any two antigenic epitopes of the present invention is (GGS)n, where n is any integer between 2 and 4.

[0022] Porcine reproductive and respiratory syndrome virus (PRRSV) structural proteins GP3, GP4, GP5, and M are important immunogenic proteins. The full-length proteins contain non-protective epitopes and immunomodulatory epitopes, which can reduce the protective immune response of vaccines. This invention truncates the proteins and selects antigenic epitopes for recombination, which can reduce the immune protective response and produce a good protective effect against PRRSV infection. Furthermore, this invention recombines the GP3, GP4, GP5, and M protein epitopes and connects them sequentially, which is more conducive to maximizing vaccine immunogenicity and achieving synergistic effects.

[0023] This invention provides a DNA fragment encoding the fusion protein described in the above-described technical solution. As one embodiment, the nucleotide sequence of the DNA fragment of this invention is shown in SEQ ID NO:7.

[0024] SEQ ID NO: 1: GRGPPCLTRQAAAQIYEPGRTLWCRIGHDRCEESHDELGFVVPPGLS; SEQ ID NO:2: DIKTNTTSASSSVVLQDISCLSHSYPASAAFRKLPQCR; SEQ ID NO:3: SHLQLIYNLTICELYGTDWLEEKFD; SEQ ID NO:4:CMSWRYSCTRYTNFSLNTKGRLYPWRSPVIIERGGKVDVGGLLIDLKKVVLDGSAATPVTKIPAEQWGRP; SEQ ID NO:5: MGSSIADFCNLE; SEQ ID NO:6:GRGPPCLTRQAAAQIYEPGRTLWCRIGHDRCEESDHDELGFVVPPGLSGGSGGSGGSDIKTNTTSASSSVVLQDISCLSHSYPASAAFRKLPQCRGGSGGSGGSSHLQLIYNLTICELYGTDWLEEKFDGGSGGSGGSCMSWRYSCTRYTNFSLNTKGRLYPWRSPVIIERGGKVDVGGLLIDLKKVVLDGSAATPVTKIPAEQWGRPGGSGGSGGSMGSSIADFCNLE; SEQ ID NO:7:GGTCGTGGTCCGCCGTGTCTGACTCGTCAGGCGGCTGCTCAAATCTACGAACCGGGTCGTACTCTGTGGTGCCGTATCGGTCACGACCGTTGCGAAGAAAGCGATCACGATGAACTGGGTTTCGTTGTTCCGCCAGGTCTTAGCGGCGGTAGCGGTGGTTCTGGCGGTTCTGACATCAAGACCAACACCACCTCTGCTTCTTCCAGCGTTGTACTGCAAGACATCAGCTGCCTGAGTCACTCTTATCCGGCTTCCGCTGCGTTCCGTAAGCTCCCGCAGTGCCGTGGTGGTTCCGGCGGATCTGGTGGCTCTTCTCACCTGCAGCTGATCTACAACCTGACCATCTGCGAACTGTACGGTACCGACTGGCTGGAAGAGAAATTCGATGGCGGCTCCGGCGGCTCTGGCGGTAGCTGCATGTCTTGGCGTTACTCTTGCACTCGTTACACCAACTTCTCGCTGAACACCAAAGGTCGTCTGTACCCTTGGCGTTCTCCGGTTATCATCGAACGTGGTGGTAAAGTTGACGTTGGTGGTCTCTTGATCGATCTGAAGAAAGTTGTTCTGGACGGTTCTGCAGCAACTCCGGTTACCAAGATCCCTGCGGAACAGTGGGGCCGTCCGGGTGGAAGCGGTGGTAGTGGTGGTAGCATGGGTAGCTCCATCGCCGATTTCTGCAACCTGGAA。

[0025] This invention provides a porcine reproductive and respiratory syndrome virus nanoparticle, the nanoparticle comprising a fusion protein and a β-cyclic peptide linked to an N-terminal flexible peptide of the fusion protein; the amino acid sequence of the β-cyclic peptide is shown in SEQ ID NO:8; the fusion protein is the fusion protein described in the above technical solution or a fusion protein encoded by a DNA fragment described in the above technical solution.

[0026] As one embodiment, the nucleotide sequence of the β-cyclic peptide of the present invention is shown in SEQ ID NO:9. As one embodiment, the amino acid sequence of the flexible peptide connecting the fusion protein and the β-cyclic peptide of the present invention is (GGS)n, where n is any integer between 2 and 4.

[0027] In one embodiment, the nanoparticles of the present invention further include an OX40L protein linked to the N-terminal flexible peptide of the β-cyclic peptide, the amino acid sequence of which is shown in SEQ ID NO:10. In another embodiment, the nucleotide sequence of the OX40L protein is shown in SEQ ID NO:11. In yet another embodiment, the amino acid sequence of the flexible peptide linking the OX40L protein and the β-cyclic peptide is SSSGSG (SEQ ID NO:20). The present invention links the β-cyclic peptide to the OX40L protein, allowing OX40L to self-assemble under the action of the β-cyclic peptide, thus obtaining a nanoparticle fusion protein and fully leveraging the OX40L protein's ability to enhance cellular immunity. If the β-cyclic peptide and OX40L protein are not linked and exist as a composite, the OX40L protein cannot effectively exert its function.

[0028] This invention provides a DNA fragment encoding the porcine reproductive and respiratory syndrome virus (PRRSV) nanoparticles described in the above-described technical solution. As one embodiment, the nucleotide sequence of the DNA fragment is shown in SEQ ID NO:12 or SEQ ID NO:13. The DNA fragment shown in SEQ ID NO:12 is a PRSV nanoparticle formed by fusing the fusion protein and β-cyclic peptide described in the above-described technical solution. The DNA fragment shown in SEQ ID NO:13 is a PRSV nanoparticle formed by fusing the fusion protein, β-cyclic peptide, and OX40L protein described in the above-described technical solution.

[0029] SEQ ID NO:8: INHVGGTGGAIMAPVAVTRQLVGS; SEQ ID NO:9: ATCAACCACGTCGGCGGTACAGGCGTGCTATCATGGCACCTGTGGCTGTGACACGTCAGCTGGTGGGAAGC; SEQ ID NO:10:MGTMKVQNNSIIINCDGFYLITLKGYFSQELSLMLQYRKGTKPLFSLYKVKSVDSVTVADVAFKDKVFPNVTTHSASCEDIQVNGGELILIHQNPGRFCVY; SEQ ID NO:11:ATGGGTACCATGAAGGTGCAGAACAATTCCATCATCATCAACTGCGACGGCTTCTACCTGATTACCCTGAAGGGCTACTTTTCCCAGGAGCTGTCCCTGATGCTGCAGTACAGGAAGGGGACGAAACCCCTGTTCTCCCTGTACAAGGTGAAGAGCGTGGACAGCGTGACCGTGGCTGATGTGGCTTTCAAGGACAAGGTGTTCCCGAACGTGACCACCCACTCCGCAAGCTGTGAGGATATCCAGGTGAACGGCGGCGAGCTGATCCTGATTCATCAGAACCCTGGCCGTTTTTGCGTGTAT; SEQ ID NO:12:ATCAACCACGTCGGCGGTACAGGCGGTGCTATCATGGCACCTGTGGCTGTGACACGTCAGCTGGTGGGAAGCGGTGGCTCTGGTGGTAGCGGCGGTTCCGGTGGATCCGGTCGTGGTCCGCCGTGTCTGACTCGTCAGGCGGCTGCTCAAATCTACGAACCGGGTCGTACTCTGTGGTGCCGTATCGGTCACGACCGTTGCGAAGAAAGCGATCACGATGAACTGGGTTTCGTTGTTCCGCCAGGTCTTAGCGGCGGTAGCGGTGGTTCTGGCGGTTCTGACATCAAGACCAACACCACCTCTGCTTCTTCCAGCGTTGTACTGCAAGACATCAGCTGCCTGAGTCACTCTTATCCGGCTTCCGCTGCGTTCCGTAAGCTCCCGCAGTGCCGTGGTGGTTCCGGCGGATCTGGTGGCTCTTCTCACCTGCAGCTGATCTACAACCTGACCATCTGCGAACTGTACGGTACCGACTGGCTGGAAGAGAAATTCGATGGCGGCTCCGGCGGCTCTGGCGGTAGCTGCATGTCTTGGCGTTACTCTTGCACTCGTTACACCAACTTCTCGCTGAACACCAAAGGTCGTCTGTACCCTTGGCGTTCTCCGGTTATCATCGAACGTGGTGGTAAAGTTGACGTTGGTGGTCTCTTGATCGATCTGAAGAAAGTTGTTCTGGACGGTTCTGCAGCAACTCCGGTTACCAAGATCCCTGCGGAACAGTGGGGCCGTCCGGGTGGAAGCGGTGGTAGTGGTGGTAGCATGGGTAGCTCCATCGCCGATTTCTGCAACCTGGAAGGCGGTTCTGGTGGTTCTCACCATCACCACCACCACCATCAC;

[0030] This invention provides a method for preparing porcine reproductive and respiratory syndrome virus (PRRSV) nanoparticles as described in the above technical solution, comprising the following steps: cloning the DNA fragment of the PRSV nanoparticles as described in the above technical solution into a vector to obtain a recombinant vector; transforming the recombinant vector into a host bacterium to obtain an engineered bacterium; and culturing, expressing, and purifying the engineered bacterium to obtain the PRSV nanoparticles.

[0031] In one embodiment, the present invention clones the DNA fragment of the porcine reproductive and respiratory syndrome virus nanoparticles described in the above-mentioned technical solution into a vector after adding a purification tag to the 3' end. In one embodiment, the purification tag is an 8×His tag. In another embodiment, the present invention utilizes a flexible peptide to link the C-terminus of the DNA fragment to the purification tag.

[0032] This invention provides a biological material comprising a recombinant vector or engineered bacteria, wherein the recombinant vector comprises a base vector and a target gene inserted into the base vector; the engineered bacteria comprises a base bacterium and a target gene introduced into the base bacterium or the recombinant vector; the target gene is the DNA fragment described in the above technical solution.

[0033] In one embodiment, the basic vector of the present invention is an *E. coli* expression vector. In another embodiment, the basic vector of the present invention is pColdⅡ. In yet another embodiment, the basic bacterium of the present invention is *E. coli*.

[0034] This invention provides the application of the fusion protein described in the above-described technical solution, or the fusion protein encoded by the DNA fragment described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles encoded by the DNA fragment described in the above-described technical solution, or the porcine reproductive and respiratory syndrome virus nanoparticles prepared by the preparation method described in the above-described technical solution, or the biomaterials described in the above-described technical solution, in the preparation of porcine reproductive and respiratory syndrome vaccines.

[0035] As one implementation method, the porcine reproductive and respiratory syndrome (PRRS) vaccine of the present invention is a porcine reproductive and respiratory syndrome nanoparticle vaccine.

[0036] This invention provides a porcine reproductive and respiratory syndrome (PRRS) vaccine, comprising an antigen and an adjuvant; wherein the antigen is a fusion protein as described in the above-described technical solution, or a fusion protein encoded by a DNA fragment as described in the above-described technical solution, or a porcine reproductive and respiratory syndrome virus nanoparticle as described in the above-described technical solution, or a porcine reproductive and respiratory syndrome virus nanoparticle encoded by a DNA fragment as described in the above-described technical solution, or a porcine reproductive and respiratory syndrome virus nanoparticle prepared by the preparation method described in the above-described technical solution.

[0037] In one embodiment, the volume ratio of antigen to adjuvant in this invention is 1:1. In another embodiment, the adjuvant in this invention is ISA 201VG adjuvant.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a fusion protein and nanoparticles of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus and their applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1 1. Synthesis of the target gene (1) A fusion protein containing the PRRSV recombinant antigenic epitope with the amino acid sequence shown in SEQ ID NO:6 was synthesized based on the PRRSV GX-3264 (GeneBank accession no: PV240231) sequence. An 8×His tag was inserted at its C-terminus, denoted as MEs. The amino acid sequence is shown in SEQ ID NO:14, and the nucleotide sequence encoding the gene is shown in SEQ ID NO:15. The structural diagram is shown in [Figure 1]. Figure 1 As shown.

[0040] SEQ ID NO:14:GGSGGSGGSGGSGRGPPCLTRQAAAQIYEPGRTLWCRIGHDRCEESDHDELGFVVPPGLSGGSGGSGGSDIKTNTTSASSSVVLQDISCLSHSYPASAAFRKLPQCRGGSGGSGGSSHLQLIYNL TICELYGTDWLEEKFDGGSGGSGGSCMSWRYSCTRYTNFSLNTKGRLYPWRSPVIIERGGKVDVGGLLIDLKKVVLDGSAATPVTKIPAEQWGRPGGSGGSGGSMGSSIADFCNLEGGSGGSHHHHHHHH; SEQ ID NO:15:

[0041] (2) OX40L was artificially synthesized. The amino acid sequence is shown in SEQ ID NO:10 and the nucleotide sequence is shown in SEQ ID NO:11.

[0042] (3) Artificially synthesized β-cyclic peptide, the amino acid sequence of which is shown in SEQ ID NO:8 and the nucleotide sequence of which is shown in SEQ ID NO:9.

[0043] 2. Construction of recombinant plasmids (1) Using the target gene obtained in step 1 as a template, the gene fragment was obtained by overlap PCR amplification using the primers shown in Table 1. Specifically: Using the aforementioned MEs and β-cyclic peptides as templates, β-MEs with nucleotide sequences as shown in SEQ ID NO:12 were obtained by overlap PCR amplification using β-MEs-F and β-MEs-R. A schematic diagram of the structure is shown below. Figure 1 As shown; Using the aforementioned β-MEs and OX40L as templates, two overlap PCR amplifications were performed using OX40L-F, OX40L-R, and β-MEs-R to obtain OX40L-β-MEs with nucleotide sequences as shown in SEQ ID NO:13. A schematic diagram of the structure is shown below. Figure 1 As shown; the primer combination used for the first overlap PCR amplification was OX40L-F and OX40L-R, and the primer combination used for the second overlap PCR amplification was OX40L-F and β-MEs-R.

[0044] The reaction system for the overlap PCR amplification was as follows: 25 μL of 2×PhantaMax Buffer, 1 μL of dNTPMix, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 100 ng of template, and PhantaRMax Super-Fidelity DNAPolyMEsras (1 U / μL). The system was then brought to a final volume of 50 μL with ddH2O. The reaction program for overlap PCR amplification was as follows: 95℃ for 5 min; 95℃ for 15 s, 65℃ for 15 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min.

[0045] Table 1 Primer sequences for overlap PCR amplification

[0046] (2) The coding gene fragments of MEs, β-MEs and OX40L-β-MEs obtained in the above steps were cloned into NdeⅠ and EcoRⅠ of the prokaryotic expression vector pColdⅡ, respectively, and identified by PCR. Figure 2 After the correct steps are taken, recombinant plasmids pColdⅡ-MEs, pColdⅡ-β-MEs, and pColdⅡ-OX40L-β-MEs are obtained.

[0047] 3. Construction of recombinant expression strains The successfully constructed recombinant plasmids pColdⅡ-MEs, pColdⅡ-β-MEs, and pColdⅡ-OX40L-β-MEs were transformed into the pG-Tf2 / BL21 expression strain containing the pG-Tf2 molecular chaperone, respectively. The strains were cultured overnight at 37°C, followed by single-clone selection, colony PCR detection, and sequencing. The correctly identified positive transformants were the recombinant expression strains, which were then preserved in 20% glycerol and stored at -80°C.

[0048] 4. Expression and purification of fusion proteins MEs, β-MEs and OX40L-β-MEs (1) Take the bacterial suspensions preserved in the above steps and inoculate them into fresh liquid LB medium at a ratio of 1:1000 (V / V) overnight. Then, take the overnight cultured bacterial suspensions and inoculate them into 1L of liquid LB medium at a ratio of 1:100 (V / V). Incubate at 37℃ and 180rpm for 3h. The liquid LB medium contains ampicillin at a final concentration of 100μg / mL, chloramphenicol at 25μg / mL, and tetracycline at 5ng / mL. When the OD600 value of the bacterial suspensions reaches 0.6~0.8, add IPTG at a final concentration of 0.4mmol / L for induction. Induce expression at 16℃ and 160rpm for 20h. Then, centrifuge the induced bacterial suspensions at 7000rpm for 10min to collect the bacterial cells.

[0049] (2) The bacterial cells collected in step (1) were resuspended in Tris-HCl buffer at a ratio of 1:10 (V / V) and disrupted by a low-temperature ultrasonic cell disruptor. The supernatant was collected by centrifugation at 12,000 rpm and 4°C for 40 min. The precipitate was resuspended in an equal volume of Tris-HCl and then detected by SDS-PAGE and Western blotting.

[0050] (3) The supernatant obtained in step (2) was filtered through a 0.22 μm filter membrane to remove cell debris. The filtered supernatant was then passed through a peristaltic pump to bind with Ni packing material (the His tag at the end of the target protein can bind to Ni). After binding, linear elution was performed using a protein purification elution instrument. The purification effect of the target protein was then detected by SDS and Western blotting. The results are as follows: Figures 3-5 As shown, MEs, β-MEs and OX40L-β-MEs fusion proteins with the expected target molecular weight were prepared.

[0051] Test Example 1 1. Nanoparticle size detection The β-MEs and OX40L-β-MEs fusion proteins prepared in Example 1 were concentrated by ultrafiltration using a 10 kDa ultrafiltration tube at 4°C, and then concentrated to 2 mL with Tris-HCl solution at pH 8.0. The concentration was then analyzed by SDS-PAGE. The concentrated protein was filtered through a 0.1 μM filter, diluted with Tri-HCl, and finally measured and analyzed using a dynamic laser scattering spectrometer. The results are as follows: Figure 6 As shown, the particle size of the β-MEs fusion protein is concentrated between 20 and 40 nm, and the particle size of the OX40L-β-MEs fusion protein is concentrated between 40 and 60 nm, indicating that the β-MEs and OX40L-β-MEs fusion proteins have high purity and good uniformity.

[0052] 2. Observation using transmission electron microscopy The β-MEs and OX40L-β-MEs fusion protein samples prepared in Example 1 were adsorbed onto copper grids and stained with 2% (w / v) sodium phosphotungstic acid solution for 30 seconds. After the copper grids were completely dry, transmission electron microscopy (Bio-TEM) was used to observe whether the proteins could form nanostructures. The results are as follows: Figure 7 and Figure 8 As shown, the fusion proteins β-MEs and OX40L-β-MEs prepared in this invention can both form nanoparticles.

[0053] Example 2 Preparation and Immunization Evaluation Trial of Porcine Reproductive and Respiratory Syndrome Virus Nanoparticle Vaccine 1. Preparation of porcine reproductive and respiratory syndrome virus nanoparticle vaccine The MEs, β-MEs and OX40L-β-MEs fusion proteins obtained in Example 1 were used as antigens, diluted with sterile PBS, and emulsified with ISA 201VG adjuvant at a volume ratio of 1:1 to prepare porcine reproductive and respiratory syndrome virus nanoparticle vaccines, with a final antigen concentration of 50 μg / mL.

[0054] 2. Animal immunization trials of porcine reproductive and respiratory syndrome virus nanoparticle vaccine Twenty-five 5-week-old piglets, negative for porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASV), classical classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), and porcine circovirus (PCV), were randomly divided into five groups: MEs group, β-MEs group, OX40L-β-MEs group, commercially available inactivated vaccine group, and PBS group, with five piglets in each group. Immunization was administered intramuscularly according to the protocol in Table 2, with each piglet receiving 100 μg. A booster immunization with the same dose was given 21 days after the initial immunization.

[0055] Table 2 Vaccine Immunization Schedule

[0056] 3. Indirect ELISA method for detecting serum specific IgG antibody levels Serum samples were collected at 0, 14, 28, and 42 days after the initial immunization in step 2. ELISA plates were coated with inactivated PRRSV GX-3264, and the specific IgG antibody levels in the immunized piglet serum were detected using an indirect ELISA method. The specific steps are as follows: (1) Coat the ELISA plate with inactivated PRRSV GX-3264, using 100 μL of coating buffer per well to coat 0.5 μg, and coat at 4℃ for 14-16 h; (2) Discard the coating buffer and add 200 μL of PBS solution to each well for washing; (3) Add 200 μL of 2% BSA solution to each well and block at 37℃ for 2 h; (4) Wash with PBST 4 times, 5 min each time; (5) Then add 100 μL of diluted serum to be tested to each well, set up positive and negative controls, and incubate at 37℃ for 1 h; (6) After thorough washing, use diluted HRP-labeled goat anti-pig IgG antibody (1:10000) and incubate at 37℃ for 1 h; (7) Wash with PBST 4 times, 5 min each time, add TMB substrate solution (50 μL each of solution A and solution B) and react in the dark for 15 min; (8) Add 50 μL of PBST to each well. The colorimetric reaction was terminated with a stop solution, and the OD450 value was then measured using a multi-functional microplate reader.

[0057] The results are as follows Figure 9 As shown, the results are the average of three repeated experiments. P Data with a value <0.05 were statistically significant. The results showed that, compared to the PBS group, piglets in all vaccine groups produced higher levels of specific IgG at 42 days post-immunization. Furthermore, the specific antibody levels in the OX40L-β-MEs and β-MEs vaccine groups were higher than those in the MEs monomer group.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion protein of recombinant antigenic epitopes of porcine reproductive and respiratory syndrome virus, characterized in that, The fusion protein comprises GP3 protein antigenic epitope, GP4 protein antigenic epitope, GP5 protein antigenic epitope-1, GP5 protein antigenic epitope-2 and M protein antigenic epitope connected sequentially by a flexible peptide. The amino acid sequence of the GP3 protein antigenic epitope is shown in SEQ ID NO:1; The amino acid sequence of the GP4 protein antigenic epitope is shown in SEQ ID NO:2; The amino acid sequence of the GP5 protein antigen epitope-1 is shown in SEQ ID NO:3; The amino acid sequence of the GP5 protein antigenic epitope-2 is shown in SEQ ID NO:4; The amino acid sequence of the M protein antigenic epitope is shown in SEQ ID NO:

5.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

6.

3. A DNA fragment encoding the fusion protein of claim 1 or 2.

4. A nanoparticle containing porcine reproductive and respiratory syndrome virus, characterized in that, Includes a fusion protein and a β-cyclic peptide linked to the N-terminal flexible peptide of the fusion protein; The amino acid sequence of the β-cyclic peptide is shown in SEQ ID NO:8; The fusion protein is the fusion protein described in claim 1 or 2, or the fusion protein encoded by the DNA fragment described in claim 3.

5. The porcine reproductive and respiratory syndrome virus nanoparticles according to claim 4, characterized in that, The nanoparticles also include an OX40L protein linked to the N-terminal flexible peptide of the β-cyclic peptide, the amino acid sequence of which is shown in SEQ ID NO:

10.

6. A DNA fragment encoding the porcine reproductive and respiratory syndrome virus nanoparticles of claim 4 or 5.

7. The method for preparing porcine reproductive and respiratory syndrome virus nanoparticles according to claim 4 or 5, characterized in that, Includes the following steps: The DNA fragment described in claim 6 is cloned into a vector to obtain a recombinant vector; The recombinant vector was transformed into the host bacteria to obtain engineered bacteria; The engineered bacteria were cultured, expressed, and purified to obtain the porcine reproductive and respiratory syndrome virus nanoparticles.

8. A biomaterial comprising a recombinant vector or engineered bacteria, characterized in that, The recombinant vector includes a base vector and a target gene inserted into the base vector; The engineered bacteria include a basic bacterium and a target gene or recombinant vector introduced into the basic bacterium; The target gene is the DNA fragment described in claim 3 or 6.

9. The application of the fusion protein of claim 1 or 2, the fusion protein encoded by the DNA fragment of claim 3, the porcine reproductive and respiratory syndrome virus nanoparticles of claim 4 or 5, the porcine reproductive and respiratory syndrome virus nanoparticles encoded by the DNA fragment of claim 6, the porcine reproductive and respiratory syndrome virus nanoparticles prepared by the preparation method of claim 7, or the biomaterial of claim 8 in the preparation of porcine reproductive and respiratory syndrome vaccines.

10. A vaccine for porcine reproductive and respiratory syndrome, characterized in that, Including antigens and adjuvants; The antigen is the fusion protein of claim 1 or 2, the fusion protein encoded by the DNA fragment of claim 3, the porcine reproductive and respiratory syndrome virus nanoparticle of claim 4 or 5, the porcine reproductive and respiratory syndrome virus nanoparticle encoded by the DNA fragment of claim 6, or the porcine reproductive and respiratory syndrome virus nanoparticle prepared by the preparation method of claim 7.

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