Epitope tandem polypeptide of porcine reproductive and respiratory syndrome and related biological materials and applications thereof

Abstract

The application discloses an epitope tandem polypeptide of porcine reproductive and respiratory syndrome and related biological materials and application thereof, and belongs to the field of viral antigen vaccines or biomedical technologies. The application provides an epitope tandem polypeptide which is composed of neutralizing antigen epitopes, Th epitopes and CTL epitopes of multiple structural proteins and non-structural proteins of PRRSV in series, and includes PNB, PTH and PCTL. The epitope tandem polypeptide provided by the application can be directly used for preparing a subunit vaccine of PRRSV, and has the advantages of simple preparation method, strong immunogenicity and good safety. The epitope tandem polypeptide can also be combined with an inactivated vaccine of PRRSV and applied to enhance cellular immunity induced by the inactivated vaccine, so that the epitope tandem polypeptide can more effectively prevent and protect pigs from PRRSV infection. The epitope tandem polypeptide composition contains common epitope sequences of seven representative strains (LV, VR-2332, CH-1a, JXA1, NADC30, NADC34 and RFLP 1-4-4) of PRRSV, and is expected to have good cross-protection for infection of different strains.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, and relates to viral antigen vaccines or biomedical technologies, specifically to an epitope tandem polypeptide composition for porcine reproductive and respiratory syndrome and its related biomaterials and applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) infection is a critical industrial problem that urgently needs to be addressed in pig production. PRRSV is one of the most serious pathogens threatening the global pig industry. First reported in the United States in the late 1980s, it now presents as an epidemic infection in pig herds in most countries. PRRSV infection can cause porcine reproductive and respiratory syndrome, also known as blue ear disease, primarily manifesting as respiratory symptoms in piglets and abortion, reproductive failure, fetal death, and congenital infection in pregnant sows.

[0003] PRRSV is an enveloped, single-stranded, non-segmented, positive-signal RNA virus. Due to its genetic structure, PRRSV exhibits high variability, making it one of the challenges in its clinical prevention and control. According to the latest classification criteria of the International Committee on Taxonomy of Viruses (ICTV) in 2022, PRRSV is divided into two independent species: Betaarterivirus suid 1 (formerly PRRSV-1, European type) and Betaarterivirus suid 2 (formerly PRRSV-2, North American type), belonging to the genus Betaarterivirus and family Arteriviridae. Since its first discovery in China in the late 1990s, PRRSV-2 has been the dominant strain in China. Representative strains include the earliest classic strain (VR-2332-like), highly pathogenic PRRSV strains (HP-PRRSVs, JXA1-like), low pathogenic strains (LP-PRRSVs, CH-1a-like), and the more recently emerging NADC30, NADC34, and QYYZ-like strains. Since its introduction in 2014, the NADC30-like strain has mutated or recombinated to form variant viruses, causing widespread outbreaks with diverse clinical manifestations. The detection rate of the NADC34-like strain in China has increased significantly since 2017, particularly in Heilongjiang Province, showing a trend of wider distribution in northern regions than in the south. Currently, PRRSV remains widespread and rapidly evolving, and controlling PRRSV infection clinically remains a significant challenge.

[0004] The use of PRRSV vaccines has become the second major challenge in its clinical prevention and control. Currently, commercially available PRRSV vaccines are mainly live attenuated vaccines, including classic strains (CH-1R, Ingelvac MLV, R98) and highly pathogenic strains (JXA1-R, PC, HuN4-F112, TJM-F92, GDr180); while inactivated vaccines have a smaller market share, with only three strains: CH-1a, M-2, and JXA1. Although classic live attenuated and inactivated vaccines have been used clinically for many years, these vaccines have corresponding problems in their application. On the one hand, the use of live attenuated vaccines can rapidly clear viremia, induce protective immunity against homologous strains, and protect animals from disease; however, it also raises a series of biosafety issues, such as vaccine-derived viral virulence reversion, strain recombination, and shedding of the virus—this is one of the "bottleneck" issues. On the other hand, inactivated vaccines have shown good safety in clinical applications, but they have also revealed problems such as poor cellular immune responses and insufficient immune protection—this is the second "bottleneck" issue. Therefore, solving the problem requires efforts in three aspects: first, reducing or eliminating the biosafety risks of live attenuated vaccines; second, enhancing the cellular immune response of inactivated vaccines; and third, developing new, safe, and effective anti-PRRSV vaccines.

[0005] Epitope peptide vaccines are a class of vaccines based on specific epitopes of pathogens. They possess high antigen specificity and high immune response specificity, while reducing the toxic side effects of conventional vaccines, making them promising novel vaccine candidates. Epitope peptide vaccines typically contain two types of antigenic epitopes: B-cell linear epitopes, which primarily enhance humoral immune responses; and T-cell linear epitopes, especially helper T lymphocyte (Th) and cytotoxic T lymphocyte (CTL) epitopes, which can elicit strong cellular immune responses and synergize with humoral immune responses to enhance the protective effect of the vaccine. Due to their high specificity and safety, epitope peptide vaccines have wide applications in the field of antiviral vaccines. For example, the IC41 vaccine candidate for hepatitis C virus (HCV) consists of five synthetic peptides derived from the HCV core, NS3, and NS4 proteins, containing HCV CD4 and CD8 T-cell epitopes. It is generally well-tolerated in healthy volunteers and elicits a Th1 / Tc1 specific immune response against HCV peptides.

[0006] The advantages of epitope peptide vaccines are: 1. High safety and specificity: They consist only of short-chain amino acids and do not contain live or attenuated pathogens. Furthermore, the removal of toxic components and decoy epitopes from viral proteins makes them particularly suitable for viral infections that produce large amounts of non-neutralizing antibodies and exhibit antibody-dependent enhancement (ADE). Moreover, they can significantly reduce cross-reactivity with host tissues, thereby lowering the risk of autoimmune reactions. 2. Easy to design and manufacture, allowing for rapid adaptation to pathogen mutations. The production process is standardized and does not require complex cell culture. 3. Generally stable and with a longer shelf life compared to live vaccines that require strict cold chain storage. 4. Epitope peptide vaccines based on conserved epitopes are beneficial for developing universal vaccines, providing cross-protection against viral mutations.

[0007] Currently, there are several types of patents related to PRRSV epitope peptide vaccines. The first type involves tandem epitopes targeting a single viral protein. For example, patent document CN118496323A discloses the conserved neutralizing epitope QT7 of the major glycoprotein GP5 of the North American porcine reproductive and respiratory syndrome virus (PRRSV-2) envelope, along with the nucleic acid molecule, expression vector, neutralizing antibody, and its applications. The second type involves tandem epitopes targeting different proteins of the same strain. For example, patent document CN103421817A discloses T-cell epitopes of GP3, GP4, GP5, M, and N proteins of the WUH3 strain and a modified GP5B-cell epitope linked by a linker. The third type involves tandem epitopes targeting single proteins of different genotypes. For example, patent document CN116655750A discloses a PRRSV M protein SLA-1*04:01:01 restricted T-cell epitope peptide and its applications. The above three types of epitope peptide vaccines do not conflict with claims 1-4 of this invention. This invention provides an epitope tandem peptide composition composed of neutralizing antigenic epitopes, Th epitopes, and CTL epitopes of multiple PRRSV structural proteins and non-structural proteins tandemly. This epitope tandem peptide composition contains common epitope sequences from seven representative PRRSV strains, covering the vast majority of different genotype sequences currently available, and exhibits broader cross-protection than other patents. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to improve the protective efficacy of PRRSV inactivated vaccines. The invention provides a PRRSV epitope tandem polypeptide, which can not only be used as a PRRSV subunit vaccine itself, but also aims to enhance the efficacy of PRRSV inactivated vaccines, especially to improve their cellular immune level, thereby solving the problems of poor cellular immune response and insufficient immune protection in the clinical application of inactivated vaccines.

[0009] Another technical problem to be solved by the present invention is to provide biomaterials for epitope tandem peptides of porcine reproductive and respiratory syndrome.

[0010] The final technical problem to be solved by this invention is to provide the application of the above-mentioned epitope tandem polypeptide in the preparation of a vaccine to prevent diseases caused by porcine reproductive and respiratory syndrome virus infection.

[0011] Technical solution: To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0012] An epitope tandem polypeptide for porcine reproductive and respiratory syndrome, wherein the epitope tandem polypeptide comprises two or three subunits: PNB, PTH, and PCTL, preferably including PNB, PTH, and PCTL subunits, or including PTH and PCTL subunits.

[0013] The PNB subunit is a polypeptide composed of the following 13 epitopes in sequence or a polypeptide with the same function, or a polypeptide composed of the following 13 epitopes in sequence and with linking peptides added between the epitopes or a polypeptide with the same function; the linking peptides include: AAY, GGPPG, EAAK and GSGGGS.

[0014] QAAAEVYEPGRS, QAAAEILEPGKS, QAATEIYEPGRS, QAARQRLEPGRN, QAAAQILEPGGS, QAAAQILEPGRS, QAAAQIYEPGRS, WSFADGN, GVSAAQEKISFG, SSHLQLIYNLT, SSTYQYIYNLT, SSHIQLIYNLT, SSHFQLIYNLT;

[0015] The PTH subunit is a polypeptide composed of the following 7 epitopes in sequence or a polypeptide with the same function, or a polypeptide composed of the following epitopes in sequence and linking peptides between the epitopes or a polypeptide with the same function. The linking peptides include: AAY, GGPPG, EAAK and GSGGGS.

[0016] RSTPAIVRWFAAHLLYELAC, PNRDRILAALAYHMKANNVSEYYASAAAILMDSCA, VQPLIVYSDDLVLYAES, WPDRLVASLRPIHKYSRA, LACAEFSLDDPVRYKHTWGFESDTAYLYEFTG, EFTQRSLVVDHVRLLHFMTPETMR, RYTNFLLDTKGRLYRWRSPVI;

[0017] The PCTL subunit is a polypeptide composed of the following 10 epitopes in sequence or a polypeptide with the same function, or a polypeptide composed of the following 10 epitopes in sequence and with linking peptides added between the epitopes or a polypeptide with the same function. The linking peptides include: AAY, GGPPG, EAAK and GSGGGS.

[0018] GIDGTLWDF, ICDAIQPDY, EVDLPDGDY, MVNTTRVTY, KVAHNLGFY, GADLAVTPY, ASDWFAPRY, AIEAETCKY, RTAIGTPVY, LSDSGRISY.

[0019] Preferably, the PNB is a polypeptide with an amino acid sequence as shown in SEQ ID No: 31, or a polypeptide with more than 80% identity to SEQ ID No: 31 and with the same function.

[0020] QAAAEVYEPGRSgpgpgQAAAEILEPGKSgpgpgQAATEIYEPGRSgpgpgQAARQRLEPGRNgpgpgQAAAQILEPGGSgpgpgQAAAQILEPGRSgpg pgQAAAQIYEPGRSkkWSFADGNgpgpgGVSAAQEKISFGeaakSSHLQLIYNLTeaakSSTYQYIYNLTeaakSSHIQLIYNLTeaakSSHFQLIYNLT;

[0021] The PTH is a polypeptide with an amino acid sequence as shown in SEQ ID No: 32, or a polypeptide with more than 80% identity to SEQ ID No: 32 and with the same function.

[0022] RSTPAIVRWFAAHLLYELACaayPNRDRILAALAYHMKANNVSEYYASAAAILMDSCAgpgpgVQPLIVYSDDLVLYAESaayWPDRLVASLRPIHKYSRAaayLACAEFSLDDPVRYKHTWGFESDTAYLYEFTGaayEFTQRSLVVDHVRLLHFMTPETMRgpgpgRYTNFLLDTKGRLYRWRSPVI;

[0023] The PCTL is a polypeptide with an amino acid sequence as shown in SEQ ID No: 33, or a polypeptide with more than 80% identity to SEQ ID No: 33 and with the same function.

[0024] GIDGTLWDFgpgpgICDAIQPDYgpgpgEVDLPDGDYaayMVNTTRVTYaayK VAHNLGFYgpgpgGADLAVTPYaayASDWFAPRYaayAIEAETCKYaayRTAIGTPV YaayLSDSGRISYaay.

[0025] The nucleotide sequence of the epitope tandem polypeptide of porcine reproductive and respiratory syndrome most preferably found in this invention is shown in SEQ ID NO:34, and the amino acid sequence is shown in SEQ ID No:35.

[0026] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the protein isolated according to the present invention, as long as they encode and have the function of the protein of the present invention, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0027] As is known to those skilled in the art, the fusion protein with epitope peptides as the main functional component refers to a fusion protein whose primary purpose is to induce a PRRSV-specific immune response in the body. This includes fusion proteins that have been linked to tag proteins to facilitate protein purification or detection; or auxiliary components that enhance immune efficacy, such as immune adjuvant molecules (interferon and GM-CSF, etc.), targeting molecules (TLR ligands and peptides targeting immune cells, etc.), or Fc molecules, nanoparticles, etc. All of these are derived from and equivalent to the proteins of this invention.

[0028] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0029] A biomaterial containing an epitope tandem polypeptide of porcine reproductive and respiratory syndrome, wherein the biomaterial is any one of the following:

[0030] A recombinant vector expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome (PRRS) is provided. The vector is known to those skilled in the art and includes, but is not limited to, plasmids, bacteriophages (such as λ phage or M13 filamentous phage), and granules (i.e., Cosmids). Any plasmid and vector can be used as long as it can stably replicate in host cells. Those skilled in the art can utilize DNA recombination technology and other techniques to construct expression vectors containing specific elements such as the nucleic acid molecules or nucleic acid molecule combinations described in this invention, suitable transcription and translation regulatory sequences, promoters, and selective marker genes.

[0031] Recombinant microorganisms expressing the epitope tandem polypeptides of porcine reproductive and respiratory syndrome (PRRS); the microorganisms may be yeast, bacteria, algae, fungi, or viruses. Among them, bacteria may be derived from *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, *Bacillus*, etc. The viruses may be retroviruses (including lentiviruses), adenoviruses, rabies viruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), baculoviruses, or vaccinia viruses.

[0032] Recombinant cells expressing the epitope tandem polypeptide of the aforementioned porcine reproductive and respiratory syndrome. The cells referred to are cells suitable for vector delivery, including but not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells), or prokaryotic cells (such as Escherichia coli or Bacillus subtilis).

[0033] Any of the following applications of the epitope tandem polypeptides for the above-mentioned reproductive and respiratory syndromes are within the scope of protection of this invention:

[0034] Application in the preparation of vaccines to prevent diseases caused by porcine reproductive and respiratory syndrome virus infection;

[0035] Application in the preparation of products for the prevention and treatment of diseases caused by porcine reproductive and respiratory syndrome virus infection;

[0036] Application in the preparation of agents or drugs for inducing an immune response to porcine reproductive and respiratory syndrome virus antigens.

[0037] The present invention also provides a porcine reproductive and respiratory syndrome (PRRS) vaccine, which uses a tandem peptide of PRRS epitopes as an antigen, or uses a tandem peptide of PRRS and a PRRSV inactivated vaccine together as an antigen.

[0038] A method for preparing a vaccine for porcine reproductive and respiratory syndrome, the method comprising expressing the fusion protein of claim 1 using a prokaryotic system.

[0039] Furthermore, the porcine reproductive and respiratory syndrome vaccine may also include an adjuvant and / or a vaccine delivery system.

[0040] The adjuvant is a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-inoculated with it. The adjuvants described herein are well known to those skilled in the art and include, but are not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant).

[0041] The vaccine delivery system described herein is a type of substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. The vaccine delivery system described herein may be a salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano-adjuvant vaccine delivery system.

[0042] As is well known to those skilled in the art, in order to enhance the immunogenicity of antigen proteins, in addition to adding compounds with immunomodulatory effects as adjuvants, gene combinations can be adjusted to express them into particulate structures; or they can be aggregated in vitro and encapsulated in liposomes or microspheres.

[0043] Beneficial effects

[0044] This invention provides an epitope tandem polypeptide, composed of multiple structural proteins of PRRSV tandemly linked with neutralizing antigenic epitopes, Th epitopes, and CTL epitopes of non-structural proteins, including PNB, PTH, and PCTL. The epitope tandem polypeptide provided by this invention can be directly used to prepare PRRSV subunit vaccines, with a simple preparation method, strong immunogenicity, and good safety. The epitope tandem polypeptide can also be used in combination with PRRSV inactivated vaccines to enhance cellular immunity induced by the inactivated vaccine, more effectively preventing and protecting pigs from PRRSV infection. The epitope tandem polypeptide composition contains common epitope sequences of seven representative PRRSV strains (LV, VR-2332, CH-1a, JXA1, NADC30, NADC34, RFLP 1-4-4), and is expected to have good cross-protection against infection by different strains. Compared to the group immunized with inactivated vaccine alone, the group immunized with PTH-PCTL-PNE and inactivated vaccine together can induce the production of neutralizing antibodies and various cytokines earlier and significantly enhance the cellular immune response, which can more effectively prevent and protect pigs from PRRSV infection. Attached Figure Description

[0045] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0046] Figure 1 This refers to the design and amino acid sequence of an epitope polypeptide PTH-PCTL-PNE in Example 1 of the present invention.

[0047] Figure 2 This is the recombinant plasmid map constructed in Example 1 of this invention;

[0048] Figure 3 This is an SDS-PAGE image of the purified PTH-PCTL-PNE recombinant protein in Example 3 of this invention;

[0049] Figure 4 This refers to the level of anti-specific antibodies detected by ELISA in Example 5 of this invention;

[0050] Figure 5 It is the neutralizing antibody titer determined by the virus neutralization experiment in Example 6 of this invention;

[0051] Figure 6 These are the levels of various cytokines related to cellular immunity measured in Example 7 of this invention. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0054] Example 1: Design and vector construction of a PRRSV epitope polypeptide composition PTH-PCTL-PNE.

[0055] The seven Th-derived epitope sequences in Table 1 were tandemly linked into a PTH, the ten CTL epitope sequences into a PCTL, and the thirteen neutralizing epitope sequences into a PNE. Then, the PTH, PCTL, and PNE were tandemly linked to form an epitope polypeptide composition. The specific tandem sequence, linking peptides, and amino acid sequences used are as follows: Figure 1 .

[0056] Table 1

[0057]

[0058]

[0059] Synthesize the coding gene as shown in SEQ ID No. 31.

[0060] The synthesized gene sequence was recombined with the expression plasmid vector pET-28a(+) to obtain the recombinant plasmid pET28a-PTH-PCTL-PNE. The map of the recombinant plasmid is shown below. Figure 2 As shown.

[0061] Example 2: Transformation and Expression of Recombinant Plasmids

[0062] The obtained recombinant plasmid was transformed into BL21 Escherichia coli and amplified in 50 mL of TB medium containing ampicillin at 37°C until the amplification reached OD. 600 = Around 0.8.

[0063] After cooling to room temperature for half an hour, add IPTG solution to a final concentration of 0.5 mM and induce expression of recombinant protein PTH-PCTL-PNE at 37°C for 4-6 hours.

[0064] Centrifuge the induced E. coli at 10,000×g for 5 minutes, discard the supernatant, and retain the bacterial precipitate for further purification.

[0065] Example 3: Purification of recombinant protein PTH-PCTL-PNE

[0066] The bacterial precipitate obtained in Example 2 was resuspended with lysis buffer and mixed at a ratio of 10 mL of lysis buffer per gram of wet bacterial precipitate. The lysis buffer formulation was as follows: 50 mM NaH₂PO₄ (pH 8.0), 300 mM NaCl, 20 mM imidazole (1-formylimidazole), 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT (dithiothreitol), 0.5 vol% Triton X-100, and the balance being water.

[0067] After resuspending the bacterial suspension, let it stand on ice for 10 minutes, and then sonicate it for 10 minutes with a 5-second sonication followed by a 5-second pause at 25% amplitude (operated on ice; these parameters are suitable for 20-40 mL of resuspended suspension) to obtain the lysed bacterial suspension.

[0068] The lysed bacterial culture was centrifuged at 4°C and 18,000×g for 10 minutes.

[0069] The obtained supernatant was added to Ni-NTA His·Bind resin and mixed by shaking at 50 rpm at room temperature for 30 min.

[0070] Transfer the supernatant containing the packing material to the column, collect the flow-through, pass it through the column again, and collect more than 20 μL of the flow-through for storage for SDS-PAGE electrophoresis analysis.

[0071] Wash with wash buffer (50 mM NaH2PO4·2H2O, 20 mM Imidazole, 50 mM NaCl) at least 20 times the volume of the packing material, and collect at least 20 μL of wash through for SDS-PAGE electrophoresis analysis.

[0072] Elute the target protein with 5 times the volume of elution buffer (50 mM NaH2PO4·2H2O, 500 mM Imidazole, 300 mM NaCl, 10% Glycerol), collecting 2 mL per tube. Preserve the fractions for SDS-PAGE electrophoresis analysis.

[0073] The results are as follows Figure 3 As shown, high concentrations of PTH-PCTL-PNE recombinant protein were obtained through prokaryotic expression and purification with Ni ion packing material.

[0074] Example 4: Porcine immunization and challenge experiments with epitope polypeptide vaccines

[0075] The immunogenicity of the PTH-PCTL-PNE recombinant protein prepared in Example 3 was verified, and the specific steps are as follows:

[0076] The piglets were grouped according to Table 2, with 5 healthy three-way crossbred weaned piglets in each group (4 weeks old, weighing 9.0±1.50 kg).

[0077] Table 2 Vaccine Immunization Groups

[0078]

[0079]

[0080] To better demonstrate the immunogenicity of the epitope peptide vaccine and its adjuvant effect on inactivated vaccines, no additional adjuvants were added during vaccination. Each group received a second immunization 21 days after the initial immunization, using the same dosage and route as the initial immunization. Blood samples were collected and serum separated after the second immunization. Blood samples were collected 21 days after the second immunization, and the titer of specific IgG antibodies was detected using ELISA, while the titer of neutralizing antibodies was determined using a neutralization assay.

[0081] Example 5: ELISA detection of specific IgG antibody levels

[0082] The serum collected in Example 4 was tested for anti-specific IgG antibody titer by ELISA, and the steps are as follows:

[0083] Coating: PRRSV N protein or the purified PTH-PCTL-PNE protein from Example 3 were added to coating buffer (0.05 mol / L carbonate buffer, pH 9.6), 100 ng / 100 μL per well, and added to 96-well plates. The plates were then blocked overnight at 4°C.

[0084] Blocking: Serially dilute the serum sample to be tested 10 times (1:100-1:1,000,000) with serum diluent (0.1% BSA), and then add 100 μL to the reaction wells of the pre-coated sample (simultaneously prepare blank wells, negative control wells, and positive control wells).

[0085] Primary antibody incubation: Wash 3 times with PBST, initially dilute the collected serum with PBS at a ratio of 1:100, serially dilute 100 μL / well, and incubate at 37°C for 1.5 h.

[0086] Secondary antibody incubation: Wash 3 times with PBST. Dilute the secondary antibody (rabbit anti-porcine IgG, HRP) 1:5000 with PBST, 100 μL / well, and add to the ELISA plate as described above. Incubate at 37°C for 30 min.

[0087] Color development and reading: Wash 5 wells with 100 μL / well of PBST. Add 100 μL / well of TMB colorimetric solution to the ELISA plate in the dark and incubate at 37°C for 10 min. Stop the reaction by adding 100 μL of stop solution (2M sulfuric acid) to each well. Measure the absorbance at OD450 nm using a microplate reader and analyze the data.

[0088] The results are as follows Figure 4 As shown, 14 days after the second immunization, serum samples were collected from pigs. ELISA results coated with PTH-PCTL-PNE protein showed that the PTH-PCTL-PNE immunization group produced high levels of specific antibodies against PTH-PCTL-PNE protein and PRRSV, with antibody levels significantly higher than the control group. ELISA results coated with PRRSV-N protein showed that both the PRRSV inactivated vaccine immunization group and the combined immunization group produced high levels of specific antibodies against PRRSV-N protein, with antibody levels significantly higher than the control group.

[0089] Example 6: Determination of Neutralizing Antibody Titer

[0090] The neutralizing antibody titer in the serum collected in Example 4 was detected by the following steps:

[0091] Cell preparation: Seed MARC-145 cells into 96-well cell culture dishes the night before.

[0092] Serum dilution: All serum samples were heat-inactivated (56°C, 30 min). Serum was serially diluted 2-fold with antibiotic-free and serum-free DMEM medium (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280). Eight 200 μL PCR tubes were used. 90 μL of DMEM was added to the first PCR tube, and 50 μL of DMEM was added to the remaining seven. 10 μL of serum was added to the first PCR tube, mixed well, and then 50 μL was added to the next PCR tube. The same method was used to dilute the serum sequentially.

[0093] 50 μL of diluted 200 TCID 50 The viral solution (CH-1α) was mixed with 50 μL of serially diluted serum and incubated at 37°C for 1 h.

[0094] Aspirate the culture medium from the 96-well plate inoculated with cells the previous night and rinse once with DMEM. Add 100 μL of the virus-serum mixture to each well according to the pre-set arrangement. Add unneutralized virus to the positive control wells, add low-dilution serum to the negative control wells, and add culture medium only to the normal cell control wells. Incubate at 37°C for 1 hour in a 5% CO2 incubator.

[0095] Aspirate the virus and serum mixture, and rinse twice with DMEM. Finally, add 100 μL of DMEM medium containing 1% P / S and 2% FBS, and incubate at 37°C for 5 days in a 5% CO2 incubator.

[0096] Cells were fixed with 4% paraformaldehyde, observed using indirect immunofluorescence, and the neutralizing antibody titer was calculated using the Reed-Muench method.

[0097] The results are as follows Figure 5 As shown, neutralizing antibodies were detectable as early as 2 weeks after the first immunization in the PTH-PCTL-PNE immunization group, while in the PRRSV inactivated vaccine immunization group, neutralizing antibodies were detectable starting 4 weeks after immunization. Although immunization with PTH-PCTL-PNE alone does not produce high titers of neutralizing antibodies, immunization with PTH-PCTL-PNE in combination with an inactivated vaccine can produce high titers of neutralizing antibodies.

[0098] Example 7: Measurement of Cellular Immune Response Level

[0099] Serum cytokine concentrations were determined using an IFN-γ, IL-2, IL-4, IL-5, IL-10, and IL-12 ELISA kit according to the manufacturer's instructions.

[0100] The results are as follows Figure 6 As shown, compared to the PRRSV inactivated vaccine group alone, both the PTH-PCTL-PNE group and the combined immunization group significantly induced immunized pigs to produce higher levels of IFN-γ, IL-2, and IL-12 cytokines. Figure 6 A, 6C, 6E), while there was no significant difference in IL-4, IL-5, and IL-10 levels. Figure 6 B, 6D, 6F). This indicates that PTH-PCTL-PNE primarily induces Th1-type cellular immunity.

[0101] In summary, this invention enables the high-level expression and purification of the epitope polypeptide tandem protein PTH-PCTL-PNE using a prokaryotic expression system. The PTH-PCTL-PNE epitope polypeptide tandem composition of this invention exhibits good immunogenicity. In adjuvant-free porcine immunization experiments, PTH-PCTL-PNE induces high levels of humoral immunity, including high levels of specific IgG antibodies and a certain titer of neutralizing antibodies; it also induces Th1 and Th2 cellular immune responses. Importantly, compared to groups immunized only with inactivated vaccines, the combined immunization group of PTH-PCTL-PNE and inactivated vaccine induces neutralizing antibodies and various cytokines earlier and significantly enhances cellular immune responses, thus more effectively preventing and protecting pigs from PRRSV infection.

[0102] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0104] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An epitope tandem polypeptide of porcine reproductive and respiratory syndrome, characterized in that: The amino acid sequence of the epitope tandem polypeptide is shown in SEQ ID No:

35.

2. A biomaterial comprising an epitope tandem polypeptide composition for porcine reproductive and respiratory syndrome, characterized in that, The biomaterial is any one of the following: Nucleotides expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome as described in claim 1; A recombinant vector expressing the epitope tandem polypeptide of claim 1; Recombinant microorganisms expressing the epitope tandem polypeptide of claim 1; Recombinant cells expressing the epitope tandem polypeptide of claim 1.

3. Any of the following applications of the epitope tandem polypeptide of porcine reproductive and respiratory syndrome as described in claim 1: Application in the preparation of vaccines to prevent diseases caused by porcine reproductive and respiratory syndrome virus infection; Application in the preparation of products for the prevention and treatment of diseases caused by porcine reproductive and respiratory syndrome virus infection; Application in the preparation of agents or drugs for inducing an immune response to porcine reproductive and respiratory syndrome virus antigens.

4. A vaccine for porcine reproductive and respiratory syndrome, characterized in that, The vaccine comprises the epitope tandem polypeptide of porcine reproductive and respiratory syndrome as described in claim 1.

5. The porcine reproductive and respiratory syndrome vaccine according to claim 4, characterized in that, The epitope tandem peptide of porcine reproductive and respiratory syndrome (PRRS) can be used as an antigen, or the epitope tandem peptide of PRRSV can be used together with the PRRSV inactivated vaccine as an antigen.

6. A method for preparing a vaccine for porcine reproductive and respiratory syndrome, characterized in that, The method includes expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome as described in claim 1 using a prokaryotic system.

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