Epitope tandem polypeptide for porcine reproductive and respiratory syndrome and related biological material and application thereof

Abstract

The invention discloses an epitope tandem polypeptide of porcine reproductive and respiratory syndrome as well as a related biological material and application thereof, and belongs to the technical field of virus antigen vaccines or biomedicine. The invention provides an epitope tandem polypeptide, which is formed by connecting a plurality of PRRSV structural proteins with neutralizing epitopes, Th epitopes and CTL epitopes of non-structural proteins in series, and comprises PNB, PTH and PCTL. The epitope tandem polypeptide provided by the invention can be directly used for preparing a subunit vaccine of the PRRSV, the preparation method is simple, the immunogenicity is strong, and the safety is good. The epitope tandem polypeptide can also be used in combination with a PRRSV inactivated vaccine, is used for enhancing cellular immunity induced by the inactivated vaccine, and can more effectively prevent and protect pigs from being infected by PRRSV. The epitope tandem polypeptide composition comprises common epitope sequences of seven PRRSV representative strains (LV, VR-2332, CH-1a, JXA1, NADC30, NADC34 and RFLP (Restriction Fragment Length Polymorphism) 1-4-4), and has relatively good cross protection on infection of different strains.

Description

Technical Field

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

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) infection is an important industrial problem that needs to be solved in the pig production process. PRRSV is one of the most serious pathogens that endanger the global pig industry. It was first reported in the United States in the late 1980s and has been an epidemic infection in pig herds in most countries. PRRSV infection can cause porcine reproductive and respiratory syndrome, also known as blue ear disease, which is mainly manifested by 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-sense RNA virus. Due to its genetic structure, PRRSV has high variability, which has become one of the difficulties in its clinical prevention and control. According to the latest classification standards 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), which belong to the genus Betaarterivirus and the family Arteriviridae. Since it was first discovered in China in late 1995, PRRSV-2 has been the main strain in China. Representative strains include the earliest classical strains (VR-2332-like), highly pathogenic strains of PRRSV (HP-PRRSVs, JXA1-like), low pathogenic strains (LP-PRRSVs, CH-1a-like), and NADC30, NADC34 and QYYZ-like strains that have appeared in recent years. Since the introduction of the NADC30-like strain in 2014, it has mutated or recombined to form a mutant virus, causing widespread epidemics with diverse clinical manifestations. The detection rate of the NADC34-like strain in China has increased significantly since 2017, especially in Heilongjiang Province, showing a trend of being more widely distributed in the north than in the south. At present, PRRSV is still widely prevalent and rapidly evolving, and there are still huge challenges in clinical prevention and control of PRRSV infection.

[0004] The use of PRRSV vaccines has become the second difficulty in its clinical prevention and control. At present, commercial 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 the market share of inactivated vaccines is relatively small, with only three strains, CH-1a, M-2, and JXA1. Although classic live attenuated vaccines 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 quickly eliminate viremia, induce protective immunity to homologous strains, and protect animals from disease; but at the same time, it has caused a series of biosafety issues, such as vaccine-derived virus virulence reversion, strain recombination, and toxin shedding, which is one of the "stuck points". On the other hand, inactivated vaccines have shown good safety in clinical applications, but they have also exposed problems such as poor cellular immune response and insufficient immune protection, which is the second "stuck point". 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 type of vaccine based on pathogen-specific epitopes. They have high antigenic specificity and high immune response specificity, while reducing the toxic side effects of conventional vaccines. They are new candidate vaccines with potential for application. Epitope peptide vaccines usually contain two antigenic epitopes, one is a B cell linear epitope, which mainly enhances humoral immune response; the other is a T cell linear epitope, especially helper T lymphocyte (Th) and cytotoxic T lymphocyte (CTL) epitopes, which can trigger a strong cellular immune response and can synergize with the humoral immune response to enhance the protective effect of the vaccine. Epitope peptide vaccines are widely used in the field of antiviral vaccines due to their high specificity and safety. For example, the IC41 vaccine candidate for hepatitis C virus (HCV) consists of five synthetic peptides derived from the core, NS3 and NS4 proteins of HCV, containing HCV CD4 and CD8 T cell epitopes, which are generally well tolerated in healthy volunteers and trigger Th1 / Tc1-specific immune responses against HCV peptides.

[0006] The advantages of epitope peptide vaccines are: 1. High safety and specificity, they are composed only of short-chain amino acids and do not contain live or attenuated pathogens; at the same time, the toxic components and bait epitopes in viral proteins are removed, making them particularly suitable for some viral infections that produce a large number of non-neutralizing antibodies and have antibody-dependent enhancement (ADE) effects; secondly, cross-reactions with host tissues can be greatly reduced, thereby reducing the risk of autoimmune reactions. 2. Easy to design and produce, allowing rapid adaptation to pathogen mutations, standardized production processes, and no need for complex cell culture. 3. Compared with live vaccines that require strict cold chain storage, they are generally stable and have a longer shelf life. 4. Epitope peptide vaccines based on conserved epitopes are conducive to the development of universal vaccines, providing cross-protection to overcome viral mutations.

[0007] There are several types of patents on PRRSV epitope polypeptide vaccines at present. The first type is to connect the epitopes of a single viral protein in series. For example, patent document CN118496323A discloses the conserved neutralizing epitope QT7, nucleic acid molecules, expression vectors, neutralizing antibodies and their applications of the main glycoprotein GP5 of the North American porcine reproductive and respiratory syndrome virus (PRRSV-2) envelope. The second type is to connect the epitopes of different proteins of the same strain in series. For example, patent document CN103421817A discloses that the T cell epitopes of GP3, GP4, GP5, M and N proteins of WUH3 strain and the modified GP5B cell epitope are connected by linker. The third type is to connect the epitopes of single proteins of different genotypes in series. For example, patent document CN116655750A discloses a PRRSV M protein SLA-1*04:01:01 restricted T cell epitope polypeptide and its application. The above three types of epitope peptide vaccines have no conflict of interest with requirements 1 to 4 of the present invention. The present invention provides an epitope tandem peptide composition, which is composed of neutralizing antigen epitopes, Th epitopes and CTL epitopes of multiple structural proteins and non-structural proteins of PRRSV in series. The epitope tandem peptide composition contains the common epitope sequences of seven representative strains of PRRSV, covering most of the different genotype sequences at present, and has a wider 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 enhance the protective power of PRRSV inactivated vaccines, and to provide a PRRSV epitope tandem polypeptide, which can not only be used as a PRRSV subunit vaccine itself, but also has the main purpose of enhancing the efficacy of PRRSV inactivated vaccines, especially improving its cellular immunity level, thereby solving the problems of poor cellular immune response and insufficient immune protection of inactivated vaccines in clinical applications.

[0009] The technical problem that the present invention needs to solve is to provide a biological material of epitope tandem polypeptides of porcine reproductive and respiratory syndrome.

[0010] The final technical problem to be solved by the present invention is to provide the application of the epitope tandem polypeptides in the preparation of vaccines for preventing diseases caused by porcine reproductive and respiratory syndrome virus infection.

[0011] Technical solution: In order to solve the above technical problems, the present invention adopts the following technical solution:

[0012] A porcine reproductive and respiratory syndrome epitope tandem polypeptide, the epitope tandem polypeptide comprises two or three of PNB subunit, PTH subunit and PCTL subunit, preferably comprises the epitope tandem polypeptide comprising PNB subunit, PTH subunit and PCTL subunit, or comprises PTH subunit and PCTL subunit.

[0013] Wherein, the PNB subunit is a polypeptide composed of the following 13 epitopes connected in series in sequence or a polypeptide having the same function as the following 13 epitopes connected in series in sequence or a polypeptide having the same function as the following 13 epitopes connected in series in sequence and a connecting peptide connected between the epitopes or a polypeptide having the same function as the following 13 epitopes connected in series in sequence; the connecting peptide includes: AAY, GPGPG, 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 seven epitopes connected in series in sequence or a polypeptide having the same function as the following seven epitopes, or a polypeptide composed of the following epitopes connected in series in sequence and a connecting peptide connected between the epitopes or a polypeptide having the same function as the following seven epitopes, wherein the connecting peptide includes: AAY, GPGPG, EAAK and GSGGGS;

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

[0017] The PCTL subunit is a polypeptide composed of the following 10 epitopes connected in series in sequence or a polypeptide having the same function as the following 10 epitopes, or a polypeptide composed of the following 10 epitopes connected in series in sequence and connected with a connecting peptide between the epitopes or a polypeptide having the same function as the following 10 epitopes, wherein the connecting peptide includes: AAY, GPGPG, EAAK and GSGGGS;

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

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

[0020] QAAAEVYEPGRSgpgpgQAAAEILEPGKSgpgpgQAATEIYEPGRSgpgpgQAARQRLEPGRNgpgpgQAAAQILEPGGSgpgpgQAAAQILEPGRSgpg pgQAAAQIYEPGRSkkWSFADGNgpgpgGVSAAQEKISFGeaakSSHLQLIYNLTeaakSSTYQYIYNLTeaakSSHIQLIYNLTeaakSSHFQLIYNLT;

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

[0022] RSTPAIVRWFAAHLLYELACaayPNRDRILAALAYHMKANNVSEYYASAAAILMDSCAgpgpgVQPLIVYSDDLVLYAESaayWPDRLVASLRPIHKYSRAaayLACAEFSLDDPVRYKHTWGFESDTAYLYEFTGaayEFTQRSLVVDHVRLLHFMTPETMRgpgpgRYTNFLLDTKGRLYRWRSPVI;

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

[0024] GIDGTLWDFgpgpgICDAIQPDYgpgpgEVDLPDGDYaayMVNTTRVTYaayK VAHNLGFYgpgpgGADLAVTPYaayASDWFAPRYaayAIEAETCKYaayRTAIGTPV YaayLSDSGRISYaay.

[0025] The most preferred porcine reproductive and respiratory syndrome epitope tandem polypeptide of the present invention has a nucleotide sequence as shown in SEQ ID NO:34, and an amino acid sequence as shown in SEQ ID NO:35.

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

[0027] It is known to those skilled in the art that the fusion protein with the epitope polypeptide as the main functional component refers to a fusion protein mainly for the purpose of inducing the body to produce a PRRSV-specific immune response. It includes fusion proteins connected to tag proteins to facilitate protein purification or detection; or auxiliary components that can enhance the immune effect by adding immune adjuvant molecules (interferon and GM-CSF, etc.), targeting molecules (TLR ligands and polypeptides targeting immune cells, etc.) or Fc molecules, nanoparticles, etc., all of which are derived from the protein of the present invention and are equivalent to the protein of the present invention.

[0028] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

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

[0030] The recombinant vector expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), any plasmid and vector can be used as long as they can stably replicate in the host cell. Those skilled in the art can use a series of techniques such as DNA recombination technology to construct an expression vector containing the nucleic acid molecule or nucleic acid molecule composition of the present invention, appropriate transcription and translation regulatory sequences, promoters, and selectable marker genes and other specific elements.

[0031] A recombinant microorganism expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome; the microorganism may be yeast, bacteria, algae, fungi or virus. Among them, the bacteria may be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. The virus may be a retrovirus (including lentivirus), adenovirus, rabies virus, adeno-associated virus, herpes virus (such as herpes simplex virus), insect baculovirus or vaccinia virus.

[0032] The recombinant cell expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome mentioned above. The cell refers to a cell that can be used to introduce a vector, 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 above-mentioned epitope tandem polypeptides of reproductive and respiratory syndrome is within the protection scope of the present invention:

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

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

[0036] Application in the preparation of a medicament or drug 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 vaccine, which uses the epitope tandem polypeptides of porcine reproductive and respiratory syndrome as antigens, or the epitope tandem polypeptides of porcine reproductive and respiratory syndrome and the PRRSV inactivated vaccine as antigens.

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

[0039] Furthermore, the porcine reproductive and respiratory syndrome vaccine may further 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 to the antigen inoculated with it. The adjuvant described herein is well known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant).

[0041] The vaccine delivery system is a type of substance that can carry antigenic substances to the body's immune system, store and exert its antigenic effects therein for a long time. The vaccine delivery system described herein can be a saline 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] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0043] Beneficial Effects

[0044] The present invention provides an epitope tandem polypeptide, which is composed of a series of neutralizing antigen epitopes, Th epitopes and CTL epitopes of multiple structural proteins and non-structural proteins of PRRSV, including PNB, PTH and PCTL. The epitope tandem polypeptide provided by the present invention can be directly used to prepare a subunit vaccine of PRRSV, and the preparation method is simple, the immunogenicity is strong, and the safety is good. The epitope tandem polypeptide can also be used in combination with an inactivated PRRSV vaccine to enhance the cellular immunity induced by the inactivated vaccine, and can more effectively prevent and protect pigs from infection with PRRSV. The epitope tandem polypeptide composition contains the common epitope sequences of seven representative strains of PRRSV (LV, VR-2332, CH-1a, JXA1, NADC30, NADC34, RFLP 1-4-4), and is intended to have good cross-protection against infection with different strains. Compared with the group immunized with inactivated vaccine only, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0046] Figure 1 The design and amino acid sequence of an epitope polypeptide PTH-PCTL-PNE in Example 1 of the present invention;

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

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

[0049] Figure 4 is the anti-specific antibody level detected by ELISA in Example 5 of the present invention;

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

[0051] Figure 6 These are the levels of various cytokines related to cellular immunity measured in Example 7 of the present invention. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0053] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0055] The 7 Th-derived epitope sequences in Table 1 were connected in series to form a PTH, the 10 CTL epitope sequences were connected in series to form a PCTL, and the 13 neutralization epitope sequences were connected in series to form a PNE. Then, PTH, PCTL and PNE were connected in series to form an epitope polypeptide composition. The specific connection sequence, the connecting peptide and the amino acid sequence used are as follows: Figure 1 .

[0056] Table 1

[0057]

[0058]

[0059] The coding gene shown in SEQ ID No.31 was synthesized.

[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 in Figure 2 shown.

[0061] Example 2: Transformation and expression of recombinant plasmid

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

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

[0064] The induced expressing E. coli was centrifuged at 10,000 × g for 5 minutes, the supernatant was discarded, and the bacterial pellet was retained for the next step of purification.

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

[0066] The bacterial precipitate obtained in Example 2 was resuspended in a lysis solution and mixed according to a ratio of 10 mL of lysis solution per gram of wet weight of bacterial precipitate. The lysis solution formula is: 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 20 mM imidazole (1-formyl imidazole), 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT (dithiothreitol), 0.5 volume % Triton X-100, and the balance is water.

[0067] The resuspended bacterial solution was placed on ice for 10 minutes and then subjected to ultrasonic disruption, with 5 seconds of ultrasonication and 5 seconds of rest, and 25% amplitude for a total of 10 minutes (operated on ice, this parameter is suitable for 20-40 mL of resuspended solution) to obtain a disrupted lysed bacterial solution.

[0068] The disrupted lysate was centrifuged at 4°C, 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] The supernatant containing the filler was transferred to the column, and the flow through was collected and passed through the column once more. More than 20 μL of the flow through was collected and saved for SDS-PAGE electrophoresis analysis.

[0071] The cells were washed with wash buffer (50 mM NaH2PO4·2H2O, 20 mM Imidazole, 50 mM NaCl) which was 20 times the volume of the filler. More than 20 μL of wash through was collected and saved for SDS-PAGE electrophoresis analysis.

[0072] The target protein was eluted with elution buffer (50 mM NaH2PO4·2H2O, 500 mM Imidazole, 300 mM NaCl, 10% Glycerol) with a volume 5 times that of the filler, and 2 mL was collected in each tube. The fractions were saved for SDS-PAGE electrophoresis analysis.

[0073] The results are as follows Figure 3 As shown, high-concentration PTH-PCTL-PNE recombinant protein was obtained by prokaryotic expression and Ni ion filler purification.

[0074] Example 4: Pig immunization and challenge experiments with epitope peptide vaccines

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

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

[0077] Table 2 Vaccine immunization groups

[0078]

[0079]

[0080] In order to better reflect the immune effect of the epitope peptide vaccine itself and its auxiliary effect on the inactivated vaccine, no additional adjuvant was added during vaccination. The above groups received a second immunization 21 days after the first immunization. The immunization dose and route were the same as the first immunization. Blood was collected and serum was separated after the second immunization. Blood was collected 21 days after the second immunization, and the specific IgG antibody titer was detected by ELISA and the neutralizing antibody titer was detected by neutralization experiment.

[0081] Example 5: ELISA to detect specific IgG antibody levels

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

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

[0084] Blocking: Use serum diluent (0.1% BSA) to dilute the serum sample to be tested 10-fold (1:100-1:1,000,000), and then add 100 μL to the reaction wells with the coated samples (blank wells, negative control wells and positive control wells are also prepared).

[0085] Incubation with primary antibody: Wash 3 times with PBST, initially dilute the collected serum with PBS at 1:100, perform 2-fold serial dilutions at 100 μL / well, and incubate at 37°C for 1.5 h.

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

[0087] Color development and reading: 100 μL / well PBST, wash 5 times. Add 100 μL / well TMB color development solution, add to the ELISA plate in the dark, and color for 10 min at 37°C. Add 100 μL stop solution (2M sulfuric acid) to stop the reaction. Detect the OD450 nm absorbance with an enzyme reader and perform data analysis.

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

[0089] Example 6. Neutralizing Antibody Titer Determination

[0090] The neutralizing antibody titer of the serum collected in Example 4 was detected as follows:

[0091] Prepare cells: MARC-145 cells were seeded in 96-well cell culture dishes the night before.

[0092] Dilute serum: Heat inactivate all sera to be tested (56°C, 30 min). Dilute the serum 2-fold in DMEM medium without antibiotics and serum (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280). Take 8 200μL PCR tubes, add 90μL DMEM to the first PCR tube, and add 50μL DMEM to the remaining 7. Take 10μL serum and add it to the first PCR tube. After mixing, take 50μL and add it to the next PCR tube. Dilute the serum in the same way.

[0093] 50 μL of the diluted 200 TCID 50 The virus solution (CH-1α) was mixed with 50 μL of 2-fold serially diluted serum and incubated at 37°C for 1 h.

[0094] The culture medium in the 96-well plate inoculated with cells the night before was removed and rinsed once with DMEM. 100 μL of the mixture of virus and serum was added to each well according to the set arrangement. Unneutralized virus was added to the positive control wells, serum with a low dilution multiple was added to the negative control wells, and only culture medium was added to the normal cell control wells, and incubated at 37°C in a 5% CO2 incubator for 1 hour.

[0095] The virus and serum mixture was removed by aspiration, and DMEM was added for rinsing twice. Finally, 100 μL of DMEM medium containing 1% P / S and 2% FBS was added, and the cells were cultured at 37°C in a 5% CO2 incubator for 5 days.

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

[0097] The results are as follows Figure 5 As shown in the figure, neutralizing antibodies can be detected in the PTH-PCTL-PNE immunization group as early as 2 weeks after the first immunization, while neutralizing antibodies can only be detected in the PRRSV inactivated vaccine immunization group 4 weeks after immunization. Although PTH-PCTL-PNE immunization alone cannot produce high-titer neutralizing antibodies, combined immunization with inactivated vaccines can produce high-titer neutralizing antibodies.

[0098] Example 7: Determination of cellular immune response levels

[0099] The concentrations of cytokines in serum were determined using IFN-γ, IL-2, IL-4, IL-5, IL-10, and IL-12 ELISA kits according to the manufacturer's instructions.

[0100] The results are as follows Figure 6 As shown in the figure, compared with the group immunized with PRRSV inactivated vaccine alone, the PTH-PCTL-PNE group and the combined immunization group could significantly induce the immunized pigs to produce higher levels of IFN-γ, IL-2 and IL-12 cytokines ( Figure 6 A, 6C, 6E), while the levels of IL-4, IL-5, and IL-10 did not differ significantly ( Figure 6 B, 6D, 6F). This indicates that PTH-PCTL-PNE mainly induces Th1 cell immunity.

[0101] In summary, the present invention can use a prokaryotic expression system to achieve high-level expression and purification of epitope polypeptide tandem protein PTH-PCTL-PNE. The PTH-PCTL-PNE epitope polypeptide tandem composition of the present invention has good immunogenicity. In the pig immunization experiment without adjuvant, PTH-PCTL-PNE induces a high level of humoral immunity, including a high level of specific IgG antibodies and a certain titer of neutralizing antibodies; at the same time, it induces Th1 and Th2 cellular immune responses. Importantly, compared with the group immunized with only inactivated vaccine, the PTH-PCTL-PNE and inactivated vaccine combined immunization group 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.

[0102] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

[0104] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A tandem polypeptide of epitopes of porcine reproductive and respiratory syndrome, characterized in that: The epitope tandem polypeptide includes a PNB subunit, a PTH subunit, and a PCTL subunit; Wherein, the PNB subunit is a polypeptide composed of epitopes shown in SEQ ID No: 1 to 13 connected in series, or a polypeptide composed of epitopes shown in SEQ ID No: 1 to 13 connected in series in series and connecting peptides between epitopes; The PTH subunit is a polypeptide consisting of epitopes shown in SEQ ID Nos: 14 to 20 connected in series in sequence, or a polypeptide consisting of epitopes shown in SEQ ID Nos: 14 to 20 connected in series in sequence and with linker peptides added between the epitopes; The PCTL subunit is a polypeptide composed of epitopes shown in SEQ ID Nos: 21 to 30 connected in series in sequence, or a polypeptide composed of epitopes shown in SEQ ID Nos: 21 to 30 connected in series in sequence and with linker peptides added between the epitopes.

2. The epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to claim 1, characterized in that: The PNB subunit is a polypeptide having an amino acid sequence as shown in SEQ ID No:

31.

3. The epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to claim 1, characterized in that: The PTH is a polypeptide having an amino acid sequence as shown in SEQ ID No:

32.

4. The epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to claim 1, characterized in that: The PCTL is a polypeptide having an amino acid sequence as shown in SEQ ID No:

33.

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

34.

6. A biomaterial of a porcine reproductive and respiratory syndrome epitope tandem polypeptide composition, characterized in that: The biological material is any of the following: A nucleotide sequence for expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to any one of claims 1 to 5. A recombinant vector expressing the epitope tandem polypeptide according to any one of claims 1 to 5; A recombinant microorganism expressing the epitope tandem polypeptide according to any one of claims 1 to 5; A recombinant cell expressing the epitope tandem polypeptide according to any one of claims 1 to 5.

7. Any of the following uses of the epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to any one of claims 1 to 5: Application in the preparation of vaccines for preventing diseases caused by porcine reproductive and respiratory syndrome virus infection; Application in the preparation of products for preventing and treating diseases caused by porcine reproductive and respiratory syndrome virus infection; Application in the preparation of a medicament or drug for inducing an immune response to porcine reproductive and respiratory syndrome virus antigens.

8. Porcine reproductive and respiratory syndrome vaccine, characterized in that The vaccine comprises the epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to any one of claims 1 to 5.

9. The porcine reproductive and respiratory syndrome vaccine according to claim 8, characterized in that The epitope tandem peptides of porcine reproductive and respiratory syndrome are used as antigens, or the epitope tandem peptides of porcine reproductive and respiratory syndrome and PRRSV inactivated vaccine are used as antigens together.

10. A method for preparing a porcine reproductive and respiratory syndrome vaccine, characterized in that: The method comprises expressing the epitope tandem polypeptide of porcine reproductive and respiratory syndrome according to any one of claims 1 to 5 by using a prokaryotic system.

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