Swine Seneca virus synthetic peptide vaccine and its preparation method and application

Abstract

The present invention discloses a synthetic peptide vaccine for porcine Seneca virus, and its preparation method and application; the synthetic peptide vaccine comprises a lipidated T cell helper epitope peptide and a B cell antigen epitope peptide related to the major structural protein VP1 of Seneca virus in series. Advantages of the present invention include: the synthetic peptide vaccine lacks infectious pathogens, ensuring absolute safety and easily distinguishing infected animals from vaccinated animals, with no risk of release during the preparation process; the synthetic peptide vaccine contains accurately molecularly divided immunogens, which can exclude harmful sequence antigens or other pathogen-related molecules, without causing virulence reversal and without the risk of genetic integration or recombination; the antigen is easy to prepare, easy to synthesize and amplify, with low requirements for equipment and preparation environment, and the vaccine cost is relatively low; the immunogenicity of the vaccine is optimized by fine-tuning the structure during design; the synthetic peptide vaccine antigen is relatively stable, and can be easily transported and stored under simple conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic peptide vaccines, in particular to a synthetic peptide vaccine for porcine Seneca virus, a preparation method and applications thereof, and in particular to a synthetic peptide vaccine for prevalent porcine Seneca virus disease, a preparation method and applications thereof. Background Art

[0002] Seneca valley virus (SVV) belongs to the genus Senecavirus of the family Picornaviridae and is the only member of this genus. SVV was first isolated from a cell culture medium contaminant in the United States in 2002 and was initially defined as an oncolytic virus, but it was later confirmed that it could infect pigs and cause primary vesicular disease in pigs. SVV infection causes vesicular lesions on the snout and coronary band of the hoof of pigs, accompanied by clinical manifestations such as lameness, anorexia, and lethargy. The clinical symptoms are indistinguishable from those caused by foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. Its transmission characteristics are similar to those of foot-and-mouth disease virus, and mixed infection with foot-and-mouth disease virus exists, which seriously interferes with the prevention and control of foot-and-mouth disease.

[0003] Since its identification in 2002, SVV has primarily spread sporadically in the United States and Canada. However, since late 2014, SVV has become widespread in several countries, including the United States (2015, 2016, and 2017), Canada (2007, 2011, 2015, and 2016), Brazil (2014 and 2015), Colombia (2016), China (2015, 2016, and 2017), and Thailand (2016). After its introduction into China in 2015, SVV has caused outbreaks in 12 provinces, including Fujian, Guangzhou, Hunan, Hubei, Anhui, Jiangsu, Zhejiang, Henan, Hebei, Liaoning, and Heilongjiang. Vaccination is a reliable and effective means of specifically preventing and controlling porcine Seneca virus disease.

[0004] SVV-001 has the typical characteristics of a picornavirus genome, with a standard L-4-3-4 layout, namely, leader protein (L), P1 (cleaved into four structural proteins VP1, VP2, VP3 and VP4), P2 (cleaved into three non-structural proteins 2A, 2B and 2C), and P3 (cleaved into four non-structural proteins 3A, 3B, 3C and 3D).

[0005] Currently under development, most SVV vaccines are either traditional inactivated vaccines or vaccines based on prokaryotically expressed recombinant proteins. However, the production process for inactivated vaccines requires virus cultivation, which places high safety requirements and production costs. Recombinant protein vaccines often express antigens as inclusion bodies, making subsequent product purification difficult. Furthermore, the expression system's post-translational processing and modification systems are imperfect, resulting in generally low biological activity. Therefore, the research and development of synthetic peptide vaccines for porcine Seneca virus is particularly important. Summary of the Invention

[0006] In order to solve the defects in the prior art, the purpose of the present invention is to provide a porcine Seneca virus synthetic peptide vaccine with immune protection efficacy against porcine Seneca virus, as well as a preparation method and application thereof.

[0007] Peptide vaccines rely on short peptide fragments to design highly targeted immune responses, avoiding allergic or reactive sequences. Based on structural biology analysis, we identify the B cell binding site located in the GH loop (approximately residues 183-213) of the SVV VP1 protein as a primary epitope that can induce neutralizing antibodies against the virus in natural hosts and animal models. During infection, T cell recruitment leads to rapid destruction and clearance of the pathogen itself or infected host cells, thereby preventing the spread of infection. The present invention designs a lipidated T cell helper peptide linked to a B cell antigen epitope in an antigenic peptide sequence with nearly uniform charge balance. This lipopeptide, comprising three palmitic acid acyl chains linked to the peptide's N-terminal cysteine, is a self-assembling molecule capable of forming peptide-functionalized supramolecular nanostructures. It also exhibits good immunogenicity in diverse genetic backgrounds. Therefore, synthetic peptides that stimulate antibody responses in animals by leveraging lipidated T cell epitopes to provide full cooperation between T cells and B lymphocytes are attractive SVV vaccine candidates. They are highly pure, structurally defined, stable, and safe to use. Furthermore, they can be prepared using modular methods and can be combined with different T cell helper peptides.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention relates to a synthetic peptide vaccine of porcine Seneca virus, comprising a lipidated T cell helper epitope polypeptide and a B cell antigen epitope polypeptide related to the major structural protein VP1 of the Seneca strain; the lipidated T cell helper epitope polypeptide and the B cell antigen epitope polypeptide are connected in series.

[0010] The lipidated T cell helper epitope polypeptide and the B cell antigen epitope polypeptide related to the main structural protein VP1 of the Seneca strain are obtained by chemical synthesis. The lipidated T cell helper epitope polypeptide and the B cell antigen epitope polypeptide are connected in series by chemical synthesis.

[0011] As one embodiment of the present invention, the tandem method specifically comprises linking the lipidated T cell helper epitope polypeptide to the B cell antigen epitope polypeptide via at least two lysines. Preferably, lysine contains two amino groups (α-amino group and ε-amino group), and the tandem method specifically comprises linking the lipidated T cell helper epitope polypeptide to the B cell antigen epitope polypeptide via ε-N-lysine-lysine, where ε-N-lysine-lysine represents the N-terminal lysine that is subsequently linked to the ε-amino group when linking other molecules.

[0012] As one embodiment of the present invention, the T cell helper epitope of the T cell helper epitope polypeptide is derived from the surface antigen of Clostridium tetani (1084-1099 polypeptide sequence), the circumspora sporozoite coat protein surface antigen of Plasmodium falciparum (16-35 polypeptide sequence), or the immunoglobulin heavy chain surface antigen (3-17 polypeptide sequence). The T cell helper epitope polypeptide is artificially combined with mutation optimization and tripalmitoylcysteine ​​lipidation modification to introduce "Pam3C" to form a lipidated T cell helper epitope polypeptide.

[0013] The lipidated T cell helper epitope polypeptide is obtained by palmitic acid lipidation optimization, and the specific method is: connecting tripalmitoyl cysteine ​​(Pam3Cys) to the N-terminus of the artificial combination mutation optimized T cell helper epitope polypeptide to lipidate the polypeptide so that the antigen polypeptide forms a water-soluble amphiphilic compound (such as Figure 1 ), has zwitterionic properties, which induce non-specific effects at higher concentrations, allowing T helper epitope polypeptides to produce stronger T cell-mediated immunity, especially enhancing the CD4+ and CD8+ T cell protective antibody responses, and preventing the immediate degradation of vaccine peptides, thereby enhancing the duration of immune responses in animals.

[0014] As one embodiment of the present invention, as shown in Table 1 below, the T cell helper epitope polypeptide optimized by artificial combination mutation includes one of Th1′, Th2′, and Th3′;

[0015] Table 1. T cell epitope peptide sequences

[0016] T cell epitope code T cell epitope sequences Th1′ (SEQ ID NO. 1) VSIDKFRIFSKALNPK Th2′ (SEQ ID NO. 2) EYLNKIQNSLSTEWSPASVT Th3′ (SEQ ID NO. 3) LSEIKGVIVHRLEGV

[0017] As shown in Table 2 below, the lipidated T cell helper epitope polypeptide includes one of Th1, Th2, and Th3;

[0018] Table 2. Lipidated T cell epitope peptide sequences

[0019] T cell epitope code T cell epitope sequences Th1 <![CDATA[Pam3CVSIDKFRIFSKALNPK]]> Th2 <![CDATA[Pam3CEYLNKIQNSLSTEWSPASVT <!-- 2 -->]]> Th3 <![CDATA[Pam3CLSEIKGVIVHRLEGV]]>

[0020] The Seneca strain includes type A Seneca virus; by comparing the three-dimensional structure of the Seneca virus VP1 protein and the amino acid sequence of the highly variable region, it is known that if one wants to design and synthesize a synthetic peptide that can widely cover the VP1 antigenic site, it is necessary to include more fragments of the ring structure and consider the variation of amino acids in key sites. In the early guinea pig immunization experiments, the present invention found that the long VP1 polypeptide antigen has stronger immune properties, and the VP1 amino acid sequence contains 1 BC loop, 1 CD loop and 1 GH loop; and the polypeptide sequence located in the GH loop (positions 183 to 213) has the strongest immune efficacy and is relatively conservative, which can stimulate the animal body to produce a specific immune response. According to the analysis of the Seneca virus gene sequence named SVA001 in NCBI, the corresponding B cell epitope polypeptide containing the GH loop sequence of amino acids 174 to 218 in VP1 was finally obtained.

[0021] SVA001 sequence: (SEQ ID NO.4)

[0022] STDNAETGVI EAGNTDTDFS GELAAPGSNH TNVKFLFDRS RLLNVIKVLE KDAVFPRPFPTQEGAQQDDG YFCLLTPRPT VASRPATRFG LYANPSGSGV LANTSLDFNF YSLACFTYFR SDLEVTVVSLEPDLEFAVGW FPSGSEYQAS SFVYDQLHVP FHFTGRTTPRA FASKGGKVSFVLPWNSVSSV LPVRWGGASKLSSATRGLPA HADWGTIYAF VPRPNEKKST AVKHVAVYIR YKNARAWCPS MLPFRSYKQK MLM.

[0023] The precise conformation of the peptide epitope within the antigen-antibody complex is crucial for antibody activity. Therefore, the present invention utilizes covalent side-chain crosslinking via disulfide bonds to constrain peptide epitopes, presenting the peptide vaccine in a conformationally correlated manner. To this end, the present invention replaces both P at position 183 and A at position 212 of the SVA001 VP1 sequence with C to form disulfide bonds, thereby enhancing the stability of the VP1 ring structure. Furthermore, an exogenous T-helper epitope is added to the N-terminus of the peptide chain to enhance T cell immunity. This combination of a T-cell epitope peptide and a B-cell epitope peptide from the VP1 primary antigenic site expands its range of action, enabling immunostimulation in animals with diverse MHC genetic backgrounds. Furthermore, the introduction of a tripalmitoylcysteine ​​(Pam3Cys) at the N-terminus of the antigen peptide enhances the hydrophobicity of the lipidated antigen sequence, thereby slowing the degradation rate of the antigen sequence by enzymes in the animal body and effectively prolonging the duration of immunity. The resulting lipidated T+B cell epitope peptide sequence is shown in Table 3 below.

[0024] Table 3. Lipidated T+B cell epitope peptide sequences

[0025]

[0026] As an embodiment of the present invention, the B cell antigen epitope sequence is:

[0027] KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY (SEQ ID NO. 5). The 10th C and the 39th C of the B cell antigen epitope sequence form a disulfide bond, and a ring structure is formed through two cysteines.

[0028] As an embodiment of the present invention, the synthetic peptide vaccine further comprises an immune adjuvant, and the lipidated T cell helper epitope polypeptide and the B cell antigen epitope tandem polypeptide are mixed with the immune adjuvant to form a water-in-oil or water-in-oil-in-water emulsion vaccine.

[0029] As an embodiment, the immune oil adjuvant is selected from SEPPIC's ISA 50Vc to form a water-in-oil emulsion vaccine;

[0030] As an embodiment, the immune oil adjuvant is selected from ISA 206 of SEPPIC Company to form a water-in-oil-in-water emulsion vaccine.

[0031] As an embodiment, the dosage of the tandem polypeptides in the vaccine is 10-50 ug / ml.

[0032] The present invention also relates to a method for preparing a porcine Seneca virus synthetic peptide vaccine, comprising the following steps:

[0033] S1. Synthesizing a B cell antigen epitope polypeptide on a solid phase support by chemical synthesis, then linking at least two lysines, then linking a T cell helper epitope polypeptide to the ε-amino group of the N-terminal lysine of the polypeptide, and then linking Fmoc-Pam2Cys-OH and removing Fmoc to palmitoylate the N-terminus to form a three-chain lipopeptide, ultimately obtaining a lipidated T cell helper epitope polypeptide and a B cell antigen epitope polypeptide tandem polypeptide linked to the solid phase support;

[0034] S2. removing the tandem polypeptides from the solid support by mixing with an organic acid solution, precipitating with ether, and washing to obtain a crude product of the lipidated T cell helper epitope mixed polypeptide and the B cell antigen epitope polypeptide tandem polypeptide;

[0035] S3, cyclizing the amino acid thiol groups on the crude tandem peptides of the lipidated T cell helper epitope peptide and the B cell antigen epitope peptide by DMSO oxidation to form intramolecular disulfide bonds (and detecting the residual amount of thiol groups by Ellman's reagent);

[0036] S4. After the cyclization is completed, the organic acid in the solution is removed by a cation exchange resin, and the effluent is collected;

[0037] S5, purification using chromatography;

[0038] S6. Concentrate and purify the solution through a tangential flow membrane system and replace the solution with water for injection to remove the organic solvent. The resulting aqueous phase concentrate is then filtered and sterilized.

[0039] S7. Under the action of shear force, the sterile liquid is mixed with the oil adjuvant to form an emulsion to obtain a vaccine.

[0040] In some embodiments, in step S5, a cyano-bonded phase is used to purify the tandem polypeptides by reverse-phase high performance liquid chromatography to remove small molecule impurities in the tandem polypeptides and obtain a pure target product organic solution with a good yield.

[0041] In some embodiments, in step S6, the solution is sterilized by filtration through a 0.22 μm filter element.

[0042] As an embodiment, in step S1, the B cell antigen epitope polypeptide sequence is: KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY.

[0043] As an embodiment, in step S1, the T cell helper epitope polypeptide includes one of Th1′, Th2′, and Th3′;

[0044] The sequence of Th1′ is: VSIDKFRIFSKALNPK;

[0045] The sequence of Th2′ is: EYLNKIQNSLSTEWSPASVT;

[0046] The sequence of Th3′ is: LSEIKGVIVHRLEGV.

[0047] The present invention also relates to the use of a porcine Seneca virus synthetic peptide vaccine for preparing a preparation for preventing and treating porcine Seneca virus disease or for preparing a preparation for distinguishing infected animals from immunized animals.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] Compared with the traditional inactivated porcine Seneca vaccine under development or the vaccine formed by recombinant protein expressed in prokaryotes, the synthetic peptide vaccine described in the present invention has the following advantages: (i) the synthetic peptide vaccine lacks infectious pathogens, which not only ensures absolute safety but also can easily distinguish infected animals from vaccinated animals, and there is no risk of release during the preparation process; (ii) the synthetic peptide vaccine contains accurately molecularly divided immunogens, which can exclude harmful sequence antigens or other pathogen-related molecules, without causing reversal of virulence and without the risk of genetic integration or recombination; (iii) the synthetic peptide vaccine antigen is easy to prepare, easy to synthesize and amplify, with low requirements for equipment and preparation environment, and the vaccine cost is relatively low; (iv) the immunogenicity of the synthetic peptide vaccine can be optimized by fine-tuning the structure during design; (v) the synthetic peptide vaccine antigen is relatively stable, so it is easy to transport and store under simple conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0051] Figure 1 Schematic diagram of the synthesis of tripalmitoylcysteine ​​(Pam3C) lipidated antigenic peptide. DETAILED DESCRIPTION

[0052] The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. For those of ordinary skill in the art, without departing from the inventive concept, some changes and improvements can also be made, and these all belong to protection scope of the present invention. The endpoints and any values ​​of the scope disclosed in this article are not limited to this precise range or value, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, between the endpoint values ​​of each range, between the endpoint values ​​of each range and a separate point value, and between the separate point value, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article. Below in conjunction with specific embodiments, the present invention is described in detail:

[0053] Example 1

[0054] A porcine Seneca virus synthetic peptide vaccine and a preparation method thereof, comprising the following steps:

[0055] A. Using a fully automatic peptide synthesizer, the synthesis of polypeptide antigens is performed by Merrifield solid-phase synthesis. 25% piperidine is used to deprotect the synthetic resin, and the fully automatic synthesizer is run to add the Fmoc / tBu protected amino acid and the reaction condensing agent to the reactor for coupling reaction. When the coupling reaction is complete, the acetylation reaction program is performed with 2.5% acetylimidazole solution to terminate the generation of the peptide chain with the wrong sequence. The deprotection reaction is then performed again to remove the F-moc protecting group at the N-terminus of the peptide resin and enter the next reaction cycle. The synthesis process is from the C-terminus to the N-terminus of the synthetic sequence, with deprotection reaction, coupling reaction, and acetylation reaction as a cycle, and is repeated continuously according to the set sequence;

[0056] The specific process of step A is as follows:

[0057] A1. Preparation of B cell antigen epitope peptides: Synthesis was performed using the Merrifield solid-phase synthesis method using an EST-150 fully automated peptide synthesizer. Rink amide MBHA resin was initially used as the solid phase support, with N-methyl 2-pyrrolidone as the resin swelling and washing solvent. The resin was deprotected using 25% piperidine. The fully automated synthesizer was then added to the reactor with an Fmoc / tBu-protected amino acid, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate as a reaction agent, and N,N-diisopropylethylamine as a basic catalyst for coupling reactions. After each coupling step was complete, an acetylation reaction was performed with 2.5% acetylimidazole solution to terminate the formation of peptide chains with incorrect sequences. A further 25% piperidine deprotection reaction was then performed to remove the Fmoc protecting group at the N-terminus of the peptide resin, allowing for the next reaction cycle. The synthesis process is from the C-terminus to the N-terminus of the synthetic sequence, with deprotection reaction, coupling reaction, and acetylation reaction as a cycle, and is continuously repeated according to the set sequence, thereby obtaining the B cell antigen polypeptide SVA001 VP1 (174-218) (Cys183-Cys212).

[0058] A2. Preparation of T cell helper surface peptides:

[0059] A portion of the synthesized B-cell antigen epitope peptide resin was removed and ligated to two lysine residues. Specifically, an Fmoc-Lys(Boc)-OH residue was first attached. After removing the N-terminal Fmoc residue, a Boc-Lys(Fmoc)-OH protected amino acid was attached. Coupling was performed using an N,N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole synthesis system. After washing with N-methyl 2-pyrrolidone, acetylation was performed with a 2.5% acetylimidazole solution to terminate the peptide chain with the incorrect sequence. The Fmoc residue on the ε-amino group of the Boc-Lys(Fmoc)-OH residue was then removed with 25% piperidine to expose the ε-amino group. Synthesis was then continued according to the sequence of the T-cell helper epitope peptide, using Fmoc / tBu protected amino acids and an N,N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole synthesis system for condensation. The synthesis process proceeded from the C-terminus to the N-terminus of the T-cell helper epitope peptide sequence, with a cycle of deprotection, coupling, and acetylation. Thus, Th1′-ε-KK-(SVA001 VP1(174-218)), Th2′-ε-KK-(SVA001 VP1(174-218)), and Th3′-ε-KK-(SVA001 VP1(174-218)) were synthesized.

[0060] A3. Preparation of lipidated T cell helper surface peptides:

[0061] The preparation method for Pam3Cys lipidated peptides involves coupling a portion of a peptide resin containing Th1′-ε-KK-(SVA001 VP1(174-218)), Th2′-ε-KK-(SVA001 VP1(174-218)), and Th3′-ε-KK-(SVA001 VP1(174-218)) antigen epitopes with Fmoc-Pam2Cys-OH and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate as a condensing agent and N,N-diisopropylethylamine as a basic catalyst. After the coupling reaction is complete, an acetylation reaction is performed with 2.5% acetylimidazole solution to terminate any peptide chains with incorrect sequences. Then, 25% piperidine was used again to remove the N-terminal Fmoc protecting group of cysteine ​​and palmitoyl chloride (Pam-Cl) and pyridine were used to catalyze the formation of palmitoylated N-terminal three-chain lipopeptides, thereby synthesizing lipidated Th1-ε-KK-(SVA001 VP1(174~218)), lipidated Th2-ε-KK-(SVA001 VP1(174~218)), and lipidated Th3-ε-KK-(SVA001 VP1(174~218)).

[0062] B. Separation of peptide from solid support: Prepare a reaction solution containing 90% trifluoroacetic acid (TFA), 4% triisopropylsilane, 5% phenol, and 1% water for injection. Add the peptide-resin and allow to react with constant stirring for 4 hours. Then, precipitate with ether solution to obtain a peptide-resin mixture. Separate the peptide solution and resin particles using a sand core funnel.

[0063] C. Cyclization reaction: The pH of the peptide solution was adjusted to 6.0-8.0 with aqueous ammonia and acetic acid solution. The cyclization reaction was carried out with DMSO to form an intramolecular disulfide bond between SVA001 VP1 (174-218) and the corresponding site (Cys183-Cys212) of the antigen peptide. The residual amount of sulfhydryl groups was detected with Ellman's reagent until the reaction was complete.

[0064] D. Desalting and purification: The cyclized polypeptide solution is treated with a strong base anion exchange resin to remove salts and organic acids in the solution, and the effluent is collected;

[0065] E. Purification: Purification was performed by reverse-phase high-performance liquid chromatography using a cyano-bonded phase, using a Sepfocus 50DAC preparative liquid chromatography system, CN-HPLC 250 mm × 50 mm, particle size 10 μm, flow rate 40 ml / min, solvent system: Solution A: aqueous solution containing 1% TFA, Solution B: acetonitrile solution containing 1% TFA, with a gradient of B from 25% to 100% B over 3 column volumes to separate the target antigen peptide from impurity peptides. Impurities in the antigen polypeptide were removed by segmented collection to obtain a pure target product organic solution with a good yield.

[0066] F. Concentration: The purified solution is concentrated by a tangential flow membrane system and the organic solvent is removed by replacing the solution with water for injection to obtain an aqueous solution of antigen peptides. The concentration of the antigen peptide solution is determined by the Lorry method;

[0067] G. Sterilization of polypeptides: After the polypeptides are purified and concentrated, they are sterilized by filtration using a sterile filter with a pore size of 0.22 μm in a clean bench to obtain sterile antigen polypeptides 1, 2, 3, 4, 5, 6, and 7 as shown in Table 4.

[0068] Table 4. B cell epitope, T+B and lipidated T+B cell epitope peptide sequences

[0069]

[0070] Example 2

[0071] A porcine Seneca virus synthetic peptide vaccine and its preparation method, using Montanide ISA 50V C The vaccine is prepared with the antigen polypeptide in Example 1, specifically comprising the following steps:

[0072] 1. Preparation of aqueous phase: Dilute the fusion antigen polypeptides 1, 2, 3, 4, 5, 6, and 7 obtained in Example 1 to 50 μg / ml using sterilized water for injection, and filter through a filter with a pore size of 0.22 μm.

[0073] 2. Oil phase preparation: Oil phase adjuvant Montanide ISA 50V C Sterilize at 120°C for 30 minutes and then bring to room temperature for later use.

[0074] 3. Emulsification: First add the oil phase into the emulsification tank, then stir at 80-100 r / min, and slowly add the water phase at the same time. The oil phase / water phase ratio is 1 / 1. After adding, stir for 2 minutes, and then stir at 8500 r / min for 6 minutes to emulsify it to form an oil-in-water emulsion, thereby obtaining the porcine Seneca virus synthetic peptide vaccine of the present invention, which is recorded as vaccines SVA001_A′V, SVA001_B′V, SVA001_C′V, SVA001_AV, SVA001_BV, SVA001_CV, and SVA001_VP1V.

[0075] Example 3

[0076] A porcine Seneca virus synthetic peptide vaccine and a preparation method thereof, wherein the vaccine is prepared using Montanide ISA 206 and the antigenic polypeptide of Example 1, and specifically comprises the following steps:

[0077] 1. Preparation of aqueous phase: Dilute the fusion antigen polypeptides 1, 2, 3, 4, 5, 6, and 7 obtained in Example 1 to 50 μg / ml using sterilized water for injection, and filter through a filter with a pore size of 0.22 μm.

[0078] 2. Oil phase preparation: Sterilize the oil phase adjuvant Montanide ISA 206 at 120°C for 30 minutes and then allow to cool to room temperature for later use.

[0079] 3. Emulsification: First add the oil phase into the emulsification tank, then stir at 40-60 r / min, and slowly add the water phase at the same time. The oil phase / water phase ratio is 1 / 1. After adding, stir for 2 minutes, and then stir at 350 r / min for 5 minutes at 31°C to emulsify it to form a water-in-oil-in-water emulsion. Keep the temperature at 20°C for 1 hour to obtain the porcine Seneca virus synthetic peptide vaccine of the present invention, which is recorded as vaccines SVA001_A′V206, SVA001_B′V206, SVA001_C′V206, SVA001_AV206, SVA001_BV206, SVA001_CV206, and SVA001 VP1V206.

[0080] Example 4

[0081] An application of a synthetic peptide vaccine for porcine Seneca virus, detecting antibody levels in pigs after immunization with Montanide ISA 50Vc and the antigen emulsified vaccine in Example 2;

[0082] (1) 40 pigs (healthy pigs weighing about 40 kg, ELISA titer not higher than 1:8) were screened and randomly divided into 8 groups, of which groups 1, 2, 3, 4, 5, 6, and 7 were immunized groups, and each pig was immunized with a vaccine emulsified with 25 μg of synthetic peptide and adjuvant, namely, the vaccine SVA001_A′V, SVA001_B′V, SVA001_C′V, SVA001_AV, SVA001_BV, SVA001_CV, and SVA001_VP1V obtained in Example 2; Group 8 was the control group, and each pig was immunized with the same volume of PBS buffer.

[0083] (2) The immune operation process is shown in Table 5 below.

[0084] Table 5: Immunization procedures of Example 4

[0085]

[0086] (3) Antibody Level Detection. ELISA antibody testing was performed on the collected serum. The results showed that the antibody levels of all vaccine groups increased significantly after immunization, and the immunogenicity was good. Among them, the immunogenicity of the SVA001_AV group was the highest, and that of the SVA001_VP1V group was the lowest. Compared with the SVA001_A′V group, the SVA001_AV group had a longer duration of immunity. The details are shown in Tables 6, 7, 8, 9, 10, 11, 12, and 13 below.

[0087] Table 6: SVA001_A′V antibody titer test results

[0088]

[0089] Table 7: SVA001_B′V antibody titer test results

[0090]

[0091] Table 8: SVA001_C′V antibody titer test results

[0092]

[0093] Table 9: SVA001 AV antibody titer test results

[0094]

[0095] Table 10: SVA001_BV antibody titer test results

[0096]

[0097] Table 11: SVA001_CV antibody titer test results

[0098]

[0099] Table 12: SVA001 VP1V antibody titer test results

[0100]

[0101] Table 13: PBS antibody titer test results

[0102]

[0103] Example 5

[0104] An application of a synthetic peptide vaccine for porcine Seneca virus, using the Montanide ISA 206 and antigen emulsified vaccine described in Example 3 to detect antibody levels in pigs;

[0105] (1) 40 pigs (healthy pigs weighing about 40 kg, ELISA titer not higher than 1:8) were screened and randomly divided into 5 groups, of which Groups 1, 2, 3, and 4 were immunization groups, and each pig was immunized with a vaccine emulsified with 25 μg of synthetic peptide and adjuvant, namely, the vaccine SVA001_AV206, SVA001_BV206, SVA001_CV206, and SVA001_VP1V206 obtained in Example 3; Group 5 was the control group, and each pig was immunized with the same volume of PBS buffer.

[0106] (2) The immune operation process is shown in Table 14 below.

[0107] Table 14: Immunization Procedures for Example 5

[0108]

[0109] (3) Antibody Level Detection. ELISA antibody testing was performed on the collected serum. The results showed that the antibody levels in all vaccine groups increased significantly after immunization, and the immunogenicity was good. Among them, the SVA001_AV206 group had the highest immunogenicity, and the SVA001_VP1V206 group had the lowest. The specific results are shown in Tables 15, 16, 17, 18, and 19 below.

[0110] Table 15: SVA001_AV206 antibody titer test results

[0111]

[0112] Table 16: SVA001 BV206 Antibody Titer Test Results

[0113]

[0114]

[0115] Table 17: SVA001 CV206 antibody titer test results

[0116]

[0117] Table 18: SVA001 VP1V206 antibody titer test results

[0118]

[0119] Table 19: PBS antibody titer test results

[0120]

[0121] Example 6

[0122] Application of a synthetic peptide vaccine for porcine Seneca virus and detection of antibody levels in pigs after immunization with vaccine samples of different antigen contents.

[0123] (1) Montanide ISA 50Vc and the SVA001_A antigen in Example 1 were emulsified and divided into three groups according to the different antigen contents, as shown in Table 20.

[0124] Table 20 SVA001_A antigen content in each group

[0125]

[0126] (2) 32 pigs (healthy pigs weighing about 40 kg, ELISA titer no higher than 1:8) were screened and randomly divided into 5 groups. Groups 1, 2, and 3 were immunized groups, and each pig was immunized with 1 mL of the corresponding vaccine according to Table 21; Group 4 was the control group, and each pig was immunized with the same volume of PBS buffer.

[0127] (3) The immunization operation process is shown in Table 21 below.

[0128] Table 21: Immunization Procedure of Example 6

[0129]

[0130] (4) Antibody Level Detection. ELISA antibody testing was performed on the collected serum. The results showed that the antibody levels of all vaccine groups increased significantly after immunization, and the immunogenicity was good. The immunogenicity of the SVA001_AV25 group was significantly higher than that of the SVA001_AV10 group, but there was no significant difference with the SVA001_AV50 group. Therefore, it is recommended to use 25 μg of SVA001_A antigen for vaccine preparation. The details are shown in Tables 22, 23, 24, and 25 below.

[0131] Table 22: SVA001_AV10 antibody titer test results

[0132]

[0133] Table 23: SVA001 AV25 antibody titer test results

[0134]

[0135]

[0136] Table 24: SVA001_AV50 antibody titer test results

[0137]

[0138] Table 25: PBS antibody titer test results

[0139]

[0140] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A porcine Seneca virus synthetic peptide vaccine, characterized in that: It includes a lipidated T cell helper epitope peptide, a B cell antigen epitope peptide related to the major structural protein VP1 of Seneca virus, and the lipidated T cell helper epitope peptide and the B cell antigen epitope peptide are connected in series; The lipidated T cell helper epitope polypeptide is selected from one of Th1, Th2, and Th3; The sequence of Th1 is: Pam3CVSIDKFRIFSKALNPK; The sequence of Th2 is: Pam3CEYLNKIQNSLSTEWSPASVT; The sequence of Th3 is: Pam3CLSEIKGVIVHRLEGV; The B cell antigen epitope polypeptide sequence is: KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY; the 10th C and the 39th C of the B cell antigen epitope polypeptide sequence form a disulfide bond, and a ring structure is formed through two cysteines; The lipidated T cell helper epitope polypeptide is linked to the B cell antigen epitope polypeptide via ε-N-lysine-lysine, and the T cell helper epitope polypeptide is linked to the ε-amino group of the most N-terminal lysine.

2. The porcine Seneca virus synthetic peptide vaccine according to claim 1, characterized in that The vaccine also includes an immune adjuvant; the lipidated T cell helper epitope polypeptide and the B cell antigen epitope polypeptide are tandemly mixed with the immune adjuvant to form a water-in-oil or water-in-oil-in-water emulsion vaccine.

3. The porcine Seneca virus synthetic peptide vaccine according to claim 2, characterized in that The immune adjuvant is ISA50V or ISA206; the dosage of the tandem polypeptide in the vaccine is 10-50ug / ml.

4. A method for preparing a porcine Seneca virus synthetic peptide vaccine according to any one of claims 1 to 3, characterized in that: The steps include: S1. Synthesizing a B cell antigen epitope polypeptide on a solid phase support by chemical synthesis, then linking two lysines, then linking a T cell helper epitope polypeptide to the ε-amino group of the N-terminal lysine of the polypeptide, and then linking Fmoc-Pam2Cys-OH and removing Fmoc to palmitoylate the N-terminus to form a tripalmitoylated cysteine ​​lipopeptide, ultimately obtaining a lipidated T cell helper epitope polypeptide and a B cell antigen epitope polypeptide tandem polypeptide linked to the solid phase support; S2. removing the tandem polypeptide from the solid support by mixing an organic acid solution, precipitating with ether, and washing to obtain a crude product of the lipidated T cell helper epitope polypeptide and the B cell antigen epitope polypeptide tandem polypeptide; S3, cyclizing the amino acid sulfhydryl groups on the crude tandem peptides of the lipidated T cell helper epitope peptide and the B cell antigen epitope peptide by DMSO oxidation to form an intramolecular disulfide bond; S4. After the cyclization is completed, the organic acid in the solution is removed by a cation exchange resin, and the effluent is collected; S5, purification using chromatography; S6. Concentrate and purify the solution through a tangential flow membrane system and replace the solution with water for injection to remove the organic solvent. The resulting aqueous phase concentrate is then filtered and sterilized. S7. Under the action of shear force, the sterile liquid is mixed with the oil adjuvant to form an emulsion to obtain a vaccine.

5. The method for preparing a porcine Seneca virus synthetic peptide vaccine according to claim 4, wherein: In step S1, the B cell antigen epitope polypeptide sequence is: KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY; the T cell helper epitope polypeptide is selected from one of Th1′, Th2′, and Th3′; The sequence of Th1′ is: VSIDKFRIFSKALNPK; The sequence of Th2′ is: EYLNKIQNSLSTEWSPASVT; The sequence of Th3′ is: LSEIKGVIVHRLEGV.

6. A use of a porcine Seneca virus synthetic peptide vaccine according to any one of claims 1 to 3, characterized in that: Used for preparing preparations for preventing porcine Seneca virus disease or for preparing preparations for distinguishing infected animals from immunized animals.

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