Antigen polypeptides of a group of porcine senecavirus and uses thereof

Abstract

The application discloses a group of antigen polypeptides of porcine Seneca Valley virus and application thereof; the antigen polypeptide of the porcine Seneca Valley virus comprises a fragment shown in sequence SEQ ID NO.1, or a sequence obtained by substituting, deleting or adding one or more amino acids to sequence SEQ ID NO.1 and having immunogenicity. Compared with a porcine Seneca Valley epitope vaccine product under research, the antigen polypeptide of the porcine Seneca Valley virus and application thereof in the field of vaccines have the following advantages: the application can stabilize the spatial conformation of the antigen polypeptide by limiting the polypeptide epitope, and can significantly improve the immunogenicity of the original antigen polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a kind of antigen polypeptide of pig Seneca virus and application thereof. BACKGROUND

[0002] Seneca valley virus (SVV) belongs to the Senecavirus genus of Picornaviridae family, and is also the only member of the genus. SVV was first isolated in cell culture medium contamination in the United States in 2002, and was initially defined as an oncolytic virus, but later confirmed to be able to infect pigs and cause primary vesicular disease in pigs. SVV infection causes vesicular lesions in the snout and coronal band of the hoof of pigs, accompanied by lameness, anorexia and lethargy and other clinical manifestations. It is difficult to distinguish from the clinical symptoms 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 there is mixed infection with foot-and-mouth disease virus, which seriously interferes with the prevention and control of foot-and-mouth disease.

[0003] SVV-001 has typical characteristics of a picornavirus genome, with a standard L-4-3-4 layout, i.e., a leader protein (L), P1 (cleavage of VP1, VP2, VP3 and VP4 four structural proteins), P2 (cleavage into 2A, 2B and 2C three non-structural proteins), P3 (cleavage into 3A, 3B, 3C and 3D four non-structural proteins).

[0004] Currently, the SVV vaccines under research are mostly traditional inactivated vaccines or vaccines formed by recombinant proteins expressed by prokaryotes. However, the inactivated vaccine production process requires virus cultivation, so the safety level is high, and the production cost is also high; the antigens of the recombinant protein vaccine are often expressed in the form of inclusion bodies, which leads to subsequent product purification difficulties, the post-translational processing and modification system of the expression system is imperfect, and the biological activity of the expression product is generally low. Therefore, it is particularly important to study the antigen polypeptide of pig Seneca virus and lay a theoretical foundation for the research and development of pig Seneca virus synthetic peptide vaccine. SUMMARY

[0005] To solve the defects in the prior art, the present application provides a group of antigen polypeptides of pig Seneca virus and applications thereof, and lays a theoretical foundation for the research and development of pig Seneca virus synthetic peptide vaccine. The precise conformation of the polypeptide epitope in the antigen-antibody complex is important for the activity of the antibody. Therefore, some methods are used to limit the peptide epitope, including covalent side chain, i.e., cross-linking of the side chain by forming a covalent restriction containing a disulfide bond, or integrating the epitope into a larger scaffold containing elements that induce the conformation of the related peptide, to present the polypeptide in a conformation-dependent manner.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The present application relates to a group of antigen polypeptides of porcine Senecavirus, which include a fragment shown in sequence SEQ ID NO.1, or a sequence with one or several amino acids substituted, deleted or added and with immunogenicity.

[0008] As an embodiment of the present application, the antigen polypeptide of porcine Senecavirus is a sequence with one cysteine added on each side of the fragment shown in SEQ ID NO.1 to form an intramolecular macrocycle, as shown in SEQ ID NO.2.

[0009] As an embodiment of the present application, the antigen polypeptide of porcine Senecavirus is a sequence with the N-terminal and C-terminal of the fragment shown in SEQ ID NO.1 connected to form an intramolecular amide ring. Such as Cyc(KGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIY).

[0010] As an embodiment of the present application, the antigen polypeptide of porcine Senecavirus is a sequence with two amino acids in the fragment shown in SEQ ID NO.1 substituted into cysteine, as shown in SEQ ID NO.3.

[0011] The present application also relates to a preparation method of a group of antigen polypeptides of porcine Senecavirus, which includes the following steps:

[0012] S1: synthesizing the antigen polypeptide of porcine Senecavirus on a solid carrier by chemical synthesis, or synthesizing the antigen polypeptide of porcine Senecavirus on a solid carrier by chemical synthesis, then treating and concentrating and drying with dilute acid, dissolving with a solvent to condense the N and C terminals of the antigen polypeptide, and precipitating the fully-protected antigen polypeptide with methanol;

[0013] S2: cutting the antigen polypeptide on the solid carrier prepared in step S1 or the fully-protected antigen polypeptide with N and C terminal cyclization by mixing an organic acid solution, precipitating with an ether, and washing to obtain an antigen polypeptide crude product;

[0014] S3: for the antigen polypeptide crude product that needs to be cyclized, cyclizing to form an intramolecular disulfide bond by DMSO oxidation of amino acid thiol;

[0015] S4: purifying the antigen polypeptide crude product by high-pressure liquid chromatography to obtain the antigen polypeptide of porcine Senecavirus.

[0016] As an embodiment of the present application, the sequence of the antigen polypeptide of porcine Senecavirus in step S1 is selected from one of the following:

[0017] KGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIY,

[0018] CKGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIYC,

[0019] Cyc(KGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIY),

[0020] KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY.

[0021] The present application also relates to a synthetic peptide vaccine for Seneca Valley virus in swine, which comprises the aforementioned antigen polypeptide and an immunological adjuvant.

[0022] As an embodiment of the present application, the immunological adjuvant is ISA 50V or ISA206.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] Compared with the conventional Seneca Valley virus inactivated vaccine or the recombinant protein epitope vaccine expressed by prokaryotes, the antigen polypeptide of Seneca Valley virus and its application in the field of vaccine have the following advantages (i) the vaccine product prepared by polypeptide 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 in the preparation process; (ii) the vaccine product prepared by polypeptide contains accurately molecularly divided immunogens, which can exclude harmful sequence antigens or other pathogen-related molecules, and there is no risk of genetic integration or recombination; (iii) the antigen polypeptide is easy to prepare, easy to synthesize and scale up, and has low requirements for equipment and preparation environment, and the cost of preparing vaccine is relatively low; (iv) the vaccine prepared by antigen polypeptide is relatively stable, so it is easy to realize transportation and storage under simple conditions. DETAILED DESCRIPTION

[0025] The following examples will facilitate further understanding of the present application for those skilled in the art, but do not limit the present application in any form. Those of ordinary skill in the art can also make several changes and improvements without departing from the concept of the present application, which are within the scope of the present application. The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. These ranges and values should be interpreted as being inclusive of values reasonably close thereto. For values whose inclusion between the stated endpoints is explicitly excluded, other explicitly stated endpoints are to be understood as being included. The present application is described in detail below with specific reference to the following examples:

[0026] Example 1

[0027] A pig Seneca virus synthetic peptide vaccine and a preparation method thereof, comprising the following steps:

[0028] A1, preparing SVA001-I antigen epitope polypeptide: using PSI-300B full-automatic peptide synthesizer, using Merrifield solid-phase synthesis method for synthesis. Starting with Rink amide MBHA resin as a solid-phase carrier for synthesis, using N-methyl 2-pyrrolidone (NMP) as a swelling resin solvent and a washing solvent, using 25% piperidine for deprotection reaction of the synthesis resin, running the full-automatic synthesizer, adding Fmoc / tBu protected amino acids and hexafluorophosphoric acid benzotriazole-1-yl-oxytripyrrolidinophosphonium reaction condensing agent and N,N-diisopropylethylamine alkaline catalyst to the reactor for coupling reaction according to the antigen polypeptide sequence, when each coupling reaction is complete, 2.5% acetylimidazole solution is used to perform acetylation reaction program to terminate the generation of peptide chain of wrong sequence. Then, 25% piperidine deprotection reaction is performed again to remove the N-terminal Fmoc protection group of the peptide resin, and the next reaction cycle is entered. The synthesis process is from the C-terminal to the N-terminal of the synthesis sequence, and the deprotection reaction, coupling reaction and acetylation reaction are cycled, and the sequence is repeatedly performed according to the set sequence, thereby obtaining the fully protected antigen polypeptide SVA001-I. The antigen peptide is cut from the resin by using high-concentration trifluoroacetic acid (TFA), the resin is removed by filtration, the filtrate is concentrated, and the antigen polypeptide is obtained by ether precipitation, and the SVA001-I antigen polypeptide with a purity of more than 95% is obtained by reverse phase high performance liquid chromatography purification.

[0029] A2, Preparation of SVA001-II Antigen Epitope Polypeptide: The PSI-300B full-automatic peptide synthesizer was used to synthesize the antigen epitope polypeptide by the Merrifield solid-phase synthesis method. Rink amide MBHA resin was used as the solid-phase carrier for synthesis, N-methyl 2-pyrrolidone (NMP) was used as the swelling resin solvent and washing solvent, and 25% piperidine was used to deprotect the synthesized resin. The full-automatic synthesizer was operated, and Fmoc / tBu-protected amino acids and hexafluorophosphoric acid benzotriazole-1-yl-oxytripyrrolidinophosphonium reaction condensing agent and N,N-diisopropylethylamine basic catalyst were added to the reactor for coupling reaction according to the antigen polypeptide sequence. After each coupling reaction was completed, 2.5% acetylimidazole solution was used to perform acetylation reaction program to terminate the generation of incorrect sequence peptide chains. Then, the 25% piperidine deprotection reaction was performed again to remove the N-terminal Fmoc protection group of the peptide resin, and the next reaction cycle was entered. The synthesis process was repeated from the C-terminal to the N-terminal of the synthesis sequence in cycles of deprotection reaction, coupling reaction, and acetylation reaction according to the set sequence, thereby obtaining the fully protected antigen polypeptide SVA001-II. The antigen peptide was then cut from the resin with high-concentration trifluoroacetic acid (TFA), the resin was removed by filtration, and the filtrate was concentrated and precipitated with ethyl ether to obtain the antigen polypeptide. The antigen polypeptide was dissolved in water for injection and oxidized with low-concentration dimethyl sulfoxide (DMSO) to form an intramolecular disulfide bond product, and then purified by reverse-phase high-performance liquid chromatography to obtain SVA001-II antigen polypeptide with a purity of more than 95%.

[0030] A3, Preparation of SVA001-III Antigen Epitope Polypeptide: The SVA001-III antigen epitope polypeptide was synthesized by using a PSI-300B fully automatic peptide synthesizer and a Merrifield solid-phase synthesis method. A 2-Chlorotrityl chloride resin was used as a solid-phase carrier for synthesis, N-methyl 2-pyrrolidone (NMP) was used as a swelling resin solvent and a washing solvent, and 25% piperidine was used to deprotect the synthesized resin. The fully automatic synthesizer was operated, and Fmoc / tBu-protected amino acids, hexafluorophosphoric acid benzotriazole-1-yl-oxytripyrrolidinophosphonium (PyBOP) reaction condensing agent, and N,N-diisopropylethylamine (DIPEA) basic catalyst were added to the reactor for coupling reaction according to the sequence of the antigen polypeptide. After washing the resin, the 25% piperidine deprotection reaction was performed again to remove the N-terminal Fmoc protection group of the peptide resin, and the next reaction cycle was entered. The synthesis process was repeated according to the set sequence from the C-terminal to the N-terminal, with deprotection reaction and coupling reaction as the cycle, so that the fully protected antigen polypeptide was obtained. The fully protected antigen peptide resin was treated with 1% trifluoroacetic acid / dichloromethane solution, and after removing the resin and concentrating, the N and C terminal exposed fully protected antigen polypeptide was precipitated with methanol. After drying, the NMP / DMSO (50 / 50, v / v) solution was added, and the condensation reaction was performed for 24 hours with PyBOP / DIPEA. The N and C terminal fully protected antigen polypeptide SVA001-III was precipitated by adding methanol. The side chain temporary protection group of the ringed antigen polypeptide was removed by using a high-concentration trifluoroacetic acid (TFA) solution, and after concentration, the crude antigen polypeptide was precipitated with tert-butyl methyl ether. The purity of the SVA001-III antigen polypeptide was greater than 95% after purification by reverse-phase high-performance liquid chromatography.

[0031] A4, Preparation of SVA001-IV antigen epitope polypeptide: using PSI-300B full-automatic peptide synthesizer, using Merrifield solid phase synthesis method. Start with Rink amide MBHA resin as a solid phase carrier for synthesis, N-methyl 2-pyrrolidone (NMP) as a swelling resin solvent and washing solvent, 25% piperidine for deprotection reaction of the synthesized resin, run the automatic synthesizer, according to the antigen polypeptide sequence, Fmoc / tBu protected amino acid and hexafluorophosphoric acid benzotriazole-1-yl-oxytripyrrolidinyl phosphorus reaction condensing agent and N,N-diisopropylethylamine basic catalyst are added to the reactor for coupling reaction, after each coupling reaction is completed, 2.5% acetylimidazole solution is used to perform acetylation reaction program to terminate the generation of peptide chain with wrong sequence. Then perform 25% piperidine deprotection reaction again to remove the N-terminal Fmoc protection group of the peptide resin, and enter the next reaction cycle. The synthesis process is from the C-terminal to the N-terminal of the synthesis sequence, and the deprotection reaction, coupling reaction and acetylation reaction are repeated according to the set sequence, so as to obtain the fully protected antigen polypeptide SVA001-IV. Then cut the antigen peptide from the resin with high-concentration trifluoroacetic acid (TFA), filter to remove the resin, and then precipitate with tert-butyl methyl ether after concentrating the filtrate to obtain the antigen polypeptide. The antigen polypeptide is dissolved in water for injection, and then oxidized with low-concentration dimethyl sulfoxide (DMSO) to form intramolecular disulfide bond product, and then purified by reverse phase high performance liquid chromatography (HPLC) to obtain SVA001-IV antigen polypeptide with a purity of more than 95%.

[0032] A total of four polypeptides, the sequences are as follows:

[0033] KGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIY, SEQ ID NO. 1;

[0034] CKGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIYC, SEQ ID NO. 2;

[0035] Cyc(KGGKVSFVLPWNSVSSVLPVRWGGASKLSSATRGLPAHADWGTIY) (N, C-terminal amide ring);

[0036] KGGKVSFVLCWNSVSSVLPVRWGGASKLSSATRGLPAHCDWGTIY, SEQ ID NO. 3;

[0037] The corresponding sequences are named SVA001-I, SVA001-II, SVA001-III, SVA001-IV.

[0038] Example 2

[0039] Immunogenicity analysis of a group of antigen polypeptides of Seneca Valley virus.

[0040] 2.1 Preparation of immunization samples. The antigen polypeptides prepared in Example 1 were diluted to 50 μg / ml with PBS, respectively, and emulsified with sterilized Montanide ISA 50V adjuvant at a volume ratio of 1:1 to prepare immunization samples, which were named V-SVA001-I, V-SVA001-II, V-SVA001-III, and V-SVA001-IV, respectively.

[0041] 2.2 Detection of antibody levels in pigs after immunization;

[0042] (1) 40 pigs (healthy pigs with a body weight of about 40 kg, ELISA titer not higher than 1:8) were randomly divided into 5 groups, of which groups 1, 2, 3, and 4 were immunization groups, and each pig was immunized with 25 μg of antigen polypeptides emulsified with adjuvant, i.e., samples V-SVA001-I, V-SVA001-II, V-SVA001-III, and V-SVA001-IV obtained in Example 1; group 5 was a control group, and each pig was immunized with the same volume of PBS buffer.

[0043] (2) The immunization operation process is shown in Table 1 below.

[0044] Table 1: Immunization operation procedure of Example 2

[0045]

[0046] (3) Antibody level detection. The collected serum was subjected to ELISA antibody detection. The results showed that the antibody levels of the vaccine groups were significantly improved after immunization, and the immunogenicity was good, among which the immunogenicity of the V-SVA001-IV group was the highest, and the immunogenicity of the V-SVA001-I group was the lowest, as shown in Tables 2, 3, 4, 5, and 6 below.

[0047] Table 2: V-SVA001-I antibody titer detection results

[0048]

[0049] Table 3: V-SVA001-II antibody titer detection results

[0050]

[0051] Table 4: V-SVA001-III antibody titer detection results

[0052]

[0053] Table 5: V-SVA001-IV antibody titer detection results

[0054]

[0055] Table 6: PBS antibody titer detection results

[0056]

[0057] In summary, the present application can significantly improve the immunogenicity of the original antigen polypeptide by limiting the spatial conformation of the polypeptide epitope to stabilize the antigen polypeptide.

[0058] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. An antigenic polypeptide of Seneca Valley virus of swine characterized in that, The amino acid sequence of the antigen polypeptide of the porcine Seneca Valley virus is shown as SEQ ID NO. 3, and the thiol groups of the two cysteines in the sequence form an intramolecular disulfide bond.

2. A method of producing an antigenic polypeptide of a Seneca Valley virus of a pig according to claim 1, characterized in that, The method comprises the following steps: S1: synthesizing the antigen polypeptide of the porcine Seneca Valley virus on a solid phase carrier by a chemical synthesis method; S2: cutting the antigen polypeptide on the solid phase carrier prepared in step S1 by mixing an organic acid solution, precipitating with an ether, and washing to obtain an antigen polypeptide crude product; S3: the antigen polypeptide crude product is oxidized by DMSO to cyclize the thiol groups of cysteines to form an intramolecular disulfide bond; S4: purifying the antigen polypeptide crude product by high-pressure liquid chromatography to obtain the antigen polypeptide of the porcine Seneca Valley virus.

3. A synthetic peptide vaccine for Seneca Valley virus in swine, characterized in that, The vaccine comprises the antigen polypeptide of claim 1 and an immunological adjuvant.

4. The synthetic Seneca Valley Virus peptide vaccine of claim 3, wherein, The immunological adjuvant is ISA50V.

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