Porcine circovirus type 3 Cap protein and porcine circovirus type 3 subunit vaccine

Abstract

The invention relates to the technical field of veterinary biological products, and particularly provides a porcine circovirus type 3 Cap protein and a porcine circovirus type 3 subunit vaccine. The amino acid in the NLS region of the Cap protein of the porcine circovirus type 3 (PCV3) has 12R-K substitution, and 75-76aa (AI) is substituted into PGGGSNPR. The amino acid sequence of the Cap protein is as shown in SEQ ID NO. 4. The invention also provides a PCV3 virus-like particle antigen which is the Cap protein of the porcine circovirus type 3 (PCV3). The virus-like particle vaccine comprises an immunizing dose of the PCV3 virus-like particle antigen and a pharmaceutically acceptable carrier. According to the invention, the correct assembly of the PCV3 virus-like particles is promoted, and the stability of the protein is improved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of veterinary biological products, and particularly provides a Cap protein of porcine circovirus type 3 and a porcine circovirus type 3 subunit vaccine. BACKGROUND

[0002] Porcine circovirus type 3 (PCV3) is a non-enveloped icosahedral single-stranded DNA (ssDNA) virus belonging to the Circovirus genus of the Circoviridae family, and PCV3 was first discovered and identified in the United States in 2016. At present, PCV3 only exists in domestic pigs and wild boars, and can be combined with other pig diseases to cause clinical symptoms and pathological manifestations mainly including reproductive disorders, PDNS, systemic inflammatory disease, respiratory disease, diarrhea and central nervous system signs. The Cap protein of PCV3 is a structural protein of the virus and is an important candidate protein for vaccine research and development. The PCV3 Cap is difficult to express in E. coli due to the N-terminal rich basic amino acids and rare codons, and the protein is prone to aggregation, so it is difficult to achieve assembly. Therefore, the existing PCV3 subunit vaccine usually has the problem of low antibody level after immunization.

[0003] Protein engineering is a technology for obtaining more ideal target products by modifying the structure or properties of a protein molecule. Traditional protein engineering methods mostly modify target proteins by introducing random mutations. With the rapid development of computer technology and bioinformatics technology, computer simulation is more and more applied to protein engineering. According to the data in the database established by analyzing the structure and function of natural proteins, the spatial structure and biological function of a certain amino acid sequence peptide chain can be predicted, and the relationship between structure and function can be directly investigated and analyzed through gene recombination experiments.

[0004] Therefore, the PCV3 Cap protein with high expression and high assembly is obtained by modifying the protein. SUMMARY

[0005] One of the purposes of the application is to provide a Cap protein of porcine circovirus type 3 (PCV3).

[0006] One of the purposes of the application is to provide a PCV3 virus-like particle antigen.

[0007] One of the purposes of the application is to provide a PCV3 virus-like particle vaccine.

[0008] One of the purposes of the application is to provide an application of the PCV3 virus-like particle antigen in the preparation of a medicine for preventing porcine circovirus-related diseases.

[0009] The application relates to a Cap protein of porcine circovirus type 3 (PCV3), wherein the NLS region of the Cap protein has 12R-K substitution and 75-76 aa (AI) is replaced by PGGGSNPR.

[0010] Further, the amino acid sequence of the Cap protein is shown in SEQ ID NO. 4.

[0011] The application relates to a PCV3 virus-like particle antigen, which is the Cap protein as described above.

[0012] The biological material related to the Cap protein described above, wherein the biological material comprises any one of the following:

[0013] (1) a nucleic acid fragment encoding the Cap protein;

[0014] (2) an expression cassette containing the nucleic acid fragment in (1);

[0015] (3) a vector containing the nucleic acid fragment in (1) or the expression cassette in (2);

[0016] (4) a recombinant cell containing the nucleic acid in (1), the expression cassette in (2) or the vector in (3);

[0017] Preferably, the nucleic acid fragment encoding the Cap protein has the nucleotide sequence shown in SEQ ID NO. 3.

[0018] The application relates to a PCV3 virus-like particle vaccine, wherein the virus-like particle vaccine comprises an immunizing amount of the PCV3 virus-like particle antigen and a pharmaceutically acceptable carrier.

[0019] As an embodiment of the application, in the virus-like particle vaccine of the application, the content of the PCV3 virus-like particle antigen is greater than or equal to 30 mu g / ml.

[0020] As an embodiment of the application, in the virus-like particle vaccine of the application, the content of the PCV3 virus-like particle antigen is 30 mu g / ml to 100 mu g / ml.

[0021] In the virus-like particle vaccine of the application, the content of the PCV3 virus-like particle antigen can also be selected from 30 mu g / ml to 50 mu g / ml, or 50 mu g / ml to 100 mu g / ml.

[0022] Further, the pharmaceutically acceptable carrier comprises at least one of an adjuvant, a freeze-drying protective agent, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant and a preservative.

[0023] Preferably, the adjuvant comprises one or several of aluminium hydrogel adjuvant, saponin, Afuvidine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide;

[0024] Preferably, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably from 30% V / V to 60% V / V, more preferably 50% V / V;

[0025] Preferably, the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine or an amino acid;

[0026] Preferably, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor or interleukin 2.

[0027] Compared with the prior art, the technical effects of the present application are:

[0028] The present application uses E. coli expression system to express PCV3 Cap protein to prepare virus-like particles. The present application first replaces the amino acid 12R-K in the NLS region by analyzing the importance of the PCV3 Cap protein nucleolus positioning region and the amino acid composition, and the result is that the expression amount of PCV3 Cap after amino acid replacement is improved. Further through sequence alignment and using the structure prediction tool Alpha-Fold to predict the structure of PCV3 Cap (12R-K) and PCV2 Cap, it is found that the biggest difference between PCV2 Cap and PCV3 Cap (12R-K) is that PCV2 Cap has an extra beta fold (82-89aa PGGGSNPR), and the corresponding amino acid position of PCV3 Cap (12R-K) is 75-76aa AI. Therefore, according to the different structures of the two, the 75-76aa (AI) of PCV3 Cap (12R-K) is replaced by PGGGSNPR, which promotes the correct assembly of PCV3 virus-like particles and improves the stability of the protein, providing a possibility for the preparation of PCV3 virus-like particle vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is an electron micrograph of PCV3 Cap (12R-K);

[0030] Figure 2 is an electron micrograph of AI-replaced Cap (12R-K);

[0031] Figure 3 is PCV2 Cap and PCV3 Cap (12R-K) Structure prediction comparison chart. DETAILED DESCRIPTION

[0032] The relevant terms in this application are explained as follows:

[0033] “Antigen” refers to a substance that can induce an immune response in an organism, i.e., can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, make them proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0034] “Virus-like particles (VLPs)” are particles assembled from one or more viral structural proteins, which have similar external structures and antigenicity to virus particles, but do not contain viral genes.

[0035] The term “vaccine”, “vaccine composition” refers to a pharmaceutical composition containing PCV3 virus-like particle antigens, which can induce, stimulate or enhance the immune response of pigs against PCV3.

[0036] The term “immunizing amount” shall be understood as “immunologically effective amount”, also known as immunoprotective amount or effective amount for generating an immune response, which is an amount of antigen effective to induce an immune response in the recipient, which is sufficient to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof. The immune response can be sufficient for diagnostic purposes or other assays, or can be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications thereof caused by infection with a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the immunogenic composition can be assessed by measuring, for example, clinical signs in the test animals, such as a decrease in healthy litters, an increase in the number of stillborns, the overall physiological condition and overall health and performance of the test animals. The immune response can include, but is not limited to, inducing cellular and / or humoral immunity.

[0037] The term "pharmaceutically acceptable carrier" refers to all components of the vaccine composition of the application other than the circovirus antigen, a carrier or diluent which does not stimulate the formation of antibodies that interfere with the activity and properties of the compounds for which it is employed, and a diluent preferably an adjuvant. The term "adjuvant" can include aluminium hydroxide adjuvant; saponins such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives selected from the group. The term "emulsion" can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from the oligomerization of alkenes, such as squalane or squalene oil, in particular iso-octene or eucalyptene; linear alkyl esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / eucate), glyceryl tri-(octanoate / eucate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearyl esters. The oil is used in combination with an emulsifying agent in order to form the emulsion. The emulsifying agent is preferably a non-ionic surfactant, in particular an ester of sorbitan, an ester of mannide (such as anhydrous mannide oleate), an ester of a fatty glycol, an ester of polyglycerol, an ester of propylene glycol and an ester of oleic, isostearic, ricinoleic or hydroxystearic acid, optionally ethoxylated, and also a polyoxypropylene-polyoxyethylene block copolymer, in particular a Pluronic product, in particular L121. See "The theory and practical application of adjuvants" edited by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94 and "Vaccine" (1997, 15: 564-570) by Todd et al. For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adiuvant approach" by Powell M and Newman M (Plenum Press, 1995) can be used.The term "polymers of acrylic or methacrylic acid" preferably are cross-linked polymers of acrylic or methacrylic acid, in particular cross-linked with polyalkenyl ethers of sugars or polyalcohols, which compounds are known under the name Carbomer (Carbopol, trade name) (Pharm. Europa, 1996, 8(2)). The skilled person can also refer to US patent 2 909 462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, in which at least 3 of the hydroxyl groups have their hydrogen atoms replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves can contain further substituents, such as methyl groups. These products are sold under the name Carbopol (BFGoodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or with allylpentaerythritol. Among these, Carbopol 974P, 934P and 971P can be mentioned, most preferably Carbopol 971P is used. The term "co-polymers of maleic anhydride and alkenyl derivatives" also contemplates co-polymers of maleic anhydride with ethylene, EMA (Monsanto), which polymers dissolve in water to produce an acidic solution, which is neutralized, preferably to physiological pH, in order to produce an adjuvant solution into which the immunogenic, immunizing or vaccine composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc. Preferably, the adjuvant comprises one or several of mineral oil, aluminium gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, co-polymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201 or Gel adjuvant.

[0038] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, allowing the genetic material carried by the vector to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, etc.

[0039] The term "assembly" refers to the process by which structural proteins (e.g., capsid proteins) of a virus or between structural proteins and nucleic acids form regular, particulate structures through various interactions, including the assembly of native virus particles and the assembly of virus-like particles.

[0040] As an embodiment of the present application, in the virus-like particle vaccine of the present application, the PCV3 virus-like particle antigen content is ≥ 30 μg / ml.

[0041] As an embodiment of the present application, in the virus-like particle vaccine of the present application, the PCV3 virus-like particle antigen content is 30 μg / ml to 100 μg / ml.

[0042] As an embodiment of the present application, in the virus-like particle vaccine of the present application, the PCV3 virus-like particle antigen content is 50 μg / ml.

[0043] In the virus-like particle vaccine of the present application, the PCV3 virus-like particle antigen content can also be selected from 30 μg / ml to 50 μg / ml, or 50 μg / ml to 100 μg / ml.

[0044] In the virus-like particle vaccine of the present application, the PCV3 virus-like particle antigen content is 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml or 100 μg / ml.

[0045] In some embodiments, the pharmaceutically acceptable carrier includes at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

[0046] Preferably, the adjuvant comprises one or several of aluminium hydrogel adjuvant, saponin, Afutidine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, Monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide.

[0047] In some embodiments, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably from 30% V / V to 60% V / V, more preferably 50% V / V.

[0048] In some embodiments, the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine or an amino acid.

[0049] Preferably, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin 2.

[0050] To prepare such a vaccine, methods known in the art can be used.

[0051] The vaccine according to the application can be prepared in an oral or non-oral dosage form.

[0052] Preferably, the non-oral dosage form is administrable by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.

[0053] The application also relates to the use of the virus-like particle antigen for the preparation of a medicament for the prevention of a porcine circovirus-related disease, wherein the porcine circovirus-related disease is a disease caused by an infection with porcine circovirus type 3.

[0054] As an embodiment of the application, the use for the preparation of a medicament for the prevention of a porcine circovirus-related disease is the use for the preparation of a medicament for the prevention of postweaning multisystemic wasting syndrome, porcine dermatitis nephropathy syndrome, proliferative haemorrhagic pneumonia, reproductive disorders and inflammatory reactions of the heart and multiple systems in pigs.

[0055] The term "porcine circovirus-related disease" as used in the present application refers to a disease caused by an infection with porcine circovirus type 3. Without being limited thereto, this includes porcine dermatitis nephropathy syndrome, reproductive disorders and inflammatory reactions of the heart and multiple systems in pigs.

[0056] The term "prevention" in relation to diseases associated with porcine circovirus type 3 infection refers to inhibiting the replication of porcine circovirus type 3, inhibiting the transmission of porcine circovirus type 3 or preventing porcine circovirus type 3 from inhabiting in its host, and alleviating the symptoms of diseases or conditions of porcine circovirus type 3 infection.

[0057] The advantages and characteristics of the present application will become more apparent with the description of specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions all fall within the protection scope of the present application.

[0058] The chemical reagents used in the embodiments of the present application are all of analytical purity and purchased from the National Pharmaceutical Group. The experimental methods described in the present application are all conventional methods unless otherwise specified; and the biological materials described in the present application can be obtained from commercial channels unless otherwise specified.

[0059] Example 1 Amino acid substitution of NLS region of PCV3 Cap

[0060] 1.1 Gene synthesis

[0061] According to the codon optimization of the Cap gene sequence of PCV3 in GenBank (GenBank: UYE91634.1), the gene synthesis was entrusted to Goldengene Biotechnology Co., Ltd.

[0062] 1.2 Primer design

[0063] According to the optimized sequence, the upstream and downstream primers of Cap (12R-K) were designed and synthesized by Goldengene Biotechnology Co., Ltd.

[0064] 1.3 Strain construction

[0065] The PCV3 Cap (12R-K) gene fragment (SEQ ID NO. 1) with enzyme digestion sites at both ends was obtained by PCR amplification, and after gel recovery, PCV3 Cap (12R-K) and pET28a vector were double-digested with NcoI / XhoI, and after gel recovery, they were connected overnight. The connection product was transformed into E. coli competent DH5α cells, and positive clones were screened to extract plasmids. The recombinant plasmid with correct sequencing was named pET28a-PCV3 Cap (12R-K).

[0066] 1.4 Protein expression and purification

[0067] The four recombinant plasmids were transformed into E. coli competent BL21(DE3) cells (kanamycin resistant), and single colonies were selected and cultured at 37°C to OD600=1. The target protein expression was induced by adding 0.5 mM final concentration of chemical inducer isopropyl thiogalactoside (IPTG) at 28°C, and an uninduced group without IPTG was set as a control. After 28°C, 16-20 h induction, the bacterial cells of the induced group and the uninduced group were collected, resuspended in 20 mM PB, 500 mM NaCl, pH 8.0, and broken by high pressure for 3-5 times at 800 bar. The supernatant was centrifuged and subjected to SDS-PAGE analysis. Compared with the supernatant band of the uninduced group, a clear target band of 27 kD appeared in the supernatant of the induced group, which was consistent with the expectation, indicating that the 12R-K substitution of Cap achieved soluble expression. The supernatant was added to a final concentration of 40% saturated ammonium sulfate, and the precipitate was collected by centrifugation after stirring at 4°C for 30 min. The precipitate was resuspended in 20 mM PB, 500 mM NaCl, pH 8.0, and the resuspension solution was centrifuged to obtain the supernatant, which was filtered through a 0.45 μm filter membrane to remove fine impurities. The filtered supernatant was subjected to routine nickel column affinity chromatography purification; the wash buffer was 50 mM imidazole, 20 mM PB, 500 mM NaCl; the elution buffer was 500 mM imidazole, 20 mM PB, 500 mM NaCl; and the eluate was dialyzed against dialysis buffer (20 mM PB, 500 mM NaCl, pH 8.0) at 4°C overnight to obtain the target protein, which was subjected to SDS-PAGE analysis. The purity of the purified protein was about 80%, and the protein concentration was adjusted to 0.25 mg / mL after BCA quantification.

[0068] 1.5 Negative staining transmission electron microscopy detection

[0069] The PCV3 Cap (12R-K) protein was diluted with ddH2O to a concentration of 0.1 mg / mL, and 10 μl of the protein was pipetted onto a clean plastic film. The carbon-coated copper grid after discharge was placed in the protein solution for 2 min, and then gently absorbed with filter paper, followed by two gentle washes with double-distilled water, absorption with filter paper, incubation with 2% uranyl acetate for 2 min, and natural air drying. The stained protein sample was observed by transmission electron microscopy to determine the size and morphology of the particles, and the results are shown in Figure 1 . It can be seen that the number of particles of PCV3 Cap (12R-K) in the field of view is small, and the particles are in aggregate form. This indicates that although PCV3 Cap (12R-K) improves the protein expression amount, it cannot improve the protein assembly problem.

[0070] Example 2 Structure prediction of PCV3 Cap (selective AI substitution)

[0071] The structure of PCV3 Cap (12R-K) amino acid sequence (SEQ ID NO. 2) was predicted using the structure prediction tool Alpha-Fold, and then compared. Then, using pymol software, the predicted structure of PCV3 Cap (12R-K) and the published PCV2 Cap structure prediction map were compared and analyzed. It was found that PCV2 Cap and PCV3 Cap (12R-K) structures mainly exist in three different regions. The largest different region is the additional beta sheet of PCV2 Cap, with amino acid positions 82-89aa (PGGGSNPR), and the corresponding PCV3 Cap (12R-K) amino acid positions are 75-76aa (AI) Figure 3 ). Therefore, it is speculated that replacing the amino acid AI of PCV3 Cap (12R-K) with PGGGSNPR can promote the assembly of Cap protein without changing the immunogenicity of Cap protein. Figure 3 In the figure, the green part is PCV2 Cap, the gold part is PCV3 Cap, the yellow part is the additional beta sheet of PCV2, and the red part is the additional alpha helix and beta sheet of PCV3.

[0072] Example 3 Expression and purification of AI-replaced PCV3 Cap (12R-K)

[0073] 3.1 Primer design

[0074] According to the sequence of PCV3 Cap (12R-K) after AI replacement, a combination of overlapping PCR primers was designed, and the primers were synthesized by Jinweizhi Company.

[0075] 3.2 Strain construction

[0076] Through two rounds of amplification by overlapping PCR, the PCV3 Cap (12R-K) gene fragment with AI replacement at both ends was obtained. The improved-PCV3 Cap (12R-K) gene fragment with AI replacement was recovered by gel recovery, and then digested with NcoI / XhoI. After gel recovery, the product was connected overnight, and the ligation product was transformed into E. coli competent DH5a cells. Positive clones were selected, and the recombinant plasmid with correct sequencing was named PET28a-PCV3 Cap (12R-K)-AIr.

[0077] 3.3 Protein expression and purification

[0078] The recombinant plasmid was transformed into E. coli competent BL21(DE3) cells (kanamycin resistant), and a single colony was selected and cultured at 37°C to OD600=1. At 28°C, 0.5 mM of the chemical inducer isopropyl thiogalactoside (IPTG) was added to induce the expression of the target protein. At the same time, an uninduced group without IPTG was set as a control. After 28°C, 16-20 h of induction, the bacterial cells of the induced group and the uninduced group were collected, resuspended in 20 mM PB, 500 mM NaCl, pH 8.0, and broken by high pressure for 3-5 times at 800 bar. The supernatant was centrifuged and subjected to SDS-PAGE analysis. Compared with the supernatant band of the uninduced group, a clear target band of 27 kD appeared in the supernatant of the induced group, which was consistent with the expectation, indicating that PCV3 Cap(12R-K)-AIr was soluble expressed. The supernatant of the induced group was added to a final concentration of 40% saturated ammonium sulfate, and the precipitate was collected by centrifugation after stirring at 4°C for 30 min. The precipitate was resuspended in 20 mM PB, 500 mM NaCl, pH 8.0, and the resuspension solution was centrifuged to obtain the supernatant. The supernatant was filtered through a 0.45 μm filter membrane to remove fine impurities, and the filtered supernatant was subjected to routine nickel column affinity chromatography purification; the wash buffer was 50 mM imidazole, 20 mM PB, 500 mM NaCl; the elution buffer was 500 mM imidazole, 20 mM PB, 500 mM NaCl; the eluate was collected and dialyzed against dialysis buffer (20 mM PB, 500 mM NaCl, pH 8.0) at 4°C overnight to obtain the target protein, which was subjected to SDS-PAGE analysis. The purity of the purified protein was about 85%, and the protein concentration was adjusted to 0.30 mg / mL after BCA quantification.

[0079] Example 4 AI replacement PCV3 Cap(12R-K) VLPs assembly

[0080] 4.1 Nanoparticle size instrument particle size detection

[0081] After the nanoparticle size instrument was opened and balanced for 30 min, the detection temperature was set to 25°C. 500 μL of sample buffer (20 mM PB, 500 mM NaCl, pH 8.0) was used to wash the cuvette twice, and then 500 μL of PCV3 Cap(12R-K)-AIr protein solution was used to wash the cuvette twice. Finally, 1.0 mL of PCV3 Cap(12R-K)-AIr protein solution was added for particle size detection. The protein was stored at 4°C after sterile filtration, and the particle size was detected at 7 days, 14 days, and 28 days. The detection results are shown in Table 1. In Table 1, Z-Average refers to the average particle size of the particles in the solution, and PDI is the polydispersity coefficient, which represents the uniformity of the particles. The smaller the PDI value, the more uniform the particles. The particle size results show that the modified Cap particles are uniform and have good stability.

[0082] Table 1 Sample Day of purification 7 days at 4°C 14 days at 4°C 28 days at 4°C Z-Average 23.3 nm 23.9 nm 24.2 nm 26.6 nm PDI 0.156 0.161 0.189 0.239

[0083] 4.2 Negative staining transmission electron microscope detection

[0084] PCV3 Cap (12R-K)-AIr protein was diluted to a concentration of 0.1 mg / mL with ddH2O, 10 μl of the protein was pipetted onto a clean plastic film, and the carbon-coated copper grid after discharge was placed in the protein solution for 2 min, and then gently absorbed with filter paper, and then gently washed twice with double distilled water, and then absorbed with filter paper, and then incubated with 2% uranyl acetate for 2 min, and then naturally air-dried. The protein sample after staining was observed by transmission electron microscope for the size and morphology of the particles, and the results are shown in Figure 2 It can be seen that Cap (12R-K)-AIr forms VLPs with a diameter of about 20 nm in the field of view, the number of particles is large, and the dispersity is good, which indicates that the AI replacement modification can improve the assembly efficiency of Cap.

[0085] Example 5 Preparation of PCV3 Cap subunit vaccine

[0086] The Cap (12R-K)-AIr protein solution was slowly added to the adjuvant, and the emulsifier was continuously stirred at a speed of 800 rpm for 12 min during the addition process. The adjuvant suitable for the present application can be known to those skilled in the art. In the present application, the adjuvant selected is a biphasic adjuvant (water-in-oil-in-water emulsion), which can be, for example, SEIWEI 201 adjuvant. The specific formula is shown in Table 2.

[0087] Table 2 PCV3 virus-like particle vaccine ratio Component Vaccine 1 Vaccine 2 Vaccine 3 PCV3 antigen (pg / ml) 30 50 100 Biphasic adjuvant (V / V %) 50％ 50％ 50％

[0088] Example 6 Immunogenicity test of PCV3 Cap subunit vaccine

[0089] Sera were collected from 21-27 day-old experimental pigs and tested for anti-PCV3 antibodies using an ELISA kit, all of which were negative. 20 antibody-negative pigs were randomly divided into 4 groups, 5 pigs per group, and immunized with PCV3 subunit vaccine. The 1st-3rd groups of piglets were immunized with vaccines 1-3, respectively, and the 4th group of piglets was not immunized as a control group. Each immunized group of piglets was injected with 2 ml of vaccine per pig, and the control group of piglets was injected with 2 ml of normal saline per pig. At 21 days after immunization, the same vaccine was used for 2 times of immunization at the same dose, and serum samples were collected from the experimental pigs 14 days after the 2nd immunization (35 days after the 1st immunization). The antibody level was detected using an indirect ELISA kit, and the antibody detection results are shown in Table 3.

[0090] Table 3 PCV3 virus-like particle vaccine antibody detection results Note: OD of negative sample 450 Average = 0.069

[0091] According to the judgment standard of the results of the classical indirect ELISA, P / N < 1.6 is antibody negative; 2.1 > P / N ≥ 1.6 is antibody suspicious; and P / N ≥ 2.1 is antibody positive. According to the above judgment standard, the immune vaccine group can reach the standard of antibody positive after the second immunization, the antibody positive rate is 100%, and the P / N value is > 10, indicating that the level of the produced antibody is high.

[0092] At the same time, 21 days after the second immunization, blood was collected from each group for challenge, and the challenge dose was Porcine Circovirus type 3, strain SG (Porcine Circovirus type 3, strain SG, preserved in China Center for Type Culture Collection, preservation number CCTCC NO. V201712, preservation date March 23, 2017, preservation address: Wuhan, Wuhan University, China), 10 5.0 TCID50 / head, after challenge, the piglets in each group were continuously observed, and all the test pigs were killed after 25 days, and the determination was made according to the clinical symptoms, pathological changes and virus detection results of the piglets in each group. The specific results are shown in Table 4.

[0093] Table 4 PCV3 subunit vaccine immunogenicity challenge test results

[0094] The results show that after the PCV3 subunit vaccine is immunized to piglets, 100% (5 / 5) protection can be provided for the piglets, and all the piglets in the challenge control group are sick after challenge. It is shown that the PCV3 subunit vaccine provided by the application has good protection.

[0095] The above only describes the preferred embodiments of the present application, and does not make any form of limitation on the present application. Although the present application has been disclosed as the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A Cap protein of porcine circovirus type 3 (PCV3), wherein the NLS region amino acids of the Cap protein have a 12R-K substitution and a 75-76aa (AI) substitution with PGGGSNPR.

2. The Cap protein according to claim 1, wherein, The amino acid sequence of the Cap protein is shown in SEQ ID NO.

4.

3. A PCV3 virus-like particle antigen, wherein the PCV3 virus-like particle antigen is the Cap protein as described in claim 1 or 2.

4. A biomaterial related to the Cap protein of claim 2, said biomaterial comprising any one of the following: (1) The nucleic acid fragment encoding the Cap protein; (2) An expression cassette containing the nucleic acid fragments in (1); (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2); (4) Recombinant cells containing the nucleic acid in (1), the expression cassette in (2), or the vector in (3).

5. The biomaterial according to claim 4, wherein, The nucleotide sequence of the nucleic acid fragment encoding the Cap protein is shown in SEQ ID NO.

3.

6. The use of the PCV3 virus-like particle antigen according to claim 3 in the preparation of a medicament for the prevention of porcine circovirus-related diseases.

7. A PCV3 virus-like particle vaccine, wherein, The virus-like particle vaccine comprises an immunizing dose of the PCV3 virus-like particle antigen and a pharmaceutically acceptable carrier.

8. The PCV3 virus-like particle vaccine according to claim 7, wherein, The content of the PCV3 virus-like particle antigen is ≥30 μg / ml; Preferably, the content of the PCV3 virus-like particle antigen is 30 μg / ml to 100 μg / ml.

9. The PCV3 virus-like particle vaccine according to claim 7, wherein, The pharmaceutically acceptable carriers include at least one of adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

10. The PCV3 virus-like particle vaccine according to claim 9, wherein, The adjuvants include one or more of the following: aluminum gel adjuvant, saponins, avrididine, DDA, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin of Escherichia coli, cholera toxin, IMS1314, and muramyl dipeptide. Preferably, the concentration range of the adjuvant is from 5% V / V to 60% V / V, more preferably from 30% V / V to 60% V / V, and even more preferably 50% V / V; Preferably, the freeze-drying protectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids; Preferably, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

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