Design and preparation method of PPV-PCV2 nanoparticle vaccine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a PPV-PCV2 nanoparticle vaccine design and a preparation method. According to the invention, PPV1VP2 protein is used as a skeleton, a SpyTag peptide fragment is inserted into a loop 2 region of the VP2 protein, and the fusion protein is expressed by using an insect cell baculovirus system and is marked as PPV-ST; a plurality of B cell epitopes of Cap protein of PCV2 and a plurality of T cell antigen epitopes of Rep protein of PCV2 are connected in series, a SpyCatcher peptide fragment is fused at an N end, and expression is performed by using a prokaryotic expression system and is marked as SC-PCV2. Finally, the PPV-ST protein and the SC-PCV2 protein are subjected to covalent binding assembly, the nanoparticle vaccine is prepared, and the obtained PPV-PCV2 vaccine has good immunogenicity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a PPV-PCV2 nanoparticle vaccine design and preparation method. Background Art

[0002] Porcine parvovirus (PPV) is the main pathogen causing SMEDI, the clinical symptoms of which are recurrent estrus, abortion, and mummification or stillbirth in sows. Porcine circovirus (PCV) is the main pathogen causing postweaning multisystemic wasting syndrome (PMWS), which refers to postweaning diarrhea, dyspnea, weight gain failure, anemia and jaundice, and wasting disease in which pigs cannot thrive. A large amount of research evidence and field surveys have found that PCV2 and PPV co-infection exists in pigs and wild boars in different countries and regions. Among them, PPV infection can provide better replication conditions for PCV2 infection, so the co-infection of these two viruses can aggravate the clinical symptoms of PMWS and other diseases, seriously affecting the fertility and survival rate of pigs, thereby restricting the economic development of the pig industry. At present, the prevention and control measures for PPV and PCV2 are mainly single vaccine vaccination. In contrast, combined vaccines have the advantages of reducing immunization procedures, reducing vaccination costs, and providing more protective efficacy. At present, the two-in-one vaccines for these two viruses are still in the research and development stage, mainly including two-in-one inactivated vaccines and two-in-one nucleic acid vaccines, but these two types of vaccines have problems such as biosafety and strong side effects. Therefore, it is of great significance to develop a safer new PPV and PCV2 two-in-one vaccine.

[0003] Soluble antigens are easily hydrolyzed by proteases due to their small size (<10nm), and cannot stimulate the body to produce a good immune response. Therefore, these small protein antigens are often coupled with nanoparticles to improve their immunogenicity. Virus-like particles (VLPs) are a natural nanoparticle with a diameter of 20 to 100nm, which can improve the immunogenicity of heterologous antigens displayed on VLPs. At present, the main methods for displaying antigens on the surface of virus-like particles are chemical methods and gene fusion methods, each of which has its advantages and disadvantages. Among them, the use of chemical cross-linking agents can form a covalent connection between antigens and reactive amino acid side chains in VLPs, but there may be multiple reactive sites in the antigen, resulting in unpredictable binding between antigens and VLPs, and the nanoparticles formed by this coupling method cannot achieve a uniform display effect. Single antigen epitope genes can generally be successfully fused to the surface of VLPs, but the capacity of VLPs to accommodate peptide segments is limited, and even if the chimerism is successful, the chimeric virus-like particles are usually unstable.

[0004] PPVVP2 protein can self-assemble to form virus-like particles. This property can be used as an antigen carrier to carry foreign genes into the body. However, the use of gene fusion methods to directly embed antigens on the surface of virus-like particles may result in unsuccessful self-assembly. The SpyTag-SpyCatcher system is an emerging protein coupling method that can covalently link antigens to virus-like particles to form virus-like particles with surface-displayed antigens, which has the advantage of being able to couple larger antigens. SpyTag and SpyCatcher in the SpyTag-SpyCatcher system can form high-yield and irreversible isopeptide bonds under commonly used protein buffer conditions (including pH, temperature or buffer, etc.). Moreover, the SpyTag peptide segment contains only 13 amino acid residues, so the fusion of this peptide segment generally does not hinder protein expression or polypeptide folding. Therefore, if a two-unit subunit vaccine for PPV and PCV2 co-infection can be constructed based on the SpyTag-SpyCatcher system, it will be of great significance for the prevention and control of PPV and PCV2. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present invention constructs a virus-like particle vaccine that displays the main antigenic epitopes of PCV2 on the surface of PPV VLPs based on the SpyTag-SpyCatcher system, laying a research foundation for the development of candidate bivalent subunit vaccines for preventing and treating mixed infections of PPV and PCV2.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a PPV-PCV2 nanoparticle vaccine design and preparation method, comprising the following steps:

[0008] S1. Preparation of PPV-ST recombinant protein: inserting SpyTag peptide or SpyCatcher peptide into loop2 region of PPVVP2 to construct recombinant protein, constructing the obtained recombinant protein into recombinant plasmid, transfecting the recombinant plasmid and baculovirus genome plasmid into insect cells to construct recombinant baculovirus, wherein the baculovirus genome plasmid includes Bacmid plasmid, and the insect cells include Sf-βX cells, and finally purifying PPV-ST recombinant protein from cells infected with recombinant baculovirus;

[0009] S2. Preparation of SC-PCV2 recombinant protein: 4-6 B cell epitopes of PCV2 ORF2, 1-3 T cell epitopes of ORF1 and 1-3 T cell epitopes of ORF3 are selected to construct recombinant protein; antigenic epitopes are connected by Linker1, and the amino acid residues of Linker1 include "KK" or "KKK"; SpyCatcher peptide segment or SpyTag peptide segment and antigenic epitope are connected by Linker2, and the amino acid residues of Linker2 include GGGSGGGTGGGSGGG; a histidine tag is added to the C-terminus of the amino acid sequence; then the recombinant protein is cloned into a plasmid vector, and the constructed plasmid is transferred into an expression strain, and the expression strain includes a BL21 strain, and a single clone containing the recombinant plasmid is expanded and cultured, and then the SC-PCV2 recombinant protein is obtained by purification;

[0010] S3. PPV-ST protein and SC-PCV2 protein are mixed and covalently assembled to obtain the product.

[0011] The amino acid sequence of PPV-ST is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; the amino acid sequence of SC-PCV2 is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6.

[0012] The present invention constructs a virus-like particle vaccine that displays the main antigenic epitopes of PCV2 on the surface of PPVVLPs based on the SpyTag-SpyCatcher system. At the same time, PPV-PCV2 vaccine was injected into mice intramuscularly at 1, 10, and 20 μg / mouse (low, medium, and high doses), and a single vaccine group and a PBS control group were set up at the same time, and booster immunization was performed 14 days later. Afterwards, the specific IgG antibody titer in the serum of the immunized mice was detected by indirect ELISA, and the level of related cytokines (IL-4 and IFN-γ) secreted by mouse spleen lymphocytes was detected by ELISpot method. PCR and WB identification showed that the recombinant baculovirus was successfully constructed and could express recombinant proteins; SDS-PAGE and WB identification showed that the prokaryotic system successfully expressed recombinant proteins. At the same time, it was found that the covalent assembly product could be specifically recognized by mouse anti-His tag antibodies, indicating that the two proteins could be covalently assembled. The specific IgG antibody titer in the serum of mice in the PPV-PCV2 medium-dose group and the high-dose group was significantly higher than that in the PBS group (P<0.05). The IL-4 levels in the PPV-PCV2 medium-dose group and high-dose group were significantly higher than those in the PBS group (P<0.05). The above results showed that the porcine parvovirus-porcine circovirus bivalent subunit vaccine prepared based on the SpyTag / SpyCatcher system had good immunogenicity.

[0013] Preferably, in S1, the recombinant protein is constructed into a recombinant plasmid using a plasmid vector, and the plasmid vector includes a pOET1 vector.

[0014] Preferably, in S2, the recombinant protein is cloned into an expression vector, and the expression vector includes a pET-28a expression vector.

[0015] Preferably, in S1, after transfection, the virus can be collected when pathological changes in Sf-βX cells are observed, and the virus can be expanded and cultured and then infected and expressed, and then the PPV-ST recombinant protein can be purified.

[0016] More preferably, the purification method of the PPV-ST recombinant protein is as follows: collecting the expressed culture supernatant, resuspending the cell pellet with a buffer after centrifugation, ultrasonically lysing the cells, collecting the lysate supernatant after centrifugation again, precipitating the target protein with a saturated ammonium sulfate solution, stirring overnight and collecting the precipitate by centrifugation, dissolving the precipitate with PBS, and using a desalting column to remove excess ammonium sulfate to obtain a protein sample. Further, the purification method of the PPV-ST recombinant protein is as follows: collecting the expressed culture supernatant, resuspending the cell pellet with a buffer after centrifugation, ultrasonically lysing the cells, collecting the lysate supernatant after centrifugation again, precipitating the target protein with a saturated ammonium sulfate solution, stirring overnight and collecting the precipitate by centrifugation, dissolving the precipitate with PBS, and using a desalting column to remove excess ammonium sulfate to obtain a protein sample.

[0017] Preferably, in S2, the specific operation of the expanded culture is: picking a single clone containing the recombinant plasmid to culture in LB containing Kana at 37°C overnight, and then adding IPTG inducer to induce expression. More preferably, the specific operation of the expanded culture is: picking a single clone containing the recombinant plasmid to culture in LB containing Kana at 37°C overnight, and then expanding the culture at a ratio of 1:100, and culturing at 37°C until OD 600 The concentration of IPTG inducer was 0.5-0.7, and then IPTG inducer was added to a final concentration of 0.4-0.6mM, and finally cultured at 16°C overnight with shaking.

[0018] More preferably, the method for purifying the SC-PCV2 recombinant protein is as follows: centrifuging the culture after expansion and discarding the supernatant, adding lysis solution to the obtained bacterial precipitate to resuspend the bacteria, breaking the bacteria, centrifuging the obtained lysis supernatant, and then purifying the target protein.

[0019] Preferably, in S3, an adjuvant is added after the PPV-ST protein and the SC-PCV2 protein are covalently assembled.

[0020] More preferably, the adjuvant includes but is not limited to ISA201 mineral oil.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] In order to prepare a bivalent subunit vaccine of porcine parvovirus-porcine circovirus, the present invention uses the VP2 protein of porcine parvovirus type 1 (PPV1) as a skeleton, inserts the SpyTag peptide segment into the loop 2 region of the VP2 protein, and uses an insect cell baculovirus system to express the fusion protein, which is recorded as PPV-ST; at the same time, multiple B cell epitopes of the Cap protein of porcine circovirus type 2 (PCV2) and multiple T cell antigen epitopes of the Rep protein are connected in series, and the SpyCatcher peptide segment is fused at the N-terminus, and expressed using a prokaryotic expression system, which is recorded as SC-PCV2. Finally, the PPV-ST protein and the SC-PCV2 protein are purified and incubated at 25°C for 6 hours at a molar ratio of 1:1 to enable covalent binding and assembly, and ISA 201 mineral oil is used as an adjuvant to prepare them into a PPV-PCV2 vaccine. The porcine parvovirus-porcine circovirus bivalent subunit vaccine constructed by the present invention based on the SpyTag-SpyCatcher system not only retains the conformation of virus-like particles, but also can display multiple PCV2 antigenic epitopes on the surface of PPVVLPs, and can induce the body to produce a cellular immune response by inducing a Th2 type reaction. It has good immunogenicity and provides a new idea for the development of combined subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of identification of PPV-ST recombinant baculovirus; A: PCR identification (M: DNA marker; 1: PPV-ST gene amplification); B: Western Blot identification (M: protein marker; 1: PPV-ST protein; 2: Sf-βX cell negative control);

[0024] Figure 2 The SDS-PAGE and electron microscopy analysis results of purified PPV-ST; left: M: protein marker; 1: PPV-ST protein; right: transmission electron microscopy observation results;

[0025] Figure 3 The results of SDS-PAGE and electron microscopy analysis of purified ST-PPV; A: M: protein marker; 1: ST-PPV protein; B: transmission electron microscopy observation results;

[0026] Figure 4 The results of purified SDS-PAGE identification of SC-PCV2 / 1 and SC-PCV2 / 2 proteins; M: protein marker; 1, 2, 3: SC-PCV2 / 1 protein; 4, 5: SC-PCV2 / 2 protein;

[0027] Figure 5Identification results of PPV-PCV2 covalent assembly; A: SDS-PAGE analysis (M: protein marker; 1: PPV-ST; 2: SC-PCV2 / 1; 3: covalent assembly); B: Western Blot analysis (M: protein marker; 1: PPV-ST; 2: SC-PCV2 / 1; 3: covalent assembly); C: Transmission electron microscopy results;

[0028] Figure 6 Identification results of ST-PPV-PCV2 / 1 covalent assembly; M: protein marker; 1: ST-PPV and SC-PCV2 / 1 covalent assembly;

[0029] Figure 7 is the specific antibody titer of mouse serum after immunization;

[0030] A: PPV-ST specific antibody titer; B: SC-PCV2 specific antibody titer.

[0031] Figure 8 The numbers of IL-4 and IFN-γ cytokines secreted by mouse spleen cells; A: IL-4 secreted by PPV-ST; B: IL-4 secreted by SC-PCV2; C: IFN-γ secreted by PPV-ST; D: IFN-γ secreted by SC-PCV2. Compared with the blank group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0034] Design and preparation method of a PPV-PCV2 nanoparticle vaccine

[0035] 1. Materials

[0036] 1.1. Strains and plasmids

[0037] Sf-βX cells (Sf9 insect cells without rhabdovirus, obtained by monoclonal screening from Super Sf-a cells in Patent CN104087549B) were isolated and preserved by our company. flashBAC TM Kits were purchased from Oxford Expression Technologies; Escherichia coli DH5α competent cells and Escherichia coli BL21(DE3) competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0038] 1.2. Reagents and Instruments

[0039] The KOD-Plus-Neo PCR kit was purchased from TOYOBO; the Ezup column viral DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Ni Sepharose packing was purchased from Cytiva; the Elispot kit was purchased from CTL; the baculoFECTIN II transfection reagent was purchased from Oxford Expression Technologies; the ultrasonic cell disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; the inverted microscope was purchased from Guangzhou Mingmei Optoelectronic Technology Co., Ltd.; the medium and high pressure chromatography system was purchased from BIO-ROD; the gel imaging analysis system was purchased from Beijing Saizhi Chuangye Technology Co., Ltd.

[0040] 1.3. Experimental Animals

[0041] SPF-grade BALB / c mice, female, 7 weeks old, were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. License number: SCXK(Yue) 2022-0063.

[0042] 2. Test Methods and Results

[0043] 2.1. Expression and Purification of PPV-ST / ST-PPV Recombinant Proteins

[0044] A foreign gene of a certain size can be inserted into the loo2 of PPV VP2 and the non-essential region at the N-terminus. Inserting a foreign gene at these sites will not affect the assembly of virus-like particles. For this purpose, the SpyTag peptide was inserted into the loop2 region or the N-terminus of PPV VP2, and vectors were constructed and expressed respectively to obtain PPV-ST (loop2 region) protein and ST-PPV (N-terminus) protein.

[0045] 2.1.1. Construction, Expression and Purification of PPV-ST Vector

[0046] (1) Vector Construction

[0047] The SpyTag peptide was inserted into the loop 2 region of PPVVP2 (denoted as PPV-ST). After the amino acid sequence was determined, the gene sequence was synthesized by Changzhou Jiyu Biotechnology Co., Ltd., and the PPV-ST sequence was inserted into the pOET1 vector (purchased from Oxford Expression Technologies) to obtain the recombinant plasmid pOET1-PPV-ST.

[0048] PPV-ST amino acid sequence (SEQ ID NO: 1):

[0049] MSENVEQHNPINAGTELSATGNESGGGGGGGGGRGAGGVGVSTGTFNNQTEFQYLGEGLVRITAHASRLIHLNMPEHETYKRIHVLNSESGVAGQMVQDDAHTQMV TPWSLIDANAWGVWFNPADWQLISNNMTEINLVSFEQAIFNVVLKTITESATSPPTKIYNNDLTASLMVALDTNNTLPYTPAAPRSETLGFYPWLPTKPTQYRYYL SCIRNLNP PT GGGGSAHIVMVDAYKPTKGGGGSTDSIQTGLHSDIMFYTIENAVPIHLLRTGDEFSTGIYHFDTKPLKLTHSWQTNRSLGLPPKLLTEPTTEGDQHPGTLPAANTRKGYHQTINNSYTEATAIRPAQVGYNTPYMNFEYSNGGPFLTPIVPTANTQYNDDEPNGAIRFTMDYQHGHLTTSSQELERYTFNP QSKCGRAPKQQFNQQAPLNLENTNNGTLLPSDPIGGKSNMHFMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPFVCKNNPPGQLFVKIAPNLTDDFNADSPQQPRIITYSNFWWKGTLTFTAKMRSSNMWNPIQQHTTTAENIGNYIPTNIGGIRMFPEYSQLIPRKLY. (The underline is loop 2 area, the wavy line is the SpyTag peptide).

[0050] PPV-ST nucleotide sequence (SEQ ID NO: 2):

[0051] ATGTCCGAAAACGTGGAGCAGCACAACCCTATCAACGCTGGTACCGAACTGAGCGCCACCGGAAACGAGAGCGGTGGTGGTGGTGGAGGTGGTGGTGGCCGTGGTGCTGGTGGTGTGGGTGTGTCCACTGGAACTTTCAACAACCAGACTGAATTCCAGTACCTGGGTGAGGGCCTGGTGCGTATCACCGCTCATGCTTCCCGCCTGATCCACCTGAACATGCCCGAGCACGAGACTTACAAGCGCATCCACGTGCTGAACTCCGAGTCCGGTGTGGCTGGACAGATGGTGCAGGACGACGCTCACACCCAGATGGTGACTCCTTGGAGCCTGATCGACGCCAACGCCTGGGGTGTTTGGTTCAACCCTGCCGACTGGCAGCTGATCTCCAACAACATGACTGAAATCAACCTGGTCAGCTTCGAGCAGGCCATCTTCAACGTGGTCCTGAAGACTATCACTGAATCCGCTACTAGCCCCCCTACCAAGATCTACAACAACGACCTGACTGCCAGCCTGATGGTCGCTCTGGACACTAACAACACTCTGCCTTACACCCCTGCTGCTCCCCGTTCAGAGACTCTGGGTTTCTACCCCTGGCTGCCCACTAAGCCCACTCAGTACCGCTACTACCTG AGCTGCATCCGCAACCTGAACCCTCCTACC

[0052] (2) Construction and identification of recombinant baculovirus

[0053] Using flashBAC TM Kits (purchased from Oxford Expression Technologies) were used to construct Bacmid recombinant baculovirus. Sf-βX insect cells (without rhabdovirus) were plated in six-well plates and cultured in a 27°C incubator using Vigor-S101S insect cell serum-free medium for 4 h. The recombinant plasmid pOET1-PPV-ST and the baculovirus genome plasmid Bacmid (flashBAC ULTRA) were co-transfected into Sf-βX cells using baculoFECTIN II transfection reagent (baculoFECTINII, purchased from Oxford Expression Technologies). After culturing at 27°C for 4 days, the cell lesions were observed. The virus could be collected when cell lesions were observed, and the virus was expanded to the P3 generation. The culture supernatant of the P3 virus was taken, and the viral DNA was extracted using a kit and then PCR reaction was performed using primers, upstream primer (SEQ ID NO: 11): TGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCC, downstream primer (SEQ ID NO: 12): CCAAAAAGCTCAACTACAAATTTGATCGCGGACGTGTTAGCCGACACA, amplification system: PCR Buffer 12.5 μL, dNTPs 5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DNA template 3 μL, Enzyme amplification enzyme 0.5 μL, ddH 2 O 3μL, total volume 20μL; reaction procedure: 94℃, 5min; denaturation: 98℃, 30s, annealing: 60℃, 30s, extension: 68℃, 240s, 30 cycles; 68℃ extension 10min. The amplified product was detected by 1% agarose gel electrophoresis. Take 200μL 1×10 6 The cells / mL of diseased cells were resuspended in PBS, 5× electrophoresis loading buffer was added, and the cells were boiled in a boiling water bath for 10 min. SDS-PAGE electrophoresis was then performed at 140 V for 50 min.

[0054] Using P0 generation PPV-ST recombinant baculovirus DNA as template, PCR identification was performed. The results are as follows Figure 1 As shown in A, the target fragment of 4185 bp was amplified. The infected cells of generation P3 were identified by Western Blot, and a specific protein with a relative molecular mass of about 65 kDa was observed ( Figure 1B) The bands indicate that the PPV-ST recombinant baculovirus was successfully constructed.

[0055] (3) Purification and analysis of PPV-ST recombinant protein

[0056] The cells (generation P3) were collected by centrifugation at 10,000 g / min for 15 min and 25 mM NaHCO 3 (pH 8.3) The cell pellet was resuspended to a density of 2×107 cells / mL, and the cells were lysed by ultrasound, and centrifuged at 15000g / min for 15min. The lysate supernatant was collected, and the target protein was precipitated with a saturated ammonium sulfate solution (pH7.0) with a final concentration of 15%, and stirred slowly at 4°C overnight. The precipitate was collected by centrifugation, dissolved with PBS at a material-liquid ratio of 1:10, and the excess ammonium sulfate was removed using a HiTrap desalting column (purchased from Cytiva). The collected protein samples were subjected to SDS-PAGE electrophoresis to identify the purification effect.

[0057] The purified protein was analyzed by SDS-PAGE. The results showed that the PPV-ST protein was purified by saturated ammonium sulfate with a final concentration of 15%, and the target band was single ( Figure 2 (as shown in the left figure), and the purity can reach more than 90% after analysis by Image J software.

[0058] 5 μL of purified PPV-ST protein was placed on a copper grid, stained with phosphotungstic acid for 5 min, excess solution was removed with filter paper, and air-dried for 15 min. The sample was placed in a transmission electron microscope and photographed at 120 kV.

[0059] Transmission electron microscopy results Figure 2 (Right figure) shows that PPV-ST recombinant protein can self-assemble to form virus-like particles with a diameter of about 25 nm.

[0060] 2.1.2. Expression and purification of ST-PPV recombinant protein

[0061] Referring to the method in step 2.1.1, the SpyTag peptide was inserted into the N-terminus of PPVVP2, and the ST-PPV vector and recombinant baculovirus were constructed first, and ST-PPV was expressed and purified. The purified sample was observed by transmission electron microscopy, and the results were as follows: Figure 4 As shown, the purified ST-PPV protein ( Figure 3 A) can also self-assemble to form virus-like particles with a diameter of about 25 nm ( Figure 3 B).

[0062] Amino acid sequence of ST-PPV (SEQ ID NO: 3):

[0063] MAHIVMVDAYKPTKSENVEQHNPINAGTELSATGNESGGGGGGGGGRGAGGVGVSTGTFNNQTEFQYLGEGLVRITAHASRLIHLNMPEHETYKRIHVLNSESGVAGQMVQDDAHTQMVTPWSLIDANAWGVWFNPADWQLISNNMTEINLVSFEQAIFNVVLKTITESATSPPTKIYNNDLTASLMVALDTNNTLPYTPAAPRSETLGFYPWLPTKPTQYRYYLSCIRNLNPPTTDSIQTGLHSDIMFYTIENAVPIHLLRTGDEFSTGIYHFDTKPLKLTHSWQTNRSLGLPPKLLTEPTTEGDQHPGTLPAANTRKGYHQTINNSYTEATAIRPAQVGYNTPYMNFEYSNGGPFLTPIVPTANTQYNDDEPNGAIRFTMDYQHGHLTTSSQELERYTFNPQSKCGRAPKQQFNQQAPLNLENTNNGTLLPSDPIGGKSNMHFMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPFVCKNNPPGQLFVKIAPNLTDDFNADSPQQPRIITYSNFWWKGTLTFTAKMRSSNMWNPIQQHTTTAENIGNYIPTNIGGIRMFPEYSQLIPRKLY. (The wavy line indicates the SpyTag peptide segment).

[0064] The nucleotide sequence of ST-PPV (SEQ ID NO: 4):

[0065]

[0066] 2.2. Design, vector construction, expression and purification of SC-PCV2 recombinant protein

[0067] (1) SC-PCV2 design and vector construction

[0068] Antigenic epitopes are divided into T cell epitopes (recognized by T cell receptors) and B cell epitopes (recognized by B cells or antibodies) according to their receptors. In this study, the IEDB Analysis Resource online website was used to predict PCV2 ORF2 and ORF1 B cell and T cell epitopes and construct different T cell and B cell combinations.

[0069] Specific method:

[0070] 1) SC-PCV2 / 1: Recombinant proteins were constructed with 5 B cell epitopes of PCV2 ORF2, 2 T cell epitopes of ORF1, and 1 T cell epitope of ORF3. "KKK" or "KK" amino acid residues were used as linkers between antigenic epitopes; the flexible linker sequence "GGGSGGGTGGGSGGG" was used as linker between the SpyCatcher peptide segment and the antigenic epitope, and a 6× histidine tag was added to the C-terminus of the amino acid sequence for protein purification and identification. The obtained amino acid sequence was sent to Changzhou Jiyu Biotechnology Co., Ltd. for synthesis, and the sequence was cloned into the pET-28a vector.

[0071] 2) SC-PCV2 / 2: Recombinant proteins were constructed with 6 B cell epitopes of PCV2 ORF2, 2 T cell epitopes of ORF1, and 1 T cell epitope of ORF3. "KKK" or "KK" amino acid residues were used as linkers between antigenic epitopes; between SpyCatcher and antigenic epitopes, a flexible linker sequence "GGGSGGGTGGGSGGG" was used as a linker, and a 6× histidine tag was added to the C-terminus of the amino acid sequence for protein purification and identification. The obtained amino acid sequence was sent to Changzhou Jiyu Biotechnology Co., Ltd. for synthesis, and the sequence was cloned into the pET-28a vector.

[0072] Table 1 B cell and T cell epitope sequences and their locations in ORF1 / ORF2 / ORF3

[0073]

[0074] Table 2 Different SC-PCV2 combinations

[0075]

[0076]

[0077] SC-PCV2 / 1 amino acid sequence (SEQ ID NO: 5):

[0078]

[0079] (The grey shading indicates SpyCatcher or different cell epitopes).

[0080] SC-PCV2 / 1 nucleotide sequence (SEQ ID NO: 6):

[0081]

[0082] (The grey shading indicates SpyCatcher or different cell epitopes).

[0083] SC-PCV2 / 2 amino acid sequence (SEQ ID NO: 7):

[0084]

[0085] (The grey shading indicates SpyCatcher or different cell epitopes).

[0086] SC-PCV2 / 2 nucleic acid sequence (SEQ ID NO: 8):

[0087]

[0088] (The grey shading indicates SpyCatcher or different cell epitopes).

[0089] SpyCatcher amino acid sequence (SEQ ID NO: 9):

[0090] MDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVET AAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI.

[0091] SpyCatcher nucleic acid sequence (SEQ ID NO: 10):

[0092] ATGGACAGCGCAACCCATATTAAATTTAGCAAACGTGACGAGGACGGCAAAGAACTGGCAGGCGCTACAATGGAACTGCGTGATAGCAGTGGCAAAACCATTAGCACCTGGATTAGTGATGGTCAGGTGAAAGATTTTTA CCTGTATCCGGGCAAATACACCTTTGTGGAAACCGCAGCACCGGATGGCTATGAAGTGGCAACCGCCATTACCTTTACCGTTAATGAACAGGGTCAGGTTACCGTTAATGGCAAAGCCACCAAAGGTGATGCACATATT.

[0093] (2) Expression of SC-PCV2 / 1 or SC-PCV2 / 2 proteins

[0094] The constructed vector was transformed into the BL21 strain, and a single clone containing the recombinant plasmid was picked and cultured overnight at 37°C in LB containing Kana. Then, the culture was expanded at a ratio of 1:100 and cultured at 37°C until OD 600 IPTG inducer was then added to a final concentration of 0.5 mM, and the cells were cultured overnight at 16°C with shaking.

[0095] (3) Purification and identification of SC-PCV2 / 1 or SC-PCV2 / 2 recombinant proteins

[0096] The culture was centrifuged at 12000g for 30 min and the supernatant was discarded. Lysis solution (20mM Tris, 150mM NaCl, pH 8.0) was added to the obtained bacterial precipitate to resuspend the bacteria, and the bacteria were broken by ultrasound. Centrifuged at 12000g for 30 min, the obtained lysis supernatant sample was filtered using a 0.45μm filter membrane. The target protein was purified by affinity chromatography using a nickel column (purchased from Cytiva), and the purified sample was identified by SDS-PAGE electrophoresis.

[0097] like Figure 4 As shown, after purification, the SC-PCV2 / 1 protein has a high purity and a size of about 28 kDa; the molecular weight of the SC-PCV2 / 2 protein is slightly larger, greater than 30 kDa.

[0098] 2.3. Covalent assembly and identification of PPV-ST and ST-PPV proteins with SC-PCV2 / 1 and SC-PCV2 / 2 proteins, respectively

[0099] SC-PCV2 / 1 protein was mixed with PPV-ST and ST-PPV protein at a molar ratio of 1:1, and allowed to stand at 25°C for 6 hours to allow for covalent assembly. 5× electrophoresis loading buffer was added, and the reaction was terminated by boiling in a water bath for 10 minutes. The assembly efficiency was then determined by SDS-PAGE electrophoresis. The PPV-PCV2 covalently linked product was identified by western-blot and observed by transmission electron microscopy. Figure 5 A SDS-PAGE analysis showed that after the SC-PCV2 / 1 protein and PPV-ST protein were mixed and incubated, a protein band with a relative molecular weight of about 92 kDa appeared, indicating that the PPV-ST protein and SC-PCV2 / 1 proteins could effectively assemble into particles. However, after the ST-PPV protein and SC-PCV2 / 1 protein were mixed and incubated, no band appeared at around 92 kDa ( Figure 6 ), indicating that ST-PPV protein and SC-PCV2 / 1 proteins cannot be effectively assembled into particles.

[0100] Since the PCV2 protein has a 6×His-Tag at the end, the covalent assembly product can also be specifically recognized by the 6×His-Tag antibody. Figure 5 The covalent assembly product shown in B was analyzed by Western-blot. Lane 3 showed that the covalent assembly product PPV-PCV2 / 1 protein (92 kDa) could be specifically recognized by the 6×His-Tag antibody. The result was in line with expectations, indicating that the PPV-ST protein and SC-PCV2 / 1 protein could be covalently linked to form the PPV-PCV2 protein. Figure 5 Transmission electron microscopy observations of the covalent assembly products of C show that the products are granular.

[0101] In addition, when SC-PCV2 / 2 was connected with PPV-ST and ST-PPV, respectively, unlike the connection with SC-PCV2 / 1 and PPV-ST, their connection products showed obvious precipitation. After identification, most of the protein was in the precipitation, and only a small amount of protein was in the supernatant, indicating that the connection effect of SC-PCV2 / 2 with PPV-ST or ST-PPV was not good.

[0102] After comprehensive evaluation, the recombinant protein (PPV-PCV2 / 1, referred to as PPV-PCV2 for subsequent immunogenicity evaluation) assembled from SC-PCV2 / 1 protein (referred to as SC-PCV2 for subsequent immunogenicity evaluation) and PPV-ST protein was selected for subsequent immunogenicity evaluation.

[0103] 2.9. Vaccine preparation and immunogenicity evaluation

[0104] The obtained recombinant protein was prepared into a vaccine with ISA 201 mineral oil as adjuvant at a ratio of recombinant protein volume: adjuvant volume = 1:1. 48 BALB / c mice were randomly divided into 8 groups, with 6 BALB / c mice in each group. The vaccine was injected intramuscularly into mice at 1, 10, and 20 μg / mouse (low, medium, and high doses). At the same time, a single vaccine group (PPV-ST, SC-PCV2 / 1) and a PBS control group were set up, and booster immunization was performed 14 days later. On days 0, 14, 21, 28, and 35, blood was collected from the eye sockets and mouse serum was separated. On day 42, blood was collected from the eye sockets and mice were killed, and the spleen was removed. The experimental groups, antigen content, related protein equivalents, immunization doses, and the number of immunized BALB / c mice are shown in Table 3. Among them, PPV-VLPs were prepared by our company in the early stage (similar to the preparation method of ST-PPV mentioned above, using flashBAC TM Kits, construct PPV-VP2 recombinant baculovirus without any exogenous protein sequence, and infect Sf-βX insect cells, and obtain self-assembling VLPs after purification), PCV2 commercial vaccine is porcine circovirus type 2 genetically engineered subunit vaccine (Escherichia coli source) (Veterinary Drug No. 160021128) purchased from Plike Biotechnology Co., Ltd.

[0105] Table 3 Grouping of mouse immunization test

[0106]

[0107] 2.10. Determination of specific antibodies

[0108] The ELISA plate was coated with PPV-ST protein or SC-PCV2 protein and left to stand overnight at 4°C; blocked with 10% newborn calf serum at 37°C for 2h; the blocking solution was discarded, and the mouse serum separated on days 14, 21, 28, and 35 was added, and incubated at 37°C for 1h; washed three times with PBST, and HRP-labeled goat anti-mouse IgG (purchased from Dingguo Changsheng Biotechnology Co., Ltd.) was added, and incubated at 37°C for 1h; washed three times, TMB color development for 20min, and after terminating the reaction with ELISA stop solution, the OD value was read at a wavelength of 450nm on the ELISA reader. Result judgment: If the OD value of the test well is >0.1 and greater than 2.1 times that of the negative control well, it is judged as positive, and the highest dilution multiple of the serum judged as positive is the serum antibody titer.

[0109] The indirect ELISA method was used to detect the PPV-ST and SC-PCV2 specific antibodies in the serum of each group of mice. The detection results of PPV-ST specific antibodies are shown in Figure 7As shown in A, on the 14th day after the first vaccination, the PPV-PCV2 medium-dose group and the PPV-PCV2 high-dose group induced high levels of PPV-ST-specific antibodies, while the low-dose group had low levels of specific antibodies. After booster immunization, the serum antibody level of the low-dose group began to rise slowly, and on the 42nd day, the serum anti-PPV-ST IgG antibody titer of the group reached a maximum of 1:2. The anti-PPV-ST IgG antibody titer of the immune serum of mice in the medium-dose and high-dose groups was at a high level, which was comparable to the antibody level of the PPV-ST single vaccine group, and the highest titer could reach 1:2. 18 and 1:2 20 .

[0110] The results of SC-PCV2 specific antibody detection are as follows Figure 7 As shown in B, after the second immunization, the specific IgG antibody level of the low-dose group remained at a low level, and the antibody titer was negative, indicating that the dose of 1 μg was too low to stimulate the mice to produce specific antibodies; the average titer of the medium-dose group and the high-dose group was at a high level, and the average titer of the two groups was close. The anti-SC-PCV2 IgG antibody titer of mouse immune serum can be as high as 1:2 18 and 1:2 19 , and there was no significant difference compared with the SC-PCV2 single seedling group.

[0111] 2.11. Determination of IL-4 and IFN-γ cytokines

[0112] Treat the mice according to the method in step 2.9. Kill the mice by cervical dislocation. Remove the spleen of the mice under sterile conditions and grind the spleen with a syringe piston. Use lymphocyte separation solution (purchased from Cytiva) to separate spleen lymphocytes and adjust the lymphocyte concentration to 1.0×10 6 / mL, add 100μL to each well of a 96-well plate, and spread 3 wells for each sample. At the same time, set up negative control, positive control and blank control. The cells were spread without protein stimulation as a negative control, the cells were spread with 1μg of concanavalin A (ConA) as a positive control, and the cells were not spread with culture medium as a blank control. 1μg of the corresponding protein was added to each well to stimulate lymphocytes for 48h. The above experimental process was performed according to the instructions of the ELISpot kit, and the ELISpot plate spots were counted using a counting instrument.

[0113] IFN-γ is secreted by Th1 cell subsets, inducing the expression of MHC class II antigens in monocytes, macrophages, dendritic cells, etc., so that they participate in the process of antigen presentation and specific immune recognition, and mainly mediates cellular immunity. IL-4 is secreted by Th2 cell subsets, promoting B cell proliferation, enhancing the ability of B cells to present antigens, and causing the immune system to respond to small amounts of antigen stimulation, and mainly mediates humoral immunity. Figure 8As shown in the figure, after stimulation with PPV-ST protein and SC-PCV2 protein, the number of spots formed by IL-4 secreted by spleen lymphocytes in the medium-dose group, high-dose group and single vaccine group of PPV-PCV2 was significantly higher than that in the PBS negative control group (P < 0.05). However, there was no significant difference in IFN-γ secreted by spleen lymphocytes in all experimental groups compared with the PBS negative control group (P > 0.05). IL-4 is one of the representative cytokines of Th2 type cellular immunity. The above results indicate that the PPV-PCV2 vaccine mainly induces the secretion of Th2 type cytokines in mice and protects the body through the Th2 type cellular immunity pathway.

[0114] In summary, the present invention constructs a virus-like particle vaccine that displays the main PCV2 antigen epitopes on the surface of PPVVLPs based on the SpyTag-SpyCatcher system. The vaccine not only retains the conformation of the virus-like particles, but also can display multiple PCV2 antigen epitopes on its surface, providing a new idea for the development of combined subunit vaccines. At the same time, based on the SpyTag-SpyCatcher system, a rapid vaccine platform can also be established, using PPV virus-like particles as nanoparticle skeletons, fusing SpyCatcher peptide segments at both ends of different antigens, and modularly assembling the two, so that PPV virus-like particle vaccines that display different antigens on the surface can be quickly developed, thereby greatly reducing the time for vaccine development and accelerating the development of virus-like particle vaccines.

[0115] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A design and preparation method of a PPV-PCV2 nanoparticle vaccine, characterized in that: The following steps are involved: S1. Preparation of PPV-ST recombinant protein: inserting SpyTag peptide or SpyCatcher peptide into loop2 region of PPV VP2 to construct recombinant protein, constructing the obtained recombinant protein into recombinant plasmid, transfecting the recombinant plasmid and baculovirus genome plasmid into insect cells to construct recombinant baculovirus, wherein the baculovirus genome plasmid includes Bacmid plasmid, and the insect cells include Sf-βX cells, and finally purifying PPV-ST recombinant protein from cells infected with recombinant baculovirus; S2. Preparation of SC-PCV2 recombinant protein: 4-6 B cell epitopes of PCV2 ORF2, 1-3 T cell epitopes of ORF1 and 1-3 T cell epitopes of ORF3 are selected to construct recombinant protein; antigenic epitopes are connected by Linker1, and the amino acid residues of Linker1 include "KK" or "KKK"; SpyCatcher peptide segment or SpyTag peptide segment and antigenic epitope are connected by Linker2, and the amino acid residues of Linker2 include GGGSGGGTGGGSGGG; a histidine tag is added to the C-terminus of the amino acid sequence; then the recombinant protein is cloned into a plasmid vector, and the constructed plasmid is transferred into an expression strain, and the expression strain includes a BL21 strain, and a single clone containing the recombinant plasmid is expanded and cultured, and then the SC-PCV2 recombinant protein is obtained by purification; S3. PPV-ST protein and SC-PCV2 protein are mixed and covalently assembled to obtain the product.

2. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: The amino acid sequence of PPV-ST is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; the amino acid sequence of SC-PCV2 is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO:

6.

3. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: In S1, the recombinant protein is constructed into a recombinant plasmid using a plasmid vector, and the plasmid vector includes a pOET1 vector.

4. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: In S2, the recombinant protein is cloned into an expression vector, wherein the expression vector includes a pET-28a expression vector.

5. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: In S1, after transfection, the virus can be collected when pathological changes in Sf-βX cells are observed. The virus can be expanded and cultured and then infected and expressed, and then the PPV-ST recombinant protein can be purified.

6. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 5, characterized in that: The purification method of PPV-ST recombinant protein is as follows: collect the expressed culture supernatant, resuspend the cell pellet with buffer after centrifugation, lyse the cells by ultrasonication, collect the lysate supernatant after centrifugation again, precipitate the target protein with saturated ammonium sulfate solution, stir overnight and collect the precipitate by centrifugation, dissolve the precipitate with PBS, and use a desalting column to remove excess ammonium sulfate to obtain a protein sample.

7. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: In S2, the specific operation of the expanded culture is: picking a single clone containing the recombinant plasmid and culturing it in LB containing Kana at 37°C overnight, and then adding IPTG inducer to induce expression.

8. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: The purification method of SC-PCV2 recombinant protein is as follows: the culture after expanded culture is centrifuged and the supernatant is discarded, the lysis solution is added to the obtained bacterial precipitate to resuspend the bacteria, the bacteria are broken, the lysis supernatant obtained after centrifugation is purified, and the target protein is obtained.

9. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 1, characterized in that: In S3, after the covalent assembly of PPV-ST protein and SC-PCV2 protein, an adjuvant was added.

10. The design and preparation method of a PPV-PCV2 nanoparticle vaccine according to claim 9, characterized in that: The adjuvant included ISA 201 mineral oil.

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