Recombinant Seneca virus for expressing porcine circovirus type 3 Cap fusion protein

Abstract

The invention provides a recombinant Seneca virus for expressing a porcine circovirus type 3 Cap fusion protein. The recombinant Seneca virus provided by the invention can efficiently and stably express the Cap protein. The expressed Cap protein is formed by connecting antigen epitope genes in series and is obtained through a prokaryotic expression system, and the truncated Cap protein with high immunogenicity is obtained through immunogenicity identification and screening. An SVA reverse genetic operating system is proved to be an important tool for research and development of genetic engineering vaccines, and the recombinant Seneca virus can be propagated on a large scale in host cells, so that large-scale production of recombinant vaccine antigens can be realized; when an inactivated vaccine prepared from the recombinant Seneca virus is used for immunizing an animal, the animal body can be stimulated to generate a Cap specific antibody and an SVA neutralizing antibody at the same time, and a vaccine candidate strain is provided for preventing the porcine circovirus type 3 and the Seneca virus.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering products, and in particular relates to a recombinant Seneca virus expressing porcine circovirus type 3 Cap fusion protein. Background Art

[0002] Porcine circovirus type 3 (PCV3) is a single-stranded circular DNA virus belonging to the genus Porcine Circovirus of the family Porcine Circoviridae. The genome size is about 2000 nt and contains three major open reading frames (ORFs). ORF2 encodes the capsid protein (Cap) of the virus antigen, which is a key protein for the host immune response. After infection, the main clinical symptoms include weaned pig multisystemic wasting syndrome (PMWS), sow reproductive failure, porcine respiratory syndrome and porcine dermatitis nephropathy syndrome (PDNS). It spreads quickly and has high pathogenicity. At present, there are still no effective measures for the prevention and control of the disease. In addition, due to the great difference in the properties of PCV2 Cap protein and PCV3 Cap protein, the commercialized PCV2 vaccine has no cross-immune protection against PCV3, so the development of a new PCV3 vaccine is crucial.

[0003] Senecavirus A (SVA) is a small RNA virus with a single-stranded positive-strand RNA structure. Infected pigs show blister-like lesions on the hoof and nose, accompanied by clinical symptoms such as lameness, anorexia and lethargy. Newborn piglets will suffer from severe diarrhea, dehydration and even death when they are infected. It is one of the main pathogens causing vesicular syndrome. In 2015, the first outbreak occurred in Guangdong, my country. Subsequently, SVA infection cases were reported in Hubei, Henan, Fujian, Gansu and other provinces, causing great economic losses to my country's pig industry. Currently, there is no commercial vaccine available.

[0004] Currently, vaccines are the most effective prevention and control measures for porcine circovirus type 3 and Seneca virus. Cap protein is an ideal target antigen for the development of PCV3 vaccine, and its antigenic epitope is the core of immune protection. The inventors previously used the Escherichia coli prokaryotic expression system to obtain a soluble truncated Cap protein with strong immunogenicity, but there are still problems such as complex protein purification process, low purification efficiency, and not conducive to large-scale production. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant Senecavirus expressing porcine circovirus type 3 Cap fusion protein and application thereof in preparing porcine circovirus type 3 vaccine.

[0006] The present invention first provides a porcine circovirus type 3 Cap fusion protein, comprising:

[0007] 1) a fusion protein having an amino acid sequence of SEQ ID NO: 3;

[0008] 2) A fusion protein derived from 1) by replacing, deleting or adding one or more amino groups in 1).

[0009] The present invention also provides a nucleic acid fragment for encoding the above fusion protein, a specific sequence of which is SEQ ID NO: 4;

[0010] The present invention also provides a use of the porcine circovirus type 3 Cap fusion protein, which is inserted into the Seneca virus genome as an antigenic polypeptide to prepare a recombinant Seneca virus;

[0011] Another aspect of the present invention provides a recombinant Seneca virus, wherein a nucleic acid fragment encoding the above fusion protein is inserted into the genome of the recombinant Seneca virus;

[0012] Furthermore, the nucleic acid fragment is inserted between the Seneca virus cDNA 2A and 2B genes.

[0013] The present invention also provides a use of the prepared recombinant Seneca virus, which is to use it as an antigen to prepare a vaccine.

[0014] The present invention also provides a virus vaccine, which uses the inactivated recombinant Seneca virus as an antigen.

[0015] The present invention provides a recombinant Senecavirus expressing porcine circovirus type 3 Cap protein, and the provided recombinant Senecavirus can express Cap protein efficiently and stably. The expressed Cap protein is composed of antigen epitope genes in series, and is a truncated Cap protein with high immunogenicity obtained by a prokaryotic expression system and screened by immunogenicity identification. The SVA reverse genetics operating system has been proven to be an important tool for the development of genetic engineering vaccines. The recombinant Senecavirus can be propagated on a large scale in host cells, which is conducive to the large-scale production of recombinant vaccine antigens. Immunizing animals with inactivated vaccines prepared with the recombinant Senecavirus can stimulate the animal body to produce Cap-specific antibodies and SVA neutralizing antibodies at the same time, providing a vaccine candidate strain for the prevention of porcine circovirus type 3 and Senecavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is the SDS-PAGE identification result of the purified Cap-I protein and Cap-II protein in Example 1, wherein M is the protein Maker, lane 1 is the Cap-I protein before purification, lane 2 is the Cap-I protein after purification, lane 3 is the Cap-II protein before purification, and lane 4 is the Cap-II protein after purification;

[0017] Figure 2 This is the identification result diagram of the recombinant plasmid pUC19-Cap-IPCR in Example 2. Lane 1 is the blank control, lane 2 is the amplification product of the Cap-I gene fragment with a size of 491 bp, and M is the BM 2000+ DNA Maker;

[0018] Figure 3 This is a schematic diagram of the construction method of the chimeric truncated PCV3 Cap foreign gene Seneca virus recombinant plasmid pSVA-PCV3-Cap-I in Example 2;

[0019] Figure 4 This is the PCR identification result diagram of the S1 fragment, S2-Cap-T2A fragment, and S3 fragment of SVA in Example 3. Lane 1 is the amplification product of the S1 fragment with a size of 2673 bp, lane 2 is the amplification product of the S2-Cap-T2A fusion fragment with a size of 2616 bp, lane 3 is the amplification product of the S3 fragment with a size of 2524 bp, lane 4 is the blank control, and M is the BM 5000+ DNA Maker;

[0020] Figure 5 This is the cytopathic effect (CPE) diagram caused by the recombinant virus rSVA-PCV3-Cap-I after infecting BHK-21 cells. A is normal BHK-21 cells, B is the cytopathic effect caused by SVA infection, and C is the cytopathic effect caused by rSVA-PCV3-Cap-I infection;

[0021] Figure 6 This is the identification result diagram of the passage stability of the recombinant virus S2-Cap-I-T2A fragment in Example 4. Lane 1 is the blank control, lane 2 is the amplification product of the S2-Cap-I-T2A fragment of the 1st generation recombinant virus, lane 3 is the amplification product of the S2-Cap-I-T2A fragment of the 5th generation recombinant virus, lane 4 is the amplification product of the S2-Cap-T2A fragment of the 10th generation recombinant virus, and lane 5 is the amplification product of the S2-Cap-I-T2A fragment of the 15th generation recombinant virus;

[0022] Figure 7 This is the identification result diagram of the passage stability of the truncated Cap fragment of the recombinant virus in Example 4. Lane 1 is the amplification product of the truncated Cap fragment of the 1st generation recombinant virus, lane 2 is the amplification product of the truncated Cap fragment of the 5th generation recombinant virus, lane 3 is the amplification product of the truncated Cap fragment of the 10th generation recombinant virus, lane 4 is the amplification product of the truncated Cap fragment of the 15th generation recombinant virus, and lane 5 is the blank control;

[0023] Figure 8The indirect immunofluorescence identification result of the recombinant virus rSVA-PCV3-Cap-I in Example 4 is shown, wherein A is the IFA identification result of the inoculated recombinant virus rSVA-PCV3-Cap-I, B is the IFA identification result of the inoculated parental SVA strain, and C is the blank control;

[0024] Fig. 9 This is a diagram showing the results of Western blot analysis of the recombinant virus rSVA-PCV3-Cap-I in Example 4;

[0025] Fig.10 This is a graph showing the results of Cap-specific antibody detection after animals were immunized with the recombinant vaccine in Example 5. DETAILED DESCRIPTION

[0026] The present invention utilizes the SVA reverse genetic operation platform to construct a recombinant plasmid fused with a truncated PCV3 Cap antigen epitope gene, transfects BHK-21 cells, and successfully rescues the recombinant virus expressing the PCV3 Cap protein. The inactivated vaccine is prepared with the recombinant plasmid to immunize animals, which can produce both anti-PCV3 and anti-SVA specific antibodies.

[0027] The construction method and application of a recombinant Seneca virus expressing porcine circovirus type 3 Cap protein described in the present invention are further introduced in detail below in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0028] Example 1: Truncated expression and immunogenicity study of PCV3 Cap protein

[0029] 1. Screening for truncated PCV3 Cap genes

[0030] According to the amino acid sequence of the Cap protein in the PCV3 CN Nanjing 2017 strain (GenBank accession number: MK580468.1) (amino acid sequence is SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is SEQ ID NO: 2), the B cell antigen epitopes, cytotoxic T cell (CTL) antigen epitopes, and helper T cell (Th) antigen epitopes of the PCV3 Cap protein were predicted using the IEDB online website (https: / / www.iedb.org / ) and the ABC pred (http: / / crdd.osdd.net / raghava / abcpred / ) online tool. The fragments with overlapping prediction results and the fragments with high scores were selected as preferred sequences, and the adjacent sequences were merged to screen out 6 dominant B cell antigen epitopes, which were the amino acid sequences of positions 42-60, 71-92, 94-105, 113-160, 169-178, and 189-204. Three dominant CTL antigen epitopes were screened out, which were the amino acid sequences at positions 95-103, 173-182, and 197-207, and one dominant Th antigen epitope was screened out, which was the amino acid sequence DDPYAESSTRKVMTS at positions 123-137.

[0031] According to the prediction results of online tools, the antigen epitopes of different types of cells were analyzed and merged, and the epitopes were analyzed and merged through Linker (EAAAK) n The three antigen epitopes were connected in a certain order to obtain two different amino acid sequences, which were named Cap-I peptide and Cap-II peptide, and their amino acid sequences were shown in SEQ ID NO: 3 and SEQ ID NO: 5, respectively. After analysis by online tools for allergenicity and antigenicity, the results showed that both Cap-I peptide and Cap-II peptide were non-allergenic, and the antigenicity prediction results of Cap-I peptide and Cap-II peptide were relatively close.

[0032] 2. Induction, expression and identification of truncated Cap proteins

[0033] According to the codon preference of Escherichia coli, the gene sequences corresponding to the truncated PCV3 Cap-I peptide and PCV3Cap-II peptide were optimized and synthesized, and the nucleotide sequences of the encoding genes were SEQ ID NO:4 and SEQ ID NO:6, respectively. In order to improve the soluble expression of the truncated Cap protein, the gene sequence of the small ubiquitin protein modification molecule SUMO was fused to the 3′ end of the truncated Cap protein gene sequence, and connected to the pET-28a vector through the restriction endonuclease EcoR I and BamH I restriction sites to obtain the recombinant expression plasmids pET-PCV3Cap-I-SUMO and pET-PCV3Cap-II-SUMO. The two recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells, coated on LB plates, cultured at 37°C overnight, and single clone colonies were picked for identification. The positive strains were inoculated with LB culture medium and cultured at 37°C and 220rpm. When the OD 600 When the value reached 0.6-0.8, IPTG was added at a final concentration of 1.0 mmol / L. The induction was continued for 6 h at 25 °C and 180 rpm. The bacterial sludge was harvested by centrifugation, the precipitate was resuspended in PBS buffer, and ultrasonically disrupted at 300 W. The recombinant protein was expressed in a soluble form. After ultrasonic disruption, the supernatant was purified by nickel column chromatography to obtain recombinant Cap-I protein and Cap-II protein. The SDS-PAGE results are shown in Figure 1 As shown, target bands of approximately 32 kDa and 26 kDa appeared.

[0034] 3. Comparison of immunogenicity of recombinant Cap-I protein and Cap-II protein subunit vaccines

[0035] Using EtEraser TM The HP high-efficiency endotoxin removal kit was used to remove endotoxin from the purified recombinant Cap-I protein and Cap-II protein. After passing the test, the PCV3 Cap protein subunit vaccine was prepared according to the IMS1313VG adjuvant instructions. The PCV3 recombinant Cap protein content was not less than 100μg / ml. The prepared Cap-I protein and Cap-II subunit vaccines were tested for appearance, sterility, stability, etc., and the results showed that they were qualified and stored at 4°C for future use.

[0036] Fifteen 7-week-old BALB / c mice were randomly divided into three groups, each with 5 mice. Two groups were subcutaneously injected with recombinant Cap-I protein and Cap-II protein subunit vaccines, respectively, and the third group was subcutaneously injected with normal saline, 0.1 ml per mouse. After 14 days, the mice were boosted with the same dose. Blood was collected on the 28th day after immunization, and serum was separated. The antibody titer was detected using a commercial ELISA kit. The results showed that the serum OD of the Cap-I protein subunit vaccine group was 1.3447 μg / mL. 450nmThe mean value (1.36) was higher than that of serum OD in the Cap-II protein subunit vaccine immunization group. 450nm The mean value was (0.73), and the difference was significant (P<0.01), indicating that Cap-I protein has stronger immunogenicity.

[0037] Example 2: Construction and identification of recombinant plasmid pCap-T2A

[0038] Based on the determination of the amino acid sequences and encoding genes of the immunogenic Cap-I protein and Cap-II protein, recombinant Seneca virus is prepared according to the conventional steps of inserting the exogenous immune antigen protein sequence into the Seneca virus genome. The following implementation steps take the insertion of Cap-I protein into the Seneca virus genome as an example.

[0039] 1. Construction and identification of recombinant plasmid pUC19-Cap

[0040] Primers were designed and synthesized based on the truncated PCV3 Cap-I gene sequence. An EcoRI restriction site was introduced into the upstream primer sequence, and a BamH I restriction site was included in the downstream primer sequence:

[0041] Cap-I F1: 5'-G GAATTC AAATATAGCACCATGAACGTGATTAGC-3' (the underline indicates the EcoR I restriction site)

[0042] Cap-I R1: 5'-GC GGATCC CACACTTCTTTGGTGCCATAAAAA-3' (the underline indicates the BamH I restriction site)

[0043] The pET-28a-PCV3Cap-I recombinant plasmid was used as a template and DNA polymerase and primers Cap-I F1 and Cap-IR1 were used for amplification. The amplification system was 25 μl: 2× Rapid Taq Master Mix 12.5 μl, 1.0 μl of upstream and downstream primers, 2.0 μl of template, and ddH 2O is completed to 25μl. The PCR amplification conditions are: pre-denaturation at 95℃ for 3min, denaturation at 95℃ for 15s, annealing at 54℃ for 15s, extension at 72℃ for 15s, for a total of 30 cycles, and further extension at 72℃ for 5min. After the PCR reaction is completed, the products are collected and identified by 1.0% agarose gel electrophoresis. The target bands are purified and recovered using a gel recovery kit. The purified and recovered PCR products are double-digested with the pUC19 plasmid using restriction endonucleases EcoRI and BamH I, respectively, and then ligated with T4 ligase. The ligated products are transformed into DH5α Escherichia coli competent cells, coated with LB plates containing ampicillin resistance and inverted for culture, positive transformants are screened, inoculated into LB liquid culture medium for expansion, and the plasmids are extracted and identified by PCR. The positive plasmids are selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The correct recombinant plasmid is named pUC19-Cap-I. The PCR identification results are as follows: Figure 2 shown.

[0044] 2. Construction and identification of recombinant plasmid pUC-Cap-I-T2A

[0045] Primers containing homology arm sequences were designed according to the position of the T2A gene fragment inserted into the pUC19-Cap-I plasmid. Both the primers and the T2A gene fragment were synthesized by Shanghai Biotech Co., Ltd. Using the synthesized T2A gene fragment as a template, the gene sequence of which is shown in SEQ ID NO:7, a T2A fragment with a homology sequence was obtained by PCR, the gene sequence of which is shown in SEQ ID NO:8, and purified and recovered using a gel recovery kit. Primer design and synthesis are as follows:

[0046] T2A-F: 5′-CACCAAAGAAGTGTG GGATCC GAGGGCAGAGGAAGTCTTCTAACA-3′ (the underline indicates the BamHI restriction site).

[0047] T2A-R: 5′-CAGGTCGACTCTAGA GGATCC AGGGCCGGGATTCCTCCA-3′ (the underline indicates the BamH I restriction site).

[0048] The pUC19-Cap-I recombinant plasmid was digested with BamH I to obtain a linearized vector. The linearized fragment was mixed with the T2A gene fragment with a homologous sequence. The recombination reaction was completed using the ClonExpress II One Step Cloning Kit to achieve in vitro circularization of the two linearized DNAs. The recombinant product was directly transformed into DH5α Escherichia coli competent cells, coated on LB plates containing ampicillin resistance for culture, and positive transformants were screened. The correct positive clones identified by PCR were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The correctly sequenced recombinant plasmid was named pUC-Cap-I-T2A.

[0049] Example 3: Construction and identification of a recombinant Seneca virus infectious clone expressing porcine circovirus type 3 Cap-I protein

[0050] Based on the full-length infectious clone rSVA of pSVA-HHS01, the Cap-I-T2A gene was inserted between 2A and 2B of its genome to construct the recombinant plasmid pSVA-PCV3-Cap. The SVA recombinant gene was divided into three fragments, S1, S2, and S3, for amplification, wherein the S2 fragment included the gene sequences encoding the VP1, 2A, 2B, and 2C proteins, and then the S2 fragment was divided into three parts, S2-F1, S2-F2, and S2-F3, for amplification, and the S2-F2 fragment was the inserted Cap-I-T2A fragment.

[0051] 1. Amplification of recombinant SVAS2 fragments

[0052] Using rSVAcDNA infectious clone plasmid as template, S2-F1-F / R and S2-F3-F / R primers were used to amplify S2-F1 and S2-F3 fragments, respectively. 2× Rapid Taq Master Mix was used for amplification. The amplification system was 25 μl: 2× Rapid Taq Master Mix 12.5 μl, upstream and downstream primers 1.5 μl, template 2.0 μl, ddH 2O was filled to 25 μl. The PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, 35 cycles in total, and 72°C extension for 5 min. After the PCR reaction was completed, the product was collected and identified by 1.0% agarose gel electrophoresis. The target band was purified and recovered using a DNA agarose gel recovery kit. According to the known positions and sequences of SVA2A and 2B, the corresponding homology arm primers were designed. The recombinant plasmid pUC-Cap-I-T2A constructed in Example 2 was used as a template, and Cap-I-T2A-F and Cap-I-T2A-R were used as primers. The Cap-I-T2A fragment with a homologous sequence was amplified by PCR, and then S2-F1-F and S2-F3-R were used as primers. Cap-I-T2A was inserted between 2A and 2B of SVA by fusion PCR to obtain the fusion fragment S2-Cap-I-T2A. The electrophoresis results of the above S2-Cap-I-T2A amplification products are as follows Figure 4 As shown, the target band size is approximately 2616 bp, which is consistent with the expected size.

[0053] 2. Amplification of recombinant SVAS1 and S3 fragments

[0054] Using rSVAcDNA infectious clone plasmid as template, S1-F / R and S3-F / R primers were used to obtain S1 and S3 fragments by PCR. The amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 58℃ annealing for 15s, 72℃ extension for 30s, a total of 35 cycles, and 72℃ extension for 5min. After the PCR reaction was completed, the products were collected and identified by 1.0% agarose gel electrophoresis. The results are as follows Figure 4 As shown, the sizes of S1 fragment and S3 fragment were approximately 2673 bp and 2524 bp, respectively, which were consistent with the expected sizes.

[0055] 3. Construction and identification of pSVA-PCV3-Cap-I recombinant plasmid

[0056] The pcDNA3.1 eukaryotic expression vector was double-digested with KpnI and NotI restriction endonucleases to obtain linearized fragments. The linearized fragments were mixed with the S1, S2-Cap-I-T2A, and S3 fragments of the SVA genome in a certain proportion and then the recombination reaction was completed by the NEBuilder high-fidelity DNA assembly cloning kit, transformed into Top10 competent cells, and coated on LB plates for overnight culture. The screened positive clones were inoculated into LB liquid culture medium for culture. After the positive clones were expanded and cultured, the plasmids were extracted using a plasmid extraction kit for PCR identification and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the correct recombinant plasmid was pSVA-PCV3-Cap-I.

[0057] Table 1: Information of primers designed and synthesized for different amplification fragments

[0058]

[0059]

[0060] Example 4: Rescue and identification of recombinant Senecavirus expressing porcine circovirus type 3 Cap protein

[0061] 1. Rescue of recombinant virus rSVA-PCV3-Cap-I

[0062] BHK-21 cells were expressed at 1×10 5 The cells were inoculated into 6-well cell culture plates at a density of 100 cells / ml and transfected when the confluence of BHK-21 cells reached 60%. TM According to the instructions of the 2000 (Invitrogen) transfection reagent, 4 μg of the correctly identified recombinant plasmid pSVA-PCV3-Cap-I was transferred into BHK-21 cells. At the same time, normal cells and liposome control groups were set up and placed at 37°C and 5% CO 2 Cultured in an incubator. 6 hours after transfection, the supernatant was discarded and replaced with fresh DMEM medium containing 2% FBS. The culture was continued and the cell pathological changes were observed. The virus was harvested when about 90% of the cells showed cytopathic effect (CPE). After repeated freezing and thawing for 3 times, the culture was continuously passaged on BHK-21 cells until the virus could stably produce pathological phenomena such as cell rounding, shedding, disintegration into fragments or vacuoles. The rescued recombinant virus was named rSVA-PCV3-Cap-I. The results are shown in Figure 5 As shown, the rescued rSVA-PCV3-Cap-I recombinant virus produced cytopathic effects similar to that of the parental SVA.

[0063] 2. Identification of recombinant virus rSVA-PCV3-Cap

[0064] 1) Stability of recombinant virus passage

[0065] The recombinant virus rSVA-PCV3-Cap-I was continuously propagated in BHK-21 cells for 15 generations. Virus fluids of the 1st, 5th, 10th and 15th generations were taken and the RNA of the recombinant virus was extracted using the TaKaRa MiniBEST Viral RNA / DNAExtraction Kit. After reverse transcription, the fusion fragment S2-Cap-I-T2A and Cap-I gene fragment were amplified as templates. The results of agarose gel electrophoresis identification were as follows: Figure 6 and Figure 7The results showed that the fusion gene fragments and Cap exogenous gene fragments inserted by the recombinant viruses of each generation were consistent with the expected size, indicating that the inserted target fragments could exist stably without deletion or mutation. The titer of the virus of each generation was 10 9.29 TCID 50 / ml~10 9.5 TCID 50 / ml, indicating that the recombinant virus proliferated stably on BHK-21 cells.

[0066] Virus titer determination method: The harvested recombinant virus was first frozen and thawed three times, centrifuged at 6000r / min for 10min, and the recombinant virus supernatant was serially diluted 10-fold with serum-free DMEM high-glucose medium. -6 ~10 -9 4 dilutions were inoculated into 96-well culture plates with monolayers of BHK-21 cells. Each dilution was inoculated into 8 replicate wells, with 0.1 ml per well. At the same time, 8 wells without virus inoculation were set as controls. The plates were placed at 37°C and 5% CO. 2 The cells were cultured in an incubator and the cytopathic effect was observed daily for 5 to 7 days. The half-cell culture infection dose (TCID) was calculated according to the Reed-Muench method. 50 ).

[0067] 2) Identification of the expression of truncated PCV3 Cap protein by indirect immunofluorescence (IFA)

[0068] When the confluence of BHK-21 cells in the 6-well plate reached about 80%, one well was infected with P15 rSVA-PCV3-Cap-I and the parental SVA strain, respectively. Normal cells were set as controls and the plates were placed at 37°C and 5% CO. 2 Culture in an incubator for 12 to 24 hours, then discard the supernatant, wash three times with sterile PBS buffer, add 4% paraformaldehyde to fix for 20 minutes, then permeabilize with 0.02% TritonX-100 for 5 minutes, wash three times with PBS buffer, add 5% BSA solution to block for 1 hour, wash three times with PBS buffer, use PCV3 Cap mouse polyclonal antibody as primary antibody, and FITC-labeled goat anti-mouse IgG as secondary antibody to identify PCV3 Cap antigen. The results are shown in Figure 8 As shown, IFA was performed using PCV3 Cap polyclonal antibody, and the recombinant virus rSVA-PCV3-Cap-I cell wells showed specific fluorescence, while the parental SVA strain infected wells and normal cell controls had no fluorescence, indicating that the recombinant virus rSVA-PCV3-Cap-I can express PCV3 Cap-I protein.

[0069] 3) Western blot identification of PCV3 Cap protein

[0070] Recombinant virus rSVA-PCV3-Cap-I, PCV3 strain, parental SVA strain and normal control group cells were collected, and protein samples were obtained according to the conventional Western blot operation method, and then SDS-PAGE electrophoresis was performed. After transfer and blocking, PCV3 Cap mouse polyclonal antibody was used as the primary antibody for overnight incubation at 4°C, PBST was washed 3 times, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. It was incubated at room temperature for 2 hours, and PBST solution was washed 3 times before color development. The results are as follows Fig. 9 As shown, the PCV3 strain showed a specific target band at about 25 kDa, the recombinant virus rSVA-PCV3-Cap-I showed a specific target band at about 18 kDa, and the parental virus SVA and the cell control did not show the target band, indicating that the recombinant virus rSVA-PCV3-Cap-I can correctly express the inserted PCV3Cap-I protein after infecting BHK-21 cells.

[0071] Example 5: Preparation and immunogenicity study of Seneca virus recombinant vaccine expressing porcine circovirus type 3 Cap protein

[0072] The gene encoding the Cap-II protein was inserted into the Seneca virus genome according to the methods of Examples 2, 3 and 4, and the recombinant virus rSVA-PCV3-Cap-II was obtained after rescue and identification.

[0073] 1. Preparation of Seneca virus recombinant vaccine

[0074] The recombinant viruses rSVA-PCV3-Cap-I and rSVA-PCV3-Cap-II were inoculated into BHK-21 cell monolayers at a ratio of 0.3%. When the cell cytopathic effect reached more than 80%, the virus titer was determined after repeated freezing and thawing for 3 times, and the TCID was calculated according to the Reed-Muench method. 50 The results showed that the virus titers were 10 9.33 TCID 50 / ml and 10 9.12 TCID 50 / ml. Inactivate with formaldehyde at a final concentration of 0.1% at 37°C for 20h. After inactivation, take samples and blindly propagate them in BHK-21 cells for 3 generations. No cytopathic effect was observed during the period, indicating that the recombinant virus solution was completely inactivated. After adding sterile Tween-80 to the inactivated recombinant virus solution and mixing it evenly, emulsify it with mineral oil adjuvant at a ratio of 1:2.5 to prepare the vaccine, aseptically package it in quantitative form, and store it at 4°C. Use it after the vaccine passes the sterility test.

[0075] 2. Immunogenicity test of Seneca virus recombinant vaccine

[0076] The two qualified recombinant virus inactivated vaccines were injected intramuscularly into 5 healthy piglets at 35 days of age, 2.0 ml each, and boosted with the same dose 21 days later. Another 5 piglets were set up as a non-immunized control group. Before immunization, the SVA neutralizing antibody titer of all pigs was less than 1:4, and the PCV3 antibody was negative (OD 450 nm<0.3). Blood was collected on the 14th, 21st, 28th and 60th day after immunization, and serum was separated. The serum SVA neutralizing antibody titer was detected by cell neutralization test, and the serum Cap specific antibody titer was detected by indirect ELISA method. The results of SVA neutralizing antibody are shown in Table 2.

[0077] Table 2: SVA neutralizing antibody test results

[0078]

[0079]

[0080] The results showed that SVA neutralizing antibodies could be detected 14 days after the second immunization of the two recombinant virus inactivated vaccines, and SVA antibodies reached their peak 60 days after the second immunization. Fig.10 As shown, the antibody titers of the two recombinant virus inactivated vaccines reached the highest peak 28 days after the second immunization, among which the antibody titer of the immunization group 1 was significantly higher than that of the immunization group 2, indicating that the rSVA-PCV3-Cap-I recombinant virus inactivated vaccine has stronger immunogenicity.

[0081] The above results indicate that the recombinant vaccine prepared with the rSVA-PCV3-Cap-I recombinant virus can induce the animal body to produce high levels of SVA neutralizing antibodies and Cap-specific antibodies at the same time, and has good immunogenicity.

Claims

1. A porcine circovirus type 3 Cap fusion protein, characterized in that The fusion protein comprises: 1) A fusion protein having an amino acid sequence of SEQ ID NO: 3; 2) A fusion protein derived from 1) by replacing, deleting or adding one or more amino groups in 1).

2. A nucleic acid fragment, characterized in that The nucleic acid fragment is used to encode the fusion protein according to claim 1.

3. The nucleic acid fragment according to claim 2, characterized in that The nucleic acid fragment is characterized in that the sequence of the nucleic acid fragment is SEQ ID NO:

4.

4. Use of the fusion protein of claim 1 as an antigenic polypeptide inserted into the genome of Seneca virus to prepare recombinant Seneca virus.

5. A recombinant Seneca virus, characterized in that: A nucleic acid fragment encoding the fusion protein according to claim 1 is inserted into the genome of the recombinant Seneca virus.

6. The recombinant Senecavirus according to claim 5, characterized in that The nucleic acid fragment is inserted between the Seneca virus cDNA 2A and 2B genes.

7. Use of the recombinant Seneca virus according to claim 5 as an antigen for preparing a vaccine.

8. A virus vaccine, characterized in that: The virus vaccine is prepared using the inactivated recombinant Seneca virus of claim 5 as an antigen.

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