Porcine pestivirus and porcine parvovirus bivalent subunit vaccine and preparation method thereof

Abstract

The application discloses a CSFV-PPV bivalent subunit vaccine and a preparation method thereof. The vaccine comprises a first recombinant protein encoded by a first gene, a second recombinant protein encoded by a second gene and a pharmaceutically acceptable carrier. The first gene has a sequence shown in SEQ ID NO:1 or an increased or reduced sequence thereof. The second gene has a sequence shown in SEQ ID NO:2 or an increased or reduced sequence thereof. The application takes the antigen E2 protein of CSFV (CSFV) and the VP2 protein of PPV (PPV) as double targets, expresses the recombinant SC-E2 protein with a SpyCatcher label and the recombinant ST-VP2 protein with a SpyTag label in insect cells through a recombinant baculovirus vector, realizes in-vitro covalent assembly of the double antigens, and constructs the bivalent subunit vaccine which can simultaneously prevent and control two diseases, and has the advantages of high safety, strong immunogenicity, high prevention and control efficiency and easiness in large-scale production.

Description

TECHNICAL FIELD

[0001] The application specifically relates to a porcine parvovirus and classical swine fever recombinant baculovirus double subunit vaccine and a preparation method thereof, and belongs to the technical field of animal immunological drugs. BACKGROUND

[0002] Classical Swine Fever (CSF) is an acute infectious disease caused by CSFV, with high pathogenicity and high mortality, which can cause acute death, growth stagnation and reproduction disorders in pigs, and pigs of all ages are susceptible. Porcine Parvovirus (PPV) is a reproductive disorder disease caused by PPV, mainly infecting breeding sows, leading to abortion, mummified fetus, stillbirth or weak piglets, and the initial production of sows is susceptible, and the reproduction disorder symptom lasts for 1-2 breeding cycles. In actual breeding scenarios, CSFV and PPV often occur in combined infection, forming "double infection", which is significantly more harmful than single pathogen infection, further aggravating breeding losses.

[0003] At present, the commercial vaccines for CSFV and PPV are mainly single disease vaccines, including live CSF vaccine, inactivated PPV vaccine or subunit vaccine, etc., but the existing vaccines have some defects, mainly as follows:

[0004] First, the protection range is limited and cannot cope with mixed infection. The existing vaccines are mostly single pathogen vaccines, which can only prevent and control one of CSF or PPV, and cannot resist the combined infection of CSFV and PPV. However, "double infection" frequently occurs in actual breeding, and different vaccines need to be inoculated multiple times, increasing the operation cost of breeding, and frequent immunization can easily cause stress reaction in pigs, affecting production performance.

[0005] Second, there is a biological safety risk. Although the existing live CSF vaccine (such as CN116036257B) has strong immunization effect, it has potential risks of recombination with wild virus and interference of immune suppression, which may lead to the spread of vaccine strains in pig populations or cause clinical symptoms, aggravate the symptoms of latent infection pigs and short-term virus shedding, and pose a threat to the biological safety of specific pathogen-free (SPF) pigs and purification farms, which does not meet the strict requirements of modern pig farming on vaccine safety.

[0006] Thirdly, the ability to cope with variant strains is weak. The protective efficacy of the CSFV rabbitized attenuated vaccine and the gene-deleted attenuated vaccine is highly dependent on the homology of the epidemic strain. Although the mutation rate of CSFV is relatively slow, in recent years, genetic subtype differentiation has occurred, and when the wild epidemic strain undergoes antigenic drift, such live vaccines cannot effectively induce specific neutralizing antibodies, and have basically no protection against heterologous strains, which may lead to immune failure in pig populations, causing regional acute death, reproduction disorders and other serious losses. The PPV subunit vaccine based on the prokaryotic expression system targets the VP2 protein, and if amino acid mutations (such as hemagglutination active site mutations) occur, it will directly lead to antigenic changes, and the matching degree of the original antigen epitope of the vaccine and the variant strain will be greatly reduced.

[0007] Fourthly, the vaccine immunogenicity is insufficient. Some subunit vaccines against PPV (such as subunit vaccines based on prokaryotic expression systems) have problems such as protein folding errors and weak immunogenicity, and toxic proteins and endotoxins are easily left over in the prokaryotic expression process, which may cause local inflammation or allergic reactions in pigs, and the short protection period of the vaccine due to low immunogenicity requires frequent booster immunization, increasing the breeding burden. SUMMARY

[0008] The main purpose of the present application is to provide a CSFV-PPV bivalent subunit vaccine and a preparation method thereof to overcome the deficiencies in the prior art.

[0009] To achieve the aforementioned purposes of the application, the following technical solutions are used.

[0010] The first aspect of the present application provides a recombinant protein composition, comprising:

[0011] a first recombinant protein encoded by a first gene;

[0012] a second recombinant protein encoded by a second gene.

[0013] In one embodiment, the molar ratio of the first recombinant protein and the second recombinant protein is about 1:1.

[0014] In one embodiment, the first recombinant protein and the second recombinant protein are covalently linked, and the second recombinant protein self-assembles into virus-like particles (VLPs).

[0015] The second aspect of the present application provides a gene composition, comprising:

[0016] a first gene for encoding the first recombinant protein;

[0017] a second gene for encoding the second recombinant protein.

[0018] The third aspect of the present application provides a recombinant vector composition, comprising:

[0019] a first recombinant vector comprising a first gene;

[0020] a second recombinant vector comprising a second gene.

[0021] In one embodiment, the first recombinant vector or the second recombinant vector comprises, but is not limited to, pFastBac 1, pVL1393, pFastBac dual, or pDEST8, etc., preferably pFastBac 1.

[0022] The fourth aspect of the present application provides a combination of host cells, comprising:

[0023] a first host cell comprising a first gene;

[0024] a second host cell comprising a second gene.

[0025] In one embodiment, the first host cell or the second host cell comprises, but is not limited to, Sf9, HighFive, or Sf21 cell, preferably Sf9 cell.

[0026] The fifth aspect of the present application provides a recombinant protein conjugate, which is covalently linked by a first recombinant protein and a second recombinant protein; wherein the first recombinant protein is encoded by a first gene, and the second recombinant protein is encoded by a second gene.

[0027] In one embodiment, the molar ratio of the first recombinant protein to the second recombinant protein is about 1:1.

[0028] In one embodiment, the second recombinant protein in the conjugate self-assembles into a virus-like particle.

[0029] The first gene in the present application can have the sequence shown in SEQ ID NO: 1 or an increased or truncated sequence thereof, in particular a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 1. More preferably, the sequence of the first gene is shown in SEQ ID NO: 1.

[0030] The second gene in the present application can have the sequence shown in SEQ ID NO: 2 or an increased or truncated sequence thereof, in particular a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 2. More preferably, the sequence of the second gene is shown in SEQ ID NO: 2.

[0031] The sixth aspect of the present application provides an immunological composition, comprising: the recombinant protein composition or the recombinant protein conjugate; and a pharmaceutically acceptable carrier.

[0032] In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of two or more of MONTANIDE ISA 206VG, MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and phytohemagglutinin, preferably white oil.

[0033] In one embodiment, the recombinant protein composition comprises the first recombinant protein and the second recombinant protein in a molar ratio of about 1:1.

[0034] The seventh aspect of the present application provides a method for preparing a recombinant protein, comprising:

[0035] providing a combination of the host cells;

[0036] culturing the first host cell and the second host cell under suitable conditions, respectively, and then isolating to obtain the first recombinant protein and the second recombinant protein.

[0037] In one embodiment, the method for preparing specifically comprises:

[0038] S1, respectively preparing a nucleic acid molecule for encoding the first recombinant protein and the second recombinant protein;

[0039] S2, constructing a recombinant vector, cloning the nucleic acid molecule of step S1 into a shuttle vector, respectively, to obtain a recombinant shuttle vector containing the target gene;

[0040] S3, transforming the recombinant shuttle vector into a competent cell to obtain a recombinant plasmid, and then transfecting a host cell, respectively, to obtain a recombinant baculovirus;

[0041] S4, inoculating the recombinant baculovirus into a host cell to obtain an expression product, i.e., the first recombinant protein and the second recombinant protein.

[0042] Exemplarily, the method for preparing comprises:

[0043] S1, respectively preparing a nucleic acid molecule for encoding the first recombinant protein (recombinant SC-E2 protein) and the second recombinant protein (recombinant ST-VP2 protein);

[0044] S2, constructing a recombinant vector, cloning the nucleic acid molecule of step S1 into a shuttle vector (e.g., pFastBac1), respectively, to obtain a recombinant shuttle vector (e.g., pF-SC-E2 and pF-ST-VP2) containing the target gene;

[0045] S3, transforming the recombinant shuttle vector into DH10Bac competent cells, obtaining recombinant plasmids (e.g., Re-Bacmid-AP33 recombinant plasmid and Re-Bacmid-AP65 recombinant plasmid) through blue-white spot screening and PCR identification, integrating the target gene into Bacmid through transposition, obtaining recombinant baculovirus vectors (e.g., Bacmid-SC-E2 and Bacmid-ST-VP2) through screening and identification, and then transfecting Sf9 insect cells, respectively, to obtain recombinant baculoviruses;

[0046] S4, purifying the recombinant baculoviruses and determining the titer, inoculating Sf9 cells, and obtaining expression products of recombinant SC-E2 protein and recombinant ST-VP2 protein.

[0047] Further, the preparation method can further include the steps of isolating and purifying the first recombinant protein and the second recombinant protein, and the optional isolation and purification method includes but is not limited to chromatography, dialysis and other methods known in the art.

[0048] In one embodiment, the preparation method can further include:

[0049] S5, mixing the purified recombinant SC-E2 protein and the purified recombinant ST-VP2 protein at an appropriate ratio (e.g., a molar ratio of about 1:1), incubating, covalently combining the two to achieve dual antigen assembly, and obtaining an assembled recombinant protein composition.

[0050] In one embodiment, the preparation method can further include:

[0051] S6, mixing the assembled recombinant protein composition with a pharmaceutically acceptable carrier.

[0052] An eighth aspect of the present application provides the use of the recombinant protein composition, the genomic composition, the recombinant protein covalent conjugate or the immunological composition in the production of a medicament for inducing an immune response against porcine pestivirus and / or porcine parvovirus infection in a subject animal or a medicament for preventing an animal from being infected with porcine pestivirus and / or porcine parvovirus.

[0053] A ninth aspect of the present application provides the use of the recombinant protein composition, the recombinant protein covalent conjugate or the immunological composition in the preparation of a porcine pestivirus and porcine parvovirus bivalent subunit vaccine.

[0054] A tenth aspect of the present application provides a porcine pestivirus and porcine parvovirus bivalent subunit vaccine, wherein the vaccine comprises the recombinant protein composition or the recombinant protein covalent conjugate. Further, the vaccine can further comprise a pharmaceutically acceptable carrier.

[0055] The recombinant subunit vaccine against classical swine fever virus and / or porcine parvovirus infection provided in this invention only requires administration of an effective dose to the animal. The "effective dose" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0056] The eleventh aspect of the present invention provides a method for preparing the classical swine fever and porcine parvovirus bivalent subunit vaccine.

[0057] For example, the method may include the following steps:

[0058] S10. Target selection and antigen design, including:

[0059] S11. Screening of core protective antigens: Screening for the core protective E2 protein responsible for inducing neutralizing antibodies from classical swine fever virus, and screening for the key immunogen VP2 protein that can self-assemble into virus-like particles from porcine parvovirus. Both antigens have good inter-strain immune correlation.

[0060] S12. Recombinant Antigen Construction: The N-terminus of the classical swine fever virus (CSV) E2 protein-coding gene was fused with a SpyCatcher tag-coding sequence to construct the first gene (SC-E2 gene); the N-terminus of the porcine parvovirus (PSV) VP2 protein-coding gene was fused with a SpyTag tag-coding sequence to construct the second gene (ST-VP2 gene). Dual antigen assembly was achieved using the specific response of SpyCatcher / SpyTag, and the self-assembly properties of VP2 enhanced the immune response.

[0061] In some cases, the SpyCatcher / SpyTag covalent tag pair can be replaced with SnoopCatcher / SnoopTag, Isopeptag / Pilin-C, or other specific binding tag systems, but the following requirements must be met: ① the key neutralizing antigen epitopes are not masked when the tag is fused with the antigen protein for expression; ② the folding efficiency of E2 and VP2 proteins and the efficiency of dual antigen assembly (≥80%) are not affected, and the in vitro assembly product can still induce high-titer antibodies.

[0062] S20, Construction of recombinant baculovirus vector, including:

[0063] S21. Transfer Vector Construction: The SC-E2 gene was synthesized and cloned into the pUC-17 vector to obtain the pUC-SC-E2 plasmid vector. Specific amplification primers for the SC-E2 gene were designed based on the vector sequence. The pFastBac1 plasmid vector and the purified product were digested with enzymes and ligated, then transformed into DH5α competent cells to obtain the recombinant transfer vector pF-SC-E2. Similarly, the recombinant transfer vector pF-ST-VP2 was obtained using a similar method. Colony PCR and sequencing verification were performed to ensure the correct gene sequence and insertion direction.

[0064] S22. Construction of recombinant Bacmid: Two recombinant transfer vectors were transformed into DH10Bac competent cells, and the target gene was integrated into Bacmid through transposition. The first recombinant vector and the second recombinant vector (recombinant baculovirus vectors Bacmid-SC-E2 and Bacmid-ST-VP2) were obtained after screening and identification.

[0065] S30. Expression and purification of recombinant antigens, including:

[0066] S31. Preparation of recombinant baculovirus: Two recombinant baculovirus vectors were transfected into Sf9 insect cells, and after culture, P1 generation recombinant baculoviruses rBac-SC-E2 and rBac-ST-VP2 were harvested and amplified to obtain high-titer virus stock solutions.

[0067] S32. Antigen Expression and Purification: Sf9 cells were inoculated with high-titer recombinant baculovirus for large-scale expression. After harvesting the culture products, the first recombinant protein (recombinant SC-E2 protein, hereinafter referred to as SC-E2 protein) and the second recombinant protein (recombinant ST-VP2 protein, hereinafter referred to as ST-VP2 protein) were purified using SP-FF ion exchange chromatography and other techniques to ensure that the protein purity met the standards.

[0068] In some cases, Sf9 insect cells can be replaced with other commonly used insect cell lines such as Sf21 and High Five (Hi5). For example, Sf9 cells can be replaced with Hi5 cells.

[0069] In some cases, the recombinant baculovirus-insect cell expression system can be replaced with the Pichia pastoris eukaryotic expression system, but the design scheme of the E2 and VP2 protein encoding genes needs to be adjusted to suit the yeast expression preferences.

[0070] In some cases, the aforementioned purification methods can also employ nickel column affinity chromatography, which has higher specificity and can improve protein purity to over 95%, but requires the addition of a His tag sequence during the gene construction stage.

[0071] S40. Covalent assembly and identification of dual antigens, including:

[0072] S41. In vitro assembly: The two purified recombinant proteins are mixed and incubated in an appropriate ratio, and the dual antigens are assembled through the covalent binding of SpyCatcher and SpyTag.

[0073] S42. Assembly Identification: The specific binding of the two recombinant proteins was verified by observing changes in characteristic bands using SDS-PAGE and by combining Western blotting to confirm successful assembly.

[0074] S50. Vaccine formulation preparation: The assembled recombinant protein composition is used as an immunogen and mixed with adjuvants to prepare a vaccine formulation. After passing the test, it can be used for animal immunization.

[0075] The adjuvants used can be oil-emulsion adjuvants such as Montanide ISA 206 and ISA 61VG, or aluminum adjuvants such as aluminum hydroxide gel. Oil-emulsion adjuvants can prolong antibody duration. Provided the vaccine emulsion stability is maintained, aluminum adjuvants can also be used, which are more suitable for primary immunization of piglets.

[0076] A twelfth aspect of the present invention provides a method for inducing an immune response against classical swine fever virus and / or porcine parvovirus infection or for protecting a test animal from classical swine fever virus and / or porcine parvovirus infection, the method comprising administering the classical swine fever and porcine parvovirus bivalent subunit vaccine to the test animal.

[0077] The thirteenth aspect of the present invention also provides a vaccine suitable for inducing an immune response against classical swine fever virus and / or porcine parvovirus infection in test animals, said vaccine comprising: the recombinant protein composition and an adjuvant. As used in this specification, "adjuvant" means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen encoded by said gene. Further, the adjuvant may be an oil-emulsion adjuvant, such as the water-in-oil adjuvant produced by Suzhou Womei Biotechnology Co., Ltd., to improve vaccine efficacy.

[0078] The vaccine of this invention can be formulated as a single-dose or two-dose bivalent prophylactic vaccine, administered via intramuscular or subcutaneous injection in the neck for routine immunization of piglets over one month of age, gilts, and pregnant sows (before mating), thereby effectively preventing acute death and reproductive disorders caused by CSFV, and abortion and mummified fetuses caused by PPV. Compared to existing single vaccines, it effectively reduces the difficulty of controlling "dual infection" in large-scale pig farms.

[0079] The vaccine described in this invention can also be used as an auxiliary tool for disease eradication in large-scale pig farms. It can be used to immunize CSFV / PPV-negative pig herds in CSFV / PPV-eradicated farms to establish a stable immune barrier; it can also be used to rapidly restore the resistance of pig herds after culling infected pigs in positive farms. Generally, serological monitoring (ELISA to detect antibodies) can be used to assess the eradication effect.

[0080] The vaccine described in this invention contains no intact virus or genetic material, does not interfere with pathogen detection results, and can effectively help pig farms build CSFV / PPV disease-free communities.

[0081] The fourteenth aspect of the present invention also provides a kit comprising the recombinant protein composition, the gene composition, a combination of the recombinant vectors, a combination of the host cells, the recombinant protein covalent linker, or the immune composition.

[0082] Furthermore, the kit may also include containers or instruments, such as syringes, for packaging or administering the recombinant protein composition, the gene composition, the combination of recombinant vectors, the combination of host cells, or the immune composition to animals.

[0083] For example, the recombinant protein composition of this application has high protein purity and strong specificity. When used as a porcine virus antibody detection reagent, it can significantly improve the accuracy of the detection reagent (error ≤ 5%) and avoid problems such as large batch-to-batch differences in existing antigens.

[0084] More specifically, the purified SC-E2 recombinant protein and ST-VP2 recombinant protein can be used as specific antigens to develop CSFV antibody ELISA detection kits and PPV antibody HI detection kits, replacing existing viral culture antigens.

[0085] Compared with the prior art, the technical solution provided by the embodiments of the present invention has at least the following advantages:

[0086] (1) By using the CSFV core protective antigen E2 protein and the PPV key immunogen VP2 protein as dual targets, a bivalent subunit vaccine was prepared through the synergistic use of a recombinant baculovirus system and a SpyTag / SpyCatcher system, achieving "one injection to prevent two diseases". This design can directly address the "double infection" that frequently occurs in actual breeding, reduce the number of immunizations, reduce labor costs and pig stress, and avoid immune fatigue caused by multiple immunizations.

[0087] (2) Two antigen proteins were produced using a baculovirus-insect cell eukaryotic expression system: On the one hand, insect cells have eukaryotic protein modification (such as glycosylation) mechanisms, which can ensure that E2 and VP2 proteins fold correctly and ensure effective exposure of antigen epitopes; on the other hand, by adding a SpyCatcher tag to the N-terminus of E2 protein and a SpyTag tag to the N-terminus of VP2 protein, the two proteins can be covalently assembled, and VP2 protein can self-assemble into virus-like particles (VLPs), mimicking the structure of natural viruses, significantly improving immunogenicity and solving the problem of weak immunogenicity caused by prokaryotic expression systems.

[0088] (3) The recombinant baculovirus bivalent subunit vaccine provided contains purified recombinant E2 protein and recombinant VP2 protein as its active ingredients. It does not contain complete virus particles and genetic material of CSFV and PPV, thus fundamentally eliminating the risk of virulence reversion and transmission of vaccine strains in existing live vaccines. At the same time, baculoviruses are non-pathogenic to mammals, and the expression products are free of exogenous contaminants after purification. After vaccination, there are no safety hazards to pigs (including piglets and pregnant sows), and its safety is significantly better than that of existing vaccines. Attached Figure Description

[0089] Figure 1 This is a gel electrophoresis result of PCR amplification of the SC-E2 gene;

[0090] Figure 2 This is a plasmid map of the baculovirus transfer vector pF-SC-E2;

[0091] Figure 3 This is a gel electrophoresis result of the ST-VP2 gene after PCR amplification;

[0092] Figure 4 This is a plasmid map of the baculovirus transfer vector pF-ST-VP2;

[0093] Figure 5 This is an amplification and identification diagram of the recombinant baculovirus plasmid rBac-SC-E2;

[0094] Figure 6 This is an amplification and identification diagram of the recombinant baculovirus plasmid rBac-ST-VP2;

[0095] Figure 7 This is a graph obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the SC-E2 protein recombinant baculovirus F1 generation strain.

[0096] Figure 8 This is an SDS-PAGE image of ST-VP2 protein expression in recombinant baculovirus F1 generation strain;

[0097] Figure 9 This is a graph showing the results of Western blot analysis of SC-E2 protein.

[0098] Figure 10 This is a graph showing the results of Western blot analysis of ST-VP2 protein.

[0099] Figure 11 This is a fluorescence detection image of Sf9 cells inoculated with empty baculovirus and recombinant baculovirus;

[0100] Figure 12 This is a graph showing the SDS-PAGE results of SC-E2 eluted with different concentrations of NaCl;

[0101] Figure 13 This is a graph showing the SDS-PAGE results of ST-VP2 eluted with different concentrations of NaCl.

[0102] Figure 14 This is an image showing the SDS-PAGE results of the E2-VP2 covalently linked product;

[0103] Figure 15 This is a graph showing the immunoblotting results of the E2-VP2 covalently linked product;

[0104] Figure 16 This is a transmission electron microscope (TEM) image of the E2-VP2 covalently linked product. Detailed Implementation

[0105] The present invention is further illustrated below by way of examples. All reagents and raw materials used in the following examples are commercially available, and experimental methods not specifically described are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0106] Example 1: Construction and identification of transfer vector pF-SC-E2

[0107] 1. SC-E2 gene amplification and purification

[0108] The SC-E2 gene (SEQ ID NO:1) was codon optimized and synthesized at Suzhou Genewise Biotechnology Co., Ltd., and cloned into the pUC-17 vector to obtain the pUC-SC-E2 plasmid vector. Specific amplification primers for the SC-E2 gene were designed based on the vector sequence.

[0109] The sequence of the upstream primer SC-E2-F is: 5'-CCTCTAGAGCCACCATGGTCACCACCCTGTCTGG-3';

[0110] The downstream primer SC-E2-R is: 5'-CCAAGCTTTTAGGCAGGCAGGCAGCCAG-3'.

[0111] The sequences of the upstream primer SC-E2-F and the downstream primer SC-E2-R are numbered SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0112] Using pUC-SC-E2 plasmid as a template, SC-E2-F and SC-E2-R were used as upstream and downstream primers for PCR amplification. The amplification system is shown in Table 1.

[0113] Table 1 SC-E2 gene amplification system

[0114]

[0115] The reaction conditions were as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 1 minute 45 seconds, 30 cycles; 72℃ extension for 5 minutes, storage at 2–8℃. The PCR products were then subjected to gel electrophoresis to verify the size of the target gene. Figure 1 As shown, the target band appeared at the 1437bp position, indicating successful amplification of the target gene. The gene was then purified using a gel extraction and purification kit. Figure 1 In the middle, number 1 is the SC-E2 gene, number 2 is the negative control, and M is the DNA molecular quality standard (DNA Marker).

[0116] 2. Enzyme digestion and purification

[0117] The pFastBac1 plasmid vector and the purified PCR product SC-E2 gene were digested with Xba I and Hind III at 37℃ for 3 hours. The specific digestion reaction system is shown in Tables 2 and 3. The digestion products were subjected to gel electrophoresis, and the digested pFastBac1 vector and SC-E2 gene fragment were purified using a gel extraction and purification kit, respectively.

[0118] Table 2 SC-E2 gene restriction enzyme digestion system

[0119]

[0120] Table 3 pFastBac1 vector digestion system

[0121]

[0122] 3. Connection

[0123] The digested pFastBac1 vector and SC-E2 gene digestion products were ligated using T4 DNA ligase in a 16°C water bath overnight. The specific ligation reaction system is shown in Table 4.

[0124] Table 4. Ligation system of SC-E2 gene and pFastBac1 plasmid

[0125]

[0126] 4. Transformation

[0127] Add 10 µl of the ligation product to 100 µl of DH5α competent cells and mix well. Incubate at 42°C for 90 seconds under heat shock, followed by an ice bath for 2 minutes. Add 900 µl of LB liquid medium without Amp and incubate at 37°C for 1 hour. Take 1.0 ml of the bacterial culture, concentrate it to 100 µl, and spread it onto LB solid medium containing Amp. Incubate at 37°C for 16 hours.

[0128] 5. Colony PCR and sequencing identification

[0129] Six single colonies from the plate were inoculated into LB broth and incubated at 37°C for 2 hours. Colony PCR was performed using the bacterial culture as a template and SC-E2-F and SC-E2-R as primers. The PCR products were verified by gel electrophoresis to determine the size of the target gene. The correctly identified bacterial cultures were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Strains with correct sequence and ligation direction were selected for preservation. A schematic diagram of the constructed baculovirus transfer vector pF-SC-E2 containing the target gene is shown below. Figure 2 .

[0130] Example 2: Construction and Identification of Transfer Vector pF-ST-VP2

[0131] 1. ST-VP2 gene amplification and purification

[0132] The ST-VP2 gene (SEQ ID NO:2) was codon optimized and synthesized at Suzhou Genewise Biotechnology Co., Ltd., and cloned into the pUC-17 vector to obtain the pUC-ST-VP2 plasmid vector. Specific amplification primers for the ST-VP2 gene were designed based on the vector sequence.

[0133] The upstream primer ST-VP2-F is: 5'-CCTCTAGAGCCACCATGCGTGGTGGTGTCCCG-3';

[0134] The downstream primer ST-VP2-R is: 5'-CCAAGCTTTTACTACCTTTACGGAGACATG-3'.

[0135] The sequences of the upstream primer ST-VP2-F and the downstream primer ST-VP2-R are numbered SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0136] Using pUC-ST-VP2 plasmid as a template, and ST-VP2-F and ST-VP2-R as upstream and downstream primers, PCR amplification was performed. The amplification system is shown in Table 5.

[0137] Table 5 ST-VP2 gene amplification system

[0138]

[0139] The reaction conditions were as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 1 minute 45 seconds, 30 cycles; 72℃ extension for 5 minutes, and storage at 4℃. The PCR products were then subjected to gel electrophoresis to verify the size of the target gene. Figure 3 As shown, the target band appeared at the 1818bp position, indicating successful amplification of the target gene. The gene was then purified using a gel extraction and purification kit. Figure 3 In the table, number 1 represents the ST-VP2 gene, number 2 represents the negative control, and M represents the DNA molecular quality standard.

[0140] 2. Enzyme digestion and purification

[0141] The pFastBac1 plasmid vector and the purified PCR product ST-VP2 gene were digested with Xba I and Hind III at 37℃ for 3 hours. The specific digestion reaction system is shown in Tables 6 and 7. The digestion products were subjected to gel electrophoresis, and the digested pFastBac1 vector and ST-VP2 gene fragment were purified using a gel extraction and purification kit, respectively.

[0142] Table 6 ST-VP2 gene digestion system

[0143]

[0144] Table 7 pFastBac1 vector digestion system

[0145]

[0146] 3. Connection

[0147] The digested pFastBac1 vector and ST-VP2 gene digestion products were ligated using T4 DNA ligase in a 16°C water bath overnight. The specific ligation reaction system is shown in Table 8.

[0148] Table 8. Ligation system of ST-VP2 gene and pFastBac1 plasmid

[0149]

[0150] 4. Transformation

[0151] Add 10 µl of the ligation product to 100 µl of DH5α competent cells and mix well. Incubate at 42°C for 90 seconds under heat shock, followed by an ice bath for 2 minutes. Add 900 µl of LB liquid medium without Amp and incubate at 37°C for 1 hour. Take 1.0 ml of the bacterial culture, concentrate it to 100 µl, and spread it onto LB solid medium containing Amp. Incubate at 37°C for 16 hours.

[0152] 5. Colony PCR and sequencing identification

[0153] Six single colonies from the plate were inoculated into LB broth and incubated at 37°C for 2 hours. Colony PCR was performed using the bacterial culture as a template and ST-VP2-F and ST-VP2-R as primers. The PCR products were verified by gel electrophoresis to determine the size of the target gene. The correctly identified bacterial cultures were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Strains with correct sequence and ligation direction were selected for preservation. The constructed baculovirus transfer vector pF-ST-VP2 containing the target gene is shown below. Figure 4 As shown.

[0154] Example 3 Construction of recombinant baculovirus plasmids Bacmid-SC-E2 and Bacmid-ST-VP2

[0155] 1. DH10Bac transformation

[0156] Take 1 μL each of pF-SC-E2 and pF-ST-VP2 plasmids from Examples 1 and 2, add them to 100 μL of DH10Bac competent cells, mix well, incubate on ice for 30 minutes, subject to heat shock at 42°C for 90 seconds, then incubate on ice for 2 minutes. Add 900 μL of LB liquid medium without Amp and incubate at 37°C for 5 hours. Take 100 μL of bacterial culture, dilute it 81 times, and spread 100 μL of the diluted bacterial culture onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG. Incubate at 37°C for 48 hours.

[0157] 2. Monoclonal selection and identification

[0158] Large white colonies were picked using an inoculation needle and streaked onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG. The cultures were incubated at 37°C for 48 hours. Single colonies were then picked and inoculated onto LB liquid medium containing gentamicin, kanamycin, and tetracycline for further culture. The bacterial strain was preserved, and plasmids were extracted. Recombinant plasmids Bacmid-SC-E2 and Bacmid-ST-VP2 were obtained. After extracting the genome of positive Bacmid-SC-E2 and Bacmid-ST-VP2, PCR amplification was performed using universal primers M12-F / R, as shown below. Figure 5 , Figure 6 As shown, a specific band appears at approximately 3762 bp for Bacmid-SC-E2 and at approximately 4143 bp for Bacmid-ST-VP2, consistent with the expected results, indicating that the two genes have been integrated into the recombinant baculovirus Bacmid.

[0159] Figure 5 In the table, M represents the molecular weight standard of DNA, 1-5 represent rBac-SC-E2, and 6 represents the negative control.Figure 6 In the table, M represents the molecular weight standard of DNA, 1-5 represent rBac-ST-VP2, and 6 represents the negative control.

[0160] Example 4: Transfection with recombinant baculovirus

[0161] In a six-well plate, each well is inoculated with 0.8 × 10⁸ g of seed. 6 Sf9 cells were collected, with a confluence of 50%–70%. For each well, the following complex was prepared: 4 μL of Cellfectin transfection reagent was diluted with 100 μL of transfection medium T1 and briefly vortexed; 3 μg of recombinant Bacmid-SC-E2 and recombinant Bacmid-ST-VP2 plasmids from Example 3 were diluted with 100 μL of transfection medium T1, and the diluted transfection reagents and plasmids were mixed separately and gently blown to prepare the transfection mixture. A control was prepared by transfecting Bacmid plasmids without the target gene. After cell attachment, the above transfection complex was added, and the cells were incubated at 27°C for 5 hours. The supernatant was removed, 2 mL of fresh SF-SFM medium was added, and the cells were incubated at 27°C for 4–5 days before harvesting the supernatant. Recombinant baculoviruses rBac-SC-E2 and rBac-ST-VP2 were obtained, respectively. The viral titers of the harvested P1 generation recombinant baculoviruses were determined using the MTT relative potency assay. The viral titer of rBac-SC-E2 was 7.5 × 10⁻⁶. 8.3 The pfu / mL and rBac-ST-VP2 virus titer were 3.7 × 10⁻⁶ pfu / mL. 8.5 pfu / mL. Recombinant baculoviruses rBac-SC-E2 and rBac-ST-VP2 were amplified as seed viruses for later use.

[0162] Example 5: SDS-PAGE Detection

[0163] SDS-PAGE analysis was performed on the cell cultures of rBac-SC-E2 and rBac-ST-VP2 harvested in Example 4 and the control group. Sf9 cells infected with empty baculovirus were used as a negative control. The specific procedure was as follows: 40 μL of harvested cell culture was added to 10 μL of 5× loading buffer, incubated in boiling water for 5 minutes, centrifuged at 12000 rpm for 1 minute, and the supernatant was used for SDS-PAGE gel electrophoresis (12% concentration gel). After electrophoresis, the gel was stained, destained, and the target bands were observed. The results are shown below. Figure 7 , 8 As shown, the expression levels of the two fusion proteins in this embodiment are relatively high. Figure 7In the table, 1 represents 500 μg / ml BSA, 2 represents 250 μg / ml BSA, 3 represents 125 μg / ml BSA, 4 represents 62.5 μg / ml BSA, 5 represents 31.25 μg / ml BSA, M represents the molecular weight standard of the pre-stained protein, 6 represents the supernatant of normal cell culture, 7 represents the supernatant of empty virus culture, and 8 represents the culture of F1 generation virus. Figure 8 In the table, 1 represents 500 μg / ml BSA, 2 represents 250 μg / ml BSA, 3 represents 125 μg / ml BSA, 4 represents 62.5 μg / ml BSA, 5 represents 31.25 μg / ml BSA, M represents the molecular weight standard of the pre-stained protein, 6 represents the supernatant of normal cell culture, 7 represents the supernatant of empty virus culture, and 8 represents the culture of F1 generation virus.

[0164] Example 6: Detection by Western blotting

[0165] The product obtained from SDS-PAGE electrophoresis in Example 5 was transferred onto an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with porcine anti-E2 and VP2 positive sera for 2 hours each, rinsed, incubated with HRP-labeled goat anti-porcine polyclonal antibody secondary antibody for 2 hours, rinsed, and then an enhanced chemiluminescent fluorescent substrate was added. Images were then taken using a chemiluminescence imaging system. The results are shown below. Figure 9 , 10 As shown, the recombinant baculovirus expression sample had the target band, while the negative control did not, indicating that the target protein was correctly expressed in Sf9 cells. Figure 9 In the table, M represents the molecular weight standard (Marker) of the pre-stained protein, 1 represents the supernatant of normal cell culture, 2 represents the supernatant of empty virus culture, and 3 represents the F1 generation virus culture. Figure 10 In the table, M represents the molecular weight standard of the pre-stained protein, 1 represents the supernatant of normal cell culture, 2 represents the supernatant of empty virus culture, and 3 represents the F1 generation virus culture.

[0166] Example 7 Indirect Immunofluorescence Detection

[0167] Add 100 µl of Sf9 cell suspensions transfected with rBac-SC-E2 and rBac-ST-VP2 to each well of a 96-well cell culture plate (cell concentration 2.5 × 10⁻⁶ cells / well). 5 ~4.0×10 5 Sf9 cells / ml were seeded in 4 wells and incubated at 27°C for 15 minutes to allow them to adhere to the bottom of the culture plate. Then, 10 µL of a 10-fold diluted virus was added to each well. A blank cell control was also included. After seeding, the cells were incubated at 27°C for 72–96 hours. The culture medium was discarded, and the cells were fixed with cold methanol / acetone (1:1). The cells were first reacted with porcine anti-E2 and anti-VP2 polyclonal antibodies, and then with FITC-labeled goat anti-porcine IgG. The results were observed under an inverted fluorescence microscope. Figure 11As shown, no fluorescence was observed in Sf9 cells inoculated with empty baculovirus, while fluorescence was observed in Sf9 cells inoculated with recombinant baculovirus, indicating that the target antigen protein was correctly expressed in Sf9 cells and the recombinant baculovirus was correctly constructed. Figure 11 In the table, 1 represents rBac-SC-E2, 2 represents rBac-ST-VP2, and 3 represents an empty baculovirus control.

[0168] Example 8: Serum-free suspension culture of insect cells in a bioreactor

[0169] Sf9 insect cells were aseptically cultured in 1000 mL shake flasks for 3–4 days until the concentration reached 3 × 10⁻⁶. 6 ~5×10 6 When the cells are at a concentration of 3 × 10⁶ cells / ml and viability is greater than 95%, they are seeded into a 5L bioreactor at a seeding concentration of 3 × 10⁶ cells / ml. 5 ~8×10 5 Cells / ml. When the cell concentration reaches 3 × 10⁶ cells / ml. 6 ~5×10 6 When the cell density reaches 100 cells / ml, the cells are seeded into a 50L bioreactor and allowed to grow to a concentration of 3×10⁻⁶ cells / ml. 6 ~5×10 6 Cells / ml were seeded into a 500L bioreactor, and the cell concentration was increased to 2×10⁻⁶ cells / ml. 6 ~5×10 6 At a cell / ml concentration, rBac-SC-E2 was inoculated, and the reactor culture conditions were pH 6.0–6.5, temperature 25–27°C, dissolved oxygen 30%–80%, and stirring speed 40–80 r / min. Considering the optimal conditions for cell culture, the preferred settings were pH 6.2, cell culture temperature 27°C, dissolved oxygen 50%, and stirring speed 50–70 r / min. After culturing for 5–9 days post-infection, a final concentration of BEI (1 / 1000) was added, and the mixture was incubated at 37°C for 48 hours. Then, a final concentration of Na2S2O3 (2 / 1000) was added to terminate the inactivation. The cell culture supernatant was harvested by centrifugation or hollow fiber filtration and stored as rBac-SC-E2 protein stock solution at 2–8°C. Simultaneously, protein stock solutions expressing rBac-ST-VP2 and various control groups were prepared using the same method.

[0170] Example 9 Protein purification and assembly

[0171] 1. Protein purification

[0172] The sample supernatant was used for SP-FF ion exchange and gradient elution of proteins to determine the optimal elution concentration and maximum loading capacity. The aforementioned rBac-SC-E2 and rBac-ST-VP2 protein stock solutions were centrifuged at 9000 rpm and 4°C for 30 min, and the supernatant was collected. For gradient elution with ion exchange, the equilibration buffer was 10 mM PB, pH 6.0. After loading 5 CV, the sample was washed with the equilibration buffer for 10 CV. The elution gradient was 30 mM NaCl, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, 250 mM NaCl, 500 mM NaCl, and 1 M NaCl. CIP regeneration of the packing material was performed according to the instruction manual. The sample was then subjected to SDS analysis. Figure 12 , 13 As shown, there are no obvious bands in the 30mM~100mM elution (lanes 8-10), while the target protein is present in the 150mM~1M elution (lanes 11-14) with high purity. Figure 12 In the table, 1 represents 500 μg / ml BSA, 2 represents 250 μg / ml BSA, 3 represents 125 μg / ml BSA, 4 represents 62.5 μg / ml BSA, 5 represents 31.25 μg / ml BSA, M represents the molecular weight standard of the pre-stained protein, 6 represents the rBac-SC-E2 protein stock solution, 7 represents the flow-through buffer, 8 represents the 30 mM NaCl elution buffer, 9 represents the 50 mM NaCl elution buffer, 10 represents the 100 mM NaCl elution buffer, 11 represents the 150 mM NaCl elution buffer, 12 represents the 250 mM NaCl elution buffer, and 13 represents the 500 mM NaCl elution buffer. Figure 13 In the table, 1 represents 500 μg / ml BSA, 2 represents 250 μg / ml BSA, 3 represents 125 μg / ml BSA, 4 represents 62.5 μg / ml BSA, 5 represents 31.25 μg / ml BSA, M represents the molecular weight standard of the pre-stained protein, 6 represents the rBac-ST-VP2 protein stock solution, 7 represents the flow-through buffer, 8 represents the 30 mM NaCl elution buffer, 9 represents the 50 mM NaCl elution buffer, 10 represents the 100 mM NaCl elution buffer, 11 represents the 150 mM NaCl elution buffer, 12 represents the 250 mM NaCl elution buffer, and 13 represents the 500 mM NaCl elution buffer.

[0173] 2. Covalent assembly of SC-E2 and ST-VP2 proteins

[0174] The purified SC-E2 and ST-VP2 proteins were mixed at a molar ratio of 1:1 and incubated at 25°C for 6 hours. Then, 5× loading buffer was added, and the reaction was terminated by boiling in a water bath for 10 minutes. Assembly efficiency was assessed using 4%–20% SDS-PAGE, and the results are as follows: Figure 14As shown. The E2-VP2 covalently linked product was identified by Western blotting [transfer, 90V for 120 min; blocking overnight with 10% skim milk at 4℃; adding His-tag mouse monoclonal antibody (1:1000 dilution), incubating at 37℃ for 1 h; adding HRP-labeled goat anti-mouse IgG (1:1000 dilution), incubating at room temperature for 1 h]. The results are as follows. Figure 15 As shown, the E2-VP2 covalently linked product was observed by transmission electron microscopy, and the results are as follows. Figure 16 As shown.

[0175] Figure 14 In the table, 1 represents 500 μg / ml BSA, 2 represents 250 μg / ml BSA, 3 represents 125 μg / ml BSA, 4 represents 62.5 μg / ml BSA, 5 represents 31.25 μg / ml BSA, M represents the molecular weight standard of the pre-stained protein, 6 represents SC-E2 protein, 7 represents ST-VP2 protein, and 8 represents E2-VP2 protein. Figure 15 In the table, M represents the molecular weight standard of the pre-stained protein, 1 represents the primary antibody (pig anti-E2 serum), 2 represents the primary antibody (pig anti-VP2 serum), and 3 represents the negative control.

[0176] Example 10: Agar Spectroscopy Detection

[0177] The titers of expressed SC-E2 and ST-VP2 proteins were detected using the agarose gel diffusion method. A quincunx pattern of wells was created on an agarose gel plate. CSFV and PPV standard sera were added to the center of each well, and the expressed antigens diluted to the powers of 2 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) were added around the wells. After incubation upside down for 72 hours, the precipitation line was observed. The highest dilution ratio at which the precipitation line appeared was the agarose gel diffusion titer. The agarose gel diffusion titer results were as follows: SC-E2 protein titer was 1:256, and ST-VP2 protein titer was 1:128.

[0178] Example 11 Vaccine Preparation

[0179] The E2-VP2 protein stock solution harvested in Example 9 was used to prepare a vaccine. The specific operation was as follows: an appropriate amount of recombinant protein stock solution was mixed in equal proportion and added to Montanide ISA 206 adjuvant. The mixture was emulsified at a speed of 800-1200 r / min and at 28-32°C for 20-30 minutes, so that the protein concentration in the final emulsified vaccine was 50 µg / ml. After emulsification and quality inspection, the vaccine was stored at 2-8°C.

[0180] Example 12 Immunopotency Test

[0181] 1. Antibody level detection

[0182] Twenty gilts aged 150–160 days were randomly divided into four groups: A: E2-VP2 combined vaccine immunization group; B: E2 single vaccine immunization group; C: VP2 single vaccine immunization group; D: control group. In group A, each gilt received an intramuscular injection of 2 mL of the prepared combined vaccine in the neck; in group B, each gilt received an intramuscular injection of 2 mL of vaccine prepared from a single E2 protein in the neck (preparation method as in Example 11); in group C, each gilt received an intramuscular injection of 2 mL of vaccine prepared from a single VP2 protein in the neck; and in group D, the control group received 2 mL of physiological saline. All gilts were isolated and raised under the same conditions. A booster immunization was administered three weeks after the initial immunization, using the same dose and route. Blood was collected from the vena cava before the initial immunization and at 14, 28, and 42 days after the initial immunization. Serum was separated and analyzed using TCID50. 50 The CSFV neutralizing antibody titers of each group were detected using the method, and the results are shown in Table 9. The E2-VP2 combination vaccine immunization group and the E2 monotherapy group showed positive neutralizing antibody titers 14 days after immunization. 42 days after the first immunization, the neutralizing antibody titer (1:256) in the E2-VP2 combination vaccine immunization group was higher than that in the E2 monotherapy group (1:128). The PPV HA titers of each group were detected using the HA-HI method. The antibody titers of the E2-VP2 combination vaccine immunization group and the VP2 monotherapy group were positive 14 days after immunization. 42 days after the first immunization, the neutralizing antibody titer (1:256) in the E2-VP2 combination vaccine immunization group was higher than that in the VP2 monotherapy group (1:128). The results are shown in Table 10.

[0183] CSFV neutralizing antibody titer detection method:

[0184] ① ST cell suspension preparation: Confluent, well-formed ST cells were digested with 0.25% trypsin to prepare a cell suspension, and the cell density was adjusted to 3.0 × 10⁻⁶ cells / year. 5 ~5.0×10 5 cells / ml.

[0185] ② Serum inactivation: Place the serum to be tested, negative serum and positive serum in a 56℃ water bath for 30 minutes.

[0186] ③ Serum dilution and neutralization: Take a 96-well cell culture plate and add 50 µl of DMEM culture medium containing 5% bovine serum to each well. Then, add 50 µl of the serum to be tested to the first well and mix well. Take another 50 µl and add it to the second well, mix well, and then take another 50 µl and add it to the third well, and so on, until the tenth well (discard the 50 µl mixture). The serum dilutions are 1:2, 1:4, 1:8 to 1:1024. Each serum dilution should be performed in quadruplicate. Add 200 TCID50 solution to each well. 50 Add 0.1 ml of cytotoxic virus solution of classical swine fever rabbit-attenuated live vaccine strain C to serum wells of different dilutions (to determine potency before use), 50 µl / well, and incubate at 37°C for 1 hour.

[0187] ④ Control: Set up 4 viral regression controls at 100 TCID titers. 50 / 0.1ml, 10TCID 50 / 0.1ml, 1TCID 50 / 0.1ml, 0.1TCID 50 / 0.1ml), each titer is prepared in 4 wells, with 100µl of virus solution per well. Simultaneously, 4–8 wells are set up as blank cell controls, with 100µl of maintenance solution added to each well. Negative and positive serum controls are also included (50µl of undiluted serum sample mixed with an equal volume of DMEM maintenance solution), repeated in 4 wells, and incubated at 37°C for 1 hour.

[0188] ⑤ Inoculation: After incubation, add 100µl of ST cell suspension to each well using a multichannel pipette, and incubate in a 37.0℃±0.5℃ 5% CO2 incubator for 72-96 hours, and perform immunofluorescence assay.

[0189] ⑥ Cell fixation: Discard the culture medium in the cell plate, wash the cells once with PBS (0.01 mol / L, pH 7.20-7.40), 200 µl / well, 5 minutes each time; add pre-cooled methanol / acetone (methanol:acetone = 1:1) fixative, 100 µl / well, and incubate at 2-8℃ for 40 minutes.

[0190] ⑦ Add primary antibody: Discard the fixative, wash 3 times with PBS, 200 µl / well, 5 minutes each time. Add an appropriate concentration of primary antibody (swine fever virus monoclonal antibody), 50 µl / well, and incubate at 37°C in a humidified chamber for 90 minutes.

[0191] ⑧ Add secondary antibody: Discard the liquid in the wells, wash 3 times with PBS containing 5% skim milk, 200 µl / well, 5 minutes each time. Add an appropriate concentration of secondary antibody (FITC-labeled goat anti-mouse antibody), 50 µl / well, incubate at 37°C in a humidified chamber for 1 hour, discard the liquid in the wells, and wash 5 times with PBS.

[0192] ⑨ Microscopic observation: The cell culture plates were observed under a fluorescence microscope. Wells with specific green fluorescence were identified as virus-infected wells, and wells without specific green fluorescence were identified as uninfected wells.

[0193] ⑩ Experimental conditions: (1) 100 TCID 50 / 0.1ml should show a specific green fluorescence throughout, 0.1TCID 50(1) No green specific fluorescence should appear in 0.1 ml; (2) No green specific fluorescence should appear in the cell control wells; (3) No green specific fluorescence should appear in the positive serum cell wells; (4) Green specific fluorescence should appear in the negative serum cell wells. The test is then valid. Record the number of wells with and without green specific fluorescence at each dilution of serum sample, and calculate the serum neutralizing antibody titer according to the half-maximal effect (Reed-Muench method). The neutralizing antibody titer in the non-immunized control group should not be higher than 1:4, and the neutralizing antibody titer in the immunized group should not be lower than 1:32.

[0194] 2) PPV HI antibody titer detection method:

[0195] ①PPV HA potency testing:

[0196] Add 50 µl of PBS to each well of a 96-well V-plate. Add 50 µl of PPV stock solution to well 1, mix thoroughly, then add 50 µl to well 2. Serially dilute up to well 11, discarding the 50 µl solution at each dilution. Well 12 serves as the PBS control well. Add 50 µl of 1% guinea pig red blood cell suspension to each well, mix well on a micro-shaker, and incubate at room temperature for 60 minutes before observing the results. Results are determined when no red blood cell agglutination occurs in the PBS control wells. The hemagglutination titer of the sample is the highest dilution factor at which 100% red blood cell agglutination occurs.

[0197] Result determination:

[0198] 100% agglutination (++++): Agglutinated red blood cells form a thin film that evenly covers the bottom of the pore; when agglutinated strongly, they shrink into clumps.

[0199] 75% agglutination (+++): Agglutinated red blood cells cover the bottom of the well relatively evenly, with very few red blood cells settling.

[0200] 50% agglutination (++): Agglutinated red blood cells cover the bottom of the well, but a small number of red blood cells settle into small dots in the center.

[0201] 25% agglutination (+): Red blood cells settle in the center of the well bottom, but there are still scattered red blood cell agglutinations around them.

[0202] 0% agglutination (-): All red blood cells are settled in the center of the bottom of the well, with no scattered red blood cell agglutination around them.

[0203] ②Preparation of 4HA unit antigen:

[0204] Dilute the antigen solution for determining HA titer to 4HA / 25µl. If the HA titer is 1:256, the dilution factor for the four hemagglutinin units of antigen should be 1:64. Add 0.1ml of hemagglutinin antigen to 6.3ml of PBS to achieve a final concentration of 1:64. Take 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, and 0.6ml of PBS (0.01mol / L, pH 7.40), and add 0.1ml of the prepared 1:64 virus dilution solution to achieve final concentrations of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7, respectively. Take 25µl of the virus dilution solution from each dilution, and add 25µl of PBS (0.01mol / L, pH 7.20–7.40) and 25µl of 0.5% guinea pig red blood cell suspension sequentially. Mix well and let stand at room temperature for 30–60 minutes. Observe the agglutination endpoint and adjust the hemagglutinin dilution appropriately based on the results to ensure that the working solution is 4 HA units.

[0205] ③ Hemagglutination inhibition test:

[0206] Add 25 µl of PBS (0.01 mol / L, pH 7.20–7.40) to wells 2–11 of a 96-well V-type microplate, and 100 µl of PBS (0.01 mol / L, pH 7.20–7.40) to well 12. Add 25 µl of the treated serum sample to well 1 (if well 1 is positive, the HI titer of the serum is 1:8). Add another 25 µl of serum to well 2, mix thoroughly, and then add 25 µl to well 3. Serially dilute up to well 10, discarding 25 µl from well 10. Set up one row of negative controls and two rows of positive controls, treated and diluted in the same way as the serum samples. Add 25 µl of viral antigen solution containing 4 HA units to wells 1–11 and incubate at room temperature for 60 minutes. Add 25 µl of 1% guinea pig red blood cell suspension to wells 1–12, mix gently, and let stand at room temperature for about 120 minutes. Determine the result when the control red blood cells settle to the bottom of the well in a button-like shape. The highest dilution of serum that completely inhibits the four hemagglutination units of antigen is taken as the hemagglutination inhibition (HI) titer of the test serum.

[0207] The test result is valid only if the serum titer of the negative serum control well is not higher than 1:8, and the serum titer error between the two rows of positive serum control wells does not exceed one titer. The highest dilution of serum in which erythrocyte agglutination is completely inhibited is taken as the hemagglutination inhibition (HI) titer of that serum. A negative test is defined as an HI titer not higher than 1:8, and a positive test is defined as an HI titer lower than 1:16.

[0208] Result determination:

[0209] 00% Inhibition (++++): All red blood cells settled in the center of the bottom of the well, with no scattered red blood cell aggregation around them.

[0210] 75% Inhibition (+++): Red blood cells settle in the center of the well bottom, but there are still scattered red blood cell agglutinations around them.

[0211] 50% Inhibition (++): Agglutinated red blood cells cover the bottom of the well, but a small number of red blood cells settle into small dots in the center.

[0212] 25% Inhibition (+): Agglutinated red blood cells cover the bottom of the well relatively evenly, with very few red blood cells settling.

[0213] 0% Inhibition (-): Agglutinated red blood cells form a thin film that evenly covers the bottom of the pore; when there is strong agglutination, they shrink into clumps.

[0214] Table 9. CSFV neutralizing antibody titer

[0215]

[0216] Table 10 PPV HI antibody titer

[0217]

[0218] As can be seen in the embodiments of this application, the bivalent vaccine design based on CSFV-E2 and PPV-VP2 dual antigens achieves a CSFV neutralizing antibody titer of 1:256 and a PPV HI antibody titer of 1:256 42 days after vaccination, with protection rates exceeding 90% against both single and mixed infections. This represents an efficiency improvement of over 50% compared to "single vaccine combined immunization," achieving highly efficient synergistic prevention and control. This is highly beneficial for reducing the number of immunizations, avoiding stress reactions caused by frequent vaccinations, and reducing labor and vaccine procurement costs, thereby significantly reducing the burden on farmers.

[0219] Meanwhile, in this application embodiment, the immunogenicity enhancement technology of SpyCatcher / SpyTag covalent assembly combined with VLPs is adopted. Eukaryotic expression ensures correct protein folding, and VLPs and covalent assembly fully expose the antigen epitopes, thereby obtaining high immunogenicity and stable antigen structure. The agar amplification titer reaches SC-E2 1:256 and ST-VP2 1:128. The antibody production time is earlier than that of prokaryotic expression vaccines, and the covalent connection avoids the separation of two antigens. The quality stability is better than that of existing mixed bivalent vaccines.

[0220] Furthermore, this application employs a baculovirus-insect cell system for the soluble secretory expression of recombinant proteins. Combined with the eukaryotic secretion mechanism of Sf9 insect cells, codon optimization of the recombinant proteins allows for the natural secretion of recombinant SC-E2 protein (containing a SpyCatcher tag) and recombinant ST-VP2 protein (containing a SpyTag tag) into the cell culture supernatant after expression. By optimizing cell culture conditions and harvesting points, the target protein crude product can be obtained directly from the supernatant without cell lysis, reducing interference from impurities caused by cell lysis and significantly improving the purity of the crude product. This simplifies subsequent purification steps, requiring only ion exchange chromatography, greatly reducing the difficulty and cost of the process. Moreover, relying on the eukaryotic modification capabilities of insect cells, the secreted protein can achieve correct folding and necessary modifications, ensuring full exposure of its antigenic epitopes and effectively guaranteeing protein activity. This provides a solid foundation for subsequent dual-antigen covalent assembly and vaccine immunogenicity.

[0221] In summary, this invention successfully constructed a bivalent subunit vaccine by expressing recombinant CSFV-E2 and recombinant PPV-VP2 antigens separately using a recombinant baculovirus system and employing SpyCatcher / SpyTag covalent assembly technology. The use of an insect cell eukaryotic expression system ensured the correct folding of the antigen proteins, while the self-assembly of the VP2 protein to form virus-like particles (VLPs) significantly enhanced the immunogenicity of the vaccine. The vaccine of this invention possesses multiple advantages, including high safety (containing no intact virus or genetic material), strong immunogenicity (inducing high-titer neutralizing antibodies), high prevention and control efficiency (one injection prevents two diseases), controllable process (suitable for large-scale suspension culture), and simple production. In particular, it can stimulate specific humoral immunity against classical swine fever virus (CSFV) and porcine parvovirus (PPV), effectively preventing single and mixed infections of the two viruses, thus providing a highly efficient disease prevention solution for large-scale pig farms.

[0222] It should be understood that the above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A recombinant protein covalent linker, characterized in that: The linker is formed by covalent connection of a first recombinant protein and a second recombinant protein in a molar ratio of 1:1, wherein the second recombinant protein self-assembles into a virus-like particle; the first recombinant protein is encoded by a first gene with a sequence as shown in SEQ ID NO: 1, the second recombinant protein is encoded by a second gene with a sequence as shown in SEQ ID NO: 2, and the first recombinant protein and the second recombinant protein are both expressed by a baculovirus-insect cell expression system.

2. The method of producing a recombinant protein conjugate according to claim 1, wherein, The method comprises: S1, respectively preparing nucleic acid molecules for encoding a first recombinant protein and a second recombinant protein; S2, constructing a recombinant vector, cloning the nucleic acid molecules of step S1 into a shuttle vector respectively to obtain a recombinant shuttle vector containing a target gene, the shuttle vector comprising pFastBac 1, pVL1393, pFastBac dual or pDEST8; S3, transforming competent cells with the recombinant shuttle vector to obtain a recombinant plasmid, and then transfecting host cells respectively to obtain a recombinant baculovirus, the host cells comprising Sf9, High Five or Sf21 cells; S4, inoculating the recombinant baculovirus into host cells to obtain an expression product, i.e., the first recombinant protein and the second recombinant protein, and then mixing the first recombinant protein and the second recombinant protein in a molar ratio of 1:

1.

3. An immunological composition, characterized in that, The method comprises: The recombinant protein covalent linker of claim 1; and a pharmaceutically acceptable carrier.

4. The immunological composition of claim 3, wherein: The pharmaceutically acceptable carrier comprises any one or a combination of two or more of MONTANIDE ISA 206VG, MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, plant cell agglutinin.

5. Use of the recombinant protein covalent linker of claim 1 or the immunological composition of any one of claims 3-4 in the manufacture of a medicament for inducing an immune response in a subject animal against infection with porcine pestivirus and / or porcine parvovirus.

6. Use of the recombinant protein covalent linker of claim 1 or the immunological composition of any one of claims 3-4 in the manufacture of a medicament for preventing infection of an animal with porcine pestivirus and / or porcine parvovirus.

7. Use of the recombinant protein covalent linker of claim 1 or the immunological composition of any one of claims 3-4 in the preparation of a porcine pestivirus and porcine parvovirus bivalent subunit vaccine.

Images