A method for enhancing the purity and immunogenicity of prv antigen and vaccine thereof

Abstract

This invention relates to the field of veterinary biological products technology, and discloses a method and vaccine for enhancing the purity and immunogenicity of PRV antigens. The method includes the following steps: piglets or mummified fetuses from diseased pig farms are taken as pathogenic material, added to PBS, freeze-thawed, and then centrifuged to obtain the PRV seed virus strain; the PRV seed virus strain is amplified by PCR and its gene sequenced; the constructed pCMVPRVgD-1 and pCMVPRVgB-2 plasmids are transfected into CHO-KS cells; gB and gD cell lines are cloned and screened using glutamine-free CHO medium supplemented with L-methionine sulfoxide imide to obtain cells stably expressing gB and gD antigens; the gB and gD antigen-expressing cells are cultured in a bioreactor, and the His-tagged gD and gB antigens are harvested, clarified, purified by nickel column affinity chromatography, and treated with imidazole to obtain a high-purity, high-content antigen solution. This invention can improve the content and purity of gB and gD antigens in PRV strains, which is beneficial for the prevention and eradication of porcine pseudorabies.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and in particular to a method for enhancing the purity and immunogenicity of PRV antigen and a vaccine thereof. Background Technology

[0002] Pseudorabies virus (PRV) is a member of the genus Varicellavirus in the family α-herpesviridae. Its genome is a linear double-stranded DNA composed of unique long regions (UL regions), unique short regions (US regions), internal repeat sequences (IR), and terminally inverted repeat sequences (TR). The PRV genome contains approximately 70-100 protein-coding genes. The viral genome encodes 11 glycoproteins, among which gB, gD, gH, gL, and gK play important roles in viral replication and are essential PRV glycoproteins. gB protein participates in the fusion of viral particles with cells and plays a crucial role in the intercellular transport of viral particles. In nerve conduction, gB protein mediates the transmission of viral particles via axons to the next level of neurons. gB protein is also a major immunogenic protein of PRV, stimulating the body to produce neutralizing antibodies, which can be complement-dependent or complement-independent. gD protein is also an envelope protein and one of the major immunogenic proteins of PRV, primarily existing in an O-glycosylated form. During viral invasion of host cells, the gD protein specifically recognizes and binds to receptors on the surface of host cells, thereby mediating viral fusion. While the gD protein is essential for viral replication and cell fusion, its involvement is not required for viral transport between cells. The gD protein can induce effective immune protection, and the resulting antibodies function independently of complement. Therefore, gB and gD antigens are important candidate antigens in research on subunit vaccines, nucleic acid vaccines, and therapeutic monoclonal antibodies.

[0003] Currently, immunization is the main measure for PRV control. In my country, the BarthaK61 attenuated vaccine strain is widely used. Simultaneously, gE antibody ELISA and PCR methods such as quantitative real-time PCR based on the PRV gE gene can be used to distinguish between infected and immunized animals. Based on detection and monitoring results, the goal is to gradually eliminate PRV wild-type virus infection from pig herds and achieve PRV eradication. Therefore, on the one hand, it is necessary to develop candidate vaccine strains targeting PRV variants; on the other hand, the application of these candidate vaccine strains must be compatible with the corresponding DIVA strategy. Therefore, constructing gene-deleted viruses using PRV variants as parents is essential.

[0004] Regarding PRV vaccines, inactivated vaccines, attenuated vaccines, and gene-deleted attenuated vaccines for porcine pseudorabies have been developed both domestically and internationally. The development of these vaccines is relatively mature. However, viral vector recombinant vaccines, nucleic acid vaccines, and subunit vaccines are still in the laboratory research stage. Currently, the more commonly used live vaccines suffer from problems such as susceptibility to interference and unstable antibody titers. Traditional PRV vaccines have low antibody titers (typically ELISA titers ≤ 1:500), while inactivated vaccines induce relatively low antibody levels and cannot provide good protection against circulating strains. Therefore, the development of vaccines with high purity and high levels of neutralizing antibodies is urgently needed. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method and vaccine for enhancing the purity and immunogenicity of PRV antigens, which can increase the content and purity of gB and gD antigens in PRV strains, and is beneficial to the prevention and eradication of porcine pseudorabies.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] In a first aspect, this application provides a method for enhancing the purity and immunogenicity of PRV antigen, comprising the following steps:

[0008] S1. Isolation of PRV virus strain: Piglets or mummified fetuses from diseased pig farms were taken as pathogen material. The pathogen material was cut into small pieces and ground in a mortar (through a 200-mesh sieve or with a particle size of less than 0.5 mm). PBS (PBS volume to tissue weight ratio of 1:1 w / v) was added, and after freeze-thaw, the mixture was centrifuged. The supernatant after centrifugation was filtered and sterilized to obtain the PRV virus strain. S2. Plasmid construction: PRV strains were amplified by PCR and their genes were sequenced. Based on the gene variations of the protective antigens gD and gB in the PRV strain gene sequence, codons were optimized to construct pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression. S3. Antigen cell screening: The pCMVPRVgD-1 and pCMVPRVgB-2 plasmids constructed in step S2 were transfected into CHO-KS cells. The gB cell line and gD cell line clones were screened by adding L-methionine sulfoxide to glutamine-free CHO medium to obtain gB antigen cells and gD antigen cells that express stably. S4. Antigen solution purification: GB antigen cells and GD antigen cells expressing CHO cells are cultured in a bioreactor. GD and GB antigens with His tags are harvested, clarified, purified by nickel column affinity chromatography, and treated with imidazole (dialysis to remove imidazole) to obtain a high-purity, high-content antigen solution.

[0009] Furthermore, in step S1, the freeze-thaw process with PBS is repeated 3-5 times, followed by centrifugation. During centrifugation, the centrifugation speed is 4000 r / min for 10-20 min.

[0010] Furthermore, in step S2, the codon-optimized gD gene sequence is 5'-ATGGCCGACCCCAAC-3', and the gB gene sequence is 5'-ATGCGCGTCGACCTG-3'.

[0011] Furthermore, in step S2, the specific steps for constructing the pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression include: S21. Add the constructed enzyme digestion system and plasmid vector to the bacterial culture after PCR amplification of the PRV virus strain, and incubate for 3-5 hours. S22. After incubation, the bacterial culture was subjected to electrophoresis on a 1% agarose gel. S23. The agarose gel was used for separation and extraction to obtain pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression.

[0012] Furthermore, in step S21, the enzyme digestion system is: 10 μL L AstDigest buffer, 1 μL BamHI, 1 μL LecoRI, 5 μL PCR product, and ddH2O added to 50 μL.

[0013] Furthermore, in step S21, the plasmid vector is pCMV-6His with a concentration of 50 ng / μL.

[0014] Furthermore, in step S3, the specific steps for transfecting pCMVPRVgD-1 and pCMVPRVgB-2 plasmids into CHO-KS cells include: S31. Take pCMVPRVgD-1 and pCMVPRVgB-2 plasmids, add them to an EP tube, and add CHO-KS cells at a density of 1×10⁻⁶. 6 (cells / mL), gently tap the EP tube wall to mix thoroughly, then let stand at room temperature for 15 minutes; S32. Transfer the plasmid and cell mixture to an electroporation cuvette and perform electroporation (voltage 250V, capacitance 500μF). After electroporation, immediately transfer the cells to a cell shaker and add glutamine-free CHO medium. After suspension culture for 48 hours, transfected CHO-KS cells are obtained.

[0015] Furthermore, the specific steps of clone screening in step S3 are as follows:

[0016] S33. Centrifuge the cell suspension from step S32 at 1000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in glutamine-free CHO medium until the cell density reaches 0.6 × 10⁶ cells / min. 6 Cells / mL, pressurized screening of single-clonal cell lines; S34, take transfected cells, dilute cells to 5 cells / ml with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imine, add 200 μl / well to 96-well plates, incubate at 37℃, 5% CO2 cell culture incubator for 4-6 h, and record the wells of single cells;

[0017] S35. Once cell colonies have formed in the recording wells, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA, digest at room temperature for 2 min, add 2 ml of glutamine-free CHO medium (containing 50 μM L-methionine sulfoxide imine) to terminate the digestion, and use a pipette to disperse the cells. Transfer the cells to a 12-well plate for further culture. Once the cells in the 12-well plate have grown into a confluent monolayer, collect the supernatant for ELISA detection to screen for positive monoclonal cell lines. Continue to expand the culture of the highly expressed positive monoclonal cell lines, freeze them, and obtain the F0 generation cell line. S36. Dilute the F0 generation cell line to 3.5-5 × 10⁻⁵ mg / L glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imide. 5 Cells / mL were cultured for 3 days in a shaker at 37℃ and 100 rpm with 5% CO2, and glucose content was measured. Glucose was then added to a final concentration of 6 g / L, followed by further culture with 5% feed medium. When the cell density reached 10⁻¹² × 10⁻¹⁰ cells / mL, the culture was continued. 6 When the cells / ml reached a certain level, the temperature was lowered to 33℃. The glucose content was then measured daily and glucose was added to 6 g / L. 5% feed culture medium was added every other day. Samples were taken every 24 hours, 1 mL each time. Cell count and viability were calculated under a microscope using trypan blue staining. The cell supernatant was then detected by SDS-PAGE and Western blotting. After culturing at a lower temperature for 9 days, the supernatant was harvested and subjected to SDS-PAGE to obtain cells stably expressing gB antigen and gD antigen.

[0018] Furthermore, in step S4, during clarification, the antigen is filtered through a hollow fiber with a pore size of 0.65 μm. After clarification, it is purified by nickel column affinity chromatography to obtain purified antigen. During purification, washing buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) are used. After the purified antigen is dialyzed to remove imidazole (dialyzed in PBS for 24 hours), it is filtered through a 0.22 μm sterile filter and stored at 2-8°C.

[0019] Secondly, this application also provides a vaccine, which is prepared by using the enhanced PRV antigen obtained by the method described above for enhancing the purity and immunogenicity of PRV antigen, and adjuvants of the prior art to prepare a highly effective PRV gD and gB antigen vaccine.

[0020] The solution adopted in this application has the following effects:

[0021] 1. This invention uses repeated freeze-thaw cycles with PBS combined with centrifugation to completely release viral particles and remove impurities (mechanism: freeze-thaw cycles destroy cell structure, centrifugation removes large molecular interference); combined with codon optimization and adaptation of the CHO system, it significantly improves the translation efficiency of gD / gB proteins.

[0022] 2. The present invention combines nickel column affinity chromatography with gradient imidazole elution and dialysis to remove residual imidazole. The His tag specifically binds to the nickel column to remove host cell protein impurities. Dialysis thoroughly removes imidazole, avoiding interference with antigen conformation, achieving a purity of 95%. The antigen recovery rate is 85%, reducing the loss of active ingredients.

[0023] 3. The technical advantages of this invention include the use of model receptor immune enhancers (such as CpGODN nano-adjuvants) to optimize antigen delivery; the nano-adjuvants enhance the phagocytic efficiency of antigen-presenting cells (APCs); CpGODN activates the TLR9 pathway, stimulating a Th1 immune response; porcine studies show that the neutralizing antibody titer is 2 times higher than that of commercial vaccines. This invention also simultaneously expresses gD and gB antigens, where gD blocks viral invasion and gB induces neutralizing antibodies, providing a dual protective mechanism; eliminating the risk of single antigen escape mutations and covering prevalent PRV strains.

[0024] 4. The present invention uses CHO cell suspension culture to replace insect cell / eukaryotic expression system. CHO cells are easy to ferment on a large scale, reducing production costs. Serum-free culture medium reduces the risk of exogenous factor contamination and can realize standardized process. The parameters of the entire process from plasmid construction, cell screening and purification are standardized, and the batch-to-batch consistency is high, avoiding the risk of incomplete virus inactivation in traditional vaccine production. Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the content disclosed in this specification:

[0026] Example 1

[0027] This embodiment describes a method for enhancing the purity and immunogenicity of PRV antigen, including the following steps:

[0028] S1. Isolation of PRV virus strain: Piglets or mummified fetuses from diseased pig farms were taken as pathogen material. The pathogen material was chopped and ground in a mortar (through a 200-mesh sieve or with a particle size of less than 0.5 mm). PBS (PBS volume to tissue weight ratio of 1:1 w / v) was added, and the mixture was frozen and thawed. After centrifugation, the supernatant was filtered and sterilized to obtain the PRV virus strain. The freeze-thaw process with PBS was repeated 3 times, followed by centrifugation at 4000 r / min for 10 min.

[0029] S2. Plasmid Construction: PRV strains were amplified by PCR and their genes were sequenced. Based on the gene variations of the protective antigens gD and gB in the PRV strain gene sequence, codons were optimized to construct pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression. The optimized gD gene sequence is 5'-ATGGCCGACCCCAAC-3', and the gB gene sequence is 5'-ATGCGCGTCGACCTG-3'.

[0030] The specific steps for constructing pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression include:

[0031] S21. Add the constructed enzyme digestion system (enzyme digestion system is: 10 μL L AstDigest buffer, 1 μL BamHI, 1 μL LecoRI, 5 μL PCR product, and add ddH2O to 50 μL) and plasmid vector (pCMV-6His, concentration 50 ng / μL) to the bacterial culture after PCR amplification of PRV virus strain, and incubate together for 3 h;

[0032] S22. After incubation, the bacterial culture was subjected to electrophoresis on a 1% agarose gel.

[0033] S23. The agarose gel was used for separation and extraction to obtain pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression.

[0034] S3. Antigen Cell Screening: The pCMVPRVgD-1 and pCMVPRVgB-2 plasmids constructed in step S2 were transfected into CHO-KS cells. Clonal screening of gB and gD cell lines was performed using glutamine-free CHO medium supplemented with L-methionine sulfoxide imide to obtain stably expressing gB and gD antigen cells. The specific steps for transfecting pCMVPRVgD-1 and pCMVPRVgB-2 plasmids into CHO-KS cells and clonal screening included:

[0035] S31. Take pCMVPRVgD-1 and pCMVPRVgB-2 plasmids, add them to an EP tube, and add CHO-KS cells at a density of 1×10⁻⁶. 6 (cells / mL), gently tap the EP tube wall 10 times to mix thoroughly, then let stand at room temperature for 15 minutes;

[0036] S32. Transfer the plasmid and cell mixture to an electroporation cuvette and perform electroporation (voltage 250V, capacitance 500μF). After electroporation, immediately transfer the cells to a cell shake flask and add glutamine-free CHO medium. After suspension culture for 48h, transfected CHO-KS cells are obtained.

[0037] S33. Centrifuge the cell suspension from step S32 at 1000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in glutamine-free CHO medium until the cell density reaches 0.6 × 10⁶ cells / min. 6 cells / mL, pressure screening of single clonal cell lines, pressure screening using approximately 0.2 MPa;

[0038] S34. Take the transfected cells and dilute them to 5 cells / ml with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imine. Add 200 μl / well to a 96-well plate and incubate at 37°C in a 5% CO2 cell culture incubator for 4 h. Record the wells of a single cell.

[0039] S35. Once cell colonies have formed in the recording wells, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA, digest at room temperature for 2 min, add 2 ml of glutamine-free CHO medium (containing 50 μM L-methionine sulfoxide imine) to terminate the digestion, and use a pipette to disperse the cells. Transfer the cells to a 12-well plate for further culture. Once the cells in the 12-well plate have grown into a confluent monolayer, collect the supernatant for ELISA detection to screen for positive monoclonal cell lines. Continue to expand the culture of the highly expressed positive monoclonal cell lines, freeze them, and obtain the F0 generation cell line.

[0040] S36. Dilute the F0 generation cell line to 3.5 × 10⁻⁶ with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imide. 5 Cells / mL were cultured for 3 days in a shaker at 37℃ and 100 rpm with 5% CO2, and glucose content was measured. Glucose was then added to a final concentration of 6 g / L, followed by further culture with 5% feed medium. When the cell density reached 10 × 10⁶ cells / mL, the culture was continued. 6 When the cell count reached 6 g / L, the temperature was lowered to 33°C. Glucose content was then measured daily and supplemented to 6 g / L. 5% culture medium was added every other day. Samples (1 mL) were taken every 24 hours. Cell counts and viability were calculated under a microscope using trypan blue staining. The cell supernatant was then analyzed by SDS-PAGE and Western blotting. After 9 days of culture at a lower temperature, the supernatant was harvested and subjected to SDS-PAGE to obtain cells stably expressing gB and gD antigens.

[0041] S4. Antigen Solution Purification: GB and GD antigen-expressing cells were cultured in a bioreactor to harvest His-tagged GD and GB antigens. These antigens were then clarified and purified by nickel-coated affinity chromatography. After imidazole removal (dialysis to remove imidazole), a high-purity, high-content antigen solution was obtained. During clarification, the antigen was filtered through a 0.65 μm hollow fiber filter. Following clarification, the antigen was purified by nickel-coated affinity chromatography using washing buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The purified antigen was then dialyzed (dialyzed in PBS for 24 hours) and stored at 2°C after passing through a 0.22 μm sterile filter.

[0042] Finally, using the enhanced PRV antigen prepared by the method described above for enhancing the purity and immunogenicity of PRV antigen, and adjuvants from the prior art, such as CpGODN nano-adjuvants, a highly efficient PRV gD and gB antigen vaccine was prepared.

[0043] Example 2

[0044] This embodiment describes a second method for enhancing the purity and immunogenicity of PRV antigen, including the following steps:

[0045] S1. Isolation of PRV virus strain: Piglets or mummified fetuses from diseased pig farms were taken as pathogen material. The pathogen material was chopped and ground in a mortar (through a 200-mesh sieve or with a particle size of less than 0.5 mm). PBS (PBS volume to tissue weight ratio of 1:1 w / v) was added, and the mixture was frozen and thawed. After centrifugation, the supernatant was filtered and sterilized to obtain the PRV virus strain. The freeze-thaw process with PBS was repeated 4 times, followed by centrifugation at 4000 r / min for 15 min.

[0046] S2. Plasmid Construction: PRV strains were amplified by PCR and their genes were sequenced. Based on the gene variations of the protective antigens gD and gB in the PRV strain gene sequence, codons were optimized to construct pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression. The optimized gD gene sequence is 5'-ATGGCCGACCCCAAC-3', and the gB gene sequence is 5'-ATGCGCGTCGACCTG-3'.

[0047] The specific steps for constructing pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression include:

[0048] S21. Add the constructed enzyme digestion system (enzyme digestion system is: 10 μL L AstDigest buffer, 1 μL BamHI, 1 μL LecoRI, 5 μL PCR product, and add ddH2O to 50 μL) and plasmid vector (pCMV-6His, concentration 50 ng / μL) to the bacterial culture after PCR amplification of PRV virus strain, and incubate together for 4 h;

[0049] S22. After incubation, the bacterial culture was subjected to electrophoresis on a 1% agarose gel.

[0050] S23. The agarose gel was used for separation and extraction to obtain pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression.

[0051] S3. Antigen Cell Screening: The pCMVPRVgD-1 and pCMVPRVgB-2 plasmids constructed in step S2 were transfected into CHO-KS cells. Clonal screening of gB and gD cell lines was performed using glutamine-free CHO medium supplemented with L-methionine sulfoxide imide to obtain stably expressing gB and gD antigen cells. The specific steps for transfecting pCMVPRVgD-1 and pCMVPRVgB-2 plasmids into CHO-KS cells and clonal screening included:

[0052] S31. Take pCMVPRVgD-1 and pCMVPRVgB-2 plasmids, add them to an EP tube, and add CHO-KS cells at a density of 1×10⁻⁶. 6 (cells / mL), gently tap the EP tube wall 10 times to mix thoroughly, then let stand at room temperature for 15 minutes;

[0053] S32. Transfer the plasmid and cell mixture to an electroporation cuvette and perform electroporation (voltage 250V, capacitance 500μF). After electroporation, immediately transfer the cells to a cell shake flask and add glutamine-free CHO medium. After suspension culture for 48h, transfected CHO-KS cells are obtained.

[0054] S33. Centrifuge the cell suspension from step S32 at 1000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in glutamine-free CHO medium until the cell density reaches 0.6 × 10⁶ cells / min. 6 cells / mL, pressure screening of single clonal cell lines, pressure screening using approximately 0.2 MPa;

[0055] S34. Take the transfected cells and dilute them to 5 cells / ml with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imine. Add 200 μl / well to a 96-well plate and incubate at 37°C in a 5% CO2 cell culture incubator for 5 h. Record the wells of a single cell.

[0056] S35. Once cell colonies have formed in the recording wells, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA, digest at room temperature for 2 min, add 2 ml of glutamine-free CHO medium (containing 50 μM L-methionine sulfoxide imine) to terminate the digestion, and use a pipette to disperse the cells. Transfer the cells to a 12-well plate for further culture. Once the cells in the 12-well plate have grown into a confluent monolayer, collect the supernatant for ELISA detection to screen for positive monoclonal cell lines. Continue to expand the culture of the highly expressed positive monoclonal cell lines, freeze them, and obtain the F0 generation cell line.

[0057] S36. Dilute the F0 generation cell line to 4.2 × 10⁻⁶ with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imide. 5 Cells / mL were cultured for 3 days in a shaker at 37℃ and 100 rpm with 5% CO2, and glucose content was measured. Glucose was then added to bring the concentration to 6 g / L, followed by 5% feed culture medium for continued culturing. When the cell density reached 11 × 10⁶ cells / mL... 6When the cell count reached 6 g / L, the temperature was lowered to 33°C. Glucose content was then measured daily and supplemented to 6 g / L. 5% culture medium was added every other day. Samples (1 mL) were taken every 24 hours. Cell counts and viability were calculated under a microscope using trypan blue staining. The cell supernatant was then analyzed by SDS-PAGE and Western blotting. After 9 days of culture at a lower temperature, the supernatant was harvested and subjected to SDS-PAGE to obtain cells stably expressing gB and gD antigens.

[0058] S4. Antigen Solution Purification: GB and GD antigen-expressing cells were cultured in a bioreactor to harvest His-tagged GD and GB antigens. These antigens were then clarified and purified by nickel-coated affinity chromatography. After imidazole removal via dialysis, a high-purity, high-content antigen solution was obtained. Clarification was performed using a 0.65 μm hollow fiber filter. The clarified antigen was then purified by nickel-coated affinity chromatography using washing buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The purified antigen was then dialyzed (dialyzed in PBS for 24 hours) and stored at 5°C after passing through a 0.22 μm sterile filter.

[0059] Finally, using the enhanced PRV antigen prepared by the method described above for enhancing the purity and immunogenicity of PRV antigen, and the adjuvant of the prior art, CpGODN nano-adjuvant, a highly efficient PRV gD and gB antigen vaccine was prepared.

[0060] Example 3

[0061] This embodiment describes method three for enhancing the purity and immunogenicity of PRV antigen, including the following steps:

[0062] S1. Isolation of PRV virus strain: Piglets or mummified fetuses from diseased pig farms were taken as pathogen material. The pathogen material was chopped and ground in a mortar (through a 200-mesh sieve or with a particle size of less than 0.5 mm). PBS (PBS volume to tissue weight ratio of 1:1 w / v) was added, and the mixture was frozen and thawed. After centrifugation, the supernatant was filtered and sterilized to obtain the PRV virus strain. The freeze-thaw process with PBS was repeated 5 times, followed by centrifugation at 4000 r / min for 20 min.

[0063] S2. Plasmid Construction: PRV strains were amplified by PCR and their genes were sequenced. Based on the gene variations of the protective antigens gD and gB in the PRV strain gene sequence, codons were optimized to construct pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression. The optimized gD gene sequence is 5'-ATGGCCGACCCCAAC-3', and the gB gene sequence is 5'-ATGCGCGTCGACCTG-3'.

[0064] The specific steps for constructing pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression include:

[0065] S21. Add the constructed enzyme digestion system (enzyme digestion system is: 10 μL L AstDigest buffer, 1 μL BamHI, 1 μL LecoRI, 5 μL PCR product, and add ddH2O to 50 μL) and plasmid vector (pCMV-6His, concentration 50 ng / μL) to the bacterial culture after PCR amplification of PRV virus strain, and incubate together for 5 h;

[0066] S22. After incubation, the bacterial culture was subjected to electrophoresis on a 1% agarose gel.

[0067] S23. The agarose gel was used for separation and extraction to obtain pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression.

[0068] S3. Antigen Cell Screening: The pCMVPRVgD-1 and pCMVPRVgB-2 plasmids constructed in step S2 were transfected into CHO-KS cells. Clonal screening of gB and gD cell lines was performed using glutamine-free CHO medium supplemented with L-methionine sulfoxide imide to obtain stably expressing gB and gD antigen cells. The specific steps for transfecting pCMVPRVgD-1 and pCMVPRVgB-2 plasmids into CHO-KS cells and clonal screening included:

[0069] S31. Take pCMVPRVgD-1 and pCMVPRVgB-2 plasmids, add them to an EP tube, and add CHO-KS cells at a density of 1×10⁻⁶. 6 (cells / mL), gently tap the EP tube wall 10 times to mix thoroughly, then let stand at room temperature for 15 minutes;

[0070] S32. Transfer the plasmid and cell mixture to an electroporation cuvette and perform electroporation (voltage 250V, capacitance 500μF). After electroporation, immediately transfer the cells to a cell shake flask and add glutamine-free CHO medium. After suspension culture for 48h, transfected CHO-KS cells are obtained.

[0071] S33. Centrifuge the cell suspension from step S32 at 1000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in glutamine-free CHO medium until the cell density reaches 0.6 × 10⁶ cells / min. 6 cells / mL, pressure screening of single clonal cell lines, pressure screening using approximately 0.2 MPa;

[0072] S34. Take the transfected cells and dilute them to 5 cells / ml with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imine. Add 200 μl / well to a 96-well plate and incubate at 37°C in a 5% CO2 cell culture incubator for 6 h. Record the wells of a single cell.

[0073] S35. Once cell colonies have formed in the recording wells, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA, digest at room temperature for 2 min, add 2 ml of glutamine-free CHO medium (containing 50 μM L-methionine sulfoxide imine) to terminate the digestion, and use a pipette to disperse the cells. Transfer the cells to a 12-well plate for further culture. Once the cells in the 12-well plate have grown into a confluent monolayer, collect the supernatant for ELISA detection to screen for positive monoclonal cell lines. Continue to expand the culture of the highly expressed positive monoclonal cell lines, freeze them, and obtain the F0 generation cell line.

[0074] S36. Dilute the F0 generation cell line to 5 × 10⁻⁶ mcg using glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imide. 5 Cells / mL were cultured for 3 days in a shaker at 37℃ and 100 rpm with 5% CO2, and glucose content was measured. Glucose was then added to a final concentration of 6 g / L, followed by further culture with 5% feed medium. When the cell density reached 12 × 10⁶ cells / mL, the culture was continued. 6 When the cell count reached 6 g / L, the temperature was lowered to 33°C. Glucose content was then measured daily and supplemented to 6 g / L. 5% culture medium was added every other day. Samples (1 mL) were taken every 24 hours. Cell counts and viability were calculated under a microscope using trypan blue staining. The cell supernatant was then analyzed by SDS-PAGE and Western blotting. After 9 days of culture at a lower temperature, the supernatant was harvested and subjected to SDS-PAGE to obtain cells stably expressing gB and gD antigens.

[0075] S4. Antigen Solution Purification: GB and GD antigen-expressing cells were cultured in a bioreactor to harvest His-tagged GD and GB antigens. These antigens were then clarified and purified by nickel-coated affinity chromatography. After imidazole removal (dialysis to remove imidazole), a high-purity, high-content antigen solution was obtained. During clarification, the antigen was filtered through a 0.65 μm hollow fiber filter. Following clarification, the antigen was purified by nickel-coated affinity chromatography using washing buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The purified antigen was then dialyzed (dialyzed in PBS for 24 hours) and stored at 2°C after passing through a 0.22 μm sterile filter.

[0076] Finally, using the enhanced PRV antigen prepared by the method described above for enhancing the purity and immunogenicity of PRV antigen, and the adjuvant of the prior art, CpGODN nano-adjuvant, a highly efficient PRV gD and gB antigen vaccine was prepared.

[0077] The antigens prepared in Examples 1-3 were tested for the following indicators, and the results are as follows:

[0078]

[0079] As can be seen from the above results, this invention achieves a four-dimensional improvement in purity, potency, efficiency, and safety through seed virus separation optimization, cell screening innovation, purification process upgrade, and adjuvant synergistic design, providing core technical support for PRV purification.

[0080] The above provides a detailed description of a method for enhancing the purity and immunogenicity of PRV antigens provided by this invention. The specific embodiments are provided only to aid in understanding the method and its core principles. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.

[0081] It should be noted that: for experimental steps or conditions not specified in the examples, the procedures and conditions described in conventional experimental procedures in the literature of this art can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0082] The above examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

Claims

1. A method for enhancing the purity and immunogenicity of PRV antigen, characterized in that, Includes the following steps: S1. Isolation of PRV virus strain: Piglets or mummified fetuses from diseased pig farms were taken as pathogen material. The pathogen material was cut into pieces, ground in a mortar and pestle, then PBS was added and frozen and thawed, and centrifuged. The supernatant after centrifugation was filtered and sterilized to obtain the PRV virus strain. S2. Plasmid construction: PRV strains were amplified by PCR and their genes were sequenced. Based on the gene variations of the protective antigens gD and gB in the PRV strain gene sequence, codons were optimized to construct pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression. S3. Antigen cell screening: The pCMVPRVgD-1 and pCMVPRVgB-2 plasmids constructed in step S2 were transfected into CHO-KS cells. The gB cell line and gD cell line clones were screened by adding L-methionine sulfoxide to glutamine-free CHO medium to obtain gB antigen cells and gD antigen cells that express stably. S4. Antigen solution purification: GB antigen cells and GD antigen cells expressing CHO cells are cultured in a bioreactor. GD and GB antigens with His tags are harvested, clarified, purified by nickel column affinity chromatography, and treated with deimidazole to obtain a high-purity, high-content antigen solution.

2. The method for enhancing PRV antigen purity and immunogenicity according to claim 1, characterized in that, In step S1, the freeze-thaw process with PBS is repeated 3-5 times, followed by centrifugation. During centrifugation, the centrifugation rate is 4000 r / min for 10-20 min.

3. The method for enhancing PRV antigen purity and immunogenicity according to claim 1, characterized in that, In step S2, the codon-optimized gD gene sequence is 5'-ATGGCCGACCCCAAC-3', and the gB gene sequence is 5'-ATGCGCGTCGACCTG-3'.

4. The method for enhancing PRV antigen purity and immunogenicity according to claim 1 or 3, characterized in that, The specific steps for constructing pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression include: S21. Add the constructed enzyme digestion system and plasmid vector to the bacterial culture after PCR amplification of the PRV virus strain, and incubate for 3-5 hours. S22. After incubation, the bacterial culture was subjected to electrophoresis on a 1% agarose gel. S23. The agarose gel was used for separation and extraction to obtain pCMVPRVgD-1 and pCMVPRVgB-2 plasmids with His tag fusion expression.

5. The method for enhancing PRV antigen purity and immunogenicity according to claim 4, characterized in that, In step S21, the enzyme digestion system is: 10 μL L AstDigest buffer, 1 μL BamHI, 1 μL LecoRI, 5 μL PCR product, and ddH2O added to 50 μL.

6. The method for enhancing PRV antigen purity and immunogenicity according to claim 4, characterized in that, In step S21, the plasmid vector is pCMV-6His with a concentration of 50 ng / μL.

7. The method for enhancing PRV antigen purity and immunogenicity according to claim 1, characterized in that, In step S3, the specific steps for transfecting pCMVPRVgD-1 and pCMVPRVgB-2 plasmids into CHO-KS cells include: S31, taking pCMVPRVgD-1 and pCMVPRVgB-2 plasmids, adding them to an EP tube, increasing the CHO-KS cell density, gently tapping the EP tube wall to mix thoroughly, and then incubating at room temperature for 15 minutes; S32, transferring the plasmid-cell mixture to an electroporation cuvette and performing electroporation. After electroporation, immediately transfer the cells to a cell shake flask and add glutamine-free CHO medium. After suspension culture for 48 hours, transfected CHO-KS cells are obtained.

8. The method for enhancing PRV antigen purity and immunogenicity according to claim 7, characterized in that, The specific steps for clone screening are as follows: S33. Centrifuge the cell suspension from step S32 at 1000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in glutamine-free CHO medium until the cell density reaches 0.6 × 10⁶ cells / min. 6 cells / mL, pressure screening for single-clonal cell lines; S34. Take the transfected cells and dilute them to 5 cells / ml with glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imine. Add 200 μl / well to a 96-well plate and incubate at 37°C in a 5% CO2 cell culture incubator for 4-6 h. Record the wells of single cells. S35. Once cell colonies have formed in the recording wells, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA and digest at room temperature for 2 min. Add 2 ml of glutamine-free CHO medium to stop the digestion, and use a pipette to disperse the cells. Transfer the cells to a 12-well plate for further culture. Once the cells in the 12-well plate have grown into a confluent monolayer, collect the supernatant for ELISA detection to screen for positive monoclonal cell lines. Continue to expand the culture of the highly expressed positive monoclonal cell lines, freeze them, and obtain the F0 generation cell line. S36. Dilute the F0 generation cell line to 3.5-5 × 10⁻⁵ mg / L glutamine-free CHO medium containing 50 μM L-methionine sulfoxide imide. 5 Cells / mL were cultured for 3 days in a shaker at 37℃ and 100 rpm with 5% CO2, and glucose content was measured. Glucose was then added to a final concentration of 6 g / L, followed by further culture with 5% feed medium. When the cell density reached 10⁻¹² × 10⁻¹⁰ cells / mL, the culture was continued. 6 When the cells / ml reached a certain level, the temperature was lowered to 33℃. The glucose content was then measured daily and glucose was added to 6 g / L. 5% feed culture medium was added every other day. Samples were taken every 24 hours, 1 mL each time. Cell count and viability were calculated under a microscope using trypan blue staining. The cell supernatant was then detected by SDS-PAGE and Western blotting. After culturing at a lower temperature for 9 days, the supernatant was harvested and subjected to SDS-PAGE to obtain cells stably expressing gB antigen and gD antigen.

9. The method for enhancing the purity and immunogenicity of PRV antigen according to claim 1, characterized in that, In step S4, during clarification, the antigen is filtered through a hollow fiber with a pore size of 0.65 μm. After clarification, it is purified by nickel column affinity chromatography to obtain purified antigen. During purification, washing buffer and elution buffer are used. After dialysis and deimidazole treatment, the purified antigen is filtered through a 0.22 μm sterile filter and stored at 2-8℃.

10. A vaccine, characterized in that, An enhanced PRV antigen, prepared using the method for enhancing the purity and immunogenicity of PRV antigen as described in any one of claims 1-9, and an adjuvant, are used to prepare a highly efficient PRV gD and gB antigen vaccine.

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