A porcine pseudorabies virus antigen, a porcine pseudorabies subunit vaccine, and a preparation method and application thereof

Abstract

This invention relates to the field of veterinary vaccines, specifically to a porcine pseudorabies virus antigen, a porcine pseudorabies subunit vaccine, its preparation method, and its application. The porcine pseudorabies virus antigen of this invention is the porcine pseudorabies virus gD protein, and the amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO:1. The porcine pseudorabies subunit vaccine prepared using the porcine pseudorabies virus antigen has a simple preparation method, high neutralizing antibody value, and strong antigenic immunogenicity. It can effectively activate the body's immune response and provide excellent immunoprotection against porcine pseudorabies.

Description

Technical Field

[0001] This invention relates to the field of veterinary vaccines, specifically to a porcine pseudorabies virus antigen and subunit vaccine, its preparation method, and its application. Background Technology

[0002] Porcine pseudorabies is an acute infectious disease of animals caused by pseudorabies virus (PRV). It not only affects pigs but also poses a potential threat to other animals and humans. While existing technologies, such as patents CN116814561A and CN113425839A, have developed corresponding vaccines to combat porcine pseudorabies, environmental factors, natural viral evolution, gene mutations, gene recombination, and the selective pressure exerted on the virus by frequent vaccine use have led to continuous viral mutations to evade immune mechanisms. Therefore, vaccines prepared from existing classic PRV strains and variant strains are no longer effective in controlling the currently prevalent porcine pseudorabies.

[0003] Therefore, finding new porcine pseudorabies virus antigens and developing new porcine pseudorabies subunit vaccines is of great practical significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a porcine pseudorabies virus antigen.

[0005] The second technical problem to be solved by the present invention is to provide the encoding gene of the above-mentioned porcine pseudorabies virus antigen.

[0006] The third technical problem to be solved by the present invention is to provide an expression carrier.

[0007] The fourth technical problem to be solved by this invention is to provide a recombinant cell,

[0008] The fifth technical problem to be solved by the present invention is to provide a method for preparing porcine pseudorabies virus antigen.

[0009] The sixth technical problem to be solved by the present invention is to provide the use of the porcine pseudorabies virus antigen in the preparation of a subunit vaccine for the prevention and / or treatment of porcine pseudorabies.

[0010] The seventh technical problem to be solved by the present invention is to provide a subunit vaccine for porcine pseudorabies.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0012] A porcine pseudorabies virus antigen, wherein the porcine pseudorabies virus antigen is the porcine pseudorabies virus gD protein;

[0013] The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO:1.

[0014] Furthermore, the nucleotide sequence of the gene encoding the porcine pseudorabies virus gD protein is shown in SEQ ID NO:2.

[0015] Expression vectors containing the encoding gene of the porcine pseudorabies virus gD protein are also within the scope of protection of this invention.

[0016] Recombinant cells containing the encoding gene of the porcine pseudorabies virus gD protein, or containing the expression vector, are also within the scope of protection of this invention.

[0017] A method for preparing porcine pseudorabies virus antigen specifically involves cloning the nucleotide sequence of the porcine pseudorabies virus gD protein into a eukaryotic expression vector to construct a recombinant plasmid, then transfecting the recombinant plasmid into expression cells, followed by culture, screening, protein expression, and purification to obtain the porcine pseudorabies virus antigen.

[0018] The eukaryotic expression vector is any one of pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, with pcDNA3.1 being the preferred eukaryotic expression vector.

[0019] The expressing cells are CHO cells, which are mammalian cells.

[0020] Specifically, the culture involves culturing the cells at 36℃-38℃ in a cell culture incubator containing 5% CO2 for 48 hours, then discarding the culture medium and replacing it with DMEM complete medium containing 0.8 mg / ml G418 for further culture.

[0021] The screening process involves detecting the protein expression yield of monoclonal cell lines using ELISA, and screening monoclonal cells with a protein expression yield greater than 1 g / L.

[0022] The protein expression involved seeding monoclonal cells into commercially available CHO serum-free culture medium at a cell density of 1.0 × 10⁶ cells / year. 5 Cells were cultured at 36℃-38℃ in a constant temperature shaker containing 5% CO2 at a speed of 100 r / min. Cell density, viability, and glucose content were measured every 24 hours. When glucose was below 2.5 g / L, glucose was added to 3-4 g / L. Feeding was performed on the 4th and 9th days of fermentation, with the added volume being 10% of the original culture medium. The temperature was lowered to 32℃ on the 5th day of fermentation. On the 12th day, the cell fermentation broth was collected.

[0023] The use of the porcine pseudorabies virus antigen in the preparation of a subunit vaccine for the prevention and / or treatment of porcine pseudorabies is also within the scope of protection of this invention.

[0024] A porcine pseudorabies subunit vaccine comprising the porcine pseudorabies virus antigen is also within the scope of protection of this invention.

[0025] The content of the porcine pseudorabies virus antigen is 30–125 μg / ml.

[0026] In some embodiments of the present invention, the content of the porcine pseudorabies virus gD protein is 40 μg / mL.

[0027] The porcine pseudorabies subunit vaccine includes an adjuvant, preferably ISA 201VG.

[0028] Specifically, the preparation method of the above-mentioned porcine pseudorabies subunit vaccine involves preparing the porcine pseudorabies virus antigen into an antigen solution, mixing it with an adjuvant, emulsifying it, and then stirring it at 34–36°C and 400–500 rpm for 15–20 minutes.

[0029] The antigen solution concentration is 30–125 μg / mL, and the adjuvant is ISA201.

[0030] In some embodiments of the present invention, the antigen solution concentration is 40 μg / mL, and the adjuvant is ISA201.

[0031] Specifically, the mass ratio of the antigen solution to the adjuvant is 1:1.

[0032] Beneficial effects:

[0033] This invention provides a porcine pseudorabies virus antigen, the amino acid sequence of which is shown in SEQ ID NO:1. Using this porcine pseudorabies virus antigen, this invention prepares a porcine pseudorabies virus subunit vaccine. The preparation method is simple, the vaccine has a high neutralizing antibody value, and the antigen exhibits strong immunogenicity, effectively activating the body's immune response and providing excellent immunoprotection against porcine pseudorabies. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0035] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0036] Example 1: Construction of cells expressing porcine pseudorabies virus gD protein

[0037] 1. gD gene synthesis

[0038] The gD genes of the classic strain (JF797217.1) and the currently prevalent strain (KP257591.1) were selected from GenBank as research subjects. After sequence optimization and modification, a signal peptide sequence (METDTLLLWVLLLWVPGSTGD) was added to the N-terminus of the optimized gD sequence, and a His tag was added, resulting in 5 sequences encoding the gD gene, labeled gD-1, gD-2, gD-3, and gD-4. The synthesis of all gD genes was completed by General Biotechnology Co., Ltd. The amino acid sequences of gD-1, gD-2, gD-3, and gD-4 are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively, and the corresponding nucleotide sequences are shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively.

[0039] 2. Construction of recombinant plasmids

[0040] The encoding gene of the porcine pseudorabies virus gD recombinant protein from step 1 was cloned into the eukaryotic expression vector pcDNA3.1. After verification, recombinant plasmids pcDNA3.1-gD-1, pcDNA3.1-gD-2, pcDNA3.1-gD-3, and pcDNA3.1-gD-4 were obtained, respectively.

[0041] 3. Cell transfection

[0042] (1) Passage and amplify the purchased CHO cells. At this time, the cells are in good condition and growing vigorously.

[0043] (2) One day before transfection (Day – 1), seed the CHO cells from step 1 to achieve a final CHO cell density of 3.5 × 10⁻⁶. 6 One live cell / mL, allowing cells to grow overnight.

[0044] (3) On the following day (day 0), the viable cell density and survival rate percentage were measured. When the cell density reached approximately 7–10 × 10⁻⁶ cells / day... 6 1 live cells / mL. Transfection can continue only when the survival rate is 95-99%.

[0045] (4) Dilute the cells from step 3 to a final density of 6 × 10⁶ cells using fresh ExpiCHO expression medium preheated to 37°C. 6 Live cells / mL. Gently shake the culture flask to mix the cells.

[0046] Note: Discard the remaining cells; never reuse high-density cells for routine passage.

[0047] (5) Prepare the ExpiFectamineCHO / plasmid DNA complex using cold reagent (4°C). It is not necessary to place the reagent on ice during the complexation process. Simply remove the reagent from the freezer and begin DNA complexation. The specific procedure is as follows:

[0048] A. Gently invert the ExpiFectamineCHO reagent bottle four times to mix thoroughly.

[0049] B. Dilute 20 μL of recombinant plasmid DNA with 1 mL of cold OptiPRO medium. Shake or invert the test tube to mix.

[0050] C. Dilute 80 μL of ExpiFectamineCHO reagent with 920 μL of OptiPRO medium. Shake or invert the test tube or gently blow on it 2-3 times to mix.

[0051] D. Add the diluted ExpiFectamineCHO reagent to the diluted recombinant plasmid DNA. Shake or invert the test tube to mix thoroughly, ensuring that the ExpiFectamineCHO reagent and recombinant plasmid DNA come into full contact and form an ExpiFectamineCHO / plasmid DNA complex.

[0052] (6) Incubate the ExpiFectamineCHO / plasmid DNA complex obtained in step 5 at room temperature for 2 minutes, and then slowly transfer the solution to the culture flask in step 4, gently shaking the culture flask during the addition process.

[0053] (7) After culturing in a cell culture incubator containing 5% CO2 at 36-38℃ for 48 hours, discard the culture medium and replace it with DMEM complete culture medium containing 0.8 mg / ml G418 for further culture.

[0054] 4. Screening of monoclonal cells

[0055] Remove cells from the 6-well plate from the incubator, discard the culture medium, and perform pressure screening with DMEM complete medium (containing 0.8 mg / ml G418). When the negative control cells are almost completely dead, start single-clonal cell screening.

[0056] Adjust the live cell density to 1.0 cells / well, seed 200 μL into each well of a 96-well plate, and incubate at 36℃-38℃ (preferably 37℃). Once the single cell line in the 96-well plate has expanded to 80-100% or higher, detect the protein expression yield of the monoclonal cell line using ELISA. Digest cells with high expression levels and transfer them to 24-well plates. When the 24-well plates are confluent, collect the supernatant and detect the protein expression yield of the monoclonal cell line using ELISA. Screen monoclonal cells with a protein expression yield greater than 1 g / L.

[0057] 5. Protein expression

[0058] Monoclonal cells were seeded into commercially available CHO serum-free culture medium at a cell density of 1.0 × 10⁶ cells / year. 5 Cells were cultured at 36–38°C in a constant-temperature shaker containing 5% CO2 at a speed of 100 rpm. Cell density, viability, and glucose content were monitored every 24 hours. When glucose fell below 2.5 g / L, glucose was added to bring the concentration up to 3–4 g / L. Feeding was performed on days 4 and 9 of fermentation, with the added volume being 10% of the original culture medium. The temperature was lowered to 32°C on day 5 of fermentation. On day 12, the cell fermentation broth was collected. After purification, five cell lines were obtained to express proteins, which were named gD1, gD2, gD3, and gD4, respectively.

[0059] Example 2: Immunogenicity assay

[0060] Five different protein fragments obtained in Example 1 were used to prepare antigen solutions with PBS buffer as solvent. The antigen solutions were mixed with ISA 201VG at a mass of 40 μg / mL and emulsified. The mixture was stirred at 34°C and 500 rpm for 15 min to prepare different groups of vaccine samples. KM mouse immunization experiments were conducted in the laboratory.

[0061] Sixty healthy KM mice (purchased from Xi'an Yifengda Biotechnology Co., Ltd.) weighing approximately 20g were randomly divided into six groups. Groups 1 to 5 were immunized with vaccine samples of different antigen groups (subcutaneous injection, 0.2mL / mouse), with 10 mice not immunized serving as a control group. Twenty-one days post-immunization, blood was collected from the orbital sinus, and serum was separated under sterile conditions to determine the neutralizing antibody against porcine pseudorabies virus. The protein group with the highest neutralizing antibody value was found to be gD1 protein in group 1. Specific experimental results are shown in Table 1.

[0062] Table 1. Grouping and Results of Immunization Experiments in KM Mice

[0063]

[0064] Example 3: Evaluation of Immunization Efficacy and Safety

[0065] The immunogenicity and safety of the gD1 protein obtained in Example 2 were evaluated. The specific steps were as follows:

[0066] The gD1 protein was purified using hollow fiber ultrafiltration to remove cell debris and other impurities. The purified gD1 protein was then quantified using a protein quantification kit. The quantified gD1 protein was used to prepare an antigen solution with PBS buffer. A 40 μg / mL antigen solution was then mixed with ISA201 adjuvant to prepare the vaccine. Specifically, the emulsification ratio of the aqueous phase (antigen solution) to the oil phase (ISA201 adjuvant) was 1:1 by mass. The oil phase was first added to a beaker with slow stirring, followed by the gradual addition of the aqueous phase. After the aqueous phase was completely added, a shear emulsification device was started, and the mixture was emulsified for 15 minutes to form a stable water-in-oil emulsion, thus obtaining a vaccine emulsion with good physical stability. After emulsification, the vaccine emulsion was aseptically dispensed into 50 mL vials, sealed, and stored at 2–8°C to obtain the porcine pseudorabies virus subunit vaccine.

[0067] Healthy, well-developed 21-day-old piglets that were not infected with porcine pseudorabies virus were selected as experimental animal models. After immunizing the 21-day-old piglets with the porcine pseudorabies virus subunit vaccine (intramuscular injection behind the ear and neck, 2 mL / head), blood samples were collected, and the level of neutralizing antibodies in the serum was quantitatively analyzed. The results showed that the neutralizing antibody level at 21 days was not lower than 1:362.

[0068] To further verify the actual protective effect of the vaccine, immunized pigs were challenged with both the classic virulent strain (SC strain) and the currently circulating strain (JS strain). The challenge results showed that the vaccine could effectively prevent both the classic virulent strain (SC strain) and the currently circulating strain (JS strain) simultaneously, with a protection rate of 100%. No fever or abnormal clinical symptoms were observed in any of the immunized pigs (Table 2).

[0069] Table 2 Experimental Groups and Results

[0070]

[0071] This invention provides a concept and method for developing porcine pseudorabies virus antigen, porcine pseudorabies subunit vaccine, and their preparation and application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A porcine pseudorabies virus antigen, characterized in that, The porcine pseudorabies virus antigen is the porcine pseudorabies virus gD protein; wherein, the amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO:

1.

2. The gene encoding the porcine pseudorabies virus gD protein according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. An expression carrier, characterized in that, It contains the encoding gene as described in claim 2.

4. A recombinant cell, characterized in that, It contains the coding gene as described in claim 2, or the expression vector as described in claim 3.

5. The method for preparing porcine pseudorabies virus antigen according to claim 1, characterized in that, The recombinant plasmid was constructed by cloning the gene encoding the porcine pseudorabies virus gD protein into a eukaryotic expression vector. The recombinant plasmid was then transfected into expression cells, and after culturing, screening, protein expression, and purification, the porcine pseudorabies virus antigen was obtained.

6. The preparation method according to claim 5, characterized in that, The eukaryotic expression vector is any one of pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, and the expression cell is CHO cell, a mammalian cell.

7. The use of the porcine pseudorabies virus antigen of claim 1 in the preparation of a subunit vaccine for the prevention of porcine pseudorabies.

8. A subunit vaccine for porcine pseudorabies, characterized in that, The porcine pseudorabies subunit vaccine includes the porcine pseudorabies virus antigen as described in claim 1.

9. The porcine pseudorabies subunit vaccine according to claim 8, characterized in that, The content of the porcine pseudorabies virus antigen is 30~125μg / ml.

10. The porcine pseudorabies subunit vaccine according to claim 8, characterized in that, The porcine pseudorabies subunit vaccine includes an adjuvant.

11. The porcine pseudorabies subunit vaccine according to claim 10, characterized in that, The adjuvant is ISA201VG.

Images