A stably expressed porcine pseudorabies virus gD recombinant protein, vaccine composition, and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically to a stably expressed porcine pseudorabies virus gD recombinant protein, a vaccine composition, its preparation method, and its applications. The invention provides a stably expressed porcine pseudorabies virus gD recombinant protein, the amino acid sequence of which is shown in SEQ ID NO:7. The porcine pseudorabies virus gD recombinant protein is stably expressed in monoclonal cells, exhibiting stable expression levels after 48 consecutive passages. This lays a solid foundation for its subsequent applications in the preparation of stable porcine pseudorabies subunit vaccine compositions or in porcine pseudorabies virus vaccine efficacy testing formulations.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a stably expressed porcine pseudorabies virus gD recombinant protein, a vaccine composition, its preparation method, and its application. Background Technology

[0002] Porcine pseudorabies, also known as swine fever scrapy disease or swine infectious encephalomyelitis, is an acute infectious disease caused by the porcine pseudorabies virus (PRV) that infects pigs, cattle, sheep, dogs, cats, rabbits, and even humans.

[0003] Although vaccines against porcine pseudorabies are already available on the market, such as the JS-A1 strain, Bartha-K61 strain, HB-98 strain, and HB2000 strain, the viral strains are constantly evolving. To adapt to viral mutations, further improve vaccine efficacy, achieve precise control and eradication of porcine pseudorabies, and promote the development of vaccines and diagnostic technologies, it is necessary to continuously explore and prepare new recombinant proteins of porcine pseudorabies virus that can be stably expressed to cope with emerging viral strains. 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 stably expressed porcine pseudorabies virus gD recombinant protein.

[0005] The second technical problem to be solved by the present invention is to provide the encoding gene of the stable expressed porcine pseudorabies virus gD recombinant protein.

[0006] The third technical problem to be solved by the present invention is to provide a recombinant expression vector containing the above-mentioned coding gene.

[0007] The fourth technical problem to be solved by the present invention is to provide a recombinant cell containing the above-mentioned coding gene or the above-mentioned recombinant expression vector.

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

[0009] The sixth technical problem to be solved by the present invention is to provide the use of a stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of a subunit vaccine composition 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 porcine pseudorabies virus subunit vaccine composition.

[0011] The eighth technical problem to be solved by the present invention is to provide the application of a stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of a porcine pseudorabies virus vaccine efficacy testing agent.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0013] A stably expressed porcine pseudorabies virus gD recombinant protein, the amino acid sequence of which is shown in SEQ ID NO:7.

[0014] The recombinant porcine pseudorabies virus gD protein showed no decrease in protein expression level after 48 consecutive passages in monoclonal cells, maintaining a good level of protein expression.

[0015] The coding gene for the porcine pseudorabies virus gD recombinant protein has the nucleotide sequence shown in SEQ ID NO:8.

[0016] Specifically, the encoding gene was obtained by selecting the gD gene of the classic strain (JF797217.1) and the currently popular strain (KP257591.1) from GenBank as the research object. 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.

[0017] A recombinant expression vector containing the encoding gene of the porcine pseudorabies virus gD recombinant protein is also within the scope of protection of this invention.

[0018] A recombinant cell containing the encoding gene of the porcine pseudorabies virus gD recombinant protein or a recombinant expression vector is also within the scope of protection of this invention.

[0019] A method for preparing stably expressed porcine pseudorabies virus gD recombinant protein involves transfecting a recombinant plasmid expressing porcine pseudorabies virus gD recombinant protein into mammalian cells, followed by culture, pressure screening, monoclonal cell screening, protein expression and purification.

[0020] The recombinant plasmid expressing the porcine pseudorabies virus gD recombinant protein is obtained by cloning the coding gene of the porcine pseudorabies virus gD recombinant protein into a eukaryotic expression vector.

[0021] Specifically, the eukaryotic expression vector includes, but is not limited to, pcDNA3.1. All eukaryotic expression vectors that can be used for cloning porcine pseudorabies virus gD recombinant protein in the prior art are within the scope of protection of this invention, such as pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.

[0022] The mammalian cells are preferably CHO cells, with a cell confluence of 80% to 90% during transfection.

[0023] The culture conditions are as follows: 36℃~38℃, cultured in a cell culture incubator containing 5% CO2 for 48 hours.

[0024] The pressure screening mentioned above refers to screening for positive cell lines using G418.

[0025] Specifically, screening was performed under pressure in DMEM complete medium containing 0.8 mg / mL G418.

[0026] The monoclonal cell screening process begins after the negative control cells have been largely eliminated through pressure screening. The viable cell density is adjusted to 1.0 cells / well, and 200 μL is seeded into each well of a 96-well plate. The plates are incubated at 36℃–38℃. Once the single cell line in the 96-well plate has expanded to 80–100% or higher, the protein expression yield of the monoclonal cell line is detected by ELISA. Cells with high expression levels are digested and transferred to 24-well plates. When the 24-well plates are confluent, the supernatant is collected for analysis, and the protein expression yield of the monoclonal cells is detected by ELISA.

[0027] 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°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 was below 2.5 g / L, glucose was added to bring the glucose level up 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°C on the 5th day of fermentation. On the 12th day, the cell fermentation broth was collected.

[0028] The use of the stably expressed porcine pseudorabies virus gD recombinant protein in the preparation of subunit vaccine compositions for the prevention and / or treatment of porcine pseudorabies is also within the scope of protection of this invention.

[0029] A porcine pseudorabies virus subunit vaccine composition comprising an immunizing dose of porcine pseudorabies virus gD recombinant protein.

[0030] The application of the porcine pseudorabies virus gD recombinant protein in the preparation of porcine pseudorabies virus vaccine efficacy testing agents is also within the scope of protection of this invention.

[0031] Beneficial Effects: This invention provides a recombinant porcine pseudorabies virus gD protein (i.e., gD4) that can be stably expressed, the amino acid sequence of which is shown in SEQ ID NO:7. It is obtained by transfecting a recombinant plasmid expressing the porcine pseudorabies virus gD protein into mammalian cells, followed by culture, pressure selection, monoclonal cell selection, and protein expression. The monoclonal cells expressing the porcine pseudorabies virus gD recombinant protein exhibit stable passage numbers; even after 48 consecutive passages, gD4 still maintains a good protein expression level. This lays a solid foundation for the subsequent preparation of stable porcine pseudorabies subunit vaccine compositions or efficacy testing agents for porcine pseudorabies virus vaccines. Attached Figure Description

[0032] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0033] Figure 1 The results show the identification of gD4 protein expression in cells at different passages. M represents the marker; N represents the negative control; P represents the positive control; 1 represents passage F10; 2 represents passage F16; 3 represents passage F22; 4 represents passage F38; and 5 represents passage F48. 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 per well in a 96-well plate, and incubate at 36–38°C (preferably 37°C). When the single cell line in the 96-well plate expands to 80–100% or more, detect the protein expression yield of the monoclonal cell line by 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 cells by ELISA. Select 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. Five monoclonal cell expression proteins were obtained after purification and named gD1, gD2, gD3, and gD4, respectively.

[0059] Example 2: Stability evaluation of gD protein cells

[0060] Four types of gD protein-expressing cells frozen in Example 1 were seeded in 60 mL of basal culture medium to achieve a cell density of 1.0 × 10⁻⁶ cells / year. 5 Cells / mL were cultured in suspension at 36–38°C in a shaker containing 5% CO2. When the cell density reached 3 × 10⁻⁶ cells / mL... 5 When the cell count reached 90% or higher, the cells were passaged every 2-3 days for continuous passage. Cells from F10, F16, F22, F38 and F48 generations were used for protein expression. On day 12, the cell culture supernatant was harvested and the protein expression level was detected. The results are detailed in Table 1. Figure 1 The results of gD4 protein expression identification at different cell generations.

[0061] Table 1. Expression levels of gD protein in cells of different generations

[0062]

[0063] From Table 1 and Figure 1 As can be seen, the gD4 protein maintained good protein expression levels even after 48 consecutive passages, indicating that its passage count was stable. This lays a good foundation for the subsequent preparation of stable porcine pseudorabies subunit vaccines or efficacy testing agents for porcine pseudorabies virus vaccines.

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

[0065] The immunogenicity and safety of gD4 protein expressed in the gD-4 cell line were evaluated. The specific procedures are as follows:

[0066] The gD4 protein was purified using hollow fiber ultrafiltration to remove cell debris and other impurities. The purified gD4 protein was then quantified using a protein quantification kit. The quantified gD4 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 1:102. This result fully demonstrates that the porcine pseudorabies virus subunit vaccine can effectively stimulate the piglets to produce high levels of neutralizing antibodies, providing a strong immune defense against the invasion of porcine pseudorabies virus.

[0068] To verify the actual protective effect of the vaccine, a challenge phase was included in the experiment. 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 infection from both the classic virulent strain (SC strain) and the currently circulating strain (JS strain) simultaneously, achieving a 100% protection rate. No fever or abnormal clinical symptoms were observed in any of the immunized pigs.

[0069] In summary, the porcine pseudorabies virus subunit vaccine of this invention can induce high levels of neutralizing antibodies in 21-day-old piglets after immunization, and also demonstrates good protective efficacy and safety in challenge experiments (Table 2).

[0070] Table 2 Experimental Groups and Results

[0071]

[0072] This invention provides a stable expression of porcine pseudorabies virus gD recombinant protein, a vaccine composition, and its preparation and application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. 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 stably expressed porcine pseudorabies virus gD recombinant protein, characterized in that, The amino acid sequence of the porcine pseudorabies virus gD recombinant protein is shown in SEQ ID NO:

7.

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

8.

3. A recombinant expression vector, 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 recombinant expression vector as described in claim 3.

5. The method for preparing the porcine pseudorabies virus gD recombinant protein according to claim 1, characterized in that, The recombinant plasmid containing the encoding gene of the porcine pseudorabies virus gD recombinant protein was transfected into mammalian cells, and the protein was obtained by culturing, pressure screening, single-clone cell screening, protein expression and purification.

6. The preparation method according to claim 5, characterized in that, The recombinant plasmid containing the encoding gene of the porcine pseudorabies virus gD recombinant protein is obtained by cloning the encoding gene of the porcine pseudorabies virus gD recombinant protein into a eukaryotic expression vector.

7. The use of the stably expressed porcine pseudorabies virus gD recombinant protein of claim 1 in the preparation of a subunit vaccine composition for the prevention of porcine pseudorabies.

8. A porcine pseudorabies virus subunit vaccine composition, characterized in that, The porcine pseudorabies virus subunit vaccine composition comprises the porcine pseudorabies virus gD recombinant protein as described in claim 1.

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