Pseudorabies virus gD protein of pig, and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically to porcine pseudorabies virus gD protein, its preparation method, and its applications. This invention provides a porcine pseudorabies virus gD protein with high purity, the amino acid sequence of which is shown in SEQ ID NO:7. After expression, the porcine pseudorabies virus gD protein was purified by protein ultrafiltration in six batches, and its protein purity was increased by at least 246.2% compared to the unpurified form. It can be used to develop high-purity subunit vaccines, thereby contributing to the control and eradication of porcine pseudorabies.

Description

Technical Field

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

[0002] Porcine pseudorabies is an acute infectious disease of pigs caused by the pseudorabies virus (PrV), and it is widely distributed worldwide. It has an extremely high mortality rate not only in piglets but also affects adult pigs, causing reproductive disorders such as abortion, stillbirth, and mummified fetuses. Vaccination is an effective means of controlling the incidence of porcine pseudorabies.

[0003] The gD protein is one of the main immunogenic proteins of porcine pseudorabies virus, playing a crucial role in viral structure, infection mechanism, immune response, and vaccine development. Currently, although many gD vaccines against porcine pseudorabies are available on the market, the continuous evolution of pseudorabies virus strains means that existing vaccines may not provide complete protection. Therefore, it is necessary to continuously explore and develop new vaccines to effectively address viral mutations.

[0004] In view of this, the present invention provides a novel pseudorabies virus gD protein. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a porcine pseudorabies virus gD protein, which addresses the shortcomings of the prior art.

[0006] Another technical problem to be solved by the present invention is to provide the encoding gene of the above-mentioned porcine pseudorabies virus gD protein.

[0007] Another technical problem to be solved by the present invention is to provide a recombinant expression vector and recombinant cells containing the above-mentioned coding genes.

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

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

[0010] The final technical problem to be solved by this invention is to provide a porcine pseudorabies virus subunit vaccine.

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

[0012] A porcine pseudorabies virus gD protein, the amino acid sequence of which is shown in SEQ ID NO:7.

[0013] The coding gene for the porcine pseudorabies virus gD protein is shown in SEQ ID NO:8.

[0014] Specifically, the encoding gene was obtained by selecting the gD gene of the classic strain (JF797217.1) and the currently prevalent 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.

[0015] A recombinant expression vector containing the encoding gene of the porcine pseudorabies virus gD protein, or a recombinant cell, is also within the scope of protection of this invention.

[0016] The method for preparing the porcine pseudorabies virus gD protein specifically involves transfecting a recombinant plasmid containing the encoding gene of the porcine pseudorabies virus gD protein into mammalian cells, followed by culture, pressure screening, monoclonal cell screening, protein expression, and protein ultrafiltration purification to obtain the porcine pseudorabies virus gD protein.

[0017] 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.

[0018] 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.

[0019] The mammalian cells are preferably CHO cells.

[0020] The culture conditions are as follows: 36℃~38℃, cultured in a cell culture incubator containing 5% CO2 for 48 hours.

[0021] The pressure screening is performed by pressure screening in DMEM complete medium containing 0.8 mg / mL G418.

[0022] 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.

[0023] 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 a density of 100 cells / mL in a constant-temperature shaker containing 5% CO2 at 36–38°C. Cell density, viability, and glucose content were monitored every 24 hours. When glucose levels fell below 2.5 g / L, glucose was added to bring the levels 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. On day 5 of fermentation, the temperature was lowered to 32°C. On day 12, the cell fermentation broth was collected.

[0024] Specifically, the protein ultrafiltration purification involves purifying the cell fermentation broth obtained by protein expression of porcine pseudorabies virus gD protein using hollow fiber ultrafiltration.

[0025] Specifically, the hollow fiber ultrafiltration purification process involves 1 to 8 batches of ultrafiltration fluid exchange, preferably 6 to 8 batches, and more preferably 6 batches.

[0026] In some embodiments of the present invention, after the cell fermentation broth obtained by protein expression of the porcine pseudorabies virus gD protein was purified by hollow fiber ultrafiltration for 6 batches, the purity of the porcine pseudorabies virus gD protein was increased by at least 246.2% compared with that before purification.

[0027] The use of the porcine pseudorabies virus gD protein 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.

[0028] A porcine pseudorabies virus subunit vaccine is also within the scope of protection of this invention.

[0029] Specifically, the porcine pseudorabies virus subunit vaccine contains the porcine pseudorabies virus gD protein.

[0030] Beneficial Effects: This invention provides a porcine pseudorabies virus gD protein (i.e., gD4) with high protein purity. The amino acid sequence of this recombinant porcine pseudorabies virus gD protein is shown in SEQ ID NO:7. After expression, the porcine pseudorabies virus gD protein was purified by protein ultrafiltration in six batches, and its protein purity was increased by at least 246.2% compared with that before purification. The porcine pseudorabies virus gD protein can be used to develop high-purity subunit vaccines, which is beneficial for the control and eradication of porcine pseudorabies. Attached Figure Description

[0031] 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.

[0032] Figure 1 The HPLC chromatogram of the gD4 protein purified sample. Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0035] Example 1: Construction of cells expressing porcine pseudorabies virus gD protein

[0036] 1. gD gene synthesis

[0037] 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 five sequences encoding the gD gene, labeled gD-1, gD-2, gD-3, gD-4, and gD-5. The synthesis of all gD genes was completed by General Biotechnology Co., Ltd. The amino acid sequences of gD-1, gD-2, gD-3, gD-4, and gD-5 are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9, respectively, and the corresponding nucleotide sequences are shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, respectively.

[0038] 2. Construction of recombinant plasmids

[0039] 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, pcDNA3.1-gD-4, and pcDNA3.1-gD-5 were obtained, respectively.

[0040] 3. Cell transfection

[0041] (1) Passage and amplify the purchased CHO cells. At this time, the cells are in good condition and growing vigorously.

[0042] (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.

[0043] (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%.

[0044] (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.

[0045] Note: Discard the remaining cells; never reuse high-density cells for routine passage.

[0046] (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:

[0047] A. Gently invert the ExpiFectamineCHO reagent bottle four times to mix thoroughly.

[0048] B. Dilute 20 μL of recombinant plasmid DNA with 1 mL of cold OptiPRO medium. Shake or invert the test tube to mix.

[0049] 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.

[0050] 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.

[0051] (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.

[0052] (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.

[0053] 4. Screening of monoclonal cells

[0054] 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.

[0055] 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℃ (preferably 37℃). 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.

[0056] 5. Protein expression

[0057] 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. On day 5, the temperature was lowered to 32°C. On day 12, the cell fermentation broth was collected. After purification, five monoclonal cell expression proteins were obtained and named gD1, gD2, gD3, gD4, and gD5, respectively.

[0058] 6. Establishment of protein purification process

[0059] Each batch of cell fermentation broth from step 5 was purified by hollow fiber ultrafiltration. The specific purification process is as follows:

[0060] (1) Clarification: Wash the hollow fiber ultrafiltration system with 1×PBS buffer, add 1000mL of cell fermentation broth to the medium exchange tank, turn on the stirrer and circulation pump, and set the shear rate to 4000s. -1 After circulating for a certain period of time, the solution was filtered through a 0.2μm hollow fiber microfiltration column, and 900mL of permeate was collected.

[0061] (2) Washing and filtration of the retentate: Add an equal volume of 1×PBS buffer to the remaining 100 mL of cell fermentation broth in the culture tank, turn on the stirrer and circulation pump, and set the shear rate to 4000 s. -1 Collect the permeate to obtain 100 mL of washing filtrate, which is then set aside. Mix the permeate and washing filtrate thoroughly to obtain 1200 mL of clarified cell supernatant.

[0062] (3) Liquid exchange and determination: The harvested cell supernatant was concentrated and exchanged using a 10kD hollow fiber membrane. The liquid exchange buffer was PBS. The liquid exchange was carried out by continuous flow equal volume exchange (i.e., the flow rate of the feeding end and the permeation end was the same). The liquid exchange volume was 5 times. Samples were taken during the liquid exchange for HPLC detection. After the liquid exchange was completed, the antigen solution was collected and stored at 2-8℃.

[0063] The purity of each batch of cell fermentation broth and protein solution purified by hollow fiber ultrafiltration was determined by HPLC, and the results are shown in Table 1.

[0064] As shown in Table 1, with the increase of the replacement fluid volume, the number and area of ​​impurity peaks gradually decreased, while the area of ​​the main peak remained basically unchanged. This indicates that ultrafiltration not only gradually improves the purity of gD protein but also minimizes the loss of effective protein. Furthermore, the purity of the unpurified gD protein ranged from 25% to 30%. After six ultrafiltration replacements, the final purity of the gD protein was consistently no less than 70%, and further replacements could not significantly improve antigen purity. Therefore, the final ultrafiltration replacement fluid volume was determined to be five times the volume after concentration. After six batches of ultrafiltration replacements, the purity of gD4 protein showed the most significant improvement, increasing by at least 246.2% compared to before purification. Figure 1 The HPLC chromatogram of the gD4 protein purified sample.

[0065] Table 1. Results of clarification and purification of gD antigen

[0066]

[0067]

[0068] Example 2: Evaluation of Immunization Efficacy and Safety

[0069] The immunogenicity and safety of gD4 protein expressed in the gD-4 cell line were evaluated. The specific procedures are as follows:

[0070] The purified gD4 protein from Example 1 was quantitatively analyzed using a protein quantification kit. The quantified gD4 protein was used to prepare an antigen solution in 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.

[0071] 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 1:91.

[0072] To 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).

[0073] Table 2 Experimental Groups and Results

[0074]

[0075]

[0076] This invention provides a method and approach for the preparation and application of porcine pseudorabies virus gD protein. 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 gD protein, characterized in that, The amino acid sequence of the porcine pseudorabies virus gD protein is shown in SEQ ID NO:

7.

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

8.

3. A recombinant expression vector or recombinant cell containing the encoding gene as described in claim 2.

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

5. The preparation method according to claim 4, characterized in that, The recombinant plasmid was obtained by cloning the gene encoding the porcine pseudorabies virus gD protein into a eukaryotic expression vector.

6. The preparation method according to claim 4, characterized in that, The protein ultrafiltration purification specifically involves purifying the cell fermentation broth obtained by protein expression of porcine pseudorabies virus gD protein using hollow fiber ultrafiltration.

7. The preparation method according to claim 6, characterized in that, The hollow fiber ultrafiltration purification process involves ultrafiltration fluid replacement in batches of 1 to 8.

8. The preparation method according to claim 7, characterized in that, After purifying six batches of cell fermentation broth obtained from the expression of porcine pseudorabies virus gD protein using hollow fiber ultrafiltration, the purity of porcine pseudorabies virus gD protein was increased by at least 246.2% compared to before purification.

9. The use of the porcine pseudorabies virus gD protein of claim 1 in the preparation of a subunit vaccine for the prevention of porcine pseudorabies.

10. A porcine pseudorabies virus subunit vaccine, characterized in that, The porcine pseudorabies virus subunit vaccine contains the porcine pseudorabies virus gD protein as described in claim 1.

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