Porcine pseudorabies virus gD protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a porcine pseudorabies virus gD protein as well as a preparation method and application thereof. The invention provides a porcine pseudorabies virus gD protein with high protein purity. The amino acid sequence of the porcine pseudorabies virus gD protein is shown as SEQ ID NO: 7. After the porcine pseudorabies virus gD protein is expressed, protein ultrafiltration purification is performed for 6 batches, the protein purity is at least improved by 246.2% compared with that before purification, and the porcine pseudorabies virus gD protein can be used for developing high-purity subunit vaccines, so that control and elimination of porcine pseudorabies are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a gD protein of porcine pseudorabies virus, a preparation method thereof, and an application thereof. Background Art

[0002] Porcine pseudorabies is an acute infectious disease of pigs caused by porcine pseudorabies virus (PrV), which is widely distributed worldwide. It not only has a very high lethality rate for piglets, but also has a certain impact on adult pigs, such as causing reproductive disorders, including abortion, stillbirth, mummified fetus, etc. Vaccine immunization is an effective means to control the incidence of porcine pseudorabies.

[0003] The gD protein is one of the main immunogenic proteins of porcine pseudorabies virus and plays an important role in the structure, infection mechanism, immune response, and vaccine development of the virus. At present, although there are many gD vaccines against porcine pseudorabies on the market, due to the continuous evolution of pseudorabies virus strains, the existing vaccines may not provide complete protection, which requires continuous exploration of the research and development of new vaccines to effectively respond to virus mutations.

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

[0005] The technical problem to be solved by the present invention is to provide a gD protein of porcine pseudorabies virus in view of the deficiencies of the prior art.

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

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

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

[0009] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned gD protein of porcine pseudorabies virus in the preparation of a subunit vaccine for preventing and / or treating porcine pseudorabies.

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

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

[0013] The coding gene encoding the porcine pseudorabies virus gD protein has a nucleotide sequence as shown in SEQ ID NO:8.

[0014] Specifically, this coding gene selects the gD genes of classical strains (JF797217.1) and current prevalent strains (KP257591.1) from GenBank as the research objects. After sequence optimization and modification, a signal peptide sequence (METDTLLLWVLLLWVPGSTGD) is added to the N-terminus of the optimized gD sequence, and a His tag is attached.

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

[0016] The preparation method of the porcine pseudorabies virus gD protein is specifically to transfect the recombinant plasmid cloned with the coding gene of the porcine pseudorabies virus gD protein into mammalian cells, and through cultivation, pressure screening, monoclonal cell screening, protein expression and protein ultrafiltration purification, the porcine pseudorabies virus gD protein is prepared.

[0017] Among them, 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. Eukaryotic expression vectors that can be used for cloning the porcine pseudorabies virus gD recombinant protein in the prior art are all within the scope of protection of the present invention, such as pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.

[0019] Among them, the mammalian cell is preferably a CHO cell.

[0020] Among them, for the cultivation, the cultivation conditions are: 36°C to 38°C, and cultivate in a cell culture incubator containing 5% CO 2 for 48 hours.

[0021] Among them, the pressure screening is carried out by pressure screening in DMEM complete medium containing 0.8 mg / mL G418.

[0022] Among them, for the monoclonal cell screening, when the negative control cells are basically all dead after pressure screening, the monoclonal cell screening begins. Adjust the viable cell density to 1.0 cell / well, inoculate a 96-well plate, 200 μL per well, and place it at 36°C to 38°C. When a single cell strain in the 96-well plate has expanded to more than 80 - 100%, detect the expression yield of the monoclonal cell strain protein by ELISA method. Digest the cells with a higher expression level and transfer them to a 24-well plate. When the 24-well plate is full, take the supernatant for detection, and detect the protein expression yield of the monoclonal cells by ELISA method.

[0023] Among them, for the protein expression, the monoclonal cells are respectively inoculated into a commercial CHO serum-free medium, and the cell density is 1.0×10 5 cells / mL, and cultured in a constant temperature shaker at 36 - 38°C with 5% CO 2 at a rotation speed of 100 r / min; detect the cell density, viability and glucose content every 24 hours. When the glucose is lower than 2.5 g / L, supplement glucose to 3 - 4 g / L; perform feeding on the 4th and 9th days of fermentation, and add a volume of 10% of the original medium; cool down to 32°C on the 5th day of fermentation; on the 12th day, collect the cell fermentation broth.

[0024] Among them, for the ultrafiltration purification of the protein, specifically, the cell fermentation broth obtained by protein expression of the porcine pseudorabies virus gD protein is purified by hollow fiber ultrafiltration.

[0025] Specifically, for the hollow fiber ultrafiltration purification, the number of ultrafiltration buffer exchange batches is 1 - 8 batches, preferably 6 - 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 is purified by hollow fiber ultrafiltration for 6 batches, the purity of the porcine pseudorabies virus gD protein is increased by at least 246.2% compared with that before purification.

[0027] The application of the porcine pseudorabies virus gD protein in the preparation of a subunit vaccine for preventing and / or treating porcine pseudorabies is also within the scope of protection of the present invention.

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

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

[0030] Beneficial effects: The present invention provides a porcine pseudorabies virus gD protein (i.e., gD4) with high protein purity. The amino acid sequence of the recombinant porcine pseudorabies virus gD protein is shown in SEQ ID NO:7. After the porcine pseudorabies virus gD protein is expressed and ultrafiltered and purified 6 batches, its protein purity is increased by at least 246.2% compared with that before purification. The porcine pseudorabies virus gD protein can be used to develop a subunit vaccine with high purity, which is beneficial to the control and elimination of porcine pseudorabies. Description of the Drawings

[0031] The following further specifically describes the present invention with reference to the drawings, and the above and / or other advantages of the present invention will become clearer.

[0032] Figure 1 It is the HPLC chromatogram of the purified sample of gD4 protein. Detailed Embodiments

[0033] The following further specifically describes the present invention with reference to the detailed embodiments, and the above and / or other advantages of the present invention will become clearer.

[0034] In the following experimental methods described in the following examples, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels.

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

[0036] 1. Synthesis of gD gene

[0037] The gD genes of the classical strain (JF797217.1) and the currently prevalent strain (KP257591.1) were selected from GenBank as the research objects. 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 attached. Finally, 5 sequences encoding the gD gene were obtained, which were respectively labeled as gD-1, gD-2, gD-3, gD-4, and gD-5. The synthesis of all gD genes was completed by General Biosystems Co., Ltd. Among them, 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] Clone the coding gene of the porcine pseudorabies virus gD recombinant protein in step 1 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 are obtained respectively.

[0040] 3. Cell transfection

[0041] (1) Passage and amplify the purchased CHO cells. At this time, the cell state is good and the growth is vigorous.

[0042] (2) One day before transfection (day -1), seed the CHO cells in step 1 so that the final density of the CHO cells is 3.5×10 6 viable cells / mL, and let the cells grow overnight.

[0043] (3) The next day (day 0), measure the viable cell density and the percentage of viability. When the cell density reaches about 7 - 10×10 6 viable cells / mL and the viability is 95 - 99%, then transfection can continue.

[0044] (4) Dilute the cells in step 3 to a final density of 6×10 6 viable cells / mL using fresh pre-warmed (37°C) ExpiCHO expression medium. Gently shake the culture flask to mix the cells evenly.

[0045] Note: Discard the remaining cells; do not use high-density cells for routine passage again.

[0046] (5) Prepare the ExpiFectamine CHO / plasmid DNA complex using cold reagents (4°C). During the complexation process, there is no need to place the reagents on ice. Just take the reagents out of the refrigerator and start the DNA complexation. The specific process is as follows:

[0047] A. Gently invert the ExpiFectamine CHO reagent bottle 4 times up and down to mix well.

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

[0049] C. Dilute 80 μL of ExpiFectamine CHO reagent with 920 μL of OptiPRO medium. Shake or invert the test tube or gently pipette 2 - 3 times to mix well.

[0050] D. Add the diluted ExpiFectamine CHO reagent to the diluted recombinant plasmid DNA. Shake or invert the tube up and down to mix well so that the ExpiFectamine CHO reagent is in full contact with the recombinant plasmid DNA and form the ExpiFectamine CHO / plasmid DNA complex.

[0051] (6) Incubate the ExpiFectamine CHO / 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) Culture in a cell culture incubator at 36 - 38 °C with 5% CO 2 After culturing for 48 hours, discard the liquid, and replace it with DMEM complete medium containing 0.8 mg / ml G418 for further culture.

[0053] 4. Screening of monoclonal cells

[0054] Take out the cells in the 6-well plate from the incubator, discard the liquid, and perform pressure screening with DMEM complete medium (containing 0.8 mg / ml G418). When the negative control cells are basically all dead during the pressure screening, start the screening of monoclonal cells.

[0055] Adjust the viable cell density to 1.0 cell / well, inoculate into a 96-well plate, 200 μl per well, and place it at 36 °C - 38 °C (preferably 37 °C). When a single cell strain in the 96-well plate has expanded to more than 80 - 100%, detect the protein expression yield of the monoclonal cell strain by ELISA method. Digest the cells with a higher expression level and transfer them to a 24-well plate. When the 24-well plate is full, take the supernatant for detection, detect the protein expression yield of the monoclonal cells by ELISA method, and screen for monoclonal cells with a protein expression yield greater than 1 g / L.

[0056] 5. Protein expression

[0057] Inoculate the monoclonal cells into a commercial CHO serum-free medium respectively, with a cell density of 1.0×10 5 cells / mL, and culture in a constant temperature shaker at 36 - 38 °C with 5% CO 2 at a rotation speed of 100 r / min; detect the cell density, viability and glucose content every 24 hours. When the glucose is lower than 2.5 g / L, supplement glucose to 3 - 4 g / L; perform feeding on the 4th and 9th days of fermentation, with the added volume being 10% of the original medium; lower the temperature to 32 °C on the 5th day of fermentation; on the 12th day, collect the cell fermentation broth. After purification respectively, obtain the expressed proteins of 5 monoclonal cells, named gD1, gD2, gD3, gD4, gD5 in sequence.

[0058] 6. Establishment of Protein Purification Process

[0059] Ultrafiltration purification of each batch of cell fermentation broth in step 5 was carried out through hollow fiber, and the specific purification process is as follows:

[0060] (1) Clarification: Wash the hollow fiber ultrafiltration system with 1×PBS buffer. Add 1000 mL of cell fermentation broth into the liquid exchange pool, turn on the stirrer and circulation pump, and set the shear rate to 4000 s -1 , after circulating for a certain time, filter through a 0.2 μm hollow fiber microfiltration column, and collect 900 mL of the permeate.

[0061] (2) Wash-filtration of the retentate: Add an equal volume of 1×PBS buffer to the remaining 100 mL of cell fermentation broth in the liquid exchange pool, turn on the stirrer and circulation pump, and set the shear rate to 4000 s -1 , collect the permeate to obtain 100 mL of wash filtrate for later use. Mix the above permeate and wash filtrate evenly to obtain 1200 mL of clarified cell supernatant.

[0062] (3) Liquid exchange and determination: Concentrate and exchange the harvested cell supernatant with a 10 kD hollow fiber membrane. The liquid exchange buffer is PBS. Perform liquid exchange in a continuous flow equal volume manner (i.e., the feeding end and the permeate end have the same flow rate), and the liquid exchange volume is 5 times. During the liquid exchange, samples are taken for HPLC detection. After the liquid exchange is completed, collect the antigen solution and store it at 2 - 8°C.

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

[0064] As can be seen from Table 1, with the increase of the replacement liquid volume, the number and area of impurity peaks gradually decrease, and the area of the main peak remains basically unchanged. That is, through ultrafiltration liquid exchange, not only can the purity of gD protein be gradually improved, but also the loss of effective protein is small. In addition, the purity of the unpurified gD protein is between 25 - 30%; after 6 times of ultrafiltration liquid exchange, the final purity of the gD protein is not less than 70%, and further liquid exchange cannot significantly improve the antigen purity. Therefore, it is finally determined that the ultrafiltration liquid exchange volume is set to 5 times the concentrated volume. After 6 batches of ultrafiltration liquid exchange, the purity of gD4 protein is improved most significantly, and its protein purity is increased by at least 246.2% compared with that before purification. Figure 1 It is the HPLC chromatogram of the purified gD4 protein sample.

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

[0066]

[0067]

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

[0069] The immunization effect and safety of the gD4 protein expressed by the gD-4 cell line were evaluated. The specific operations were carried out according to the following steps:

[0070] The purified gD4 protein in Example 1 was quantitatively analyzed using a protein quantification kit. The quantified gD4 protein was prepared into an antigen solution with PBS buffer as the solvent, and then a vaccine was prepared by mixing the 40 μg / mL antigen solution with ISA201 adjuvant. Specifically, the emulsification ratio of the aqueous phase (antigen solution) to the oil phase (ISA201 adjuvant) was 1:1 by mass. First, the oil phase was introduced into a beaker and stirred slowly, and the aqueous phase was slowly added. After the addition of the aqueous phase was completed, a shear emulsification device was started, and the mixture was emulsified for 15 min to form a stable water-in-oil emulsion, thereby obtaining a vaccine emulsion with good physical stability. After emulsification was completed, the vaccine emulsion was aseptically and quantitatively dispensed (50 mL / bottle), sealed with a cap, and a porcine pseudorabies virus subunit vaccine was obtained, which was stored at 2-8 °C.

[0071] Healthy and well-developed 21-day-old piglets that had not been 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 was collected respectively, and the neutralizing antibody level in the serum was quantitatively analyzed. The results showed that the 21-day neutralizing antibody was not less than 1:91.

[0072] To verify the actual protective effect of the vaccine, the immunized pigs were challenged with classical virulent strains (SC strain) and currently prevalent strains (JS strain) respectively. The challenge results showed that the vaccine could effectively prevent the attacks of both classical virulent strains (SC strain) and currently prevalent strains (JS strain) at the same time, and the protection rate reached 100%. All immunized pigs showed no increase in body temperature and abnormal clinical symptoms (Table 2).

[0073] Table 2 Experimental Grouping and Results

[0074]

[0075]

[0076] The present invention provides an idea and method for a porcine pseudorabies virus gD protein, its preparation method and application. There are many specific methods and ways to implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this example can be implemented by 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 recombinant 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:

8.

3. A recombinant expression vector or recombinant cell containing the coding gene according to claim 2.

4. The method for preparing the pseudorabies virus gD protein according to claim 1, characterized in that: The recombinant plasmid cloned with the gene encoding the porcine pseudorabies virus gD protein according to claim 1 is transfected into mammalian cells, and the porcine pseudorabies virus gD protein is prepared through culture, pressure screening, monoclonal cell screening, protein expression and protein ultrafiltration purification.

5. The preparation method according to claim 4, characterized in that: The recombinant plasmid is obtained by cloning the coding gene of 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 liquid obtained by protein expression of the pseudorabies virus gD protein using hollow fiber ultrafiltration.

7. The preparation method according to claim 6, characterized in that: The hollow fiber ultrafiltration purification has 1 to 8 ultrafiltration liquid exchange batches, preferably 6 to 8 batches.

8. The preparation method according to claim 7, characterized in that: After 6 batches of cell fermentation liquid obtained by protein expression of the porcine pseudorabies virus gD protein were purified by hollow fiber ultrafiltration, the purity of the porcine pseudorabies virus gD protein was at least increased by 246.2% compared with that before purification.

9. Use of the pseudorabies virus gD protein of claim 1 in the preparation of a subunit vaccine for preventing and / or treating pseudorabies in pigs.

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

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