Method for producing porcine pseudorabies virus recombinant protein by fed-batch culture of bioreactor

Abstract

The invention provides a method for producing porcine pseudorabies virus recombinant protein by using bioreactor fed-batch culture, and relates to the technical field of biology. The culture method comprises the following steps: inoculating a recombinant CHO cell containing a gD protein coding gene into a basic culture medium, and controlling the temperature to be 35-37 DEG C; feeding a first fed-batch culture medium and a second fed-batch culture medium every 1-2 days from 2-4 days after inoculation, reducing the culture temperature after the exponential growth period of the cells is ended, and continuously culturing for a certain time to obtain gD protein; the basic culture medium is a Shanghai brachycarpus leaf base Eden B100S culture medium, the first feed culture medium is a Shanghai brachycarpus leaf base Eden F100a culture medium, and the second feed culture medium is a Shanghai brachycarpus leaf base Eden F100b culture medium. According to the culture method, domestic raw materials are adopted, so that the stability of a culture process is improved on the basis of reducing the cost, and meanwhile, the problem of low expression quantity of gD protein is solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for producing porcine pseudorabies virus recombinant protein by using a bioreactor fed-batch culture. Background Art

[0002] Pseudorabies is a highly contagious disease of pigs caused by the pseudorabies virus (PRV). It primarily affects pigs but can also infect a variety of other domestic and wild animals. The disease can cause miscarriage, stillbirth, and mummification in pregnant sows, as well as neurological symptoms and death in piglets, resulting in significant economic losses for the pig industry. PRV has a strong ability to mutate, which challenges the protective efficacy of traditional vaccines and increases the difficulty of pseudorabies prevention and control. The development of new pseudorabies vaccines is a current research hotspot, with genetically engineered subunit vaccines and DNA vaccines being two of the most sought-after new vaccines. These new vaccines offer advantages such as high safety and effective immune responses, providing new options for the prevention and control of pseudorabies.

[0003] The reactor antigen production of the gD protein of the porcine pseudorabies subunit vaccine currently adopts a high-density culture Fed-Batch process constructed with a CHO cell line, using imported basal culture medium and feed culture medium. The high price limits its application and there may be a risk of supply chain disruption, which may affect the stability of large-scale continuous production and increase the price of the finished vaccine. Summary of the Invention

[0004] To address these issues, the present invention tested cell growth and protein expression in various domestic culture media, using imported culture media as a control group. After selecting the culture media, the authors explored and optimized the inoculation density, feeding strategy, cooling time and temperature, reactor settings (pH, DO, rotational speed), and harvesting time during the Fed-Batch culture process to achieve higher protein expression.

[0005] The first object of the present invention is to provide a method for producing porcine pseudorabies virus recombinant protein using a bioreactor fed-batch culture, comprising the following steps:

[0006] S1. Inoculate recombinant CHO cells containing the gene encoding the recombinant protein of the porcine pseudorabies virus in basal culture medium and control the temperature at 35-37°C;

[0007] S2. Starting from 2 to 4 days after inoculation, the first feed medium and the second feed medium are added every 1 to 2 days. After the exponential growth phase of the cells, the culture temperature is lowered and the culture is continued for a certain period of time to harvest the recombinant pseudorabies virus protein.

[0008] The basal medium is Eden B100S basal medium produced by Shanghai Beianji Biotechnology Co., Ltd. (or a composition having the same composition as the basal medium or containing the composition);

[0009] The first feed medium is Eden F100a fed-batch medium produced by Shanghai Bio-Technology Co., Ltd. (or a composition having the same composition as the first feed medium or containing the first feed medium);

[0010] The second feed medium is Eden F100b fed-batch medium of Shanghai Beianji Biotechnology Co., Ltd. (or a composition having the same composition as the second feed medium or containing the second feed medium);

[0011] The recombinant protein of porcine pseudorabies virus is porcine pseudorabies virus gD protein.

[0012] Furthermore, the nucleotide sequence of the gene encoding the porcine pseudorabies virus gD protein is shown in SEQ ID NO.1.

[0013] Furthermore, the host cells of the recombinant CHO cells include but are not limited to CHO-K1, CHO-dhfr, CHO-DXB11, CHO-DG44, CHO-S and other cell lines.

[0014] Furthermore, in step S1, the inoculation amount of recombinant CHO cells was 0.3×10 6 ~0.5×10 6 cells / mL.

[0015] Furthermore, in step S2, starting from the 2nd to 4th day after inoculation and every 1 to 2 days, the first feed medium with a volume ratio of 4% to 6% and the second feed medium with a volume ratio of 0.4% to 0.6% are fed.

[0016] Furthermore, in step S2, starting from the third day after inoculation and every other day, the first feed medium with a volume ratio of 4% to 6% and the second feed medium with a volume ratio of 0.4% to 0.6% are fed.

[0017] Furthermore, in step S2, the culture temperature is lowered from the 4th to 5th day after inoculation; the culture temperature is lowered to 31-33°C.

[0018] Furthermore, in step S2, the harvesting time is determined according to the cell viability. Specifically, the recombinant porcine pseudorabies virus protein is harvested when the cell viability is lower than 75%.

[0019] Furthermore, during the culture process, the pH is controlled at 6.95±0.2 to 7.05±0.2, the dissolved oxygen content is controlled at 30% to 50%, and the rotation speed is controlled at 150 to 350 rpm.

[0020] Furthermore, the culture process also includes a step of supplementing glucose, specifically: when the glucose concentration in the culture medium is lower than 3 g / L, glucose is added to 5-7 g / L.

[0021] In the present invention:

[0022] CHO cells (Chinese Hamster Ovary cells) are a mammalian cell line commonly used in recombinant protein production. They have many advantages, such as: (1) CHO cells are not susceptible to human virus infection, which can reduce the risk of viral contamination and ensure the purity and quality of the production process and products. This makes them very popular in the biopharmaceutical field, because safety is one of the important considerations in the manufacture of biopharmaceutical products; (2) CHO cells have similar post-translational modification characteristics to human cells, and can achieve correct glycosylation, folding and other post-modifications, so they can produce recombinant proteins with good biological activity and stability. This similarity makes CHO cells an ideal matrix for synthesizing complex proteins, which can ensure the quality and activity of recombinant proteins. Compared with other cell lines, CHO cells have a higher cell density and a longer culture life, and can stably produce high-quality recombinant proteins. Therefore, as a carrier for recombinant protein production, CHO cells are considered to be a safe, effective and efficient choice, and have broad application prospects in the biopharmaceutical field. It is precisely these advantages that make CHO cells an ideal choice for recombinant protein production, and the use of bioreactor fed-batch culture technology (Fed-Batch) is an important process method for optimizing the production of recombinant proteins in CHO cells.

[0023] In the fed-batch process, initial cell growth is maintained in a basal medium, followed by the addition of feed medium during fed-batch culture to continuously replenish nutrients and extend the culture time. This approach can avoid nutrient limitations and promote higher recombinant protein yields. However, during fed-batch culture, it is necessary to understand the characteristics of cell growth and cell metabolism, as well as the impact of various process parameters on recombinant protein expression and quality attributes.

[0024] Therefore, the present invention optimizes the culture process for cell growth and metabolism, including basal culture medium and feed culture medium, inoculation density and feeding strategy, cooling time and temperature, pH, DO, stirring speed, protein harvesting time, etc. Among them:

[0025] Nutrients have an important influence on the growth and protein expression of CHO cells. The culture medium runs through the upstream cell amplification culture and product expression of CHO protein drug production. It is closely related to the product yield and quality and directly affects the downstream separation and purification. It is a key raw material for recombinant protein production. Moreover, for different recombinant proteins, specific culture medium formulas are required to support their synthesis and folding, thereby optimizing their expression and yield. For example, amino acids are one of the key components of feed culture medium, and their concentration and properties will affect the yield and quality of recombinant proteins in fed batch culture. In addition, glucose is the main carbon source for CHO cells in the exponential growth phase, and its concentration and consumption rate will also directly affect cell growth and yield. In addition to amino acids and carbon sources, inorganic salts, trace elements, vitamins, lipids and other components also affect the productivity of CHO cells and protein quality attributes;

[0026] Feeding strategies play a key role in improving cell growth and productivity. Different protein types require different feeding strategies to optimize nutrient uptake rates and reduce the accumulation of inhibitory waste metabolites. By optimizing the feed medium and feeding method, the cell growth environment can be improved, the accumulation of inhibitory substances can be controlled, and growth and protein productivity can be improved.

[0027] In fed-batch culture, the culture environment is crucial. Parameters such as temperature, pH, and dissolved oxygen all influence CHO cell growth and recombinant protein expression. By properly controlling these parameters, the yield and quality of recombinant proteins can be improved.

[0028] In summary, different protein types respond differently to the Fed-Batch strategy and require different culture media, feeding strategies, and environmental conditions to achieve optimal cell growth, protein expression, and product quality, so specific optimization is necessary.

[0029] Preferably, the specific steps of the method of the present invention include:

[0030] The recombinant CHO cells containing the recombinant protein encoding gene of the porcine pseudorabies virus were cultured at 0.3×10 6 cells / mL in a basal culture medium, the temperature was controlled at 37°C, the pH was 6.95±0.2, the dissolved oxygen content was 50%, and the rotation speed was 250 rpm. Starting from the third day after inoculation and every other day, a first feed medium with a volume ratio of 4% and a second feed medium with a volume ratio of 0.4% were added. On the fifth day after inoculation, the culture temperature was lowered to 31°C and the culture was continued. When the cell viability was lower than 75% (day 13-15, preferably day 13), the porcine pseudorabies virus recombinant protein was harvested;

[0031] The basal culture medium is Eden B100S basal culture medium produced by Shanghai Bio-Technology Co., Ltd.

[0032] The first feed medium is Eden F100a fed-batch medium produced by Shanghai Bio-Technology Co., Ltd.

[0033] The second feed medium is Eden F100b fed-batch medium from Shanghai Bio-Technology Co., Ltd.

[0034] The recombinant protein of porcine pseudorabies virus is porcine pseudorabies virus gD protein.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention uses genetically engineered subunit vaccines to optimize the Fed-Batch process constructed by CHO cell lines for PRV-gD recombinant protein: a group of alternative culture media with higher protein expression and lower cost are screened, the reactor culture and process parameters are adjusted and optimized, the cell expression process is stable, and the replacement of imported culture media with domestic culture media reduces culture costs and supply chain risks, which can completely replace the existing production technology. The protein expression amount is 1.3 to 1.7 times that of the original process, and the culture medium cost can be reduced by 5 to 6 times when produced in a 200L reactor. In the subsequent seedling preparation, the total cost can be reduced by 6 to 10 times by combining the improved protein expression amount and culture medium price, which greatly reduces the production pressure and cost expenditure of the enterprise. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0038] The main reagents, instruments, etc. used in the following examples and their purchase information and material sources are as follows:

[0039] (1) Reagents

[0040] ExpiCHO Stable Production Medium (SPM medium) was purchased from Gibco, catalog number: A3711101,

[0041] CellBoost TM 7A was purchased from Cytiva, product number: SH31026.01,

[0042] Cell Boost TM 7B was purchased from Cytiva, product number: SH31027.02CN,

[0043] CD CHO 045 culture medium was purchased from Jianshun Biotechnology Co., Ltd., catalog number: DP1633.

[0044] TK002 culture medium was purchased from Jianshun Biotechnology Co., Ltd., catalog number: 22061-377.

[0045] CD Feed 002 culture medium was purchased from Jianshun Biotechnology Co., Ltd., catalog number: 99014-302.

[0046] CHO CD04 Medium was purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number: A11004.

[0047] CHO CD06 Medium was purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number: A11006.

[0048] CHO Feed (1 set contains 1L 02A and 0.1L B02) was purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number: A11902.

[0049] Eden B101S basal culture medium was purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0115603.

[0050] Eden B100S basal culture medium was purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0116402.

[0051] Eden F100a fed-batch culture medium was purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0116502.

[0052] Eden F101a fed-batch culture medium was purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0122102.

[0053] Eden F100b fed-batch culture medium was purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0116602.

[0054] CHO-500 was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1010,

[0055] CHO-200 was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1004,

[0056] Mate Cell Feed-200A was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1005.

[0057] Mate Cell Feed-200B was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1006.

[0058] Mate Cell Feed-500A was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1011.

[0059] Mate Cell Feed-500B was purchased from Sipeng Biotechnology (Suzhou) Co., Ltd., catalog number: L1012.

[0060] (2) Instruments and equipment

[0061] The pH meter was purchased from Mettler-Toledo, the CountStar cell counter was purchased from Shanghai Ruiyu Biotechnology Co., Ltd., the sterile pipe machines were purchased from Sartorius and TERUMO, the 2L bioreactor was purchased from Applikon, the biochemical analyzer was purchased from Silman Biological Company, and the freezing point osmometer was purchased from GENOTEC.

[0062] Example 1 Construction of a fully suspended monoclonal cell line CHO-gD cells

[0063] The specific construction process of the full suspension monoclonal cell line CHO_gD cells used in this study is as follows:

[0064] The synthesized gene sequence encoding the gD protein was cloned into the eukaryotic transfer vector pcDNA3.1 via the Mlu I and Hind III sites. The ligation product was obtained by overnight ligation at 16°C using T4 DNA ligase. The product was transformed with competent Escherichia coli DH5α and plated on LB plates containing ampicillin. After overnight incubation at 37°C, positive colonies were selected and cultured in LB medium containing ampicillin for plasmid extraction. The correct recombinant plasmid, pcDNA3.1-gD, was obtained by PCR, double enzyme digestion, and sequencing.

[0065] The recombinant plasmid pcDNA3.1-gD was transfected into well-growing whole suspension CHO-S cells to obtain recombinant cells CHO_gD.

[0066] The coding sequence of gD protein is as follows (SEQ ID NO.1):

[0067]

[0068] Example 2 Optimization of Fed-Batch Process

[0069] (1) Culture medium screening:

[0070] The recombinant CHO_gD cells were inoculated into different culture media for fermentation screening. The results are shown in Table 1. CHO CD04Medium, Eden B101S, and Eden B100S performed the best, so these four culture media were subsequently used for further process optimization.

[0071] Table 1 Grouping of culture medium screening experiments

[0072]

[0073]

[0074] The present invention further used basal medium SPM and feed medium Cell Boost 7A / 7B as controls, and used 4 domestic culture media CD CHO 045, CHO CD04 Medium, Eden B101S, and Eden B100S were used as basal media and four corresponding feed media were screened. The specific steps were as follows: CHO_gD cells prepared in Example 1 were inoculated into the basal medium at a certain inoculation density. During the culture process, the temperature was controlled at 37°C, the dissolved oxygen (DO) was 40%, and the rotation speed was 150 rpm. The pH was maintained within the range of 6.95±0.2 by CO2 and Na2CO3. When glucose was lower than 3 g / L, glucose was fed to 6 g / L. The feed medium was then fed according to the feeding strategy, and the temperature was lowered to 32°C on the fourth day. The feeding strategy was as follows: For example, in BR01, 3% feed medium CD Feed 002 was fed on day 4 after inoculation, 6% CD Feed 002 was fed on day 6, 3% CD Feed 002 was fed on day 8, 6% CD Feed 002 was fed on day 10, and 3% CD Feed 002 was fed on day 12. For example, in BR02, 4% Feed 02ASupplement and 0.4% Feed B02Supplement were fed on days 3, 5, 7, 9, 10, and 11 after inoculation, respectively. The same was true for the other groups. Grouping and parameter settings are shown in Table 2:

[0075] Table 2 Grouping of culture medium rescreening experiment

[0076]

[0077] The results are shown in Table 3. Regarding cell growth during culture, the four domestic culture media showed higher viable cell densities in the CHO CD04 Medium and Eden B100S groups than in the imported SPM medium, while those in the CD CHO 045 and Eden B101S groups were lower. Regarding protein expression, Eden B100S showed higher protein expression than the original imported medium, while CD CHO 045 and CHOCD04 Medium showed similar protein expression differences compared to the original imported medium. However, Eden B101S showed lower protein expression than the original imported medium. Because Eden B100S performed best in both cell growth and protein expression, the BR04 group (Eden B100S) was ultimately selected as the basal medium, with Eden F100a and Eden F100b as the feed medium.

[0078] Table 3 Culture medium rescreening results

[0079]

[0080]

[0081] (2) Optimization of inoculation density and feeding strategy:

[0082] During cell culture, the inoculation density will affect the cell growth density, integral viable cell density (IVCD) and culture time during the entire culture process, and thus affect the protein expression level. It is necessary to effectively control the inoculation density. Too much or too little feed medium added will have a certain impact on cell growth and protein expression. In order to obtain a high-yield and stable quality cell culture process, it is necessary to optimize the cell inoculation density and the amount of feed medium added to obtain the optimal parameters and operating space (based on BR04 in step (1)). After screening the initial and feed medium, optimize the cell inoculation density and the amount of feed medium added. The inoculation density optimization range is 0.3~1.0×10 6 cells / mL, and the seeding density was 0.3×10 6 cells / mL、0.5×10 6 cells / mL、0.7×10 6 cells / mL, 1.0×10 6 cells / mL. The feeding strategy was to feed 4%-6% Eden F100a and 0.4%-0.6% Eden F100b on D3 / D5 / D7 / D9 / D11 / D13, with feed rates of 4% / 0.4%, 5% / 0.5%, and 6% / 0.6%, respectively. Fed-Batch expression was performed. Experimental groups are shown in Table 4:

[0083] Table 4 Inoculation density and feeding strategy optimization experimental groups

[0084]

[0085] The inoculation density and feeding strategy of the selected alternative culture media were optimized. The experimental data are shown in Table 5. The results show that different inoculation densities affect cell growth. The higher the cell inoculation density, the faster the cell growth and the higher the cell density. The inoculation density is 0.3×10 6 cells / mL and 0.5×10 6 cells / mL, increasing the feed ratio has little effect on cell growth and protein expression. The inoculation density is 0.7×10 6 cells / mL and 1.0×10 6 cells / mL, different feed ratios had little effect on early cell growth, but significantly reduced protein expression. Increasing the seeding density (0.7×10 6 cells / mL, 1.0×10 6 cells / mL) Although the cells grew faster in the early stage, the maintenance of cell viability in the later stage of culture was poor, resulting in reduced protein expression. Considering the comprehensive cell growth and protein expression, the seeding density was selected to be 0.3-0.5×10 6 cells / mL, and the feeding strategy was to feed on D3 / D5 / D7 / D9 / D11 / D13 at a volume ratio of 4% / 0.4% to 6% / 0.6%.

[0086] Table 5 Optimization of inoculation density and feeding strategy

[0087]

[0088]

[0089] (3) Optimization of cooling temperature and cooling time:

[0090] The middle and late stages of cell growth are the protein expression period. Lowering the culture temperature, controlling the cell growth rate, and extending the cell culture cycle are beneficial to increasing the amount of protein expression. Therefore, when performing Fed-Batch, changing the culture process temperature during the cell culture process can increase the early cell growth rate to obtain a sufficient number of cells, providing a basis for late protein expression. The optimum temperature for the exponential growth phase of cells is 37°C, and no optimization is required. The present invention studies the temperature parameters and cooling time for the middle and late stages of cell growth and expression (based on BR01 in step 2). The cooling temperature range studied is 31°C to 35°C, the cooling time is D4 to D5, and the experimental groups are shown in Table 6:

[0091] Table 6 Optimization groups of cooling temperature and cooling time

[0092] Experiment number BR01 BR02 BR03 BR04 BR05 BR06 Cultivation temperature 37-31 37-33 37-35 37-31 37-33 37-35 Cooling time D4 D4 D4 D5 D5 D5

[0093] During the culture process, the cell cooling temperature and time processes were studied. The results are shown in Table 7. The maximum viable cell density during the D5 cooling process was higher than that during the D4 cooling process, but the difference in protein expression was small, indicating that the maximum viable cell density and protein yield are not completely proportional. Cooling to different temperatures during the culture process had little effect on cell growth overall. Protein expression was slightly lower at 35°C, and the differences in protein expression at the other two temperatures were small. Different cooling time cultures had little effect on the expression of the target protein, and the subsequent cooling culture process was determined to be cooling to 31-33°C from D4 to D5.

[0094] Table 7 Optimization of cooling temperature and cooling time

[0095]

[0096] (4) Optimization of reactor culture process parameters

[0097] Reactor parameter optimization includes control parameters such as pH, DO, temperature, and stirring speed, all of which will affect cell growth and protein expression.

[0098] ① pH is a key process parameter in cell culture, regulating the activity of intracellular enzymes. To produce consistent product quality, the pH operating range was studied. Fed-Batch expression was performed using the following pH settings: 6.95±0.2, 7.00±0.2, and 7.05±0.2 (based on BR04 in Step 3).

[0099] The results are shown in Table 8. There is no significant difference in cell growth and protein expression in the three pH ranges. During the expression process, the pH range can be set from 6.95±0.2 to 7.05±0.2 to achieve higher protein expression levels.

[0100] Table 8 pH screening experiment

[0101]

[0102] ②DO and rotation speed are related to shear force. The rotation speed and bubble size will affect cell growth and thus protein yield. The DO and rotation speed ranges were studied, and the optimized rotation speed range was 150rpm, 250rpm, and 350rpm; the DO range was 30%, 40%, and 50%. The combined experimental groups are shown in Table 9:

[0103] Table 9 DO and speed screening experiment

[0104] Experiment number BR01 BR02 BR03 BR04 BR05 BR06 BR07 Speed ​​(rpm) 150 250 350 250 150 250 350 DO (%) 30 30 30 40 50 50 50

[0105] The results are shown in Table 10. The higher the rotation speed, the faster the cell growth. There is little difference in DO protein expression between different rotation speeds. Therefore, DO of 30-50% and rotation speed of 150-350 rpm can be used in the future.

[0106] Table 10 DO and speed screening experiment

[0107]

[0108] (5) Determination of reactor harvest time:

[0109] When performing Fed-Batch expression, different harvest times can affect protein yield and quality. It is important to research the optimal harvest time. Cultivate cells according to the parameters for the BR06 group in step 4 above. Once cell viability in the 2L reactor falls below 90%, begin daily protein measurement. Determine the harvest time based on cell status and protein expression.

[0110] The results of the study on different harvesting times of the 2L reactor culture medium are shown in Table 11. The expression level continued to increase from the 8th to the 13th day of culture, and the protein expression level decreased slightly from D14. It was determined that the 2L reactor was harvested when the cell viability was lower than 75% or on D13.

[0111] Table 11 Harvest time screening experiment

[0112]

[0113] Example 3 Cultivation process parameter confirmation and scale-up

[0114] The optimized process parameters were expressed in a Fed-Batch format using a 2L reactor. Based on the equal P / V principle, the process was scaled up to a 50L reactor to verify the feasibility of process expression and scale-up. The process scale-up parameters are shown in Table 12. Three batches of experiments were conducted using a 50L reactor. The viable cell density at the time of cell cooling was 10.80-13.00×10 6 cells / mL, and the highest viable cell density was 14.10~15.70×10 6 cells / mL, and the harvested protein expression level was 2360-2615 mg / L. The process was relatively stable.

[0115] Table 12 Process scale-up parameters

[0116]

[0117] In summary, the present invention has screened out an alternative culture medium with higher protein expression and lower cost. The cell expression process is stable and the parameters have been optimized. It can completely replace the existing production technology. The protein expression is 1.3 to 1.7 times that of the original process (using imported basal culture medium and feed culture medium). When produced in a 200L reactor, the culture medium cost can be reduced by 5 to 6 times. In the subsequent seedling preparation, the total cost can be reduced by 6 to 10 times by combining the improved protein expression and culture medium price, which greatly reduces the production pressure and cost expenditure of enterprises. The specific data are as follows:

[0118] Table 12 Cost comparison

[0119]

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing porcine pseudorabies virus recombinant protein using bioreactor fed-batch culture, characterized in that: The following steps are involved: S1. Inoculate recombinant CHO cells containing the gene encoding the recombinant protein of the porcine pseudorabies virus in basal culture medium and control the temperature at 35-37°C; S2. Starting from 2 to 4 days after inoculation, the first feed medium and the second feed medium are added every 1 to 2 days. After the exponential growth phase of the cells, the culture temperature is lowered and the culture is continued for a certain period of time to harvest the recombinant pseudorabies virus protein. The basal culture medium is Eden B100S basal culture medium produced by Shanghai Bio-Technology Co., Ltd. The first feed medium is Eden F100a fed-batch medium produced by Shanghai Bio-Technology Co., Ltd. The second feed medium is Eden F100b fed-batch medium from Shanghai Bio-Technology Co., Ltd. The recombinant protein of porcine pseudorabies virus is porcine pseudorabies virus gD protein.

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

1.

3. The method according to claim 1, characterized in that The host cells of the recombinant CHO cells include CHO-K1 cell strain, CHO-dhfr cell strain, CHO-DXB11 cell strain, CHO-DG44 cell strain or CHO-S cell strain.

4. The method according to claim 1, wherein In step S1, the inoculum size of recombinant CHO cells was 0.3 × 10 6 ~0.5×10 6 cells / mL.

5. The method according to claim 1, wherein In step S2, starting from the 2nd to 4th day after inoculation and every 1 to 2 days, a first feed medium with a volume ratio of 4% to 6% and a second feed medium with a volume ratio of 0.4% to 0.6% are fed. Preferably, in step S2, starting from the third day after inoculation and every other day, the first feed medium with a volume ratio of 4% to 6% and the second feed medium with a volume ratio of 0.4% to 0.6% are fed.

6. The method according to claim 1, characterized in that In step S2, the culture temperature is lowered from the 4th to 5th day after inoculation; Preferably, the culture temperature is lowered to 31-33°C.

7. The method according to claim 1, characterized in that In step S2, the recombinant protein of pseudorabies virus is harvested when the cell viability is lower than 75%.

8. The method according to claim 1, characterized in that During the culture process, the pH is controlled at 6.95±0.2 to 7.05±0.2, the dissolved oxygen content is controlled at 30% to 50%, and the rotation speed is controlled at 150 to 350 rpm.

9. The method according to claim 1, characterized in that The culture process also includes the step of supplementing glucose; Preferably, when the glucose concentration in the culture medium is lower than 3 g / L, glucose is added to a concentration of 5 to 7 g / L.

10. The method according to claim 1, characterized in that The specific steps include: The recombinant CHO cells containing the recombinant protein encoding gene of the porcine pseudorabies virus were cultured at 0.3×10 6 The cells were inoculated into a basal culture medium at an inoculum size of 10 cells / mL. The temperature, pH, dissolved oxygen content, and rotation speed were controlled at 37°C, 6.95±0.2, 50%, and 250 rpm. Starting from the third day after inoculation, a first feed medium with a volume ratio of 4% and a second feed medium with a volume ratio of 0.4% were added every other day. On the fifth day after inoculation, the culture temperature was lowered to 31°C and culture was continued. The porcine pseudorabies virus recombinant protein was harvested when the cell viability was lower than 75%.

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