Preparation method and application of pseudorabies virus gB protein

Abstract

The invention provides a preparation method and application of a pseudorabies virus gB protein, and relates to the technical field of biology. The preparation method comprises the following steps: culturing CHO cells expressing the pseudorabies virus gB protein by adopting a fed-batch culture mode; a basic culture medium for fed batch culture comprises a CD CHO 045 culture medium, and a fed culture medium is a CD Feed 002 culture medium. The preparation method solves the problem of high production cost of the pseudorabies virus gB protein in the prior art, and improves the yield of the pseudorabies virus gB protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method of pseudorabies virus gB protein and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Pseudorabies (PR) in pigs is an infectious disease caused by the pseudorabies virus (PRV). It can infect pigs of all ages and can cause miscarriage, stillbirth, and mummification in pregnant sows, as well as neurological symptoms and death in piglets, severely hindering the healthy development of the swine industry. Furthermore, PRV infection has a low mortality rate in fattening or breeding pigs, and can even present as an asymptomatic infection. Pigs are the only species that can survive infection with PRV, making them a significant source of PR. The virus has a broad spectrum of infection, infecting mammals including cattle, sheep, dogs, and cats, with an extremely high mortality rate. Therefore, the prevention and control of pseudorabies in pigs is of vital importance to public health.

[0004] Pseudorabies virus (PRV) has 11 glycoproteins. Glycoproteins gB, gC, and gD are the primary viral antigenic components that induce protective neutralizing antibodies. Glycoprotein B (gB) is a key protein for PRV entry into host cells. It binds to receptors on the host cell membrane, triggering membrane fusion and enabling the viral genome to enter the host cell. During membrane fusion, the fusion loop of gB inserts into the lipid bilayer of the host cell membrane, facilitating fusion of the viral envelope and the host cell membrane. gB can induce both complement-dependent and complement-independent neutralizing antibodies, making it a promising candidate for subunit vaccine development.

[0005] Mammalian cell cultures, such as Chinese hamster ovary (CHO) cells, are widely used in the commercial production of therapeutic biomolecules, such as monoclonal antibodies and proteins. CHO cells offer significant advantages, including efficient expression, high safety, and ease of culture operation. Antigen production for the gB protein of the pseudorabies virus subunit vaccine currently utilizes a high-density fed-batch (fed-batch) process developed with CHO cell lines, using imported basal and feed media. The high cost of imported media limits its application, and the potential for supply chain disruptions can impact the stability of large-scale continuous production and increase the price of the finished vaccine. Furthermore, the Fed-batch process requires large amounts of culture medium and complex antigen components, potentially impacting cell growth, metabolism, and expression during the culture process. Therefore, reducing the production cost of the pseudorabies virus gB protein and increasing protein expression remain unresolved challenges.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The object of the present invention is to provide a method for preparing pseudorabies virus gB protein, so as to alleviate the problems in the prior art of high production cost of pseudorabies virus gB protein and the need to improve protein production.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, a method for preparing pseudorabies virus gB protein is provided, the method comprising culturing CHO cells expressing pseudorabies virus gB protein in a fed-batch culture method; The basal culture medium for the fed-batch culture includes CD CHO 045 culture medium, and the feed culture medium is CD Feed002 culture medium.

[0009] In an optional embodiment, the preparation method comprises culturing CHO cells under stirring conditions.

[0010] In an optional embodiment, the stirring speed is 150~350 rpm; and / or the dissolved oxygen content is 30~50%.

[0011] In an optional embodiment, the seeding density of CHO cells in the basal culture medium is 0.7-1.0×10 6 cells / mL, preferably 1.0×10 6 cells / mL.

[0012] In an optional embodiment, the preparation method comprises supplementing the culture system with feed medium starting from the 2nd to 4th day after the start of culture, and then supplementing the feed medium every 1 to 2 days until the end of culture.

[0013] In an optional embodiment, the preparation method comprises supplementing the culture system with feed medium starting from the fourth day after the start of culture, and then supplementing the feed medium every other day until the end of culture.

[0014] In an optional embodiment, the preparation method uses a feeding method to supplement the feed culture medium.

[0015] In an optional embodiment, the amount of the feed medium added is 2-6% of the volume of the culture medium containing the CHO cells during feeding, preferably 4%.

[0016] In an optional embodiment, the preparation method includes lowering the culture temperature after the exponential growth phase of the CHO cells ends.

[0017] In an optional embodiment, the culture temperature from the start of culture to before the culture temperature is lowered is 36.5-37.5°C.

[0018] In an optional embodiment, lowering the culture temperature includes lowering the temperature to 31-33°C.

[0019] In an optional embodiment, the preparation method comprises lowering the temperature to 31-33°C on the 4th to 5th day from the start of culture, preferably lowering the temperature to 33°C on the 5th day.

[0020] In an optional embodiment, the pH of the culture medium during the CHO cell culture process is 6.93-7.07.

[0021] In an optional embodiment, the culture is terminated when the CHO cell viability is lower than 80%; and / or, the culture is terminated on the 12th day from the start of culture.

[0022] In an optional embodiment, the preparation method comprises: inoculating CHO cells expressing pseudorabies virus gB protein at a seeding density of 0.7-1.0×10 6 cells / mL are inoculated into a basal culture medium CD CHO 045, cultured at 36.5-37.5°C, and cooled to 31-33°C on the 4th-5th day after the start of culture; and feed medium CD Feed 002 is supplemented by fed addition starting on the 2nd-4th day after the start of culture, and then fed every 1-2 days until the CHO cell viability is less than 80%; and / or, the culture is terminated on the 12th day after the start of culture; the culture is cultured under the conditions of a stirring speed of 150-350 rpm, a dissolved oxygen content of 30-50%, and a pH of 6.93-7.07.

[0023] In a second aspect, a method for preparing the pseudorabies virus gB protein according to the first aspect is provided for preparing a vaccine for preventing pseudorabies virus infection.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention selects a basal medium comprising CD CHO 045 medium and uses CD Feed 002 medium as a feed medium for fed-batch culture of CHO cells expressing pseudorabies virus gB protein. This method can achieve higher expression of pseudorabies virus gB protein and lower production costs. The selected culture requires only one feed, making operation simpler. Its addition reduces CO2 infusion, making the cell expression process more stable and enabling a complete replacement for existing production technologies. In a preferred embodiment, pseudorabies virus gB protein expression is 1.3-1.6 times that of the original process, while reducing costs by 4-5 times. Combining the increased protein expression with the culture medium price during subsequent vaccine preparation can reduce total costs by 5-8 times, significantly reducing production pressure and costs for enterprises. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] It should be noted that: Unless otherwise specified, all embodiments and preferred implementation methods mentioned herein can be combined with each other to form new technical solutions. All technical features and preferred features mentioned herein can be combined with each other to form new technical solutions. The components or preferred components involved can be combined with each other to form new technical solutions.

[0027]

[0046] "Range" disclosed herein in the form of lower limits and upper limits can have one or more lower limits, and one or more upper limits, respectively.

[0028] Herein, unless otherwise indicated, the various reactions or process steps may be performed sequentially or in any non-sequential manner. Preferably, the reaction methods herein are performed sequentially.

[0029] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual relationship, order, or importance between these entities or actions, such as numbering first or second.

[0030] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0031] Herein, the terms "comprise" or "comprising" are intended to imply the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

[0032] Unless otherwise indicated, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to this article.

[0033] In a first aspect, a method for preparing pseudorabies virus gB protein is provided, the method comprising culturing CHO cells expressing pseudorabies virus gB protein in a fed-batch culture manner.

[0034] As used herein, fed-batch culture refers to a culture method in which cells are first inoculated into a basal medium and then fed with feed medium during the culture process. During the addition of feed medium (e.g., before and / or during the addition of feed medium), a certain amount of medium from the original culture system may or may not be removed to maintain the total volume of medium in the entire culture system within a predetermined range.

[0035] In the preparation method provided by the first aspect of the present invention, the basal culture medium includes CD CHO 045 culture medium, and the feed culture medium is CD Feed 002 culture medium.

[0036] Chinese Hamster Ovary (CHO) cells expressing pseudorabies virus gB protein are recombinant cells containing a gene encoding the pseudorabies virus gB protein. The gene encoding the pseudorabies virus gB protein may or may not be integrated into the CHO cell genome. For example, the recombinant CHO cells contain a vector carrying the gene encoding the pseudorabies virus gB protein. CHO cell lines expressing pseudorabies virus gB protein can be conventional and known in the art for expressing exogenous proteins. Exemplary CHO cell lines include, but are not limited to, the CHO-K1 cell line, the CHO-dhfr cell line, the CHO-DXB11 cell line, the CHO-DG44 cell line, or the CHO-S cell line.

[0037] In an optional embodiment, the preparation method includes culturing the CHO cells under stirring conditions. Further optionally, the CHO cells are cultured in a stirred bioreactor to achieve cultivation under stirring conditions.

[0038] In an optional embodiment, the stirring speed is 150-350 rpm, for example, but not limited to 150, 200, 250, 300 or 350 rpm.

[0039] In an optional embodiment, the dissolved oxygen content (DO) is 30-50%, for example, but not limited to, 30, 35, 40, 45 or 50%.

[0040] In an optional embodiment, the seeding density of CHO cells in the basal culture medium is 0.7-1.0×10 6 cells / mL, for example, but not limited to, 0.7, 0.8, 0.9 or 1.0×10 6 cells / mL, preferably 1.0×10 6 cells / mL.

[0041] In an optional embodiment, the preparation method includes supplementing the culture system with feed medium starting on day 2 to 4 (for example, but not limited to day 2, 3, or 4) after the start of culture, and then supplementing the feed medium every 1 to 2 days until the end of culture. Herein, the start of culture refers to the time when the CHO cells are inoculated into the basal medium.

[0042] In an alternative embodiment, the preparation method includes supplementing the culture system with feed medium starting on day 4 from the start of culture, and then supplementing the feed medium every other day until the end of culture. Taking a culture period of 12 days as an example, the preparation method includes supplementing the feed medium on days 4, 6, 8, 10, and 12 from the start of culture.

[0043] In an optional embodiment, the preparation method uses a feeding method to supplement the feed medium. Supplementing the feed medium by feeding means adding the feed medium to the culture container at a certain speed.

[0044] In an optional embodiment, the amount of feed medium added is 2-6% of the volume of the culture medium in which the CHO cells are located during feeding, for example, but not limited to 2, 3, 4, 5 or 6%, preferably 4%.

[0045] In an optional embodiment, the preparation method includes lowering the culture temperature after the exponential growth phase of the CHO cells ends.

[0046] In an optional embodiment, the culture temperature from the start of culture to before the culture temperature is lowered is 36.5-37.5°C, preferably 37°C.

[0047] In an optional embodiment, lowering the culture temperature includes lowering the temperature to 31-33°C, for example, but not limited to 31, 32 or 33°C.

[0048] In an optional embodiment, the preparation method comprises lowering the temperature to 31-33°C on the 4th to 5th day from the start of culture, preferably lowering the temperature to 33°C on the 5th day.

[0049] In an optional embodiment, the pH of the culture medium during the CHO cell culture process is 6.93-7.07, for example, but not limited to, 6.95, 7.0, 7.05 or 7.07.

[0050] In an optional embodiment, the culture is terminated when the CHO cell viability is lower than 80%; and / or, the culture is terminated on the 12th day from the start of culture.

[0051] In some specific embodiments, the method for preparing the pseudorabies virus gB protein comprises: CHO cells expressing pseudorabies virus gB protein were seeded at a density of 0.7-1.0×10 6 cells / mL are inoculated into a basal culture medium CD CHO 045, cultured at 36.5-37.5°C, and cooled to 31-33°C on the 4th-5th day after the start of culture; and feed medium CD Feed 002 is supplemented by fed addition starting on the 2nd-4th day after the start of culture, and then fed every 1-2 days until the CHO cell viability is less than 80%; and / or, the culture is terminated on the 12th day after the start of culture; the culture is cultured under the conditions of a stirring speed of 150-350 rpm, a dissolved oxygen content of 30-50%, and a pH of 6.93-7.07.

[0052] It is understood that the method for preparing the pseudorabies virus gB protein provided in the first aspect further includes one or more steps of isolating, enriching, purifying, drying, sterilizing, packaging, and storing the pseudorabies virus gB protein after the completion of the culture of the CHO cells expressing the pseudorabies virus gB protein. The specific process selection, as well as the specific operating steps, process parameters, and equipment involved in the selected process, can be carried out according to methods well known in the art, such as those described in various general and more specific textbooks, references, process manuals, product specifications, standard documents, and equipment manuals, and the present invention is not limited thereto.

[0053] In a second aspect, the method for preparing the pseudorabies virus gB protein described in the first aspect is used to prepare a vaccine for preventing pseudorabies virus infection. Applying the method described in the first aspect to the preparation of a vaccine for preventing pseudorabies virus infection can significantly reduce the raw material cost of the vaccine.

[0054] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0055] The detailed information of the culture medium used in the following examples is as follows: CD CHO 045 medium: purchased from Gansu Jianshun Biotechnology Co., Ltd., catalog number DP-1633; CD CHO 031 medium: purchased from Gansu Jianshun Biotechnology Co., Ltd., catalog number 88031-585; QuaCell® CHO CD04 Medium: purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number: A11004; Eden B101S culture medium: purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0115603; SPM medium: ExpiCHO Stable Production Medium, purchased from Gibco, product number: A3711101.

[0056] CD Feed 002: purchased from Jianshun Biotechnology Co., Ltd., catalog number 99014-302; ALLY CHO 300a / ALLY CHO 100b: purchased from Jianshun Biotechnology Co., Ltd., catalog number 99190-24068 / 99169-1473; QuaCell® CHO Feed02 / QuaCell® CHO A12904: purchased from Zhongshan Kangsheng Biotechnology Co., Ltd., catalog number A12902 / A12904; Eden F101aS / Eden F100bS: purchased from Shanghai Bio-Technology Co., Ltd., catalog number: FG0122102 / FG0116602; Cell Boost TM 7a / Cell Boost TM 7b, purchased from Cytiva, catalog number: SH31026.01 / SH31027.02CN.

[0057] The detailed information of the equipment used in the following examples is as follows: 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 2 L bioreactor was purchased from Applikon, the biochemical analyzer was purchased from Silman Biotechnology, and the freezing point osmometer was purchased from GENOTEC.

[0058] In the following examples, the CHO cell line for the pseudorabies virus gB protein is CHO-S; The following examples all adopt the Fed-Batch method for fed-batch culture, that is, when adding feed medium, the feed medium is added to the culture container at a certain speed, and the original medium in the culture system is not discharged.

[0059] Example 1 Culture medium screening: When expressing proteins in cell culture, a chemically defined medium free of animal or plant components is required. This medium supports high-density cell culture and has the advantage of increasing yield. This example screened the original process medium, SPM, using four domestically produced culture media (CD CHO 045, CD CHO 031, CHO CD04 Medium, and Eden B101S) as basal media, along with four corresponding feed media. The grouping and parameter settings are shown in Table 1.

[0060] Table 1 Grouping of culture medium screening experiment

[0061] Among the four domestic culture media, CD CHO 031 and CD CHO 045 showed higher protein expression than the original imported culture medium. Eden B101S showed little difference in protein expression compared to the original imported culture medium, while CHO CD04 Medium showed lower protein expression than the original imported culture medium. Because CD Feed 002 feed medium is simple to operate, CD CHO 031, CHO CD04 Medium, and Eden B101S each require two feed media, and one of these feed media significantly increases the pH of the culture medium, potentially impacting subsequent cell growth, CD CHO 045 was ultimately selected as the alternative basal medium, with CD Feed 002 serving as the feed medium. The results are shown in Table 2.

[0062] Table 2 Results of culture medium screening experiment

[0063] Example 2 Optimization of different types of reactors Using different reactor types during cell culture and expression can significantly impact cell growth and expression processes. Based on the culture conditions in Experiment BR01 of Example 1, fed-batch expression was performed using a stirred-flow bioreactor, a turbulent flow bioreactor, and a rocking bioreactor. The stirred-flow bioreactor performed better in terms of both cell growth and protein expression, and was ultimately selected. The results for different reactor types are shown in Table 3.

[0064] Table 3: Results of different types of reactors

[0065] Example 3 Inoculation density optimization 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. Therefore, effective control of the inoculation density is necessary. Based on the culture conditions of Experiment BR01 in Example 1, the cell inoculation density was further optimized. A stirred reactor was used, and the inoculation density was optimized in the range of 0.3-1.5×10 6 cells / mL, and the seeding densities were: 0.3, 0.5, 0.7, 1.0, 1.2, and 1.5×10 6 cells / mL, and Fed-Batch expression was performed. The experimental groups are shown in Table 4: Table 4 Inoculation density and optimization experiment grouping

[0066] The experimental data are shown in Table 5. The results show that different seeding densities affect cell growth. The higher the cell seeding density, the faster the cell growth and the higher the cell density. The seeding density is 0.7~1.2×10 6 cells / mL. The seeding density is 0.3×10 6 cells / mL and 1.5×10 6 cells / mL. The optimal seeding density is 0.7~1.0×10 6 cells / mL.

[0067] Table 5 Inoculation density optimization results

[0068] Example 4 Optimization of feeding strategy: During cell culture and expression, adding too much or too little feed medium 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 amount of feed medium added to the cells during the fed-batch culture process to obtain the optimal parameters and operating space. Based on the culture conditions of Experiment No. BR01 in Example 1, a stirred reactor was used with an inoculation density of 1.0×10 6 Based on the cells / mL, the optimized feeding strategy was to feed 2%~8% every other day from D4 to D12 for Fed-Batch expression, and finally the feeding strategy of 4% on D4 / D6 / D8 / D10 / D12 was selected.

[0069] Table 6 Grouping and results of feeding strategy experiments

[0070] Example 5 Optimization of cooling temperature and cooling time: During Fed-Batch, the cell culture process usually changes the culture process temperature. In order to increase the early growth rate of cells and obtain a sufficient number of cells to provide a basis for later protein expression, the temperature of cells in the exponential growth phase is generally higher than that in the middle and late expression period. The middle and late stages of cell growth are generally the protein expression period. Lowering the culture temperature, controlling the cell growth rate, and extending the cell culture cycle are conducive to increasing the amount of protein expression. The optimum temperature for the exponential growth phase of cells is 37°C. No optimization is required. The temperature parameters and cooling time for the middle and late stages of cell growth and expression are studied. The cooling temperature range studied is 31°C~35°C, and the cooling time is D4~D5. For specific experimental conditions, please refer to Table 7. For other culture conditions, refer to Example 1. Experiment No. BR01 group culture conditions, using a stirred reactor, the inoculation density is 1.0×10 6 cells / mL. The experimental results are shown in Table 7. There was little difference in protein expression between the D5 and D4 cooling processes. Cooling to different temperatures had little effect on cell growth overall. Protein expression was slightly lower at 35°C, and there was little difference in protein expression at the other two temperatures. Different cooling time cultures had little effect on target protein expression, so the subsequent cooling process was determined to be cooling to 31-33°C from D4 to D5.

[0071] Table 7 Cooling time and temperature experimental groups

[0072] Example 6 Optimization of reactor culture process parameters Reactor parameter optimization includes control parameters such as pH, DO, temperature, stirring speed, and ventilation, all of which will affect cell growth and protein expression. pH is one of the important process parameters for cell culture and has a certain regulatory effect on the activity of intracellular enzymes. Under normal circumstances, cells can grow and express normally within the pH range of 6.80-7.20. In order to produce products of consistent quality, the operating range of pH was studied. The specific experimental conditions are grouped in Table 8. Other culture conditions refer to the culture conditions of Experiment No. BR01 in Example 1. A stirred reactor was used with an inoculation density of 1.0×10 6 The experimental results are shown in Table 8, which show that the effect varies slightly from 6.95 to 7.05 ± 0.2.

[0073] Table 8 Cell culture pH experiment groups and experimental results

[0074] Dissolved oxygen concentration (DO) and rotation speed are related to shear force. The rotation speed and bubble size affect cell growth and thus protein yield. The DO and rotation speed ranges were studied, and the optimized rotation speed range was 150 rpm, 250 rpm, and 350 rpm; the DO range was 30%, 40%, and 50%. The specific experimental conditions are grouped in Table 9. Other culture conditions refer to the culture conditions of Experiment BR01 in Example 1. A stirred reactor was used with an inoculation density of 1.0×10 6 cells / mL. The experimental results are shown in Table 9. The results show that the higher the rotational speed, the faster the cell growth. When the rotational speed and DO are combined, the difference in protein expression between the different rotational speeds and DO is small. Therefore, a DO of 30-50% and a rotational speed of 150-350 rpm can be used in the future.

[0075] Table 9 Dissolved oxygen concentration (DO) and speed experimental groups and experimental results

[0076] Example 7 Determination of reactor harvest time: When performing Fed-Batch expression, different harvest times can have a certain impact on protein yield and quality. It is necessary to study the harvest time of the culture to determine the optimal harvest time. When the cell viability in the 2L reactor drops below 90%, protein levels should be measured daily. The harvest time should be determined based on cell status and protein expression. For specific experimental condition groups, see Table 10. For other culture conditions, refer to the culture conditions of Experiment BR01 in Example 1. A stirred tank reactor was used, and the inoculation density was 1.0 × 10 6 The results of the study on different harvesting times of the 2 L reactor culture medium are shown in Table 10. The expression level continued to increase from day 8 to day 15 of culture, and the protein expression level decreased slightly from day 13. This may be due to protein degradation caused by long culture time. The 2 L reactor was harvested when the cell viability was lower than 80% or on day 12.

[0077] Table 10 Harvest time experimental groups

[0078] In summary, the existing technology (see Experimental Group BR05 in Example 1) uses imported basal culture medium and feed medium. The above examples demonstrate that using CD CHO 045 medium as the basal culture medium and CD Feed 002 medium as the feed medium for fed-batch culture of CHO cells expressing pseudorabies virus gB protein can achieve higher protein expression levels and lower production costs. Furthermore, the selected culture requires only one feed, making operation simpler and reducing CO2 intake. The cell expression process is stable and can completely replace existing production technologies. Protein expression levels are 1.3-1.6 times higher than the original process. When produced in a 200 L reactor, the culture medium cost can be reduced by 4-5 times. The combined effect of increased protein expression and culture medium price during subsequent seedling preparation can reduce the total cost by 5-8 times, significantly reducing production pressure and costs for enterprises. Specific data are as follows: Table 11 Effect comparison

[0079] 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 preparing pseudorabies virus gB protein, characterized in that: This includes culturing CHO cells expressing pseudorabies virus gB protein using a fed-batch culture method; The basal culture medium for the fed-batch culture includes CD CHO 045 culture medium, and the feed culture medium is CD Feed 002 culture medium.

2. The preparation method according to claim 1, characterized in that including culturing CHO cells under agitated conditions; Optionally, the stirring speed is 150-350 rpm; and / or the dissolved oxygen content is 30-50%.

3. The preparation method according to claim 1, characterized in that The seeding density of CHO cells in the basal culture medium is 0.7-1.0×10 6 cells / mL, preferably 1.0×10 6 cells / mL.

4. The preparation method according to claim 1, characterized in that The preparation method comprises the steps of supplementing the culture system with feed medium starting from the 2nd to 4th day after the start of culture, and then supplementing the feed medium every 1 to 2 days until the end of culture; Optionally, the preparation method comprises supplementing the culture system with feed medium starting from the fourth day after the start of culture, and then supplementing the feed medium every other day until the end of culture; Optionally, the preparation method uses a feeding method to supplement the feed culture medium.

5. The preparation method according to claim 1, characterized in that The amount of feed medium added is 2-6% of the volume of the culture medium containing the CHO cells during feeding, preferably 4%.

6. The preparation method according to claim 1, characterized in that The preparation method comprises lowering the culture temperature after the exponential growth phase of the CHO cells; Optionally, the culture temperature from the start of culture to before lowering the culture temperature is 36.5-37.5°C; Optionally, lowering the culture temperature includes lowering the temperature to 31-33°C; Optionally, the preparation method comprises lowering the temperature to 31-33° C. on the 4th to 5th day from the start of culture, preferably lowering the temperature to 33° C. on the 5th day.

7. The preparation method according to claim 1, characterized in that The pH of the culture medium during CHO cell culture is 6.93~7.

07.

8. The preparation method according to claim 1, characterized in that The culture is terminated when the CHO cell viability is lower than 80%; and / or, the culture is terminated on the 12th day from the start of culture.

9. The preparation method according to any one of claims 1 to 8, characterized in that include: CHO cells expressing pseudorabies virus gB protein were seeded at a density of 0.7-1.0×10 6 cells / mL are inoculated into a basal culture medium CD CHO 045, cultured at 36.5-37.5°C, and cooled to 31-33°C on the 4th-5th day after the start of culture; and feed medium CD Feed 002 is supplemented by fed addition starting on the 2nd-4th day after the start of culture, and then fed every 1-2 days until the CHO cell viability is less than 80%; and / or, the culture is terminated on the 12th day after the start of culture; the culture is cultured under the conditions of a stirring speed of 150-350 rpm, a dissolved oxygen content of 30-50%, and a pH of 6.93-7.

07.

10. Use of the method for preparing the pseudorabies virus gB protein according to any one of claims 1 to 9 in preparing a vaccine for preventing pseudorabies virus infection.

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