CHO cell culture method for expressing hog cholera virus E2 fusion protein

Abstract

The invention discloses a CHO cell culture method for expressing hog cholera virus E2 fusion protein. The CHO cell culture method comprises the following steps: subculture; and carrying out gradient cooling type fed-batch culture. The inoculation day is the 0th day, 3% of the material is supplemented on the 3rd day, 3% of the material is supplemented on the 4th day, 4% of the material is supplemented on the 5th day, 3% of the material is supplemented on the 6th day, 3% of the material is supplemented on the 7th day, 3% of the material is supplemented on the 8th day, 3% of the material is supplemented on the 10th day, and 3% of the material is supplemented on the 12th day; culturing in a gradient cooling manner, when the initial culture temperature is 36.5-37.5 DEG C and the cell density is 10.0-18.0 * 10 < 6 > cells / mL, cooling to 34-35 DEG C, cooling to 32-33 DEG C after 24 hours, cooling to 30-31 DEG C after 48 hours, maintaining until the 14th day or the cell viability is lower than 70%, harvesting supernatant, and finishing the culture. According to the culture process disclosed by the invention, the cell culture density and motility rate are improved, and the expression quantity of the E2 protein is remarkably improved, so that the production efficiency is improved, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary vaccine cell culture, and in particular to a CHO cell culture method for expressing classical swine fever virus E2 genetic engineering fusion protein. Background Art

[0002] Classical Swine Fever (CSF) is one of the most serious viral diseases that endanger the pig industry. Since the emergence of CSF, it has caused significant economic losses to the pig industry. Under natural conditions, the Classical Swine Fever Virus (CSFV) invades the pig's body through the nose, sometimes enters the body through the conjunctiva of the eye, the genital mucosa or skin wounds, and can also infect pigs through the oral and non-intestinal routes. CSF clinically manifests as an acute type with a very high mortality rate or a subacute type with an uncertain mortality rate, a chronic type, a latent type and a persistent infection type. The main symptoms are high fever and degeneration of small blood vessel walls in the affected pigs, causing extensive bleeding, infarction and necrosis of various organs and tissues. The World Organization for Animal Health (OIE) lists CSF as one of the most important notifiable infectious diseases, and it is listed as a Class I infectious disease in my country.

[0003] CSFV is an enveloped virus with a genome length of about 123kb and only one large open reading frame (ORF). This ORF is translated into a polyprotein with 3898 amino acid residues and a molecular weight of about 438kDa, and is further processed into mature proteins under the action of viral and host cell proteases. Among all the structural and non-structural proteins of CSFV, E0 and E2 are the most valuable for immune prevention and treatment research, especially E2 protein, which is currently the preferred protein for subunit vaccines.

[0004] At present, most countries in the world rely on live attenuated vaccines and subunit vaccines to prevent and control swine fever. For live attenuated vaccines, in serum antibody testing, it is impossible to distinguish whether the immune response is caused by natural infection or vaccination. After vaccination with live attenuated vaccines, swine fever viruses can still infect animals, leaving a certain level of low virus in the animal's body.

[0005] The preparation of swine fever subunit vaccines by genetic engineering mainly focuses on the E2 antigen region, and the E2 antigen vaccine is currently an important means of controlling the disease. Although there are already genetic engineering subunit vaccines expressing swine fever E2 antigens on the market, such as Escherichia coli, baculovirus, and insect cells, people are still seeking new low-cost methods for preparing long-acting swine fever E2 antigen vaccines.

[0006] Prior patent CN107974432B discloses a method for culturing a CHO cell line that secretes and expresses swine fever E2 protein or BVDV-E2 protein, which uses two imported basal culture media (Dynamis and EX-Cell 302 culture media, where Dynamis is a product of Thermo Fisher and Ex-cell 302 is a product of Merck), has high liquid preparation risk (the preparation methods of the two culture media are different, whether they are prepared separately and then mixed for use, or solid powder is dissolved in a container during preparation, there is currently no standard), is expensive, and has high production costs. Moreover, the method in the prior patent is used to ferment and produce swine fever E2 protein, and the highest protein expression level can only reach 2.18g / L. There is no case in the prior art solution where the reactor culture yield reaches 3.5g / L or more and can achieve production. Summary of the invention

[0007] The object of the present invention is to provide a CHO cell culture method combining controlled culture temperature with a feeding mode.

[0008] By optimizing the gradient cooling combined with the medium supplement method, the expression of CHO cells for classical swine fever E2 protein is increased. The medium composition is not changed, the operation is simple, no additional chemical reagents are added, and the risk is low. It is at a high level in the field of using CHO cells to express E2 protein, and can be mass-produced in reactors, which greatly improves production efficiency. The use of domestic culture media also further reduces production costs.

[0009] The objective of the present invention is achieved through the following technical solutions: A method for culturing CHO cells expressing classical swine fever virus E2 fusion protein comprises the following steps: S1. Subculture: inoculating CHO seed cells that secrete and express the fusion protein of classical swine fever virus E2 into the basal culture medium for subculture; S2, Gradient cooling fed-batch culture: CHO seed cells (CHO-E2 cells) after subculture in S1 were cultured at 0.5-1.0×10 6 cells / mL were inoculated into the basal medium, and the feed medium was added in batches for feed culture. The temperature was gradually lowered, and the initial culture temperature was 36.5-37.5℃; the cell density was 10.0-18.0×10 6 When the cell viability is less than 70%, the supernatant is harvested and the culture is terminated.

[0010] As an embodiment of the present invention, in step S2, when feed culture medium is added in batches for fed culture, the day of inoculation is taken as day 0, 3% of the initial working volume (or mass) is fed on day 3, 3% on day 4, 4% on day 5, 3% on day 6, 3% on day 7, 3% on day 8, 3% on day 10, and 3% on day 12.

[0011] As an embodiment of the present invention, the basal culture medium is a CHO cell serum-free culture medium.

[0012] Furthermore, the CHO cell serum-free culture medium is at least one of CM-013, AltairCHO, SagiCHO, StarCHO, CDCHO 031, CD CHO 041, and CD CHO 045.

[0013] As an embodiment of the present invention, in step S2, the feed medium is a CHO cell batch feed medium, specifically at least one group selected from DF-013 and DF-003, AltairCHO Feed and CDFS36, AF183 and CDFS36, XYZF1.5 and XYZ F7.

[0014] Preferably, in step S2, the CHO cell batch fed culture medium is DF-013 and DF-003, and the dosage ratio (mass ratio or volume ratio) of DF-013 and DF-003 is 10:1.

[0015] As an embodiment of the present invention, the classical swine fever virus E2 fusion protein includes the extracellular region of the classical swine fever virus envelope glycoprotein E2, and the amino acid sequence of the extracellular region of the classical swine fever virus envelope glycoprotein E2 is shown in Sequence ID.NO.3.

[0016] As an embodiment of the present invention, the classical swine fever virus E2 fusion protein further includes a signal peptide and / or a histidine tag.

[0017] As an embodiment of the present invention, the amino acid sequence of the signal peptide is shown in Sequence ID.NO.2; And / or, the amino acid sequence of the histidine tag is shown in Sequence ID.NO.4.

[0018] As one embodiment of the present invention, the amino acid sequence of the classical swine fever virus E2 fusion protein is as shown in Sequence ID.NO.1, or is an amino acid sequence as shown in Sequence ID NO.1 including an immunogenic derivative protein that has been substituted, deleted or added with one or several amino acids.

[0019] As an embodiment of the present invention, in step S1, the temperature during the subculture is 36.5-37.5°C; and / or, CO 2 The content is 5~8%; and / or, humidity is 75-85%; And / or, the rotation speed is 100~130rpm.

[0020] Preferably, in step S1, the temperature during the subculture is 37°C; and / or, CO 2 The content is 5%; and / or, humidity is 80%; and / or, the rotation speed is 120 rpm (amplitude 50 mm).

[0021] As an embodiment of the present invention, in step S1, the subculture is performed in a shaking incubator.

[0022] As an embodiment of the present invention, in step S1, during the subculture, the cells are subcultured every 2 days, and 12.5-50 μM methionine sulfoximine (MSX) is added to the basal culture medium starting from the second generation.

[0023] As an embodiment of the present invention, in step S2, before cooling to 30-31°C, the pH is 7.0±0.4; And / or, after cooling to 30-31°C, the pH is 7.0±0.2. Because cell growth metabolism, nutrient composition and metabolite concentrations are constantly changing, the pH changes dynamically within the set range during the entire culture process.

[0024] As an embodiment of the present invention, in step S2, the fed-batch culture rotation speed is 180-250 rpm; the fed-batch culture is carried out in a glass reactor, and the rotation speed of the glass reactor is 180-250 rpm; and / or, the dissolved oxygen is controlled at 40% to 60%; And / or, when the glucose concentration is lower than 4 g / L, supplement glucose to 4-5 g / L.

[0025] Preferably, the culture speed is 220 rpm; And / or, the dissolved oxygen is controlled at 50%.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1) The highest cell density during the culture process reached 30.0×10 6 cells / mL, and the cell viability is above 98%; 2) Gradient cooling combined with fed-batch culture can maintain high cell growth, and the cell density is maintained at 20.0×10 on day 14. 6 cells / mL or more, with a viability of more than 90%; 3) Using the method of the present invention, the concentration of the classical swine fever virus E2 subunit fusion protein expressed by the cell line can reach 3.56 g / L, which greatly increases the protein expression amount of the classical swine fever virus E2 subunit fusion protein; 4) By combining gradient cooling with commercial feed supplementation, the culture medium composition is not changed, the operation is simple, no additional chemical reagents are added, and the risk is low; 5) It can be mass-produced in reactors, which greatly improves production efficiency, and the use of domestic culture media further reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 It is a curve diagram of cell culture density change of each embodiment and comparative example; Figure 2 The cell culture activity change curves of various embodiments and comparative examples are shown; Figure 3 It is a comparison chart of protein expression levels when the supernatant is harvested after cultivation of each embodiment and comparative example; Figure 4 The figure is the result of SDS-PAGE electrophoresis after dilution of the supernatant of Example 1, wherein lane M is a marker, lane 1 is the supernatant of Example 1 on the 10th day; lane 2 is the supernatant of Example 1 on the 11th day; lane 3 is the supernatant of Example 1 on the 12th day; lane 4 is the supernatant of Example 1 on the 13th day; and lane 5 is the supernatant of Example 1 on the 14th day.

[0028] Figure 5 It is a graph showing the result of SDS-PAGE electrophoresis after dilution of the supernatant of Example 2, wherein lane M is a marker, lane 1 is the supernatant of Example 2 on the 7th day; lane 2 is the supernatant of Example 2 on the 8th day; lane 3 is the supernatant of Example 2 on the 9th day; lane 4 is the supernatant of Example 2 on the 10th day; lane 5 is the supernatant of Example 2 on the 11th day, lane 6 is the supernatant of Example 2 on the 12th day, lane 7 is the supernatant of Example 2 on the 13th day, lane 8 is the supernatant of Example 2 on the 14th day, and lane 9 is the supernatant of Example 1 on the 14th day. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all belong to the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the reagents and instruments used in the following examples can be obtained through conventional commercial channels.

[0030] The serum-free basal medium CM-013 used is a product of Suzhou Womei Biological Co., Ltd., and the feed medium DF-013 and DF-003 are both products of Suzhou Womei Biological Co., Ltd. The serum-free basal medium for CHO cells (CM-013, AltairCHO, SagiCHO, StarCHO, CD CHO 031, CD CHO 041, CD CHO 045) and CHO feed medium (DF-013, DF-003, AltairCHO Feed, AF183, CDFS36, CDFS36, XYZF1.5, XYZ F7) used in the present invention can be purchased from Suzhou Womei Biological Co., Ltd., Shanghai Aopma Biotechnology Co., Ltd., Jianshun Biotechnology (Nantong) Co., Ltd. and other companies. Other reagents are all domestically produced commercial products. The CHO-E2 cell line used in the embodiment is the Chinese hamster ovary cell SLPCB05 with a deposit number of CCTCC NO: C2022313, which was deposited in the China Center for Type Culture Collection on October 12, 2022, and the test result on October 18, 2022 was survival.

[0031] Example 1 (1) Seed subculture: Take out a frozen CHO-E2 seed cell (CHO cell expressing classical swine fever virus E2 fusion protein) from the liquid nitrogen tank, thaw it in a 37°C water bath for 2.5 min, and transfer it to a 125 mL shake flask containing 30 mL CM-013 medium after thawing. Incubate at 37°C, 5% CO 2 , 80% humidity, 120rpm conditions, subculture every 2 days, add 50μM MSX from the 2nd generation, and use 1L shaking flask to expand the cells to 250mL for later use.

[0032] (2) Fed-batch culture: The seed cells cultured in step (1) were cultured at a rate of 0.5 × 10 6cells / mL was inoculated into a 3L glass reactor containing CM-013 basal medium, and batch fed culture was started. The feed medium was DF-013 and DF-003. The two culture media were added at the same time. The ratio of the two usage was DF-013:DF-003=10:1 (volume ratio or mass ratio is acceptable, in this embodiment, it is volume ratio). The following feed amount expressed in percentage during feed is the usage of DF-013, which was added according to the initial working volume. The day of inoculation was day 0; 3% on day 3, 3% on day 4, 4% on day 5, 3% on day 6, 3% on day 7, 3% on day 8, 3% on day 10, and 3% on day 12; the initial culture temperature was 37°C, and the culture was continued until day 5 (the density reached 10.0~18.0×10 6 cells / mL), the temperature was lowered to 35℃, and then lowered to 33℃ again after 24h, and then continued to lower to 31℃ after 48h, and maintained until the end of the culture; the pH was set to 7.0±0.4 before cooling to 31℃, and to 7.0±0.2 after cooling to 31℃ (because the cell growth metabolism, nutrient components and metabolite concentrations are constantly changing, the pH changes dynamically within the set range during the entire culture process); the reactor speed was 220rpm, and the dissolved oxygen was controlled at 50%. During the culture process, when the glucose concentration was lower than 4g / L, glucose was supplemented to 4~5g / L (the glucose in the feed was included, and the glucose concentration also changed dynamically during the feed culture process), and the supernatant was harvested on the 14th day.

[0033] Figure 4 The figure is the result of SDS-PAGE electrophoresis after dilution of the supernatant of Example 1, wherein lane M is a marker, lane 1 is the supernatant of Example 1 on the 10th day; lane 2 is the supernatant of Example 1 on the 11th day; lane 3 is the supernatant of Example 1 on the 12th day; lane 4 is the supernatant of Example 1 on the 13th day; and lane 5 is the supernatant of Example 1 on the 14th day. Figure 4 As a result, it can be seen that as the culture days increase, the expression level of the target protein increases.

[0034] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0035] The results showed that Example 1 reached a maximum cell density of 30.01×10 6 cells / mL, the viability was 98.17% at this time; the viability on the 14th day was 90.58%, and the density was 22.75×10 6 cells / mL, and the protein expression level was 3.56g / L.

[0036] Example 2 (1) Seed subculture: Take out a frozen CHO-E2 seed cell (CHO cell expressing classical swine fever virus E2 fusion protein) from the liquid nitrogen tank, thaw it in a 37°C water bath for 2.5 min, transfer it to a 125 mL shake flask containing 30 mL Altair CHO medium, and incubate it at 37°C, 5% CO 2 , 80% humidity, 120rpm conditions, subculture every 2 days, add 50μM MSX from the 2nd generation, and use 1L shake flask to expand the cells to 300mL for later use.

[0037] (2) Fed-batch culture: The seed cells cultured in step (1) were cultured at a rate of 0.5 × 10 6 The density of cells / mL was inoculated into a 3L glass reactor containing AltairCHO basal medium, and batch feeding culture was started. The feed medium was AF183 and CDFS36. The two culture media were added at the same time. The ratio of the usage (surface volume) of the two media was AF183:CDFS36=10:1. The feed amount expressed in percentage during the following feeding is the usage of AF183, which was added according to the initial working volume. The day of inoculation was day 0; the feed was 3% on the 3rd day, 3% on the 4th day, 4% on the 5th day, 3% on the 6th day, 3% on the 7th day, 3% on the 8th day, 3% on the 10th day, and 3% on the 12th day; the initial culture temperature was 37°C, and the culture was continued until the 5th day (the density reached 10.0-18.0×10 6 cells / mL), the temperature was lowered to 35℃, and then lowered to 33℃ again after 24h, and then continued to lower to 31℃ after 48h, and maintained until the end of the culture; the pH was set to 7.0±0.4 before cooling to 31℃, and to 7.0±0.2 after cooling to 31℃ (because the cell growth metabolism, nutrient components and metabolite concentrations are constantly changing, the pH changes dynamically within the set range during the entire culture process); the reactor speed was 220rpm, and the dissolved oxygen was controlled at 50%. During the culture process, when the glucose concentration was lower than 4g / L, glucose was supplemented to 4~5g / L (the glucose in the feed was included, and the glucose concentration also changed dynamically during the feed culture process), and the supernatant was harvested on the 14th day.

[0038] Figure 5The figure is the result of SDS-PAGE electrophoresis after dilution of the supernatant of Example 2, wherein lane M is a marker, lane 1 is the supernatant of Example 2 on the 7th day; lane 2 is the supernatant of Example 2 on the 8th day; lane 3 is the supernatant of Example 2 on the 9th day; lane 4 is the supernatant of Example 2 on the 10th day; lane 5 is the supernatant of Example 2 on the 11th day, lane 6 is the supernatant of Example 2 on the 12th day, lane 7 is the supernatant of Example 2 on the 13th day, lane 8 is the supernatant of Example 2 on the 14th day, and lane 9 is the supernatant of Example 1 on the 14th day. Figure 5 As a result, it can be seen that as the culture days increase, the expression level of the target protein increases.

[0039] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0040] The results showed that Example 2 reached a maximum cell density of 28.5×10 6 cells / mL, the viability was 97.9% at this time; the viability on the 14th day was 88.9%, and the density was 19.1×10 6 cells / mL, and the protein expression level was 3.3g / L.

[0041] Comparative Example 1 (1) Seed subculture: proceed as per step (1) of Example 1.

[0042] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6 cells / ml was inoculated into a 3L glass reactor containing CM-013 basal medium, and fed-batch culture was started (the feed medium was DF-013 and DF-003, and the two culture media were added at the same time, and the ratio of the two used amounts was DF-013:DF-003 = 10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the amount of DF-013 used, added according to the initial working volume. The day of inoculation was day 0; the feed was 4% on the 3rd day, and 4% every other day thereafter; the initial culture temperature was 37°C, and when the culture reached day 5 (cell density was 12.0-18.0×10 6 cells / mL), the culture temperature was adjusted to 34℃ and maintained until the end of the culture; the pH was set to 7±0.2 during the whole culture process, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 14th day.

[0043] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0044] The results showed that the cell density of Comparative Example 1 at the end of the 14th day of culture was 14.75×10 6 cells / mL, the cell viability was 75.55%, and the protein expression level was 2.6 g / L. The highest cell density reached 26.22×10 6 cells / mL, and the cell viability was 97.81%.

[0045] Comparative Example 2 (1) Seed subculture: proceed as in step (1) of the example.

[0046] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6 The density of cells / ml was inoculated into a 3L glass reactor containing CM-013 basal medium, and batch fed culture was started. The feed medium was DF-013 and DF-003. The two culture media were added at the same time. The ratio of the two usage was DF-013:DF-003 = 10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the usage of DF-013, which was added according to the initial working volume. The day of inoculation was day 0; 4% was fed on the 3rd day, and 4% was fed every other day thereafter; the initial culture temperature was 37°C, and when the culture reached the 5th day (cell density was 12.0-18.0×10 6 cells / mL), the culture temperature was adjusted to 32°C and maintained until the end of the culture; the pH was set to 7±0.2, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 14th day.

[0047] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0048] The results showed that the cell density of Comparative Example 2 at the end of the 14th day of culture was 16.55×10 6 cells / mL, the cell viability was 86.54%, and the protein expression level was 2.3 g / L. The highest culture density was reached on the 7th day, which was 25.31×106 cells / mL, and the cell viability was 98.38%.

[0049] Comparative Example 3 (1) Seed subculture: proceed as in step (1) of the example.

[0050] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6 The density of cells / ml was inoculated into a 3L glass reactor containing CM-013 basal medium, and batch fed culture was started. The feed medium was DF-013 and DF-003. The two culture media were added at the same time. The ratio of the two usage was DF-013:DF-003 = 10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the usage of DF-013, which was added according to the initial working volume. The day of inoculation was day 0; 3% was fed on the 3rd day, and 3% was fed every other day thereafter; the initial culture temperature was 37°C, and when the culture reached the 6th day (cell density was 12.0~18.0×10 6 cells / mL), the culture temperature was adjusted to 32°C and maintained until the end of the culture; the pH was set to 7±0.2, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 14th day.

[0051] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0052] The results showed that the cell density of Comparative Example 3 at the end of the 14th day of culture was 14.24×10 6 cells / mL, the cell viability was 77.15%, and the protein expression was 2.1 g / L. The highest culture density was reached on the 8th day, which was 22.35×10 6 cells / mL, and the cell viability was 98.22%.

[0053] Comparative Example 4 (1) Seed subculture: proceed as in step (1) of the example.

[0054] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6The cells / ml were inoculated into a 3L glass reactor containing CM-013 medium, and fed-batch culture was started. The feed medium was DF-013 and DF-003. The two culture media were added at the same time. The ratio of the two media was DF-013:DF-003 = 10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the amount of DF-013 used, which was added according to the initial working volume. The day of inoculation was day 0; the feed was 4% on the 3rd day, and 4% every other day thereafter; the initial culture temperature was 37°C. When the culture reached day 7 (cell density 18.0-25.0×10 6 cells / mL), the culture temperature was adjusted to 32°C and maintained until the end of the culture; the pH was set to 7±0.2, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 14th day.

[0055] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0056] The results showed that the cell density of Comparative Example 4 at the end of the 14th day of culture was 20.10×10 6 cells / mL, cell viability was 86.50%, and protein expression was 2.4 g / L. The highest culture density was 25.02×10 6 cells / mL, and the cell viability was 98.20%.

[0057] Comparative Example 5 (1) Seed subculture: proceed as in step (1) of the example.

[0058] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6 The cells / ml were inoculated into a 3L glass reactor containing CM-013 basal medium, and fed-batch culture was started. The feed medium was DF-013 and DF-003. The two media were added at the same time. The ratio of the two media was DF-013:DF-003 = 10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the amount of DF-013 used, which was added according to the initial working volume. The day of inoculation was day 0; 4% was fed on the 3rd day, and 4% was fed every other day thereafter; the initial culture temperature was 37°C, and the culture was continued until day 8 (or the cell density was greater than 25.0×10 6cells / mL), the culture temperature was adjusted to 32°C and maintained until the end of the culture; the pH was set to 7±0.2, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 14th day.

[0059] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0060] The results showed that the cell density of comparative example 5 at the end of the 14th day of culture was 11.88×10 6 cells / mL, the cell viability was 72.91%, and the protein expression level was 1.7 g / L; the highest culture density was 23.11×10 6 cells / mL, and the cell viability was 97.05%.

[0061] Comparative Example 6 (1) Seed subculture: proceed as in step (1) of the example.

[0062] (2) Fed-batch culture: The seed cells obtained in step (1) were cultured at a rate of 0.5 × 10 6 The density of cells / ml was inoculated into a 3L glass reactor containing CM-013 basal medium, and batch fed culture was started. The feed medium was DF-013 and DF-003. The two culture media were added at the same time, and the ratio of the two usage was DF-013:DF-003=10:1 (volume ratio). The feed amount expressed in percentage during the following feeding is the usage of DF-013, which was added according to the initial working volume. The day of inoculation was day 0; 4% was fed on the 3rd day, and 4% was fed every other day thereafter; the culture temperature was 37°C and maintained until the end of the culture; the pH was set to 7±0.2, the reactor speed was 180rpm, and the dissolved oxygen was controlled at 50%; when the glucose concentration was lower than 4g / L during the whole culture process, glucose was supplemented to 4~5g / L (including the glucose in the feed), and the supernatant was harvested on the 12th day.

[0063] The cell culture density and its changes are shown in Tables 1 and Figure 1 ; The cell culture viability and changes are shown in Tables 2 and Figure 2 After the culture was completed, the protein concentration at the time of harvest was detected by HPLC. The results are shown in Table 3 and Figure 3 .

[0064] The results showed that the cell density of Comparative Example 6 at the end of the 12th day of culture was 8.56×106 cells / mL, the cell viability was 55.28%, and the protein expression was 1.1 g / L. The highest culture density was 22.60×10 6 cells / mL, and the cell viability was 97.52%.

[0065] According to the comparison of the results, the examples of the present application have the highest cell density and activity at the end of culture, and the protein expression level is significantly higher than that of the comparative examples. The highest cell density and viability during culture are also higher than those of the comparative examples.

[0066] Table 1 The viable cell density (1×10 6 cells / ml) table

[0067] Table 2 Cell viability (%) during fed-batch culture of each embodiment and comparative example

[0068] Table 3 Target protein expression at the end of cultivation in the examples and comparative examples (g / L)

[0069] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for culturing CHO cells expressing classical swine fever virus E2 fusion protein, characterized in that: The steps include: S1. Subculture: inoculating CHO seed cells that secrete and express the fusion protein of classical swine fever virus E2 into the basal culture medium for subculture; S2, Gradient cooling fed-batch culture: CHO seed cells after subculture in S1 were cultured at 0.5~1.0×10 6 cells / mL were inoculated into the basal medium, and the feed medium was added in batches for feed culture. The temperature was gradually lowered, and the initial culture temperature was 36.5-37.5℃; the cell density was 10.0-18.0×10 6 When the cell viability is less than 70%, the supernatant is harvested and the culture is terminated.

2. The CHO cell culture method according to claim 1, characterized in that: In step S2, when feed culture medium is added in batches for fed culture, the day of inoculation is taken as day 0, 3% is fed on the 3rd day, 3% is fed on the 4th day, 4% is fed on the 5th day, 3% is fed on the 6th day, 3% is fed on the 7th day, 3% is fed on the 8th day, 3% is fed on the 10th day, and 3% is fed on the 12th day.

3. The CHO cell culture method according to claim 1, characterized in that: The basal culture medium is a serum-free culture medium for CHO cells, and the serum-free culture medium for CHO cells is at least one of CM-013, AltairCHO, SagiCHO, StarCHO, CD CHO031, CD CHO 041, and CD CHO 045.

4. The CHO cell culture method according to claim 1, characterized in that: The classical swine fever virus E2 fusion protein comprises the extracellular region of the classical swine fever virus envelope glycoprotein E2, and the amino acid sequence of the extracellular region of the classical swine fever virus envelope glycoprotein E2 is shown in Sequence ID.NO.

3.

5. The CHO cell culture method according to claim 4, characterized in that: The amino acid sequence of the classical swine fever virus E2 fusion protein is as shown in Sequence ID.NO.1, or is an amino acid sequence as shown in Sequence ID NO.1 including an immunogenic derivative protein that has been substituted, deleted or added with one or several amino acids.

6. The CHO cell culture method according to claim 1, characterized in that: In step S1, during the subculture, the temperature is 36.5-37.5°C; and / or, CO2 content is 5~8%; and / or, humidity is 75-85%; And / or, the rotation speed is 100~130rpm.

7. The CHO cell culture method according to claim 1, characterized in that: In step S1, during the subculture, the cells are subcultured every 2 days, and 12.5-50 μM methionine sulfoximine is added to the basal medium starting from the second generation.

8. The CHO cell culture method according to claim 1, characterized in that: In step S2, the feed medium is at least one of DF-013 and DF-003, AltairCHO Feed and CDFS36, AF183 and CDFS36, XYZF1.5 and XYZ F7.

9. The CHO cell culture method according to claim 1, characterized in that: In step S2, before cooling to 30-31°C, the pH is 7.0±0.4; and / or, after cooling to 30-31°C, the pH is 7.0±0.

2.

10. The CHO cell culture method according to claim 1, characterized in that: In step S2, the fed-batch culture speed is 180-250 rpm; and / or, the dissolved oxygen is controlled at 40% to 60%; And / or, when the glucose concentration is lower than 4 g / L, supplement glucose to 4-5 g / L.

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