Culture medium for high-density serum-free suspension culture of BHK-21 cells and application of culture medium

Abstract

The invention relates to a culture medium for high-density serum-free suspension culture of BHK-21 cells and application of the culture medium. The culture medium contains inorganic salt, amino acid, vitamins, energy substances, a buffer substance, an antioxidant, sterol, polyamine, an additive and an indicator, and the antioxidant comprises dihydrolipoic acid and S-acetyl-L-glutathione. According to the invention, the culture medium for serum-free suspension culture of the BHK-21 cells, which is clear in components and remarkable in culture effect, can be provided. The added dihydrolipoic acid and S-acetyl-L-glutathione are combined as antioxidants of the culture medium, so that the survival rate of BHK-21 cells cultured in the culture medium is improved while the oxidation resistance of the serum-free suspension culture medium is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological products, in particular, to a culture medium for serum-free high-density suspension culture of BHK-21 cells and its application in porcine Senecavirus vaccine. BACKGROUND

[0002] BHK-21 cell is a baby hamster kidney cell, which is abbreviated as BHK-21 cell. The cell belongs to fibroblast cells, and mainly grows in adhesion in vitro. After suspension domestication, the cell can grow in suspension. BHK-21 cell is sensitive to a variety of economic animal viruses, and can be widely applied to preparation of newcastle disease virus, porcine encephalitis B, foot-and-mouth disease, bovine nodular skin disease virus, pseudorabies virus, goat pox virus, porcine Senecavirus and other virus vaccines.

[0003] The use of serum-free suspension cell culture process brings hope for the development of high-quality vaccines. The use of serum increases the difficulty of downstream separation and purification and increases the production cost. On the other hand, single cell suspension culture can be free from the limitation of cell growth surface, and is more conducive to high-density growth of cells, and is easy to realize large-scale production of cells and related products in a bioreactor. Therefore, the development and research of serum-free suspension culture system of BHK-21 cells with clear composition, serum-free and animal-derived components have great significance.

[0004] Senecavirus A (SVA) is a pathogen causing idiopathic vesicular disease (IVD) in pigs. The clinical symptoms of porcine Senecavirus disease are similar to those of vesicular stomatitis infection and foot-and-mouth disease virus infection, which can cause vesicles in the mouth and nose of live pigs, and also cause ulceration. SVA mainly infects newborn piglets, which can cause acute death of newborn piglets, and the mortality rate of piglets of 1-4 days old is the highest, about 30%-70%. Due to the lack of effective therapeutic drugs, vaccination is still the most effective method to control and prevent infectious diseases. SUMMARY

[0005] The present application provides a culture medium for serum-free suspension culture of BHK-21 cells and its application in Senecavirus culture.

[0006] According to the first aspect of the present application, a culture medium for serum-free suspension culture of BHK-21 cells with clear composition and significant culture effect is provided. Dihydrolipoic acid and S-acetyl-L-glutathione are added as an antioxidant combination of the culture medium in a certain proportion, which improves the antioxidant performance of the serum-free suspension culture medium and also improves the cell survival rate of BHK-21 cells cultured in the culture medium.

[0007] Specifically, the culture medium for serum-free suspension culture of BHK-21 cells in the present application contains: Inorganic salts: the inorganic salts include compounds containing iron, magnesium, potassium, sodium, zinc, and calcium; Amino acids: the amino acids include L-glutamine, L-arginine hydrochloride, L-cystine hydrochloride, L-glutamic acid hydrochloride, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; Vitamins: the vitamins include nicotinamide, D-calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and folic acid; Energy substances: the energy substances include D-glucose, sodium pyruvate, inositol, and L-glutamine; Buffering substances: the buffering substances include sodium bicarbonate, HEPES, and MOPS; Antioxidants: the antioxidants include dihydrolipoic acid and S-acetyl-L-glutathione; Sterols: the sterols include cholesterol; Polyamines: the polyamines include putrescine, spermine, and spermidine; Additives: the additives include polyvinyl alcohol, F68, dextran sulfate, choline chloride, ethanolamine, and thymidine; Indicators: the indicators include phenol red sodium.

[0008] The culture medium for serum-free suspension culture of BHK-21 cells in the present application contains the following components in the final concentration of mg / L: Iron nitrate nonahydrate 10-80, dihydrolipoic acid 0.01-0.1, magnesium sulfate 10-50, choline chloride 10-100, potassium chloride 100-500, sodium selenite 10-30, sodium bicarbonate 1000-2000, ethanolamine 1-10, sodium chloride 1000-5000, ferric citrate 70-80, zinc sulfate heptahydrate 0.1-1, polyvinyl alcohol 10-30, sodium phosphate monobasic 10-50, F6 1000-2000, L-glutamine 100-500, zinc sulfate 10-50, L-arginine hydrochloride 100-500, cholesterol 2-20, L-cystine hydrochloride 10-50, S-acetyl-L-glutathione 1-5, L-glutamic acid hydrochloride 100-1000, dextran sulfate 10-100, glycine 10-80, folic acid 5-20, L-histidine hydrochloride 10-80, inositol 5-30, L-isoleucine 100-200, nicotinamide 5-20, L-leucine 100-200, D-pantothenic acid calcium 5-20, L-lysine hydrochloride 100-200, pyridoxal hydrochloride 5-20, L-methionine 100-200, pyridoxine hydrochloride 5-20, L-phenylalanine 100-250, riboflavin 0.2-1, L-serine 10-100, thiamine hydrochloride 2-20, L-threonine 100-250, D-glucose 1000-5000, L-tryptophan 10-100, thymidine 20-80, L-tyrosine 10-100, phenol red sodium 10-30, L-valine 10-100, pyruvic acid sodium 10-100, putrescine 0.1-1, HEPES 1000-2000.

[0009] Preferably, the final concentrations of each component of the medium in mg / L are: Iron nitrate 50, dihydrolipoic acid 0.05, magnesium sulfate 30, choline chloride 50, potassium chloride 300, sodium selenite 25, sodium bicarbonate 1500, ethanolamine 10, sodium chloride 4000, ferric citrate 75, zinc sulfate 0.5, transferrin 15, sodium phosphate 30, F68 1000, L-glutamine 400, recombinant insulin 10, L-arginine hydrochloride 500, cholesterol 15, L-cystine hydrochloride 40, S-acetyl-L-glutathione 4, L-glutamic acid hydrochloride 800, dextran sulfate 80, glycine 40, folic acid 10, L-histidine hydrochloride 80, inositol 15, L-isoleucine 150, nicotinamide 10, L-leucine 150, D-pantothenic acid calcium 10, L-lysine hydrochloride 150, pyridoxal hydrochloride 10, L-methionine 100, pyridoxine hydrochloride 10, L-phenylalanine 100, riboflavin 0.5, L-serine 100, thiamine hydrochloride 10, L-threonine 200, D-glucose 5000, L-tryptophan 50, thymidine 60, L-tyrosine 50, phenol red sodium 20, L-valine 50, pyruvic acid sodium 50, putrescine 1, HEPES 1000.

[0010] According to the second aspect of the present application, a method for domesticating a non-suspended BHK-21 cell line into a suspended BHK-21 cell line is provided, comprising: domesticating the non-suspended BHK-21 cell line in the aforementioned culture medium of the present application.

[0011] The method comprises the steps of resuscitating the cells, subculturing the cells, serum-free adherent culturing the cells, and serum-free suspended culturing the cells, In the step of serum-free suspended culturing the cells, the serum-free culture medium of the present application is used.

[0012] In the step of serum-free full-suspended culturing the BHK-21 cells, the cell density of the last generation is adjusted to 1×10 6 cells / mL, and the cells are cultured at 37℃ with 5% CO2 and 140 r / min shaking; the cells are subcultured by centrifugation and medium replacement, and the cells are not subcultured, a large number of cells will aggregate and die in the initial suspended stage, and this method can prevent the enzymes released after the cells are broken from affecting the growth of the cells and prevent the cells from growing due to the low initial density. When the cell density reaches 3×10 6 cells / mL, the cell density is adjusted to 5×10 5 cells / mL with the serum-free full-suspended culture medium, and the cells are cultured at 37℃ with 5% CO2 and 140 r / min shaking, and after 15-20 generations of subculture, the cells are stable in proliferation, uniform in size, and free of cell aggregation, and the survival rate is above 95%. The domesticated serum-free full-suspended culture type BHK-21 cell line is named BHK-21-S.

[0013] According to a third aspect of the present application, the present application provides a method for preparing a Senecavirus using a suspension cell line, wherein the method comprises the following steps: (1) domesticating a non-suspension BHK-21 cell line into a suspension BHK-21 cell line using the culture medium prepared by the present application and the method for domesticating a BHK-21 cell line into a suspension BHK-21 cell line according to the present application; (2) preparing a Senecavirus using the suspension BHK-21 cell line obtained in step (1).

[0014] In some embodiments, the Senecavirus according to the present application can be selected from the group consisting of SVV-001 strain, CH-HN strain, CH-GD strain, CH-01-2015, CH-HB-2017, CH-04-2015 strain, SCH-DB-11-2015 strain, CH-DL-01-2016 strain, CH-ZW-01-2016 strain, HB-CH-2016 strain, THA / 2016 strain, VNM / 2019 strain, KS15-01 strain, BRA / RS / 2016 strain.

[0015] Preferably, the virus is Newcastle disease virus, porcine encephalitis virus, foot-and-mouth disease virus, bovine dermatophilus virus, pseudorabies virus, goat pox virus, porcine Senecavirus.

[0016] In some embodiments, the preparation of Senecavirus in step (2) comprises the following steps: BHK-21 cells are inoculated at a density of 0.5-1 x 10 6 cell / ml, after 48 hours, the cell density is grown to 5-6 x 10 6 cell / ml, fresh culture medium is added to adjust the cell density to 1-2 x 10 6 cell / ml, the pH value of the cell solution is adjusted, Senecavirus is inoculated, and the virus is harvested when the cell viability is less than 40%. In some embodiments, the culture temperature of the suspension BHK-21-S cell line in step (2) is 33-37°C; in some embodiments, the culture rotation speed is 80-120 rpm. In some embodiments, the pH value of the cell solution to be inoculated in step (2) is adjusted to 7.0-7.6. In some embodiments, the initial cell density inoculated in step (2) is 1-2 x 10 6 cell / ml. Then, Senecavirus is inoculated at a dose of 0.1-1% of the volume ratio. In some embodiments, the pH of the cell solution to be infected can be adjusted using various alkaline reagents, NaHCO3 solution, NaOH solution, KOH solution, Na2CO3 solution; In some embodiments, the pH of the cell solution to be infected is adjusted using a NaHCO3 solution. In some embodiments, the concentration of the NaHCO3 solution is 2% to 8% (w / v).

[0017] The preparation of Senecavirus using the method of the present application realizes the technical breakthrough of suspension culture of Senecavirus and industrial production of Senecavirus vaccine. The culture of Senecavirus using the method effectively improves the production capacity, virus titer and quality stability uniformity of the obtained virus, reduces the batch-to-batch difference, and lays a foundation for accelerating the large-scale production of suspension-prepared Senecavirus vaccine.

[0018] The serum-free and fully-suspension-cultured BHK-21 cell line provided by the present application can be used in the following aspects: 1. Virus isolation and preparation 2. Application in detecting viruses for non-diagnostic treatment purposes 3. Product preparation for detecting viruses 4. Vaccine product preparation 5. Application in drug screening, the drug being a drug for preventing or treating diseases caused by viral infection The present application develops a serum-free medium suitable for BHK-21 suspension cells, and obtains a suspension BHK-21-S cell line by domestication from a non-suspension BHK-21 cell line, with a cell density of 1.2×10 7 cell / ml and a cell viability of more than 95%.

[0019] The BHK21 cells obtained using the medium of the present application have increased infectivity to Senecavirus, and the effective antigen content of the virus is significantly improved, thereby realizing the goal of high-density suspension culture of BHK-21 cells and improving the production efficiency of Senecavirus. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Microscope image of BHK-21 adherent cells; Figure 2 Microscope image of BHK-21 suspension cells; Figure 3 Growth curve of BHK-21 suspension cells; Figure 4 Cytopathic effect of BHK-21 suspension cells after inoculation with porcine Senecavirus; DETAILED DESCRIPTION

[0021] For purposes of the present application, the technical solutions and advantages will be more clearly described below, and the technical solutions in the present application will be clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. In the examples provided in the present specification, the specific techniques or conditions are not specified, and the techniques or conditions described in the literature in the art or according to the product manual are used. The reagents or instruments not specified by the manufacturer are conventional products that can be purchased from a regular channel.

[0022] Example 1: BHK-21 cell serum-free suspension culture medium The BHK-21 cell serum-free suspension culture medium of the present application is prepared by the following method.

[0023] The components of the serum-free cell culture medium are weighed, premixed, pulverized, finally mixed, and subpackaged to obtain a dry powder. The dry powder is dissolved in purified water at 20-25°C, the pH value is adjusted to 7.2, and the volume is adjusted to obtain the BHK-21 cell serum-free suspension culture medium. The final concentration of each component is as follows (see Table 1).

[0024] Table 1

[0025] The BHK-21 cell serum-free suspension culture medium provided by the present application can obtain high-density and high-viability BHK-21 serum-free suspension cells according to a certain cell suspension domestication method. Moreover, the serum-free suspension culture medium and the domesticated BHK-21 cells are used to carry out cell reactor scale-up suspension culture, complete virus culture preparation, and the cell culture passage operation is simple, and the culture scale linear amplification is easy to realize, which lays a foundation for large-scale production of corresponding viral vaccines.

[0026] In addition, the medium of Table 1 components is shown in Example 1, but each component can be floated around the concentration shown in Example 1, such as using the concentration range shown in Table 2, which can also achieve the technical effects of the present application.

[0027] Table 2

[0028] Example 2: Serum-free suspension domestication of adherent BHK-21 cells I. Experimental materials 1. Cells: BHK-21 adherent cells purchased from American Type Culture Collection (ATCC) 2. Medium and reagent: calcium and magnesium free PBS, DMEM medium from Zhongshengtianxin Biotech Co., Ltd. (Wuxi), Trpzyme® recombinant trypsin digestion solution from Gbico, newborn calf serum from Rongye Biotech Co., Ltd. 3. Culture vessel: T75 cell culture bottle, 125 mL, 250 mL, 1000 mL shake flask, all purchased from Corning Life Sciences Co., Ltd. II. Experimental steps 1. Serum reduction domestication of BHK-21 adherent cells 1) Revive cells: Take one BHK-21 adherent cell cryopreservation tube from the liquid nitrogen tank, shake it in the 37°C warm water to quickly melt it, and then transfer it to a 10 ml centrifuge tube under sterile conditions. Centrifuge at 1000 rpm for 5 min to remove the supernatant, resuspend with DMEM + 10% NBS, and inoculate the cells into a T25 culture bottle. Incubate at 37°C, 5% CO2, and the adherent cell morphology is shown in Figure 1 , the cells are spindle-shaped and the morphology is full; 2) Cell subculture: After the cells grow to form a monolayer, discard the culture medium, wash with calcium and magnesium free PBS, add 1 ml of recombinant trypsin digestion solution, discard the digestion solution after the cells are dispersed, and terminate the digestion with DMEM + 10% NBS. Subculture at a ratio of 1:5, and subculture the BHK-21 cells for 3 times according to the same method.

[0029] 3) Cell serum reduction adherent culture: The adherent culture type BHK-21 cells obtained above are cultured with DMEM medium containing 5%, 2%, and 1% newborn calf serum, respectively, and subcultured at a ratio of 1:5. Incubate in a 37°C, 5% CO2 cell incubator. The medium containing 5% and 2% newborn calf serum is each subcultured for 2-3 generations, and the medium containing 1% newborn calf serum is each subcultured for 5-8 generations, so that the cells gradually adapt to low serum adherent culture.

[0030] 4) Cell serum-free suspension culture: Adjust the cell density obtained in the last step to 1×10 6 cells / mL with serum-free whole suspension culture medium, and culture at 37°C, 5% CO2, and 140 r / min on a shaker. Centrifuge and replace the medium for subculture, without subculture, until the cell density reaches 3×10 6 cells / mL. Adjust the cell density to 5×10 5 cells / mL with serum-free whole suspension culture medium, and culture at 37°C, 5% CO2, and 140 r / min on a shaker. After 15-20 generations of subculture, the cells proliferate stably without cell aggregation, and the viability reaches more than 95%. The domesticated serum-free whole suspension culture type BHK-21 cell line is named BHK-21-S. 5) Cell cryopreservation and library construction: Obtain suspension cells that grow stably in serum-free suspension medium. Centrifuge the culture at 1000 rpm for 5 min to collect cells, resuspend in BHK-21 cell serum-free medium + 7% DMSO, and adjust the cell density to 3 × 10⁶ cells / mL. 7 The fractions were dispensed into cryovials and placed in an ultra-low temperature freezer at -80°C overnight before being transferred to a liquid nitrogen tank for long-term storage. III. Experimental Results like Figure 2 As shown, the serum-free suspension cells of BHK-21 obtained through domestication appeared as single, round, suspended cells under an inverted microscope, without clumping or adherent cells. In contrast, Figure 1 The adherent cells shown are spindle-shaped and attached to the surface.

[0031] Figure 3 The growth of these serum-free suspension cells in serum-free suspension medium is shown. Culture conditions were 37°C, 5% CO2, and 130 rpm. Peak cell density was reached after 48 hours of culture, with cell proliferation increasing 12-fold in 48 hours, exceeding that of commercially available culture media.

[0032] Example 3 The only difference between Example 3 and Example 1 is that the concentration of some of the dihydrolipoic acid in the culture medium was replaced from 0.05 mg / L to 4 mg / L, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 0.05 mg / L.

[0033] Example 4 The only difference between Example 4 and Example 1 is that the concentration of some of the dihydrolipoic acid in the culture medium was replaced from 0.05 mg / L to 2.025 mg / L, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 2.025 mg / L.

[0034] Comparative Example Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that some of the dihydrolipoic acid in the culture medium was replaced with lipoic acid.

[0035] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that S-acetyl-L-glutathione was not added to the culture medium, and the concentration of some dihydrolipoic acid was replaced from 0.05 mg / L to 4 mg / L.

[0036] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that no dihydrolipoic acid was added to the culture medium, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 2.025 mg / L.

[0037] Culture medium performance test Example 1, 3, 4 and Comparative Example 1, 2, 3 acclimated serum-free suspension culture type BHK-21 cells were inoculated into 10 L bioreactor at 0.5 x 10 6 cells / ml respectively, and the reactor parameters were set as follows: temperature 37℃, rotation speed 90 rpm / min, dissolved oxygen 60%, pH value 7.0. The cell count and cell viability were checked every 24 h.

[0038] Table 3 shows the cell density and cell viability of Example 1, 3, 4 acclimated serum-free suspension culture type BHK-21 cells cultured for 4 days.

[0039] Table 3

[0040] Table 4 shows the cell density and cell viability of Comparative Example 1-3 acclimated serum-free suspension culture type BHK-21 cells cultured for 4 days.

[0041] Table 4

[0042] According to Figure 2 , Figure 3 and Table 3, the BHK-21 suspension cells acclimated in the medium of Example 1 component concentration appeared as single round cells under the inverted microscope, no cell clumping was observed, and no adherent cells were observed. The BHK-21 suspension cells acclimated in the medium of Example 1 component concentration were inoculated at a cell density of 0.5 x 10 6 cells / ml, and the BHK-21 suspension cells grew to a density of 6 x 10 6 cells / ml after 48 hours, with a 12-fold proliferation, and the cell morphology was good, with a viability of 98%, indicating that the BHK-21 cells acclimated in the serum-free suspension culture medium of the present application grew well, with a high proliferation density, which can meet the large-scale production needs of BHK-21 cells.

[0043] According to Reference Example 1, 3, 4 and Table 3, the growth cell density increase and cell viability of Example 1 acclimated BHK-21 suspension cells in 4 days were better than those of Example 2 and Example 3.

[0044] As can be seen from Reference Example 1, Comparative Examples 1-3, Table 3 and Table 4, the growth cell density increase and cell viability of the BHK-21 suspension cells domesticated in the medium with the component concentration of Example 1 are superior to those of Comparative Examples 1, 2 and 3, which is probably due to the fact that the thioctic acid of Comparative Example 1 is not hydrogenated, which is not conducive to the synergistic antioxidant effect with S-acetyl-L-glutathione. Comparative Example 2 lacks the antioxidant capacity of extracellular reactive oxygen scavenging due to the lack of S-acetyl-L-glutathione, and Comparative Example 3 lacks the antioxidant capacity of intracellular reactive oxygen scavenging due to the lack of partially hydrogenated thioctic acid, which all result in poor increase density and cell viability of the medium domesticated BHK-21 suspension cells.

[0045] The present application adopts the serum-free whole-suspension medium for BHK-21 cells, so that the cells are in a high-density and high-viability proliferation state in suspension, which avoids the operation process of adherent cells needing to be digested and is easy to realize large-scale culture of BHK-21 cells.

[0046] Example 5 Preparation of porcine Senecavirus After the BHK-21 suspension cells are grown for 2 days, the cell density is adjusted to 1.5-2×10 6 / ml with the serum-free suspension medium, and the cells are inoculated with 0.1% (v / v) virus and cultured on a 120 r / min, 37℃, 5% CO2 shaker, and then harvested when the cell viability is lower than 40% after 24 h of culture, and the cytopathic effect is observed. As shown in the table, there are many cell fragments, and the cells are aggregated and have different sizes. The virus liquid after harvesting is repeatedly frozen and thawed three times at-80℃, and the supernatant virus liquid is collected by centrifugation at 5000 rpm for 10 min and then detected. Figure 4

[0047] The virus titer is 10 50 TCID 8.2 / 0.1 mL by the Reed-Muench method and TCID 50 detection. It can be seen that the serum-free whole-suspension culture type BHK-21 cell line domesticated in suspension of the present application is sensitive to porcine Senecavirus. It is shown that the serum-free suspension culture medium for BHK-21 cells of the present application has a good application prospect in the production of porcine Senecavirus vaccine.

[0048] The above are only examples of the present application and are not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the scope of claims of the present application.​

Claims

1. A culture medium for serum-free suspension culture of BHK-21 cells, said culture medium comprising: inorganic salts: said inorganic salts include compounds containing iron, magnesium, potassium, sodium, zinc, calcium; amino acids: said amino acids include L-glutamine, L-arginine hydrochloride, L-cystine hydrochloride, L-glutamic acid hydrochloride, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine; vitamins: said vitamins include nicotinamide, calcium D-pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, folic acid; energy substances: said energy substances include D-glucose, sodium pyruvate, inositol, L-glutamine; buffer substances: said buffer substances include sodium bicarbonate, HEPES or MOPS; antioxidants: said antioxidants include dihydrolipoic acid, S-acetyl-L-glutathione; sterols: said sterols include cholesterol; polyamines: said polyamines include putrescine or spermine and spermidine; additives: said additives include polyvinyl alcohol, F68, dextran sulfate, choline chloride, ethanolamine, thymidine; indicators: said indicators include phenol red sodium. The final concentration of each component contained in the culture medium is as follows (in mg / L) : ferric nitrate nonahydrate 10-80, dihydrolipoic acid 0.01-0.1, magnesium sulfate 10-50, choline chloride 10-100, potassium chloride 100-500, sodium selenite 10-30, sodium bicarbonate 1000-2000, ethanolamine 1-10, sodium chloride 1000-5000, ferric citrate 70-80, zinc sulfate heptahydrate 0.1-1, polyvinyl alcohol 10-30, sodium dihydrogen phosphate 10-50, F68 1000-2000, L-glutamine 100-500, zinc sulfate 10-50, L-arginine hydrochloride 100-500, cholesterol 2-20, L-cystine hydrochloride 10-50, S-acetyl-L-glutathione 1-5, L-glutamic acid hydrochloride 100-1000, dextran sulfate 10-100, glycine 10-80, folic acid 5-20, L-histidine hydrochloride 10-80, inositol 5-30, L-isoleucine 100-200, nicotinamide 5-20, L-leucine 100-200, calcium D-pantothenate 5-20, L-lysine hydrochloride 100-200, pyridoxal hydrochloride 5-20, L-methionine 100-200, pyridoxine hydrochloride 5-20, L-phenylalanine 100-250, riboflavin 0.2-1, L-serine 10-100, thiamine hydrochloride 2-20, L-threonine 100-250, D-glucose 1000-5000, L-tryptophan 10-100, thymidine 20-80, L-tyrosine 10-100, phenol red sodium 10-30, L-valine 10-100, sodium pyruvate 10-100, putrescine 0.1-1, HEPES 1000-2000. ​ ​ ​ ​ ​ ​ ​ ​ ​ 2. The medium of claim 1, wherein, ​ ​ 3. The medium of claim 2, wherein each component of the medium is in a final concentration of mg / L: Iron nitrate nonahydrate 50, dihydrolipoic acid 0.05, magnesium sulfate 30, choline chloride 50, potassium chloride 300, sodium selenite 25, sodium bicarbonate 1500, ethanolamine 10, sodium chloride 4000, ferric citrate 75, zinc sulfate heptahydrate 0.5, transferrin 15, sodium phosphate dibasic 30, F68 1000, L-glutamine 400, recombinant insulin 10, L-arginine hydrochloride 500, cholesterol 15, L-cystine hydrochloride 40, S-acetyl-L-glutathione 4, L-glutamic acid hydrochloride 800, dextran sulfate 80, glycine 40, folic acid 10, L-histidine hydrochloride 80, inositol 15, L-isoleucine 150, nicotinamide 10, L-leucine 150, D-pantothenic acid calcium 10, L-lysine hydrochloride 150, pyridoxal hydrochloride 10, L-methionine 100, pyridoxine hydrochloride 10, L-phenylalanine 100, riboflavin 0.5, L-serine 100, thiamine hydrochloride 10, L-threonine 200, D-glucose 5000, L-tryptophan 50, thymidine 60, L-tyrosine 50, phenol red sodium 20, L-valine 50, sodium pyruvate 50, putrescine 1, HEPES 1000.

4. A method for domesticating a non-suspended BHK-21 cell line into a suspended BHK-21 cell line, comprising: a step of domesticating a non-suspended BHK-21 cell line in culture with the medium of any one of claims 1-3.

5. The method of claim 4, wherein, the method comprises steps of resuscitating cells, subculturing cells, serum-free adherent culturing cells, and serum-free suspension culturing cells, in the step of serum-free suspension culturing cells, the medium of any one of claims 1-3 is used.

6. The method of claim 5, comprising: In the cell serum-free suspension culture step, the cell density obtained in the last generation of the step is adjusted to 1×10 6 cells / mL, and the cells are cultured at 37℃ with 5% CO2 and 140r / min shaking; the cells are centrifuged and replaced with a medium, and are not subcultured until the cell density reaches 3×10 6 cells / mL, and then the cell density is adjusted to 5×10 5 cells / mL with the medium of any one of claims 1-3, and the cells are cultured at 37℃ with 5% CO2 and 140r / min shaking, and after 15-20 generations of subculture, a serum-free fully-suspended culture type BHK-21 cell line is obtained.

7. A method for preparing a Senecavirus, comprising: (1) domesticating a non-suspended BHK-21 cell line into a suspended BHK-21 cell line using the method of any one of claims 4-6; (2) preparing a Senecavirus using the suspended BHK-21 cell line obtained in step (1).

8. The method of claim 7, wherein, the culture temperature of the suspended BHK-21-S cell line in step (2) is 33-37℃, the culture rotation speed is 80-120 rpm, and the pH value is adjusted to 7.0-7.6, Inoculate initial cell density 1-2 x 10 6 cell / ml, then inoculate Seneca Valley virus at 0.1-1% of the volume of the inoculum.

9. The method of claim 7, wherein, The preparation of Senecavirus in step (2) comprises the following steps: BHK-21 cells are inoculated at a density of 0.5-1×10 6 cell / ml, after 48 hours, the cell density grows to 5-6×10 6 cell / ml, fresh culture medium is added to adjust the cell density to 1-2×10 6 cell / ml, the pH value of the cell solution is adjusted, Senecavirus is inoculated, and after the cell viability is less than 40%, the virus is harvested.

10. The method of any one of claims 7-9, wherein, The Senecavirus is selected from the group consisting of SVV-001 strain, CH-HN strain, CH-GD strain, CH-01-2015, CH-HB-2017, CH-04-2015 strain, SCH-DB-11-2015 strain, CH-DL-01-2016 strain, CH-ZW-01-2016 strain, HB-CH-2016 strain, THA / 2016 strain, VNM / 2019 strain, KS15-01 strain, BRA / RS / 2016 strain.

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