A method for preparing a vero cell influenza virus vaccine

Abstract

The present application relates to a kind of preparation methods of Vero cell influenza virus vaccine, belong to the technical field of virus culture.In the culture of influenza virus, medium is added with red algae protein and soybean protein hydrolysate, the former is precipitated by low-temperature ammonium sulfate and purified by PD-10 desalting column, maintains native conformation, can activate integrin-FAK-Src and IGF-1R signal pathway, promotes cell adhesion and proliferation;The latter contains soybean protein isolate, polysaccharide and RGD motif peptide, can form adhesive film on the substrate and adhere through integrin mediation.Both of them improve cell adhesion, metabolic homeostasis and antioxidant capacity, so that Vero cell can grow in high density under serum-free condition and efficiently replicate influenza virus, virus titer (TCID 50 ) reaches 8.5 log 10 / mL, suitable for industrialized vaccine production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus culture, and particularly relates to a preparation method of Vero cell influenza virus vaccine. BACKGROUND

[0002] At present, the existing influenza vaccine production mainly relies on the method of culturing viruses in chicken embryos. This method is mature in process, but has problems such as long cycle, limited production scale, sensitivity to production environment and raw materials, and possible introduction of chicken-derived allergens, which is difficult to meet the demand of large-scale and rapid production.

[0003] Vero cells are widely used in the production of various virus vaccines, such as inactivated polio vaccine and rabies vaccine, due to their high genetic stability, strong adaptability and lack of interferon production capacity. Under serum-free culture conditions, Vero cell culture can avoid potential safety risks brought by animal serum and improve process controllability. However, the growth rate, adhesion ability and virus yield of Vero cells under serum-free conditions are often lower than those under serum-containing culture conditions, which is particularly prominent in the efficient preparation of influenza viruses.

[0004] In order to improve the proliferation capacity of Vero cells for influenza viruses under serum-free conditions, researchers have tried to add various nutritional ingredients and growth factors, such as transferrin, insulin, epidermal growth factor, etc., to the culture medium to improve cell metabolic state and virus yield. However, there is still a lack of mature technical solution to significantly improve influenza virus titer while ensuring high-density cell culture.

[0005] CN114540277A discloses a serum-free culture medium for culturing Vero cells and a preparation method thereof, which records plant hydrolyzed protein and up to 80 additives. The batch difference control is difficult, and it is difficult to ensure the stability of each component.

[0006] Therefore, there is an urgent need for an improved serum-free culture method and medium formula for Vero cells, which can significantly improve the virus production level of Vero cells for influenza viruses under serum-free conditions, and has stability and controllability for industrialization, thereby providing a more optimal process basis for large-scale production of influenza vaccines. SUMMARY

[0007] The present application aims to provide a serum-free culture method based on Vero cells and a special medium formula, which is used for efficient preparation of influenza viruses, to solve the problem of poor growth state of Vero cells under serum-free conditions and low virus yield in the preparation of existing influenza vaccines.

[0008] To achieve the above object, the technical scheme provided by the application comprises adding protein components of specific sources and proportions in a serum-free culture medium of Vero cells, wherein the protein components comprise red algae protein and / or soybean protein hydrolysate, the red algae protein is added in an amount of 100-500 mg / L, the soybean protein hydrolysate is added in an amount of 100-500 mg / L, and the red algae protein and the soybean protein hydrolysate can be used in combination in proportion.

[0009] In the method of the application, the culture medium can be a modified formula of a commercial serum-free culture medium, and by adding the above-mentioned protein components in a certain amount, and by using microcarrier adhesion culture, the Vero cells can be grown in high density under serum-free conditions. Subsequently, the influenza virus is inoculated, and the culture is carried out under suitable temperature, pH and dissolved oxygen conditions, the supernatant is harvested, and the purification is carried out through steps such as clarification, ultrafiltration and chromatography, so that the high-titer influenza virus stock solution can be obtained, which is suitable for subsequent vaccine preparation.

[0010] Specifically, a preparation method of Vero cell influenza virus vaccine comprises the following steps:

[0011] Step S1: Vero cell culture

[0012] After the Vero cells are subcultured for 1-2 generations in a culture medium containing 3-10 wt% serum, the Vero cells are transferred into a serum-free Vero cell culture medium for subculture for 3-8 generations, so as to establish a serum-free Vero cell bank;

[0013] Step S2: inoculation and propagation of influenza virus

[0014] After the influenza virus strain is subcultured for 1-2 generations in an avian embryo or other suitable host, the virus is adapted by subculture for 3-8 generations in a serum-free Vero cell culture medium, so as to establish a virus seed bank;

[0015] In a bioreactor, 5-15 g / L of microcarriers are added to the serum-free Vero cells for high-density suspension culture, and when the cell density reaches 0.5×10 6 -2.0×10 6 cells / mL, the virus is inoculated; the culture conditions are as follows: temperature 35-38℃, pH 7.0-7.5, dissolved oxygen keeping 30%-60% saturated air, stirring speed 50-120 rpm; and the culture medium is a serum-free Vero cell culture medium;

[0016] Step S3: purification and inactivation of virus

[0017] A precipitant is added to the virus liquid in an amount of 2-6 wt%, and the virus precipitate is collected by centrifugation after standing at 4℃ for 8-24 h; after sterilization by membrane filtration, an inactivator is added in a mass ratio of 1:3000-1:5000, and the inactivator is inactivated at 2-8℃ for 48-96 h, and is hydrolyzed at 30-40℃ for 2-5 h.

[0018] The vaccine stock solution is obtained by ion exchange chromatography and ultrafiltration concentration.

[0019] More specifically, the purification and inactivation of the virus in step S3 is as follows: PEG-6000 is added as a precipitant to a final concentration of 4wt%, and the mixture is shaken thoroughly, then placed at 4℃ for 12h, and then centrifuged at 8000xg for 30min to collect the virus precipitate; the virus is separated by centrifugation at 10000xg for 60min; the virus is sterilized by 0.22μm ultrafiltration, and β-propiolactone is added as an inactivator at a final concentration of 1:4000 by mass, and then inactivated at 4℃ for 72h, and then hydrolyzed at 37℃ for 3h.

[0020] More specifically, the ion exchange chromatography and ultrafiltration concentration in step S3 is as follows: anion exchange chromatography is performed after equilibrating the chromatography column with 0.1mol / L PBS buffer containing 1.0mol / L NaCl, the salt concentration of the sample is adjusted to 1.0mol / L NaCl, and the ion exchange chromatography is performed, and the flow-through peak is collected; the vaccine stock solution is obtained by ultrafiltration concentration with a 500KD membrane and clarification filtration with a 0.25μm filter.

[0021] Preferably, the serum-free Vero culture medium in steps S1, S2 and S3 comprises a basic culture medium containing non-essential amino acids, at least one functional protein, and a plurality of additive components, the concentration of the functional protein is 200-1000mg / L, and the additive components include but are not limited to transferrin 1-10mg / L, recombinant epidermal growth factor 0.001-0.1mg / L, fibroblast growth factor 0.1-5mg / L, recombinant insulin 1-20mg / L, recombinant fibronectin 0.5-5mg / L, poly-L-lysine 0.5-5mg / L, sodium selenite 0.001-0.1mg / L, zinc sulfate 0.001-0.05mg / L, myo-inositol 10-100mg / L, ethanolamine 5-20mg / L, polyether F-68 0.1-1mg / L, and sodium pyruvate 1-10mg / L. Further preferably, the concentration of the functional protein in the additive components is 800mg / L, the concentration of transferrin is 6mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01mg / L, the concentration of fibroblast growth factor (FGF) is 2mg / L, the concentration of recombinant insulin is 8mg / L, the concentration of recombinant fibronectin is 2.5mg / L, the concentration of poly-L-lysine is 1.4mg / L, the concentration of sodium selenite is 0.03mg / L, the concentration of zinc sulfate is 0.01mg / L, the concentration of myo-inositol is 40mg / L, the concentration of ethanolamine is 11mg / L, the concentration of polyether F-68 is 0.3mg / L, and the concentration of sodium pyruvate is 4mg / L.

[0022] Preferably, the functional protein is at least one of agar protein, soybean protein hydrolysate, and wheat protein hydrolysate.

[0023] Preferably, the red algae protein is prepared by the following method: the red algae is mixed and cultured in an acidic condition in a photobioreactor to the end of the exponential growth phase, the algal cells are harvested and washed; the algal cells are suspended in a salt-containing buffer, mechanically broken and centrifuged, and the supernatant is collected as a crude extract; the crude extract is subjected to ammonium sulfate fractionation and desalting treatment to obtain a protein solution; the protein solution is subjected to heat inactivation treatment and centrifuged to remove the precipitate, and the obtained supernatant is freeze-dried to obtain the red algae protein.

[0024] Further preferably, the preparation method of the red algae protein is as follows: the culture medium is BG-11 culture medium with pH 2.0-3.0, mixed and cultured in a flat panel airlift photobioreactor with a volume of 5-10 L, the temperature is 35-40℃, the light intensity is 100-200 μmol photons·m⁻²·s⁻¹, the aeration amount is 100-200 NL / h, and the washed algal cells are harvested at the end of the exponential growth phase; the algal cells are broken by bead beating in a 0.05-0.20 mol / L, pH 6.5-7.5 PBS buffer containing 300-700 mmol / L salt, and centrifuged, and the supernatant is collected; the obtained crude extract is subjected to ammonium sulfate precipitation with a saturation degree of 40%-100%, centrifugation, and desalting treatment to obtain a protein solution; the protein solution is inactivated by heating at 65-75℃ for 10-30 minutes, centrifuged to remove the precipitate, and the supernatant is freeze-dried to obtain the red algae protein.

[0025] The present application maintains the natural conformation of the red algae protein through low-temperature ammonium sulfate precipitation and PD-10 desalting column buffer replacement; at the same time, the red algae protein activates the integrin-mediated cell adhesion and proliferation pathway through phosphorylation of focal adhesion kinase and Src kinase, and up-regulates the expression of IGF-1 receptor, triggers the mitogenic signal cascade reaction, and improves the reproduction efficiency of cells.

[0026] Preferably, the soybean protein hydrolysate is prepared by the following method: the soybean protein is dispersed in water for heat pretreatment and then cooled; alkaline protease is added under suitable pH and temperature conditions for enzymatic reaction; after the reaction is completed, the enzyme is inactivated by heating, cooled and centrifuged to remove the insoluble matter; and the obtained supernatant is dried to obtain the soybean protein hydrolysate.

[0027] Further preferably, the preparation method of the soybean protein hydrolysate is as follows: 15-25 g of soybean protein is added to 80-120 mL of water, heated at 80-95 ℃ for 5-15 min, and then cooled to 45-55 ℃; 0.5-1.5 g of alkaline protease is added, and enzymolysis is carried out at 150-250 rpm, pH 7.0-8.0, and 45-55 ℃ for 8-16 h; the reaction solution is heated at 90-105 ℃ for 5-15 min to inactivate the enzyme, and after being cooled to room temperature, centrifugation is carried out at 6000-10000 rpm for 15-25 min, the precipitate is discarded, and the supernatant is collected; the supernatant is freeze-dried to obtain the soybean protein hydrolysate.

[0028] The adherent proteins (such as soybean protein isolate SPI) and polysaccharides in the soybean protein hydrolysate of the application can form a film on the surface of the culture medium, which contains specific structures capable of interacting with cell surface receptors, directly promoting cell adhesion, and effectively improving the adsorption effect of cells on glass / microcarriers. Furthermore, the active peptides in the soybean protein hydrolysate may contain a gelatin-like RGD (arginine-glycine-aspartic acid) motif, directly mediating adhesion through integrin receptors; at the same time, it can change the surface charge of the substrate, optimize protein adsorption and cell integrin binding through electrostatic interaction, and indirectly improve the adhesion efficiency.

[0029] The application has the following advantages:

[0030] Compared with the prior art, the application has the following advantages:

[0031] (1) In the combined formula of the application, the red algae protein and the soybean protein hydrolysate play complementary roles in cell signal activation, microenvironment stabilization, adhesion promotion, and nutrition supplementation: the red algae protein provides growth signals and anti-stress protection for cells, and the soybean protein hydrolysate improves the physical binding of cells to the substrate and provides low molecular peptides that can be quickly utilized, and the two work together to enable Vero cells to grow at a high density and high activity under serum-free conditions, and significantly improve the virus infection efficiency and replication capacity.

[0032] It has been verified that under the same culture conditions, the influenza virus titer of the culture medium combined with the addition of red algae protein and soybean protein hydrolysate can reach 8.5 log10 TCID 50 / mL, which is significantly improved compared with single components or conventional commercial serum-free culture medium. This effect is due to the functional synergy of the two natural non-animal source proteins, which not only solves the problem of insufficient cell adhesion and growth in serum-free culture, but also eliminates the potential risk of animal source components, and is suitable for popularization and application in industrialized scale vaccine production.

[0033] (2) The protein used in the application is derived from plants or algae, avoiding the immunogenicity and safety risks that may be caused by animal-derived components (such as bovine serum albumin), and complying with the regulatory trend of serum-free and animal-free vaccine production.

[0034] (3) The components used in the preparation method of the application are easy to obtain and have good batch stability, and can be stably operated in large-scale culture systems such as stirred reactors, which are suitable for industrial production of influenza vaccines. DETAILED DESCRIPTION

[0035] The parameters and sources of some raw materials in the examples are as follows:

[0036] The influenza virus strain is A / Vietnam / 1194 / 2004 (H5N1 original strain);

[0037] The DMEM culture solution containing 6wt% bovine serum refers to uniformly mixing 6g bovine serum into 94g DMEM culture solution. The DMEM culture solution is a brand of Punsai, and the model number is PM150210.

[0038] VirusPro® VP SFM serum-free culture solution contains 4mmol / L L-glutamine and 3.9g / L glucose.

[0039] The MEM basic medium containing non-essential amino acids is a brand of Punsai, and the model number is PM150410.

[0040] Alkaline protease with an enzyme activity of 500,000 U / g.

[0041] pH 2.5 BG-11 culture medium: 0.28 mol glucose is added to 950 mL BG-11 culture medium and mixed uniformly, 0.1 mol / L sulfuric acid is used to adjust the pH to 2.5, and the BG-11 culture medium is diluted to 1 L.

[0042] Example 1: A preparation method of Vero cell influenza virus vaccine, comprising the following steps:

[0043] Step S1 Vero cell culture

[0044] Establishing a cell bank

[0045] Vero cells (ATCC CCL-81) are used, and are passed for 2 generations in DMEM culture solution containing 6wt% fetal bovine serum; then are passed for 5 generations in Vero cell serum-free culture medium A, to establish a master cell bank, i.e. a serum-free Vero cell bank;

[0046] Step S2 inoculation and proliferation of influenza virus

[0047] The influenza virus strain is passed through chicken embryos for 2 generations, and then inoculated into the serum-free Vero cells of step S1 in a serum-free culture medium A, and subcultured for 5 generations to establish a virus stock, i.e. a Vero cell culture virus stock;

[0048] The microcarriers are washed and autoclaved, and added to the bioreactor at a concentration of 10 g / L; the serum-free Vero cells of step S1 are used as host cells, and high-density suspension culture is carried out in the bioreactor by using the microcarriers Cytodex-1, when the cell density reaches 1.0 x 10 6 cells / mL, virus inoculation is carried out, and the inoculation amount MOI is 0.001;

[0049] The parameters during the culture are as follows:

[0050] Temperature: constant temperature 37℃

[0051] pH value: maintained in the range of 7.2-7.4

[0052] Dissolved oxygen (DO): kept at 40%-50% saturated air

[0053] Stirring speed: 80 rpm

[0054] Serum-free culture medium: Vero cell serum-free culture medium A is selected;

[0055] Step S3 Purification of the virus

[0056] PEG-6000 is added to a final concentration of 4wt%, and after being placed at 4℃ for 12h, the virus precipitate is collected by centrifugation at 8000 x g for 30min; the virus is separated by centrifugation at 10000 x g for 60min; 0.22μm ultrafiltration is used for sterilization, β-propiolactone is added at a mass ratio of 1:4000 to a final concentration, inactivated at 4℃ for 72h, and hydrolyzed at 37℃ for 3h;

[0057] Anion exchange chromatography: after equilibrating the chromatography column with 0.1mol / L pH7.0 PBS buffer containing 1.0mol / L NaCl, the salt concentration of the sample is adjusted to 1.0mol / L NaCl, ion exchange chromatography is carried out, and the flow-through peak is collected; 500KD membrane package ultrafiltration is used for concentration, and 0.25μm filter membrane is used for clarification, to obtain the vaccine stock solution; the sample is subjected to HA content detection and total protein content detection.

[0058] Comparative Example 1: a preparation method of a Vero cell influenza virus vaccine

[0059] It is basically the same as Example 1, and the only difference is that the serum-free culture medium of Vero cells in Example 1 is replaced by a commercially available VirusPro® VP SFM serum-free culture solution.

[0060] Example 2: A method of preparing a Vero cell influenza virus vaccine

[0061] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium B.

[0062] Example 3: A method of preparing a Vero cell influenza virus vaccine

[0063] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium C.

[0064] Comparative Example 2: A method of preparing a Vero cell influenza virus vaccine

[0065] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium D.

[0066] Example 4: A method of preparing a Vero cell influenza virus vaccine

[0067] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium E.

[0068] Comparative Example 3: A method of preparing a Vero cell influenza virus vaccine

[0069] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium F.

[0070] Comparative Example 4: A method of preparing a Vero cell influenza virus vaccine

[0071] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium G.

[0072] Example 5: A method of preparing a Vero cell influenza virus vaccine

[0073] Essentially identical to Example 1, with the exception that the Vero cell serum- free medium of Example 1 is replaced with Vero cell serum-free medium H.

[0074] Example 6: Preparation of Vero cell serum-free media A-H

[0075] Vero cell serum-free culture medium A, including MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of phycoerythrin is 800 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0076] A method for preparing phycoerythrin, comprising the following steps:

[0077] (1) Red algae (Galdieria sulphuraria 074G) is cultured in a mixed culture mode, and the culture medium is pH 2.5 BG-11 medium; the culture is carried out in a 6L flat plate airlift photobioreactor, and the control conditions are: temperature 38℃, light intensity 150 μmol photons·m -2 ·s -1 , ventilation volume 150NL / h; the biomass is harvested at the end of the exponential growth, the dry weight is about 9.5g / L, and the cells are collected and washed twice with distilled water to remove the residual medium under the conditions of 4℃ and 4000×g centrifugation for 15 minutes;

[0078] (2) Cell disruption

[0079] The cell disruption is carried out by bead beating method using 0.5mm glass beads;

[0080] Parameters:

[0081] Biomass / glass bead ratio: 1:10 w / w;

[0082] Disruption conditions: 2100rpm, 30 seconds each time, interval 2 minutes, -20℃ cooling, a total of 6 cycles;

[0083] Buffer: 0.1mol / L pH 7.0 PBS buffer containing 500mmol / L NaCl;

[0084] Through cascade extraction, three cycles including disruption→centrifugation→resuspension, after disruption, centrifugation at 4℃ and 4000×g for 10 minutes, the supernatant is collected, and microscopic observation shows that the cell disruption rate reaches 85-90%;

[0085] (3) Protein extraction and concentration

[0086] The crude extract was centrifuged at 11000xg for 2 hours at 4℃, and the supernatant was discarded, and the precipitate was reserved; 60% saturated ammonium sulfate aqueous solution was added under the condition of 200rpm stirring, and the mixture was incubated at 4℃ overnight; the mixture was centrifuged at 11000xg for 2 hours at 4℃, and the supernatant was discarded, and the precipitate was reserved;

[0087] Desalting treatment: the precipitate was resuspended with 50mmol / L pH 7.0 PBS buffer, and residual ammonium sulfate was removed by a PD-10 desalting column (Sephadex G-25 resin) to obtain a desalted protein solution;

[0088] (4) Heat inactivation treatment

[0089] The desalted protein solution was heated in a 70℃ water bath for 20 minutes, and then centrifuged at 13000xg for 20 minutes at 4℃ to remove the heat denaturation precipitate. The supernatant was freeze-dried to obtain the red algae protein.

[0090] The Vero cell serum-free culture medium B includes a MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of soybean protein hydrolysate is 800mg / L, the concentration of transferrin is 6mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01mg / L, the concentration of fibroblast growth factor (FGF) is 2mg / L, the concentration of recombinant insulin is 8mg / L, the concentration of adhesion factor-recombinant fibronectin is 2.5mg / L, the concentration of adhesion factor-poly-L-lysine is 1.4mg / L, the concentration of sodium selenite is 0.03mg / L, the concentration of zinc sulfate is 0.01mg / L, the concentration of myo-inositol is 40mg / L, the concentration of ethanolamine is 11mg / L, the concentration of polyether F-68 is 0.3mg / L, and the concentration of sodium pyruvate is 4mg / L.

[0091] A method for preparing soybean protein hydrolysate, comprising the following steps:

[0092] 1) 20g of soybean protein was added to 100mL of water, heated at 90℃ for 10 minutes, and cooled to 50℃ for standby;

[0093] 2) 1g of alkaline protease was added, and the mixture was enzymatically hydrolyzed at 200rpm, pH 7.5, and 50℃ for 12h; the enzyme activity was inactivated by boiling at 100℃ for 10min; the mixture was centrifuged at 8000rpm for 20min after cooling to room temperature, and the precipitate was discarded, and the supernatant was collected; after freeze-drying, the soybean protein hydrolysate was obtained.

[0094] Vero cell serum-free medium C, including MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of phycobiliprotein is 400 mg / L, the concentration of soybean protein hydrolysate is 400 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0095] Vero cell serum-free medium D, including MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of bovine serum albumin is 800 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0096] Vero cell serum-free medium E, including MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of wheat protein hydrolysate is 800 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0097] A method for preparing wheat protein hydrolysate, comprising the following steps:

[0098] 1) 20 g of wheat protein was weighed and added to 100 mL of water, heated at 90°C for 10 min, and cooled to 50°C for standby;

[0099] 2) 1 g of alkaline protease was added, and the enzyme hydrolysis was carried out at 200 rpm, pH 8.0, and 50°C for 12 h;

[0100] 3) boiling for 10 min at 100℃ to inactivate the enzyme, cooling to room temperature, and centrifuging at 8000 rpm for 20 min to collect the supernatant;

[0101] 4) freeze-drying the supernatant to obtain the wheat protein hydrolysate.

[0102] The serum-free culture medium F for Vero cells comprises a MEM basic medium containing non-essential amino acids and an additive component; in the additive component, the concentration of perilla seed protein is 800 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of myo-inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0103] The preparation method of perilla seed protein comprises the following steps:

[0104] 1) 50 g of perilla seed powder is added to 500 mL of petroleum ether, and defatted for 4 h at room temperature with stirring, filtered, and the process is repeated twice to obtain defatted perilla seed powder;

[0105] 2) the defatted perilla seed powder is added to 500 mL of deionized water, and the pH is adjusted to 9.0, and the mixture is stirred for 2 h at room temperature;

[0106] 3) centrifuging at 8000 rpm for 20 min to collect the supernatant;

[0107] 4) adjusting the pH of the supernatant to 4.5, and standing for 30 min to precipitate the protein;

[0108] 5) centrifuging at 8000 rpm for 20 min to discard the supernatant and collect the precipitate;

[0109] 6) resuspending the precipitate with a small amount of deionized water, adjusting the pH to 7.0, and freeze-drying to obtain perilla seed protein.

[0110] Vero cell serum-free medium G, comprising MEM basic medium containing non-essential amino acids and an addition component; in the addition component, the concentration of soybean protein is 800 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of myo-inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0111] Vero cell serum-free medium H, comprising MEM basic medium containing non-essential amino acids and an addition component; in the addition component, the concentration of phycochromine is 400 mg / L, the concentration of soybean protein is 400 mg / L, the concentration of transferrin is 6 mg / L, the concentration of recombinant epidermal growth factor (rhEGF) is 0.01 mg / L, the concentration of fibroblast growth factor (FGF) is 2 mg / L, the concentration of recombinant insulin is 8 mg / L, the concentration of recombinant fibronectin is 2.5 mg / L, the concentration of poly-L-lysine is 1.4 mg / L, the concentration of sodium selenite is 0.03 mg / L, the concentration of zinc sulfate is 0.01 mg / L, the concentration of myo-inositol is 40 mg / L, the concentration of ethanolamine is 11 mg / L, the concentration of polyether F-68 is 0.3 mg / L, and the concentration of sodium pyruvate is 4 mg / L.

[0112] The phycochromine is prepared according to the method for preparing the phycochromine in the Vero cell serum-free medium A.

[0113] Test Example 1

[0114] The infectious titer (TCID50) of the Vero cell culture virus obtained in step S2 in the examples and comparative examples is shown in Table 1. 50 )as shown in Table 1.

[0115] Table 1 Infectious titer (TCID 50 )as shown in Table 1.

[0116]

[0117] The infectious titer (7.5 log 10 / mL) was significantly better than that of Example 2 (6.8), Example 4 (6.1), Example 5 (6.7), and Comparative Examples 1 (5.4), 2 (5.5), 3 (5.8), and 4 (5.6). The reason is that the low-temperature ammonium sulfate precipitation + PD-10 desalting column buffer replacement was used in the preparation process of the red algae protein, which maximized the maintenance of its natural conformation and active domain. This enables it to retain the ability to bind to cell membrane receptors and activate signals after entering the culture medium, enhance cell adhesion and proliferation, and provide antioxidant protection, thereby significantly improving the virus replication efficiency under serum-free conditions. Soybean, wheat, and perilla seed-derived plant proteins and their hydrolysates mainly provide RGD motifs, which are biased towards integrin-mediated adhesion, and have limited ability to activate growth signaling pathways such as IGF-1R. Bovine serum albumin mainly plays a role in osmotic pressure regulation and protein protection, and does not directly activate cell proliferation signals.

[0118] Example 3 (8.5 log 10 / mL) was significantly better than that of Examples 1-2. The reason is that the medium C of Example 3 contains both red algae protein and soybean protein hydrolysate, which play a synergistic role in cell signal activation, microenvironment stabilization, adhesion promotion, and nutrient supply, allowing Vero cells to maintain a higher density and activity, thereby achieving higher virus yield. It is worth noting that during the establishment of the virus seed bank, the virus titer (TCID 50 ) > 7 log 10 / mL was achieved after 5 passages of adaptive passage in serum-free Vero cell culture medium A / C, reducing the time of passage culture and improving the culture efficiency.

[0119] Test Example 2

[0120] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium A in a 35 mm culture dish at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were digested, collected, and counted, and the same method was used for continuous passage culture for two times.

[0121] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium B in a 35 mm culture dish at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were digested, collected, and counted, and the same method was used for continuous passage culture for two times.

[0122] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium C in 35 mm culture dishes at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were collected by digestion and counted. The same method was used for subculture for two times.

[0123] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium D in 35 mm culture dishes at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were collected by digestion and counted. The same method was used for subculture for two times.

[0124] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium E in 35 mm culture dishes at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were collected by digestion and counted. The same method was used for subculture for two times.

[0125] Vero cells (ATCC CCL-81) were cultured in Vero cell serum-free medium F in 35 mm culture dishes at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were collected by digestion and counted. The same method was used for subculture for two times.

[0126] Vero cells (ATCC CCL-81) were cultured in VirusPro® VP SFM serum-free medium in 35 mm culture dishes at a cell seeding density of 1 x 10 4 / cm 2 , and the culture conditions were 37°C, 5% CO2. After 72 hours of culture, the cells were collected by digestion and counted. The same method was used for subculture for two times.

[0127] The cell viability was detected by CCK-8 method, and the test results were as follows:

[0128] Table 2 Vero cell viability

[0129]

[0130] From the cell viability data in Table 2, it can be seen that the cell viability of Example 3 (92.8%) is the highest, further proving the advantages of the combination of double proteins in improving cell state, delaying decline and improving toxin production capacity; the cell viability of Example 1 (85.6%) and Example 2 (84.3%) is better than that of other single protein or animal source protein group, indicating that the biological compatibility and promotion effect of functional proteins of plant / algal origin in serum-free culture are better.

[0131] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and changes without requiring creative efforts based on the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application should be within the protection scope defined by the claims.

Claims

1. A method of preparing a Vero cell influenza virus vaccine, characterized by, The method comprises the following steps: Step S1 Vero cell culture After the Vero cells are subcultured for 1-2 generations in a culture medium containing 3-10 wt% serum, the Vero cells are subcultured for 3-8 generations in a serum-free Vero cell culture medium to establish a serum-free Vero cell bank; Step S2 inoculation and propagation of influenza virus After the influenza virus strain is subcultured for 1-2 generations in a chicken embryo, the virus is subcultured for 3-8 generations in a serum-free Vero cell culture medium to establish a virus stock; In the bioreactor, 5-15 g / L microcarriers were added to serum-free Vero cells for high-density suspension culture, and when the cell density reached 0.5×10 6 -2.0×10 6 cells / mL, the virus was inoculated; the culture conditions were temperature 35-38℃, pH 7.0-7.5, dissolved oxygen maintained at 30%-60% saturated air, stirring speed 50-120 rpm; the culture medium was serum-free Vero cell culture medium; Step S3 virus purification and inactivation 2-6 wt% of a precipitant is added to the virus solution, and the virus precipitate is collected after the solution is placed at 4℃ for 8-24 h and centrifuged; after bacteria are removed by membrane filtration, an inactivator is added at a mass ratio of 1:3000-1:5000, and the solution is inactivated at 2-8℃ for 48-96 h and hydrolyzed at 30-40℃ for 2-5 h; The vaccine bulk is obtained through ion exchange chromatography and ultrafiltration concentration; The serum-free Vero cell culture medium comprises a MEM basic culture medium containing non-essential amino acids and an additive component; In the additive component, the concentration of the red algae protein is 400 mg / L, the concentration of the soybean protein hydrolysate is 400 mg / L, the concentration of the transferrin is 6 mg / L, the concentration of the recombinant epidermal growth factor is 0.01 mg / L, the concentration of the fibroblast growth factor is 2 mg / L, the concentration of the recombinant insulin is 8 mg / L, the concentration of the recombinant fibronectin is 2.5 mg / L, the concentration of the poly-L-lysine is 1.4 mg / L, the concentration of the sodium selenite is 0.03 mg / L, the concentration of the zinc sulfate is 0.01 mg / L, the concentration of the inositol is 40 mg / L, the concentration of the ethanolamine is 11 mg / L, the concentration of the polyether F-68 is 0.3 mg / L, and the concentration of the sodium pyruvate is 4 mg / L; The preparation method of the red algae protein is as follows: the culture medium is BG-11 culture medium with pH 2.0-3.0, the mixed culture is carried out in a flat plate airlift photobioreactor with a volume of 5-10 L, the temperature is 35-40 DEG C, the light intensity is 100-200 mu mol photons m-2s-1, the aeration amount is 100-200 NL / h, and the washed algal cells are harvested at the end of the exponential growth phase; the algal cells are repeatedly broken by bead beating in a PBS buffer with 0.05-0.20 mol / L, pH 6.5-7.5 and containing 300-700 mmol / L salt, and centrifuged to collect the supernatant; the obtained crude extract is subjected to ammonium sulfate fractionation precipitation at a saturation degree of 40%-100% and desalting treatment to obtain a protein solution. -2 ·s -1 , the aeration amount is 100-200 NL / h, and the washed algal cells are harvested at the end of the exponential growth phase; the algal cells are repeatedly broken by bead beating in a PBS buffer with 0.05-0.20 mol / L, pH 6.5-7.5 and containing 300-700 mmol / L salt, and centrifuged to collect the supernatant; the obtained crude extract is subjected to ammonium sulfate fractionation precipitation at a saturation degree of 40%-100% and desalting treatment to obtain a protein solution. The protein solution is heated at 65-75℃ for 10-30 min to inactivate the solution, the precipitate is removed by centrifugation, and the supernatant is freeze-dried to obtain the red algae protein; the algal cells are red algae Galdieria sulphuraria 074G; The preparation method of the soybean protein hydrolysate is as follows: 15-25 g of soybean protein is added to 80-120 mL of water, heated at 80-95℃ for 5-15 min, and then cooled to 45-55℃; 0.5-1.5 g of alkaline protease is added, and the solution is enzymolyzed at 150-250 rpm, pH 7.0-8.0, and 45-55℃ for 8-16 h; the reaction solution is heated at 90-105℃ for 5-15 min to inactivate the enzyme activity, cooled to room temperature, and centrifuged at 6000-10000 rpm for 15-25 min; the precipitate is discarded, and the supernatant is collected; the supernatant is freeze-dried to obtain the soybean protein hydrolysate.

2. The preparation method of the Vero cell influenza virus vaccine according to claim 1, characterized in that, In the step S3, the virus is purified and inactivated by adding PEG-6000 to a final concentration of 4 wt%, fully shaking, placing at 4℃ for 12 h, centrifuging at 8000×g for 30 min to collect the virus precipitate, centrifuging at 10000×g for 60 min to separate the virus, filtering bacteria through 0.22 μm ultrafiltration, adding an inactivator β-propiolactone at a final concentration of 1:4000, inactivating at 4℃ for 72 h, and hydrolyzing at 37℃ for 3 h.

3. The method for preparing the Vero cell influenza virus vaccine as described in claim 1, characterized in that, The ion exchange chromatography and ultrafiltration concentration in step S3 specifically comprises: anion exchange chromatography, equilibrating the chromatography column with 0.1 mol / L PBS buffer containing 1.0 mol / L NaCl, adjusting the salt concentration of the sample to 1.0 mol / L NaCl, performing ion exchange chromatography, collecting the flow-through peak; and using 500KD membrane to perform ultrafiltration concentration, clarifying the filtrate through a 0.25 μm filter, thereby obtaining the vaccine stock solution.

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