Preparation method of Vero cell influenza virus vaccine

Abstract

The invention relates to a preparation method of a Vero cell influenza virus vaccine, and belongs to the technical field of virus culture. In the culture of the influenza virus, red algae protein and soybean protein hydrolysate are added into a culture medium at the same time, the red algae protein and soybean protein hydrolysate are subjected to low-temperature ammonium sulfate precipitation and PD-10 desalination column purification, natural conformation is kept, integrin-FAK-Src and IGF-1R signal channels can be activated, and cell adhesion and proliferation are promoted; the latter contains soybean protein isolate, polysaccharide and RGD motif peptide, and can form an adhesion film on a substrate and mediate adhesion through integrin. According to the invention, the Vero cells and the recombinant plasmid are combined to improve cell adherence, metabolic homeostasis and oxidation resistance, so that the Vero cells grow at high density and efficiently replicate influenza viruses under a serum-free condition, the virus titer (TCID50) reaches 8.5 log10 / mL, and the Vero cells and the recombinant plasmid are suitable for industrial vaccine production.

Description

Technical Field

[0001] This invention relates to the field of virus culture technology, and in particular to a method for preparing a Vero cell influenza virus vaccine. Background Technology

[0002] Currently, the production of existing influenza vaccines mainly relies on the method of culturing viruses in chicken embryos. This method is mature, but it has problems such as long cycle, limited production scale, sensitivity to production environment and raw materials, and the possibility of introducing chicken-derived allergens, making it difficult to meet the needs of large-scale and rapid production.

[0003] Vero cells are widely used in the production of various viral vaccines, such as inactivated polio vaccine and rabies vaccine, due to their high genetic stability, strong adaptability, and lack of interferon production capacity. Serum-free culture of Vero cells avoids the potential safety risks associated with animal serum and improves 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 evident in the efficient preparation of influenza virus.

[0004] To enhance the influenza virus proliferation capacity of Vero cells under serum-free conditions, researchers have attempted to add various nutrients and growth factors, such as transferrin, insulin, and epidermal growth factor, to the culture medium to improve cell metabolism and virus yield. However, a mature technical solution for significantly increasing influenza virus titers while maintaining high-density cell culture remains lacking.

[0005] CN114540277A discloses a serum-free culture medium for culturing Vero cells and its preparation method, which describes plant hydrolyzed protein and up to 80 kinds of additives. It is difficult to control batch-to-batch differences 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 culture medium formulation for Vero cells that can significantly improve the viral load of Vero cells for influenza under serum-free conditions, and possess stability and controllability that can be scaled up industrially, thus providing a better technological basis for the large-scale production of influenza vaccines. Summary of the Invention

[0007] The purpose of this invention is to provide a serum-free culture method based on Vero cells and a special culture medium formulation for the efficient preparation of influenza virus, so as to solve the problems of poor growth status and low virus yield of Vero cells under serum-free conditions in the preparation of existing influenza vaccines.

[0008] To achieve the above objectives, the technical solution provided by the present invention includes: adding protein components of a specific source and proportion to a serum-free culture medium for Vero cells, wherein the protein components include red algae protein and / or soybean protein hydrolysate, wherein the amount of red algae protein added is 100-500 mg / L, the amount of soybean protein hydrolysate added is 100-500 mg / L, and they can be used in combination in proportion.

[0009] In the method of this invention, the culture medium can be a modified formulation of a commercial serum-free culture medium. By quantitatively adding the aforementioned protein components and combining it with microcarrier adherent culture, Vero cells can achieve high-density growth under serum-free conditions. Subsequently, influenza virus is inoculated, cultured under suitable temperature, pH, and dissolved oxygen conditions, and the supernatant is harvested. After purification through clarification, ultrafiltration, and chromatography, a high-titer influenza virus stock solution can be obtained, suitable for subsequent vaccine preparation.

[0010] Specifically, a method for preparing a Vero cell influenza virus vaccine includes the following steps: Step S1: Vero cell culture After passage of Vero cells for 1-2 generations in a medium containing 3-10 wt% serum, they were transferred to a serum-free Vero cell medium and passaged for 3-8 generations to establish a serum-free Vero cell bank. Step S2: Influenza virus inoculation and replication After passage of influenza virus strains for 1-2 generations in avian embryos or other suitable hosts, they are adaptively passaged for 3-8 generations in serum-free Vero cell culture medium to establish a virus stock bank. In a bioreactor, serum-free Vero cells were cultured in high-density suspension with 5-15 g / L microcarriers. When the cell density reached 0.5 × 10⁻⁶ cells / L... 6 -2.0×10 6 Inoculate with virus at a concentration of cells / mL; culture conditions are: temperature 35-38℃, pH 7.0-7.5, dissolved oxygen maintained at 30%-60% saturated air, stirring speed 50-120 rpm; culture medium is serum-free Vero cell culture medium; Step S3: Virus purification and inactivation Add 2-6 wt% precipitant to the virus solution, let it stand at 4℃ for 8-24 h, and then centrifuge to collect the virus precipitate; after sterilization by membrane filtration, add inactivating agent at a mass ratio of 1:3000-1:5000, inactivate at 2-8℃ for 48-96 h, and hydrolyze at 30-40℃ for 2-5 h; The vaccine stock solution was obtained by ion exchange chromatography and ultrafiltration concentration.

[0011] More specifically, the purification and inactivation of the virus in step S3 is as follows: PEG-6000 precipitant is added to a final concentration of 4wt%, shaken thoroughly, placed at 4℃ for 12h, and then centrifuged at 8000×g for 30min to collect the virus precipitate; the virus is separated by centrifugation at 10000×g for 60min; sterilized by 0.22μm ultrafiltration, and β-propiolactone extinguishing agent is added at a final concentration of 1:4000 by mass, inactivated at 4℃ for 72h, and hydrolyzed at 37℃ for 3h.

[0012] More specifically, the ion exchange chromatography and ultrafiltration concentration in step S3 are as follows: anion exchange chromatography is performed by equilibrating the chromatography column with 0.1 mol / L pH7.0 PBS buffer containing 1.0 mol / L NaCl, adjusting the sample salt concentration to 1.0 mol / L NaCl, and collecting the flow-through peak; ultrafiltration concentration is performed using a 500KD membrane, followed by clarification and filtration through a 0.25 μm filter membrane to obtain the vaccine stock solution.

[0013] Preferably, the serum-free Vero culture medium in steps S1, S2, and S3 includes a basal culture medium containing non-essential amino acids, at least one functional protein, and multiple additives. The concentration of the functional protein is 200-1000 mg / L, and the additives include, but are not limited to, transferrin 1-10 mg / L, recombinant epidermal growth factor 0.001-0.1 mg / L, fibroblast growth factor 0.1-5 mg / L, recombinant insulin 1-20 mg / L, recombinant fibronectin 0.5-5 mg / L, poly-L-lysine 0.5-5 mg / L, sodium selenite 0.001-0.1 mg / L, zinc sulfate 0.001-0.05 mg / L, inositol 10-100 mg / L, ethanolamine 5-20 mg / L, polyether F-68 0.1-1 mg / L, and sodium pyruvate 1-10 mg / L. More preferably, the added components contain the following concentrations: functional protein 800 mg / L, transferrin 6 mg / L, recombinant epidermal growth factor (rhEGF) 0.01 mg / L, fibroblast growth factor (FGF) 2 mg / L, recombinant insulin 8 mg / L, recombinant fibronectin 2.5 mg / L, poly-L-lysine 1.4 mg / L, sodium selenite 0.03 mg / L, zinc sulfate 0.01 mg / L, inositol 40 mg / L, ethanolamine 11 mg / L, polyether F-68 0.3 mg / L, and sodium pyruvate 4 mg / L.

[0014] Preferably, the functional protein is at least one of red algae protein, soybean protein hydrolysate, and wheat protein hydrolysate.

[0015] Preferably, the red algae protein is prepared by the following method: red algae are cultured in a photobioreactor under acidic conditions until the end of the exponential growth phase, and the algal cells are harvested and washed; the algal cells are suspended in a salt-containing buffer solution, mechanically broken and centrifuged, and the supernatant is collected as a crude extract; the crude extract is subjected to ammonium sulfate fractionation precipitation and desalting treatment to obtain a protein solution; the protein solution is heated to inactivate and centrifuged to remove the precipitate, and the obtained supernatant is freeze-dried to obtain the red algae protein.

[0016] Further preferably, the preparation method of the red algal protein is as follows: the culture medium is BG-11 medium with pH 2.0-3.0, and it is cultured in a 5-10L flat-plate airlift photobioreactor with a temperature of 35-40℃, a light intensity of 100-200 μmol photons·m⁻²·s⁻¹, and an aeration rate of 100-200 NL / h. The washed algal cells are harvested at the end of the exponential growth phase. The algal cells are then cultured in a 0.05-0.20 mol / L medium with a pH of 100-300 μmol / L. In PBS buffer containing 300-700 mmol / L salt at 6.5-7.5 mmol / L, the protein was repeatedly broken down by bead milling (1800-2400 rpm, 20-40 seconds each time, 4-8 cycles) and centrifuged. The supernatant was collected. The crude extract was precipitated with 40%-100% saturated ammonium sulfate, centrifuged, and desalted to obtain a protein solution. The protein solution was heated at 65-75℃ for 10-30 minutes to inactivate the protein, centrifuged to remove the precipitate, and the supernatant was freeze-dried to obtain red algae protein.

[0017] This invention maintains the native conformation of red algal protein through low-temperature ammonium sulfate precipitation and PD-10 desalting column buffer replacement; at the same time, red algal protein activates integrin-mediated cell adhesion and proliferation pathways by phosphorylating focal adhesion kinase and Src kinase, and upregulates IGF-1 receptor expression, triggering a mitotic signaling cascade reaction and improving cell proliferation efficiency.

[0018] Preferably, the soybean protein hydrolysate is prepared by the following method: dispersing soybean protein in water and pre-treating it by heating, then cooling it; adding alkaline protease under suitable pH and temperature conditions to carry out enzymatic hydrolysis; after the reaction is completed, heating to inactivate the enzyme, cooling and centrifuging to remove insoluble matter; and drying the obtained supernatant to obtain the soybean protein hydrolysate.

[0019] A further preferred method for preparing the soybean protein hydrolysate is as follows: 15-25g of soybean protein is added to 80-120mL of water and heated at 80-95℃ for 5-15 minutes, then cooled to 45-55℃; 0.5-1.5g of alkaline protease is added, and enzymatic hydrolysis is carried out at 150-250rpm, pH 7.0-8.0, and 45-55℃ for 8-16 hours; the reaction solution is heated at 90-105℃ for 5-15 minutes to inactivate enzyme activity, cooled to room temperature, and centrifuged at 6000-10000rpm for 15-25 minutes, the precipitate is discarded, and the supernatant is collected; the supernatant is freeze-dried to obtain the soybean protein hydrolysate.

[0020] The adhesive proteins (such as soy protein isolate SPI) and polysaccharides in the soybean protein hydrolysate of this invention can form a thin film on the substrate surface of the culture medium. Their specific structural domains can interact with cell surface receptors, directly promoting cell adhesion and effectively improving cell adsorption on glass / microcarriers. Furthermore, the active peptides in the soybean protein hydrolysate may contain gelatin-like RGD (arginine-glycine-aspartic acid) motifs, directly mediating adhesion through integrin receptors; simultaneously, they may alter the substrate surface charge, optimizing protein adsorption and integrin binding through electrostatic interactions, indirectly enhancing adhesion efficiency.

[0021] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following significant advantages: (1) In the combined formulation of the present invention, red algae protein and soybean protein hydrolysate play complementary roles in cell signal activation, microenvironment stabilization, adhesion promotion and nutrient supplementation: red algae protein provides growth signals and anti-stress protection for cells, while soybean protein hydrolysate improves the physical binding between cells and substrate and provides readily available low molecular weight peptides. The synergistic effect of the two enables Vero cells to maintain high density and high activity growth under serum-free conditions, and significantly improves viral infection efficiency and replication capacity.

[0022] It has been verified that, under the same culture conditions, a culture medium supplemented with a combination of red algae protein and soybean protein hydrolysate can achieve an influenza virus titer of 8.5 log10 TCID. 50 The concentration of [amount] / mL is significantly higher than that of single-component or conventional commercial serum-free culture media. This effect stems from the synergistic function of two natural non-animal-derived proteins, which not only solves the problem of insufficient cell adhesion and growth in serum-free culture, but also eliminates the potential risks of animal-derived components, making it suitable for widespread application in industrial-scale vaccine production.

[0023] (2) The proteins used in this invention are derived from plants or algae, which avoids the immunogenicity and safety risks that may be caused by animal-derived components (such as bovine serum albumin), and conforms to the regulatory trend of serum-free and animal-free vaccine production.

[0024] (3) The components used in the preparation method of the present invention are easy to obtain and have good batch stability. They can be stably operated in large-scale culture systems such as stirred reactors and are suitable for the industrial production of influenza vaccines. Detailed Implementation

[0025] The parameters and sources of some raw materials in the examples are as follows: The influenza virus strain is A / Vietnam / 1194 / 2004 (the original H5N1 strain). DMEM culture medium containing 6 wt% bovine serum refers to the medium prepared by adding 6 g of bovine serum to 94 g of DMEM culture medium and mixing thoroughly. DMEM culture medium, brand: Pronosai; catalog number: PM150210.

[0026] VirusPro® VP SFM serum-free culture medium containing 4 mmol / L L-glutamine and 3.9 g / L glucose.

[0027] MEM basal culture medium containing non-essential amino acids, brand: Pronosai; catalog number: PM150410.

[0028] Alkaline protease, enzyme activity 500,000 U / g.

[0029] For pH 2.5 BG-11 medium, add 0.28 mol of glucose to 950 mL of BG-11 medium and mix well. Adjust the pH to 2.5 with 0.1 mol / L sulfuric acid and bring the volume to 1 L with BG-11 medium.

[0030] Example 1: A method for preparing a Vero cell influenza virus vaccine, comprising the following steps: Step S1: Vero cell culture Establish a cell bank Vero cells (ATCC CCL-81) were passaged twice in DMEM medium containing 6 wt% fetal bovine serum; then passaged five times in serum-free Vero cell medium A to establish a master cell bank, i.e., a serum-free Vero cell bank. Step S2: Influenza virus inoculation and replication The influenza virus strain was passaged twice in chicken embryos, and then the serum-free Vero cells from step S1 were inoculated in serum-free Vero cell culture medium A. After passage five times, a virus stock bank was established, namely, Vero cell cultured virus stock. The microcarriers were cleaned and autoclaved, and then added to the bioreactor at a concentration of 10 g / L. Serum-free Vero cells from step S1 were used as host cells and cultured in high-density suspension in the bioreactor using the Cytodex-1 microcarrier. The cell density was increased to 1.0 × 10⁻⁶ cells / L. 6 When the viral load reaches 0.001 cells / mL, virus inoculation is performed at an MOI of 0.001. The parameters during the cultivation process are as follows: Temperature: Constant at 37℃ pH value: Maintain within the range of 7.2-7.4. Dissolved oxygen (DO): Maintain 40%-50% saturated air Stirring speed: 80 rpm Serum-free culture medium: Vero cell serum-free culture medium A was selected; Step S3 Virus purification Add PEG-6000 to a final concentration of 4wt%, shake well, and incubate at 4℃ for 12h. Collect the virus precipitate by centrifugation at 8000×g for 30min. Separate the virus by centrifugation at 10000×g for 60min. Sterilize by ultrafiltration at 0.22μm, add β-propiolactone at a final concentration of 1:4000 by mass, inactivate at 4℃ for 72h, and hydrolyze at 37℃ for 3h. Anion exchange chromatography was performed by equilibrating the column with 0.1 mol / L pH 7.0 PBS buffer containing 1.0 mol / L NaCl, adjusting the sample salt concentration to 1.0 mol / L NaCl, and collecting the flow-through peak. The solution was then concentrated by ultrafiltration using a 500 kDa membrane and clarified by filtration through a 0.25 μm filter to obtain the vaccine stock solution. The HA content and total protein content of the sample were then determined.

[0031] Comparative Example 1: A method for preparing a Vero cell influenza virus vaccine The study was essentially the same as in Example 1, except that the serum-free culture medium for Vero cells in Example 1 was replaced with commercially available VirusPro® VP SFM serum-free culture medium.

[0032] Example 2: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium B for Vero cells.

[0033] Example 3: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium for Vero cells C.

[0034] Comparative Example 2: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium D for Vero cells.

[0035] Example 4: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium for Vero cells E.

[0036] Comparative Example 3: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium for Vero cells F.

[0037] Comparative Example 4: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium for Vero cells G.

[0038] Example 5: A method for preparing a Vero cell influenza virus vaccine It is basically the same as Example 1, except that the serum-free culture medium for Vero cells in Example 1 is replaced with serum-free culture medium for Vero cells H.

[0039] Example 6: Preparation of serum-free culture medium AH for Vero cells Vero cell serum-free culture medium A includes MEM basal medium containing non-essential amino acids and additives. The additives contain 800 mg / L red algae protein, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0040] The preparation method of red algae protein includes the following steps: (1) Red algae (Galdieria sulphuraria 074G) were cultured in a mixed culture mode using pH 2.5 BG-11 medium. The culture was carried out in a 6L flat-plate airlift photobioreactor under the following conditions: temperature 38℃, light intensity 150 μmol photons·m -2 ·s -1 The aeration rate was 150 NL / h; the biomass was harvested at the end of the exponential growth stage, with a dry weight of about 9.5 g / L. The cells were collected by centrifugation at 4℃ and 4000×g for 15 minutes and washed twice with distilled water to remove culture medium residue. (2) Cell disruption Cell disruption was performed using a bead milling method with 0.5mm glass beads; parameter: Biomass / glass beads ratio: 1:10 w / w; Crushing conditions: 2100 rpm, 30 seconds each time, 2 minutes interval, -20℃ cooling, 6 cycles in total; Buffer solution: 0.1 mol / L pH 7.0 PBS buffer containing 500 mmol / L NaCl; The cell disruption was achieved through a cascade extraction process involving three cycles: disruption, centrifugation, and resuspension. After disruption, the cells were centrifuged at 4°C and 4000×g for 10 minutes, and the supernatant was collected. Microscopic observation showed that the cell disruption rate reached 85-90%. (3) Protein extraction and concentration The crude extract was centrifuged at 11000×g at 4℃ for 2 hours, the supernatant was discarded, and the precipitate was retained; 60% saturated ammonium sulfate aqueous solution was added under stirring at 200 rpm, and incubated overnight at 4℃; the supernatant was discarded and the precipitate was retained after centrifugation at 11000×g at 4℃ for 2 hours. Desalting treatment: The precipitate was resuspended in 50 mmol / L pH 7.0 PBS buffer and the residual ammonium sulfate was removed by passing it through a PD-10 desalting column (Sephadex G-25 resin) to obtain a desalted protein solution; (4) Heat inactivation treatment The desalted protein solution was heated in a 70°C water bath for 20 minutes, then centrifuged at 13000×g at 4°C for 20 minutes to remove the heat-denatured precipitate. The supernatant was then freeze-dried to obtain red algae protein.

[0041] Vero cell serum-free culture medium B includes MEM basal medium containing non-essential amino acids and additives. The additives contain 800 mg / L soybean protein hydrolysate, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L adherent factor-recombinant fibronectin, 1.4 mg / L adherent factor-poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0042] The preparation method of soybean protein hydrolysate includes the following steps: 1) Add 20g of soy protein to 100mL of water, heat at 90℃ for 10 minutes, and cool to 50℃ for later use; 2) Add 1g of alkaline protease and hydrolyze for 12h at 200rpm, pH 7.5 and 50℃; boil at 100℃ for 10min to inactivate enzyme activity; cool to room temperature and centrifuge at 8000rpm for 20min, discard the precipitate and collect the supernatant; freeze dry to obtain soybean protein hydrolysate.

[0043] Vero cell serum-free medium C includes MEM basal medium containing non-essential amino acids and additives. The additives contain 400 mg / L red algae protein, 400 mg / L soybean protein hydrolysate, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0044] Vero cell serum-free medium D includes MEM basal medium containing non-essential amino acids and additives. The additives contain bovine serum albumin at a concentration of 800 mg / L, transferrin at a concentration of 6 mg / L, recombinant epidermal growth factor (rhEGF) at a concentration of 0.01 mg / L, fibroblast growth factor (FGF) at a concentration of 2 mg / L, recombinant insulin at a concentration of 8 mg / L, recombinant fibronectin at a concentration of 2.5 mg / L, poly-L-lysine at a concentration of 1.4 mg / L, sodium selenite at a concentration of 0.03 mg / L, zinc sulfate at a concentration of 0.01 mg / L, inositol at a concentration of 40 mg / L, ethanolamine at a concentration of 11 mg / L, polyether F-68 at a concentration of 0.3 mg / L, and sodium pyruvate at a concentration of 4 mg / L.

[0045] Vero cell serum-free culture medium E includes MEM basal medium containing non-essential amino acids and additives. The additives contain 800 mg / L wheat protein hydrolysate, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0046] The preparation method of wheat protein hydrolysate includes the following steps: 1) Weigh 20g of wheat protein and add it to 100mL of water. Heat at 90℃ for 10 minutes and cool to 50℃ for later use. 2) Add 1g of alkaline protease and hydrolyze for 12h at 200rpm, pH 8.0, and 50℃; 3) Boil at 100℃ for 10 min to inactivate the enzyme, cool to room temperature, centrifuge at 8000 rpm for 20 min, discard the precipitate, and collect the supernatant; 4) Freeze-dry the supernatant to obtain wheat protein hydrolysate.

[0047] Vero cell serum-free culture medium F includes MEM basal medium containing non-essential amino acids and additives. The additives contain 800 mg / L perilla seed protein, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0048] The preparation method of perilla seed protein includes the following steps: 1) Take 50g of perilla seed powder, add 500mL of petroleum ether, stir and defatted at room temperature for 4 hours, filter, repeat twice, discard the filtrate to obtain defatted perilla seed powder; 2) Add defatted perilla seed powder to 500mL of deionized water, adjust the pH to 9.0, and extract at room temperature for 2 hours with stirring. 3) Centrifuge at 8000 rpm for 20 min and collect the supernatant; 4) Adjust the pH of the supernatant to 4.5 and let it stand for 30 minutes to allow the protein to precipitate; 5) Centrifuge at 8000 rpm for 20 min, discard the supernatant, and collect the precipitate; 6) Resuspend the precipitate in a small amount of deionized water, adjust the pH to 7.0, and freeze dry to obtain perilla seed protein.

[0049] Vero cell serum-free culture medium G includes MEM basal medium containing non-essential amino acids and additives. The additives contain 800 mg / L soy protein, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0050] Vero cell serum-free culture medium H includes MEM basal medium containing non-essential amino acids and additives. The additives contain 400 mg / L red algae protein, 400 mg / L soybean protein, 6 mg / L transferrin, 0.01 mg / L recombinant epidermal growth factor (rhEGF), 2 mg / L fibroblast growth factor (FGF), 8 mg / L recombinant insulin, 2.5 mg / L recombinant fibronectin, 1.4 mg / L poly-L-lysine, 0.03 mg / L sodium selenite, 0.01 mg / L zinc sulfate, 40 mg / L inositol, 11 mg / L ethanolamine, 0.3 mg / L polyether F-68, and 4 mg / L sodium pyruvate.

[0051] The preparation method for red algae protein is the same as that in Vero cell serum-free culture medium A.

[0052] Test Example 1 The infectivity titer (TCID) of the Vero cell culture virus obtained in step S2 of the examples and comparative examples 50 As shown in Table 1.

[0053] Table 1 Infectious titers (TCID) 50 )

[0054] Example 1 used Vero cells cultured in serum-free medium A with added red algae protein to obtain a virus strain with an infectivity titer of 7.5 log10. 10The serum albumin ( / mL) was significantly superior to that of Examples 2 (6.8), 4 (6.1), 5 (6.7), and Comparative Examples 1 (5.4), 2 (5.5), 3 (5.8), and 4 (5.6). This is because the preparation of the red algae protein involved low-temperature ammonium sulfate precipitation followed by PD-10 desalting column replacement buffer, which maximally maintained its native conformation and active domains. This allowed it to retain its ability to bind to cell membrane receptors and activate signals after entering the culture medium, enhancing cell adhesion and proliferation, while also providing antioxidant protection, thus significantly improving viral replication efficiency under serum-free conditions. Plant proteins and their hydrolysates derived from soybeans, wheat, and perilla seeds mainly provide RGD motifs, tending towards integrin-mediated adhesion, with limited activation capacity for growth signaling pathways such as IGF-1R. Bovine serum albumin primarily functions as an osmotic regulator and protein protectant, without directly activating cell proliferation signals.

[0055] Example 3 (8.5 log) 10 The viral load ( / mL) in Example 3 was significantly better than in Examples 1-2. This is because culture medium C in Example 3 contains both red algae protein and soybean protein hydrolysate. These two substances work synergistically in cell signal activation, microenvironment stabilization, adhesion promotion, and nutrient supply, maintaining higher density and activity of Vero cells, thus achieving a higher viral yield. It is noteworthy that in the establishment of the virus strain libraries in Examples 1 and 3, adaptive passage for 5 generations in serum-free Vero cell culture medium A / C achieved the desired viral titer (TCID). 50 ) > 7 log 10 / mL, reducing the time for subculture and improving culture efficiency.

[0056] Test Example 2 Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell culture medium A at a seeding density of 1 × 10⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0057] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell culture medium B at a cell seeding density of 1 × 10⁻⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0058] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell medium C, with a cell seeding density of 1 × 10⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0059] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell medium D, with a cell seeding density of 1 × 10⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0060] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell medium E at a seeding density of 1 × 10⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0061] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using serum-free Vero cell medium F, with a cell seeding density of 1 × 10⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0062] Vero cells (ATCC CCL-81) were cultured in 35 mm culture dishes using VirusPro® VP SFM serum-free medium at a seeding density of 1 × 10⁻⁶ cells / mL. 4 / cm 2 Culture conditions: 37℃, 5% CO2. After culturing for 72 hours, the cells were digested, collected, and counted. The cells were then passaged twice more using the same method.

[0063] Cell viability was assessed using the CCK-8 assay, and the results are as follows: Table 2 Vero cell viability

[0064] As can be seen from the cell viability data in Table 2, the cell viability of Example 3 (92.8%) was the highest, further confirming the advantages of the dual-protein combination in improving cell state, delaying cell death, and enhancing toxin production capacity. The cell viability of Example 1 (85.6%) and Example 2 (84.3%) was better than that of other single-protein or animal-derived protein groups, indicating that the functional proteins derived from plants / algae have better biocompatibility and promoting effect in serum-free culture.

[0065] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a Vero cell influenza virus vaccine, characterized in that, Includes the following steps: Step S1: Vero cell culture After passage of Vero cells for 1-2 generations in a medium containing 3-10 wt% serum, they were transferred to a serum-free Vero cell medium and passaged for 3-8 generations to establish a serum-free Vero cell bank. Step S2: Influenza virus inoculation and replication After passage of influenza virus strains in chicken embryos for 1-2 generations, they are adaptively passaged for 3-8 generations in serum-free Vero cell culture medium to establish a virus stock bank. In a bioreactor, serum-free Vero cells were cultured in high-density suspension with 5-15 g / L microcarriers. When the cell density reached 0.5 × 10⁻⁶ cells / L... 6 -2.0×10 6 Inoculate with virus at a concentration of cells / mL; culture conditions are: temperature 35-38℃, pH 7.0-7.5, dissolved oxygen maintained at 30%-60% saturated air, stirring speed 50-120 rpm; culture medium is serum-free Vero cell culture medium; Step S3: Virus purification and inactivation Add 2-6 wt% precipitant to the virus solution, let it stand at 4℃ for 8-24 h, and then centrifuge to collect the virus precipitate; after sterilization by membrane filtration, add inactivating agent at a mass ratio of 1:3000-1:5000, inactivate at 2-8℃ for 48-96 h, and hydrolyze at 30-40℃ for 2-5 h; The vaccine stock solution was obtained by ion exchange chromatography and ultrafiltration concentration. The serum-free Vero medium in steps S1, S2, and S3 includes a basal medium containing non-essential amino acids, at least one functional protein, and various additives. The concentration of the functional protein is 200-1000 mg / L, and the additives include, but are not limited to, transferrin 1-10 mg / L, recombinant epidermal growth factor 0.001-0.1 mg / L, fibroblast growth factor 0.1-5 mg / L, recombinant insulin 1-20 mg / L, recombinant fibronectin 0.5-5 mg / L, poly-L-lysine 0.5-5 mg / L, sodium selenite 0.001-0.1 mg / L, zinc sulfate 0.001-0.05 mg / L, inositol 10-100 mg / L, ethanolamine 5-20 mg / L, polyether F-68 0.1-1 mg / L, and sodium pyruvate 1-10 mg / L. The functional protein is at least one of red algae protein, soybean protein hydrolysate, and wheat protein hydrolysate.

2. The method for preparing the Vero cell influenza virus vaccine as described in claim 1, characterized in that, The red algae protein was prepared by the following method: red algae were cultured in a photobioreactor under acidic conditions until the end of the exponential growth phase, and the algal cells were harvested and washed; the algal cells were suspended in a salt-containing buffer solution, mechanically broken and centrifuged, and the supernatant was collected as the crude extract; the crude extract was subjected to ammonium sulfate fractionation precipitation and desalting treatment to obtain the protein solution. The protein solution was heated to inactivate it and centrifuged to remove the precipitate. The resulting supernatant was then freeze-dried to obtain the red algae protein.

3. The method for preparing the Vero cell influenza virus vaccine as described in claim 2, characterized in that, The preparation method of the red algae protein is as follows: The culture medium is BG-11 medium with a pH of 2.0-3.0, and the culture is carried out in a 5-10 L flat-plate airlift photobioreactor with a temperature of 35-40℃ and a light intensity of 100-200 μmol photons·m⁻¹. -2 ·s -1 The aeration rate was 100-200 NL / h, and the washed algal cells were harvested at the end of the exponential growth phase. The algal cells were then repeatedly broken up by bead milling in 0.05-0.20 mol / L PBS buffer (pH 6.5-7.5) containing 300-700 mmol / L salt, followed by centrifugation. The supernatant was collected. The crude extract was subjected to fractional precipitation with 40%-100% saturated ammonium sulfate and desalting to obtain a protein solution. The protein solution was inactivated by heating at 65-75℃ for 10-30 minutes, then centrifuged to remove the precipitate, and the supernatant was freeze-dried to obtain red algae protein.

4. The method for preparing the Vero cell influenza virus vaccine as described in claim 1, characterized in that, The soybean protein hydrolysate was prepared by the following method: soybean protein was dispersed in water and pretreated by heating, then cooled; alkaline protease was added under suitable pH and temperature conditions to carry out enzymatic hydrolysis; after the reaction was completed, the enzyme was inactivated by heating, cooled and centrifuged to remove insoluble matter; the supernatant was dried to obtain the soybean protein hydrolysate.

5. The method for preparing the Vero cell influenza virus vaccine as described in claim 4, characterized in that, The preparation method of the soybean protein hydrolysate is as follows: 15-25g of soybean protein is added to 80-120mL of water and heated at 80-95℃ for 5-15 minutes, then cooled to 45-55℃; 0.5-1.5g of alkaline protease is added and enzymatically hydrolyzed at 150-250rpm, pH 7.0-8.0, and 45-55℃ for 8-16 hours; the reaction solution is heated at 90-105℃ for 5-15 minutes to inactivate enzyme activity, cooled to room temperature, and centrifuged at 6000-10000rpm for 15-25 minutes, the precipitate is discarded, and the supernatant is collected; the supernatant is freeze-dried to obtain the soybean protein hydrolysate.

6. The method for preparing the Vero cell influenza virus vaccine as described in claim 1, characterized in that, The purification and inactivation of the virus in step S3 is as follows: PEG-6000 precipitant is added to a final concentration of 4wt%, shaken thoroughly, and placed at 4℃ for 12h. The virus precipitate is collected by centrifugation at 8000×g for 30min. The virus is separated by centrifugation at 10000×g for 60min. The virus is then sterilized by ultrafiltration at 0.22μm. β-propiolactone inactivator is added at a final concentration of 1:4000 by mass, and the virus is inactivated at 4℃ for 72h and hydrolyzed at 37℃ for 3h.

7. 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 are as follows: Anion exchange chromatography is performed by equilibrating the chromatography column with 0.1 mol / L pH7.0 PBS buffer containing 1.0 mol / L NaCl, adjusting the sample salt concentration to 1.0 mol / L NaCl, and collecting the flow-through peak; ultrafiltration concentration is performed using a 500 KD membrane, followed by clarification and filtration through a 0.25 μm filter membrane to obtain the vaccine stock solution.

Images