Serum-free suspension culture method of feline parvovirus

Abstract

This invention relates to the field of culture methods, specifically to a serum-free, fully suspended culture method for feline parvovirus. The invention prepares a quercetin-chicory extract, which is added to the culture medium. The raw materials for preparing the quercetin-chicory extract include the following components in parts by weight: 8-12 parts chicory extract, 2-3 parts 3-aminophenylboronic acid, and 1-2 parts quercetin. The quercetin-chicory extract can improve cell viability and enhance cell sensitivity. The invention also prepares a modified dextran, which is added to the culture medium after inoculation to enhance viral infection efficiency and increase viral yield.

Description

Technical Field

[0001] This invention relates to the field of virus culture technology, specifically to a serum-free, fully suspended culture method for feline parvovirus. Background Technology

[0002] Feline parvovirus (FPV), also known as feline panleukopenia virus or feline distemper virus, is highly contagious and can be transmitted through direct contact, indirect contact, and mother-to-child transmission. It can occur year-round, but is most prevalent in winter and spring. It mutates rapidly and has a wide distribution. Cats infected with FPV may exhibit symptoms such as high fever, vomiting, diarrhea (even bloody stools), and severe dehydration. Complications may include pancreatitis, and the virus can also damage the cerebellum, leading to motor dysfunction. Due to its high infectivity, high mortality rate, and the threat it poses to kittens, feline distemper has become a disease that cat owners and animal welfare organizations need to pay special attention to and prevent. Vaccination and good hygiene practices can effectively reduce the infection and mortality rates of feline distemper. Therefore, increasing FPV virus production has become a pressing technical challenge. Increasing FPV virus production requires a deeper understanding of the interaction between the virus and cells, balancing the nutritional needs of cell growth during the toxin-producing period with viral amplification, and improving the economics of vaccine production. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a serum-free, fully suspended culture method for feline parvovirus.

[0004] This invention is achieved through the following technical solution:

[0005] A method for serum-free complete suspension culture of feline parvovirus includes the following steps:

[0006] S1: Remove the cryovials of the frozen cat kidney cells from liquid nitrogen, thaw them in a 37°C water bath, resuspend the cells in serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in serum-free medium until the cell density is 0.8-1.2 × 10⁻⁶ cells / mL. 6 cells / mL, incubated in a 5% CO2, 37℃ incubator;

[0007] S2: After culturing for 48 h in step S1, add serum-free medium until the cell density reaches 0.8-1.2 × 10⁻⁶ cells / year. 6 The cells / mL were passaged and cultured in a 5% CO2 incubator at 37°C.

[0008] S3: After culturing for 48 h in step S2, adjust the cell density to 2.0 × 10⁶ cells / h using serum-free medium. 6 cells / mL, inoculated at an MOI of 0.1-0.01, for 10 [cells / mL].8.33 TCID 50 Feline parvovirus seed strains were inoculated at a concentration of 1 / mL, and then 1-2 mg / mL of modified dextran was added. The mixture was then cultured in a 5% CO2 incubator at 37°C.

[0009] Furthermore, the serum-free culture medium uses CatVax-S100S culture medium purchased from Shanghai Beianji Biotechnology Co., Ltd. as the basic component. The CatVax-S100S culture medium also includes the following components: D-glucose 1.5-2 g / L, quercetin-chicory extract 1-2 g / L, and amino acids 0.6-1.2 g / L.

[0010] Further, the amino acids are L-arginine hydrochloride 0.06-0.14 g / L, L-glutamine 0.15-0.22 g / L, L-cysteine ​​hydrochloride 0.12-0.28 g / L, L-asparagine 0.09-0.2 g / L, L-leucine 0.02-0.05 g / L, L-threonine 0.07-0.16 g / L, L-methionine 0.06-0.15 g / L, and L-tryptophan 0.03-0.1 g / L.

[0011] Furthermore, the raw materials for preparing the quercetin-chicory extract include the following components in parts by weight: 8-12 parts chicory extract, 2-3 parts 3-aminophenylboronic acid, and 1-2 parts quercetin.

[0012] Furthermore, the preparation method of the quercetin-chicory extract includes the following steps:

[0013] A1: Dry the chicory at 50℃, pulverize it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat at 80℃ for 2-3 hours to extract 2-3 times, combine the filtrates, concentrate under reduced pressure to 20% of the original volume, add 3 times the volume of anhydrous ethanol, let stand at 4℃ for 24 hours, centrifuge at 5000 r / min for 15-20 min, and vacuum dry the precipitate at 50℃.

[0014] A2: The vacuum-dried product obtained in step A1 was added to distilled water to prepare a solution with a concentration of 10 mg / mL. After removing protein five times using the Sevage method, the solution was evaporated and concentrated to 20% of its original volume. Three times the volume of ethanol was added, and the solution was allowed to stand at 4°C for 24 h. After centrifugation at 5000 r / min for 15-20 min, the precipitate was placed in deionized water and dialyzed through a 3000 Da dialysis bag for 48 h, with the water changed every 6 h. The dialysate was freeze-dried to obtain chicory extract.

[0015] A3: Freshly prepared 0.4 mol / L sodium periodate solution was added dropwise to 8% w / v chicory extract solution and stirred at 150-200 rpm for 4-5 h at room temperature in the dark to obtain a mixture; ethylene glycol was added to the mixture and stirred for 30 min; the mixture was dialyzed with deionized water for 2 days, with the water changed every 6 h; the dialysate was freeze-dried to obtain oxidized chicory extract;

[0016] A4: Add 3-aminophenylboronic acid to PBS solution at pH 7.4, sonicate at 300-400W for 15-20 min, add quercetin, heat in a 60℃ water bath, stir continuously at 600 rpm for 2-3 h, add the oxidized chicory extract obtained in step A3, stir at 70-80℃ and 300 rpm for 1-2 h, cool to room temperature, filter, wash the filter cake with PBS solution, freeze dry to obtain quercetin-chicory extract.

[0017] Furthermore, in step A3, the volume ratio of the sodium periodate solution to the chicory extract solution is 1:1.

[0018] Further, in step A3, the volume ratio of ethylene glycol to the mixture is 1:50.

[0019] Further, in step A4, the mass concentration of the 3-aminophenylboronic acid in the PBS solution is 10-15 mg / mL.

[0020] Furthermore, the preparation method of the modified dextran includes the following steps: mixing dextran, p-toluenesulfonic acid and amino acids, adding them to dimethyl sulfoxide (DMSO), stirring and refluxing at 120°C for 22-25 h, filtering, washing the filter cake with ethanol, and vacuum drying to obtain the modified dextran.

[0021] Furthermore, the ratio of the amount of dextran, p-toluenesulfonic acid, amino acids and DMSO is 1 g:0.1 g:2 g:100 mL.

[0022] Furthermore, the amino acid is one or more combinations of arginine, histidine, and lysine.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention discloses a serum-free, fully suspended culture method for feline parvovirus, avoiding the use of animal serum, optimizing the basal culture medium formula, adding a quercetin-chicory complex, and combining it with amino acid-modified dextran, providing an efficient and safe technical solution for large-scale vaccine production, and the preparation process is relatively simple. This invention uses serum-free culture medium CatVax-S100S as the basic component, adding D-glucose, quercetin-chicory extract, and amino acids. D-glucose provides sufficient energy for cells, maintaining normal physiological metabolic activities. Amino acids, such as L-arginine hydrochloride and L-glutamine, meet the different amino acid requirements during cell growth. This invention extracts polysaccharides from chicory and uses sodium periodate to oxidize the chicory extract. Sodium periodate can selectively oxidize the vicinal diol groups in the polysaccharide structure of the chicory extract, causing them to break down and form aldehyde groups, generating oxidized chicory extract. 3-aminophenylboronic acid is ultrasonically dispersed in PBS solution and then mixed with quercetin and heated to react. The boric acid group in the extract reacts with the ortho-dihydroxy group in the quercetin molecule to form a complex, increasing the solubility of quercetin. The aldehyde group in the oxidized chicory extract is then linked to the amino group of 3-aminophenylboronic acid in the resulting complex via a Schiff base reaction to form a stable bond, yielding a quercetin-chicory extract. Combining quercetin and chicory extract synergistically exerts antioxidant effects, improves the solubility of quercetin, enhances bioavailability, helps maintain cell stability and activity, regulates the microenvironment surrounding cells, maintains normal cellular physiological functions, promotes cell growth and reproduction, and facilitates the proliferation of feline parvovirus in cells. It can also improve cell survival rate and growth rate in serum-free culture environments, enhance cell viability, and increase viral infection efficiency. This invention involves adding modified dextran after viral inoculation. Modified dextran can bind to viral particles through electrostatic interactions, promoting viral binding to the cell membrane and increasing viral infection efficiency, thereby helping to increase viral titer and playing an important role in increasing viral yield. The modified dextran is obtained by reacting dextran with positively charged amino acids under the catalysis of p-toluenesulfonic acid. The carboxyl active groups in the amino acids react with the hydroxyl groups on the dextran molecules, thereby modifying the dextran to form a modified dextran with specific structure and function. Modified dextran has positively charged amino groups, while feline parvovirus particles have a negatively charged surface. When modified dextran is added to the culture system after inoculation, the positively charged modified dextran will combine with the negatively charged regions on the surface of the virus through electrostatic attraction, forming a protective microenvironment around the virus particles. This can stabilize the structure of the virus particles, reduce damage to the virus from external factors, and promote the assembly and release of virus particles within the cell. At the same time, modified dextran can also regulate the cellular microenvironment, enabling the cells to continuously and efficiently produce viruses, thereby significantly increasing the yield of feline parvovirus. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 To assess the biosafety of the quercetin-chicory extract and modified dextran described in this invention;

[0027] Figure 2 The fold increase refers to the proliferation ratio of the culture methods described in Examples 1-3 and Comparative Examples 1-3 of this invention;

[0028] Figure 3 The virus yield is the result of the culture method described in Examples 1-3 and Comparative Examples 1-3 of this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0030] Example 1: A method for serum-free whole suspension culture of feline parvovirus, comprising the following steps:

[0031] S1: Remove the cryovials of the frozen cat kidney cells from liquid nitrogen, thaw them in a 37°C water bath, resuspend the cells in serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in serum-free medium until the cell density reaches 1.2 × 10⁻⁶ cells / mL. 6 cells / mL, incubated in a 5% CO2, 37℃ incubator;

[0032] S2: After culturing for 48 h in step S1, serum-free medium was added until the cell density reached 1.2 × 10⁻⁶ cells / h. 6 The cells / mL were passaged and cultured in a 5% CO2 incubator at 37°C.

[0033] S3: After culturing for 48 h in step S2, adjust the cell density to 2.0 × 10⁶ cells / h using serum-free medium. 6 cells / mL, at an MOI of 0.01, inoculated at 10 8.33 TCID 50 Feline parvovirus seed strain of / mL was inoculated, and after inoculation, 2 mg / mL of modified dextran was added. The culture was carried out in an incubator with 5% CO2 and 37℃.

[0034] The serum-free culture medium used CatVax-S100S medium purchased from Shanghai Beianji Biotechnology Co., Ltd. as the basic component. The CatVax-S100S medium also includes the following components: D-glucose 2 g / L, quercetin-chicory extract 2 g / L, and amino acids 1.2 g / L.

[0035] The amino acids are: L-arginine hydrochloride 0.06 g / L, L-glutamine 0.15 g / L, L-cysteine ​​hydrochloride 0.12 g / L, L-asparagine 0.09 g / L, L-leucine 0.02 g / L, L-threonine 0.07 g / L, L-methionine 0.06 g / L, and L-tryptophan 0.03 g / L.

[0036] The raw materials for preparing quercetin-chicory extract include the following components in parts by weight: 12 parts chicory extract, 3 parts 3-aminophenylboronic acid, and 2 parts quercetin.

[0037] The preparation method of quercetin-chicory extract includes the following steps:

[0038] A1: Dry the chicory at 50℃, pulverize it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80℃ for 3 hours and extract it 3 times. Combine the filtrates, concentrate them under reduced pressure to 20% of the original volume, add 3 times the volume of anhydrous ethanol, let it stand at 4℃ for 24 hours, centrifuge at 5000 r / min for 20 minutes, and dry the precipitate under vacuum at 50℃.

[0039] A2: The vacuum-dried product obtained in step A1 was added to distilled water to prepare a solution with a concentration of 10 mg / mL. After removing protein five times using the Sevage method, the solution was evaporated and concentrated to 20% of its original volume. Three times the volume of ethanol was added, and the solution was allowed to stand at 4°C for 24 h. After centrifugation at 5000 r / min for 20 min, the precipitate was placed in deionized water and dialyzed through a 3000 Da dialysis bag for 48 h, with the water changed every 6 h. The dialysate was freeze-dried to obtain chicory extract.

[0040] A3: 150 mL of freshly prepared 0.4 mol / L sodium periodate solution was added dropwise to 150 mL of 8% w / v chicory extract solution. The mixture was stirred at 200 rpm for 5 h at room temperature in the dark to obtain a mixture. 6 mL of ethylene glycol was added to the mixture and stirred for 30 min. The mixture was dialyzed with deionized water for 2 days, with the water changed every 6 h. The dialysate was freeze-dried to obtain oxidized chicory extract.

[0041] A4: Add 3 g of 3-aminophenylboronic acid to 200 mL of PBS solution at pH 7.4, sonicate at 400 W for 20 min, add 2 g of quercetin, heat in a water bath at 60 °C, stir continuously at 600 rpm for 3 h, add the oxidized chicory extract obtained in step A3, stir at 80 °C and 300 rpm for 2 h, cool to room temperature, filter, wash the filter cake with PBS solution, freeze dry to obtain quercetin-chicory extract.

[0042] The method for preparing modified dextran includes the following steps: 1 g of dextran, 0.1 g of p-toluenesulfonic acid and 2 g of arginine are mixed and added to 100 mL of dimethyl sulfoxide (DMSO). The mixture is stirred and refluxed at 120 °C for 25 h, filtered, the filter cake is washed with ethanol and dried under vacuum to obtain modified dextran.

[0043] Example 2: A method for serum-free whole suspension culture of feline parvovirus, comprising the following steps:

[0044] S1: Remove the cryovials of the frozen cat kidney cells from liquid nitrogen, thaw them in a 37°C water bath, resuspend the cells in serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in serum-free medium until the cell density reaches 0.8 × 10⁻⁶ cells / mL. 6 cells / mL, incubated in a 5% CO2, 37℃ incubator;

[0045] S2: After culturing for 48 h in step S1, serum-free medium was added until the cell density reached 0.8 × 10⁻⁶ cells / h. 6 The cells / mL were passaged and cultured in a 5% CO2 incubator at 37°C.

[0046] S3: After culturing for 48 h in step S2, adjust the cell density to 2.0 × 10⁶ cells / h using serum-free medium. 6 cells / mL, at an MOI of 0.1, inoculated at 10 8.33 TCID 50 Feline parvovirus seed strain of / mL was inoculated, and after inoculation, 1 mg / mL of modified dextran was added. The mixture was then cultured in a 5% CO2 incubator at 37°C.

[0047] The serum-free culture medium used CatVax-S100S medium purchased from Shanghai Beianji Biotechnology Co., Ltd. as the basic component. The CatVax-S100S medium also includes the following components: D-glucose 1.5 g / L, quercetin-chicory extract 1 g / L, and amino acids 0.6 g / L.

[0048] The amino acids are: L-arginine hydrochloride 0.14 g / L, L-glutamine 0.22 g / L, L-cysteine ​​hydrochloride 0.28 g / L, L-asparagine 0.2 g / L, L-leucine 0.05 g / L, L-threonine 0.16 g / L, L-methionine 0.15 g / L, and L-tryptophan 0.1 g / L.

[0049] The raw materials for preparing quercetin-chicory extract include the following components in parts by weight: 8 parts chicory extract, 2 parts 3-aminophenylboronic acid, and 1 part quercetin.

[0050] The preparation method of quercetin-chicory extract includes the following steps:

[0051] A1: Dry the chicory at 50℃, pulverize it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80℃ for 2 h to extract it twice, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add 3 times the volume of anhydrous ethanol, let it stand at 4℃ for 24 h, centrifuge it at 5000 r / min for 15 min, and dry the precipitate under vacuum at 50℃.

[0052] A2: The vacuum-dried product obtained in step A1 was added to distilled water to prepare a solution with a concentration of 10 mg / mL. After removing protein five times using the Sevage method, the solution was evaporated and concentrated to 20% of its original volume. Three times the volume of ethanol was added, and the solution was allowed to stand at 4°C for 24 h. After centrifugation at 5000 r / min for 15 min, the precipitate was placed in deionized water and dialyzed through a 3000 Da dialysis bag for 48 h, with the water changed every 6 h. The dialysate was freeze-dried to obtain chicory extract.

[0053] A3: 100 mL of freshly prepared 0.4 mol / L sodium periodate solution was added dropwise to 100 mL of 8% w / v chicory extract solution. The mixture was stirred at 150 rpm for 4 h at room temperature in the dark to obtain a mixture. 4 mL of ethylene glycol was added to the mixture and stirred for 30 min. The mixture was dialyzed with deionized water for 2 days, with the water changed every 6 h. The dialysate was freeze-dried to obtain oxidized chicory extract.

[0054] A4: Add 2 g of 3-aminophenylboronic acid to 200 mL of PBS solution at pH 7.4, sonicate at 300 W for 15 min, add 1 g of quercetin, heat in a water bath at 60 °C, stir continuously at 600 rpm for 2 h, add the oxidized chicory extract obtained in step A3, stir at 70 °C and 300 rpm for 1 h, cool to room temperature, filter, wash the filter cake with PBS solution, freeze dry to obtain quercetin-chicory extract.

[0055] The preparation method of modified dextran includes the following steps: 1 g of dextran, 0.1 g of p-toluenesulfonic acid and 2 g of histidine are mixed and added to 100 mL of dimethyl sulfoxide (DMSO). The mixture is stirred and refluxed at 120 °C for 22 h, filtered, the filter cake is washed with ethanol and dried under vacuum to obtain modified dextran.

[0056] Example 3: A method for serum-free suspension culture of feline parvovirus, comprising the following steps:

[0057] S1: Remove the cryovials of the frozen cat kidney cells from liquid nitrogen, thaw them in a 37°C water bath, resuspend the cells in serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in serum-free medium until the cell density is 1×10⁻⁶. 6 cells / mL, incubated in a 5% CO2, 37℃ incubator;

[0058] S2: After culturing for 48 h in step S1, add serum-free medium until the cell density reaches 1×10⁻⁶. 6 The cells / mL were passaged and cultured in a 5% CO2 incubator at 37°C.

[0059] S3: After culturing for 48 h in step S2, adjust the cell density to 2.0 × 10⁶ cells / h using serum-free medium. 6 cells / mL, at an MOI of 0.05, inoculated at 10 8.33 TCID 50 Feline parvovirus seed strain of / mL was inoculated, and after inoculation, 1.5 mg / mL of modified dextran was added, and cultured in a 5% CO2 incubator at 37℃.

[0060] The serum-free culture medium used CatVax-S100S medium purchased from Shanghai Beianji Biotechnology Co., Ltd. as the basic component. The CatVax-S100S medium also includes the following components: D-glucose 1.8 g / L, quercetin-chicory extract 1.5 g / L, and amino acids 0.9 g / L.

[0061] The amino acids are: L-arginine hydrochloride 0.1 g / L, L-glutamine 0.18 g / L, L-cysteine ​​hydrochloride 0.18 g / L, L-asparagine 0.15 g / L, L-leucine 0.04 g / L, L-threonine 0.1 g / L, L-methionine 0.1 g / L, and L-tryptophan 0.05 g / L.

[0062] The raw materials for preparing quercetin-chicory extract include the following components in parts by weight: 10 parts chicory extract, 2.5 parts 3-aminophenylboronic acid, and 1.5 parts quercetin.

[0063] The preparation method of quercetin-chicory extract includes the following steps:

[0064] A1: Dry the chicory at 50℃, pulverize it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80℃ for 2.5 h to extract it 3 times, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add 3 times the volume of anhydrous ethanol, let it stand at 4℃ for 24 h, centrifuge it at 5000 r / min for 18 min, and dry the precipitate under vacuum at 50℃.

[0065] A2: The vacuum-dried product obtained in step A1 was added to distilled water to prepare a solution with a concentration of 10 mg / mL. After removing protein five times using the Sevage method, the solution was evaporated and concentrated to 20% of its original volume. Three times the volume of ethanol was added, and the solution was allowed to stand at 4°C for 24 h. After centrifugation at 5000 r / min for 18 min, the precipitate was placed in deionized water and dialyzed through a 3000 Da dialysis bag for 48 h, with the water changed every 6 h. The dialysate was freeze-dried to obtain chicory extract.

[0066] A3: 125 mL of freshly prepared 0.4 mol / L sodium periodate solution was added dropwise to 125 mL of 8% w / v chicory extract solution. The mixture was stirred at 180 rpm for 4.5 h at room temperature in the dark to obtain a mixture. 5 mL of ethylene glycol was added to the mixture and stirred for 30 min. The mixture was dialyzed with deionized water for 2 days, with the water changed every 6 h. The dialysate was freeze-dried to obtain oxidized chicory extract.

[0067] A4: Add 2.5 g of 3-aminophenylboronic acid to 200 mL of PBS solution at pH 7.4, sonicate at 350 W for 18 min, add 1.5 g of quercetin, heat in a 60°C water bath, stir continuously at 600 rpm for 2.5 h, add the oxidized chicory extract obtained in step A3, stir at 75°C and 300 rpm for 1.5 h, cool to room temperature, filter, wash the filter cake with PBS solution, freeze dry to obtain quercetin-chicory extract.

[0068] The method for preparing modified dextran includes the following steps: 1 g of dextran, 0.1 g of p-toluenesulfonic acid and 2 g of lysine are mixed and added to 100 mL of dimethyl sulfoxide (DMSO). The mixture is stirred and refluxed at 120 °C for 24 h, filtered, the filter cake is washed with ethanol and dried under vacuum to obtain modified dextran.

[0069] The only difference between Comparative Example 1 and Example 1 is that chicory extract is used instead of quercetin-chicory extract.

[0070] The only difference between Comparative Example 2 and Example 1 is that quercetin is used instead of quercetin-chicory extract.

[0071] The only difference between Comparative Example 3 and Example 1 is that dextran is used instead of modified dextran.

[0072] Experimental Example 1: Blood from SD rats was centrifuged at 2000 rpm for 15 min, the supernatant was removed, and the blood cells were washed with physiological saline. The supernatant was discarded after centrifugation, and this process was repeated several times until no obvious red color was observed in the supernatant. Lower layer red blood cells (RBCs) were obtained. The RBCs were diluted with physiological saline to a 4% (v / v) solution. Quercetin-chicory extract and modified dextran were prepared into 1 mg / mL and 0.1 mg / mL physiological saline solutions, respectively. 1 mL of each of these solutions was mixed with 400 μL of the 4% RBC physiological saline solution, incubated at 37°C for 4 h, centrifuged at 2000 rpm for 15 min, and the OD value of the supernatant at 545 nm was read using a microplate reader. The hemolysis rate was calculated. Results are as follows: Figure 1 As shown.

[0073] Figure 1 The results showed that both quercetin-chicory extract and modified dextran had low hemolysis rates. At 0.1 mg / mL, the hemolysis rates of quercetin-chicory extract and modified dextran were 0.47% and 0.31%, respectively, and at 1 mg / mL, the hemolysis rates were 0.71% and 0.51%, respectively. Regardless of the concentration of the two materials, whether it was 0.1 mg / mL or 1 mg / mL, the hemolysis rate was less than 1%, indicating that the two materials had high biocompatibility.

[0074] Experimental Example 2: Cat kidney cells were cultured according to the methods of Examples 1-3 and Comparative Examples 1-2. After passage, the cells were cultured for 48 h, and the fold increase was calculated. The results are as follows: Figure 2 As shown.

[0075] Figure 2 The results showed that the cell proliferation rates of Examples 1-3 were superior to those of Comparative Examples 1-2. The proliferation rates of Examples 1-3 were 5.56, 5.55, and 5.57, respectively, which were significantly higher than those of Comparative Examples 1-2 (5.39 and 5.21). Comparative Example 1 used chicory extract instead of quercetin-chicory extract, and Comparative Example 2 used quercetin instead of quercetin-chicory extract, but no synergistic effect was achieved. These results indicate that the culture method of the present invention can enhance the viability of cat kidney cells and increase virus yield.

[0076] Experimental Example 3: After inoculation with the virus in Step S3 of Examples 1-3 and Comparative Examples 1-3 for 72 h, the viral titer of each group was measured, and the results are as follows: Figure 3 As shown.

[0077] Figure 3 The results showed that the viral titer in Examples 1-3 was 10. 9.67TCID50 / mL, 10 9.65 TCID50 / mL and 10 9.66 TCID50 / mL was significantly higher than that of comparative examples 1-3. 9.28 TCID50 / mL, 10 8.97 TCID50 / mL and 10 8.73 With a TCID50 / mL, Comparative Example 1 used chicory extract instead of quercetin-chicory extract, without binding quercetin to chicory extract, resulting in decreased viral sensitivity and reduced viral yield in feline kidney cells; Comparative Example 2 used quercetin instead of quercetin-chicory extract, resulting in decreased sensitivity and reduced viral titer in feline kidney cells; Comparative Example 3 used dextran instead of modified dextran, as unmodified dextran is a neutral compound without cationicity, leading to decreased viral infection efficiency and reduced viral titer.

[0078] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for serum-free complete suspension culture of feline parvovirus, characterized in that, Includes the following steps: S1: Thaw the frozen cat kidney cells, resuspend the cells in serum-free medium to adjust the cell density, and then incubate them in an incubator. S2: After culturing in step S1, add serum-free medium, adjust the cell density for passage, and place in an incubator for culture; S3: After culturing in step S2, adjust the cell density with serum-free medium, inoculate with feline parvovirus seed strain, add 1-2 mg / mL of modified dextran, and culture in an incubator; The serum-free culture medium uses CatVax-S100S medium as its base component and also includes D-glucose 1.5-2 g / L, quercetin-chicory extract 1-2 g / L, L-arginine hydrochloride 0.06-0.14 g / L, L-glutamine 0.15-0.22 g / L, L-cysteine ​​hydrochloride 0.12-0.28 g / L, L-asparagine 0.09-0.2 g / L, L-leucine 0.02-0.05 g / L, L-threonine 0.07-0.16 g / L, L-methionine 0.06-0.15 g / L, and L-tryptophan 0.03-0.1 g / L. The preparation method of the quercetin-chicory extract includes the following steps: A1: Dry, pulverize and sieve the chicory, extract it with distilled water, concentrate the filtrate under reduced pressure, add anhydrous ethanol, let stand, centrifuge, and vacuum dry the precipitate. A2: The vacuum-dried product obtained in step A1 was subjected to protein removal by the Sevage method, evaporated and concentrated, ethanol was added, allowed to stand, centrifuged, dialyzed, and freeze-dried to obtain chicory extract. A3: Add sodium periodate solution dropwise to 8-12 parts by weight of the solution prepared with chicory extract, stir at room temperature in the dark to obtain a mixture, add ethylene glycol, stir, dialyze, freeze dry to obtain oxidized chicory extract; A4: Add 2-3 parts by weight of 3-aminophenylboronic acid to PBS solution, sonicate, add 1-2 parts by weight of quercetin, heat in a water bath, stir, add the oxidized chicory extract obtained in step A3, stir, cool, filter, wash the filter cake, freeze dry, and obtain quercetin-chicory extract. The method for preparing the modified dextran includes the following steps: mixing dextran, p-toluenesulfonic acid and amino acids, adding them to DMSO, stirring and refluxing, filtering, washing the filter cake, and drying to obtain the modified dextran, wherein the amino acids are one or more combinations of arginine, histidine and lysine.

2. The method for serum-free complete suspension culture of feline parvovirus according to claim 1, characterized in that, In step A3, the volume ratio of sodium periodate solution to chicory extract solution is 1:1; the volume ratio of ethylene glycol to the mixture is 1:

50.

3. The method for serum-free complete suspension culture of feline parvovirus according to claim 2, characterized in that, In step A4, the mass concentration of 3-aminophenylboronic acid in the PBS solution is 10-15 mg / mL.

4. The method for serum-free complete suspension culture of feline parvovirus according to claim 3, characterized in that, The ratio of dextran, p-toluenesulfonic acid, amino acids, and DMSO is 1 g:0.1 g:2 g:100 mL.

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