Serum-free full-suspension culture method for cat parvovirus

Abstract

The invention relates to the technical field of culture methods, in particular to a serum-free full-suspension culture method for cat parvovirus. A quercetin-cichorium intybus extract is prepared and added into a culture medium, the quercetin-cichorium intybus extract is prepared from the following raw materials in parts by weight: 8-12 parts of a cichorium intybus extract, 2-3 parts of 3-aminophenylboronic acid and 1-2 parts of quercetin, and the quercetin-cichorium intybus extract can improve the cell activity and enhance the cell sensitivity. Modified glucan is also prepared and added into a culture medium after virus inoculation, so that the virus infection efficiency is enhanced, and the virus yield is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of virus culture, and particularly relates to a serum-free full suspension culture method for feline parvovirus. Background Art

[0002] Feline Parvovirus (FPV), also known as feline panleukopenia virus and feline distemper virus, has strong transmissibility and can be transmitted through direct contact, indirect contact, and mother-to-child transmission. It can occur throughout the year, especially in winter and spring. It mutates rapidly and is widely distributed. Cats infected with feline distemper may show symptoms such as high fever, vomiting, diarrhea (even bloody stools), severe dehydration, and may also be complicated by pancreatitis. The virus can also erode and damage the cerebellum, leading to motor dysfunction. Due to its high infectivity, high mortality rate, and the threat it poses to the health of kittens, feline distemper has become a disease that needs special attention and prevention by cat-owning families and animal protection organizations. Through vaccination and good hygiene management, the infection rate and mortality rate of feline distemper can be effectively reduced. Based on this, how to increase the yield of FPV virus has become an urgent technical problem to be solved. To increase the yield of FPV virus, it is necessary to deeply understand the interaction between the virus and cells, balance the nutritional supply requirements between cell growth and virus amplification during the virus-producing period, and improve the economy of vaccine production. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention proposes a serum-free full suspension culture method for feline parvovirus.

[0004] The present invention is achieved through the following technical solutions: A serum-free full suspension culture method for feline parvovirus, comprising the following steps: S1: Take out the cryopreserved tube of feline kidney cells from liquid nitrogen, thaw it in a 37°C water bath, resuspend the cells with serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add serum-free medium to resuspend the cells to a cell density of 0.8 - 1.2×10 6 cells / mL, and place it in an incubator with 5% CO2 at 37°C for culture; S2: After culturing in step S1 for 48 h, add serum-free medium until the cell density reaches 0.8 - 1.2×10 6 cells / mL for subculture, and place it in an incubator with 5% CO2 at 37°C for culture; S3: After culturing in step S2 for 48 h, adjust the cell density to 2.0×10 6 cells / mL with serum-free medium, and inoculate 10 8.33 TCID 50For the feline parvovirus seed strain at [X] / mL, after inoculation, 1 - 2 mg / mL of modified dextran is added, and it is cultured in an incubator at 37°C with 5% CO₂.

[0005] Furthermore, the serum-free medium uses the CatVax-S100S medium purchased from Shanghai Bei'anji Biotechnology Co., Ltd. as the basic component. The CatVax-S100S 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.

[0006] Furthermore, 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.

[0007] Furthermore, the raw materials for preparing the quercetin-chicory extract include the following components in parts by weight: chicory extract 8 - 12 parts, 3-aminophenylboronic acid 2 - 3 parts, and quercetin 1 - 2 parts.

[0008] Furthermore, the preparation method of the quercetin-chicory extract includes the following steps: A1: Dry chicory at 50°C, crush it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80°C for 2 - 3 h for extraction 2 - 3 times, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add 3 volumes of absolute ethanol, let it stand at 4°C for 24 h, centrifuge at 5000 r / min for 15 - 20 min, and dry the precipitate under vacuum at 50°C; A2: Dissolve the vacuum-dried product obtained in step A1 in distilled water to prepare a solution with a concentration of 10 mg / mL. After removing proteins 5 times by the Sevage method, evaporate and concentrate it to 20% of the original volume, add 3 volumes of ethanol, let it stand at 4°C for 24 h, centrifuge at 5000 r / min for 15 - 20 min, dialyze the precipitate through a 3000 Da dialysis bag in deionized water for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain the chicory extract; A3: Slowly add the freshly prepared 0.4 mol / L sodium periodate solution drop by drop into the 8% w / v chicory extract solution, stir at 150 - 200 rpm in the dark at room temperature for 4 - 5 h to obtain a mixed solution; add ethylene glycol to the obtained mixed solution, stir for 30 min, dialyze with deionized water for 2 days, change the water every 6 h, and freeze-dry the dialysate to obtain oxidized chicory extract; A4: Add 3-aminophenylboronic acid to the PBS solution with pH 7.4, ultrasonicate at 300 - 400 W for 15 - 20 min, add quercetin, heat in a water bath at 60 °C, continuously stir at 600 rpm for 2 - 3 h, add the oxidized chicory extract obtained in step A3, stir at 70 - 80 °C and 300 rpm for 1 - 2 h, cool to room temperature, filter by suction, wash the filter cake with PBS solution, and freeze-dry to obtain quercetin-chicory extract.

[0009] Further, in step A3, the volume ratio of the sodium periodate solution to the chicory extract solution is 1:1.

[0010] Further, in step A3, the volume ratio of the ethylene glycol to the mixed solution is 1:50.

[0011] Further, in step A4, the mass concentration of the 3-aminophenylboronic acid in the PBS solution is 10 - 15 mg / mL.

[0012] Further, the preparation method of the modified dextran includes the following steps: Mix dextran, p-toluenesulfonic acid, and amino acid, add them to dimethyl sulfoxide (DMSO), stir and reflux at 120 °C for 22 - 25 h, filter by suction, wash the filter cake with ethanol, and dry in vacuo to obtain modified dextran.

[0013] Further, the dosage ratio of the dextran, p-toluenesulfonic acid, amino acid, and DMSO is 1 g:0.1 g:2 g:100 mL.

[0014] Further, the amino acid is one or a combination of more than one of arginine, histidine, and lysine.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a serum-free suspension culture method for feline parvovirus, which avoids the use of animal serum, optimizes the formula of the basal medium, adds quercetin-chicory complex, and combines with dextran modified by amino acids, providing an efficient and safe technical solution for large-scale vaccine production, and the preparation process is relatively simple. The present invention selects the serum-free medium CatVax-S100S as the basic component, and adds D-glucose, quercetin-chicory extract and amino acids. D-glucose provides an abundant energy source for cells and maintains the normal physiological metabolic activities of cells. Amino acids such as L-arginine hydrochloride and L-glutamine meet the requirements of cells for different amino acids during the growth process. In the present invention, polysaccharides are extracted from chicory, and the chicory extract is oxidized by sodium periodate. Sodium periodate can selectively oxidize the vicinal diol groups in the polysaccharide structure of the chicory extract, causing it to break and form aldehyde groups, generating oxidized chicory extract. After 3-aminophenylboronic acid is ultrasonically dispersed in PBS solution, it is mixed and heated with quercetin. The boronic acid group in 3-aminophenylboronic acid will react with the vicinal dihydroxy groups in the quercetin molecule to form a complex, improving the solubility of quercetin. The aldehyde groups in the oxidized chicory extract and the amino groups of 3-aminophenylboronic acid in the obtained complex are stably connected through Schiff base reaction to obtain quercetin-chicory extract. The quercetin and chicory extract are compounded to synergistically exert an antioxidant effect, enhance the solubility of quercetin, increase the bioavailability, help maintain the stability and activity of cells, regulate the microenvironment around cells, maintain the normal physiological functions of cells, promote the growth and reproduction of cells, are beneficial to the proliferation of feline parvovirus in cells, can improve the survival rate and growth rate of cells in a serum-free culture environment, enhance cell viability, and improve the virus infection efficiency. The present invention adds modified dextran after virus inoculation. The modified dextran can bind to virus particles through electrostatic interaction, promote the binding of the virus to the cell membrane, increase the virus infection efficiency, thereby helping to increase the virus titer, and plays an important role in increasing the virus yield. The modified dextran is based on dextran and is obtained by reacting 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 molecule, thereby modifying the dextran to form a modified dextran with specific structure and function. The modified dextran has positively charged amino groups, while the surface of feline parvovirus particles is negatively charged. When the modified dextran is added to the culture system after virus inoculation, the positively charged modified dextran will bind to the negatively charged region on the virus surface through electrostatic attraction, forming a protective microenvironment around the virus particles, which can stabilize the structure of the virus particles, reduce the damage to the virus by external factors, promote the assembly and release of virus particles in cells, and at the same time, the modified dextran can also regulate the cell microenvironment, enabling the cells to continuously and efficiently produce the virus, thereby significantly increasing the yield of feline parvovirus. Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the attached drawings required in the description of the embodiments or the prior art. Obviously, the attached drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other attached drawings can also be obtained based on these drawings.

[0017] Figure 1 For the biosafety of the quercetin-chicory extract and modified dextran described in the present invention; Figure 2 For the proliferation multiple of the culture methods described in Examples 1-3 and Comparative Examples 1-3 of the present invention; Figure 3 For the virus yield of the culture methods described in Examples 1-3 and Comparative Examples 1-3 of the present invention. Detailed implementation manners

[0018] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with specific embodiments. However, the present invention is not limited to the following embodiments. It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0019] Example 1: A serum-free full suspension culture method for feline parvovirus, comprising the following steps: S1: Take out the cryopreserved tube of feline kidney cells from liquid nitrogen, thaw it in a 37°C water bath, resuspend the cells with serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add serum-free medium to resuspend the cells to a cell density of 1.2×10 6 cells / mL, and place it in a 5% CO2, 37°C incubator for culture; S2: After culturing in step S1 for 48 h, add serum-free medium to subculture at a cell density of 1.2×10 6 cells / mL, and place it in a 5% CO2, 37°C incubator for culture; S3: After culturing in step S2 for 48 h, adjust the cell density to 2.0×10 6 cells / mL, inoculate the feline parvovirus seed strain at an MOI of 0.01 and 10 8.33 TCID 50 / mL, and after virus inoculation, add 2 mg / mL of modified dextran and culture in a 5% CO2, 37°C incubator.

[0020] The serum-free medium uses the CatVax-S100S medium purchased from Shanghai Bei'anji 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.

[0021] 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.

[0022] The preparation raw materials of the quercetin-chicory extract include the following components in parts by weight: chicory extract 12 parts, 3-aminophenylboronic acid 3 parts, and quercetin 2 parts.

[0023] The preparation method of the quercetin-chicory extract includes the following steps: A1: Dry chicory at 50 °C, crush it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80 °C for 3 h and extract 3 times, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add 3 times the volume of absolute ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 20 min, and vacuum-dry the precipitate at 50 °C; A2: Dissolve the vacuum-dried product obtained in step A1 in distilled water to prepare a solution with a concentration of 10 mg / mL. After removing proteins 5 times by the Sevage method, evaporate and concentrate it to 20% of the original volume, add 3 times the volume of ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 20 min, dialyze the precipitate against deionized water through a 3000 Da dialysis bag for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain the chicory extract; A3: Gradually add 150 mL of freshly prepared 0.4 mol / L sodium periodate solution dropwise to 150 mL of 8% w / v chicory extract solution, stir it at 200 rpm in the dark at room temperature for 5 h to obtain a mixed solution; add 6 mL of ethylene glycol to the obtained mixed solution, stir for 30 min, dialyze it against deionized water for 2 days, change the water every 6 h, and freeze-dry the dialysate to obtain the oxidized chicory extract; A4: Add 3 g of 3-aminophenylboronic acid to 200 mL of PBS solution with pH 7.4, sonicate for 20 min at 400 W, add 2 g of quercetin, heat in a water bath at 60 °C, continuously stir 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 by suction, wash the filter cake with PBS solution, and freeze-dry to obtain quercetin-chicory extract.

[0024] The preparation method of modified dextran includes the following steps: Mix 1 g of dextran, 0.1 g of p-toluenesulfonic acid, and 2 g of arginine, add them to 100 mL of dimethyl sulfoxide (DMSO), stir and reflux at 120 °C for 25 h, filter by suction, wash the filter cake with ethanol, and dry in vacuum to obtain modified dextran.

[0025] Example 2: A serum-free suspension culture method for feline panleukopenia virus, including the following steps: S1: Take out the cryopreserved tube of feline kidney cells from liquid nitrogen, thaw it in a 37 °C water bath, resuspend the cells with serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add serum-free medium to resuspend the cells to a cell density of 0.8×10 6 cells / mL, and place them in an incubator with 5% CO2 at 37 °C for culture; S2: After culturing in step S1 for 48 h, add serum-free medium until the cell density reaches 0.8×10 6 cells / mL for passage, and place them in an incubator with 5% CO2 at 37 °C for culture; S3: After culturing in step S2 for 48 h, adjust the cell density to 2.0×10 6 cells / mL with serum-free medium, inoculate 10 8.33 TCID 50 / mL of feline panleukopenia virus seed strain. After inoculation with the virus, add 1 mg / mL of modified dextran, and culture in an incubator with 5% CO2 at 37 °C.

[0026] The serum-free medium uses CatVax-S100S medium purchased from Shanghai Bei'anji Biotechnology Co., Ltd. as the basic component, and the CatVax-S100S medium also includes the following components: 1.5 g / L of D-glucose, 1 g / L of quercetin-chicory extract, and 0.6 g / L of amino acids.

[0027] The amino acids are 0.14 g / L of L-arginine hydrochloride, 0.22 g / L of L-glutamine, 0.28 g / L of L-cysteine hydrochloride, 0.2 g / L of L-asparagine, 0.05 g / L of L-leucine, 0.16 g / L of L-threonine, 0.15 g / L of L-methionine, and 0.1 g / L of L-tryptophan.

[0028] The raw materials for the preparation of quercetin-chicory extract include the following components in parts by weight: 8 parts of chicory extract, 2 parts of 3-aminophenylboronic acid, and 1 part of quercetin.

[0029] The preparation method of quercetin-chicory extract includes the following steps: A1: Dry chicory at 50 °C, crush it through a 100-mesh sieve, mix it with distilled water at a mass ratio of 1:30, heat it at 80 °C for 2 h and extract twice, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add 3 volumes of absolute ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 15 min, and vacuum-dry the precipitate at 50 °C; A2: Dissolve the vacuum-dried product obtained in step A1 in distilled water to prepare a solution with a concentration of 10 mg / mL. After removing proteins 5 times by the Sevage method, evaporate and concentrate it to 20% of the original volume, add 3 volumes of ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 15 min, dialyze the precipitate in deionized water through a 3000 Da dialysis bag for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain chicory extract; A3: Gradually add 100 mL of freshly prepared 0.4 mol / L sodium periodate solution dropwise to 100 mL of 8% w / v chicory extract solution, stir it at 150 rpm in the dark at room temperature for 4 h to obtain a mixed solution; add 4 mL of ethylene glycol to the obtained mixed solution, stir for 30 min, dialyze it with deionized water for 2 days, change the water every 6 h, and freeze-dry the dialysate to obtain oxidized chicory extract; A4: Add 2 g of 3-aminophenylboronic acid to 200 mL of PBS solution with pH 7.4, ultrasonicate it at 300 W for 15 min, add 1 g of quercetin, heat it in a water bath at 60 °C, continuously stir 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 it to room temperature, filter it by suction, wash the filter cake with PBS solution, and freeze-dry it to obtain quercetin-chicory extract.

[0030] The preparation method of modified dextran includes the following steps: Mix 1 g of dextran, 0.1 g of p-toluenesulfonic acid, and 2 g of histidine, add them to 100 mL of dimethyl sulfoxide (DMSO), stir and reflux at 120 °C for 22 h, filter it by suction, wash the filter cake with ethanol, and vacuum-dry it to obtain modified dextran.

[0031] Example 3: A serum-free suspension culture method for feline parvovirus, comprising the following steps: S1: Take out the cryopreserved tube of feline kidney cells from liquid nitrogen, thaw it in a 37°C water bath, resuspend the cells with serum-free medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add serum-free medium to resuspend the cells to a cell density of 1×10 6 cells / mL, and place it in an incubator with 5% CO2 at 37°C for culture; S2: After culturing in step S1 for 48 h, add serum-free medium until the cell density reaches 1×10 6 cells / mL for passage, and place it in an incubator with 5% CO2 at 37°C for culture; S3: After culturing in step S2 for 48 h, adjust the cell density to 2.0×10 6 cells / mL, inoculate 10 8.33 TCID 50 / mL of feline parvovirus seed strain. After inoculation, add 1.5 mg / mL of modified dextran and culture it in an incubator with 5% CO2 at 37°C.

[0032] The serum-free medium uses CatVax-S100S medium purchased from Shanghai Biyuanji Biotechnology Co., Ltd. as the basic component, and the CatVax-S100S medium also includes the following components: D-glucose 1.8 g / L, quercetin-chicory extract 1.5 g / L, amino acids 0.9 g / L.

[0033] 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, L-tryptophan 0.05 g / L.

[0034] The preparation raw materials of the quercetin-chicory extract include the following components in parts by weight: chicory extract 10 parts, 3-aminophenylboronic acid 2.5 parts, quercetin 1.5 parts.

[0035] The preparation method of the quercetin-chicory extract includes the following steps: A1: Dry chicory at 50 °C, crush it and sieve through a 100-mesh sieve. Mix it with distilled water at a mass ratio of 1:30, heat it at 80 °C for 2.5 h for extraction three times, combine the filtrates, concentrate it under reduced pressure to 20% of the original volume, add three volumes of absolute ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 18 min, and dry the precipitate under vacuum at 50 °C. A2: Dissolve the vacuum-dried product obtained in step A1 in distilled water to prepare a solution with a concentration of 10 mg / mL. Remove proteins five times by the Sevage method, then evaporate and concentrate to 20% of the original volume, add three volumes of ethanol, let it stand at 4 °C for 24 h, centrifuge at 5000 r / min for 18 min. Dialyze the precipitate against deionized water through a 3000 Da dialysis bag for 48 h, changing the water every 6 h. Freeze-dry the dialysate to obtain chicory extract. A3: Gradually add 125 mL of freshly prepared 0.4 mol / L sodium periodate solution dropwise to 125 mL of 8% w / v chicory extract solution, stir at 180 rpm in the dark at room temperature for 4.5 h to obtain a mixed solution. Add 5 mL of ethylene glycol to the obtained mixed solution, stir for 30 min, dialyze against deionized water for 2 days, changing the water every 6 h. Freeze-dry the dialysate to obtain oxidized chicory extract. A4: Add 2.5 g of 3-aminophenylboronic acid to 200 mL of PBS solution with pH 7.4, sonicate at 350 W for 18 min, add 1.5 g of quercetin, heat in a water bath at 60 °C, continuously stir 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 by suction, wash the filter cake with PBS solution, and freeze-dry to obtain quercetin-chicory extract.

[0036] The preparation method of modified dextran includes the following steps: Mix 1 g of dextran, 0.1 g of p-toluenesulfonic acid and 2 g of lysine, add them to 100 mL of dimethyl sulfoxide (DMSO), stir and reflux at 120 °C for 24 h, filter by suction, wash the filter cake with ethanol, and dry under vacuum to obtain modified dextran.

[0037] The difference between Comparative Example 1 and Example 1 is only that chicory extract is used instead of quercetin-chicory extract.

[0038] The difference between Comparative Example 2 and Example 1 is only that quercetin is used instead of quercetin-chicory extract.

[0039] The difference between Comparative Example 3 and Example 1 is only that dextran is used instead of modified dextran.

[0040] Experimental Example 1: SD rat blood was taken and centrifuged at 2000 rpm for 15 min in a centrifuge. The supernatant was removed, and the obtained blood cells were washed with physiological saline. After centrifugation, the supernatant was discarded and repeated multiple times until no obvious red color was observed in the supernatant. The lower-layer red blood cells (RBCs) were obtained and diluted with physiological saline to a 4% (v / v) solution. Quercetin-chicory extract and modified dextran were made into physiological saline solutions at 1 mg / mL and 0.1 mg / mL. 1 mL of each of the above solutions was taken and mixed with 400 μL of 4% RBCs 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 with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the hemolysis rate. The results are as Figure 1 shown.

[0041] 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. At 1 mg / mL, the hemolysis rates of quercetin-chicory extract and modified dextran were 0.71% and 0.51%, respectively. Whether the concentration of the two materials was 0.1 mg / mL or 1 mg / mL, their hemolysis rates were both lower than 1%, indicating that the two materials had high biosafety.

[0042] Experimental Example 2: According to the methods of Examples 1-3 and Comparative Examples 1-2, feline kidney cells were cultured, and after passage, cultured for 48 h, and the proliferation multiple was calculated. The results are as Figure 2 shown.

[0043] Figure 2 The results showed that the cell proliferation multiples of Examples 1-3 were better than those of Comparative Examples 1-2. The proliferation multiples of the groups of Examples 1-3 were 5.56, 5.55, and 5.57, respectively, which were significantly higher than 5.39 and 5.21 of Comparative Examples 1-2. In Comparative Example 1, chicory extract was used to replace quercetin-chicory extract, and in Comparative Example 2, quercetin was used to replace quercetin-chicory extract, and the synergistic effect was not achieved. The above results indicated that the culture method of the present invention could enhance the viability of feline kidney cells and increase the virus yield.

[0044] Experimental Example 3: After culturing for 72 h after virus inoculation in step S3 of Examples 1-3 and Comparative Examples 1-3, the virus titers of each group were measured. The results are as Figure 3 shown.

[0045] Figure 3 The results showed that the virus titers of Examples 1-3 were 10 9.67 TCID50 / mL, 10 9.65 TCID50 / mL, and 10 9.66TCID50 / mL, which is significantly higher than 10 of Comparative Examples 1-3 9.28 TCID50 / mL, 10 8.97 TCID50 / mL and 10 8.73 TCID50 / mL. In Comparative Example 1, chicory extract was used to replace quercetin-chicory extract, and quercetin was not combined with chicory extract, resulting in a decrease in the virus sensitivity of feline kidney cells and a reduction in virus production; in Comparative Example 2, quercetin was used to replace quercetin-chicory extract, leading to a decrease in the sensitivity of feline kidney cells and a reduction in virus titer; in Comparative Example 3, dextran was used to replace modified dextran. Unmodified dextran is a neutral compound without cations, resulting in a decrease in virus infection efficiency and a reduction in virus titer.

[0046] Those of ordinary skill 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 present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A feline parvovirus serum-free full suspension culture method, characterized in that: The following steps are involved: S1: Thaw the frozen cat kidney cells, resuspend the cells in serum-free medium to adjust the cell density, and culture them in an incubator; S2: After culturing in step S1, serum-free culture medium is added, cell density is adjusted for subculturing, and the cells are cultured in an incubator; S3: After culturing in step S2, the cell density is adjusted with serum-free medium, the feline parvovirus seed strain is inoculated, 1-2 mg / mL of modified dextran is added, and the cells are cultured in an incubator; The serum-free culture medium uses CatVax-S100S culture medium as a basic component, and also includes 1.5-2 g / L of D-glucose, 1-2 g / L of quercetin-chicory extract, and 0.6-1.2 g / L of amino acids; The preparation method of the quercetin-chicory extract comprises the following steps: A1: Dry the chicory, grind it and sieve it, heat and extract it with distilled water, concentrate the filtrate under reduced pressure, add anhydrous ethanol, let it stand, centrifuge it, and vacuum dry the precipitate; A2: removing protein from the vacuum dried product obtained in step A1 by Sevage method, evaporating and concentrating, adding ethanol, standing, centrifuging, dialyzing, and freeze-drying to obtain chicory extract; A3: adding sodium periodate solution dropwise to a solution prepared with 8-12 parts by weight of chicory extract, stirring at room temperature in the dark to obtain a mixed solution, adding ethylene glycol, stirring, dialyzing, and freeze-drying to obtain an oxidized chicory extract; A4: adding 2-3 parts by weight of 3-aminophenylboronic acid to a PBS solution, ultrasonicating, adding 1-2 parts by weight of quercetin, heating in a water bath, stirring, adding the oxidized chicory extract obtained in step A3, stirring, cooling, filtering, washing the filter cake, and freeze-drying to obtain a quercetin-chicory extract; The preparation method of the modified dextran comprises the following steps: mixing dextran, p-toluenesulfonic acid and amino acid, adding into DMSO, stirring and refluxing, filtering, washing the filter cake and drying to obtain the modified dextran.

2. The feline parvovirus serum-free full suspension culture method according to claim 1, characterized in that: 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.

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

50.

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

5. The feline parvovirus serum-free full suspension culture method according to claim 4, characterized in that: The dosage ratio of the dextran, p-toluenesulfonic acid, amino acid and DMSO is 1 g:0.1 g:2 g:100 mL; when preparing the modified dextran, the amino acid is one or more combinations of arginine, histidine and lysine.

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