Method for full-suspension serum-free efficient production of canine distemper virus by using MDCK cell line and application

Abstract

The invention discloses a method for full-suspension serum-free efficient production of canine distemper virus by adopting an MDCK cell line and application, and belongs to the technical field of virus amplification. In order to overcome the technical defects existing in production of the canine distemper virus by using adherent growth chick embryo fibroblasts or Vero cells (E6), the invention provides a method for producing the canine distemper virus by inoculating the canine distemper virus into an MDCK (Madin Darby Canine Kidney) cell serum-free full-suspension cell line and using a bioreactor, and the method is subjected to process optimization, so that the canine distemper virus can be produced. According to the optimized method, the production cost is remarkably reduced, the production period is short, the virus content is high, the oxygen transfer coefficient in the culture process is increased, the production time consumption is reduced, and the virus production efficiency and the vaccine yield and quality are improved. The method provided by the invention can meet the large-scale efficient production of the canine distemper virus, and has a better application prospect in the production of canine distemper virus vaccines.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus amplification, and particularly relates to a method for efficiently producing canine distemper virus by using a MDCK cell line in a whole-suspension serum-free manner and application thereof. BACKGROUND

[0002] Canine distemper (CD) is an acute and highly contagious disease caused by canine distemper virus (CDV), mainly infecting canids, mustelids and some felines, and the mortality rate of puppies can be as high as 80% or more. The disease is prevalent worldwide, and the clinical manifestations are typical characteristics of biphasic fever, anorexia, conjunctivitis, gastroenteritis and neurological symptoms, which seriously threatens the healthy development of the dog industry and fur animal breeding industry in China and even the world. At present, vaccination is still the main means of preventing canine distemper, and the key to vaccine production is to efficiently and stably culture viruses, so it is of great significance to optimize the production process of canine distemper virus.

[0003] At present, the cultivation of canine distemper virus mainly relies on adherent monolayer cells, and the commonly used cell substrates include chicken embryo fibroblasts (CEF) and Vero cells (such as E6 cell line). However, there are certain defects whether CEF or Vero E6 cells are selected as cell substrates. For CEF, although it has been widely used for the production of canine distemper virus, its preparation process is complex and tedious, and it needs to extract primary cells from specific chicken embryos, which has large batch-to-batch differences and limited cell passage ability, making it difficult to achieve large-scale continuous production. In addition, CEF has low sensitivity to canine distemper virus, and the virus titer is usually difficult to reach the ideal level, resulting in low vaccine production efficiency and high cost. Although the adherent culture system represented by Vero E6 cells can support the proliferation of canine distemper virus, there are still the following problems: 5%-10% fetal bovine serum (FBS) needs to be added for traditional culture to maintain cell growth, but the cost of serum is high, and the composition has large batch-to-batch differences, which affects the stability and consistency of virus production; serum may carry viruses (such as bovine viral diarrhea virus BVDV), mycoplasma and even prions, increasing the safety hazards of biological products; the heterologous proteins in serum will be mixed into the virus culture system, increasing the complexity of the subsequent purification process and affecting the purity and quality of the vaccine.

[0004] Considering the defects of adherent culture of canine distemper virus, more efficient, more controllable and low-cost serum-free suspension culture process has been paid more attention. However, the serum-free suspension culture technology of canine distemper virus is not mature, and it is urgent to break through the technical bottleneck. For example, most canine distemper virus sensitive cells (such as Vero cells) need to be domesticated to adapt to serum-free suspension growth, which makes the effective domestication method a key to canine distemper virus production; the effect of large-scale suspension culture is affected by many parameters, and the process conditions need to be systematically optimized to achieve good culture effect. Therefore, it is urgent to explore a full-suspension serum-free efficient production method suitable for canine distemper virus. SUMMARY

[0005] In order to solve the technical defects in the production of canine distemper virus by using adherent chicken embryo fibroblasts or Vero cells, the present application provides a method for producing canine distemper virus by inoculating canine distemper virus into MDCK cell serum-free suspension cell line and using a bioreactor, and the method is optimized. The optimized method can meet the large-scale and efficient production of canine distemper virus, and has good application prospect in the production of canine distemper virus vaccine.

[0006] In order to solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a method for efficiently producing canine distemper virus by using MDCK cell line in full-suspension and serum-free manner, which comprises inoculating canine distemper virus into MDCK cell serum-free suspension cell line and culturing by using a bioreactor.

[0007] In an embodiment of the present application, the preparation method of the MDCK cell serum-free suspension cell line comprises the following steps: (1) inoculating MDCK adherent cells into culture medium containing 8% serum for culture, and then inoculating the obtained cells into culture medium with gradually reduced serum content for culture, to obtain MDCK suspension cells cultured in low serum; (2) selecting low serum-adapted MDCK adherent cells with full monolayer, digesting and inoculating into serum-free full-suspension culture medium, and then normally diluting and passing every 72 h, so that the viable cell density doubles and the viability is higher than 90% when continuously maintained for 48 h, to obtain the MDCK cell serum-free suspension cell line.

[0008] In one embodiment of the present application, step (1) is specifically as follows: first stage culture: MDCK adherent cells are inoculated in DMEM culture medium containing 8% serum at a ratio of 1:4, and subcultured for 3-5 generations; second stage culture: the serum concentration in the culture medium is reduced to 4.5%-5.5%, and the cells obtained in the first stage are inoculated and subcultured for more than 5 generations; third stage culture: the serum concentration in the culture medium is reduced to 1.5%-2.5%, and the cells obtained in the second stage are inoculated and subcultured for more than 5 generations.

[0009] In one embodiment of the present application, the serum concentration in the culture medium used in the second stage culture is 5%, and the serum concentration in the culture medium used in the third stage culture is 2%.

[0010] In one embodiment of the present application, step (2) is specifically as follows: ①: MDCK adherent cells that have adapted to low serum culture and have grown into a full monolayer are digested, and the digested cells are inoculated in serum-free MDCK cell whole-suspension culture medium for culture, so as to control the cell density to be 1.0×10 6 ~2.0×10 6 cells / mL; ②: step ① is repeated until the viable cell density doubles and the viability is higher than 90% when normally diluted and subcultured every 72 h for 48 h, i.e., a serum-free suspension cell line of MDCK cells is obtained.

[0011] In one embodiment of the present application, the control of the cell density is performed when the digested cells are inoculated in serum-free MDCK cell whole-suspension culture medium for culture for 48 h.

[0012] In one embodiment of the present application, the digestion is performed by discarding the supernatant of MDCK adherent cells that have adapted to low serum culture and have grown into a full monolayer, washing 2-4 times with 2-5 mL PBS, discarding the PBS, adding 0.25% trypsin, discarding the trypsin after action at 37°C for 2-5 min, and adding serum-free MDCK cell whole-suspension culture medium to terminate the digestion, and then blowing the cells evenly for use.

[0013] In one embodiment of the present application, the culture comprises the following steps: S1, inoculating the serum-free suspension cell line of MDCK cells in a 500 mL shake flask at an initial cell density of 1.0×10 6 ~2.0×10 6 cells / mL, with a working volume of 150 mL, and placing the shake flask in a shaking incubator at 37°C, 5% CO2, and 120 rpm for culture for 48-72 h; S2. Using a bipartitioning method for inoculation, the shake-flask cells obtained in S1 were divided into two halves, and each half was inoculated with fresh culture medium containing canine distemper virus at an inoculation rate of 5‰~1%. Canine distemper virus was produced using an Eppendon DASGIP 4-in-1 parallel bioreactor and a Sartorius stedim BIOSTAT C plus bioreactor under the conditions of pH 7.0~7.2, 33℃, 5% CO2, 80~140 rpm, and dissolved oxygen of 40%~50%. The virus solution was harvested when the cell viability dropped to 40%~50% or after culturing for 96 h, thus obtaining canine distemper virus.

[0014] In one embodiment of the present invention, the steps for producing canine distemper virus using the Eppendon DASGIP 4-in-1 parallel bioreactor are as follows: ① The cells obtained from S1 were seeded in a 1.5 L Eppendon DASGIP 4-in-1 parallel bioreactor with a working volume of 1.2 L and an initial seeding density of 1.0 × 10⁻⁶ cells / year. 6 ~2.0×10 6 Cells / mL were cultured for 72 h at pH 7.0–7.2, 37℃, 5% CO2, 140 rpm, and dissolved oxygen of 40%–50%, until a cell density of 6 × 10⁻⁶ was achieved. 6 ~8×10 6 The cell count / mL is above 90%, and the cells are viable for inoculation. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰~1% of the total culture medium volume in step ①. Control the reaction conditions of the Eppendon DASGIP 4-link parallel bioreactor as follows: pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 140 rpm, and dissolved oxygen of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50% or after culturing for 96 hours to obtain canine distemper virus.

[0015] In one embodiment of the present invention, the steps for producing canine distemper virus using the Sartorius stedim BIOSTAT C plus bioreactor are as follows: ① The cells obtained from S1 were seeded in a 50 L Sartorius stedim BIOSTAT C plus bioreactor with a working volume of 30 L and an initial seeding density of 1.0 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 Cells / mL were cultured for 72 h at pH 7.0–7.2, 37℃, 5% CO2, 80 rpm, and dissolved oxygen of 40%–50%, until a cell density of 6 × 10⁻⁶ was achieved. 6 ~8×106 The cell count / mL is above 90%, and the cells are viable for inoculation. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰~1% of the total culture medium volume in step ①. Control the reaction conditions of the Sartorius stedim BIOSTAT C plus bioreactor as follows: pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 80 rpm, and dissolved oxygen content of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50% or after culturing for 96 h, and the canine distemper virus is obtained.

[0016] A second objective of this invention is to provide the application of the above-described method in the preparation of canine distemper virus vaccines.

[0017] The beneficial effects of this invention are: This invention provides a method for producing canine distemper virus by inoculating a serum-free suspension MDCK cell line into a bioreactor. The method has been optimized, resulting in significantly reduced production costs, a shorter production cycle, higher virus content, improved oxygen transfer coefficient during culture, reduced production time, and increased virus production efficiency, vaccine yield, and quality. The method provided by this invention can meet the requirements for large-scale, high-efficiency production of canine distemper virus and has promising application prospects in the production of canine distemper virus vaccines. Attached Figure Description

[0018] Figure 1 Image of serum-free MDCK suspension cells; Figure 2 Growth curve of MDCK suspension cells cultured in low serum; Figure 3 This is a comparison image of cytopathic effect (CPE) in MDCK suspension cells cultured in the Sartorius stedim BIOSTAT C plus bioreactor for 96 h after viral inoculation, and a control group of normal, uninoculated MDCK suspension cells. Figure 3 A in the image represents a normal, uninfected MDCK suspension cell image; Figure 3 B in the figure represents the CPE produced by MDCK suspension cells 96 hours after infection; Figure 4 The figure shows the statistical results of the determination of viral load of canine distemper virus in suspension culture using E6 cell monolayer and MDCK cell monolayer, respectively. Figure 5 The graph shows the detection results of virus content corresponding to different doses of the virus. Figure 6 The graph shows the detection results of virus content corresponding to different cell densities. Figure 7 This is a graph showing the detection results of virus content corresponding to different virus collection times. Detailed Implementation

[0019] 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 and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0020] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. The materials, reagents, culture media, and instruments used are all conventional materials, reagents, culture media, and instruments in the art, and can be obtained commercially by those skilled in the art. Unless otherwise specified, the molecular biology experimental procedures involved in this invention are conventional experimental procedures in the art or can be performed according to the product instructions of the corresponding reagents.

[0021] The canine distemper virus used in this invention is the CDV-YBR strain, preserved by Vico Biotechnology Co., Ltd., Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The method provided by this invention is applicable to all canine distemper viruses.

[0022] The MDCK adherent cells used in this invention were preserved by Vico Biotechnology Co., Ltd., Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The method provided by this invention is applicable to all MDCK adherent cells.

[0023] The MDCK S3 cell suspension culture medium used in this invention was purchased from Zhejiang Yishengke Biotechnology Co., Ltd., catalog number Y1074i13.

[0024] Example 1: Preparation of serum-free suspension cell line MDCK (1) After resuscitation, the MDCK adherent cells that have grown into a monolayer were passaged in DMEM medium containing 8% FBS at a ratio of 1:4. After passage 4 times, the FBS concentration in the culture medium was reduced to 5%, and passage was continued for 5 consecutive passages. Then the FBS concentration in the culture medium was reduced to 2%, and passage was continued for more than 5 consecutive passages to obtain MDCK cell lines adapted to low serum culture.

[0025] (2) Select MDCK adherent cells that have grown into a monolayer and are adapted to low serum culture and passage them in DMEM medium containing 2% FBS at a ratio of 1:4. After culturing for 3 days, the cells grow into a monolayer. Discard the supernatant of the cells that have grown into a monolayer, add 3 mL PBS to wash twice, discard the PBS, add 0.25% trypsin, incubate at 37℃ for 3 min, discard the trypsin, add serum-free MDCK cell suspension medium to stop digestion, blow the cells evenly for later use, and seed the digested cells into MDCK cell suspension medium.

[0026] (3) Sampling and counting should be performed every 24 hours. When the counted viable cell density is greater than 1.0 × 10⁻⁶, the count should be completed. 6 If the viable cell density is 3.0 × 10⁶ cells / mL, continue culturing for 48 h. If the viable cell density is higher than 3.0 × 10⁶ cells / mL after 48 h, continue culturing. 6 If the cells / mL is high, then dilute the cells to 1.0 × 10⁶ cells / mL using serum-free, fully suspended medium. 6 Continue culturing; if the viable cell density is less than 1.0 × 10⁶ cells / mL after 48 h... 6 If the cell density reaches 1.0 × 10⁶ cells / mL, after settling, discard part of the supernatant and adjust the cell density to 1.0 × 10⁶ cells / mL using serum-free full suspension culture medium. 6 The cells / mL were cultured continuously; when samples were taken for counting after 24 h, the density of viable cells was less than 1.0 × 10⁻⁶. 6 At a cell density of 1.0 × 10⁶ cells / mL, after settling for 2 hours, part of the supernatant was discarded, and the cell density was adjusted to 1.0 × 10⁶ cells / mL using serum-free full suspension culture medium. 6 Continue culturing for 48 hours. If the viable cell density is higher than 2.0 × 10⁶ cells / mL after 48 hours... 6 Cells / mL, diluted to 1.0 × 10⁶ cells / mL using serum-free complete suspension medium. 6 Continue culturing; if the cell density is below 1.0 × 10⁶ cells / mL after 48 hours... 6 Cells / mL, after settling, part of the supernatant was discarded, and the cell count was adjusted to 1.0 × 10⁶ cells / mL using serum-free full suspension medium. 6 cells / mL, continue culturing.

[0027] (4) Repeat step (3) until normal dilution and passage are performed every 72 h. After 5 consecutive passages, the viable cell density doubles (i.e., 2.0 × 10⁻⁶) after 48 h. 6 A serum-free suspension cell line of MDCK cells was obtained by achieving a cell count (cells / mL) and a viability greater than 90%. With increasing passage numbers, the cells became increasingly adapted to the serum-free medium. An image of serum-free MDCK suspension cells is shown below. Figure 1 As shown in the figure, the growth curve of MDCK suspension cells cultured in low serum is as follows: Figure 2 As shown.

[0028] Example 2: Preparation of serum-free suspension cell line MDCK The difference between this embodiment and Embodiment 1 lies only in step (3), as follows: Sampling and counting are performed every 24 hours. When the counted viable cell density is greater than the inoculation amount of digested cells in step (2), culture continues for 48 hours. If the viable cell density is higher than 4.0 × 10⁻⁶ cells after 48 hours, the culture is continued. 6 If the cells / mL is high, then use serum-free, fully suspended culture medium to dilute the cells to 2.0 × 10⁶ cells / mL. 6 Continue culturing; if the cell density is below 2.0 × 10⁶ cells / mL after 48 hours... 6 If the cell density reaches 2.0 × 10⁶ cells / mL, after settling, discard part of the supernatant and adjust the cell density to 2.0 × 10⁶ cells / mL using serum-free full suspension culture medium. 6 Continue culturing at 100 cells / mL; when the count of viable cells is less than the amount of cells inoculated in step (2) when sampling and counting is performed after 24 h, discard part of the supernatant after standing for 4 h, and adjust the cell density to 2.0 × 10⁻⁶ cells / mL using serum-free full suspension medium. 6 Continue culturing for 48 h; if the viable cell density is higher than 4.0 × 10⁶ cells / mL after 48 h... 6 If the cells / mL is high, then use serum-free, fully suspended culture medium to dilute the cells to 2.0 × 10⁶ cells / mL. 6 Continue culturing; if the cell density is below 2.0 × 10⁶ cells / mL after 48 hours... 6 If the cell count reaches 2.0 × 10⁶ cells / mL, after allowing the cells to settle, discard part of the supernatant and adjust the cell count to 2.0 × 10⁶ cells / mL using serum-free, fully suspended culture medium. 6 Continue culturing at cells / mL.

[0029] Example 3: Method for culturing canine distemper virus using MDCK serum-free suspension cell line (1) The MDCK serum-free suspension cell line obtained in Example 2 was subjected to a concentration of 1.0 × 10⁻⁶ cells / mL. 6Initial cell density of cells / mL was seeded in 500 mL shake flasks with a working volume of 150 mL, and cultured in a shaking incubator at 37℃, 5% CO2, and 120 rpm for 72 hours. (2) The cell culture in the shake flask was divided into two parts by using the dichotomy method. The cells were cultured for 72 hours and then divided into two parts. The MDCK serum-free suspension cell line was expanded and cultured in the Sartorius stedim BIOSTAT C plus bioreactor, respectively.

[0030] The cultivation process of the Eppendon DASGIP 4-parallel bioreactor is as follows: ① The serum-free MDCK suspension cells obtained in step (1) were seeded into a 1.5 L Eppendon DASGIP 4-in-1 parallel bioreactor with a working volume of 1.2 L and an initial seeding density of 1.0 × 10⁻⁶ cells / year. 6 The cell density was controlled in the Eppendon DASGIP quadruple parallel bioreactor as follows: pH 7.0–7.2, 37°C (33°C after inoculation), 5% CO2, rotation speed 140 rpm, dissolved oxygen 40%–50%, and cultured at 37°C for 72 h to achieve a cell density of 6 × 10⁶ cells / mL. 6 ~8×10 6 If the cell count / mL is controlled to be above 90%, then the cells can be used for infection testing. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰ of the total culture medium volume in step ①. The reaction conditions of the bioreactor are pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 140 rpm, and dissolved oxygen of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50%, and store the canine distemper virus at -20℃.

[0031] The cultivation process of Sartorius stedim's BIOSTAT C plus bioreactor is as follows: ① The obtained serum-free MDCK suspension cells were seeded into a 50 L Sartorius stedim BIOSTAT Cplus bioreactor, with a working volume of 30 L and an initial seeding density of 1.0 × 10⁻⁶ cells / year. 6 The cell density was controlled at 6 × 10⁶ cells / mL in the bioreactor under the following conditions: pH 7.0–7.2, 37°C, 5% CO₂, rotation speed 80 rpm, dissolved oxygen 40%–50%, and cultured at 37°C for 72 hours. 6 ~8×10 6If the cell count / mL is controlled to be above 90%, then the cells can be used for infection testing. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰ of the total culture medium volume in step ①. The reaction conditions of the bioreactor are pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 80 rpm, and dissolved oxygen of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50% and store the canine distemper virus at -20℃.

[0032] Example 4: Method for culturing canine distemper virus using MDCK serum-free suspension cell line (1) The serum-free suspension cell line of MDCK obtained in Example 2 was subjected to a concentration of 2.0 × 10⁻⁶ cells / mL. 6 Initial cell density of cells / mL was seeded in 500 mL shake flasks with a working volume of 150 mL, and cultured in a shaking incubator at 37℃, 5% CO2, and 120 rpm for 48 hours. (2) The cell culture in the shake flask was divided into two parts by using the dichotomy method. The cells were cultured for 48 hours and then divided into two parts. The MDCK serum-free suspension cell line was expanded and cultured in the Sartorius stedim BIOSTAT C plus bioreactor, respectively.

[0033] The cultivation process of the Eppendon DASGIP 4-parallel bioreactor is as follows: ① The serum-free MDCK suspension cells obtained in step (1) were seeded into a 1.5 L Eppendon DASGIP 4-in-1 parallel bioreactor with a working volume of 1.2 L and an initial seeding density of 2.0 × 10⁻⁶ cells / year. 6 The cell density was controlled in the Eppendon DASGIP quadruple parallel bioreactor as follows: pH 7.0–7.2, 37°C (33°C after inoculation), 5% CO2, rotation speed 140 rpm, dissolved oxygen 40%–50%, and cultured at 37°C for 72 h to achieve a cell density of 6 × 10⁶ cells / mL. 6 ~8×10 6 If the cell count / mL is controlled to be above 90%, then the cells can be used for infection testing. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰ of the total culture medium volume in step ①. The reaction conditions of the bioreactor are pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 140 rpm, and dissolved oxygen of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50%, and store the canine distemper virus at -20℃.

[0034] The cultivation process of Sartorius stedim's BIOSTAT C plus bioreactor is as follows: ① The obtained serum-free MDCK suspension cells were seeded into a 50 L Sartorius stedim BIOSTAT Cplus bioreactor with a working volume of 30 L and an initial seeding density of 2.0 × 10⁻⁶ cells / year. 6 The cell density was controlled at 6 × 10⁶ cells / mL in the bioreactor under the following conditions: pH 7.0–7.2, 37°C, 5% CO₂, rotation speed 80 rpm, dissolved oxygen 40%–50%, and cultured at 37°C for 72 hours. 6 ~8×10 6 If the cell count / mL is controlled to be above 90%, then the cells can be used for infection testing. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰ of the total culture medium volume in step ①. The bioreactor reaction conditions are: pH 7.0-7.2, 33℃, 5% CO2, rotation speed 80 rpm, and dissolved oxygen 40%-50%. Harvest the virus solution when the cell viability drops to 40%-50%, and store the canine distemper virus at -20℃. A photograph of MDCK suspension cells producing CPE after 96 hours of inoculation is shown below. Figure 3 As shown.

[0035] The effects of different culture methods on the viral load obtained from producing canine distemper virus using different cells were evaluated: 1. To determine the difference in canine distemper virus content obtained by producing canine distemper virus using MDCK adherent cells and MDCK serum-free suspension cells respectively.

[0036] The method for producing canine distemper virus using MDCK adherent cells is as follows: Discard the supernatant of confluent MDCK cells, wash twice with 3 mL PBS, discard the PBS, add 0.25% trypsin, incubate at 37℃ for 3 min, discard the trypsin, and homogenize the cells with DMEM medium containing 8% FBS. Passage the cells at a 1:4 ratio into flasks, and simultaneously inoculate one flask with canine distemper virus at a 0.5% volume ratio. Incubate at 33℃ in a 5% CO2 incubator. Harvest the virus when CPE reaches 70%–80%. Store at -20℃.

[0037] This culture method was used in two parallel experiments, and the viruses obtained from the two parallel experiments were denoted as adherent CDV-1 and adherent CDV-2, respectively.

[0038] Canine distemper virus was produced using serum-free suspension cells of MDCK obtained by culturing in shake flasks, Eppendon DASGIP 4-in-1 parallel bioreactors, and Sartorius stedim's BIOSTAT C plus bioreactor.

[0039] The process of culturing serum-free MDCK suspension cells in shake flasks is as shown in step (1) of Example 3. When the cell density reaches 6 × 10⁶ cells / year, the cells are cultured until the cell density reaches 6 × 10⁶ cells / year. 6 ~8×10 6 Cells / mL, with cell viability above 90%, were inoculated with virus. The inoculation and culture process is as follows: A bipartition method was used for inoculation. The cells in the shake flask were divided in half. One flask of cells was inoculated with fresh culture medium containing canine distemper virus at a ratio of 5‰ of the total culture medium volume (working volume: 150 mL). The flask was incubated in a shaking incubator at 33℃, 5% CO2, and 120 rpm. The virus solution was harvested when cell viability dropped to 40%–50%. The harvested canine distemper virus was then frozen at -20℃. The obtained virus was designated as suspension CDV-shake flask.

[0040] The process of amplifying and culturing serum-free suspension cells of MDCK and producing canine distemper virus using the Eppendon DASGIP 4-link parallel bioreactor is shown in Example 3. The obtained virus is denoted as suspension CDV-4-link.

[0041] The process of amplifying and culturing serum-free suspension cells of MDCK and producing canine distemper virus using the Sartorius stedim BIOSTAT C plus bioreactor is shown in Example 3. The obtained virus is designated as suspension CDV-50L.

[0042] The contents of canine distemper virus obtained by the above production methods are shown in Table 1.

[0043] Table 1. Results of CDV production using adherent cells and serum-free suspension cells from MDCK.

[0044] 2. The viral load of canine distemper virus in suspension culture was determined using E6 cell monolayers and MDCK cell monolayers, respectively. The specific method for determining the content of canine distemper virus in suspension culture using E6 cell monolayers is as follows: The CDV strain in suspension culture was serially diluted 10-fold using DMEM maintenance medium containing 2% FBS. 10... -6 10 -7 10 -8 10 -9Four dilutions were seeded into well-grown E6 cell monolayers in 96-well cell culture plates at 100 µL / well, with four replicates for each dilution. A normal cell control was also included. Cells were incubated at 33°C in a 5% CO2 incubator and observed for cytopathic effects (CPE) for 7 consecutive days. Total TCID was calculated using the Reed-Muench method. 50 .

[0045] The assay was performed in four parallel experiments. The viruses obtained from the four parallel experiments were named E6 monolayer-CDV-1, E6 monolayer-CDV-2, E6 monolayer-CDV-3 and E6 monolayer-CDV-4, respectively.

[0046] The method for determining canine distemper virus in suspension culture using MDCK cell monolayers is the same as the E6 monolayer method. This assay was performed in four parallel experiments, and the viruses obtained from the four parallel experiments were designated as MDCK monolayer-CDV-1, MDCK monolayer-CDV-2, MDCK monolayer-CDV-3, and MDCK monolayer-CDV-4, respectively.

[0047] The canine distemper virus content obtained by the above detection methods is shown in Table 2 and Figure 4 As shown.

[0048] Table 2. Results of canine distemper virus content determination in suspension culture using E6 cell monolayers and MDCK cell monolayers.

[0049] The above experimental results lead to the following conclusions: Compared to producing canine distemper virus using adherent MDCK cells, producing canine distemper virus using serum-free MDCK suspension cells resulted in a virus content increase of at least one titer, demonstrating another significant advantage of suspension culture. Compared to determining the virus content of suspension-cultured canine distemper virus using MDCK cell monolayers, determining the virus content of suspension-cultured canine distemper virus using E6 cell monolayers increased the virus content by 2-3 titers. This is because canine distemper virus exhibits better adaptability in adherent E6 cell culture. Furthermore, as the culture time of E6 cell monolayers continues to extend, they are less prone to cell clumping compared to MDCK cells, making it easier to observe CPE and control cell morphology.

[0050] The present invention provides a process optimization method for culturing canine distemper virus using the MDCK serum-free suspension cell line. MDCK serum-free suspension cells prepared in 500 mL shake flasks (preparation method is the same as step (1) in Example 3) were cultured until the cell density reached 6 × 10⁶ cells / mL. 6 ~8×10 6The virus was introduced using a method to explore the production process of canine distemper virus (cells / mL, with cell viability above 90% for inoculation). This involved determining the optimal conditions for a third parameter after establishing two parameters: different inoculation doses, initial cell densities, and harvesting times. The effects of these factors on the obtained virus content were investigated (specific parameter settings are shown in Tables 3, 4, and 5; the culture conditions after inoculation in all experimental groups were 33℃, 5% CO2, and 120 rpm, with each group replicated three times). The optimal inoculation process was determined, particularly the virus harvesting time. Because the initial cell proliferation (CPE) time of canine distemper virus is relatively slow, the harvesting time was set at 96 h. The virus content detection results are shown in Tables 3, 4, and 5. Figure 5 , Figure 6 and Figure 7 .

[0051] Table 3. Detection results of virus content corresponding to different infection doses.

[0052] Table 4. Detection results of virus content corresponding to different cell densities after inoculation.

[0053] Table 5. Detection results of virus content at different collection times.

[0054] Based on the above experimental results, the optimal process conditions for canine distemper virus suspension culture were determined to be: an inoculation dose of 5‰, an initial inoculation cell density of 6 million to 8 million, and a virus harvesting time of 96 hours or when the cell viability drops to 40% to 50%.

[0055] Example 5: Application of canine distemper virus produced from MDCK serum-free suspension cells in the production of live canine distemper vaccine. The preparation method of canine distemper live vaccine is as follows: The canine distemper virus solution prepared in Example 4 using the Sartorius stedim BIOSTAT C plus bioreactor was melted, and a freeze-drying protectant was added to the virus solution. After thorough mixing, the mixture was quantitatively dispensed and freeze-dried under vacuum to obtain the live vaccine (a total of 3 parallel experiments were conducted). The freeze-drying protectant was gelatin and sucrose; the volume ratio of virus solution to protectant was 9:1.

[0056] 1. Conduct relevant tests in accordance with the current Chinese Veterinary Pharmacopoeia. The results of testing the prepared finished vaccines according to the current Chinese Veterinary Pharmacopoeia are shown in Table 6.

[0057] Table 6. Test Results of Finished Vaccines

[0058] 2. Safety Inspection Five healthy beagle dogs aged 1-2 months (with CDV serum neutralizing antibody ratio not higher than 1:2) were selected. Each dog was given 10 doses of vaccine intramuscularly and observed for 14 consecutive days. The clinical symptoms of each dog were recorded. The results are detailed in Table 7.

[0059] Table 7 Safety test results of finished vaccines

[0060] 3. Validity verification (1) Virus content determination Each of the 10 vials of vaccine was serially diluted 10-fold, and 10 were taken from each vial. -5 10 -6 10 -7 10 -8 Four dilutions were established, and each dilution was seeded into a monolayer of well-grown E6 cells. The cells were incubated at 33°C with 5% CO2 for 7 days. CPE was observed and recorded daily, and TCID was calculated using the Reed-Muench method. 50 The results are detailed in Table 8.

[0061] As can be seen from the results in Table 8, the measured viral loads of the vaccines were all higher than the established standard for viral load in finished vaccines (viral load ≥ 10 mmol / L per dose). 6.5 TCID 50 ).

[0062] Table 8 Results of vaccine virus content determination

[0063] (2) Serological test Five healthy beagle dogs aged 1-2 months (with CDV serum neutralizing antibody ratios not exceeding 1:2) were selected. Each dog was injected intramuscularly with one dose of vaccine via the neck. Blood samples were collected from each dog 21 days post-immunization to separate serum. The serum was inactivated by placing it in a 56°C water bath for 1 hour. The serum was then serially diluted with DMEM to prepare CDV indicator virus solution (200 TCID50). 50 Serum at different dilutions was mixed with an equal volume of indicator virus solution and incubated at 37°C for 1 hour. The mixture was then inoculated into E6 cell monolayers, with blank and virus controls provided. The cells were incubated at 37°C with 5% CO2 for 96–120 hours. Interpretation was performed under controlled conditions: 100% CPE in the cell culture wells of the tested serum was considered negative; more than 50% of cells showed protection; and the serum dilution at which 50% of cells did not produce CPE was the antibody titer of that serum sample. The test results are detailed in Table 9.

[0064] Table 9. Serological test results of finished vaccines

[0065] The experimental results show that the SN antibody titers of all 5 dogs were above 1:64. Literature indicates that SN antibody titers above 1:64 can resist the attack of corresponding virulent viruses. SN antibody titers are positively correlated with immune protection after challenge, that is, high SN antibody levels provide good immune protection against virulent virus challenge, and vice versa.

[0066] Conclusion: When healthy 1-2 month old beagle dogs were immunized with vaccines that met the viral content standards of finished vaccines, blood samples were collected 21 days after immunization to separate serum. The SN antibody titers were all above 1:64, which can achieve the protective effect on dogs.

[0067] The effect of the virus obtained in Example 3 was the same as that of the virus obtained in Example 4.

[0068] In summary, this application utilizes bioreactor-based fully suspended cell culture technology to replace traditional adherent cell culture technology for the production of live canine distemper virus vaccine. This enables large-scale and efficient culture of canine distemper virus, significantly increasing the virus content compared to adherent cell culture, thereby greatly improving the quality of the semi-finished product and thus enhancing vaccine quality, while ensuring the safety and efficacy of the vaccine.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for high-efficiency production of canine distemper virus using MDCK cell line in full suspension without serum, characterized in that, The method involves inoculating canine distemper virus into a serum-free suspension cell line of MDCK cells and culturing it using a bioreactor.

2. The method according to claim 1, characterized in that, The method for preparing the serum-free suspension cell line of MDCK cells includes the following steps: (1) MDCK adherent cells were seeded in culture medium containing 8% serum and cultured. The obtained cells were then seeded in culture medium with decreasing serum concentrations to obtain MDCK suspension cells cultured with low serum concentration. (2) Select MDCK adherent cells that have grown into a monolayer and are adapted to low serum culture. After digestion, they are seeded in serum-free suspension medium until normal dilution and passage are performed every 72 h. When the cell density doubles and the viability is higher than 90% after 48 h, the serum-free suspension cell line of MDCK cells is obtained.

3. The method according to claim 2, characterized in that, Step (1) is as follows: First stage culture: MDCK adherent cells are seeded in DMEM culture medium containing 8% serum at a ratio of 1:4 and passaged for 3 to 5 generations; Second stage culture: The serum concentration in the culture medium is reduced to 4.5% to 5.5%, and the cells obtained in the first stage are passaged for more than 5 generations; Third stage culture: The serum concentration in the culture medium is reduced to 1.5% to 2.5%, and the cells obtained in the second stage are passaged for more than 5 generations.

4. The method according to claim 2, characterized in that, Step (2) is as follows: ① Select MDCK adherent cells that have grown into a monolayer and are adapted to low serum culture, digest them, and then seed the digested cells into serum-free MDCK cell suspension culture medium, controlling the cell density to be 1.0 × 10⁻⁶. 6 ~2.0×10 6 cells / mL; ② Repeat step ① until normal dilution and passage are performed every 72 h. When the viable cell density doubles and the viability is higher than 90% after 48 h, the serum-free suspension cell line of MDCK cells is obtained.

5. The method according to claim 4, characterized in that, The cell density was controlled when the digested cells were seeded into serum-free MDCK cell suspension culture medium and cultured for 48 h.

6. The method according to claim 2, characterized in that, The digestion process involves discarding the supernatant of MDCK adherent cells adapted to low-serum culture and forming a monolayer, washing 2-4 times with 2-5 mL of PBS, discarding the PBS, adding 0.25% trypsin, incubating at 37°C for 2-5 minutes, discarding the trypsin, adding serum-free MDCK cell suspension medium to terminate the digestion, and then mixing the cells thoroughly for later use.

7. The method according to claim 1, characterized in that, The cultivation process includes the following steps: S1. The MDCK cell line was suspended in serum-free solution at 1.0 × 10⁻⁶ cells / cells. 6 ~2.0×10 6 Initial cell density of cells / mL was seeded into 500 mL shake flasks with a working volume of 150 mL, and cultured in a shaking incubator at 37℃, 5% CO2, and 120 rpm for 48–72 h to obtain shake flask cells; S2. Using a bipartitioning method for inoculation, the shake-flask cells obtained in S1 were divided into two halves, and each half was inoculated with fresh culture medium containing canine distemper virus at an inoculation rate of 5‰~1%. Canine distemper virus was produced using an Eppendon DASGIP 4-in-1 parallel bioreactor and a Sartorius stedim BIOSTATC plus bioreactor under the conditions of pH 7.0~7.2, 33℃, 5% CO2, 80~140 rpm, and dissolved oxygen of 40%~50%. The virus solution was harvested when the cell viability dropped to 40%~50% or after culturing for 96 h, thus obtaining canine distemper virus.

8. The method according to claim 7, characterized in that, The steps for producing canine distemper virus using the Eppendon DASGIP quadruple parallel bioreactor are as follows: ① The cells obtained from S1 were seeded in a 1.5 L Eppendon DASGIP 4-in-1 parallel bioreactor with a working volume of 1.2 L and an initial seeding density of 1.0 × 10⁻⁶ cells / year. 6 ~2.0×10 6 Cells / mL were cultured for 72 h at pH 7.0–7.2, 37℃, 5% CO2, 140 rpm, and dissolved oxygen of 40%–50%, until a cell density of 6 × 10⁻⁶ was achieved. 6 ~8×10 6 The cell count / mL is above 90%, and the cells are viable for inoculation. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰~1% of the total culture medium volume in step ①. Control the reaction conditions of the Eppendon DASGIP 4-link parallel bioreactor as follows: pH 7.0~7.2, 33℃, 5% CO2, rotation speed of 140 rpm, and dissolved oxygen of 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50% or after culturing for 96 hours to obtain canine distemper virus.

9. The method according to claim 7, characterized in that, The steps for producing canine distemper virus using the Sartorius stedim BIOSTAT C plus bioreactor are as follows: ① The cells obtained from S1 were seeded in a 50 L Sartorius stedim BIOSTAT C plus bioreactor with a working volume of 30 L and an initial seeding density of 1.0 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 Cells / mL were cultured for 72 h at pH 7.0–7.2, 37℃, 5% CO2, 80 rpm, and dissolved oxygen of 40%–50%, until a cell density of 6 × 10⁻⁶ was achieved. 6 ~8×10 6 The cell count / mL is above 90%, and the cells are viable for inoculation. ② Inoculate fresh culture medium containing canine distemper virus at a volume ratio of 5‰~1% of the total culture medium volume in step ①. Control the reaction conditions of the Sartorius stedim BIOSTAT C plus bioreactor as follows: pH 7.0~7.2, 33℃, 5% CO2, rotation speed 80 rpm, dissolved oxygen 40%~50%. Harvest the virus solution when the cell viability drops to 40%~50% or after culturing for 96 h, and the canine distemper virus is obtained.

10. The use of the method according to any one of claims 1-9 in the preparation of canine distemper virus vaccine.

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