CN105816869A - Preparation method of mink canine distemper virus live vaccine and vaccine prepared by same - Google Patents

Abstract

The present invention relates to the field of veterinary biological products, in particular to a method for preparing a live vaccine of mink canine distemper virus, comprising: inoculating sensitive cells for seedling preparation in a bioreactor and culturing them with microcarriers; to be described Sensitive cells are cultured until more than 50% of them grow into a dense monolayer, and then inoculated the canine distemper virus into the bioreactor for proliferation and culture; harvesting the virus culture medium and microcarriers, removing the microcarriers and cell debris after freezing and thawing to obtain the virus liquid, The vaccine is obtained after the virus liquid is prepared. By perfecting the reaction conditions of each step and optimizing the production process, the invention achieves the technical effects of short production cycle, high virus titer, stable product quality, improved production efficiency and small side reactions.

Description

Method for preparing mink canine distemper virus live vaccine and vaccine prepared by the method

technical field

The invention relates to the field of veterinary biological products, in particular to a method for preparing mink canine distemper virus live vaccine and the vaccine prepared by the method.

Background technique

Mink distemper, also known as mink distemper, is an acute, febrile and highly infectious highly contagious infection caused by Canine distempervirus (CDV) of the family Paramyxoviridae and the genus Morbillivirus. Sexually transmitted diseases are one of the main infectious diseases in mink farming.

Reactor suspension culture technology has been widely used in the production of biopharmaceuticals in the world, and has continued to develop in the construction of high-expression cell lines and the development of personalized culture media. A large number of new reactors and the increase in product expression have led to increased reactor capacity. Oversupply, the market in developed countries is saturated and expanding to developing countries. In China, although the cell suspension technology started relatively late, after decades of research and practice, the large-scale culture technology of animal cells has developed rapidly. With the further development of this technology, the use of bioreactors for high-density cell culture to produce veterinary vaccines is an inevitable trend in the development of the industry. With the further improvement of the production process and the more reasonable design of the bioreactor, the suspension cell culture will become easier and easier, and the mature large-scale animal cell culture technology will also more effectively promote the large-scale production of veterinary vaccines development of.

At present, foreign countries have used microcarriers to suspend culture sensitive cells in bioreactors to produce some viral vaccines, such as Sanofi Pasteur's polio vaccine, rabies vaccine, and Baxter's influenza vaccine. Domestic Liaoning Chengda Biological Co., Ltd. has introduced from abroad the technology of high-density cultivation of sensitive cells in bioreactors to produce vaccines, which has been used to prepare human rabies vaccines on a large scale. Nanjing Merial uses microcarriers to cultivate sensitive cells in bioreactors The production of chicken infectious bursa vaccine has achieved good social and economic benefits. But there is still a blank about the use of microcarriers to cultivate sensitive cells in bioreactors to produce canine distemper vaccine.

Existing mink distemper vaccines are all produced by the traditional spinner bottle cultivation process. There are many disadvantages in the traditional spinning bottle process, such as: low degree of automation, high labor intensity; uncontrollable environment for culturing cells, easy to be polluted by the environment; long time-consuming, low efficiency, high production cost, difficult to expand production; large quality differences between different batches ; involving biosafety and public health issues.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a kind of method for suspension culture sensitive cell production mink canine distemper virus live vaccine, described method compares with traditional mink canine distemper virus live vaccine production process, production period is short, virus titer is high, The quality of the product produced by the method is stable, the production efficiency is improved, and the side reaction is small.

A method for preparing live mink distemper virus vaccine, comprising the steps of:

1) Inoculate the sensitive cells for seedling production in a bioreactor and cultivate them with microcarriers;

2), after the sensitive cells are cultivated to more than 50% and grow into a dense monolayer, inoculate the canine distemper virus into the bioreactor for proliferation and culture;

3) Harvesting virus culture fluid and microcarriers, removing microcarriers and cell fragments after freezing and thawing to obtain virus fluid, and preparing the virus fluid to obtain a vaccine.

The cell microcarrier suspension culture is easier to replace the culture medium, and provides a larger surface area for cell adherent growth, thus making the cells reach a higher culture density; the bioreactor used in the application is specifically a stirred reactor (microcarrier For culture), combined with microcarrier suspension culture technology, it can achieve the technical effects of less space occupation, high cell yield (and thus high virus titer), and low production cost. In this field, it is the first time that the suspension culture method is used to produce the live vaccine of mink canine distemper virus. By perfecting the reaction conditions of each step and optimizing the production process, the present invention achieves short production cycle, stable product quality, improved production efficiency and less side effects. Response to small technical effects.

Preferably, the method for the above-mentioned mink distemper virus live vaccine preparation:

The sensitive cells for seedling production are Vero, MDCK, Marc-145;

The strain of the canine distemper virus is CDV3-CL strain, which is preserved in CGMCC, General Microorganism Center of China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC NO.10768.

The preservation address of CDV3-CL strain is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is: June 10, 2015.

Preferably, the method for preparing the above-mentioned mink distemper virus live vaccine, in step 1):

The cell density of the inoculated sensitive cells for seedling production is 4×10 ⁵ -2×10 ⁶ cells/mL;

The microcarrier used for culturing is Cytodex microcarrier, and its use density is 3-10 g/L.

Microcarrier culture (microcarrier culture) is a practical technique for high-yield culture of adherent cells. Cytodex is specially used for culturing various animal cells, and its culture volume can range from a few milliliters to more than 6000 liters. Using Cytodex microcarrier technology, simple suspension culture of adherent cells can be achieved, and millions of cells can be obtained per milliliter of culture medium. Microcarriers are suitable for various culture systems such as shake flasks, spinner bottles, stirred tanks, and WAVE bioreactors. The microcarriers used in the present invention can specifically be Cytodex-1, 2, 3, preferably Cytodex-1, purchased from GE (General Electric).

Further preferably, the method for preparing the above-mentioned mink distemper virus live vaccine, in step 1):

The cell culture fluid used when cultivating the sensitive cells for making seedlings is: DMEM culture fluid containing 5%-8% neonatal bovine serum;

The culture conditions when culturing with microcarriers are: temperature 36-38°C, _CO2 content 4.8-5.2%, stirring speed 55-65rpm, dissolved oxygen 45-55%, pH 7.2-7.4, reactor automatic control culture .

Preferably, the method for preparing live mink distemper virus vaccine as described above, in step 1), when cultivating the sensitive cells in a bioreactor, includes single-stage culture or scale-up culture;

The single-stage cultivation is a 5-14L bioreactor single-stage cultivation mode;

The scale-up culture is a 40-140L scale-up culture mode; the scale-up culture operation is to digest the cells grown on the microcarriers after single-stage culture as initial cells in a closed container with trypsin, and filter through a 90-120 μm filter screen. Transfer the cell suspension into the next larger bioreactor to continue culturing, and use the cultured cells as the initial cells for the next scale-up culture operation and repeat the above-mentioned scale-up culture operation, gradually scaling up to a production scale of 40-140L.

The digestion amplification mode of the present invention adopts a self-made digestion device (that is, the airtight container) to digest cells, which avoids the tedious operation of digesting cells in large-scale production, and the cells are not easily polluted. Use 90-120 μm stainless steel mesh filter to separate the digested cells from the original culture carrier, avoiding the uneven attachment of cells to the old and new balls during the culture process.

The self-made digestion device can be placed in a water bath for heating. The top of the main body of the container contains a three-way hole, one end of which is connected with trypsin digestion solution with a concentration of 0.25% trypsin-0.02% EDTA preheated at 37°C. Connect one end to the drain bottle.

Further preferably, the method for preparing live mink distemper virus vaccine as described above, the operation in step 2) specifically includes:

After the sensitive cells are cultured for 20-48 hours, the canine distemper virus is inoculated for proliferation and culture, and the inoculation amount is 0.001-0.01 MOI;

Or, after the sensitive cells are cultured for 48-72 hours, stop stirring, and discharge all the cell culture fluid after the microcarriers settle to the bottom of the tank; add cell maintenance fluid and inoculate canine distemper virus for proliferation and culture, and the inoculum size is 0.001-0.01 MOI.

More preferably, the canine distemper virus can be inoculated when the cell density is 1×10 ⁶ to 3×10 ⁶ cells/mL after cell culture for 20 to 48 hours.

Preferably, the method for the above-mentioned mink distemper virus live vaccine preparation:

The formula of the cell maintenance solution is: DMEM culture solution containing 1.8-2.2% newborn bovine serum;

The conditions for the proliferation culture are: temperature 33-35° C., CO ₂ content 4.8-5.2%, stirring speed 55-65 rpm, dissolved oxygen 45-55%, pH 6.9-7.2, reactor automatic control culture.

The temperature of proliferation culture is not the most suitable temperature for cell growth, but multiple experiments by the applicant have proved that a lower growth temperature is more conducive to the proliferation of viruses. The culture temperature of 33-35°C is adopted for the proliferation culture, and the sensitive cells for seedling production can be used to inoculate the virus after the shortest cultivation of 20-28 hours. The virus titers obtained by culturing are the same, but the time for cultivating sensitive cells for making seedlings is greatly reduced, which saves culturing time, shortens the production cycle, and reduces the production cost.

Preferably, the method for the above-mentioned mink distemper virus live vaccine preparation:

In step 1), the sensitive cells for seedling production are inoculated in a bioreactor and cultured with microcarriers as batch culture or continuous perfusion culture;

In step 2), the culture mode of the proliferation culture is batch culture.

Batch culture means that the cells and the culture medium are put into the reactor at one time for cultivation. The cells continue to grow and the products are also formed continuously. After a period of cultivation, the cultivation is terminated.

In perfusion culture, the cells remain in the reactor system and the culture is harvested while fresh medium is continuously added. The main advantage of perfusion culture is that the continuous perfusion medium can provide sufficient nutrients and take away metabolites; at the same time, the cells remain in the reactor system and can reach a high cell density. Compared with other methods, the yield of perfusion culture can be increased by an order of magnitude, and labor consumption can be greatly reduced.

Preferably, the method for the above-mentioned mink canine distemper virus live vaccine preparation, in step 3), the timing of the harvest virus culture fluid and microcarriers is:

More than 80% of sensitive cells showed typical cytopathic effects.

In specific operations, a significant increase in DO (dissolved oxygen content) value is also an indicator signal for harvesting virus culture medium and microcarriers, which can be combined with cytopathic effect (CPE) to achieve a more accurate estimate. CPE refers to the phenomenon that the virus proliferates in a large number of host cells, leading to cell disease or even death.

The mink canine distemper virus live vaccine prepared by the above-mentioned method for preparing the mink canine distemper virus live vaccine.

Compared with prior art, the beneficial effect of the present invention is:

1), the present invention can achieve the technical effects of short production cycle, high virus titer, stable product quality, improved production efficiency, and small side reactions by perfecting the reaction conditions of each step and optimizing the production process.

2) The application of bioreactors to produce vaccines has a high degree of automation, simple and stable production process, easy operation, large output and small footprint, easy to quickly expand the production scale; batch-to-batch quality is balanced and stable.

3) The digestion amplification mode of the present invention adopts a self-made digestion device to digest cells, which avoids the tedious operation of digesting cells in large-scale production, and the cells are not easily polluted. Use 100μm stainless steel mesh filter to separate the digested cells from the original culture carrier, avoiding the uneven attachment of cells to the old and new balls during the culture process.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

The canine distemper virus strain CDV3-CL strain provided by this application is preserved in CGMCC, General Microorganism Center of China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC NO.10768. The preservation address is: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation time is: June 10, 2015. The test result is survival.

Fig. 1 is a process flow diagram of the present invention;

Fig. 2 is the microcarrier cell picture of 24h after cell inoculation in embodiment 3 step 303;

Fig. 3 is the microcarrier cell picture of 48h after cell inoculation in embodiment 1 step 102;

Fig. 4 is the picture of microcarrier cells 72h after cell inoculation in Example 4.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example 1

A method for the preparation of mink distemper virus live vaccine is provided in Embodiment 1 of the present invention, comprising the following steps:

Step 101: Inoculate Marc-145 cells for seedling production in a 5L bioreactor at a density of 4×10 ⁵ cells/mL and culture them with microcarriers; the microcarriers used for cultivation are Cytodex microcarriers, and the density used is 3g/L; The cell culture fluid used when cultivating the sensitive cells for seedling preparation is: DMEM culture fluid containing 5% newborn calf serum; the culture condition when culturing with microcarriers is: temperature 36°C, _CO content 4.8%, the stirring speed is 55rpm, the dissolved oxygen is 45%, the pH is 7.2, and the reactor is automatically controlled for cultivation;

Step 102: After the sensitive cells are cultured for 48 hours, more than 70% of them grow into a dense monolayer, and the canine distemper virus CDV3-CL strain is inoculated into the bioreactor according to the ratio of 0.01MOI for proliferation and culture; The formula of cell maintenance solution is: DMEM culture solution containing 1.8% newborn bovine serum; the conditions of proliferation culture are: temperature 33°C, _CO content 4.8%, stirring speed 55rpm, dissolved oxygen 45%, pH 6.9, reaction The device automatically controls the cultivation;

Step 103: Harvest the virus culture medium and microcarriers when more than 80% of the sensitive cells show typical cytopathic effects among the cells to be proliferated and cultured, remove the microcarriers and cell fragments after freezing and thawing to obtain the virus solution, and use it for vaccine preparation .

Example 2

A method for the preparation of mink canine distemper virus live vaccine is provided in Embodiment 2 of the present invention, comprising the following steps:

Step 201: Inoculate MDCK cells for seedling production in a 5L bioreactor at a density of 2×10 ⁶ cells/mL and culture them with microcarriers; the microcarriers used for culturing are Cytodex microcarriers, and the density used is 10 g/mL L; the cell culture fluid used when cultivating the sensitive cells for seedling production is: DMEM culture fluid containing 8% newborn calf serum; the culture conditions when culturing with microcarriers are: temperature 38°C, _CO Content 5.2% , The stirring speed is 65rpm, the dissolved oxygen is 55%, pH7.4, the reactor is automatically controlled for cultivation; the method of culturing cells is batch cultivation;

Step 202: After the sensitive cells described in step 201 are cultured for 20 hours, more than 50% of them grow into a dense monolayer, and the canine distemper virus CDV3-CL strain is inoculated into the bioreactor according to the ratio of 0.001 MOI for proliferation and culture; proliferation The culture medium used for culturing is still the cell culture medium described in step 201; the conditions for proliferation culture are: temperature 35°C, _CO content 5.2%, stirring speed 65rpm, dissolved oxygen 55%, pH 7.2, reactor Automatically control culture; the mode of proliferation culture is batch culture or continuous perfusion culture;

Step 203: Harvest the virus culture medium and microcarriers when more than 80% of the sensitive cells show typical cytopathic effects among the cells to be proliferated and cultured, remove the microcarriers and cell fragments after freezing and thawing to obtain the virus solution, and use it for vaccine preparation .

Example 3

In order to describe the technical solution of the present application in more detail, on the basis of Examples 1 and 2, the present invention obtains Example 3 through further refinement and limitation of each operation, including the following steps, please refer to Figure 1:

Equipment and reagents used:

Bioreactor: Qizhi Bioengineering Equipment Co., Ltd. 14L bioreactor;

Microcarrier: GE Cytodex-1;

Canine distemper virus for seedling production: CDV3-CL strain, identified, kept and supplied by the Institute of Special Products of the Chinese Academy of Agricultural Sciences;

DMEM medium (dry powder): GIBCO company;

Newborn bovine serum: Inner Mongolia Jinyuankang Bioengineering Co., Ltd.;

Trypsin (dry powder): GIBCO company.

Step 301: Preparation of bioreactor and microcarrier

Thoroughly clean the tank body of the bioreactor, connect the inlet and outlet pipelines and gas lines of the reactor, calibrate the pH and DO electrodes and install them on the tank body, and perform leak detection; At room temperature, aseptically connect the liquid inlet and outlet pipes, discharge the high-pressure liquid, pretreat the microcarriers according to the instructions of GE Company, and introduce them into the bioreactor together with the sterile filtered cell growth medium. The density of the microcarriers is 6g/L; set the culture parameters, turn on the reactor temperature, stirring and ventilation control for pre-cultivation, overnight for use.

Step 302: Preparation of Sensitive Cells

Take well-grown Vero cells, discard the cell culture medium, wash the cell surface with PBS buffer solution of pH 7.2, add 0.25% trypsin-EDTA solution to digest the cell digestion solution, add DMEM containing 6% serum concentration for cell culture solution to stop the digestion, subculture the cells at a ratio of 1:3, culture at 37°C, and turn at a rotation speed of 7-9r/min.

Step 303: Bioreactor Microcarrier Cultivation

Digest the cells cultured in step 302 according to the method described in step 302, collect the cell suspension, count the cells and inoculate them in a bioreactor at a cell density of 9×10 ⁵ cells/mL. The cell culture medium is: DMEM culture medium containing 6% newborn bovine serum; the setting parameters of the bioreactor culture are culture temperature 37°C, stirring speed 60rpm, dissolved oxygen 50%, pH 7.2-7.4, reactor automatic Controlled cultivation. The picture of the cell state of the cells cultured for 24h is shown in Fig. 3 . Batch culture was adopted for culturing.

Step 304: Proliferation and cultivation of the virus liquid in the bioreactor

After the cells in step 303 were cultured for 24 hours, more than 50% of them grew into a dense monolayer, and the microcarriers were basically covered with cells under a microscope, and the cell count was 2×10 ⁶ cells/mL, and then inoculated with the virus solution, with the inoculum amount being 0.005 MOI Inoculate the canine distemper virus strain (CDV3 strain), and still use the cell culture medium described in step 302 to cultivate after inoculating the virus. The setting parameters of the bioreactor cultivation are cultivation temperature 33° C., stirring speed 60 rpm, dissolved oxygen 50%, pH 6.9-7.2, and reactor automatic control cultivation. Batch culture was adopted for culturing.

Step 305: Harvesting virus culture fluid and microcarriers

After 20 hours of poisoning, the microcarriers in the reactor were taken every 4 hours to observe the cytopathic changes. When more than 80% of the sensitive cells on the microcarriers showed typical cytopathic effects, and the DO value showed an obvious upward trend, the culture was terminated. Harvest the virus culture fluid and microcarriers, freeze and thaw twice at -20°C, remove the microcarriers and cell debris by centrifugation or filtration, harvest the virus fluid, and determine the virus content to be 6.8logTCID ₅₀ /mL.

Step 306: Vaccine Preparation

Mix the qualified virus liquid and freeze-drying protective agent in an appropriate ratio, add appropriate antibiotics, and mix thoroughly. At the same time, use 0.1mol/L NaHCO3 to adjust the pH value to 7.4, mix fully, and quantitatively distribute and freeze-dry to make a finished product.

Next, in order to make the method for producing the live vaccine of mink canine distemper virus by the suspension culture sensitive cells of the embodiment 3 of the present invention to be better applied, the present invention also provides embodiment 4 on the basis of the above-mentioned embodiment 3, implement Example 4 is a further limitation and increase of Example 3, adding the specific steps of amplified culture, and will now be elaborated and explained in detail:

Example 4

Perform operations according to steps 301-303 in Embodiment 3. The difference is that in step 303, after the cells have been cultured for 72 hours, more than 80% of the cells have grown into a dense monolayer, and the operation of step 403.5 is performed when the cell density reaches above 4×10 ⁶ cells/mL.

Step 403.5: Scale-up culture of microcarriers (not shown in Figure 1):

The agitation of the bioreactor was stopped, the microcarriers were allowed to settle naturally, the supernatant culture solution was discharged, washed twice with PBS buffer solution of pH 7.2, and the microcarriers covered with cells were collected into a kind of self-made airtight container (digestion device )middle. The self-made digestion device is shown in Figure 3. The container can be heated in a water bath. There is a three-way hole at the top of the main body of the container, and one end is connected with 0.25% trypsin-0.02% EDTA preheated at 37°C. Trypsin digestion solution, the other end is connected to the drain bottle.

After injecting trypsin digestion solution, wait for the microcarriers to settle naturally, discharge the supernatant containing PBS cleaning solution and trypsin digestion solution, digest for 5-20 minutes, add cell growth solution to terminate digestion, and dissolve the mixture of cells and microcarriers into a 14L bioreactor and start stirring. Aseptically connect the 14L bioreactor pipeline and the 40L bioreactor pipeline, filter the cell suspension through a 90-120μm stainless steel mesh, inject the cell suspension into the 40L bioreactor, and use 3g/ml of microcarriers Add new carrier, parameter setting culture process conditions as temperature 37°C, _CO2 content 5%, stirring speed 60rpm, dissolved oxygen 50%, pH 7.2, reactor automatic control culture; observe cell growth and measure glucose content , to determine whether the medium needs to be changed, and cultured for 28 hours.

Step 404: Proliferation and cultivation of the virus liquid in the bioreactor

After the cells were cultured for 28 hours, when most of the cells on the microcarriers were in a good growth state under a microscope, the virus solution was inoculated with an inoculation volume of 0.001-0.01 MOI to inoculate the canine distemper virus strain (CDV3 strain), and the parameter set for the bioreactor culture was the culture temperature 35°C, stirring speed 60rpm, dissolved oxygen 50%, pH 7.0, reactor automatic control culture.

Step 405: Harvesting virus culture fluid and microcarriers

After 20 hours of inoculation, take the microcarriers in the reactor every 4 hours to observe the cell changes. When 80% of the cells on the microcarriers have detached, and the DO value is obviously on the rise, stop the culture, harvest the virus culture medium and microcarriers, and store them at -20°C Freezing and thawing was repeated twice, microcarriers and cell debris were removed by centrifugation or filtration, and the virus liquid was harvested. The virus content was determined to be 6.5logTCID ₅₀ /mL, and the harvested liquid was stored at -20°C for later use.

Step 406: Vaccine preparation

Mix the qualified virus liquid and freeze-drying protective agent in an appropriate ratio, add appropriate antibiotics, and mix thoroughly. At the same time, adjust the pH value to 7.4 with 0.1mol/L NaHCO ₃ , mix fully, and quantitatively distribute and freeze-dry to make a finished product.

Experimental example

The technical process of the traditional spin bottle method in the production of mink distemper virus live vaccine can generally be briefly described as:

Take well-grown Vero cells, discard the cell culture medium, wash the cell surface with PBS buffer solution with pH 7.2, add 0.25% trypsin-EDTA solution to digest the cell digestion solution, add MEM containing 8% serum concentration for cell culture solution to stop the digestion, subculture the cells according to the ratio of 1:3, culture at 37°C, and the rotation speed is 7-9rmp/min.

The virus seeds used for production were diluted 5 times with MEM cell maintenance solution, inoculated at a ratio of 5% in Vero cell spinner bottles that had grown into a good monolayer, adsorbed at 37°C for 1 hour, and then added cell maintenance solution. The cell culture spinner bottle was placed under the condition of 35-37°C for rotation culture, and the observation was continued for 4 days. The cells can be harvested when the CPE reaches more than 70%, and stored at -20°C.

Embodiment 4 is compared with traditional spinning bottle method, and comparison result is as shown in table 1:

Table 1 embodiment 4 compares with traditional spinning bottle method production mink canine distemper virus live vaccine

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. the method that prepared by a mink canine distemper disease virus live vaccine, it is characterised in that comprise the steps:

1) seedling sensitive cells it is inoculated in bioreactor and cultivates with microcarrier；

2), treat that the cultivation of described sensitive cells grows up to inoculation canine distemper virus in the fine and close backward described bioreactor of monolayer to more than 50% and carries out enrichment culture；

3), gather in the crops virus-culturing fluid and microcarrier, remove microcarrier after freeze thawing and cell debris obtains virus liquid, after being prepared by described virus liquid, obtain vaccine.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 1, it is characterised in that:

Described seedling sensitive cells is Vero, MDCK, Marc-145；

The strain of described canine distemper virus is CDV3-CL strain, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number CGMCCNO.10768.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 2, it is characterised in that in step 1) in:

The cell density of the seedling sensitive cells inoculated is 4 × 10⁵～2 × 10⁶Individual/mL；

Microcarrier for cultivating is Cytodex microcarrier, and it uses density to be 3～10g/L.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 3, it is characterised in that in step 1) in:

The cell culture fluid used when cultivating described seedling sensitive cells is: the DMEM culture fluid of the new-born calf serum containing 5%～8%；

Condition of culture when cultivating with microcarrier is: temperature 36～38 DEG C, CO₂Content 4.8～5.2%, mixing speed be 55～65rpm, dissolved oxygen is 45～55%, pH7.2～7.4, reactor automatically control cultivation.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 3, it is characterised in that in step 1) in, include when bioreactor is cultivated described sensitive cells that single-stage is cultivated or amplification culture；

It is 5～14L bioreactor single-stage training modes that described single-stage is cultivated；

Described amplification culture is 40～140L amplification culture patterns；The operation of amplification culture is:

The cell of the grown on microcarriers after single-stage being cultivated uses trypsinization as initial cell in hermetic container, strainer filtering by 90～120 μm, cell suspension is accessed and the bioreactor that next stage is bigger continues cultivate, cultivation is obtained the initial cell that cell operates as amplification culture next time the operation repeating above-mentioned amplification culture, gradual magnification to 40～the production scale of 140L.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 5, it is characterised in that step 2) in operation specifically include:

After described sensitive cells cultivates 20～48h, inoculation canine distemper virus carries out enrichment culture, and inoculum concentration is 0.001～0.01MOI；

Or, cultivate after 48～72h until described sensitive cells, stop stirring, until microcarrier be deposited to tank bottoms after discharge all cells culture fluid；Adding cell maintenance medium and inoculate canine distemper virus and carry out enrichment culture, inoculum concentration is 0.001～0.01MOI.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 6, it is characterised in that:

The formula of described cell maintenance medium is: containing 1.8～2.2% DMEM culture fluid of new-born calf serum；

The condition of described enrichment culture is: temperature 33～35 DEG C, CO₂Content 4.8～5.2%, mixing speed be 55～65rpm, dissolved oxygen is 45～55%, pH6.9～7.2, reactor automatically control cultivation.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 1, it is characterised in that:

In step 1) in, described seedling sensitive cells is inoculated in bioreactor and carries out the training method cultivated for batch cultivating or continuous perfusion culture with microcarrier；

In step 2) in, the training method of described enrichment culture is batch cultivation.

Method prepared by mink canine distemper disease virus live vaccine the most according to claim 1, it is characterised in that in step 3) in, the opportunity of described results virus-culturing fluid and microcarrier is:

There is typical CPE in the sensitive cells of more than 80%.

10. mink canine distemper disease virus live vaccine prepared by the method prepared with the mink canine distemper disease virus live vaccine described in any one of claim 1～9.