CN104004720B - A kind of large scale and high density produces the method for porcine circovirus 2 type antigen - Google Patents

Abstract

The invention relates to a method for large-scale high-density production of porcine circovirus type 2 antigen. The present invention uses the bioreactor microcarrier suspension culture process to replace the existing spinner bottle culture process to produce porcine circovirus type 2 antigen. The method can greatly reduce production costs and increase production, and the unit antigen cost is reduced by 80% to 80% compared with the spinner bottle process. 90%, the production cycle is reduced by 7 days, and the output is increased by 3 to 10 times; because it can harvest antigens multiple times, the unit antigen cost is reduced by 40% to 50% compared with the traditional reactor culture process, and the output is increased by 3 to 5 times. The produced antigen has no serum residue, and the produced vaccine is safer. At the same time, the batch-to-batch difference of the product is small, the quality is stable and easy to control, and the output and quality of the product can be significantly improved.

Description

A method for large-scale high-density production of porcine circovirus type 2 antigen

technical field

The invention relates to a method for large-scale and high-density production of porcine circovirus type 2 vaccine antigen by applying bioreactor microcarrier suspension culture technology, and belongs to the field of veterinary biological products.

Background technique

At present, the production of porcine circovirus 2 (Porcine circovirus2, PCV2) vaccine antigen mainly adopts the cell spinner bottle culture method, and a few adopts the cell suspension culture method, and the semi-finished antigen virus content of the spinner bottle process is low, only 10 ^4.5-6.0 TCID ₅₀ /ml, unable to provide stable qualified antigen (qualified antigen per ml antigen content should be ≥ 10 ^5.5 TCID ₅₀ ), high concentration is required. Moreover, the spinning process is labor-intensive. A batch of 100-200 spinning bottles is required to produce a batch, and multiple people are required to carry out long-term digestion and passage operations at the same time, which increases the risk of contamination; the production cycle is long, requiring 10 days; the efficiency is low, and each cultivation It can only be harvested once; the culture medium contains a certain concentration of bovine serum, and there are many antigenic impurities, which can easily cause stress reactions; the production cost is high, and the cost of labor, site and raw materials is high; the requirements for the environment are high, and large Site and GMP workshop; there are large differences between different production batches and spinner bottles of the same production batch, which may easily cause differences in the quality of antigens within and between batches, thereby affecting the stability of vaccine quality; spinner bottle cleaning requires a lot of water , and the toxic wastewater produced needs to be specially treated, which is easy to pollute the environment. If it is not handled properly, it will cause biosafety and public health problems. The common cell microcarrier suspension culture process has improved compared with the spinner bottle process, but there is still a large room for improvement. The traditional microcarrier suspension culture process still needs 6 to 7 days for its production cycle, and each culture can only harvest 1 times; still rely on spinner bottle to provide toxic cells, cannot avoid the toxic waste water produced by spinner bottle cleaning, and traditional spinner bottle process and traditional reactor microcarrier suspension culture process, there is a high content of serum in the antigen produced by it, so Production of vaccines will cause adverse reactions in animals to varying degrees.

Contents of the invention

The object of the invention is to overcome the deficiencies of the existing spinner bottle culture method and the traditional cell suspension microcarrier culture method, and provide a method for large-scale high-density production of porcine circovirus type 2 vaccine antigen using reactor microcarrier suspension culture technology method. This method can greatly reduce the production cost and increase the output. Compared with the spin bottle process, the unit antigen cost is reduced by 80% to 90%, the production cycle is reduced by 7 days, and the output is increased by 3 to 10 times; compared with the traditional reactor culture process, the unit antigen cost is reduced by 40%. % to 50%, the yield is increased by 3 to 5 times, because it can harvest the antigen multiple times. Moreover, the process occupies a small area, can quickly carry out large-scale production, has low environmental requirements, high degree of automation, low labor cost, small difference between production batches, stable quality and easy control, and can significantly improve the output and quality of products.

In order to realize above-mentioned index, the present invention has taken following technical scheme:

A method for large-scale high-density production of porcine circovirus type 2 antigen, the method comprising the steps of:

(1) Preparation of seed cells Preheat sterile normal saline, use the preheated normal saline to wash the healthy clone cells PK-15W covered with a single layer as seed cells, add 20-40ml trypsin for digestion, during digestion , observe the changes of the cell layer at all times, and when the cell layer becomes white mist and partly falls off, add the cell growth medium containing concanavalin A and D-glucosamine with serum low-sugar DMEM, the seed cells stop digestion, and shake quickly to transfer bottle, to make the cells on the wall fall off, after repeated blowing and blowing to disperse the cells, divide the bottles according to the passage ratio of 1:3 to 1:5;

(2) Preparation of seedling cells Weigh the Cytodex-1 microcarriers according to the final culture volume to make the final concentration 5-10g/L, soak and wash with PBS, and use the cells in a stirred bioreactor after sterilization Balance the growth solution and set it aside; select seed cells in good growth state to inoculate the bioreactor with an inoculation density of 1-5×10 ⁵ cells/ml;

(3) After inoculating the virus, inoculate the PCV2 kind of virus with 3%～8% of the final culture volume; 80～120r/min;

(4) Harvesting and content determination of the virus After the cells were cultured in the cell growth medium for 48-72 hours under the aforementioned conditions, they were allowed to stand still, the supernatant was discharged, and serum-free low-sugar DMEM cells containing concanavalin A and hydrolyzed casein were added For maintenance solution, adjust the control conditions of the reactor as follows: pH 7.0-7.4, DO 20%-80%, stirring speed 100r/min-180r/min, continue to maintain for 48-72h, let it stand still, and perform the first supernatant Harvest, observe the state of the cells, if the state of the cells is good, add the same amount of fresh cell maintenance solution as the harvested supernatant, at pH 7.0-7.4, DO is 20%-80%, stirring speed is 100r/min-180r/min Continue to culture under the condition of 48～72h after standing still, carry out the supernatant harvest for the second time, repeat this process until the cells on the microcarriers all fall off, end the whole culture process, after harvesting the supernatant, the microcarriers are recycled, and The harvested antigens were repeatedly frozen and thawed three times as semi-finished products, and the virus content was determined.

(5) Prepare a vaccine with the above-mentioned porcine circovirus type 2 antigen semi-finished product.

(6) the method for preparing vaccine with the porcine circovirus type 2 antigen semi-finished product described above, comprising: PCV2 strain is proliferated in a large amount in PK-15W cell, adds common adjuvant after formaldehyde inactivation and carries out emulsification, prepares conventional Adjuvanted vaccines.

detailed description

The bioreactor is used as the culture equipment, the microcarrier is used as the carrier to support cell growth, the cloned cell PK-15W (formerly known as PK-15B1, CCTCC No.C200936) is used as the seedling cell, and PCV2 is used as the seedling poison; The low-sugar DMEM of globulin A and D-glucosamine is used as the seedling cell growth liquid; the serum-free low-sugar DMEM containing concanavalin A and hydrolyzed casein is used as the seedling cell maintenance liquid; and comprises the following steps:

(1) Seed cell culture

Wash the overgrown seed cell monolayer once with preheated sterile saline, add 20-40ml of trypsin digestion solution to digest, keep rotating the spinner bottle until all the cells fall off, add cell growth solution to stop digestion, mix well, follow 1 : 3 to 1:5 passage ratio bottle;

(2) Preparation of seed cells for inoculation

1) Preparation of the bioreactor Select a suitable bioreactor according to the volume of the culture, connect various pipelines, feeding materials and auxiliary tanks, change the liquid interface, connect the temperature, pH, DO electrodes, and calibrate the electrodes;

2) Preparation of microcarriers Weigh Cytodex-1 microcarriers into a siliconized glass bottle according to the culture volume and concentration of microcarriers, add 40 to 50 times the mass of microcarriers in PBS buffer and soak for 1 hour, then replace with fresh PBS The buffer solution continued to soak overnight; replace 30 times the volume of PBS buffer solution in glass bottles or bioreactors to prepare for sterilization;

3) Sterilization Before sterilization, carefully check the pipes and joints to ensure that the connection is intact, then perform an air tightness test, and prepare for sterilization after the test is passed; according to the scale of the bioreactor, sterilize offline below 10L, in a pressure cooker at 121°C High pressure for 30 minutes, and natural cooling after sterilization; reactors over 10L are sterilized in place.

4) Seedling cell culture Set the concentration of Cytodex-1 microcarrier to 5-10g/L in the bioreactor, and use the seedling cell growth solution to balance for standby; select seed cells with good growth status to inoculate the bioreactor , the inoculation density was 1-5×10 ⁵ cells/ml, and cultured at 37°C (see panel A in Figure 2).

(3) poisoning

About 8 to 10 hours after the cells are inserted into the reaction tank and cultured for 8 to 10 hours, when it can be seen from the sampling microscope that all the inserted cells are attached to the microcarriers (see Figure B in Figure 2), inoculate and inoculate according to 3% to 5% of the volume of the cell culture solution. Inject porcine circovirus type 2 seed virus; after inoculation, use seedling cell growth medium to continue culturing under the conditions of 37°C, pH7.0-7.4, DO20%-80%, and stirring speed 80-120r/min;

(4) Virus harvest and assay

After the cells were cultured for 48-72 hours in the above-mentioned cell growth solution for seedling preparation, pH7.0-7.4, DO20%-80%, stirring speed 80-120r/min (see Figure C and Figure D in Figure 2), statically place, release the supernatant, replace the seedling cell maintenance solution, and continue to cultivate for 48-72h under the conditions of pH 7.0-7.4, DO50%-70%, stirring speed 100-180r/min, after standing still, release the supernatant and harvest , observe that if most of the cells have fallen off, harvest them together with the microcarriers. If the cells are in good condition, continue to harvest the supernatant, and add the seedling cell maintenance solution again to continue culturing for 48-72 hours, then let it stand still, suck out the supernatant to harvest, and repeat the process. The process is until most of the cells fall off from the microcarriers, and the harvested antigens are repeatedly frozen and thawed for 2 to 3 times as a semi-finished product for virus content determination.

The virus content was determined by IFA assay, that is, the PCV2 virus solution was diluted 10 ^-1 to 10 ^-8 times, and then inoculated with the maintenance solution on the PK15-W cells covered with a single layer, 4 wells for each dilution, each well 0.2ml, set up a negative control at the same time, after culturing in an incubator containing 5% _CO2 at 37°C for 48h, fix the cells with absolute ethanol, and use indirect immunofluorescence (IFA) to determine the presence of PCV2 positive cells (green fluorescence) in each dilution The number of wells was calculated according to Karber's method to calculate the TCID ₅₀ of the virus.

Vaccine preparation with the porcine circovirus type 2 antigen semi-finished product described above

The method for preparing a vaccine with the porcine circovirus type 2 antigen described above comprises: proliferating PCV2 strains in large quantities in PK-15W cells, adding an adjuvant to emulsify after formaldehyde inactivation, and preparing an adjuvant inactivated vaccine.

The bioreactor described above is a bioreactor that can automatically control temperature, pH, dissolved oxygen, and stirring speed parameters, and is suitable for microcarrier cultivation, with a volume of 2L to 1000L; the microcarrier is Cytodex-1. The concentration used is 5-10g/L, the culture temperature is 30-38°C, the pH is 7.0-7.4, the dissolved oxygen is 20%-80%, and the stirring speed is 80-180r/min. Correct, prepare, sterilize.

The composition (V/V) of the seed cell growth liquid of the present invention is: low-sugar DMEM (AXF42461DC HYCLONE) 89%～94%, newborn bovine serum (NBCS, 130502 Zhejiang Tianhang Biotechnology Co., Ltd.) 5%～10%, Double antibody 1%.

The concentration of trypsin in the trypsin digestion solution of the present invention is 0.25%, and the concentration of EDTA is 0.02%.

The composition (V/V) of seedling cell growth liquid of the present invention is: low-sugar DMEM85%～92%, NBCS5%～10%, double antibody 1%, D-glucosamine (C4875SIGMA) 0.5%～2%, with Concanavalin A (C5275SIGMA) 0.5% to 2%.

The composition (V/V) of seedling cell maintenance liquid of the present invention is: low-sugar DMEM95%～98%, concanavalin A0.5%～2%, hydrolyzed casein (BCBL0573V Switzerland FLUKA) 0.5%～2% %, double antibody 1%.

The double antibody of the present invention is a solution containing 10000 IU/ml of penicillin and 10 mg/ml of streptomycin.

The composition and content of physiological saline of the present invention are: NaCl0.9g/L, pH is 7.0～7.4

The composition and content of the PBS buffer solution of the present invention are: NaCl8g/L, _KCl0.2g /L; _KH2PO40.24g /L; _{Na2HPO4 · 12H2O3.65g} /L, pH 7.2～7.4 .

The biological materials and reagents used in the present invention are all commercial products unless the sources are indicated, purchased from domestic reagent stores.

Microorganism resource information involved in the present invention

The microbial resources involved in the present invention include: Porcine circovirus type 2 (Porcine circovirus2, PCV2) SH strain (preservation number is CGMCC No.2389, provided by Professor Jiang Ping of Nanjing Agricultural University); PK-15W (formerly known as PK-15B1, Preservation number: CCTCC No.C200936, provided by Professor Jiang Ping of Nanjing Agricultural University).

Beneficial effects of the present invention

The invention relates to a method for large-scale high-density production of porcine circovirus type 2 antigen. The present invention uses the bioreactor microcarrier cell suspension culture process to replace the existing spinner bottle cell culture process to produce porcine circovirus type 2 antigen. The method can greatly reduce production costs and increase production, and the unit antigen cost is reduced by 80% compared with the spinner bottle process. % to 90%, the production cycle is reduced by 7 days, and the output is increased by 3 to 10 times; because it can harvest antigens multiple times, the unit antigen cost is reduced by 40% to 50% compared with the traditional reactor culture process, and the output is increased by 3 to 5 times. The batch-to-batch difference of the products is small, the quality is stable and easy to control, and the output and quality of the products can be obviously improved.

(1) The present invention uses bioreactor microcarrier cell suspension culture technology to replace existing spinner bottle cell culture technology to produce porcine circovirus type 2 antigen, which can solve the problems of low production efficiency, unstable product quality, low virus content, cost reduction, To reduce the stress response of finished vaccines, through changes in production technology and process, the quality and yield of vaccines can be improved in an all-round way, and the safety of vaccines can be improved.

(2) The present invention uses a bioreactor for vaccine production, which has a higher level of automation than the spinner bottle process, reduces labor costs, simple and stable production process, simple and easy operation and easy expansion, and reduces the number of cell passages and pancreatic enzymes compared with the traditional bioreactor process. Damage to the cells, serum-free culture, increase the number of virus harvests, and add concanavalin A to the seedling cell maintenance solution to increase the virus content, thereby increasing the production and titer of antigens, and further reducing the cost of seedling production .

(3) The virus content of the product of the present invention is 10-30 times higher than the traditional spinner bottle cell culture method, and the virus content can be stabilized between 10 ^6.5-7.0 TCID ₅₀ /ml; the antigen quality is stable, and the virus antigens harvested in each tank are uniform, It is easy to produce on a large scale, and the poisonous part of the whole production process is completely carried out in closed tanks and pipelines, which are not open, and do not involve other biosafety and other problems existing in the traditional spinner bottle production process and traditional reactor production process. public health issues.

Description of drawings

Fig. 1: preparation process flow chart of the present invention

Figure 2: The growth of the seedling cells after they are inserted into the reaction tank. In Figure A, the cells have just been inserted into the culture tank and the cells are suspended in the culture medium between the microcarriers; in Figure B, the cells have been cultured on the carrier for 8-10 hours Adhering to the wall, there are no free cells in the solution; in Figure C, the state of cell culture for 48 hours; in Figure D, a small amount of cells have fallen off the carrier at 72 hours, and the medium was changed at this time.

Example

The following examples are to further illustrate the present invention, but these examples do not constitute a limitation to the present invention.

Example 1

A method for large-scale high-density production of PCV2 antigens, comprising the steps of:

(1) Culture of seeded cells on roller bottles

Wash the seed cells of PK-15W (formerly known as PK-15B1, CCTCCNo.C200936, provided by Professor Jiang Ping, Nanjing Agricultural University) covered with monolayer with preheated sterile saline, add 30ml trypsin digestion solution to digest , continuously rotate the spinner bottle until all the cells fall off, add seed cell growth solution to stop digestion, mix well, and divide the bottles according to the passage ratio of 1:3;

(2) Inoculation culture of seed cells on bioreactor

In the bioreactor, the density of the Cytodex-1 microcarrier (GE Healthcare product) is set to 5g/L, balances with the seedling cell growth fluid, makes the microcarrier fully infiltrate in the cell growth fluid; then insert (1 ) cultured PK-15W cells in a good growth state, the inoculation density is 4×10 ⁵ cells/ml, inoculated with PCV2 seed virus according to 5% (V/V) of the cell culture medium (preservation number is CGMCC No.2389, provided by Nanjing Professor Jiang Ping, Agricultural University) seed solution; after inoculation, cell growth solution was used to cultivate at pH 7.2, DO (dissolved oxygen) 50%, and stirring speed 80r/min;

(3) Virus harvest and assay

After the cells were cultured under the aforementioned conditions for 48 hours and left to stand, release the supernatant, add the cell maintenance solution, and adjust the control conditions of the reactor as follows: pH 7.2, DO 50%, stirring speed 120r/min and continue to maintain After 48 hours, let stand for the first supernatant harvest, observe the cell state, the cell state is good, add the same amount of fresh cell maintenance solution as the harvest supernatant, at pH 7.2, DO is 50%, stirring speed is 120r/ Continue to culture for 72 hours under the condition of 1 min, then let it stand still, harvest the supernatant for the second time, observe the cell state, the cell state is good, add the same amount of fresh cell maintenance solution as the harvested supernatant, at pH 7.2, DO is 50% , under the condition of stirring speed of 120r/min, continue to cultivate for 72h, then let it stand still, and harvest the supernatant for the third time, observe the cell state, the cell state is good, and add fresh cell maintenance solution equal to the harvested supernatant, at pH7. 2. Continue to culture for 48 hours under the condition of DO is 50%, stirring speed is 120r/min, and the cells are basically shed. After harvesting the supernatant, recover the microcarriers, mix the antigens harvested several times and freeze them repeatedly. After thawing for 3 times, the cell debris was removed by filtration, the virus content was determined, and frozen at -20°C as a semi-finished product.

Semi-finished product inspection

The virus content of the semi-finished product was determined by IFA assay, that is, the PCV2 virus solution was diluted ^10-1 to ^10-8 times with the maintenance solution and then inoculated into PK15-W cells that were covered with a monolayer, with 4 wells for each dilution, and each Well 0.2ml, set up a negative control at the same time, after cultivating in an incubator containing 5% _CO2 at 37°C for 48h, fix the cells with absolute ethanol, and use indirect immunofluorescence (IFA) to determine the presence of PCV2 positive cells (green fluorescence) in each dilution According to Karber's method, calculate the virus TCID ₅₀ .

According to the national standard, the virus content should be ≥10 ^5.5 TCID ₅₀ /ml. The virus content of the semi-finished product of porcine circovirus type 2 in this example is 10 ^6.66 TCID ₅₀ /ml.

In the above technical scheme, the bioreactor is a bioreactor capable of automatically controlling parameters such as temperature, pH, DO (dissolved oxygen), and stirring speed, and is suitable for microcarrier cultivation, with a volume of 7.5L.

In the above technical solution, the composition (V/V) of the seed cell growth solution is: 89% low-sugar DMEM, 10% NBCS, and 1% double antibody.

In the above technical scheme, the concentration of trypsin in the trypsin digestion solution is 0.25%, and the concentration of EDTA is 0.02%.

In the above technical solution, the components (V/V) of the seedling cell growth solution are: low-sugar DMEM 87%, NBCS 10%, double antibody 1%, D-glucosamine 1%, Concanavalin A 1%.

In the above technical solution, the composition (V/V) of the seedling cell maintenance solution is: 97% low-sugar DMEM, 1% concanavalin A, 1% hydrolyzed casein, and 1% double antibody.

In the above technical solution, the double antibody is a solution containing 10000 IU/ml penicillin and 10 mg/ml streptomycin.

In the above technical solution, the composition and content of the physiological saline is: NaCl 0.9g/L, and the pH of the physiological saline is 7.0-7.4

In the above technical scheme, the composition and content of the PBS buffer solution are: NaCl8g/L, _KCl0.2g /L; _KH2PO40.24g /L; _{Na2HPO4 · 12H2O3.65g} /L, the The pH of the PBS buffer was 7.2.

Example 2

A method for large-scale high-density production of porcine circovirus type 2 antigen, comprising the steps of:

(1) Culture of seeded cells on roller bottles

Use preheated sterile saline to wash the healthy PK-15W cells covered with a single layer once, add 30ml of trypsin digestion solution to digest, rotate the spinner bottle until all the cells fall off, add seed cell growth solution to stop digestion, mix well, Divide vials according to the passage ratio of 1:3;

(2) Inoculation culture of seed cells on bioreactor

In the bioreactor, the density of the Cytodex-1 microcarrier is set to 6g/L, and the seedling cell growth solution is used to balance the standby; the PK-15W seed cells with good growth status are selected to be inoculated in the bioreactor, and the inoculation density is 2.5×105 cells/ml, inoculate PCV2 seed solution according to ⁵ % of the cell culture volume; after inoculation, use seedling cell growth solution to continue culturing at pH 7.2, DO50%, stirring speed 80r/min;

(3) Virus harvest and assay

After the cells were cultured for 72 hours under the aforementioned conditions, they were allowed to stand still, the supernatant was discharged, and the cell maintenance solution was added, and the control conditions of the reactor were adjusted to pH 7.2, DO was 50%, and the stirring speed was 150 r/min, and then maintained for 72 hours. Stand still, harvest the supernatant for the first time, observe the cell state, the cell state is good, add the same amount of fresh cell maintenance solution as the harvested supernatant, at pH 7.2, DO is 50%, and the stirring speed is 120r/min Continue to culture under the conditions for 72 hours, then let it stand still, harvest the supernatant for the second time, observe the cell state, the cell state is good, add the same amount of fresh cell maintenance solution as the harvested supernatant, at pH 7.2, DO is 50%, stir Continue to culture at a speed of 150r/min for 72 hours, then let it stand still, and harvest the supernatant for the third time to observe the cell state. The cell state is good, and the same amount of fresh cell maintenance solution as the harvested supernatant is added. At pH 7.2, The DO is 50%, the stirring speed is 150r/min, and the culture is continued for 48 hours, and the cells are basically shed. Thaw 3 times, filter to remove cell debris, then measure the virus content, and store at -20°C as a semi-finished product.

Semi-finished product inspection and vaccine manufacturing, finished product inspection

1. Determination of virus content in semi-finished products: according to the national standard, the virus content should be ≥ 10 ^5.5 TCID ₅₀ /ml, and the virus content of the semi-finished products in this example was determined to be 10 ^7.0 TCID ₅₀ /ml.

2. Sterility test: According to the "Quality Standards for Veterinary Biological Products of the People's Republic of China" appendix 301, aseptic growth.

3. Finished product inspection: The finished product inspection is carried out in accordance with the "Quality Standards for Porcine Circovirus Type 2 Inactivated Vaccine (SH Strain)". The result is sterile, free of mycoplasma, and the antibody titer is 1:6400, which meets the requirements.

In the above technical solution, the bioreactor is a bioreactor capable of automatically controlling parameters such as temperature, pH, dissolved oxygen, and stirring speed, and is suitable for microcarrier cultivation, with a volume of 100L.

In the above technical solution, the composition (V/V) of the seed cell growth solution is: 89% low-sugar DMEM, 10% NBCS, and 1% double antibody.

In the above technical scheme, the mass fraction of trypsin in the trypsin digestion solution is 0.25%, and the mass fraction of EDTA is 0.02%.

In the above technical solution, the components (V/V) of the seedling cell growth solution are: low-sugar DMEM 85%, NBCS 10%, double antibody 1%, D-glucosamine 2%, Concanavalin A 2%.

In the above technical solution, the composition (V/V) of the cell maintenance solution for seedling production is: 97% of low-sugar DMEM, 1% of concanavalin A, 1% of hydrolyzed casein, and 1% of double antibody.

In the above technical solution, the double antibody is a solution containing 10000 IU/ml of penicillin and 10 mg/ml of streptomycin.

In the above technical solution, the composition and content of the physiological saline is: NaCl 0.9g/L, and the pH of the physiological saline is 7.0-7.4

In the above technical scheme, the composition and content of the PBS buffer are: _NaCl8g /L, _KCl0.2g /L; _KH2PO40.24g /L _{; Na2HPO4.12H2O3.65g} /L, the The pH of the PBS buffer was 7.2.

Example 3

——Comparison of porcine circovirus 2 vaccine antigens produced by different processes

Table 1 Comparison of porcine circovirus 2 vaccine antigens produced by different processes

The results in Table 1 show that the TCID ₅₀ of the inventive process is obviously 0.5 to 1 titer higher than that of the spinning bottle process, and the TCID ₅₀ of the suspension process is _more stable than that of the spinning bottle, and the difference is smaller. The antigens produced by each process were made into finished vaccines and injected into mice, and the serum was taken to measure antibodies by ELISA. The results showed that the antibody level of the vaccines made by the inventive process was higher than that of spinner bottles.

The "embodiment 1" and "embodiment 2" mentioned in this specification refer to describing specific features, structures or characteristics in combination with the embodiment. Furthermore, when a specific feature, structure or characteristic is described in combination with any example, it is claimed that the realization of such feature, structure or characteristic in combination with other examples also falls within the scope of the present invention.

Although the present invention has been described here with reference to the examples of the present invention, it should be understood that those skilled in the art can devise many other modifications and implementations, and these modifications and implementations will fall within the scope and spirit of the principles disclosed in the application Inside. More specifically, within the scope of the disclosure and claims of the present application, various modifications and improvements can be made to the components and/or layout of the subject combination layout.

Claims (1)

1. a method for producing porcine circovirus type 2 vaccine, the method may further comprise the steps: (1) Preparation of seed cells: preheat sterile normal saline, use the preheated normal saline to wash the healthy clone cells PK-15W covered with a single layer as seed cells, add 20-40ml of trypsin to digest, digest During this period, observe the changes of the cell layer at all times. When the cell layer becomes white mist and partially falls off, add the seed cell growth solution containing concanavalin A and D-glucosamine with serum low-sugar DMEM, and the seed cells stop digestion and rapidly Shake the spinner bottle to make the cells on the wall fall off. After repeated blowing and blowing to disperse the cells, divide the bottles according to the passage ratio of 1:3 to 1:5; (2) Preparation of seedling cells: Weigh Cytodex-1 microcarriers according to the final culture volume to make the final concentration 5-10g/L, soak and wash with PBS, and use in a stirred bioreactor after sterilization The seedling cell growth solution was balanced and set aside; the seed cells in good growth state were selected to inoculate the bioreactor, and the inoculation density was 1-5×10 ⁵ cells/ml; (3) Inoculation: Subsequently, PCV2 seed virus was inoculated with 3% to 8% of the final culture volume, and the preservation number was CGMCC No.2389; after inoculation, the reactor control conditions were set to be: pH 7.0 to 7.4, DO 20 %～80%, the stirring speed is 80～120r/min; (4) Harvesting and content determination of the virus: After the cells were cultured in the seedling cell growth medium for 48-72 hours under the aforementioned conditions, they were allowed to stand still, the supernatant was discharged, and a serum-free low-sugar solution containing concanavalin A and hydrolyzed casein was added. DMEM seedling cell maintenance solution, adjust the control conditions of the reactor as follows: pH 7.0-7.4, DO 20%-80%, stirring speed 100r/min-180r/min, continue to maintain for 48-72h and then stand still for the first Harvest the supernatant and observe the cell state. If the cell state is good, add fresh seedling cell maintenance solution equal to the harvested supernatant. The pH is 7.0-7.4, the DO is 20%-80%, and the stirring speed is 100r/min. Under the condition of ~180r/min, continue to cultivate for 48 ~ 72h, then let it stand still, and harvest the supernatant for the second time. Repeat this process until all the cells on the microcarriers fall off, and end the whole culture process. After harvesting the supernatant, the microcarriers are Recycled, and the harvested antigen was repeatedly frozen and thawed 3 times as a semi-finished product, and its virus content was determined; (5) add adjuvant to carry out emulsification with above porcine circovirus type 2 antigen semi-finished product through formaldehyde inactivation, be prepared into conventional adjuvant vaccine; The ingredients (V/V) of the above-mentioned seed cell growth solution are: low-sugar DMEM 89%-94%, NBCS 5%-10%, double antibody 1%; The ingredients (V/V) of the seedling cell growth solution mentioned above are: 85%-92% of low-sugar DMEM, 5%-10% of NBCS, 1% of double antibody, 0.5%-2% of D-glucosamine, and Soy protein A 0.5% ~ 2%; The ingredients (V/V) of the seedling cell maintenance solution mentioned above are: 95%-98% low-sugar DMEM, 0.5%-2% concanavalin A, 0.5%-2% hydrolyzed casein, 1% double antibody .