CN117659137A - Method for simultaneously producing high-titer Senicavirus and swine fever virus antigens - Google Patents
Method for simultaneously producing high-titer Senicavirus and swine fever virus antigens Download PDFInfo
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Abstract
The invention discloses a method for simultaneously producing high-titer Senicavirus and classical swine fever virus antigens. The method comprises the following steps: preparing a suspended BHK-21 cell strain by using a bioreactor, respectively inoculating the Senica cereal seed virus and the swine fever virus, setting control parameters of the bioreactor to DO 30-60%, pH 7.0-7.4, stirring at 50-100 rpm/min and at 36.5-37.5 ℃ after the inoculation, sampling at regular time during the period, and observing cytopathic conditions by using 0.4% trypan blue staining, and harvesting the suspended culture virus liquid when the cell activity rate is less than or equal to 20%. The invention realizes the large-scale propagation culture of the swine fever virus and the Senicague virus at the same time, and the process has the advantages of simple process, low cost, stable toxin production effect, high titer and the like, and lays a foundation for the large-scale production of the later-stage bivalent live vaccine.
Description
Technical Field
The invention belongs to the field of biological products for animals, and particularly relates to a method for simultaneously producing high-titer Senicavirus and swine fever virus antigens by using BHK-21 cells.
Background
SVV is a newly discovered acute pathogenic microorganism capable of causing swine idiopathic vesicular disease (swine idiopathic vesicular disease, SIVD)) and epidemic temporary newborn piglet loss (epidemic transient neonatal losses, ETNL), and has the advantages of high morbidity, fast disease course, high transmission speed, lower mortality rate of piglets and high mortality rate of piglets. Clinically, the Chinese herbal medicine composition mainly shows the symptoms of rhinoscope, hoof coronary bubble, ulcer, lameness, somnolence, diarrhea of newborn piglets, acute death and the like. There is currently no biological vaccine preparation available for the prevention and management of this disease. At present, the prevention and control of the swine Senicague disease depend on effective management of a pig farm, but pig raising modes are complex and various, and the pig farm management is relatively backward, so that the development of corresponding biological products for preventing and controlling outbreaks of the epidemic disease is urgent.
Swine fever is a highly acute, febrile, contagious disease caused by the swine fever virus (CSFV) that occurs in pigs. In recent years, swine fever still occurs, and is one of the most serious infectious diseases endangering the pig industry. Quality control of vaccines has not been solved so far, among the reasons for this is the weak proliferation of Classical Swine Fever Virus (CSFV) in cells, the lack of cytopathic effect, and the very high number of interfering factors in the production process. Therefore, the production of high-quality swine fever vaccines has high technical difficulty, and the quality control of production and inspection raw materials is the key of the production process technology. Some pig farms also feel that the antigen content in the vaccine is indeed related to the antibody production rate, the immune effect during the screening practice. The swine fever prevention and control conditions are complex, and experts consider that the swine fever with toxicity and other immunosuppressive diseases of the swine farm are combined, and if the swine farm is severely polluted, relatively efficient (relatively high in antigen content) vaccines are recommended.
Disclosure of Invention
First, the technical problem to be solved
The invention aims at providing a method for simultaneously producing high-titer Senicavirus and swine fever virus antigens by using BHK-21 cells, which lays a foundation for the mass production of the late-stage bivalent inactivated vaccine.
(II) technical scheme:
the invention is realized by the following technical scheme:
the invention provides a method for simultaneously producing high-titer Senicavirus and classical swine fever virus antigens by using BHK21 cells. The method comprises the following specific steps:
the specific preparation process of the swine saika valley virus and swine fever virus antigen comprises the following steps: preparing suspended BHK-21 cells with a bioreactor until the cell density is 3.0-5.0X10 6 The preparation method comprises the steps of respectively inoculating a Senica cereal seed virus and a classical swine fever virus to cells/ml at a MOI of 0.001-0.1 dose, setting control parameters of a bioreactor after inoculation to DO of 30-60%, pH of 7.0-7.4, stirring rotation speed of 80-100 rpm/min, temperature of 36.5-37.5 ℃, sampling at regular time during the period, staining with 0.4% trypan blue to observe cytopathic condition, and harvesting suspension culture virus liquid when the cell activity rate is less than or equal to 20%.
The suspension BHK-21 cell strain is BHK-21-BP-2; the Senica valley virus is SVV-ZM-201801 strain; the swine fever virus is a swine fever lapinized virulent strain CVCC AV1412.
In the invention, the suspension culture medium used for preparing the suspension BHK-21 cells is a serum-free full suspension culture medium, comprising a BHK201 culture medium, a BS-SFM V culture medium, a CD BHK-21 culture medium,Any one or more of VM12-SFM medium and BHK-LSM medium.
The culture medium isCommercially available commercial media: BH201 (Shenzhen Co., yishenke, product code 10501), BS-SFM V Medium (Walmer Biotechnology Co., ltd., product number BSS 203142); CD BHK-21 Medium (gibco Co., ltd., product No. A16277-03),VM12 (SFM) (Jianshun, cat 77017-198), BHK-LSM (Beijing ruilin and technology Co., ltd., cat LS-1).
Preferably, the inoculation amount of the virus is 0.01-0.05MOI, the inoculation mode is incubation inoculation, the inoculation condition is that cells are firstly settled and then are respectively inoculated with the Senica cereal seed virus and the classical swine fever virus, and the incubation is carried out for 2 hours at the temperature of 37 ℃ and the rotating speed rate of 60 rpm/min.
In a preferred embodiment of the invention, the incubation is carried out at a cell density of 3 to 5X 10 6 When the cell/ml has the activity rate of more than 95%, standing the cells for 5-8 hours, discarding 50% of culture medium, respectively inoculating the Senica cereal seed virus and the classical swine fever virus with the inoculum size of MOI=0.05, and supplementing 50% of fresh culture medium after 2 hours at 37 ℃ and 60 rpm/min.
Further, the culture parameters (pH, DO, stirring speed) are pH 7.0-7.4, such as pH 7.1, 7.2, 7.3, 7.4 or any value between any two of the above ranges. DO values are 30% to 60%, such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any value between any two ranges above. The stirring rotation speed is 80-100 r/min, such as 80r/min, 90r/min, 100r/min or any value between any two value ranges.
(III) beneficial effects
1) The invention can simultaneously produce high-titer Seneca valley virus and swine fever virus antigens by using BHK21 cells.
2) The strain is SVV-ZM-201801 strain of Senica valley virus and hog cholera lapinized virus strain (CVCC AV 1412), so that the use of virulent strain is avoided, and the safety and the immunogenicity are reliable.
3) The virus liquid is cultured by adopting suspension cells, the virus titer is high, the batch-to-batch difference is small, and a large amount of antigens can be harvested at one time. Compared with the adherent cell culture, the BHK-21-BP-2 cell can adapt to serum-free full suspension culture, and has high antigen yield and small batch-to-batch difference. The content of the virus is high, and the cell has obvious lesions.
4) Serum-free suspension culture, BVDV pollution risk and cost saving;
5) The virus content is greatly improved and the antigen harvesting time is shortened through the optimization of the virus receiving dosage, the virus receiving mode and the reactor control parameters.
6) The mixed antigen prepared by the invention has good safety and effectiveness, and can induce animals to generate stronger neutralizing antibodies after immunization. As no biological products related to the Sainika vaccine exist in the markets at home and abroad, the vaccine can effectively prevent the epidemic and the transmission of SVV and CSFV in pig groups, reduce the economic loss caused by the disease and has wide application prospect.
In a word, the invention realizes the large-scale propagation culture of the swine fever virus and the Senicague virus at the same time, and the process has the advantages of simple and convenient process, low cost, stable toxin production effect, high titer and the like, and lays a foundation for the large-scale production of the late-stage bivalent live vaccine.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Example 1 preparation of Selaginella and Swine fever Virus antigen
1. Preparation of Su Nica Gu Bing venom and Swine fever venom
1. BHK-21 suspension cell resuscitation
Frozen BHK-21 suspension cell strain (BHK-21-BP-2, preservation number is CGMCC No.17588, and the cell strain BHK-21-BP-2 can be seen in CN 110157659A) is placed in a water bath at 37 ℃ for rapid thawing. Cell addition after thawingAdding fresh serum-free whole suspension culture medium with initial density of 0.5-0.8X10 6 cells/ml were inoculated into cell shake flasks, 2% fresh bovine serum was added, and incubated at 37℃in a constant temperature shaker at 120 rpm/min. The samples were periodically stained with 0.4% trypan blue and observed under a microscope for cell viability and cell doubling rate. And when the cell culture reaches the normal multiplication rate and the cell activity reaches more than 95%, carrying out normal subculture.
2. Cell passage
The cell density reaches 3 to 5 multiplied by 10 6 When the cell viability is more than 95% per ml. Diluting and passaging with serum-free whole suspension culture medium, and obtaining final concentration of cells of 0.5-0.8X10 6 The cells/ml were placed in a constant temperature shaker at 37℃and 120rpm/min to perform expansion culture of the cell seeds.
3. Step-by-step amplification culture of BHK-21 suspension cells in bioreactor
1) Suspension culture of BHK-21 suspension cells in 10L bioreactor
Shake flask to expand cell seed, inoculating into 10L bioreactor with initial cell density of 0.5-0.8X10 6 The process parameters of the reactor are set to 36.5-37.5 ℃, pH 7.0-7.2, DO30-60 percent, stirring speed is 80-100 rpm, and the cell viability and the cell density are observed by regular sampling. When the cell density reaches 3 to 5 multiplied by 10 6 When the cell viability is more than 95% per ml, the culture is further expanded.
2) Continuous amplified culture of BHK-21 suspension cells in 10L-50L-100L bioreactor
Transferring and inoculating 10L of BHK-21 suspension cells cultured in a bioreactor into a 50L bioreactor, wherein the initial density of cells is 0.5-0.8X10 6 The process parameters of the reactor are set to 36.5-37.5 ℃, pH 7.0-7.2, DO30-60 percent, stirring speed is 80-100 rpm, and the cell viability and the cell density are observed by regular sampling. When the cell density reaches 3 to 5 multiplied by 10 6 When the cell viability is more than 95% per ml, the culture is further expanded. Cells were grown in a 100L bioreactor with the same culture procedure.
3) Production of 100L grade reactor for swine Senicague disease and swine fever virus
The 100L suspension culture cells prepared by the method are subjected to virus inoculation incubation and virus culture until the swine saiikagaa valley virus suspension culture virus liquid and swine fever virus liquid are obtained:
when the cell density reaches 3 to 5 multiplied by 10 6 When the cell/ml and the activity rate reach more than 95%, the Senica valley virus seed virus (SVV-ZM-201801, the preservation number is CGMCC No.16396, the Senica valley virus SVV-ZM-201801 can be seen in CN 109554352A) and the classical swine fever lapinized strain CVCC AV1412 are inoculated by MOI of 0.001-0.1, the reactor technological parameters are set to be 36.5-37.5 ℃, pH 7.0-7.4, DO of 30-60%, the stirring speed is 80-100 rpm, and the cell activity rate and the cell density are observed by regular sampling. When the cell viability is less than or equal to 20%, the virus liquid is harvested, and the virus titer is detected by sampling. The SVV virus content is more than or equal to 10 9.0 TCID 50 Per ml, CSFV virus content is not less than 10 6.0 TCID 50 /ml。
The serum-free full suspension culture medium can be BHK201 culture medium, BS-SFM V culture medium, CD BHK-21 culture medium,Any one or more of VM12-SFM medium and BHK-LSM medium. The present example uses BS-SFM V medium.
The above media are commercially available: BH201 (Shenzhen Co., yishenke, product code 10501), BS-SFM V Medium (Walmer Biotechnology Co., ltd., product number BSS 203142); CD BHK-21 Medium (gibco Co., ltd., product No. A16277-03),VM12 (SFM) (Jianshun, cat 77017-198), BHK-LSM (Beijing ruilin and technology Co., ltd., cat LS-1).
Further, the culture parameters (pH, DO, stirring speed) are any values between pH7.0 and 7.3, such as pH 7.1, 7.2, 7.3 or any value between any two of the above ranges. DO values are 40% to 50%, such as 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any number between any two ranges above. The stirring rotation speed is 80-100 r/min, such as 80r/min, 90r/min, 100r/min or any value between any two value ranges.
Example 2, suspension culture condition optimization of Sernikaguvirus and classical swine fever virus
For the 100L virus culture process prepared in the embodiment 1, important process parameters are further optimized, and meanwhile, the influence of the virus receiving process and the virus receiving dosage on the virus production effect is compared:
1. optimization of virus-receiving process
100L of suspension culture cells, the culture medium is commercially available commercial culture medium: BS-SFM V Medium (Womei Biotechnology Co., ltd.) for cell density of 3.about.5X10 6 When the cell/ml and the activity rate reach more than 95%, respectively inoculating Senica valley virus seed virus (SVV-ZM-201801, the preservation number is CGMCC No.16396, the Senica valley virus SVV-ZM-201801 can be seen in CN 109554352A) and classical swine fever lapinized strain CVCC AV1412 with the inoculum size of MOI=0.05, adopting the processes of direct inoculation culture and incubation inoculation culture (37 ℃,60rpm/min,2 h) for inoculation culture, setting the technological parameters of a reactor to be 36.5-37.5 ℃, pH 7.0-7.4, DO 30-60%, stirring speed of 80-100 rpm, and culturing until the cell activity rate is less than or equal to 20%, and obtaining the virus liquid.
The direct inoculation culture method comprises the following steps: the cell density reaches 3 to 5 multiplied by 10 6 When the cell/ml and the activity rate reach more than 95%, respectively inoculating two viruses with the inoculum size of MOI=0.05;
incubation inoculation culture method: the cell density reaches 3 to 5 multiplied by 10 6 When the cell/ml activity reaches more than 95%, standing the cells for 5-8 hours, discarding 50% of culture medium, inoculating two viruses respectively with the inoculum size of MOI=0.05, and supplementing 50% of fresh culture medium after the inoculation amount reaches 37 ℃,60rpm/min and 2 hours.
The effect of different virus-receiving processes on the harvested virus results are shown in table 1 below:
TABLE 1 influence of different virus-receiving processes on the harvesting of viruses
According to the test comparison result, the virus harvesting time and the virus content can be effectively shortened by optimizing the virus receiving process.
2. Toxin-receiving agent amount optimization
100L of suspension culture cells, the culture medium is commercially available commercial culture medium: BS-SFM V Medium (Walmei Biotechnology Co., ltd., product number BSS 203142). When the cell density reaches 3 to 5 multiplied by 10 6 When the cell/ml and the activity rate reach more than 95%, respectively inoculating Senica valley virus seed virus (SVV-ZM-201801, the preservation number is CGMCC No.16396, senica valley virus SVV-ZM-201801 can be seen in CN 109554352A) and classical swine fever lapinized strain CVCC AV1412 with the inoculum sizes of MOI 0.001, 0.01, 0.05 and 0.1 respectively, incubating the strain-inoculating culture (shown in step 1, 37 ℃,60rpm/min and 2 h), carrying out strain-inoculating culture, and comparing the antigen production effects of several batches of different inoculum sizes MOI (0.001, 0.01, 0.05 and 0.1) of the seed viruses with the stirring speed of 100rpm, wherein the reactor technological parameters are set to be 337 ℃, pH7.2, DO 50 percent, and the results are shown in the following table 2:
TABLE 2 influence of different amounts of toxin-receiving agent of seed viruses on the amount of cultured virus
According to the test comparison result, the virus receiving amount has obvious influence on the virus receiving time and the virus content, when the virus receiving amount is controlled within the range of 0.01-0.1, the virus harvesting time can be shortened, and the SVV virus content is further increased to 10 9.5 TCID 50 Above/ml, CSFV virus content is increased to 10 6.0 TCID 50 And the ratio of the total amount of the components to be mixed is more than/ml, which is beneficial to shortening the time cost of the routine production.
Example 3, combined culture of Sernikagu virus and Swine fever Virus and Single antigen price/price ratio
100L of suspension culture cells prepared as described above were used in the method of example 1, and after virus inoculation incubation and virus culture, swine fever virus liquid and swine fever virus liquid were harvested:
to be cell denseThe degree reaches 4 multiplied by 10 6 When the cell/ml and the activity rate reach more than 95%, the MOI 0.05 is used for inoculating the Senica valley virus seed virus (SVV-ZM-201801, the preservation number is CGMCC No.16396, the Senica valley virus SVV-ZM-201801 can be seen in CN 109554352A) and/or the classical swine fever rabbit attenuated strain CVCC AV1412 (the two viruses are inoculated simultaneously or respectively), the inoculation method is to incubate and connect the virus, the reactor technological parameters are set at 37 ℃, pH7.2, DO 50 percent, the stirring speed is 100rpm, and the cell activity rate is cultivated to be less than or equal to 20 percent, and the virus content is detected respectively. The method can be used for simultaneously culturing the swine Senicavirus and the swine fever virus, so that the titer of the swine Senicavirus and the titer of the swine fever virus can be obviously improved, and the swine fever virus has better effect than single culture. Specifically, the results are shown in Table 3.
TABLE 3 comparison of virus contents of Sernikaguvirus, swine fever Virus, mixed culture and Single culture
Example 4 preparation of live vaccine
100L of suspension culture cells prepared as described above were used in the method of example 1, and after virus inoculation incubation and virus culture, swine fever virus liquid and swine fever virus liquid were harvested:
to a cell density of 4X 10 6 When the cell/ml and the activity rate reach more than 95%, the MOI 0.05 is used for inoculating the Senica valley virus seed virus (SVV-ZM-201801, the preservation number is CGMCC No.16396, the Senica valley virus SVV-ZM-201801 can be seen in CN 109554352A) and the swine fever lapinized strain CVCC AV1412, the reactor technological parameters are set to be 37 ℃, pH7.2, DO 50 percent and the stirring speed is 100rpm, and when the cell activity rate is less than or equal to 20 percent, the virus liquid is obtained.
Fully and uniformly mixing the harvested swine Senicavirus antigen and swine fever virus liquid with a sucrose gelatin protective agent according to a ratio of 7:1 (virus liquid: protective agent), and carrying out freeze vacuum drying to prepare the bivalent live vaccine.
Example 5 safety test of live vaccine
The live vaccine prepared in the example 4 is subjected to a safety evaluation test, 4-head immune live vaccines of swine fever virus antibody negative and swine Senicavirus antibody negative are screened, the live vaccine is observed for 5-7 days before inoculation, each body temperature is measured once every day in the morning and afternoon, each head is 5ml (10 parts), and 2 heads of the control group are not vaccinated to serve as negative control. Body temperature is measured every day after immunization for 0 to 7 days; continuously observing the feeding condition of the swinery for 14 days; observing and touching whether the local part of the injection part has a tumor or not.
Table 4 thermometer after live vaccine immunization
The results are shown in Table 4, no obvious body temperature rise phenomenon appears after the live vaccine is used for immunizing piglets, and the pig flock is normal in feeding and good in mental state; the vaccine injection part has no pain feeling when touched and has no tumor, which indicates that the vaccine has good safety to pigs.
Example 6 monitoring of antibody levels at various times after immunization with live vaccine
For evaluating the immune effect of the inactivated vaccine, 4 pigs are selected for carrying out an immune test, and 1 ml/head of live vaccine is injected into the neck muscle; the 4 control pigs are not vaccinated, serum is collected at 0d, 7d, 14d, 21d, 28d and 35d after immunization as a blank control, and the production level of anti-swine Senicarbazin antibody in the serum and the commercial Kernel Rapid swine fever virus antibody detection kit are used for measuring the swine fever virus antibody level.
The specific steps of the swine Senicavirus antibody assay are as follows:
1) Placing all the pig serum to be detected in a 56 ℃ water bath for incubation for 30min, and carrying out inactivation treatment;
2) Serum was used in 1:2, 1:4, 1 in 96 well cell culture plates: 8. … 1:2048 fold dilutions were made, each 4 replicates, and each well contained 100 μl of diluted serum and tested with SVV neutralizing antibody assay.
3) Diluting virus solution with cell maintenance solutionReleased to 200TCID 50 0.1mL, adding the diluted virus solution into the diluted serum obtained in the above step, mixing well at 37deg.C with 5% CO 2 Incubate in incubator for 1h.
4) BHK21 cells with good growth status were passaged 1:3 into 96-well plates at 100. Mu.l/well. And simultaneously setting a positive control group and a negative control group of the virus liquid and the non-added virus liquid. Placing at 37deg.C and 5% CO 2 Culturing in an incubator, observing cytopathic condition every day, recording pathological condition, and calculating serum neutralizing antibody condition of each group according to a Reed-Muench method. The data were collated after serum neutralization antibody titers were determined and the results are shown in table 5.
TABLE 5 production of neutralizing antibody levels by Senicarbazin after immunization with inactivated vaccine
Swine fever virus antibody level assays were performed in accordance with commercial kits. See Kernel Rapid swine fever virus antibody detection kit instructions.
Negative positive determination criteria:
the blocking rate is more than or equal to 40 percent, the blocking rate is less than 40 percent, the positive is judged,
TABLE 6 antibody levels produced by classical swine fever virus after live vaccine immunization
As shown in tables 5-6, the bigeminal live vaccine can generate relatively high antibodies, the highest SVV antibodies can reach 1:1024 after 35 days of immunization, and the antibodies start to transfer positive after 14 days of immunization. The control group was not vaccinated and no neutralizing antibodies against swine saiikague virus and no swine fever antibodies were detected. The data lays a certain theoretical and practical foundation for clinical use of swine Senicavirus disease and swine fever live vaccine.
The present invention is not limited to the preferred embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.
Claims (7)
1. A preparation method of a swine Senicavirus antigen and a swine fever virus antigen comprises the following steps:
preparing a suspension BHK-21 cell strain by a bioreactor until the cell density is 3.0-5.0x10 6 The cells/ml are inoculated with the Senica cereal seed virus and the classical swine fever virus respectively at the MOI dosage of 0.001-0.1, the control parameters of the bioreactor after the inoculation are set to DO of 30-60%, the pH of 7.0-7.4, the stirring rotation speed of 50-100 rpm/min and the temperature of 36.5-37.5 ℃, sampling is carried out regularly during the period, the cytopathic condition is observed by 0.4% trypan blue staining, and the suspension culture virus liquid is harvested when the cell activity rate is less than or equal to 20%.
2. The method according to claim 1, characterized in that: the inoculation amount of the virus is 0.01-0.05MOI, the inoculation mode is incubation inoculation, the inoculation condition is that cells are firstly settled and then are respectively inoculated with the Senica cereal seed virus and the classical swine fever virus, and the incubation is carried out for 2 hours at the temperature of 37 ℃ and the rotating speed rate of 60 rpm/min.
3. The method according to claim 1, characterized in that: the suspension BHK-21 cell strain is BHK-21-BP-2; the Senica valley virus is Senica valley virus strain SVV-ZM-201801, and the hog cholera virus is hog cholera lapinized virus strain CVCC AV1412.
4. The method of claim 1, wherein the medium used to prepare the suspension of BHK-21 cells is serum-free total suspension medium.
5. The method of claim 4, wherein the serum-free total suspension medium is BHK201 medium, BS-SFM V medium, CD BHK-21 medium,Any one or more of VM12-SFM medium and BHK-LSM medium.
6. Use of the method according to claims 1-5 for the preparation of a bivalent vaccine against swine celecoxib valley virus and swine fever virus.
7. The use according to claim 6, wherein the bivalent vaccine is a bivalent live vaccine.
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