CN106399261A - Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers - Google Patents
Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers Download PDFInfo
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- CN106399261A CN106399261A CN201610861307.XA CN201610861307A CN106399261A CN 106399261 A CN106399261 A CN 106399261A CN 201610861307 A CN201610861307 A CN 201610861307A CN 106399261 A CN106399261 A CN 106399261A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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Abstract
The invention discloses a method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers. The method comprises the following steps: reviving BHK21 cells, carrying out adherent culture on the cells in a square bottle, digesting the cells, amplifying the cells and cultivating the cells by transferring the cells into another bottle; adhering the BHK21 cells to the wall to gradually carry out enlarge cultivation until needed cell density is reached in a reactor; adding the treated microcarriers into the reactor, wherein dosage of the microcarriers is 1 to 5g/L; digesting the BHK21 cells, transferring the BHK21 cells into a reactor to cultivate through the microcarriers, wherein sampling, digesting and counting density is 1 to 4*10<9>/L, viruses are inoculated by 1 to 5 percent, cell activity is less than or equal to 5 percent; harvesting a culture solution, refrigerating the culture solution at a low temperature for later use; and after receiving the virus through a microcarrier reactor, detecting LD50. The method has the characteristics of high degree of automation, high virus titer of seed virus, good seed virus consistency, great yield, and solves the problems such as poor product quality stability and low yield in production of the foot-and-mouth disease seed virus.
Description
Technical field
The present invention relates to field of cell culture is and in particular to a kind of microcarrier suspension culture BHK21 cell produces foot and mouth disease
The method planting poison.
Background technology
Preparation kind poison is the important process link of production of vaccine.The method preparing vaccine kind poison at present includes the life of rolling bottle technique
Produce and suspension process produces two kinds：Rolling bottle technique preparation kind poison, a steady quality, malicious valency preferably but complex operation, consume a large amount of people
Work, specific yield is little, and endotoxin is difficult to control to；Suspension process can be mass-produced vaccine kind poison, operates relative ease, but malicious valency
Relatively low it is difficult to reach ideal effect.
Content of the invention
It is an object of the invention to the problem above overcoming prior art to exist, provide microcarrier suspension culture BHK21 thin
The method that born of the same parents produce foot and mouth disease kind poison, the present invention adopts bioreactor microcarrier culture vaccine kind poison, the micro- load of bioreactor
Body culture viral vaccine kind poison, has high degree of automation, kind poison poison valency is high, it is good to plant poison concordance, and the big feature of yield.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions：
A kind of method that microcarrier suspension culture BHK21 cell produces foot and mouth disease kind poison, it comprises the following steps：
S101 is by BHK21 cell recovery, square vase adhere-wall culture, then amplifies spinner culture through digestion；
S102 BHK21 cell attachment progressively amplification culture, to cell density needed for reactor；
The microcarrier that S103 reactor addition was processed, microcarrier consumption is 1-5g/L；
S104 BHK21 cell dissociation accesses the culture of reactor microcarrier, and sampling digestion count density reaches 1-4 × 109
Individual/L, connects malicious 1-5%, cell viability≤5%, harvests culture fluid, deepfreeze is standby；
After S105 microcarrier reactor receives poison, detect LD50.
Further include, in S101, BHK21 cell, after recovery, is carried out using MEM+8% new-born calf serum culture medium
Ferrum wall is cultivated, and is amplified to spinner culture through pancreatin digestion.
Further include, in S102, cell counting, 1.8-7 × 108/ L cell concentration is inoculated in 5L bioreactor.
Further include, in S103, control the condition of reactor：36-38 DEG C, pH 6.8-7.2, DO20-50%, inoculation
After 48h, sampling, violet staining carries out cell counting.
Further include, in S104, BHK21 cell includes in the step of reactor suspension amplification culture：
In S201 5L reactor, when cell density reaches 1-4 × 109During/L, settle microcarrier, discharge culture medium, use
PBS washs, each 2L, washs 2 times, adds pancreatin Digestive system, digests while stirring, determines that after reaching digestion effect, addition contains
Blood serum medium terminates pancreatin digestion, and proceeds in 100L reactor；
1-5g/L containing microcarrier in S202 100L reactor, volume of culture 70-100L, control reactor condition：36-38
DEG C, pH6.8-7.2, DO20-50%, in the above conditions, cultivated, and after inoculation 48h, sampling, violet staining is entered
Row cell counting, calculates cell growth state, when cell density reaches 1-4 × 109During/L, digested with same mode of operation and connect
Plant to 500L reactor；
1-5g/L containing microcarrier in S203 500L reactor, volume of culture 300-500L, control reactor condition：36-38
DEG C, pH6.8-7.2, DO20-50%, in the above conditions, cultivated, and after inoculation 48h, sampling, violet staining is entered
Row cell counting, calculates cell growth state, when cell density reaches 1-4 × 109During/L, foot and mouth disease kind is inoculated with 0.5-2%
Poison.
Further include, in S105, in 500L reactor inoculation kind poison after, the condition setting reactor as 36-38 DEG C,
PH is 7.2-7.5, DO20-50%, carries out kind of a poison and increases poison, timing sampling is observed, during DANGMING explict occurrence cytopathy, increasing poison of receiving
Product, is placed in -20--80 DEG C of freezen protective, carries out LD50 detection to product, and LD50 reaches 10^7～7.75/0.2ml.
Further include, step S101-105 is used for producing any foot-and-mouth disease vaccine kind poison.
Further include, step S101-105 is used for producing any hypotype foot-and-mouth disease vaccine kind poison.
The invention has the beneficial effects as follows:
The easily operated control of the method for the present invention, has high degree of automation, kind poison poison valency is high, it is good to plant poison concordance, and
The big feature of yield, and solve the problems, such as foot and mouth disease kind poison produce in product quality stability difference and yield poorly.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of description, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
The specific embodiment of the present invention is shown in detail in by following examples and its accompanying drawing.
Brief description
In order to be illustrated more clearly that the technical scheme in embodiment of the present invention technology, below will be in the description of embodiment technology
The accompanying drawing of required use be briefly described it should be apparent that, drawings in the following description be only the present invention some are real
Apply example, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to these accompanying drawings
Obtain other accompanying drawings.
Fig. 1 is method of the present invention flow chart.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation description is it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of not making creative work
Embodiment, broadly falls into the scope of protection of the invention.
Embodiment
Produce foot and mouth disease kind poison with reference to shown in Fig. 1, disclosing a kind of microcarrier suspension culture BHK21 cell in the present embodiment
Method, it comprises the following steps：
S101 BHK21 cell, after recovery, carries out ferrum wall culture using MEM+8% new-born calf serum culture medium, and warp
Cross pancreatin digestion and be amplified to spinner culture.
S102 BHK21 cell attachment progressively amplification culture, to cell density needed for reactor, cell counting, 1.8-7 ×
108/ L cell concentration is inoculated in 5L bioreactor.
The microcarrier that S103 reactor addition was processed, microcarrier consumption is 1-5g/L；Wherein, control the bar of reactor
Part：36-38 DEG C, pH 6.8-7.2, DO20-50%, after inoculation 48h, sampling, violet staining carries out cell counting.
S104 BHK21 cell dissociation accesses the culture of reactor microcarrier, and sampling digestion count density reaches 1-4 × 109
Individual/L, connects malicious 1-5%, cell viability≤5%, harvests culture fluid, deepfreeze is standby.
In S104, BHK21 cell includes in the step of reactor suspension amplification culture：
In S201 5L reactor, when cell density reaches 1-4 × 109During/L, settle microcarrier, discharge culture medium, use
PBS washs, each 2L, washs 2 times, adds pancreatin Digestive system, digests while stirring, determines that after reaching digestion effect, addition contains
Blood serum medium terminates pancreatin digestion, and proceeds in 100L reactor；
1-5g/L containing microcarrier in S202 100L reactor, volume of culture 70-100L, control reactor condition：36-38
DEG C, pH6.8-7.2, DO20-50%, in the above conditions, cultivated, and after inoculation 48h, sampling, violet staining is entered
Row cell counting, calculates cell growth state, when cell density reaches 1-4 × 109During/L, digested with same mode of operation and connect
Plant to 500L reactor；
1-5g/L containing microcarrier in S203 500L reactor, volume of culture 300-500L, control reactor condition：36-38
DEG C, pH6.8-7.2, DO20-50%, in the above conditions, cultivated, and after inoculation 48h, sampling, violet staining is entered
Row cell counting, calculates cell growth state, when cell density reaches 1-4 × 109During/L, foot and mouth disease kind is inoculated with 0.5-2%
Poison.
After S105 inoculates kind of poison in 500L reactor, as 36-38 DEG C, pH is 7.2-7.5 to the condition setting reactor,
DO20-50%, carries out kind of a poison and increases poison, timing sampling is observed, during DANGMING explict occurrence cytopathy, the malicious product of increasing of receiving, be placed in-
20--80 DEG C of freezen protective, carries out LD50 detection to product, and as shown in table 1, LD50 reaches 10^7～7.75/0.2ml.
Above-mentioned steps S101-105 are used for producing any foot-and-mouth disease vaccine kind poison, or produce any hypotype foot-and-mouth disease vaccine kind
Poison.
Table 1 rolling bottle kind poison and kind of the Conium maculatum L. number that suspends compare
The easily operated control of said method, has high degree of automation, kind poison poison valency is high, it is good to plant poison concordance, and yield
Big feature, and solve the problems, such as foot and mouth disease kind poison produce in product quality stability difference and yield poorly.
Described above to the disclosed embodiments, makes professional and technical personnel in the field be capable of or uses the present invention.
Multiple modifications to these embodiments will be apparent from for those skilled in the art, as defined herein
General Principle can be realized without departing from the spirit or scope of the present invention in other embodiments.Therefore, the present invention
It is not intended to be limited to the embodiments shown herein, and be to fit to and principles disclosed herein and features of novelty phase one
The scope the widest causing.
Claims (8)
1. a kind of microcarrier suspension culture BHK21 cell produces the method for foot and mouth disease kind poison it is characterised in that it includes following step
Suddenly：
S101 is by BHK21 cell recovery, square vase adhere-wall culture, then amplifies spinner culture through digestion；
S102 BHK21 cell attachment progressively amplification culture, to cell density needed for reactor；
The microcarrier that S103 reactor addition was processed, microcarrier consumption is 1-5g/L；
S104 BHK21 cell dissociation accesses the culture of reactor microcarrier, and sampling digestion count density reaches 1-4 × 109Individual/L, connects
Malicious 1-5%, cell viability≤5%, harvest culture fluid, deepfreeze is standby；
After S105 microcarrier reactor receives poison, detect LD50.
2. the method that microcarrier suspension culture BHK21 cell according to claim 1 produces foot and mouth disease kind poison, its feature exists
In, in S101, BHK21 cell, after recovery, carries out ferrum wall culture using MEM+8% new-born calf serum culture medium, and passes through
Pancreatin digestion is amplified to spinner culture.
3. the method that microcarrier suspension culture BHK21 cell according to claim 1 produces foot and mouth disease kind poison, its feature exists
In, in S102, cell counting, 1.8-7 × 108/ L cell concentration is inoculated in 5L bioreactor.
4. the method that microcarrier suspension culture BHK21 cell according to claim 1 produces foot and mouth disease kind poison, its feature exists
In, in S103, the condition of control reactor：36-38 DEG C, pH 6.8-7.2, DO20-50%, after inoculation 48h, sampling, crystal violet
Dyeing carries out cell counting.
5. the method that microcarrier suspension culture BHK21 cell according to claim 1 produces foot and mouth disease kind poison, its feature exists
In, in S104, BHK21 cell includes in the step of reactor suspension amplification culture：
In S201 5L reactor, when cell density reaches 1-4 × 109During/L, settle microcarrier, discharge culture medium, washed using PBS
Wash, each 2L, wash 2 times, add pancreatin Digestive system, digest while stirring, determine after reaching digestion effect, add containing serum training
Foster base terminates pancreatin digestion, and proceeds in 100L reactor；
1-5g/L containing microcarrier in S202 100L reactor, volume of culture 70-100L, control reactor condition：36-38 DEG C,
PH6.8-7.2, DO20-50%, in the above conditions, are cultivated, and after inoculation 48h, sampling, violet staining is carried out carefully
Born of the same parents count, and calculate cell growth state, when cell density reaches 1-4 × 109During/L, digested with same mode of operation and be seeded to
500L reactor；
1-5g/L containing microcarrier in S203 500L reactor, volume of culture 300-500L, control reactor condition：36-38 DEG C,
PH6.8-7.2, DO20-50%, in the above conditions, are cultivated, and after inoculation 48h, sampling, violet staining is carried out carefully
Born of the same parents count, and calculate cell growth state, when cell density reaches 1-4 × 109During/L, with 0.5-2% inoculation foot and mouth disease kind poison.
6. the method that microcarrier suspension culture BHK21 cell according to claim 1 produces foot and mouth disease kind poison, its feature exists
In, in S105, after inoculation kind poison in 500L reactor, as 36-38 DEG C, pH is 7.2-7.5 to the condition setting reactor,
DO20-50%, carries out kind of a poison and increases poison, timing sampling is observed, during DANGMING explict occurrence cytopathy, the malicious product of increasing of receiving, be placed in-
20--80 DEG C of freezen protective, carries out LD50 detection to product, and LD50 reaches 10^7～7.75/0.2ml.
7. the method that the microcarrier suspension culture BHK21 cell according to any one of claim 1-6 produces foot and mouth disease kind poison,
It is characterized in that, step S101-105 is used for producing any foot-and-mouth disease vaccine kind poison.
8. the method that the microcarrier suspension culture BHK21 cell according to any one of claim 1-6 produces foot and mouth disease kind poison,
It is characterized in that, step S101-105 is used for producing any hypotype foot-and-mouth disease vaccine kind poison.
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| CN201610861307.XA CN106399261A (en) | 2016-09-29 | 2016-09-29 | Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers |
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| CN201610861307.XA CN106399261A (en) | 2016-09-29 | 2016-09-29 | Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111979201A (en) * | 2020-08-24 | 2020-11-24 | 天信和(苏州)生物科技有限公司 | Method for improving foot-and-mouth disease virus expression level |
| CN115386534A (en) * | 2022-10-26 | 2022-11-25 | 天信和(苏州)生物科技有限公司 | Method for culturing BHK21 cells by using serum-free culture medium and application of BHK21 cells in preparation of vaccines |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111979201A (en) * | 2020-08-24 | 2020-11-24 | 天信和(苏州)生物科技有限公司 | Method for improving foot-and-mouth disease virus expression level |
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| CN115386534A (en) * | 2022-10-26 | 2022-11-25 | 天信和(苏州)生物科技有限公司 | Method for culturing BHK21 cells by using serum-free culture medium and application of BHK21 cells in preparation of vaccines |
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