CN111575246A - Method for producing porcine pseudorabies virus by serum-free full-suspension cell culture - Google Patents
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Abstract
The invention relates to a method for producing porcine pseudorabies virus by serum-free full-suspension cell culture, which comprises the following steps of inoculating the pseudorabies virus into BHK-21 host cells cultured in the serum-free suspension culture, and obtaining a large amount of porcine pseudorabies virus after culturing for 48 hours. Compared with the prior art, the pseudorabies virus obtained by the invention has no serum residue, and the titer of the pseudorabies virus is stable at 108.5TCID50More than ml, high virus titer, cost saving, mature technology, simple operation, easy scale up, easy popularization and mass production.
Description
Technical Field
The invention relates to the technical field of production of porcine pseudorabies viruses, in particular to a method for producing the porcine pseudorabies viruses by serum-free full-suspension cell culture.
Background
Pseudorabies (PR), also known as Aujeszky's Disease (AD), is a viral disease caused by Pseudorabies virus (PRV) and characterized primarily by fever and encephalomyelitis. The virus can infect various domestic animals and wild animals, including pigs, dogs, cats, cattle, sheep, mice, minks, foxes, rabbits, etc., and is 100% lethal to domestic animals and wild animals except pigs, which are natural hosts and carriers of PRV. The outbreak of pseudorabies epidemic brings huge economic loss to the farm, and the disease is considered to be one of the main diseases threatening the production of pig breeding and causing death of pigs at present.
The virus production by animal cell culture is the most common means in the production process of virus vaccines at home and abroad, and the traditional spinner flask cell culture process has the defects of low cell density, low virus yield, high production cost, long production period, high labor intensity and the like, and is gradually replaced by the emerging cell suspension culture technology. The cell suspension culture technology overcomes a plurality of defects of the traditional spinner flask culture technology by the advantages of automation, scale, simplification of cell culture, safety of operation, batch homogenization and the like, and solves a plurality of problems of labor, cost, field, production period, raw material control, unstable quality and the like in the actual production process.
PRV is omnitropic and can be cultured and propagated in tissue cells of various origins. The BHK-21 cells have wide sources and high biological safety, are sensitive to the pseudorabies virus, have high virus titer of the harvested virus and are ideal host cells for producing the pseudorabies virus. However, the applicants have found that: to achieve efficient amplification of pseudorabies virus, not only does a sensitive and ideal host cell be required, but the selection of some key parameters that influence viral amplification is also crucial. If the MOI parameter in the virus inoculation process is selected, the MOI is too high, the probability of infection of the host cell by the virus is greatly improved, and synchronous infection of a plurality of viruses may exist, so that the inoculated virus can be rapidly proliferated in the host cell to cause premature death of the host cell; at low MOI, however, only a subset of the cells may be infected by the virus, resulting in an extended viral replication cycle, resulting in a significant portion of the energy in the culture system being used for growth metabolism of the host cells rather than for virus propagation, resulting in a lower virus propagation efficiency. The selection of cell density during virus inoculation is also important for the efficient amplification of PRV, the density is too low, and the virus proliferation efficiency is reduced; too high a density, too much of the nutrients in the medium are consumed prematurely, making the virus titer difficult to increase.
In addition, the applicant also found that the existing process basically uses culture solution containing low serum to prepare the pseudorabies virus, and the titer of the produced pseudorabies virus is lower and is only 106.0-7.5TCID50About/ml, it is necessary to add the virus solutionHigh-power concentration and long production period, and the addition of serum not only increases the production cost, but also increases the risk to the safety of the vaccine and influences the quality and the stability of the vaccine in batches.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for producing porcine pseudorabies virus by serum-free full-suspension cell culture. Solves the problems of low virus titer and difficult large-scale amplification for virus culture in the prior art when the cell is adopted to proliferate the virus.
The purpose of the invention can be realized by the following technical scheme:
a method for producing porcine pseudorabies virus by serum-free full-suspension cell culture comprises the following steps:
(1) expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 0.8-1.2 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 120-;
(2) BHK-21 cells to be suspended without serum grow to 8-10 × 106At the cell density of cells/ml, BHK-21 cells were inoculated by dilution, and the cell density after inoculation was controlled to be 2-4 × 106cells/ml；
(3) Inoculating the porcine pseudorabies virus, and placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 120-140rpm, and harvesting the virus to obtain the porcine pseudorabies virus.
Preferably, in step (1), the fresh medium is a commercial medium commercially available for BHK-21 serum-free suspension culture.
Preferably, in the step (1), the fresh culture medium is serum-free culture medium for BHK cells of Chinese patent CN 201610365272.0.
Preferably, in step (1), the BHK-21 seed cells are cultured at 1.0 × 106cells/ml were seeded into fresh medium.
Preferably, in step (1), the culture time is 48 h.
Preferably, the step (A)2) The cell density after inoculation is controlled to be 3 × 106cells/ml。
Preferably, in step (2), the BHK-21 cells (amplified) are inoculated at a dilution ratio of 1 (2. + -. 0.5).
Preferably, in step (3), the MOI of the inoculated virus is 0.005 to 0.00005.
Preferably, in the step (3), the time for culturing after inoculating the porcine pseudorabies virus is 48 hours.
Preferably, the harvested virus is observed for cytopathic effects by seeding adherent cells ST and calculating TCID according to the Reed-Muench method50The virus content of each 1.0ml of the virus liquid is more than or equal to 108.5TCID50。
Virus TCID50The determination steps comprise observing the growth condition of adherent cells ST in a square bottle under an inverted microscope, digesting with 0.25% pancreatin-EDTA solution when the confluency reaches above 90%, adding a proper amount of DMEM culture medium containing 10% serum after about 3min to stop digestion, sampling and counting after mixing, and preparing the adherent cells containing 2-3 × 105cell/ml ST cell suspension (DMEM + 10% FBS), 100. mu.l per well in 96-well cell culture plates, together with PRV virus solution (no equal adherent cells) were inoculated, and PRV was performed 10-fold using serum-free DMEM-1、10-2、10-3To 10-9After serial dilutions, 100 μ l was inoculated per well, 4 replicates per dilution were made, and non-inoculated normal ST adherent cells were set up as control wells. Finally, the 96-well plate was placed in a 5% carbon dioxide incubator at 37 ℃. After 5 days, the cell lesion was observed, and then TCID was calculated from the lesion of the cells in the well plate50。
Compared with the prior art, the invention has the following beneficial effects:
the method has the advantages of simple process, large growth amount, high yield, and low cost, and the virus content of the cultured virus solution is not less than 108.5TCID50The titer of the porcine pseudorabies virus is obviously higher than that of the porcine pseudorabies virus cultured by the prior art. The used host cell BHK-21 is a cell commonly used in the market, has sufficient source, low acquisition cost, mature culture technology and easy operationThe production is carried out.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
The preparation method for producing the porcine pseudorabies virus by serum-free full-suspension cell culture comprises the following steps:
1. expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 0.8 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in 120rpm constant temperature incubator for 48 hr with cell density of 8 × 106cells/ml。
2. BHK-21 cells to be serum-free suspended grow to 8 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 2 × 106cells/ml, the liquid loading is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.005. Placing in 5% CO2Culturing at 37 deg.C and 120rpm in a constant temperature incubator, inoculating virus, and culturing for 48h to obtain virus.
Example 2
The preparation method for producing the porcine pseudorabies virus by serum-free full-suspension cell culture comprises the following steps:
1. expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 1.0 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9.0 × 106cells/ml。
2. BHK-21 cells to be suspended without serum grow to 9.0 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 3.0 × 106cells/ml, the liquid loading volume is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.0005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Example 3
The preparation method for producing the porcine pseudorabies virus by serum-free full-suspension cell culture comprises the following steps:
1. expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 1.0 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9.0 × 106cells/ml。
2. BHK-21 cells to be suspended without serum grow to 9.0 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 3.0 × 106cells/ml, the liquid loading is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.0001. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Example 4
1. Expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 1.0 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9.0 × 106cells/ml。
2. BHK-21 cells to be suspended without serum grow to 9.0 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 3.0 × 106cells/ml, liquid loading volume of 30ml, pseudorabies virus, and virus inoculation MOI of 0.00005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Example 5
1. Expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 1.2 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C and 140rpm in constant temperature incubator for 48 hr with cell density of 10 × 106cells/ml。
2. Serum-freeSuspension of BHK-21 cells grown to 10 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to be 4 × 106cells/ml, the liquid loading volume is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.0005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 140rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Comparative example 1
1. Expanding the cells, and carrying out continuous passage for more than 3 times on the BHK-21 seed cells with the ratio of 1 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9 × 106cells/ml。
2. BHK-21 cells to be serum-free suspended grow to 9 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 3 × 106cells/ml, the liquid loading is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.05. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Comparative example 2
1. Expanding the cells, and carrying out continuous passage for more than 3 times on the BHK-21 seed cells with the ratio of 1 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9 × 106cells/ml。
2. BHK-21 cells to be serum-free suspended grow to 9 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 3 × 106cells/ml, liquid loading volume of 30ml, pseudorabies virus, inoculation MOI of virus 0.000005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Comparative example 3
1. Expanding cells, and carrying out continuous passage 3The BHK-21 seed cells were cultured at 1 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9 × 106cells/ml。
2. BHK-21 cells to be serum-free suspended grow to 9 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 1 × 106cells/ml, the liquid loading volume is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.0005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Comparative example 4
1. Expanding the cells, and carrying out continuous passage for more than 3 times on the BHK-21 seed cells with the ratio of 1 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 deg.C in a constant temperature incubator at 130rpm for 48h with a cell density of 9 × 106cells/ml。
2. BHK-21 cells to be serum-free suspended grow to 9 × 106At a cell density of cells/ml, BHK-21 cells were diluted and inoculated into 125ml shake flasks, and the cell density after inoculation was controlled to 6 × 106cells/ml, the liquid loading volume is 30ml, pseudorabies virus is inoculated, and the MOI of the inoculated virus is 0.0005. Placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 130rpm, inoculating the virus, and culturing for 48h to obtain the virus.
Step 2, the expanded seed cells are inoculated according to the dilution ratio of 1 (2 +/-0.5), in each example and each comparative example, about 20ml of cell culture fluid is discarded, and about 20ml of fresh culture medium is supplemented to a shake flask with the liquid loading of 30 ml.
After harvesting the virus, storing in a refrigerator at-80 ℃, freezing and thawing for 1-2 times, centrifuging at 4000rpm for 10min, taking the supernatant, and storing in the refrigerator at-80 ℃.
Test example 1:
the serum-free full-suspension cell culture production porcine pseudorabies virus titer of examples 1-5 and comparative examples 1-4 is detectedToxic TCID50The determination steps comprise observing the growth condition of adherent ST cells in a square bottle under an inverted microscope, digesting with 0.25% pancreatin-EDTA solution when the confluency reaches above 90%, adding a proper amount of DMEM culture medium containing 10% serum after about 3min to stop digestion, sampling and counting after mixing, and preparing the product containing 2.5 × 105cell/ml ST cell suspension (DMEM + 10% FBS), 100. mu.l per well in 96-well cell culture plates, together with PRV virus solution (no equal adherent cells) were inoculated, and PRV was performed 10-fold using serum-free DMEM-1、10-2、10-3To 10-9After serial dilutions, 100ul of each well was inoculated, 4 replicates per dilution were made, and non-inoculated normal ST adherent cells were set up as control wells. Finally, the 96-well plate was placed in a 5% carbon dioxide incubator at 37 ℃. After 5 days, the cell lesion was observed, and then TCID was calculated from the lesion of the cells in the well plate50. The results are shown in Table 1 below.
TABLE 1
| Group of | Viral content (TCID)50/ml) |
| Example 1 | 108.5 |
| Example 2 | 108.67 |
| Example 3 | 108.67 |
| Example 4 | 108.5 |
| Example 5 | 108.5 |
| Comparative example 1 | 106.33 |
| Comparative example 2 | 106.5 |
| Comparative example 3 | 106.67 |
| Comparative example 4 | 106.33 |
As can be seen from the above table, the serum-free full-suspension cell culture production of porcine pseudorabies virus in examples 1-5 of the invention has high titer which is more than or equal to 108.5TCID50Per ml; comparative examples 1-4 serum-free full-suspension cell culture production of porcine pseudorabies virus has low titer which is less than or equal to 106.5TCID50The fact that the pseudorabies virus amplified by the technology has the advantages of high virus titer, cost saving, mature technology, simplicity in operation, easiness in scale amplification, easiness in popularization and mass production and the like is shown in ml.
The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A method for producing porcine pseudorabies virus by serum-free full-suspension cell culture is characterized by comprising the following steps:
(1) expanding the cells, and continuously passaging BHK-21 seed cells for more than 3 times at a ratio of 0.8-1.2 × 106cells/ml were seeded into fresh medium at a cell density of 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 120-;
(2) BHK-21 cells to be suspended without serum grow to 8-10 × 106At the cell density of cells/ml, BHK-21 cells were inoculated by dilution, and the cell density after inoculation was controlled to be 2-4 × 106cells/ml；
(3) Inoculating the porcine pseudorabies virus, and placing in 5% CO2Culturing at 37 ℃ in a constant-temperature incubator at 120-140rpm, and harvesting the virus to obtain the porcine pseudorabies virus.
2. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (1), the fresh medium is a commercial medium commercially available for BHK-21 serum-free suspension culture.
3. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 2, wherein in step (1), the fresh medium is serum-free medium for BHK cells of Chinese patent CN 201610365272.0.
4. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (1), the BHK-21 seed cells are cultured at 1.0 × 106cells/ml were seeded into fresh medium.
5. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein the culture time in step (1) is 48 h.
6. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (2), the cell density after seeding is controlled to be 3 × 106cells/ml。
7. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (2), BHK-21 cells are inoculated at a dilution ratio of 1 (2 +/-0.5).
8. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (3), the MOI of the inoculated virus is 0.005-0.00005.
9. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein in step (3), the culture time after the porcine pseudorabies virus is inoculated is 48 h.
10. The method for producing porcine pseudorabies virus in serum-free full-suspension cell culture according to claim 1, wherein the harvested virus is used for observing cytopathic effect by inoculating adherent cells ST, and TCID is calculated according to Reed-Muench method50The virus content of each 1.0ml of the virus liquid is more than or equal to 108.5TCID50。
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