CN104630159A - Method for producing hog cholera C-strain virus by culturing ST Cells in low serum - Google Patents
Method for producing hog cholera C-strain virus by culturing ST Cells in low serum Download PDFInfo
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Abstract
The invention discloses a method for producing a hog cholera C-strain virus by culturing ST Cells in low serum, and belongs to the technical field of veterinary medicine and biology. The method comprises the following steps: S1, preparing a cell subculture and culture solution; S2, domesticating cells to adapt to a low-serum culture medium; S3, domesticating cells to adapt to a low-serum culture environment; S4, breeding a cell virus strain; S5, breeding prepared venom. The method for producing the hog cholera C-strain virus by culturing ST Cells in low serum provided by the invention can remarkably reduce the production cost and can also improve the downstream purifying efficiency, and can quickly and stably expand production scale, so that the quality is easy to balance and stabilize.
Description
Technical field
The invention belongs to technical field of veterinary biology, be specifically related to the method for low serum free culture system ST cells produce swine fever C strain virus.
Background technology
ST clone pig testis (swine testis) cell, vitro culture is adherent growth, breed comparatively slow, and cell dissociation is easily agglomerating in going down to posterity in culturing process.ST cell is more responsive to multiple virus, as Pestivirus suis (CSFV), pig parvoviral (PPV), Pseudorabies virus etc.At present, cultivate ST cells produce vaccine many employings rolling bottle technique, use DMEM, MEM substratum to add the serum free culture system of 10%.
At present, no matter domestic swine Fever Vaccine production technology is traditional spinner culture technology, or the bio-reactor suspension culture techniques of advanced person, all adopt high serum content cell culture technology.This traditional technology efficiency is low, production cost is high; Significant difference between different serum batch; Amplify circulation ratio poor; High to downstream purification processing requirement; Easily occur the vaccine quality hidden danger that serum goes out complicated ingredient and causes relating to Biosafety and public health problem.In addition, current global serum supply is becoming tight, and this have impact on the production efficiency of vaccine enterprise greatly.Therefore the application of low serum free culture system technology is imperative.Through retrieval, the pertinent literature about low serum free culture system ST cells produce swine fever C strain virus is not had to report.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, the method of low serum free culture system ST cells produce swine fever C strain virus is provided, the method significantly can reduce production cost, the efficiency of downstream purification can be improved simultaneously, and can the expanding the scale of production of fast and stable, quality is easy to realize equalization stable.
Object of the present invention is achieved through the following technical solutions: the method for low serum free culture system ST cells produce swine fever C strain virus, and it comprises the following steps:
S1. the going down to posterity and cultivation of preparation cell:
Get T75 bottle and cultivate confluent monolayers ST cell, through EDTA-pancreatin cell dispersal liquid peptic cell, cell dispersion is blown and beaten with cell growth medium, add 20ml cell growth medium, ST cell being placed in temperature is that the CO2gas incubator of 37 ± 2 DEG C cultivates 72h, during good cell monolayer to be formed, carry out amplification culture, above-mentioned cell growth medium is DMEM or the MEM nutrient solution of 10% serum content;
S2. cell adapted low blood serum medium is tamed: comprise following sub-step:
S21. the domestication of first-generation cell:
The ST cell of step S1 enlarged culturing is inoculated in the low blood serum medium of the first-generation and tames, the low blood serum medium of the described first-generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 2:1, and wherein the content of serum is 8.3%;
S22. the domestication of s-generation cell:
The first-generation ST cell of domestication is inoculated in the low blood serum medium of the s-generation and tames, the low blood serum medium of the described s-generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:1, and wherein the content of serum is 7.5%;
S23. the domestication of third generation cell:
The s-generation ST cell of domestication is inoculated in the low blood serum medium of the third generation and tames, the low blood serum medium of the described third generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:2, and wherein the content of serum is 6.7%;
S24. the domestication of forth generation cell:
Be inoculated in the low blood serum medium of forth generation by the third generation ST cell of domestication and tame, the low blood serum medium of described forth generation is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 5%;
S3. cell adapted low serum free culture system environment is tamed:
Use T75 cell bottle, the forth generation ST cell of 37 DEG C of static cultivation domestications, nutrient solution is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 1 ~ 5%, is Seed cells;
S4. the breeding of cell seed culture of viruses:
With virus-culturing fluid swine fever C strain virus kind poison is inoculated in Seed cells individual layer in the ratio of 0.5 ~ 1% (v/v) and cultivates, after cultivation 72h, gather in the crops venom; Using this venom as kind of a poison, Cells for production carries out cell adaptation continuous passage, until TCID
50stable, the venom now gathered in the crops is as production kind of poison;
S5. the breeding of venom is prepared:
Seed cell to be produced forms individual layer, produces with kind of a poison in the ratio access of 0.5 ~ 1% (v/v), is placed in that culture temperature is 37 DEG C, the CO2gas incubator of 5% is cultivated, 72h results virus after inoculation.
Further, the mixed solution of described EDTA-pancreatin cell dispersal liquid to be massfraction be Hank ' the s liquid of the pancreatin of 0.10 ~ 0.25% and the EDTA of 0.02%.
Further, cell growth medium described in step S1 is the mixed solution of low blood serum medium and serum, and wherein the content of serum is 1 ~ 5%.
Further, the disease described in step S4 and S5
poison poisonplanting nutrient solution is low blood serum medium, serum and antibiotic mixed solution, and wherein, the content of serum is 0 ~ 1%, and the content of antibiotic is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4.
In technique scheme, low blood serum medium is the low blood serum medium of Merck MD series dry powder, substratum before use, need wiring solution-forming to specifications, concrete grammar is as follows: 1) all pour in a container by a bag culture medium, with a small amount of water for injection, remaining medium in bag is washed down, be incorporated to container.Inject with water (water temperature 20 DEG C ~ 30 DEG C) to 17.5 liters, gentle agitation is dissolved; 2) 23 grams of sodium bicarbonates are added; 3) gentle agitation is dissolved, and injects water to 20 liters; 4) if necessary, pH to 7.2-7.4 is adjusted with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid soln; 5) degerming with 0.2 μm of filter membrane positive press filtration; 6) solution should keep in Dark Place at 2 DEG C-8 DEG C.
The present invention has the following advantages: the present invention's low serum free culture system ST cells produce swine fever C strain virus is produced in vaccine and used low blood serum medium effectively can reduce bovine serum usage quantity in nutrient solution, and can improve the productive rate of cell density and biological products; ST cell of the present invention is even better than at the growing state of low blood serum medium the conventional medium adding 10% bovine serum, and cell density, raising virus titer can be improved, obtain same cell concentration, with low blood serum medium by than the time shortening nearly 1/3 with conventional medium, can enhance productivity.Therefore, the method that the invention provides low serum free culture system ST cells produce swine fever C strain virus significantly can reduce production cost, can improve the efficiency of downstream purification simultaneously, and can the expanding the scale of production of fast and stable, and quality is easy to realize equalization stable.
Accompanying drawing explanation
fig. 1for the growth result of ST cell when serum adding proportion is 1% in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 2for the growth result of ST cell when serum adding proportion is 2% in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 3for the growth result of ST cell when serum adding proportion is 3% in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 4for the growth result of ST cell when serum adding proportion is 4% in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 5for the growth result of ST cell when serum adding proportion is 5% in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 6for the growth result of control group ST cell in embodiment 3
figure, wherein,
figure aincubation time be 24h,
figure bincubation time be 48h,
figure cincubation time be 72h;
fig. 7for pathology situation after the ST cell Pigs Inoculated pestivirus of serum free culture system liquid domestication low in embodiment 4, wherein,
figure aserum adding proportion be 1%,
figure bserum adding proportion be 2%,
figure cserum adding proportion be 3%,
figure dfor control group,
figure eserum adding proportion be 4%,
figure fserum adding proportion be 5%.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
Embodiment 1: the method for low serum free culture system ST cells produce swine fever C strain virus, it comprises the following steps:
S1. the going down to posterity and cultivation of preparation cell:
Get T75 bottle and cultivate confluent monolayers ST cell, through EDTA-pancreatin cell dispersal liquid peptic cell, the mixed solution of EDTA-pancreatin cell dispersal liquid to be massfraction be Hank ' the s liquid of the pancreatin of 0.15% and the EDTA of 0.02%; Cell dispersion is blown and beaten with cell growth medium, add 20ml cell growth medium, ST cell being placed in temperature is that the CO2gas incubator of 37 ± 2 DEG C cultivates 72h, during good cell monolayer to be formed, carry out amplification culture, above-mentioned cell growth medium is the DMEM nutrient solution of 10% serum content;
S2. cell adapted low blood serum medium is tamed: comprise following sub-step:
S21. the domestication of first-generation cell:
The ST cell of step S1 enlarged culturing is inoculated in the low blood serum medium of the first-generation and tames, the low blood serum medium of the described first-generation is the mixed solution of the DMEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 2:1, and wherein the content of serum is 8.3%;
S22. the domestication of s-generation cell:
The first-generation ST cell of domestication is inoculated in the low blood serum medium of the s-generation and tames, the low blood serum medium of the described s-generation is the mixed solution of the DMEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:1, and wherein the content of serum is 7.5%;
S23. the domestication of third generation cell:
The s-generation ST cell of domestication is inoculated in the low blood serum medium of the third generation and tames, the low blood serum medium of the described third generation is the mixed solution of the DMEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:2, and wherein the content of serum is 6.7%;
S24. the domestication of forth generation cell:
Be inoculated in the low blood serum medium of forth generation by the third generation ST cell of domestication and tame, the low blood serum medium of described forth generation is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 5%;
S3. cell adapted low serum free culture system environment is tamed:
Use T75 cell bottle, the forth generation ST cell of 37 DEG C of static cultivation domestications, nutrient solution is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 1 ~ 5%, is Seed cells;
S4. the breeding of cell seed culture of viruses:
With virus-culturing fluid swine fever C strain virus kind poison is inoculated in Seed cells individual layer in the ratio of 0.5 ~ 1% (v/v) and cultivates, after cultivation 72h, gather in the crops venom; Using this venom as kind of a poison, Cells for production carries out cell adaptation continuous passage, until TCID
50stable, the venom now gathered in the crops is as production kind of poison; Sick
poison poisonkind of nutrient solution be DMEM, 5% serum and antibiotic mixed solution, wherein, the content of serum is 0.5%, and antibiotic content is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4;
S5. the breeding of venom is prepared:
Seed cell to be produced forms individual layer, produces with kind of a poison in the ratio access of 0.5 ~ 1% (v/v), is placed in that culture temperature is 37 DEG C, the CO2gas incubator of 5% is cultivated, and after inoculation, 72h results virus, sick
poison poisonkind of nutrient solution be DMEM, 5% serum and antibiotic mixed solution, wherein, the content of serum is 0.5%, and antibiotic content is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4.
The process of results virus liquid: the virus liquid of results is after two to three freeze thawing, centrifugal or cross and filter cell debris and be placed on-70 DEG C and save backup.
Embodiment 2: the method for low serum free culture system ST cells produce swine fever C strain virus, it comprises the following steps:
S1. the going down to posterity and cultivation of preparation cell:
Get T75 bottle and cultivate confluent monolayers ST cell, through EDTA-pancreatin cell dispersal liquid peptic cell, the mixed solution of EDTA-pancreatin cell dispersal liquid to be massfraction be Hank ' the s liquid of the pancreatin of 0.15% and the EDTA of 0.02%, cell dispersion is blown and beaten with cell growth medium, add 20ml cell growth medium, ST cell being placed in temperature is that the CO2gas incubator of 37 ± 2 DEG C cultivates 72h, during good cell monolayer to be formed, carry out amplification culture, above-mentioned cell growth medium is the MEM nutrient solution of 10% serum content;
S2. cell adapted low blood serum medium is tamed: comprise following sub-step:
S21. the domestication of first-generation cell:
The ST cell of step S1 enlarged culturing is inoculated in the low blood serum medium of the first-generation and tames, the low blood serum medium of the described first-generation is the mixed solution of the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 2:1, and wherein the content of serum is 8.3%;
S22. the domestication of s-generation cell:
The first-generation ST cell of domestication is inoculated in the low blood serum medium of the s-generation and tames, the low blood serum medium of the described s-generation is the mixed solution of the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:1, and wherein the content of serum is 7.5%;
S23. the domestication of third generation cell:
The s-generation ST cell of domestication is inoculated in the low blood serum medium of the third generation and tames, the low blood serum medium of the described third generation is the mixed solution of the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:2, and wherein the content of serum is 6.7%;
S24. the domestication of forth generation cell:
Be inoculated in the low blood serum medium of forth generation by the third generation ST cell of domestication and tame, the low blood serum medium of described forth generation is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 5%;
S3. cell adapted low serum free culture system environment is tamed:
Use T75 cell bottle, the forth generation ST cell of 37 DEG C of static cultivation domestications, nutrient solution is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 1 ~ 5%, is Seed cells;
S4. the breeding of cell seed culture of viruses:
With virus-culturing fluid swine fever C strain virus kind poison is inoculated in Seed cells individual layer in the ratio of 0.5 ~ 1% (v/v) and cultivates, after cultivation 72h, gather in the crops venom; Using this venom as kind of a poison, Cells for production carries out cell adaptation continuous passage, until TCID
50stable, the venom now gathered in the crops is as production kind of poison; Sick
poison poisonkind of nutrient solution be MEM, 5% serum and antibiotic mixed solution, wherein, the content of serum is 0.5%, and antibiotic content is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4;
S5. the breeding of venom is prepared:
Seed cell to be produced forms individual layer, produces with kind of a poison in the ratio access of 0.5 ~ 1% (v/v), is placed in that culture temperature is 37 DEG C, the CO2gas incubator of 5% is cultivated, 72h results virus after inoculation; Sick
poison poisonkind of nutrient solution be MEM, 5% serum and antibiotic mixed solution, wherein, the content of serum is 0.5%, and antibiotic content is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4.
The process of results virus liquid: the virus liquid of results is after two to three freeze thawing, centrifugal or cross and filter cell debris and be placed on-70 DEG C and save backup.
Embodiment 3: low blood serum medium domestication ST cell effect
Use T75 cell bottle, 37 DEG C of static cultivation adapt to low blood serum medium forth generation ST cell, and serum adding proportion is 1%, 2%, 3%, 4%, 5% 5 gradient, and set up use serum content be 10% the ST cell cultivated of DMEM liquid be control group, result
as Fig. 1,
fig. 2,
fig. 3,
fig. 4,
fig. 5,
fig. 6shown in, in cell cultivation process, each index is shown in
table 1.(the low blood serum medium of Merck MD series dry powder comprises DMEM and MEM)
table 1: MD series low blood serum medium domestication ST cell effect
By
table 1known, the average harvest yield of cell of experimental group, cell average activity and average specific growth rate are all higher than control group.
The low blood serum medium of embodiment 4:MD series tames bioactivity after the ST cell Pigs Inoculated epidemic diarrhea virus obtained
The low blood serum medium of MD series cultivates ST cell Pigs Inoculated epidemic diarrhea virus.Connecing malicious ratio is 1% (v/v), and contrast (serum content is the DMEM of 10%) papova maintenance medium serum-concentration is 2%, and test group virus maintenance medium serum-concentration is 0.5%, and after connecing poison, 72h gathers in the crops virus, experimental result
as Fig. 7shown in, three times test-results mean value is shown in
table 2.
table 2: ST cell proliferation hog cholera after domestication
poison feelingscondition
Embodiment 5: cost estimating and comparing
For 100L volume of culture, to before serum price increase and use cost before and after low serum technique, carry out estimating and comparing, concrete outcome is as follows:
table 3: cost estimating
In above-described embodiment, MD series is the low blood serum medium of Merck MD series.
Claims (4)
1. the method for low serum free culture system ST cells produce swine fever C strain virus, it is characterized in that, it comprises the following steps:
S1. the going down to posterity and cultivation of preparation cell:
Get T75 bottle and cultivate confluent monolayers ST cell, through EDTA-pancreatin cell dispersal liquid peptic cell, cell dispersion is blown and beaten with cell growth medium, add 20ml cell growth medium, ST cell being placed in temperature is that the CO2gas incubator of 37 ± 2 DEG C cultivates 72h, during good cell monolayer to be formed, carry out amplification culture, above-mentioned cell growth medium is DMEM or the MEM nutrient solution of 10% serum content;
S2. cell adapted low blood serum medium is tamed: comprise following sub-step:
S21. the domestication of first-generation cell:
The ST cell of step S1 enlarged culturing is inoculated in the low blood serum medium of the first-generation and tames, the low blood serum medium of the described first-generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 2:1, and wherein the content of serum is 8.3%;
S22. the domestication of s-generation cell:
The first-generation ST cell of domestication is inoculated in the low blood serum medium of the s-generation and tames, the low blood serum medium of the described s-generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:1, and wherein the content of serum is 7.5%;
S23. the domestication of third generation cell:
The s-generation ST cell of domestication is inoculated in the low blood serum medium of the third generation and tames, the low blood serum medium of the described third generation is the mixed solution of DMEM or the MEM nutrient solution containing 10% serum and the low serum free culture system liquid of Merck MD containing 5% serum, DMEM or MEM nutrient solution and Merck MD low serum free culture system liquid volume ratio are 1:2, and wherein the content of serum is 6.7%;
S24. the domestication of forth generation cell:
Be inoculated in the low blood serum medium of forth generation by the third generation ST cell of domestication and tame, the low blood serum medium of described forth generation is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 5%;
S3. cell adapted low serum free culture system environment is tamed:
Use T75 cell bottle, the forth generation ST cell of 37 DEG C of static cultivation domestications, nutrient solution is the mixed solution of the low blood serum medium of Merck MD and serum, and the content of serum is 1 ~ 5%, is Seed cells;
S4. the breeding of cell seed culture of viruses:
With virus-culturing fluid by swine fever C strain virus kind poison in 0.5 ~ 1%(v/v) ratio be inoculated in Seed cells individual layer and cultivate, gather in the crops venom after cultivation 72h; Using this venom as kind of a poison, Cells for production carries out cell adaptation continuous passage, until TCID
50stable, the venom now gathered in the crops is as production kind of poison;
S5. the breeding of venom is prepared:
Seed cell to be produced forms individual layer, in 0.5 ~ 1%(v/v) ratio access produce with kind of a poison, be placed in that culture temperature is 37 DEG C, the CO2gas incubator of 5% is cultivated, 72h results virus after inoculation.
2. the method for low serum free culture system ST cells produce swine fever C strain virus as claimed in claim 1, is characterized in that, the mixed solution of described EDTA-pancreatin cell dispersal liquid to be massfraction be Hank ' the s liquid of the pancreatin of 0.10 ~ 0.25% and the EDTA of 0.02%.
3. the method for low serum free culture system ST cells produce swine fever C strain virus as claimed in claim 1, it is characterized in that, cell growth medium described in step S1 is the mixed solution of low blood serum medium and serum, and wherein the content of serum is 1 ~ 5%.
4. the method for low serum free culture system ST cells produce swine fever C strain virus as claimed in claim 1, it is characterized in that, viral seed culture of viruses nutrient solution described in step S4 and S5 is low blood serum medium, serum and antibiotic mixed solution, wherein, the content of serum is 0 ~ 1%, the content of antibiotic is 100 ~ 200IU/mL, and the pH value of mixed solution is 7.2 ~ 7.4.
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| CN106367399A (en) * | 2016-08-30 | 2017-02-01 | 齐鲁动物保健品有限公司 | Method for producing porcine parvovirus infection vaccines by adopting full suspension technology |
| CN109517800A (en) * | 2018-12-05 | 2019-03-26 | 江苏省农业科学院 | The reconstruct ST cell and its construction method and purposes that one plant of reinforcement cholesterol is endogenously synthesized |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104940922A (en) * | 2015-07-02 | 2015-09-30 | 瑞普（保定）生物药业有限公司 | Preparation method of swine fever live vaccine |
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| CN106367399A (en) * | 2016-08-30 | 2017-02-01 | 齐鲁动物保健品有限公司 | Method for producing porcine parvovirus infection vaccines by adopting full suspension technology |
| CN106367399B (en) * | 2016-08-30 | 2019-10-11 | 齐鲁动物保健品有限公司 | A method of pig parvoviral disease vaccine is produced using full suspension technology |
| CN109517800A (en) * | 2018-12-05 | 2019-03-26 | 江苏省农业科学院 | The reconstruct ST cell and its construction method and purposes that one plant of reinforcement cholesterol is endogenously synthesized |
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Application publication date: 20150520 |