CN108570457A - A kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency - Google Patents
A kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency Download PDFInfo
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Abstract
The present invention relates to a kind of swine fever spleens to drench attenuated vaccine vaccine extracorporeal culturing method, and steps are as follows：（1）Select rabbit；（2）Production is prepared with poison；（3）Swine fever spleen drenches seedling and prepares.Including inoculation, semi-finished product are received, and smash removal of impurities to pieces, and bioreactor culture dispenses with seedling, several steps are lyophilized.The present invention, by increasing bioreactor culture link, allows swine fever virus to expand again numerous, to improve viral level in traditional swine fever spleen leaching seedling production process.The spleen leaching seedling produced through the invention is compared than traditional spleen leaching seedling, and potency is largely increased, and improves vaccine quality, is worthy to be popularized.
Description
Technical field
The invention belongs to Veterinary Medicine biological fields, and in particular to a kind of to enhance the external of swine fever spleen leaching attenuated vaccine potency
Cultural method.
Background technology
Swine fever is caused by swine fever virus, is a kind of infectious disease with high contact and high lethal, with bleeding and hair
Heat is main feature.Our country is listed as a kind of zoonosis.Prevention for the disease, the swine fever rabbitization that China develops are weak
Malicious vaccine C plants, encephalitis and viremia virusemia lesion are not generated with the pig of its inoculation, it can be with any period of pregnancy sow of safer vaccination and the food in one's mouth
It is newborn young, rate of fertilization is not influenced, stillborn foetus will not be caused.The vaccine that the pig strain is produced has swine fever rabbitization spleen leaching tissue vaccine, thin
Born of the same parents' cultivating seedling, newborn rabbit Tissue vaccine etc..
It is at present to one of most effective defensive measure of swine fever virus resistant, because of its best guarantor that swine fever spleen, which drenches attenuated vaccine,
The genetic stability, immunogenicity and safety for having held C plants, are favored by numerous raisers.But a problem is existed simultaneously,
Classical swine fever spleen drenches seedling vaccine preparation technique, and the malicious titre of production is relatively low, and the effective working cardial cell amount number of unit volume is relatively low, steady between batch
It is qualitative low.Therefore, it is the key that improve viral yield to develop new preparation method.
Invention content
The object of the present invention is to provide the extracorporeal culturing methods that a kind of enhancing swine fever spleen drenches attenuated vaccine potency, traditional
Swine fever spleen drenches in seedling production process, by increasing bioreactor culture link, allows swine fever virus to expand again numerous, to improve virus
Content reduces big rabbit and uses, not only increase animal welfare, and reduce production cost to promote vaccine valence quality.
To achieve the above object, the present invention is as follows using technical solution：
A kind of extracorporeal culturing method that can enhance swine fever spleen leaching attenuated vaccine potency.Steps are as follows for this method：
（1）Select rabbit：The rabbit of 2-3kg health is chosen, needs thermometric to observe before inoculation 3 days, body temperature need to meet the requirements.
（2）Production is prepared with poison：By fever virus lapinized Chinese Strain（China Veterinery Drug Inspection Office's identification, keeping and supply）With
20-50 times of diluted emulsion is made in sterile saline, is inoculated in rabbit ear vein injection 1ml.24 were inoculated with as a child, every 6-8
Hour thermometric is primary, selection sizing thermal response rabbit, sterile after lethal to take spleen, as production kind of a poison.It is stored in -20 DEG C.
（3）Swine fever spleen drenches seedling and prepares.It is as follows：
1）Inoculation：20-50 times of diluted emulsion is made with swine fever virus special-purpose protecting agent in production seed culture of viruses, is noted per rabbit ear vein
Penetrate 1mL；Inoculation 24 is as a child, primary every 6-8 hours thermometrics.
2）The harvest of semi-finished product：Selection sizing thermal response rabbit, it is lethal in drop beginning for 24 hours under body temperature, it is sterile to take spleen
With lymph node to get semi-finished product.
3）Smash removal of impurities to pieces：The spleen conjunction lymph node of fresh acquisition is added to the special nutritional of 1000ml-2000ml by every 100g
Liquid is smashed to pieces after five minutes with refiner, drenches seedling tissue separating filter filtering and impurity removing with spleen.
4）Bioreactor culture：Filtered nutrient solution liquid is moved into bioreactor, and big rabbit anteserum is added, if
Set parameter.Culture 2-6 hours.The big rabbit anteserum, content account for the 8-12% of added nutrient solution volume.
5）With seedling and packing：Under sterile working, mixing liquid is taken out, adds freeze drying protectant 6 ~ 9 by every liter of mixing liquid
It rises after fully stirring evenly, by 2 ~ 3ML/ bottles of quantitative separatings.
6）Freeze-drying：Vacuum freezedrying is carried out after packing rapidly, is pressed《Chinese regulations》Annex carries out, and needs
Meet regulation.
7）Product inspection：According to《People's Republic of China's regulations》Requirement be detected.
The special nutrient fluid is：Contain arginine 1g-5g, 1 g-5 g of lysine, glutamic acid 1 in every liter of nutrient solution
G-5 g, 1 g-5 g of glycine, remaining is cell growth medium（DMEM）.
The freeze drying protectant is：Contain trehalose 10-40g, sucrose 10-50g, hydrolysis breast in every liter of freeze drying protectant
Albumen 5-30g, gelatin hydrolysate 10-30g, skimmed milk power 30-50g, remaining is water for injection.
The bioreactor is stirring type bioreactor.Setup parameter is：35-38 DEG C of temperature, dissolved oxygen 40-80%,
PH6.0-8.0, mixing speed：5-30rmp.
It is described examine qualified production to plant poison must be according to《People's Republic of China's regulations》Want
It asks and is identified；Regulation should be met.
The advantage of the invention is that：The present invention will be determined as sizing heat rabbit it is sterile take spleen and lymph node after, through thin
Born of the same parents are broken, and being added to bioreactor, to continue culture 2-6 hours numerous to expand.The present invention is compared than traditional spleen leaching seedling, and potency obtains
To further increasing, vaccine quality is improved, is worthy to be popularized.
Specific implementation mode
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
A kind of extracorporeal culturing method that can enhance swine fever spleen leaching attenuated vaccine potency.Steps are as follows for this method：
（1）Select rabbit：The rabbit of 3kg health is chosen, needs thermometric to observe before inoculation 3 days, body temperature need to meet the requirements.
（2）Production is prepared with poison：By fever virus lapinized Chinese Strain（China Veterinery Drug Inspection Office's identification, keeping and supply）With
40 times of diluted emulsions are made in sterile saline, are inoculated in rabbit ear vein injection 1ml.Inoculation 24 as a child, was surveyed every 7 hours
Warm primary, selection sizing thermal response rabbit is sterile after lethal to take spleen, as production kind of a poison.It is stored in -20 DEG C.
（3）Swine fever spleen drenches seedling and prepares.It is as follows：
Inoculation：40 times of diluted emulsions are made with swine fever virus special-purpose protecting agent in production seed culture of viruses, 1mL is injected per rabbit ear vein；
Inoculation 24 is as a child, primary every 7 hours thermometrics.
Further, the harvest of semi-finished product and：Selection sizing thermal response rabbit, it is lethal in drop beginning for 24 hours under body temperature, it is sterile to adopt
Take spleen and lymph node to get semi-finished product.
Further, removal of impurities is smashed to pieces：The spleen conjunction lymph node of fresh acquisition is added to the special nutrient fluid of 2000ml by every 100g
It is smashed to pieces after five minutes with refiner, drenches seedling tissue separating filter filtering and impurity removing with spleen.
Further, bioreactor culture：Filtered liquid is moved into bioreactor, and big rabbit anteserum is added, if
Set parameter.Culture 4 hours.
Further, match seedling and packing：Under sterile working, mixing liquid is taken out, adds frozen-dried protective by every liter of mixing liquid
7 liters of agent fully stir evenly after by 2.5ML/ bottles of quantitative separatings.
Further, it is lyophilized：Vacuum freezedrying is carried out after packing rapidly, is pressed《Chinese regulations》Annex into
Row, need to meet regulation.
Further, product inspection：According to《People's Republic of China's regulations》Requirement be detected.
The special nutrient fluid is：Contain arginase 12 g, lysine 2g, glutamic acid 2g, glycine 3 in every liter of nutrient solution
G, remaining is cell growth medium（DMEM）.
The freeze drying protectant is：Contain trehalose 30g, sucrose 30g, lactoalbumin hydrolysate in every liter of freeze drying protectant
10g, gelatin hydrolysate 20g, skimmed milk power 40g, remaining is water for injection.
The biological projector setup parameter is：37 DEG C of temperature, dissolved oxygen 80%, PH6.5, mixing speed： 30rmp.
The big rabbit anteserum, content account for the 12% of added nutrient solution volume.
It is described examine qualified production to plant poison must be according to《People's Republic of China's regulations》Want
It asks and is identified；Regulation should be met.
Embodiment 2
A kind of extracorporeal culturing method that can enhance swine fever spleen leaching attenuated vaccine potency.Steps are as follows for this method：
（1）Select rabbit：The rabbit of 2kg health is chosen, needs thermometric to observe before inoculation 3 days, body temperature need to meet the requirements.
（2）Production is prepared with poison：By fever virus lapinized Chinese Strain（China Veterinery Drug Inspection Office's identification, keeping and supply）With
20 times of diluted emulsions are made in sterile saline, are inoculated in rabbit ear vein injection 1ml.Inoculation 24 as a child, was surveyed every 6 hours
It is warm primary, selection sizing thermal response rabbit, it is lethal it is sterile take spleen, as production kind of a poison.It is stored in -20 DEG C.
（3）Swine fever spleen drenches seedling and prepares.It is as follows：
Inoculation：20 times of diluted emulsions are made with swine fever virus special-purpose protecting agent in production seed culture of viruses, 1mL is injected per rabbit ear vein；
Inoculation 24 is as a child, primary every 6 hours thermometrics.
Further, the harvest of semi-finished product and：Selection sizing thermal response rabbit, it is lethal in drop beginning for 24 hours under body temperature, it is sterile
Take spleen and lymph node to get semi-finished product.
Further, removal of impurities is smashed to pieces：The spleen conjunction lymph node of fresh acquisition is added to the special nutrient fluid of 1500ml by every 100g
It is smashed to pieces after five minutes with refiner, drenches seedling tissue separating filter filtering and impurity removing with spleen.
Further, bioreactor culture：Filtered liquid is moved into bioreactor, and big rabbit anteserum is added, if
Set parameter.Culture 2 hours.
Further, match seedling and packing：Under sterile working, mixing liquid is taken out, adds frozen-dried protective by every liter of mixing liquid
9 liters of agent fully stir evenly after by 2.5ML/ bottles of quantitative separatings.
Further, it is lyophilized：Vacuum freezedrying is carried out after packing rapidly, is pressed《Chinese regulations》Annex into
Row, need to meet regulation.
Further, product inspection：According to《People's Republic of China's regulations》Requirement be detected.
The special nutrient fluid is：Contain arginine 1g, 1 g of lysine, 1 g of glutamic acid, sweet ammonia in every liter of nutrient solution
1 g of acid, remaining is cell growth medium（DMEM）.
The freeze drying protectant is：Contain trehalose 10g, sucrose 10g, lactoalbumin hydrolysate in every liter of freeze drying protectant
5g, gelatin hydrolysate 10g, skimmed milk power 30g, remaining is water for injection.
The biological projector setup parameter is：35 DEG C of temperature, dissolved oxygen 60%, pH7.0, mixing speed： 15rmp.
The big rabbit anteserum, content account for the 10% of added nutrient solution volume.
It is described examine qualified production to plant poison must be according to《People's Republic of China's regulations》Want
It asks and is identified；Regulation should be met.
Embodiment 3
A kind of extracorporeal culturing method that can enhance swine fever spleen leaching attenuated vaccine potency.Steps are as follows for this method：
（1）Select rabbit：The rabbit of 3kg health is chosen, needs thermometric to observe before inoculation 3 days, body temperature need to meet the requirements.
（2）Production is prepared with poison：By fever virus lapinized Chinese Strain（China Veterinery Drug Inspection Office's identification, keeping and supply）With
50 times of diluted emulsions are made in sterile saline, are inoculated in rabbit ear vein injection 1ml.Inoculation 24 as a child, was surveyed every 8 hours
It is warm primary, selection sizing thermal response rabbit, it is lethal it is sterile take spleen, as production kind of a poison.It is stored in -20 DEG C.
（3）Swine fever spleen drenches seedling and prepares.It is as follows：
Inoculation：50 times of diluted emulsions are made with swine fever virus special-purpose protecting agent in production seed culture of viruses, 1mL is injected per rabbit ear vein；
Inoculation 24 is as a child, primary every 8 hours thermometrics.
Further, the harvest of semi-finished product and：Selection sizing thermal response rabbit, it is lethal in drop beginning for 24 hours under body temperature, it is sterile to adopt
Take spleen and lymph node to get semi-finished product.
Further, removal of impurities is smashed to pieces：The spleen conjunction lymph node of fresh acquisition is added to the special nutrient fluid of 1000ml by every 100g
It is smashed to pieces after five minutes with refiner, drenches seedling tissue separating filter filtering and impurity removing with spleen.
Further, bioreactor culture：Filtered liquid is moved into bioreactor, and big rabbit anteserum is added, if
Set parameter.Culture 6 hours.
Further, match seedling and packing：Under sterile working, mixing liquid is taken out, adds frozen-dried protective by every liter of mixing liquid
6 liters of agent fully stir evenly after by 2.5ML/ bottles of quantitative separatings.
Further, it is lyophilized：Vacuum freezedrying is carried out after packing rapidly, is pressed《Chinese regulations》Annex into
Row, need to meet regulation.
Further, product inspection：According to《People's Republic of China's regulations》Requirement be detected.
The special nutrient fluid is：Contain arginine 5g, 5 g of lysine, 5 g of glutamic acid, sweet ammonia in every liter of nutrient solution
5 g of acid, remaining is cell growth medium（DMEM）.
The freeze drying protectant is：Contain trehalose 40g, sucrose 50g, lactoalbumin hydrolysate in every liter of freeze drying protectant
30g, gelatin hydrolysate 30g, skimmed milk power 50g, remaining is water for injection.
The biological projector setup parameter is：35 DEG C of temperature, dissolved oxygen 40%, pH7.5, mixing speed：5rmp.
The big rabbit anteserum, content account for the 8% of added nutrient solution volume.
It is described examine qualified production to plant poison must be according to《People's Republic of China's regulations》Want
It asks and is identified；Regulation should be met.
Finished product potency is examined
Test material
Test group：A groups：The vaccine prepared by 1 method of embodiment；B groups：The vaccine prepared by 2 method of embodiment；C groups：By implementation
Vaccine prepared by 3 method of example.
Control group：The 3 batches of vaccines produced by classical swine fever spleen leaching seedling；Respectively D1, D2, D3.
Test method：The finished product potency of A, B, C, D1, D2, D3 are detected respectively
1 finished product bioactivity result of table：
Note：" ++ " indicates that qualified "-" indicates unqualified.
From table 1 we it can be found that the vaccine of test group and control group is all determined as qualification.Wherein using the present invention
The finished product potency of A groups, B groups and C groups all reaches 1100 times in the spleen beautiful jade seedling test group of method production.And use produced in conventional processes
Spleen leaching seedling control group in D1 groups and D3 group finished product potency reach 950 times, D2 groups are 900 times.
In conclusion the potency for drenching attenuated vaccine using the swine fever spleen of the method for the present invention production compares produced in conventional processes really
The potency of swine fever spleen leaching attenuated vaccine want high, reduce big rabbit usage amount, not only increase animal welfare and saved and be produced into
This, also improves vaccine quality.The method of the present invention is simple, is worthy to be popularized.
Claims (6)
1. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency, it is characterised in that：Steps are as follows for this method：
（1）Select rabbit：The rabbit for choosing 2-3kg health, thermometric is observed 3 days before inoculation, and body temperature is between 38 DEG C -39.7 DEG C；
（2）Production is prepared with kind of a poison：20-50 times of diluted emulsion is made with sterile saline in fever virus lapinized Chinese Strain, is connect
Kind injects 1ml in rabbit ear vein, primary every 6-8 hours thermometrics after being inoculated with 24 hours, selection sizing thermal response rabbit, after lethal
It is sterile to take spleen, as production kind of a poison, it is stored in -20 DEG C；
（3）Swine fever spleen drenches seedling and prepares：
A is inoculated with：20-50 times of diluted emulsion is made with sterile saline in production seed culture of viruses, 1mL is injected per rabbit ear vein；It connects
Kind is primary every 6-8 hours thermometrics after 24 hours；
The harvest of B semi-finished product：The selection sizing big rabbit of thermal response, it is sterile after lethal to take spleen and lymph node to get semi-finished product；
C smashs removal of impurities to pieces：The special nutrient fluid of 1000ml-2000ml is added to use by every 100g the spleen of fresh acquisition and lymph node
Refiner is smashed to pieces after five minutes, drenches seedling tissue separating filter filtering and impurity removing with spleen；
D bioreactor cultures：Filtered special nutrient fluid liquid is moved into bioreactor, and big rabbit anteserum is added, if
Set parameter；Culture 2-6 hours；
E matches seedling and packing：Under sterile working, mixing liquid is taken out, adds 6 ~ 9 liters of freeze drying protectant to fill by every liter of mixing liquid
Divide after stirring evenly, by 2 ~ 3ML/ bottles of quantitative separatings；
F is lyophilized：Vacuum freezedrying is carried out after packing rapidly；
G product inspections：It is detected as requested.
2. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency according to claim 1, feature exist
In：Special nutrient fluid described in step C is：Contain arginine 1g-5g, 1 g-5 g of lysine, glutamic acid 1 in every liter of nutrient solution
G-5 g, 1 g-5 g of glycine, remaining is cell growth medium.
3. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency according to claim 1, feature exist
In：Bioreactor described in step D is stirring type bioreactor.
4. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency according to claim 1, feature exist
In：Bioreactor setup parameter described in step D is：35-38 DEG C of temperature, dissolved oxygen 40-80%, pH6.0-8.0, mixing speed：
5-30 rmp。
5. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency according to claim 1, feature exist
In：Big rabbit anteserum described in step D, addition account for the 8-12% of added nutrient solution volume.
6. a kind of extracorporeal culturing method of enhancing swine fever spleen leaching attenuated vaccine potency according to claim 1, feature exist
In：Freeze drying protectant described in step E is：Contain trehalose 10-40g, sucrose 10-50g, hydrolysis breast in every liter of freeze drying protectant
Albumen 5-30g, gelatin hydrolysate 10-30g, skimmed milk power 30-50g, remaining is water for injection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110179762A (en) * | 2019-07-09 | 2019-08-30 | 山西隆克尔生物制药有限公司 | A kind of preparation method of rabbit source swine fever heat resisting protective live vaccine |
| CN112843226A (en) * | 2021-01-15 | 2021-05-28 | 兆丰华生物科技(南京)有限公司 | Preparation method of hog cholera virus antigen for improving animal body virus content or reaction rate |
| CN113017899A (en) * | 2021-03-03 | 2021-06-25 | 兆丰华生物科技(福州)有限公司 | Efficient rabbit inoculation method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973931A (en) * | 2012-11-21 | 2013-03-20 | 福州大北农生物技术有限公司 | Production process for improving titer of swine fever live vaccine product |
-
2018
- 2018-05-07 CN CN201810427399.XA patent/CN108570457A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973931A (en) * | 2012-11-21 | 2013-03-20 | 福州大北农生物技术有限公司 | Production process for improving titer of swine fever live vaccine product |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110179762A (en) * | 2019-07-09 | 2019-08-30 | 山西隆克尔生物制药有限公司 | A kind of preparation method of rabbit source swine fever heat resisting protective live vaccine |
| CN112843226A (en) * | 2021-01-15 | 2021-05-28 | 兆丰华生物科技(南京)有限公司 | Preparation method of hog cholera virus antigen for improving animal body virus content or reaction rate |
| CN113017899A (en) * | 2021-03-03 | 2021-06-25 | 兆丰华生物科技(福州)有限公司 | Efficient rabbit inoculation method |
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