CN101775374B - Production method of porcine epidemic diarrhea virus - Google Patents
Production method of porcine epidemic diarrhea virus Download PDFInfo
- Publication number
- CN101775374B CN101775374B CN 201010103688 CN201010103688A CN101775374B CN 101775374 B CN101775374 B CN 101775374B CN 201010103688 CN201010103688 CN 201010103688 CN 201010103688 A CN201010103688 A CN 201010103688A CN 101775374 B CN101775374 B CN 101775374B
- Authority
- CN
- China
- Prior art keywords
- cell
- microcarrier
- reactor
- culture
- bio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a process for producing a porcine epidemic diarrhea virus by using a bioreactor microcarrier to culture an IB-RS-2 cell, which comprises production processes of different porcine epidemic diarrhea virus strains. The process comprises the following steps: (1) selecting an IB-RS-2 cell as a cell line for culturing a vaccine; (2) passaging and culturing a cell for culturing the vaccine; (3) reproducing a porcine epidemic diarrhea virus cell seed; (4) carrying out suspension culture on a microcarrier of the IB-RS-2 cell in a bioreactor; (5) producing a porcine epidemic diarrhea virus antibody; and (6) treating the obtained virus antibody solution. The invention can greatly reduce the production cost and improve the input-output ratio by 6-8 times. In addition, the invention has the advantages of short production period, small occupied area, easy and rapid expansion of production scale, little environment pollution, easy treatment, high degree of automation, few people and easy realization of balance and stability of quality. The invention can remarkably reduce the production cost and improve the yield and the quality of vaccines.
Description
Technical field
The present invention relates to a kind of applying biological reactor Microcarrier Cell Culture Techniques and produce the method for vaccine, can be used for the popular diarrhoea vaccine of suitability for industrialized production pig, substitute traditional spinner culture method production technique.
Background technology
At present, the unique dependence cell spinner bottle of domestic porcine epizootic diarrhea production of vaccine culture method is realized.This traditional technology labour intensity is large, and length consuming time, efficient are low, and production cost is high; Easily by environmental pollution; Between different production batchs or the difference between the different bottles of same production batch large; Be difficult to expanding production; The vaccine quality hidden danger that easily occurs being polluted by bacterium or other virus and cause relates to Biosafety and public health problem.
Summary of the invention
The object of the invention is to overcome the weak point that existing cell spinner bottle culture method technology exists, a kind of method of applying biological reactor micro-carriers cell culture High-efficient Production porcine epizootic diarrhea vaccine is provided.The method can reduce production costs in a large number, and with short production cycle, and occupied ground is little, is easy to expand the scale of production fast, and is low in the pollution of the environment and be easy to process, and level of automation is high, and employment is few, and quality is easy to realize equalization stable.The unit that can significantly improve Porcine epidemic diarrhea virus tires and vaccine output and quality.
For achieving the above object, the present invention has taked following technical scheme:
The production method of a kind of Porcine epidemic diarrhea virus comprises the steps:
(1) seedling going down to posterity and cultivating with cell: get the IB-RS-2 cell, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, cultivate with " cell growth medium ", culture temperature is 35-38 ℃; When forming good cell monolayer, being used for continues to go down to posterity or be inoculated in bio-reactor carries out microcarrier suspension culture;
(2) breeding of cell seed culture of viruses: the popular dysentery seed culture of viruses of pig poison is inoculated into the good cell monolayer continuation cultivation that step (1) forms, results virus liquid during to the above pathology of cell 50-100% in the 0.5-5% ratio with " virus-culturing fluid "; Again with this virus liquid as kind of a poison, carry out cell adaptation continuous passage rejuvenation at cell, the product that at every turn goes down to posterity carries out viral TCID
50With aseptic detection, reach viral TCID
50Stable and reach 10
7.5TCID
50Stop rejuvenation when/mL is above, as seeding;
(3) microcarrier suspension culture of IB-RS-2 cell in bio-reactor: the 30%-80% that bio-reactor is pressed cumulative volume adds aseptic " cell growth medium ", and press 3-10g/L concentration in every liter of aseptic cell growth medium and add microcarrier, start bio-reactor, the slow speed of revolution was stirred after 30 minutes, get well-grown IB-RS-2 cell on step (1) rolling bottle, be prepared as cell suspension through the digestion of EDTA-pancreatin cell dispersion liquid, press 1～3 * 10 after the cell counting
5The density of individual/mL is inoculated in the reactor, sets the culture parameters such as suitable temperature, PH, stirring velocity, dissolved oxygen content, carries out reactor and automatically controls cultivation.
(4) breeding of seedling venom: cell covers with 80%-90% on the microcarrier to be seen, and empty ball rate is lower than 5%,, full ball rate is greater than 80%, and cell state is good, and the cell counting result reaches 1～3 * 10
6Individual/as to connect the poison operation more than the mL, in the Porcine epidemic diarrhea virus kind poison that 0.5-5% ratio access step (2) prepares, the culture parameters such as the temperature that setting is fit to, PH, stirring velocity, dissolved oxygen content carry out reactor automatically to control cultivation; In the popular dysentery seed culture of viruses poison of the ratio of 0.1-5% access pig venom, get at regular intervals microcarrier in the reactor after connecing poison, with microscope observing cell pathology situation, and the TCID of test sample
50Cell substantially all comes off on the microcarrier to be seen, and the DO value is obvious ascendant trend, and the stopped reaction device stirs, after microcarrier all sinks to reactor bottom, and results supernatant liquor and microcarrier;
(5) processing of results virus liquid: virus liquid and microcarrier are after 1 freeze thawing, and be centrifugal or filter to remove-20 ℃ of freezing saving backup behind the cell debris.
The parameters such as in the technique scheme, bio-reactor is can A.T.C, PH, dissolved oxygen, stirring velocity are applicable to the bio-reactor of microcarrier suspension culture, and volume is 3L-3000L.
In the technique scheme, microcarrier is Cytodex series microcarrier, and microcarrier need clean before use, sterilize, and method is as follows: 1) soak microcarrier more than three hours with PBS; 2) clean microcarrier 3 times with PBS; 3) soak microcarrier with PBS, 121 ℃ of steam sterilizings 30 minutes.
In the technique scheme, " EDTA-pancreatin cell dispersion liquid " prescription is: massfraction is respectively the Hank ' s liquid of 0.25% pancreatin (1: 250), 0.02% EDTA;
In the technique scheme, the prescription of " cell growth medium " is: massfraction is respectively that 90%-98%MEM liquid, 2%-10% bovine serum, final concentration are the antibiotic of 100-200IU/mL, and PH is 6.8-7.6.
In the technique scheme, planting malicious inoculum size is 0.5-5%,
In the technique scheme, " virus-culturing fluid " prescription is: the MEM liquid, the final concentration that contain serum 1-4% are the antibiotic of 100-200IU/mL, and PH is adjusted into 7.2-7.4.
In the technique scheme, the bio-reactor setup parameter is: 33-38 ℃ of culture temperature culture temperature, PH6.5-7.68, dissolved oxygen 30%-70%, stirring velocity 30-80rpm.
Compared with prior art, the present invention has following beneficial effect:
(1) replace the rolling bottle cell culture technology to make the porcine epizootic diarrhea vaccine with the bio-reactor Microcarrier Cell Culture Techniques, can solve that production efficiency is low, unstable product quality, problem that virus titer is low, change by production technology and production technique, improve the cultivation of viral unit and tire 5-10 doubly, General Promotion vaccine quality and output, the security that improves vaccine.
(2) the present invention can reduce production costs in a large number, and with short production cycle, and each production cycle only needs 5-6 days, compares above obviously shortenings in 6-7 days of existing rolling bottle cell culture method.
(3) the applying biological reactor carries out production of vaccine, has level of automation high, and employment is few, the production technique simple and stable.The large occupied ground of easy to operate, output is little, is easy to expand the scale of production fast.Quality is easy to realize equalization stable.
(4) use present method and produce vaccine, the product virus titer improves 5-10 doubly than traditional rolling bottle cell culture method, and cost 3-4 doubly.The quality product homogeneous is easy to large-scale production, and whole production process does not relate to other biological safety and the public health problem that conventional production methods has.
Description of drawings
Fig. 1 is preparation flow figure of the present invention.
Embodiment
The invention will be further described below in conjunction with Figure of description and specific embodiment.
(1) select bio-reactor as the means of cultivating: the 75L bio-reactor.
(2) select microcarrier to attach the carrier of growth as cell: microcarrier Cytodex-3
(3) cleaning of microcarrier, sterilising method: 1) weighing Cytodex-2 microcarrier 3g/L, use 2LPBS liquid soaked three hours.2) clean 3 times with 2L PBS liquid at every turn.3) add 2L PBS liquid and soak microcarrier, 121 ℃ of steam sterilizings 30 minutes.
(4) select the IB-RS-2 cell as the seedling cell:
(5) seedling going down to posterity and cultivating with cell: above-mentioned cell is through EDTA-pancreatin cell dispersion liquid (Hank ' s liquid that contains 0.25% pancreatin (specification is 1: 250), 0.02%EDTA) had digestive transfer culture, to contain benzylpenicillin sodium and the Vetstrep of 92%MEM liquid, 8% calf serum, 200IU/ml, PH is adjusted into 7.4 cell growth medium continuation cultivation, and culture temperature is 37 ℃.When forming good individual layer, being used for continues to go down to posterity or be inoculated in bio-reactor carries out microcarrier suspension culture.
(6) breeding of cell seed culture of viruses: use cell maintenance medium, Porcine epidemic diarrhea virus kind poison is inoculated well-grown above-mentioned cell monolayer in 2% ratio in the future, continues to cultivate, and culture temperature is 33 ℃.Results virus liquid during to cell 80% above pathology; Measure the TCID of virus
50, treat that virus titer is stable and reach 10
7.5TCID
50Stop rejuvenation when/mL is above, should virus use kind of a poison as producing.
(7) microcarrier suspension culture of IB-RS-2 cell in bio-reactor: get well-grown IB-RS-2 cell on the rolling bottle, be prepared as cell suspension through the digestion of EDTA-pancreatin cell dispersion liquid, cell counting density is by 1 * 10
5In/ml reaction of inoculation the device.Bio-reactor is set as follows 37 ℃ of parameters of temperature, PH7.2, stirring velocity 60rpm, dissolved oxygen content 50% to carry out reactor and automatically controls cultivation.
(8) breeding of seedling venom: after cultivating on the microcarrier to be seen on the 3rd cell substantially cover with, and the cell counting result reaches 2 * 10
6Connect the poison operation after/ml is above.The culture parameters such as 34 ℃ of design temperatures, PH7.8, stirring velocity 70rpm, dissolved oxygen content 30% are carried out reactor and are automatically controlled cultivation.Get at regular intervals microcarrier in the reactor after connecing poison, with microscope observing cell pathology situation, and the TCID of test sample
50Cell substantially all comes off on the microcarrier to be seen, and the DO value is obvious ascendant trend, and the stopped reaction device stirs, and after microcarrier all sinks to reactor bottom, gathers in the crops respectively supernatant liquor and microcarrier.
The processing of results virus liquid: behind 1 multigelation ,-20 ℃ of freezing saving backup behind the filtration removal cell debris.
The inspection of semifinished product and vaccine manufacturing, inspection after construction
1. gather in the crops the mensuration of virus liquid poison valency: the cytopathy venom of above results is mixed rear sampling, measure malicious valency.Viral level answers 〉=10
7.5ELD
50/ ml.
Deactivation: add formaldehyde solution by 0.2% of total amount in virus liquid, 37 ℃ of condition deactivations 24 hours are put in jolting 2 minutes.Timing agitation mixing during this time.
2. the inspection of semifinished product after the deactivation
Steriling test: carry out asepsis growth by 301 pages of " People's Republic of China's veterinary biologics quality standard " appendix.
Deactivation check: after the deactivation, take a sample from inactivation of virus liquid with aseptic technique, inoculate healthy IB-RS-2 cell monolayer in 1% ratio, cultivated 3 days for 37 ℃, according to said method, in 3 generations of continuous passage, it is qualified that pathology should not appear in cell.
3. vaccine manufacturing and inspection after construction are undertaken by " porcine epizootic diarrhea, transmissible gastroenteritis bivalent inactivated vaccine are made and inspection procedure " requirement.
Claims (1)
1. the production method of a Porcine epidemic diarrhea virus is characterized in that:
Select bio-reactor as the cultivation instrument; The growing carrier of selecting microcarrier to attach as cell; Select the IB-RS-2 cell as seedling clone;
And comprise the steps:
(1) seedling going down to posterity and cultivating with cell: get the IB-RS-2 cell, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, cultivate with cell growth medium, culture temperature is 35-38 ℃; When forming good cell monolayer, being used for continues to go down to posterity or be inoculated in bio-reactor carries out microcarrier suspension culture;
(2) breeding of cell seed culture of viruses: the popular dysentery seed culture of viruses of pig poison is inoculated into the good cell monolayer continuation cultivation that step (1) forms, results virus liquid during to cell 50-100% pathology in the 0.5-5% ratio with virus-culturing fluid; Again with this virus liquid as kind of a poison, carry out cell adaptation continuous passage rejuvenation at cell, the product that at every turn goes down to posterity carries out viral TCID
50With aseptic detection, reach viral TCID
50Stable and reach 10
7.5TCID
50Stop rejuvenation when/mL is above, as seeding;
(3) microcarrier suspension culture of IB-RS-2 cell in bio-reactor: bio-reactor adds aseptic cell growth medium by the 30%-80% of cumulative volume, and press 3-10g/L concentration in every liter of aseptic cell growth medium and add microcarrier, start bio-reactor, the slow speed of revolution was stirred after 30 minutes, get well-grown IB-RS-2 cell on step (1) rolling bottle, be prepared as cell suspension through the digestion of EDTA-pancreatin cell dispersion liquid, press 1～3 * 10 after the cell counting
5The density of individual/mL is inoculated in the reactor, sets the temperature, pH, stirring velocity, the dissolved oxygen content culture parameters that are fit to, carries out reactor and automatically controls cultivation;
(4) breeding of seedling venom: cell covers with 80%-90% on the microcarrier to be seen, and empty ball rate is lower than 5%, and full ball rate is greater than 80%, and cell state is good, and the cell counting result reaches 1～3 * 10
6Individual/mL connects the poison operation, in the Porcine epidemic diarrhea virus kind poison that 0.5-5% ratio access step (2) prepares, sets the temperature, pH, stirring velocity, the dissolved oxygen content culture parameters that are fit to, carries out reactor and automatically controls cultivation; Get at regular intervals microcarrier in the reactor after connecing poison, with microscope observing cell pathology situation, and the TCID of test sample
50Cell substantially all comes off on the microcarrier to be seen, and the DO value is obvious ascendant trend, and the stopped reaction device stirs, after microcarrier all sinks to reactor bottom, and results supernatant liquor and microcarrier;
(5) processing of results virus liquid: supernatant liquor and microcarrier are after 1 freeze thawing, and be centrifugal or filter to remove-20 ℃ of freezing saving backup behind the cell debris;
Described bio-reactor is can A.T.C, pH, dissolved oxygen content, stirring velocity parameter, is applicable to the bio-reactor of microcarrier suspension culture, and volume is 3L-3000L;
Described microcarrier is Cytodex series microcarrier, and microcarrier need clean before use, sterilize, and method is as follows: 1) soak microcarrier more than three hours with PBS; 2) clean microcarrier 3 times with PBS; 3) soak microcarrier with PBS, 121 ℃ of steam sterilizings 30 minutes;
Described EDTA-pancreatin cell dispersion liquid prescription is: the quality volume fraction is respectively that 0.25% specification is the pancreatin of 1:250, the Hank ' s liquid of 0.02% EDTA;
The prescription of cell growth medium is: massfraction is respectively that 90%-98%MEM liquid, 2%-10% bovine serum, final concentration are the antibiotic of 100-200IU/mL, and pH is 6.8-7.6;
The malicious inoculum size of described kind is 0.5-5%, and the virus culture liquid formula is: the MEM liquid, the final concentration that contain serum 1-4% are the antibiotic of 100-200IU/mL, and pH is adjusted into 7.2-7.4;
The bio-reactor setup parameter is in the described step (3): culture temperature 33-38 ℃, pH 6.5-7.68, dissolved oxygen 30%-70%, stirring velocity 30-80rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010103688 CN101775374B (en) | 2010-02-01 | 2010-02-01 | Production method of porcine epidemic diarrhea virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010103688 CN101775374B (en) | 2010-02-01 | 2010-02-01 | Production method of porcine epidemic diarrhea virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101775374A CN101775374A (en) | 2010-07-14 |
| CN101775374B true CN101775374B (en) | 2013-04-24 |
Family
ID=42511960
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010103688 Active CN101775374B (en) | 2010-02-01 | 2010-02-01 | Production method of porcine epidemic diarrhea virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101775374B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002482B (en) * | 2010-12-08 | 2013-05-22 | 成都天邦生物制品有限公司 | Method for producing PRRS (Porcine Reproductive and Respiratory Syndrome) viruses |
| CN102851301B (en) * | 2012-07-06 | 2014-09-10 | 广东大华农动物保健品股份有限公司 | Pig epidemic diarrhea virus M gene full sequence amplification method |
| CN105368793A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Method of utilizing stirred bioreactor to prepare porcine epidemic diarrhea virus |
| CN107412763B (en) * | 2017-04-26 | 2020-10-16 | 广州渔跃生物技术有限公司 | Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof |
| CN107937353A (en) * | 2017-10-31 | 2018-04-20 | 广州齐志生物工程设备有限公司 | A kind of cultural method of fishes infectious Hematopoietic Necrosis's disease virus |
-
2010
- 2010-02-01 CN CN 201010103688 patent/CN101775374B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN101775374A (en) | 2010-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101748102B (en) | Method for production of porcine epidemic diarrhea virus | |
| CN102091329B (en) | Preparation method of inactivated porcine parvovirus vaccine and product thereof | |
| CN101775374B (en) | Production method of porcine epidemic diarrhea virus | |
| CN102178946B (en) | Application of baby hamster kidney(BHK)-21 cell serum-free suspension culture technology in foot-and-mouth disease vaccine production | |
| CN101979519A (en) | Method for preparing pseudorabies vaccines | |
| CN101665781B (en) | High-titer Porcine circovirus 2-type cultured cell, preparation method and use thereof | |
| CN102660510B (en) | A kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus | |
| CN101955915B (en) | Method for producing influenza virus vaccine | |
| CN103550771A (en) | Production method of transmissible gastroenteritis virus vaccine | |
| CN102861329A (en) | Production method of canine parvovirus inactivated vaccine by utilizing bioreactor | |
| CN103087995B (en) | Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells | |
| CN101716341A (en) | Human diploid cell inactivated rabies vaccine and preparation method thereof | |
| CN102133398A (en) | Method for industrially producing animal influenza vaccine by using bioreactor | |
| CN102002481B (en) | Production method of porcine reproductive and respiratory syndrome virus | |
| CN103520715B (en) | Method for producing porcine circovirus II type inactivated vaccine by utilizing WAVE bioreactor | |
| CN104027798A (en) | Method for culturing and producing PVC 2 antigen through whole suspension cells | |
| CN104004720A (en) | Method for producing porcine circovirus type 2 antigens in large scale with high density | |
| CN102038945B (en) | Method for industrially producing swine parvovirus vaccine by utilizing bioreactor | |
| CN102002482B (en) | Method for producing PRRS (Porcine Reproductive and Respiratory Syndrome) viruses | |
| CN103550772B (en) | Production method of pseudorabies virus vaccine | |
| CN102727877A (en) | Method for preparing highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) by utilizing bioreactor and application thereof | |
| CN102038943A (en) | Method for industrially producing porcine Japanese encephalitis (JE) vaccines by utilizing bioreactor | |
| CN102038944B (en) | Method for industrially producing swine fever live vaccine by using bioreactor | |
| CN105368794A (en) | Method of utilizing stirred bioreactor to produce infectious Bursal disease virus | |
| CN102657859A (en) | Method for producing avian influenza vaccine by using cells cultured by spherical microcarrier bioreactor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Patentee after: Chengdu Shiji biopharmaceutical Co.,Ltd. Address before: 610100 Ling Lu, Longquanyi economic and Technological Development Zone, Chengdu, Sichuan Patentee before: CHENGDU TECBOND BIOLOGICAL PRODUCTS CO.,LTD. |
|
| CP03 | Change of name, title or address |