CN103157105A - Method for preparing swine fever live vaccines - Google Patents
Method for preparing swine fever live vaccines Download PDFInfo
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- CN103157105A CN103157105A CN2012105781253A CN201210578125A CN103157105A CN 103157105 A CN103157105 A CN 103157105A CN 2012105781253 A CN2012105781253 A CN 2012105781253A CN 201210578125 A CN201210578125 A CN 201210578125A CN 103157105 A CN103157105 A CN 103157105A
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Abstract
The invention relates to a method for preparing swine fever live vaccines, and belongs to the field of biotechnology. The method for preparing the swine fever live vaccines includes the following steps: (1) cell inoculation, (2) culturing of cells used for vaccine preparation, (3) virus inoculation, (4) virus culture and harvest, and (5) vaccine preparation. Cells used for vaccine preparation are screened, adaptability between the cells and virus is strengthened, a riptide perfusion type bioreactor culture system is used for improving multiplication titer and harvest yield of the virus, and a whole production process does not involve other biosafety and public health problems and is suitable for large scale production.
Description
Technical field
The present invention relates to a kind of method of utilizing the torrent filling type bioreactor to prepare live vaccines of hog cholera, belong to biological technical field
Background technology
Swine fever (Classical Swine Fever, CSF) is commonly called as " rinderpest ", is called hog cholera in the U.S., and some areas, Europe are called classic swine fever.Primary disease is a kind of acute hot general septic infectious disease that is caused by swine fever virus, and various age pigs all can be fallen ill, and the course of disease is varied from acute to chronic etc.Popular throughout the year, infectiousness is extremely strong, has the height M ﹠ M, in case have destructiveness, seriously threatens the development of pig industry and affects the people's life.In swine diseases, swine fever is that harm is maximum, the most valued a kind of disease, and this disease is decided to be one of the 16 kinds of infectious disease of category-A that must examine by international veterinary hygiene rules of organization, be listed in a class infectious disease in China.
The weak poison of swine fever virus rabbitization (C strain) of China's development is internationally recognized best swine fever virus vaccine, is adopted by a lot of countries in Europe, has effectively controlled the popular of swine fever.Swine fever often has the report of immuning failure in recent years, although its reason is a lot, the quality of vaccine has very important impact.Though traditional spinner culture or the standing culture process of live vaccines of hog cholera follow the several years, be difficult to break away from and tire unstablely, the disadvantage such as differences between batches are large, and virus titer is low has greatly limited quality and the output of live vaccines of hog cholera.
In recent years, along with the development of live vaccine industry, bioreactor was utilized bioreactor to substitute the trend that traditional rolling bottle production technology has become industry development by extensive concern.Domestic existing applying biological reactor is produced the research report of live vaccines of hog cholera.From the industry overall development, the applying biological reactor is produced live vaccines of hog cholera and is had greatly improved than rolling bottle technique, but at aspects such as production technology optimization and cost control, very large development space is arranged all.The research of using torrent filling type bioreactor production live vaccines of hog cholera has no report.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the torrent filling type bioreactor to prepare live vaccines of hog cholera.Realized by following technical scheme:
A kind of method for preparing live vaccines of hog cholera, it utilizes the torrent filling type bioreactor as the cultivation instrument, and described torrent filling type bioreactor comprises the first peristaltic pump, the second peristaltic pump, the 3rd peristaltic pump, the first silica gel tube, the second silica gel tube, the 3rd silica gel tube, porose silica gel tube, scraps of paper carrier, infusion bag, vortex mixer and torrent tank; Wherein, vortex mixer is to have heating or heat insulation function, and can carry out to internal liquid the tank shape device of mixing; In described reactor, the flow direction of liquid is: infusion bag---silica gel tube---vortex mixer---silica gel tube---torrent tank---silica gel tube---infusion bag;
The live vaccine preparation is carried out according to the following steps:
A. cell inoculation
Be the infusion bag (9) that pig testis cell ST and cell growth medium mixed liquor are seeded to the torrent filling type bioreactor with cell suspension, cell is attached on polyester fiber scraps of paper carrier (8), the cell inoculum density is: every gram carrier inoculation 0.8～1.5 * 10
7Individual cell;
B. seedling cell culture
Set device parameter: temperature T 1:36.5～37.5 ℃, T2:40～42 ℃, T3:34～36 ℃, pH:7.2～7.4, DO:50%～70%, hunting speed 15～30r/min, air mass flow 100ml/min～1000ml/min, active cell is cultivated program, carries out cell culture, obtains the seedling cell;
C. virus inoculation
When cell odd-numbered day consumption sugar tends to be steady, show that cell reaches to connect malicious requirement, liquid in emptying reactor is inoculated the seedling cell with swine fever virus, and dosage of inoculation is 0.3%～1%(V/V) of effective volume of culture, adsorbs 15～30min; Add cell maintenance medium;
D. Virus culture and results
Set device parameter: temperature T 1:36～37 ℃, T2:39～41 ℃, T3:33～35 ℃, pH:7.3～7.5, DO:50%～70%, hunting speed 15～30r/min, air mass flow 400ml/min～4000ml/min starts the Virus culture program, the amplification cultivation swine fever virus;
E. virus liquid is gathered in the crops
After connecing poison, every 72h gathers in the crops once, and multiple batches of results virus liquid saves backup;
F. vaccine preparation
Add freeze drying protectant to virus liquid, packing, lyophilizing make live vaccines of hog cholera.
The above-mentioned method for preparing live vaccines of hog cholera, in described step a, seed cell is the pig testis subculture cells ST, be the cell through the screening of limiting dilution assay clone purification, use limiting dilution assay cell to be screened is carried out monoclonal, amplification culture is set up the monoclonal cell bank, by the high-adaptability monoclonal cell strain of rabbit body-shaping thermal response method screening swine fever virus vaccine.
The above-mentioned method for preparing live vaccines of hog cholera, in described step b, adopt the stage fed-batch mode to replenish cell growth medium in cell cultivation process, when the residual sugar amount during lower than 1.5g/L, stream adds additional cell growth medium, flow acceleration maintains between 1.5～2g/L with the culture fluid sugar content and is as the criterion, and when the culture fluid volume reaches effective volume of culture, changes culture fluid.
The above-mentioned method for preparing live vaccines of hog cholera in described step b, increases air mass flow day by day, and every 24h increases 100ml/min～1000ml/min.
The above-mentioned method for preparing live vaccines of hog cholera in described steps d, connects after malicious 24h the beginning Continuous-flow and adds and replenish doubly concentrated DMEM liquid of n, and flow acceleration maintains between 1～2g/L with culture fluid residual sugar amount and is as the criterion; Described n equals 2～5.
The above-mentioned method for preparing live vaccines of hog cholera, described n doubly concentrated DMEM liquid making method is: after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again (n-1) part without the Na-DMEM dehydrated medium, dissolving; Described n equals 2～5.
The above-mentioned method for preparing live vaccines of hog cholera, in described step e, harvesting approach is multiple batches of results; Every 72h gathers in the crops once, sustained yield 10～15 times, and each harvest yield is 90%～100% of culture fluid cumulative volume.
Compared with prior art, the present invention has following beneficial effect:
1. in the present invention, seedling through purification screening, has improved the sensitivity of cell to virus with cell, has guaranteed the state homogeneous of seed cell, and difference between reducing batch is guaranteed the stable of processing parameter and product quality.
2. in the present invention, compare with conventional torrent pouring bioreactor culture system, increased a vortex mixer, increase the volume of circulating system culture fluid, reduced and changed the liquid number of times, simplified operating process, optimize the Growth of Cells environment, improved virus liquid output.
3. increase gradually air mass flow in cell cultivation process of the present invention, gas circulation in the quickening system is conducive to overflowing of CO2 in culture fluid, reduces the use amount of NaHCO3.
4. the concentrated nutrient solution that uses of the present invention, the several amino acids content that both can double can obviously not increase again the osmotic pressure of culture fluid, for virus breeding provides sufficient nutrition, is conducive to improve virus titer.
5. the multiple batches of results virus liquid of the present invention had both guaranteed the titre of results virus liquids greatly to have improved again virus liquid output.
Description of drawings
Fig. 1 is that the fluid circulation system of torrent filling type bioreactor forms floor map;
12 batches of Fig. 2 AP20C results virus liquids curve chart of tiring;
10 batches of Fig. 3 AP200 results virus liquids curve chart of tiring;
In accompanying drawing or word, each list of reference numerals is: 1. the first peristaltic pump (perfusion Pump for giving-out), 2. the second peristaltic pump (perfusion liquid feeding pump), 3. the 3rd peristaltic pump, 4. the first silica gel tube, 5. the second silica gel tube, 6. the 3rd silica gel tube, 7. porose silica gel tube, 8. polyester fiber scraps of paper carrier, 9. infusion bag, 10. vortex mixer, 11. torrent tanks;
T1 is torrent pot liquid temperature, and T2 is the temperature of torrent tank heating film, and T3 is infusion bag external environment temperature, pH is torrent pot liquid pH value, DO is torrent pot liquid dissolved oxygen rate, and hunting speed is the hunting speed of torrent tank, and air mass flow is torrent tank inner air flow amount.
The specific embodiment
Below in conjunction with the specific embodiment, the present invention is described in further detail, wherein each embodiment only be used for explanation the present invention and and non-limiting scope of patent protection of the present invention.
Pig testis cell ST is available from China Veterinery Drug Inspection Office;
Swine fever virus vaccine is swine fever virus Lapinized strain (C strain), available from China Veterinery Drug Inspection Office;
The formula of cell growth medium is volumn concentration 90% DMEM in high glucose liquid, 10% new-born calf serum, and adjusting pH value is 7.4;
Cell maintenance medium is volumn concentration 96% DMEM in high glucose liquid, 4% new-born calf serum, and adjusting pH value is 7.4;
A. monoclonal cell strain preparation
A. the preparation of cell suspension
Cultivate pig testis cell ST in the cell bottle, when cell covers with monolayer into densification, clear-cut, digest with 0.025% pancreatin, make cell suspension.
B. the limiting dilution of cell
Prepared pig testis cell ST suspension is carried out cell counting, add the DMEM culture fluid diluting cells suspension of serum-free, make cell density be about 1.0 * 10
5Individual/ml; 10 times of gradient dilutions to 10 of pig testis cell ST suspension after diluting with serum-free DMEM culture fluid
3Individual/ml, then use 10 times of gradient dilutions to 10 of cell growth medium
1Individual/ml, namely every 0.1ml contains 1 cell.
C. the clone of cell cultivates
The cell suspension that above-mentioned dilution is good adds in 96 porocyte culture plates, the 0.1ml/ hole, and microscopically is observed, and discards the not good cell hole of non-single cell and cell state, is placed in 37 ℃, 5% CO
2Cultivate, every day, observation of cell growth conditions and in time change liquid, treated that cell proliferation is cloning community, chooses the good cell hole of growth conditions, digests with 0.025% pancreatin, makes suspension, repeats above-mentioned cloning process 1 ~ 2 time, until filter out the monoclonal hole of growing.
D. the amplification culture of monoclonal cell strain
Choose monoclonal growth hole, use 0.025% trypsinization, with the calf serum neutralization, resuspended with cell growth medium, be transferred to 24 porocyte culture plates and carry out amplification culture, when cell proliferation is about 80% to degree of converging, use 0.025% trypsinization, add cell growth medium, be inoculated in amplification culture in Tissue Culture Flask, cultivate 6 ~ 10 bottles, carry out the foundation of the frozen and cell bank of monoclonal cell.
B. the screening of swine fever virus vaccine high-adaptability cell strain
A. the digestion bed board of ST monoclonal cell strain
The ST monoclonal cell strain of covering with monolayer is also counted with 0.025% trypsinization, and every strain monoclonal cell paving 24 porocyte culture plates are established multiple hole, 30000 cells/well.Be placed in 37 ℃, 5% CO
2Cultivate 24h.
B. ST monoclonal cell strain Pigs Inoculated Pestivirus
Discard the cell growth medium in 24 porocyte culture plates, add 0.1M PBS, the 0.8ml/ hole is after purge 2 ~ 3 times, press V:V 3% inoculum concentration inoculation swine fever virus vaccine, the 1.0ml/ hole, 37 ℃ discard virus liquid after hatching 1h, add cell maintenance medium 1.0ml, be placed in 37 ℃, 5% CO
2Cultivate 72h, the results virus liquid.
C. the viral efficacy determinations of swine fever virus vaccine is cultivated in the strain of ST monoclonal cell
With physiological saline solution, the hog cholera venom of gathering in the crops is carried out 5 * 10
4, 1 * 10
5, 2 * 10
5, 3 * 10
5, 4 * 10
5, 5 * 10
5, 1 * 10
6, 2 * 10
6Times gradient dilution, 2 of each gradient ear vein injection body weight 2.0kg rabbit, every 1ml.After family's rabbit inoculation, respectively survey body temperature once morning and afternoon, after 48h, surveyed body temperature once every 6 hours, judges according to rabbit typing thermal response standard.Concrete steps are with reference to " People's Republic of China's regulations " in 2000 version.
C. result
A. the cultivation of ST monoclonal cell strain
Obtain ST monoclonal cell strain 47 strains by the limiting dilution assay amplification culture.Liquid nitrogen freezing is preserved, frozen 5 of every strain monoclonal cell, and every 1.5ml, density is about 5 * 10
6Individual/ml.
B. the screening of swine fever virus vaccine high-adaptability ST monoclonal cell strain.
The viral effect that infects after ST monoclonal cell strain 72h by swine fever virus vaccine detects, and filter out the ST monoclonal cell strain that 2 strains are tired the highest: the 17th strain and the 30th strain, viral effect is the highest in 47 strains, is 4 * 10
5RID/ml and 5 * 10
5RID/ml.Tire (2 * 10 with swine fever virus vaccine infects common pig testis cell ST
4～5 * 10
4RID/ml) compare, improve approximately 10 times.Thus, screening obtains the 17th strain and the 30th strain is the strain of swine fever virus vaccine high-adaptability ST monoclonal cell, and label is S17 and S30.
S17 pig testis subculture cells ST is deposited in Chinese Typical Representative culture collection center on November 14th, 2012, and the depositary institution address is Wuhan, China university, and preserving number is CCTCC NO:C2012180.Described cell is this area cell commonly used, therefore its feature description is repeated no more.
AP20C type torrent filling type bioreactor used in the present embodiment, infusion bag 9 volumes are 4.5L, and vortex mixer 10 volumes are 8L, and torrent tank 11 volumes are 15L, and infusion bag 9 contains 200g polyester fiber scraps of paper carrier 8; Theoretical effectively volume of culture is 18L.
Seedling is with the pig testis cell (ST) of cell with culture presevation in embodiment 1;
Plant poison with fever virus lapinized Chinese Strain in embodiment 1 (C strain), a certain batch of kind toxic effect valency is 5 * 10
5RID/ml;
Cell growth medium is volumn concentration 92% DMEM in high glucose liquid, 8% new-born calf serum, and adjusting pH value is 7.3;
Cell maintenance medium is volumn concentration 97% DMEM in high glucose liquid, 3% new-born calf serum, and adjusting pH value is 7.4;
Without the Na-DMEM dehydrated medium for not contain NaCl and phenol red DMEM in high glucose culture medium, by Invitrogen Corporation production and sales;
A, preparation:
The checking system air-tightness, calibration electrodes, PBS processes scraps of paper carrier 8, connects infusion bag 9, vortex mixer 10 and torrent tank 11, soaks scraps of paper carrier 8, balance sysmte with cell growth medium.
B, preparation work, concrete steps are as follows:
A. seedling is cultivated with early stage with the recovery of cell
Take out frozen pig testis cell ST recovery from liquid nitrogen container after, with cell growth medium in 37 ℃ of cultivations, grow up to after good monolayer digestion go in the 10L rolling bottle cultivate, standby.
B. the seedling inoculation of cell in bioreactor
Get 7 rolling bottles that cover with cell monolayer, use 0.025% trypsin digestion cell, preparation cell suspension 5L(contains cell and cell growth medium), total cellular score approximately 2.7 * 10
9Individual.
Two step inocalation methods, the first step: respectively pump into cell suspension 4.0L, standing adsorption 1h in infusion bag 9 from the bottom are adopted in the cell inoculation; Second step: the liquid in infusion bag 9 is pumped 1.0L, then pump into 1.0L remaining cell suspension, standing adsorption 0.5h from infusion bag 9 top deep layer pipes; Simultaneously in the preheating of the interior additional 4L cell growth medium of torrent tank 11; The cell inoculum density is: every gram carrier inoculation 1.35 * 10
7Individual cell.
C. seedling cell culture
After adsorption process is completed, device parameter is set as follows: T1:37.2 ℃, T2:41 ℃, T3:35 ℃, PH:7.3, DO:50 ~ 70%, concussion speed 30r/min, liquid circulation velocity 300ml/min, air mass flow 100ml/min.Active cell is cultivated program, carries out cell culture.
Take a sample every day and survey the residual sugar amount 2 times, calculate odd-numbered day consumption sugar amount, cell culture 36h culture fluid residual sugar amount begins stream and adds additional cell growth medium lower than 1.5g/L, and flow acceleration is 4.4ml/min; In cell culture 72h pumping out system, culture fluid pumps the culture fluid of total volume of culture 70%, pumps into growth-promoting media 3.6L, and flow acceleration is 6.2ml/min;
Cell culture fluid circumfusion speed increases gradually with the increase of incubation time, makes silica gel tube 4, the interior liquid color of silica gel tube 6 keep basically identical.Day by day increase air mass flow, every 24h increases 100ml/min.
D. swine fever virus inoculates the seedling cell
After cell inoculation consumption sugar amount reaches 2.1g/L/24h (96h) odd-numbered day on the 4th, and tends to be steady, and determines that cell density has reached to connect malicious requirement.Liquid in emptying reactor, 150ml swine fever virus liquid pump is entered in torrent bag 11, together with the 4L depletion of blood inventory times DMEM liquid in torrent bag 11, together pump in infusion bag 9, standing adsorption 20min, replenish simultaneously the DMEM nutritional solution that 6L contains serum 5% in torrent bag 11, virus inoculation dosage is about 0.83% of effective volume.
E. Virus culture and results
After adsorption process is completed, device parameter is set as follows: T1:36 ℃, T2:39 ℃, T3:35 ℃, PH:7.3, DO:50 ~ 70%, concussion speed 30rpm, liquid circulation velocity 500ml/min, ventilation 400ml/min starts the Virus culture program and begins to cultivate.
Connecing after malicious 24h the beginning Continuous-flow adds and replenishes 3 times of concentrated DMEM liquid (after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again 2 parts without the Na-DMEM dehydrated medium, dissolving forms), flow acceleration 2.5ml/min.
After connecing poison every 72h results once, for the first time, the after-crop amount is 90% of culture fluid cumulative volume, gathers in the crops complete rear additional cell maintenance medium, making the culture fluid total amount is 10L, begins stream after 24h and adds 3 times of concentrated DMEM liquid, flow acceleration is 2.6ml/min.Begin for the third time, each harvest yield is 100% of culture fluid cumulative volume, gathers in the crops complete rear additional cell maintenance medium, and making the culture fluid total amount is 10L, begins stream after 24h and adds 3 times of concentrated DMEM liquid, and flow acceleration is 2.6ml/min.Gather in the crops altogether 12 times.12 times harvest yield is respectively: 16.2L, 16.2L, 17.9L, 18L, 17.8L, 18L, 18L, 17.9L, 17.8L, 18L, 18L, 17.9L.
The venom of each results is first through the filter of 0.5 micron, after removing cell debris, in-20 ℃ of freezing preservations.
F. virus liquid efficacy test
With physiological saline solution, the hog cholera venom of gathering in the crops is carried out 5 * 10
5, 1 * 10
6, 2 * 10
6, 3 * 10
6, 4 * 10
6, 5 * 10
6, 6 * 10
6, 8 * 10
6, 1 * 10
7Times gradient dilution, 2 of each gradient ear vein injection body weight 2.0kg rabbit, every 1ml.After family's rabbit inoculation, respectively survey body temperature once morning and afternoon, after 48h, surveyed body temperature once every 6 hours, judges according to rabbit typing thermal response standard.
The virus liquid of results is measured and is tired, and 10 batches of results virus liquids tire all 〉=1 * 10
6The RID/ml(10 batch of results virus liquid curve chart of tiring seen Fig. 2)
The effect (RID/ml) of 12 batches of results virus liquids of table 1
G. packing lyophilizing
With the virus-culturing fluid that is up to the standards, mix, fully shake up by (V:V) 1:1 with sucrose skimmed milk stabilizing agent (5% sucrose and 12% skimmed milk), quantitative separating contains cell toxicant and is no less than 7500RID/ head part.
H. safety verification
Do safety check with pig, with reference to " People's Republic of China's regulations " version operation in 2000, before wean after immunity temperature of pig body, spirit, appetite and notes Seedling, without significant change, safety check is qualified.
I.. vaccine potency check
With 7500 times of every part vaccine dilutions, ear vein is injected 2 of body weight 2.0kg rabbit, every 1ml with physiological saline solution, after family's rabbit inoculation, respectively survey body temperature once morning and afternoon, after 48h, surveyed body temperature once every 6 hours, judge according to rabbit typing thermal response standard.The effect inspection is qualified.
In the present embodiment, torrent filling type bioreactor used comprises two kinds, AP20C type and AP200 type.
AP20C type torrent filling type bioreactor is with embodiment 2.
AP200 type infusion bag 9 volumes are 45L, and vortex mixer 10 volumes are 80L, and torrent tank 11 volumes are 150L, and infusion bag 9 contains 2000g polyester fiber scraps of paper carrier 8; Theoretical effectively volume of culture is 180L.
Seedling cell, kind poison are all with embodiment 2.
A, preparation
Select two kinds of torrent filling type bioreactors of AP20C type and AP200 type, do the front preparation of cultured cell.
B, preparation work, concrete steps are as follows:
A. the seedling cultivation of cell
According to embodiment 2 methods, cultivate seedling cell 96h in AP20C type bioreactor, measure to cell consumption sugar and increase to 1.9g/L/24h and when tending to be steady, with 0.025% trypsinization infusion bag 9 inner cells, make cell suspension 50L, include pig testis cell ST approximately 2.3 * 10
10Individual.The cell suspension that makes is inoculated in AP200 type bioreactor infusion bag 9 in two steps: the first step: pump into cell suspension 40L, standing adsorption 1h in infusion bag 9 from the bottom; Second step: the liquid in infusion bag 9 is respectively pumped 10L, respectively pump into 10L remaining cell suspension, standing adsorption 0.5h from infusion bag 9 top deep layer pipes; Simultaneously in the preheating of the interior additional 40L cell growth medium of torrent tank 11; The cell inoculum density is: every gram carrier inoculation 1.15 * 10
7Individual cell.
After adsorption process is completed, device parameter is set as follows: T1:37 ℃, T2:41 ℃, T3:34 ℃, PH:7.3, DO:50～70%, hunting speed 30r/min, circulation rate 3000ml/min, air mass flow 1000ml/min.Active cell is cultivated program, carries out cell culture.
Take a sample every day and survey the residual sugar amount 2 times, calculate odd-numbered day consumption sugar amount, cell culture 36h culture fluid residual sugar amount begins stream and adds additional cell growth medium lower than 1.5g/L, and flow acceleration is 44ml/min; Cell culture 72h pumps the culture fluid of total volume of culture 50%, and flow acceleration is adjusted to 60ml/min; In cell culture 96h pumping out system, culture fluid pumps the culture fluid of total volume of culture 70%, pumps into growth-promoting media 36L, and flow acceleration is 60ml/min;
Cell culture fluid circumfusion speed increases gradually with the increase of incubation time, makes silica gel tube 4, the interior liquid color of silica gel tube 6 keep basically identical.Day by day increase air mass flow, every 24h increases 1000ml/min.
B. hog cholera lapinised virus inoculation
After cell inoculation consumption sugar amount reaches 2.0g/L/24h (120h) odd-numbered day on the 5th, and tends to be steady, and determines that cell density has reached to connect malicious requirement.Liquid in emptying reactor, 1500ml hog cholera lapinised virus venom is pumped in torrent bag 11, together with the 40L depletion of blood inventory times DMEM liquid in torrent bag 11, together pump in infusion bag 9, standing adsorption 20min, replenish simultaneously the DMEM nutritional solution that 60L contains serum 5% in torrent bag 11, virus inoculation dosage is about 0.83%.
C. gather in the crops virus liquid
After adsorption process is completed, device parameter is set as follows: T1:36 ℃, T2:40 ℃, T3:33 ℃, PH:7.4, DO:50 ~ 70%, concussion speed 30rpm, liquid circulation velocity 5000ml/min, ventilation 4000ml/min starts the Virus culture program and begins to cultivate.
Connecing after malicious 24h the beginning Continuous-flow adds and replenishes 3 times of concentrated DMEM liquid (after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again 2 parts without the Na-DMEM dehydrated medium, dissolving forms), flow acceleration 25ml/min.
After connecing poison every 72h results once, for the first time, the after-crop amount is 90% of culture fluid cumulative volume, gathers in the crops complete rear additional cell maintenance medium, making the culture fluid total amount is 100L, begins stream after 24h and adds 3 times of concentrated DMEM liquid, flow acceleration is 26ml/min.Begin for the third time, each harvest yield is 100% of culture fluid cumulative volume, gathers in the crops complete rear additional cell maintenance medium, and making the culture fluid total amount is 100L, begins stream after 24h and adds 3 times of concentrated DMEM liquid, and flow acceleration is 26ml/min.Gather in the crops altogether 10 times.10 times harvest yield is respectively: 162L, 162L, 178L, 179L, 178L, 180L, 179L, 178L, 180L, 180L.
The venom of each results is first through the filter of 0.5 micron, after removing cell debris, in-20 ℃ of freezing preservations;
D. virus liquid efficacy test
With physiological saline solution, the hog cholera venom of gathering in the crops is carried out 5 * 10
5, 1 * 10
6, 2 * 10
6, 3 * 10
6, 4 * 10
6, 5 * 10
6, 6 * 10
6, 8 * 10
6, 1 * 10
7Times gradient dilution, 2 of each gradient ear vein injection body weight 2.0kg rabbit, every 1ml.After family's rabbit inoculation, respectively survey body temperature once morning and afternoon, after 48h, surveyed body temperature once every 6 hours, judges according to rabbit typing thermal response standard.
The virus liquid of results is measured and is tired, and 10 batches of results virus liquids tire all 〉=1 * 10
6The RID/ml(10 batch of results virus liquid curve chart of tiring seen Fig. 3)
The effect (RID/ml) of 10 batches of results virus liquids of table 2
| Receive inferior | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Effect RID/ | 1×10 6 | 2×10 6 | 4×10 6 | 4×10 6 | 3×10 4 | 3×10 6 | 4×10 6 | 3×10 6 | 3×10 6 | 4×10 6 |
E. packing lyophilizing
With the virus-culturing fluid that is up to the standards, mix, fully shake up by (V:V) 1:1 with sucrose skimmed milk stabilizing agent, quantitative separating contains cell toxicant and is no less than 7500RID/ head part.
F. safety verification
Do safety check with pig, with reference to " People's Republic of China's regulations " version operation in 2000, before wean after immunity temperature of pig body, spirit, appetite and notes Seedling, without significant change, safety check is qualified.
[0062]. the vaccine potency check
With 7500 times of every part vaccine dilutions, ear vein is injected 2 of body weight 2.0kg rabbit, every 1ml with physiological saline solution, after family's rabbit inoculation, respectively survey body temperature once morning and afternoon, after 48h, surveyed body temperature once every 6 hours, judge according to rabbit typing thermal response standard.The effect inspection is qualified.
Claims (7)
1. method for preparing live vaccines of hog cholera, it is characterized in that: it utilizes the torrent filling type bioreactor as the cultivation instrument, and described torrent filling type bioreactor comprises the first peristaltic pump (1), the second peristaltic pump (2), the 3rd peristaltic pump (3), the first silica gel tube (4), the second silica gel tube (5), the 3rd silica gel tube (6), porose silica gel tube (7), scraps of paper carrier (8), infusion bag (9), vortex mixer (10) and torrent tank (11); Wherein, vortex mixer (10) is to have heating or heat insulation function, and can carry out to internal liquid the tank shape device of mixing; In described reactor, the flow direction of liquid is: infusion bag (9)---silica gel tube (4)---vortex mixer (10)---silica gel tube (5)---torrent tank (11)---silica gel tube (6)---infusion bag (9);
The live vaccine preparation is carried out according to the following steps:
A. cell inoculation
Be the infusion bag (9) that pig testis cell ST and cell growth medium mixed liquor are seeded to the torrent filling type bioreactor with cell suspension, cell is attached on polyester fiber scraps of paper carrier (8), the cell inoculum density is: every gram carrier inoculation 0.8～1.5 * 10
7Individual cell;
B. seedling cell culture
Set device parameter: temperature T 1:36.5～37.5 ℃, T2:40～42 ℃, T3:34～36 ℃, pH:7.2～7.4, DO:50%～70%, hunting speed 15～30r/min, air mass flow 100ml/min～1000ml/min, active cell is cultivated program, carries out cell culture, obtains the seedling cell;
C. virus inoculation
When cell odd-numbered day consumption sugar tends to be steady, show that cell reaches to connect malicious requirement, liquid in emptying reactor is inoculated the seedling cell with swine fever virus, and dosage of inoculation is 0.3%～1%(V/V) of effective volume of culture, adsorbs 15～30min; Add cell maintenance medium;
D. Virus culture and results
Set device parameter: temperature T 1:36～37 ℃, T2:39～41 ℃, T3:33～35 ℃, pH:7.3～7.5, DO:50%～70%, hunting speed 15～30r/min, air mass flow 400ml/min～4000ml/min starts the Virus culture program, the amplification cultivation swine fever virus;
E. virus liquid is gathered in the crops
After connecing poison, every 72h gathers in the crops once, and multiple batches of results virus liquid saves backup;
F. vaccine preparation
Add freeze drying protectant to virus liquid, packing, lyophilizing make live vaccines of hog cholera.
2. prepare according to claim 1 the method for live vaccines of hog cholera, it is characterized in that, in described step a, seed cell is the pig testis subculture cells ST, be the cell through the screening of limiting dilution assay clone purification, use limiting dilution assay cell to be screened is carried out monoclonal, amplification culture is set up the monoclonal cell bank, by the high-adaptability monoclonal cell strain of rabbit body-shaping thermal response method screening swine fever virus vaccine.
3. prepare according to claim 2 the method for live vaccines of hog cholera, it is characterized in that, in described step b, adopt the stage fed-batch mode to replenish cell growth medium in cell cultivation process, during lower than 1.5g/L, stream adds additional cell growth medium when the residual sugar amount, and flow acceleration maintains between 1.5～2g/L with the culture fluid sugar content and is as the criterion, when the culture fluid volume reaches effective volume of culture, change culture fluid.
4. prepare according to claim 3 the method for live vaccines of hog cholera, it is characterized in that, in described step b, increase day by day air mass flow, every 24h increases 100ml/min～1000ml/min.
5. prepare according to claim 4 the method for live vaccines of hog cholera, it is characterized in that, in described steps d, connect after malicious 24h the beginning Continuous-flow and add and replenish doubly concentrated DMEM liquid of n, flow acceleration maintains between 1～2g/L with culture fluid residual sugar amount and is as the criterion; Described n equals 2～5.
6. prepare according to claim 5 the method for live vaccines of hog cholera, it is characterized in that, described n doubly concentrated DMEM liquid making method is: after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again (n-1) part without the Na-DMEM dehydrated medium, dissolving; Described n equals 2～5.
7. prepare according to claim 6 the method for live vaccines of hog cholera, it is characterized in that, in described step e, harvesting approach is multiple batches of results; Every 72h gathers in the crops once, sustained yield 10～15 times, and each harvest yield is 90%～100% of culture fluid cumulative volume.
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Cited By (3)
| 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 |
| CN105861452A (en) * | 2016-06-03 | 2016-08-17 | 广东海大畜牧兽医研究院有限公司 | Semi-pouring flow type culture method for duck Tembusu virus |
| CN106085965A (en) * | 2016-06-03 | 2016-11-09 | 广东海大畜牧兽医研究院有限公司 | A kind of duck tembusu virus, the vaccine prepared by this virus and preparation method thereof |
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| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN102038944A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine fever live vaccine by using bioreactor |
| CN102552896A (en) * | 2011-12-31 | 2012-07-11 | 瑞普（保定）生物药业有限公司 | Preparation method of porcine reproductive and respiratory syndrome vaccines by utilizing bioreactor |
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| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN102038944A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine fever live vaccine by using bioreactor |
| CN102552896A (en) * | 2011-12-31 | 2012-07-11 | 瑞普（保定）生物药业有限公司 | Preparation method of porcine reproductive and respiratory syndrome vaccines by utilizing bioreactor |
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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 |
| CN104940922B (en) * | 2015-07-02 | 2018-08-21 | 瑞普（保定）生物药业有限公司 | A kind of preparation method of live vaccines of hog cholera |
| CN105861452A (en) * | 2016-06-03 | 2016-08-17 | 广东海大畜牧兽医研究院有限公司 | Semi-pouring flow type culture method for duck Tembusu virus |
| CN106085965A (en) * | 2016-06-03 | 2016-11-09 | 广东海大畜牧兽医研究院有限公司 | A kind of duck tembusu virus, the vaccine prepared by this virus and preparation method thereof |
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