CN102657859A - Method for producing avian influenza vaccine by using cells cultured by spherical microcarrier bioreactor - Google Patents
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- 206010064097 avian influenza Diseases 0.000 title claims abstract description 102
- 208000002979 Influenza in Birds Diseases 0.000 title claims abstract description 40
- 229960003971 influenza vaccine Drugs 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 44
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 36
- 230000008569 process Effects 0.000 claims abstract description 21
- 229940030156 cell vaccine Drugs 0.000 claims abstract description 10
- 230000003053 immunization Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 95
- 241000700605 Viruses Species 0.000 claims description 61
- 239000001963 growth medium Substances 0.000 claims description 22
- 239000005723 virus inoculator Substances 0.000 claims description 21
- 238000011081 inoculation Methods 0.000 claims description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- 229910052760 oxygen Inorganic materials 0.000 claims description 16
- 239000001301 oxygen Substances 0.000 claims description 16
- 238000005096 rolling process Methods 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 14
- 239000002435 venom Substances 0.000 claims description 14
- 210000001048 venom Anatomy 0.000 claims description 14
- 231100000611 venom Toxicity 0.000 claims description 14
- 230000009849 deactivation Effects 0.000 claims description 13
- 238000009395 breeding Methods 0.000 claims description 12
- 230000001488 breeding effect Effects 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 241000287828 Gallus gallus Species 0.000 claims description 11
- 229960005486 vaccine Drugs 0.000 claims description 11
- 230000036039 immunity Effects 0.000 claims description 8
- 230000002779 inactivation Effects 0.000 claims description 8
- 210000000496 pancreas Anatomy 0.000 claims description 8
- 229920006395 saturated elastomer Polymers 0.000 claims description 8
- 238000009423 ventilation Methods 0.000 claims description 8
- 210000004748 cultured cell Anatomy 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 230000029087 digestion Effects 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 claims description 4
- 230000008021 deposition Effects 0.000 claims description 4
- 230000001804 emulsifying effect Effects 0.000 claims description 4
- 230000008676 import Effects 0.000 claims description 4
- 230000000644 propagated effect Effects 0.000 claims description 4
- 230000001902 propagating effect Effects 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 230000003612 virological effect Effects 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 2
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 7
- 241000712461 unidentified influenza virus Species 0.000 abstract description 6
- 239000002158 endotoxin Substances 0.000 abstract description 5
- 235000013601 eggs Nutrition 0.000 abstract description 4
- 238000003306 harvesting Methods 0.000 abstract description 3
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- 230000001580 bacterial effect Effects 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 230000000415 inactivating effect Effects 0.000 abstract 1
- 230000008901 benefit Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- 229940124873 Influenza virus vaccine Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
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Abstract
The invention provides a method for producing an avian influenza vaccine by using cells cultured by a spherical microcarrier bioreactor. The method comprises the following steps of: selecting seed cells; passaging cells reproduced in a spinner bottle; harvesting the cells in the spinner bottle; inoculating the cells; culturing the inoculated cells in the bioreactor; inoculating avian influenza virus; reproducing the avian influenza virus; collecting avian influenza virus liquid; inactivating the avian influenza virus liquid; preparing an avian influenza cell vaccine; and immunizing the avian influenza cell vaccine. By the method, the defects that the avian influenza vaccine produced by the traditional process is long in production period, large in inter-batch difference, high in endotoxin content and influenced by the supply of embryo eggs, and a tablet carrier is difficult to amplify are overcome; and the prepared avian influenza vaccine product is small in inter-batch difference, free from bacterial pollution, low in endotoxin content and formaldehyde content, stable in quality and short in the production period, and the tablet carrier is easy to amplify.
Description
Technical field
The present invention relates to the avian influenza vaccine technical field, particularly, relate to the method for utilizing spherical microcarrier bioreactor culture cells produce avian influenza vaccine.
Background technology
Past, China was always through Embryo Gallus domesticus cultivating and producing influenza vaccines.Need consume a large amount of Embryo Gallus domesticus because Embryo Gallus domesticus is produced influenza vaccines, the production cycle is long, is prone to pollute; Be not easy to control; And every fertilized eggs once can only inject a virus strains, and therefore this production method efficient is very low, is unfavorable for tackling large-scale flu outbreak.
Be that substrate prepares vaccine and has no exogenous factor and pollute, be easy to large-scale production, can keep advantages such as virus antigen is stable preferably with the mammalian cell.But, only rely on the amount of animal cell culture production virus limited, therefore the carrier culture technique is applied to the production of avian influenza vaccine, will not only keep the advantage of cell culture but also overcome the limited shortcoming of its production output undoubtedly.Spherical microcarrier is widely used in the bio-pharmaceuticals industry.Using spherical microcarrier cultured cell breeding bird flu virus to produce vaccine has the following advantages: 1) the product differences between batches are little; 2) no bacteria pollution, the product endotoxin content is low; 3) during the product deactivation, the formaldehyde use amount is low, so the product content of formaldehyde is also low; 4) constant product quality stress be little behind the chicken immune vaccine; 5) with short production cycle; 6) be easy to amplify; 7) influence that not supplied by the embryo egg.
Summary of the invention
For this reason, the invention provides a kind of method of utilizing spherical microcarrier bioreactor culture cells produce avian influenza vaccine.This method overcomes traditional handicraft and produces avian influenza vaccine, and the influence that the production cycle is long, differences between batches are big, endotoxin content is high, supplied by the embryo egg also overcomes the influence that chip carrier is difficult to amplify.The avian influenza virus vaccine product differences between batches of preparation are little, no bacteria pollution, and the product endotoxin content is low, and the product content of formaldehyde is also low, and steady quality is with short production cycle, is easy to amplify.
Technical scheme of the present invention is following: utilize the method for spherical microcarrier bioreactor culture cells produce avian influenza vaccine, comprise following concrete steps
1: select to import needed kind of cell MDCK of carrier or Vero;
2: the method that the rolling bottle propagated cell goes down to posterity: at first 1 selected kind of cell of recovery step about growth 48h, goes down to posterity according to the ratio of 1:4 in the cell bottle of T225; Passing to 1 15L rolling bottle to the cell in 4 T225 cell bottles that cover with cell then cultivates; Carrying out passage according to the ratio of 1:4 or 1:3 about 48 hours cultivates;
3: the cell in the results rolling bottle: step 2 gained cell with PBS washing 2-3 time, is added the digestion of pancreas enzyme-EDTA solution then; The 15L rolling bottle adds 100-150ml pancreas enzyme-EDTA solution, and the rolling bottle of other type adds the digestion of pancreas enzyme-EDTA solution, collecting cell then according to similar ratio;
4: behind the cell counting of results, add the ratio of 5-20 cell according to each microcarrier and carry out cell inoculation; Add 2-10 gram microcarrier in every liter of culture medium;
5: the bioreactor culture inoculating cell:
The culture medium of bioreactor culture cell is MEM (Hyclone); The serum working concentration is 7-10%; CO
2Ventilation is 5-10%; Dissolved oxygen amount is the saturated dissolved oxygen amount of the solution of 40-60%; Temperature is 37 ℃; Mixing speed is 30-55 rev/min, and pH is 6.8-7.4; The above microcarrier of 90-95% can reach the requirement of inoculation bird flu virus during 48-72h behind the cell inoculation;
6: after step 5 cultured cells reaches the requirement of inoculation bird flu virus, the inoculation bird flu virus, method is following:
6.1) clean microcarrier: deposition covers with the microcarrier of cell earlier, cleans microcarrier with PBS then;
6.2) virus inoculation: add bird flu virus-MEM solution then and carry out virus inoculation, the volume of culture medium is the volume 1/4-1 of bioreactor work in the virus inoculation process, and virus inoculation dosage is 0.00001-1.0MIO, increase serum not in the seeded process;
6.3) hatch bird flu virus and cell: after the bird flu virus inoculation; In bioreactor, supply the needed culture medium of bioreactor operate as normal; In the process that bird flu virus and cell are hatched; Stir microcarrier gently cell is fully contacted with bird flu virus, mixing speed is 30-45 rev/min, and bird flu virus and cell incubation time are 0.5-10 hour;
7: bird flu virus propagative viruses propagating culture medium is MEM (Hyclone); CO
2Ventilation is 5-10%; Dissolved oxygen amount is the saturated dissolved oxygen amount of the solution of 40-60%; Temperature is 37 ℃; Mixing speed is 30-45 rev/min, and pH is 6.5-7.2; The time of virus breeding is 24-72h;
8: centrifugal collection step 7 gained avian influenza venom, centrifugal speed is 3000-4500 rev/min, centrifugation time is 20-30 minute;
9: the bird flu virus in the inactivation step 8 gained avian influenza venom
Use the formalin-inactivated bird flu virus, the formaldehyde final concentration of adding is 0.01%-0.2%, and the time of deactivation is 24 hours, and the temperature of deactivation is 15-37 ℃; In the process of deactivation, stir and make formaldehyde and viral mixing;
10: prepare the bird flu cell vaccine according to conventional method then; Promptly according to oil phase: water is that the volume ratio of 2:1 prepares vaccine; Earlier carry out sterilization treatment to oil phase, slowly pour the avian influenza venom of step 9 gained complete inactivation in the stirring oil phase then, emulsifying makes vaccine;
11: the SPF chicken of bird flu cell vaccine immunity 21 ages in days, immunizing dose is 0.3ml, immunity was taken a blood sample respectively and is measured the titre (HI) of anti-avian influenza specific antibody in the serum in back 14 days, 21 days.
Preferably, in the step 6.3, bird flu virus and cell incubation time were not less than 1 hour;
Preferably, in the step 7, in the virus breeding process, when the virus breeding medium pH be lower than 6.5 or bird flu virus HA valency be higher than 9.0, with regard to removable parts solution.
Said microcarrier is Cytodex I or hyclone.
Beneficial effect of the present invention is: the method for the spherical microcarrier bioreactor culture of utilization according to the invention cells produce avian influenza vaccine; Bird flu virus titre (HA) is not less than 9.0; Than the method for conventional cell breeding bird flu virus, bird flu virus titre (HA) has improved and has been not less than 2 times; Produce avian influenza vaccine with traditional Embryo Gallus domesticus and compare, the production cycle shortens over half, is a method that is worthy of popularization.
The specific embodiment
Embodiment 1: microcarrier bioreactor suspension culture breeding H5 subtype avian influenza inactivated vaccine
1: select to import needed kind of cell MDCK of carrier;
2: the method that the rolling bottle propagated cell goes down to posterity: at first 1 selected kind of cell MDCK of recovery step about growth 48h, goes down to posterity according to the ratio of 1:4 in the cell bottle of T225; Passing to 1 15L rolling bottle to the cell in 4 T225 cell bottles that cover with cell then cultivates; Carrying out passage according to the ratio of 1:3 about 48 hours cultivates; The culture medium of using in this process is MEM, and serum is hyclone, and use amount is 7%;
3: the cell in the results rolling bottle: with PBS washing 2-3 time, adding concentration then is 0.25% pancreas enzyme-EDTA solution 125ml digestion, collecting cell with step 2 gained cell;
4: behind the cell counting of results, the microcarrier of selecting for use is Cytodex I, adds the ratio of 5-20 cell according to each microcarrier and carries out cell inoculation; Add 4 gram microcarriers in every liter of culture medium;
5: the condition of bioreactor culture cell is:
Select the NBS bioreactor of 7.5L for use, its working volume is 5L, and the culture medium of cultured cell is MEM (Hyclone); The serum working concentration is 10%; CO
2Ventilation is 10%; Dissolved oxygen amount is 40% the saturated dissolved oxygen amount of solution; Temperature is 37 ℃; Mixing speed is 40 rev/mins, and pH is 6.8; The cell culture time is 64h, and overgrowing with cell above the microcarrier more than 90% just can virus inoculation;
6: after step 5 cultured cells reaches the requirement of inoculation bird flu virus, the inoculation bird flu virus, method is following:
6.1) clean microcarrier: deposition covers with the microcarrier of cell earlier, cleans microcarrier with PBS then;
6.2) virus inoculation: add bird flu virus-MEM solution then and carry out virus inoculation, the volume of culture medium is the volume 1/4-1 of bioreactor work in the virus inoculation process, and virus inoculation dosage is 0.01MIO, increase serum not in the seeded process;
6.3) hatch bird flu virus and cell: after the bird flu virus inoculation; In bioreactor, supply the needed culture medium of bioreactor operate as normal; In the process that bird flu virus and cell are hatched; Stir microcarrier gently cell is fully contacted with bird flu virus, mixing speed is 40 rev/mins, and bird flu virus and cell incubation time are 2 hours;
7: bird flu virus propagative viruses propagating culture medium is MEM (Hyclone); CO
2Ventilation is 10%; Dissolved oxygen amount is 60% the saturated dissolved oxygen amount of solution; Temperature is 37 ℃; Mixing speed is 45 rev/mins, and pH is 7.2; The time of virus breeding is 56h;
8: centrifugal collection step 7 gained avian influenza venom, centrifugal speed is 3000 rev/mins, centrifugation time is 30 minutes; Its avian influenza venom harvest yield is 12L, and the HA valency is greater than 9.0;
9: the bird flu virus in the inactivation step 8 gained avian influenza venom
Use the formalin-inactivated bird flu virus, the formaldehyde final concentration of adding is 0.01%, and the time of deactivation is 24 hours, and the temperature of deactivation is 37 ℃; In the process of deactivation, stir and make formaldehyde and viral mixing;
10: prepare the bird flu cell vaccine according to conventional method then; Promptly according to oil phase: water is that the volume ratio of 2:1 prepares vaccine; Earlier carry out sterilization treatment to oil phase, slowly pour the avian influenza venom of step 9 gained complete inactivation in the stirring oil phase then, emulsifying makes vaccine;
11: the SPF chicken of bird flu cell vaccine immunity 21 ages in days, immunizing dose is 0.3ml, immunity was taken a blood sample respectively and is measured the titre (HI) of anti-avian influenza specific antibody in the serum in back 14 days, 21 days.Its result (like table 1) shows, 14 days average ACs greater than 5.0,21 days average antibody titer (HI) greater than 7.5,28 days average antibody titer (HI) greater than 8.0.
The antibody situation of change of table 1:SPF chicken inoculation H5 subtype avian influenza cell inactivated vaccine
Embodiment 2: microcarrier suspension culture breeding H9 subtype avian influenza virus
1: select to import needed kind of cell MDCK of carrier;
2: the method that the rolling bottle propagated cell goes down to posterity: at first 1 selected kind of cell MDCK of recovery step about growth 48h, goes down to posterity according to the ratio of 1:4 in the cell bottle of T225; Passing to 1 15L rolling bottle to the cell in 4 T225 cell bottles that cover with cell then cultivates; Carrying out passage according to the ratio of 1:4 about 48 hours cultivates; The culture medium of using in this process is MEM, and serum is hyclone, and use amount is 7-10%;
3: the cell in the results rolling bottle: with PBS washing 2-3 time, adding concentration then is 0.25% pancreas enzyme-EDTA solution 125ml digestion, collecting cell with step 2 gained cell;
4: behind the cell counting of results, the microcarrier of selecting for use is hyclone, adds the ratio of 5-20 cell according to each microcarrier and carries out cell inoculation; Add 4 gram microcarriers in every liter of culture medium;
5: the condition of bioreactor culture cell is:
Select the NBS bioreactor of 7.5L for use, its working volume is 5L, and the culture medium of cultured cell is MEM (Hyclone); The serum working concentration is 7%; CO
2Ventilation is 5%; Dissolved oxygen amount is 60% the saturated dissolved oxygen amount of solution; Temperature is 37 ℃; Mixing speed is 40 rev/mins, and pH is 7.4; Incubation time is 70h;
6: after step 5 cultured cells reaches the requirement of inoculation bird flu virus, the inoculation bird flu virus, method is following:
6.1) clean microcarrier: deposition covers with the microcarrier of cell earlier, cleans microcarrier with PBS then;
6.2) virus inoculation: add bird flu virus-MEM solution then and carry out virus inoculation, the volume of culture medium is the volume 1/4-1 of bioreactor work in the virus inoculation process, and virus inoculation dosage is 0.001MIO, increase serum not in the seeded process;
6.3) hatch bird flu virus and cell: after the bird flu virus inoculation; In bioreactor, supply the needed culture medium of bioreactor operate as normal; In the process that bird flu virus and cell are hatched; Stir microcarrier gently cell is fully contacted with bird flu virus, mixing speed is 40 rev/mins, and bird flu virus and cell incubation time are 2 hours;
7: bird flu virus propagative viruses propagating culture medium is MEM (Hyclone); CO
2Ventilation is 5%; Dissolved oxygen amount is 40% the saturated dissolved oxygen amount of solution; Temperature is 37 ℃; Mixing speed is 30 rev/mins, and pH is 6.5; The time of virus breeding is 56h;
8: centrifugal collection step 7 gained avian influenza venom, centrifugal speed is 3000 rev/mins, centrifugation time is 30 minutes; Its avian influenza venom harvest yield is 15L, and the HA valency is greater than 9.0;
9: the bird flu virus in the inactivation step 8 gained avian influenza venom
Use the formalin-inactivated bird flu virus, the formaldehyde final concentration of adding is 0.01%, and the time of deactivation is 24 hours, and the temperature of deactivation is 37 ℃; In the process of deactivation, stir and make formaldehyde and viral mixing;
10: prepare the bird flu cell vaccine according to conventional method then; Promptly according to oil phase: water is that the volume ratio of 2:1 prepares vaccine; Earlier carry out sterilization treatment to oil phase, slowly pour the avian influenza venom of step 9 gained complete inactivation in the stirring oil phase then, emulsifying makes vaccine;
11: the SPF chicken of bird flu cell vaccine immunity 21 ages in days, immunizing dose is 0.3ml, immunity back 14 days, 21 days, 28 days respectively blood sampling measure the titre (HI) of anti-avian influenza specific antibody in the serum.Its result (like table 2) shows, 14 days average ACs greater than 5.0,21 days average antibody titer (HI) greater than 7.3,28 days average antibody titer (HI) greater than 8.5
The antibody situation of change of table 2:SPF chicken inoculation H9 subtype avian influenza cell inactivated vaccine
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, its framework form can be flexible and changeable, can the subseries product.Just make some simple deduction or replace, all should be regarded as belonging to the scope of patent protection that the present invention is confirmed by claims of being submitted to.
Claims (4)
1. utilize the method for spherical microcarrier bioreactor culture cells produce avian influenza vaccine, it is characterized in that, comprise following concrete steps
1: select to import needed kind of cell MDCK of carrier or Vero;
2: the method that the rolling bottle propagated cell goes down to posterity: at first 1 selected kind of cell of recovery step about growth 48h, goes down to posterity according to the ratio of 1:4 in the cell bottle of T225; Passing to 1 15L rolling bottle to the cell in 4 T225 cell bottles that cover with cell then cultivates; Carrying out passage according to the ratio of 1:4 or 1:3 about 48 hours cultivates;
3: the cell in the results rolling bottle: step 2 gained cell with PBS washing 2-3 time, is added the digestion of pancreas enzyme-EDTA solution then; The 15L rolling bottle adds 100-150ml pancreas enzyme-EDTA solution, and the rolling bottle of other type adds the digestion of pancreas enzyme-EDTA solution, collecting cell then according to similar ratio;
4: behind the cell counting of results, add the ratio of 5-20 cell according to each microcarrier and carry out cell inoculation; Add 2-10 gram microcarrier in every liter of culture medium;
5: the bioreactor culture inoculating cell:
The culture medium of bioreactor culture cell is MEM (Hyclone); The serum working concentration is 7-10%; CO
2Ventilation is 5-10%; Dissolved oxygen amount is the saturated dissolved oxygen amount of the solution of 40-60%; Temperature is 37 ℃; Mixing speed is 30-55 rev/min, and pH is 6.8-7.4; The above microcarrier of 90-95% can reach the requirement of inoculation bird flu virus during 48-72h behind the cell inoculation;
6: after step 5 cultured cells reaches the requirement of inoculation bird flu virus, the inoculation bird flu virus, method is following:
6.1) clean microcarrier: deposition covers with the microcarrier of cell earlier, cleans microcarrier with PBS then;
6.2) virus inoculation: add bird flu virus-MEM solution then and carry out virus inoculation, the volume of culture medium is the volume 1/4-1 of bioreactor work in the virus inoculation process, and virus inoculation dosage is 0.00001-1.0MIO, increase serum not in the seeded process;
6.3) hatch bird flu virus and cell: after the bird flu virus inoculation; In bioreactor, supply the needed culture medium of bioreactor operate as normal; In the process that bird flu virus and cell are hatched; Stir microcarrier gently cell is fully contacted with bird flu virus, mixing speed is 30-45 rev/min, and bird flu virus and cell incubation time are 0.5-10 hour;
7: bird flu virus propagative viruses propagating culture medium is MEM (Hyclone); CO
2Ventilation is 5-10%; Dissolved oxygen amount is the saturated dissolved oxygen amount of the solution of 40-60%; Temperature is 37 ℃; Mixing speed is 30-45 rev/min, and pH is 6.5-7.2; The time of virus breeding is 24-72h;
8: centrifugal collection step 7 gained avian influenza venom, centrifugal speed is 3000-4500 rev/min, centrifugation time is 20-30 minute;
9: the bird flu virus in the inactivation step 8 gained avian influenza venom
Use the formalin-inactivated bird flu virus, the formaldehyde final concentration of adding is 0.01%-0.2%, and the time of deactivation is 24 hours, and the temperature of deactivation is 15-37 ℃; In the process of deactivation, stir and make formaldehyde and viral mixing;
10: prepare the bird flu cell vaccine according to conventional method then; Promptly according to oil phase: water is that the volume ratio of 2:1 prepares vaccine; Earlier carry out sterilization treatment to oil phase, slowly pour the avian influenza venom of step 9 gained complete inactivation in the stirring oil phase then, emulsifying makes vaccine;
11: the SPF chicken of bird flu cell vaccine immunity 21 ages in days, immunizing dose is 0.3ml, immunity was taken a blood sample respectively and is measured the titre (HI) of anti-avian influenza specific antibody in the serum in back 14 days, 21 days.
2. the method for the spherical microcarrier bioreactor culture of utilization as claimed in claim 1 cells produce avian influenza vaccine is characterized in that in the step 6.3, bird flu virus and cell incubation time were not less than 1 hour.
3. the method for the spherical microcarrier bioreactor culture of utilization as claimed in claim 1 cells produce avian influenza vaccine; It is characterized in that, in the step 7, in the virus breeding process; When the virus breeding medium pH be lower than 6.5 or bird flu virus HA valency be higher than 9.0, with regard to removable parts solution.
4. the method for the spherical microcarrier bioreactor culture of utilization as claimed in claim 1 cells produce avian influenza vaccine is characterized in that said microcarrier is Cytodex I or hyclone.
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| CN103394081A (en) * | 2013-08-12 | 2013-11-20 | 黑龙江省百洲生物工程有限公司 | Method for producing avian influenza vaccine by using WAVE bioreactor |
| CN103773741A (en) * | 2012-10-18 | 2014-05-07 | 辽宁成大生物股份有限公司 | Method for preparing influenza vaccine by using cage type ventilating and stirring bioreactor |
| CN104046588A (en) * | 2014-06-20 | 2014-09-17 | 马忠仁 | Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof |
| CN104073470A (en) * | 2014-06-30 | 2014-10-01 | 中国农业科学院上海兽医研究所 | Spinner-flask culture method for H9N2 subtype of avian influenza virus |
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| CN103773741B (en) * | 2012-10-18 | 2016-07-06 | 辽宁成大生物股份有限公司 | The method that cage air agitation bioreactor prepares influenza vaccines |
| CN103394081A (en) * | 2013-08-12 | 2013-11-20 | 黑龙江省百洲生物工程有限公司 | Method for producing avian influenza vaccine by using WAVE bioreactor |
| CN103394081B (en) * | 2013-08-12 | 2015-07-29 | 黑龙江省百洲生物工程有限公司 | WAVE wave bioreactor is utilized to produce the method for avian influenza vaccine |
| CN104046588A (en) * | 2014-06-20 | 2014-09-17 | 马忠仁 | Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof |
| CN104073470A (en) * | 2014-06-30 | 2014-10-01 | 中国农业科学院上海兽医研究所 | Spinner-flask culture method for H9N2 subtype of avian influenza virus |
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