CN104560888A - Large-scale culture method of H9N2 subtype avian influenza virus - Google Patents
Large-scale culture method of H9N2 subtype avian influenza virus Download PDFInfo
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Abstract
The invention discloses a large-scale culture method of an H9N2 subtype avian influenza virus. According to the large-scale culture method, the H9N2 virus is rapidly adapted to MDCK, the HA titer of the virus is 28, MDCK cells are cultured as seed cells by taking a 7.5L reactor as a seed cell tank and cytodex1 as a carrier, the seed cells are amplified to large-scale culture in a 40L fixed bed basket type stirring system reactor, and 80L of H9N2 viruses of which the hemagglutination titer (HA) is up to 210 can be obtained. The virus produced by using the method is high in titer and large in yield, the method is easy in standardization and is applicable to all H9 subtype influenza viruses, and the difficulty that the culture virus titer cannot meet vaccine production when the H9N2 avian influenza virus is cultured on the MDCK cells is solved.
Description
Technical field
The invention belongs to veterinary biological technical field, be specifically related to a kind of large-scale cultivation method of H9N2 subtype avian influenza virus.Method is simple, easy, and the quick adaptation of H9N2 virus on mdck cell, utilizes microcarrier to combine with chip carrier and carry out the large scale culturing of this influenza virus.
Background technology
H9N2 subtype avian influenza (Avian influenza, AI) is one of important bird transmissible disease continuing harm China and the development of world's aviculture.H9N2 subtype avian influenza is low pathogenicity bird flu.When typical case breaks out, sudden onset, infection rate are high, spread scope is large; When atypia is popular, there is no characteristic symptoms, mainly cause egg drop reduction, brood and the failure of Growing Chicken vaccine immunity.This sick infection rate is high, and spread scope wide l5 ~ 40 Japanese instar chickling is multiple; Propagate rapidly, many ages in days coexist, and large-scale chicken house, once contaminated, is difficult to be removed.H9N2 subtype avian influenza causes immunosuppression, egg drop reduction chicken group mainly to present symptom and pathologies such as breathing digestion, reproductive system and body tissue's organ hyporrhea after infecting H9N2 subtype avian influenza; Can immunity system be destroyed, cause serious immunosuppression, and the infection of easy secondary intestinal bacteria or other cause of diseases (IB, ND etc.), cause poultry M & M to rise; Be difficult to after causing laying hen egg drop reduction to recover recover previous level.H9N2 subtype avian influenza virus is extensively there is in aquatic bird, and the inapparent infection of healthy appearance.Aquatic bird is the maximum hidden danger of birds flu-preventing; H9N2, except infection fowl, has potential in threat that is reality to the lives and properties of people.Vaccine immunity is one of important measures of control H9N2 hypotype AI, and there is multiple different manufacturers research and production H9N2 hypotype AI inactivated vaccine in China, in China's widespread adoption.
The H9N2 avian influenza vaccine that China secures permission is all the inactivated vaccine in chicken embryo source.Chicken embryo is a kind of culture medium that avian influenza virus is the most conventional, commonly uses it and is separated avian influenza virus and prepares vaccine.But easily cause antigenic variation with chick embryo allantoic liquid separation or the avian influenza virus that goes down to posterity; The problem of chicken embryo quantity not sufficient and the pollution of potential exogenous virus is also there is during scale operation.Be that matrix composition vaccine has and pollutes without exogenous factor, is easy to large-scale production, can maintains the advantages such as virus antigen is stable preferably with mammalian cell; cell at present for proliferation of influenza virus mainly contains MDCK and Vero cell; wherein adapt to fast with virus on mdck cell; viral titer is high, is widely used in the research and development of avian influenza virus vaccine.
Since 1996, mammalian cell has developed into can free enlarged culturing and for suitability for industrialized production, and avian influenza vaccine also can be prepared by zooblast large scale culturing.Bio-reactor can increase the density of unit volume cell cultures effectively, thus establishes solid basis for viral vaccine scale operation.A lot of difficulty is there is: 1, virus needs adaptive process from chicken embryo to the culture medium conversion that two kinds, cell is different with H9N2 bird flu cell cultures; 2, because of the quantitative limitation of spinner culture cell, avian influenza virus HA tires can not higher than 2
6, requirement prepared by vaccine can not be met; 3, the serum free medium adding TPCK-pancreatin must be changed in the adsorption process of virus, its complex process.Therefore, research and develop new production technique, H9N2 avian influenza virus is adapted to fast on mdck cell, and the seeding tank bio-reactor being carrier from square vase, rolling bottle, cytodex1 is very important to being amplified to fixed bed simple to operate basket stirring system bio-reactor industrialization reactor scale operation restructuring H5 subtype avian influenza virus fast.
At present be mainly with cytodex1 for the Large Scale Biology reactor of production of vaccine the fixed-bed type bio-reactor be carrier stirring type bioreactor and polyester chips being carrier.Wherein cytodex1 is that carrier stirring type bioreactor is widely used, and still its technique is loaded down with trivial details can to provide amplification technique, is unfavorable for the simple operations of batch production, especially docks the influenza virus of malicious complex process.Polyester chips is the fixed-bed type bio-reactor perfusion of carrier and connects malicious technique simply, but not easily amplifies.The patent of invention specification sheets of China Patent No. 02112013.7 discloses a kind of Fibra-CeTMDisks that utilizes for the method for carrier large-scale continuous production of virus vaccine, although this technique is suitable for rabies vaccine, hemorrhagic fever, encephalitis, children's's poliovirus, be but not suitable for the influenza virus needing adaptation of virus.The patent of invention specification sheets of China Patent No. 201010596058.9 discloses and utilizes Cytodex1 for carrier is at bioreactor culture mdck cell propagation H9N2 type avian influenza virus.The patent of invention specification sheets of China Patent No. 201010159741.6 discloses the full suspension culture technique of bioreactor culture mdck cell, and propagation H9N2 virus prepares the technique of vaccine.The patent of invention specification sheets of China Patent No. 201010282155.0 discloses and utilizes Cytodex1 for carrier is in bioreactor culture MDCK or cell proliferation suitability for industrialized production animal influenza virus.
Above documents and materials have the producing and manufacturing technique relating to avian influenza virus in various degree.But the quick adaptation of H9N2 virus from chick embryo culture to mdck cell, the production amplification technique combined of cytodex1 to be carrier stirring type bioreactor and polyester chips the be basket stirring system reactor of industrialization fixed bed of carrier does not relate to.
Summary of the invention
The object of the invention is to there are provided a kind of H9N2 type avian influenza virus large-scale cultivation method, in present method, H9N2 virus adapts to fast at MDCK, and after adapting to, viral HA tires is 2
8; With 7.5L reactor for kind of a cell tank, cytodex1 is carrier cultivation mdck cell is seed cell, and be amplified to the large scale culturing of the basket stirring system reactor of 40L fixed bed, results hemagglutinative titer (HA) can reach 2
10h9N2 virus 80L.Solve current H9N2 avian influenza virus on mdck cell, cultivate the difficult problem that virus titer can not meet production of vaccine.
In order to realize above-mentioned object, the present invention by the following technical solutions:
A large-scale cultivation method for H9N2 subtype avian influenza virus, the steps include:
Avian influenza virus H9N2 adapts to mdck cell fast:
1. the quick adaptation of H9N2 on mdck cell: F60-F105 covers with individual layer in mdck cell 6 orifice plate, 36.5-37.5 DEG C, virus absorption 1-2 hour, remove virus liquid, with the DMEM in high glucose containing TPCK-pancreatin 1.0-1.5ug/ml for after viral dilution liquid cleaning mdck cell 2-4 time, change containing TPCK-pancreatin 0.3-1.8ug/ml DMEM in high glucose cell maintenance medium, get hemagglutinative titer >2
4and the highest virus liquid of viral dilution multiple or cause the most highly diluted multiple virus that cell produces pathology the latest and continue to adapt to as seed virus; Virus in 6 generations with endoadaptation mdck cell, and viral hemoagglutination to tire be 2
8.
2. viral for 40L reactor seed virus with F6-F11 generation; Its hemagglutinative titer>=2
8; EID50>=10
7.5
1.7.5L stirring reactor cultivates seed cell:
Take Cytodex1 as carrier, 2-10g/L carrier injected volume, with the PBS aquation of pH7.2 and after BALANCE CARRIERS, with tank body deactivation, changes containing 8-10%(volume ratio) new-born calf serum DMEM perfect medium.Cell inoculum size is 0.35-1.4*10
8individual cell/gram carrier, cell attachment stage mixing speed 40r/min, DO are 60%, and temperature is 36-37 DEG C, pH is 7.0-7.3.The cell cultures stage, mixing speed control 40-80r/min, according to sugared concentration 1.8-2.2g/L in reactor, cultivating adjustment perfusion rate was 5-40ml/min according to cell oxygen consumption.Cell cultures 50-70 hour cell grows to individual layer, and cell density is approximately 1.6-8.3*10
6/ ml.
2.7.5L the basket fixed-bed reactor of stirring reactor → 40L are cultivated:
1) 40L fixes the preparation of basket reactor: 40L fixes basket reactor and prepares: get FibraCel Discs carrier 1440g according to 40g/L, be placed in reactor together after sterilizing, 32%(volume ratio is added in reactor) the DMEM substratum of new-born calf serum is to 16L, make temperature-stable to 37 DEG C, pH is 7.2.
2) digest 7.5L stirring reactor seed cell and be inoculated into the basket fixed bed reaction of 40L
In 7.5L reactor, cell density reaches 5.3*10
6/ ml, stop stirring, substratum is removed after carrier precipitation, clean carrier 4 times with 37 DEG C of pre-warm PBS, after the Digestive system cleaning once of mass concentration 0.02%EDTA, 0.25% pancreatin (37 DEG C pre-warm), add the Digestive system (37 DEG C pre-warm) of 1L mass concentration 0.25% pancreatin, after 25rpm stirs 20 minutes simultaneously, add 4L DMEM fast, after mixing speed adjustment 25rpm, extracted fast in cell and DMEM mixed solution 3L to ready 40L reactor in advance by flexible pipe.Again extracting 3L DMEM adds in 7.5L reactor, after regulating mixing speed to stir 5 minutes to 40rmp, is down to 25 turns, extracts cell and DMEM mixed solution 3L fast in reactor, repeatable operation like this 5 times by flexible pipe.Regulate 40L reactor rotating speed to 75rpm, temperature to 35-37 DEG C, dissolved oxygen is 7.2 to 30-90%, pH.Adherent in cell 1 hour.
3) mdck cell is cultivated in 40L reactor
40L reactor rotating speed is regulated to be 7.2 to 75-120rpm, temperature to 37 DEG C, dissolved oxygen to 60%, pH after cell attachment.Within every 6 hours, survey sugared concentration, within about 24 hours, reactor sugar concentration reaches the ultimate value (1.5g/L) required for Growth of Cells, regulates perfusion rate at any time, makes the sugared concentration of bioreactor culture base maintain 1.8-2.8g/L.Cell reaches plateau at 72 hours, within 70 hours, is in high vigor state with inner cell, is virus inoculation Best Times.
4) virus inoculation H9N2 influenza virus in 40L reactor: before 40L bioreactor culture mdck cell virus inoculation, use PBS successively, viral dilution liquid (DMEM containing 1.2-1.5ug/ml TPCK-pancreatin) cleans cell, virus inoculation MOI is 0.01-0.00001; TPCK-pancreas enzyme concentration 1.2-1.5ug/ml, temperature 36.5-37.5 DEG C in viruses adsorption process, mixing speed 40-60r/min, DO are 60%, pH is 7.0-7.6, viruses adsorption 2 hours; Virus culture phase cell maintains and virus culture temperature is 33-35 DEG C, changes cell maintenance medium after mixing speed 60-120r/min, 4-6 hour.
5) virus multiplication and hemagglutinative titer monitoring in 40L reactor: virus culture phase cell maintains and virus culture temperature is 33-35 DEG C, cell maintenance medium (DMEM containing 0.2-0.8ug/ml TPCK-pancreatin) is changed after mixing speed 60-120r/min, 4-6 hour; It is 50-400ml/min that perfusion cultivates speed, and sugared concentration keeps 2.2-3.0g/L.Virus culture starts to monitor viral HA for 30 hours and tires, and viral hemoagglutination is tired and reached 2
2-4time, reactor enters and produces the poison stage, and adjustment temperature is 35-37 DEG C, mixing speed 60-90r/min, and sugared concentration maintenance 1.0-2.8g/L, HA tire and be up to 2
11, can 2 be gathered in the crops
10virus liquid 80L, in addition 3-4 bioreactor culture volume virus liquid hemagglutinative titer 2
7-8.
3. virus immunity effect evaluation:
1) cytotoxic deactivation and emulsification: reactor produces virus, and (it is 2 that HA tires
10) through the deactivation of 0.05-0.5% formaldehyde solution, the virus of deactivation and oil phase (Exxon Mobil Corporation) are carried out emulsification according to the volume ratio of 2:1, is prepared into oil adjuvant killed vaccine.
2) immunization experiment: the SPF chicken of getting 10 3-4 all ages, every chicken is through intramuscular injection 0.3ml vaccine immunity, and taking periodic blood surveys antibody titer at certain intervals, detects 3 months altogether.
3) antibody dynamic regularity: monitoring Serum Antibody level finds, 2 weeks blood clottings suppress (HI) to tire can 2
7.0-7.5, within 4 weeks, reach 2
8-9.3, within 11 weeks, maintain 2
7above.
Compared with prior art, the present invention has the following advantages:
The invention provides a kind of bio-reactor large-scale cultivation method of H9N2 avian influenza virus, the method has the following advantages and effect to reactor scale evaluation from H9N2 virus cell adapted:
1. with traditional influenza virus compared with the mdck cell enrichment procedure, the invention provides with six orifice plate gradient preference methods simple to operate, workload is little; Virus can adapt to mdck cell and stable propagation within 6 generations, and its HA tires and is greater than 2
8; The method is easy to stdn, is applicable to all H9 subtype influenza virus.
2. a kind of new cell cultures amplification method provided by the invention, seeding tank is marine oar stirring reactor, take cytodex1 as carrier, for pilot scale and scale operation provide MDCK seed cell, with 40L fixed-bed bioreactor for producing tank, polyester chips is carrier, large scale culturing mdck cell, propagation H9N2 influenza virus, only needs every gram of carrier to throw in 0.5-4.5 × 10
7individual cell, cell proliferation propagation fast, connects malicious technique simple, is easy to industrialization large-scale operation, solves the shortcoming that the basket fixed-bed reactor of NBS can not be sent out large.
3. method virus titer provided by the invention is high, output large, and it is 2 that a 40L reactor can provide HA to tire
10virus 80L; This viral immunogenic is good, and antibody horizontal is held time length, and after this virus liquid prepares vaccine immunity SPF chicken, tire can 2 for 14 days HI
7.0-7.5, within 4 weeks, reach 2
8-9.3, within 11 weeks, maintain 2
7above.
Accompanying drawing illustrates:
Fig. 1 is a kind of large-scale cultivation method schematic flow sheet of H9N2 avian influenza virus.
Fig. 2 is that a kind of 40L reactor is produced H9N2 influenza virus liquid HA and to be tired change curve schematic diagram.
Fig. 3 is a kind of H9N2 cell vaccine HI Fluctuation schematic diagram in SPF chicken.
Embodiment
For making the present invention easier to understand, illustrate the present invention further below in conjunction with specific embodiment.These embodiments are only not used in for the present invention and limit the scope of the invention, and NM specific experiment method in the following example, conveniently experimental technique carries out.
Embodiment 1: avian influenza virus A/Duck/Hubei/W1/2004(H9N2) strain adapts to mdck cell fast:
1) get the mdck cell of fine and close individual layer of growing up, on average spread into 6 porocyte culture plates, treat that cell covers with individual layer after cell dissociation, by chick embryo allantoic liquid protovirus venom, (its HA tires >2
5) with DMEM substratum 10 times of doubling dilutions, get 10
-3, 10
-4, 10
-5, 10
-6each extent of dilution venom 100ul, and be supplemented to 3ml with the DMEM of the 1.2ug/ml containing TPCK-pancreatin, and set up the cell hole not adding virus to be negative control.
2) under 37 DEG C of conditions, viruses adsorption removed virus liquid after 1.5 hours, after DMEM cleaning mdck cell 3 times, every hole adds the DMEM3ml of the 1.2ug/ml containing TPCK-pancreatin, every 6 hours observation cytopathies after 24 hours, survey HA to tire, after 48 hours, every 3 hours observation cytopathies, survey HA and tire, and after connecing malicious 84 hours, 6 orifice plates in-80 DEG C of freeze thawing once, all do not detect that HA tires, but 10
-3, 10
-4occur that typical influenza virus causes coming off the cytopathy of type at 70 hours respectively.
3) with 10 of 65 hours results
-4extent of dilution virus is cultivated for F1 generation continues adaptation on mdck cell.
4) get F1 generation virus with DMEM substratum 10 times of doubling dilutions, get 10
-1, 10
-2, 10
-3, 10
-4inoculate 6 orifice plate mdck cells, according to step 2) operation and observation of cell pathology find 60 hours 10
-1, 10
-2there is pathology, at 72 hours 10 in inoculation hole mdck cell
-3there is obvious pathology in extent of dilution inoculation hole, survey HA tires and is 0, with 10 of 72 hours results
-3virus is F2 generation virus, continues to adapt to cultivate on mdck cell.
5) get F2 for viral repeating step 1) method virus dilution after, get 10
-2, 10
-3, 10
-4, according to step 2) operation and observation of cell pathology find at 48 hours 10
-2, 60 hours 10
-3, 10
-4there is pathology in mdck cell, its HA tires and is respectively 2
3, 2
3, 2
2.With 10 of 72 hours results
-4virus is F3 generation virus, continues to adapt to cultivate on mdck cell.
6) F6 generation is reached continuously according to the method described above, inoculation 10
-3, 10
-4there is typical cytopathic at 55-70 hour in the mdck cell of extent of dilution virus, HA tires 2
8.
Table 1: avian influenza virus A/Duck/Hubei/W1/2004(H9N2) strain adapts to mdck cell fast
Embodiment 2:
The basket fixed-bed reactor of 7.5L stirring reactor → 40L are cultivated:
Bio-reactor: U.S. NBS company 7.5L reactor, the basket fixed-bed reactor of 40L
Carrier: cytodex-1(GE company), NBS company of the FibraCel Discs(U.S.)
Cell growth medium: volume ratio is the DMEM (GIBICO) of 8% serum
1) 7.5L stirring reactor cultivates seed cell: get cytodex144g according to the concentration of 8g/L, under room temperature, carry out aquation 24 hours with 1000mlPBS and balance 3 times making its pH7.2-7.6, to add in reactor after sterilizing together with PBS, precipitation cytodex1, extract PBS out and be replaced by volume ratio 10% new-born calf serum (folium ilicis chinensis) DMEM, regulate reactor parameter, temperature is made to be 35.5-37 DEG C, pH7.2, stirs 30rpm, DO(dissolved oxygen) be 30-80, mdck cell is inoculated, cell total amount 1.2*10 after question response device parameters is stable
9.After inoculation, culture parameters is adjusted to 37 DEG C, pH7.2, DO is 60%, stir 30rpm-80rpm, every day timing sampling, observation cell growth status, carry out cell counting and detect glucose content in substratum, when glucose concn lower than 1.8 time start perfusion, make sugared concentration in reactor maintain 1.8-2.8g/L, cultivate 90 hours, cell reaches 5.3*10
6/ ml.
2) 40L fixes basket reactor and prepares: get FibraCel Discs carrier 1440g according to 40g/L, be placed in reactor together after sterilizing, add 32%(volume ratio in reactor) the DMEM substratum of new-born calf serum to 16L, make temperature-stable to 37 DEG C.
3) 40L fixes amplification culture mdck cell in basket reactor: treat that in 7.5L reactor, cell density reaches 5.3 × 10
6/ ml, with intermediate processing, after removing substratum, clean carrier 4 times with 37 DEG C of pre-warm PBS, after the Digestive system cleaning once of mass concentration 0.02%EDTA, 0.25%EDTA pancreatin (37 DEG C pre-warm), add the Digestive system (37 DEG C pre-warm) of 500ml mass concentration 0.25% pancreatin, 25rpm stirs 20 minutes simultaneously, adding 4L DMEM fast, mixing speed adjustment 25rpm, extracting cell 3L fast to shifting to an earlier date in ready 40L reactor by flexible pipe.Again extracting 3LDMEM adds in 7.5L reactor, regulates mixing speed to stir 5 minutes to 40rmp, is down to 25 turns, extracts cell 3L fast in reactor by flexible pipe, repeatable operation like this 5 times.40L reactor rotating speed is regulated to be 7.2 to 75rpm, temperature to 37 DEG C, dissolved oxygen to 30-90%, pH.Adherent in cell 1 hour.Within every 6 hours, survey sugar consumption, within about 24 hours, reactor sugar concentration reaches the ultimate value required for Growth of Cells, regulates perfusion rate at any time, makes the sugared concentration of bioreactor culture base maintain 1.8-2.8g/L.Cell reaches plateau at 72 hours, within 70 hours, is in high vigor state with inner cell, is virus inoculation Best Times.
4)) virus inoculation 40L reactor: clean cell 2 times with PBS respectively, to clean 1 time containing TPCK-pancreas enzyme concentration for 1.2ug/mlDMEM, the DMEM cell maintenance medium 4.4L containing 1.2ug/mlTPCK-pancreatin is added in reactor, with 1L (DMEM of 1.2ug/mlTPCK-pancreatin) containing MOI0.01 virus access mdck cell, pH in reactor is now regulated to be 7.6, temperature 36.5-37 DEG C, DO are 60%.Virus absorption onto cell 2 hours.
5) in 40L reactor, virus multiplication and viral HA tire monitoring:, viruses adsorption is after 2 hours, regulate culture temperature to 33-35 DEG C, within 6 hours, change maintenance medium (TPCK-pancreas enzyme concentration 0.8ug/ml) later, within every 6 hours, survey sugared content, perfusion rate in adjustment culturing process, guarantees sugared concentration >2.8g/L in maintenance medium.Surveyed HA every 3 hours after 24 hours to tire, result as shown in Figure 2: it is 2 that about 48 hours HA tire
8, 50 hours HA tire arrival 2
9, 60 hours HA tire arrival 2
11, within 60-72 hour, HA tires and maintains 2
10, after 80 hours, HA tires and is just down to 2
8.Gathering in the crops in whole process that 80LHA tires is 2
10virus liquid.
Embodiment 3:
Virus immunity effect evaluation:
1) cytotoxic deactivation and emulsification
Reactor produces virus, and (it is 2 that HA tires
10) through formaldehyde solution deactivation, the virus of deactivation and oil phase (Exxon Mobil Corporation) are carried out emulsification according to the volume ratio of 2:1, is prepared into oil adjuvant killed vaccine.
2) immunization experiment
Get the SPF chicken in 10 3-4 age in week, every chicken is through intramuscular injection 0.3ml vaccine immunity, and the SPF chicken of simultaneously getting 10 3-4 ages in week sets up commercial seedling immunized controls.Taking periodic blood surveys antibody titer at certain intervals, detects 3 months altogether.
3) antibody dynamic regularity
See Fig. 3. after immunity shown in antibody variation curve: after this virus liquid prepares cell vaccine immunity SPF chicken, 14 days HI tire and can reach 2
7.5, within 28 days, can 2 be reached
9.2.When the 11st week, antibody is still 2
7above, commercial seedling control group 14 days HI tire and can reach 2
6.2, within 28 days, can 2 be reached
8.7.When the 11st week, antibody is still 2
6.5, show by H9N2 cell inactivation seedling significantly better than the commercial seedling that market is bought.
Claims (1)
1. a large-scale cultivation method for H9N2 subtype avian influenza virus, the steps include:
Avian influenza virus H9N2 adapts to mdck cell fast
:
the quick adaptation of H9N2 on mdck cell: F60-F105 covers with individual layer in mdck cell 6 orifice plate, 36.5-37.5 DEG C, virus absorption 1-2 hour, remove virus liquid, with the DMEM in high glucose containing TPCK-pancreatin 1.0-1.5ug/ml for after viral dilution liquid cleaning mdck cell 2-4 time, change containing TPCK-pancreatin 0.3-1.8ug/ml DMEM in high glucose cell maintenance medium, get hemagglutinative titer >2
4and the highest virus liquid of viral dilution multiple or cause the most highly diluted multiple virus that cell produces pathology the latest and continue to adapt to as seed virus; Virus in 6 generations with endoadaptation mdck cell, and viral hemoagglutination to tire be 2
8;
with F6-F11 generation virus for 40L reactor seed virus; Its hemagglutinative titer>=2
8; EID50>=10
7.5;
1).7.5L stirring reactor cultivates seed cell:
Take Cytodex1 as carrier, 2-10g/L carrier injected volume, with the PBS aquation of pH7.2 and after BALANCE CARRIERS, with tank body deactivation, changing containing volume ratio is the new-born calf serum DMEM perfect medium of 8-10%; Cell inoculum size is 0.35-1.4*10
8individual cell/gram carrier, cell attachment stage mixing speed 40r/min, DO are 60%, and temperature is 36-37 DEG C, pH is 7.0-7.3; Cell cultures stage foundation cell oxygen consumption, mixing speed control 40-80r/min, according to sugared concentration 1.8-2.2g/L in reactor, cultivating adjustment perfusion rate is 5-40ml/min; Cell cultures 50-70 hour cell grows to individual layer, and cell density is 1.6-8.3*10
6/ ml;
2)the basket fixed-bed reactor of 7.5L stirring reactor → 40L are cultivated
:
(1) 40L fixes the preparation of basket reactor: 40L fixes basket reactor and prepares: get FibraCel Discs carrier 1440g according to 40g/L, be placed in reactor together after sterilizing, the DMEM substratum of 32% new-born calf serum is added to 16L in reactor, make temperature-stable to 37 DEG C, pH is 7.2;
(2) digest 7.5L stirring reactor seed cell and be inoculated into the basket fixed bed reaction of 40L
In 7.5L reactor, cell density reaches 5.3*10
6/ ml, stop stirring, substratum is removed after carrier precipitation, clean carrier 4 times with 37 DEG C of pre-warm PBS, after the Digestive system cleaning once of mass concentration 0.02%EDTA, 0.25% pancreatin, add the Digestive system of 1L mass concentration 0.25% pancreatin, after 25rpm stirs 20 minutes simultaneously, add 4L DMEM fast, after mixing speed adjustment 25rpm, extract in cell and DMEM mixed solution 3L to ready 40L reactor in advance fast by flexible pipe; Again extracting 3L DMEM adds in 7.5L reactor, after regulating mixing speed to stir 5 minutes to 40rmp, is down to 25 turns, extracts cell and DMEM mixed solution 3L fast in reactor, repeatable operation like this 5 times by flexible pipe; Regulate 40L reactor rotating speed to 75rpm, temperature to 35-37 DEG C, dissolved oxygen is 7.2 to 30-90%, pH; Adherent in cell 1 hour;
(3) mdck cell is cultivated in 40L reactor
40L reactor rotating speed is regulated to be 7.2 to 75-120rpm, temperature to 37 DEG C, dissolved oxygen to 60%, pH after cell attachment; Within every 6 hours, survey sugared concentration, within 24 hours, reactor sugar concentration reaches the ultimate value 1.5g/L required for Growth of Cells, regulates perfusion rate at any time, makes the sugared concentration of bioreactor culture base maintain 1.8-2.8g/L; Cell reached plateau at 72 hours, within 70 hours, was in high vigor state with inner cell, was virus inoculation Best Times;
(4) virus inoculation H9N2 influenza virus in 40L reactor: before 40L bioreactor culture mdck cell virus inoculation, use PBS successively, clean cell containing the DMEM of 1.2-1.5ug/ml TPCK-pancreatin, virus inoculation MOI is 0.01-0.00001; TPCK-pancreas enzyme concentration 1.2-1.5ug/ml, temperature 36.5-37.5 DEG C in viruses adsorption process, mixing speed 40-60r/min, DO are 60%, pH is 7.0-7.6, viruses adsorption 2 hours; Virus culture phase cell maintains and virus culture temperature is 33-35 DEG C, changes cell maintenance medium after mixing speed 60-120r/min, 4-6 hour;
(5) virus multiplication and hemagglutinative titer monitoring in 40L reactor: virus culture phase cell maintains and virus culture temperature is 33-35 DEG C, changes the DMEM containing 0.2-0.8ug/ml TPCK-pancreatin after mixing speed 60-120r/min, 4-6 hour; It is 50-400ml/min that perfusion cultivates speed, and sugared concentration keeps 2.2-3.0g/L; Virus culture starts to monitor viral HA for 30 hours and tires, and viral hemoagglutination is tired and reached 2
2-4time, reactor enters and produces the poison stage, and adjustment temperature is 35-37 DEG C, mixing speed 60-90r/min, and sugared concentration maintenance 1.0-2.8g/L, HA tire and be up to 2
11, results 2
10virus liquid 80L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671002A (en) * | 2015-10-09 | 2016-06-15 | 华中农业大学 | Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain |
| CN113355297A (en) * | 2021-06-23 | 2021-09-07 | 吉林冠界生物技术有限公司 | Method for producing recombinant avian influenza virus by perfusion culture of full-suspension MDCK cells |
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| CN105671002B (en) * | 2015-10-09 | 2019-08-30 | 华中农业大学 | The building of H9N2 subtype avian influenza virus cell high yield vaccine strain and application |
| CN113355297A (en) * | 2021-06-23 | 2021-09-07 | 吉林冠界生物技术有限公司 | Method for producing recombinant avian influenza virus by perfusion culture of full-suspension MDCK cells |
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