CN106085947A - MDCK clonal cell line and application thereof - Google Patents
MDCK clonal cell line and application thereof Download PDFInfo
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Abstract
The invention discloses strain MDCK clonal cell line and an application thereof, belong to clone and the application of mdck cell.First the present invention discloses a strain MDCK clonal cell line MDCK 3D6, and its microbial preservation is numbered: CGMCC No.12040.Clonal cell line MDCK 3D6 of the present invention adapts to than parental cells fast growth and to bird flu virus height.The invention also discloses the production method of a kind of inactivated avian influenza vaccine, including: clonal cell line MDCK 3D6 is inoculated on the microcarrier of bioreactor, cultivates cell；Inoculate and breed bird flu virus, gather in the crops virus liquid, inactivation, add adjuvant mixing, emulsifying, to obtain final product.Inactivated vaccine prepared by the present invention is to chicken safety, and immunity chicken HI antibody titer is high, and the attack to bird flu virus realizes 100% immunoprotection.The present invention is for for the extensive virus of proliferation of substrate with mammalian cell and preparing avian influenza vaccine and provide foundation.
Description
Technical field
The present invention relates to MDCK clonal cell line, further relate to the application of described cell strain, belong to mdck cell clone and
Application.
Background technology
Bird flu is breaking out of the important diseases of the world of harm at present aviculture development, particularly highly pathogenicity avian influenza,
Provisions fowl industry brings destructive disaster.Vaccine immunity is the active measures and key link preventing bird flu from breaking out.
Embryo Gallus domesticus is a kind of culture matrix that bird flu virus is the most commonly used, commonly uses it and separates bird flu virus and preparation epidemic disease
Seedling, but it is long to there is the production cycle, easily pollutes, and is not easy to control, and efficiency is low, is unfavorable for that tackling large-scale flu outbreak etc. lacks
Fall into.To this end, World Health Organization (WHO) encourages developing cells culture technique to substitute current chick embryo technique runoff yield in next life influenza vaccine.
The mdck cell system in Testis et Pentis Canis source is the cell that bird flu virus is the most susceptible, has easily expansion, easily collects thin
The advantages such as born of the same parents.But, the amount relying solely on animal cell culture production virus is limited, is therefore applied to by Microcarrier Culture Techniques
The production of avian influenza vaccine, undoubtedly by the advantage not only keeping cell to cultivate but also overcome the shortcoming that its production yields is limited.
Summary of the invention
The technical problem to be solved is to provide a strain MDCK clonal cell line, its speed of growth relatively parental cells
Hurry up, and high to the adaptability of avian influenza vaccine strain；
Invention further provides one and utilize MDCK clonal cell line described in microcarrier large-scale culture, propagation fowl stream
Influenza Virus prepares the method for inactivated vaccine, for for the extensive virus of proliferation of substrate and preparing avian influenza vaccine with mammalian cell
Foundation is provided.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
First mdck cell is used limiting dilution assay to carry out single cell clone by the present invention, and it is further to choose single clone
Sub-clone, through 3 sub-clones, final acquisition 6 strain monoclonal cells, it is respectively designated as MDCK-3D2, MDCK-3D3, MDCK-
3D5, MDCK-3D6, MDCK-3D7 and MDCK-3D9.
Growth curve measurement result shows, clonal cell line MDCK-3D6 compared with parental cells, fast growth.Pass through
The Adaptability Analysis of bird flu virus is shown by clonal cell line, the clonal cell line MDCK-3D6 adaptability to bird flu virus
Height, is passaged to 11-20 generation, and virus HA reaches 256；Being passaged to 21-25 generation, virus HA reaches 512；It is significantly higher than clonal cell line
MDCK-3D2, MDCK-3D3, MDCK-3D5, MDCK-3D7 and MDCK-3D9 and parental cells.Therefore, the present invention selects fowl to flow
Influenza Virus high sensitivity adapted strain MDCK-3D6.
The MDCK clonal cell line MDCK-3D6 of acquisition is submitted to the mechanism of patent accreditation to carry out preservation by the present invention, its micro-life
Thing deposit number is: CGMCC No.12040；Classification And Nomenclature is: Madin-Darby canine kidney(cell line)；The preservation time is: on 02 02nd, 2016；Protect
Tibetan unit is: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation address is: north, Chaoyang District, Beijing City
Occasion West Road 1 institute 3, Institute of Microorganism, Academia Sinica.
MDCK clonal cell line MDCK-3D6 of the present invention can be applied to cultivate virus.Wherein, described virus includes:
Influenza virus (Influenza virus), rabies virus (Rabies virus) or parainfluenza virus (Parainfluenza
Viruses).Described influenza virus includes: bird flu virus (Avain Influenza Virus), human influenza virus (Human
Influenza Viruses) or equine influenza virus (Equine Influenza virus)；It is preferably bird flu virus.Described
Parainfluenza virus includes human parainfluenza virus (Human Parainfluenza Viruses).
The present invention further discloses the production method of a kind of inactivated avian influenza vaccine, comprise the following steps: (1) is by described
MDCK clonal cell line MDCK-3D6 is inoculated on the microcarrier in bioreactor, cultivates cell；(2) inoculation avian influenza
Poison, breeds described virus, gathers in the crops virus liquid；(3) virus liquid is inactivated, add adjuvant mixing, emulsifying, to obtain final product.
Wherein, step (1) described MDCK clonal cell line MDCK-3D6 is according to 1.5 × 105-4.5×105Individual cell/ml,
It is preferably 3.0 × 105The initial density of individual cell/ml is inoculated on microcarrier；The concentration of described microcarrier is 1-5g/L, preferably
For 3-5g/L, most preferably 3g/L；It is furthermore preferred that wherein said microcarrier is Cytodex-1.Step (1) described cultivation cell
Mixing speed be 50-120r/min, preferably 100r/min；The time of described cultivation cell is 72h；It is furthermore preferred that it is described
Cultivate cell also to include: during cell amplification culture, culture medium groundwater increment is 3-4 volume；The middle and late stage cell maintenance stage,
Culture medium groundwater increment is 1-2 volume.
Viral infection multiplicity MOI of step (2) described inoculation bird flu virus is 0.005-5；Preferably, virus infects multiple
Number MOI is 0.1.During described propagation, virus maintains the concentration of TPCK-pancreatin in liquid to be 1-10 μ g/mL, preferably 1-5 μ
G/mL, most preferably 2 μ g/mL.The time of described results virus liquid is 24-96h, preferably 48-after inoculation bird flu virus
72h, most preferably 48h.Preferably, described bird flu virus is H 5 N 1 avian influenza subtype virus strain Re-6.The inventive method pair
The concrete kind of bird flu virus is not particularly limited, it is adaptable to any obtainable bird flu virus.
Step (3) is described to be included virus liquid inactivation: is centrifuged by virus liquid, takes supernatant；By supernatant concentration, add death of monks or nuns
Agent alive, inactivation；Preferably, inactivate after supernatant is carried out 10 times of concentrations；The viral level to every 0.1ml is adjusted before inactivation of virus
≥107.0EID50, HA titre >=29, meet and join Seedling requirement.Wherein, described inactivator is formalin；According to volume basis, institute
State inactivator final concentration of virus liquid supernatant cumulative volume 0.1%；Described inactivation is 37 DEG C of inactivation 16h；The virus liquid of inactivation
Mix according to volume ratio 1:1.5 with adjuvant；Described adjuvant is oil adjuvant.
The present invention is to bioreactor culture clonal cell line MDCK-3D6 and bird flu virus increasing in the reactor
The condition of growing is optimized.Cell Initial seeding density optimum results shows, low initial density (1.5 × 105Individual cell/ml) make
Period of delay of mdck cell extends, cell growth lack initial density effect and cause growth phase to slowly, maximum cell density
Minimum.4.5×105The initial density of individual cell/ml can make mdck cell rapidly enter trophophase, but cell survival rate phase
To relatively low, this is the phenomenon owing to cell exists agglomerate and free cell does not sticks after inoculation, although distribute on single microcarrier
Number of cells a lot, but effective tactophily surface can not be provided, be unfavorable for that cell grows.3×105Individual cell/ml's
Cell initial density, can be uniformly distributed cell in each micro-carrier surface, promotes cell fast-growth, is also cell simultaneously
Abundant growing surface has been reserved in growth, the preferable culture effect of final acquisition, maintains higher cell survival rate.
Mixing speed optimum results shows, when mixing speed is 50r/min, cell density slowly rises, the highest cell
Density is only 9.3 × 105Cell/ml, it is too low that this is possibly due to mixing speed, and in rolling bottle, oxygen transfer speed can not meet cell
Consuming, therefore cell growth occurs without exponential phase；When mixing speed is 120r/min, high density is more than the former, but also
It is significantly less than the high-cell density when mixing speed is 100r/min, illustrates that excessive whipping creates serious shearing to cell
Injury.When mixing speed is 100r/min, cell rapidly enters exponential phase, during 56h cell high density be 1.5 ×
106cell/ml.Therefore, present invention determine that 100r/min is optimal mixing speed.
Microcarrier concentration optimum results shows, is not provided that when microcarrier concentration is 1g/L enough surface areas are for cell
Growth, cell is by more serious contact inhibition, and during 60h, cell density only has 1.0 × 106cell/ml；And microcarrier concentration is
During 3g/L and 5g/L, before 36h, cell density is without significant difference, and 36～60h the former cell densities are slightly above the latter, and the highest cell is close
Degree is respectively 1.5 × 106Cell/ml and 1.3 × 106cell/ml.In view of use cost, microcarrier concentration selects 3g/L.
Bioreactor cell is cultivated metabolic analysis and is shown, during mdck cell enters exponential phase, glucose contains
Amount quickly falls to minimum 5mmol/L, and metabolism lactic acid is risen rapidly to 20mmol/L by 0mmol/L, along with total cellular score connects
Nearly maximum, the glucose consumption of cell gradually tends towards stability, and lactic acid content also drops to a relatively low level simultaneously.Carefully
The intracellular growth phase, glucose utilization is slightly below lactic acid growing amount, illustrates that glucose is not that complete metabolism generates lactic acid, less than 50%
Glucose complete oxidation supply cellular energy.Therefore, during cell amplification culture, cell produces with glucose metabolism in early days
Raw a large amount of lactic acid are characterized, perfusion cultures base to control lactic acid concn for the purpose of reduced levels, groundwater increment 3～4 volume；After in
Phase survives as perfusion purpose, groundwater increment 1～2 volume with cell, gradually reduces to reach higher culture medium utilization rate.
The condition optimizing result of avian influenza mdck cell shows, virus infection multiplicity can obtain when being 0.1
The highest AIV breeds titer, reaches 108.9TCID50/ml.Additionally, 72h after the virus titer of 48h is above connecing poison after connecing poison
Virus titer, after illustrating to connect poison, 48h is results virus optimum time.When TPCK-pancreas enzyme concentration is 2.0 μ g/ml, cultivate 48-72h
Time the highest HA-HI test of AIV virus bring up to 10log2 from 8log2, be better than other process；TPCK-pancreas enzyme concentration is 7.5 and 10 μ g/
Ml, because excessive concentration is relatively big to cytoclasis effect, causes complete cell death.Therefore, the present invention breeds the virus of AIV virus
Maintaining TPCK-pancreas enzyme concentration in liquid is 2.0 μ g/ml.In the present invention, H5N1 subtype avian influenza virus breeds temperature in the reactor
It it is 38 DEG C.
The present invention uses 5L bioreactor (B5, Sartorius, Germany).The present invention kind to bioreactor
Being not particularly limited, general stirring type bioreactor is all applicable to the present invention.
The inactivated avian influenza vaccine that the inventive method produces can be applied to pre-avian influenza-prevention.Safety examination shows, connects
Planting the chicken nonsystemic reaction of the present invention each batch vaccine, injection site absorbs preferably, without observing pathological changes, it was demonstrated that vaccine of the present invention
To chicken safety.Immunization experiment result shows, vaccine immune chicken HI antibody mean titre prepared by the present invention at 7.5log2～
Between 9.4log2；After counteracting toxic substances, protective rate reaches 100%, hence it is evident that be better than similar-type products immune group.
Technical solution of the present invention compared with prior art, has the advantages that
The clonal cell line MDCK-3D6 that the present invention obtains is than parental cells fast growth, and fits bird flu virus
Ying Xinggao, is passaged to 11-20 generation, and virus HA reaches 256.Present invention optimizes and utilize microcarrier large-scale culture clonal cell line
MDCK-3D6 and the condition of propagation bird flu virus, prepared inactivated vaccine is to chicken safety, and immunity chicken HI antibody titer is high,
Attack to H5N1 subtype avian influenza virus realizes 100% immunoprotection.The present invention is with mammalian cell for the big rule of substrate
Mould virus of proliferation and prepare avian influenza vaccine and provide foundation.
The term definition that the present invention relates to
Unless otherwise defined, all technology the most used herein and scientific terminology all have with of the art
Those of ordinary skill is generally understood identical implication.
Term " single cell clone " means to cultivate a cell, is allowed to constantly divide, and forms a cell mass
Process, this group cell derived is in a common ancester cell.
Term " sub-clone " means in cell clone, to the cell cultivated, filters out again and have from original clone
The cell of certain characteristic is cultivated.
Term " passes on " and means after cell proliferation reaches certain density, then need isolate a part of cell and update battalion
The process of nutrient solution, otherwise will affect the survival of cell.
Accompanying drawing explanation
Fig. 1 is the one step growth curve figure of clonal cell line；
Fig. 2 is that the mdck cell of different initial density is at 3g/L grown on microcarriers curve；
Fig. 3 is that the mdck cell of different initial density sticks efficiency comparison when cultivating 6h on 3g/L microcarrier；
Fig. 4 is that microcarrier is cultivated the impact of mdck cell growth by mixing speed；
Fig. 5 is that microcarrier is cultivated the impact of mdck cell growth by microcarrier concentration；
Fig. 6 is the glucose of 5L bioreactor culture mdck cell, lactic acid content curve.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
1, material and reagent
1.1 strains and cell
H 5 N 1 avian influenza subtype virus strain Re-6 hemagglutinative titer is 1:512, and concentration of virus particles is about 3 × 108PFU/mL,
Given by Academy of Agricultural Sciences of state Harbin veterinary institute swine diseases molecular diagnostic laboratories；MDCK(Madin-Darbin canine
Kidney) cell is purchased from the vast Imtech in Beijing, cultivates to 89 generations at the present inventor's laboratory passage.
1.2 capital equipments and reagent
5L bioreactor (B5, Sartorius, Germany)；TPCK-pancreatin is purchased from Sigma company；Cytodex-1 purchases
From GE Healthcare company；DMEM is purchased from Life technology；Other reagent are import or domestic analytical pure product.
The clone of embodiment 1 MDCK clonal cell line
1, experimental technique
The clone of 1.1 MDCK clonal cell lines
(1) kind of a cell MDCK is selected；
(2) passage: first plant cell in the cell bottle of T25 selected by recovery step (1), grow about 48h,
Pass on according to the ratio of 1:4, continue before clone to pass on 20 times；
(3) limiting dilution assay is used to carry out MDCK single cell clone in 96 orifice plates, after postdigestive mdck cell counting
It is diluted to 1 cells/ml, the cell diluted is laid on 96 orifice plates, 0.1ml/ hole, 37 DEG C of 5%CO2Cultivate in incubator,
Observe 10；
(4) choose the further sub-clone of single clone, through 3 sub-clones, choose monoclonal strain and be enlarged cultivating, cultivate
Liquid is DMEM (containing 10%FBS), then, identifies clone cell further.
The 1.2 MDCK clonal cell lines Adaptability Analysis to bird flu virus
Cultivate MDCK clonal cell line, take the ratio of 1:10 to pass on after 15 generations, every 5 generations frozen once until
25 generations.Monoclonal cell is inoculated in Tissue Culture Flask (T25Corning), 37 DEG C, 5%CO2After cultivating 48h formation monolayer,
Discarding culture medium, PBS (pH 7.2) rinses twice, adds avian influenza virus H 5 N 1 RE-6 according to infection multiplicity (MOI)=0.1
Strain, 37 DEG C, 5%CO2Absorption 1h in incubator, addition 9ml DMEM maintenance liquid (containing final concentration 5 μ g/ml TPCK-pancreatin,
0.5%FBS), 37 DEG C, 5%CO2Continue to cultivate, observe day by day.When pathological changes reaches 80% (in 5 days), multigelation 2 times, 2,
000rpm 4 DEG C gathers in the crops supernatant after centrifugal 20 minutes, carries out HA detection.Measure the HA of different generation MDCK clonal cell line respectively,
With maternal mdck cell for comparison.
2, experimental result
The acquisition of 2.1 MDCK clonal cell lines
The present invention uses limiting dilution assay to carry out MDCK single cell clone, then chooses the further sub-clone of single clone,
Through 3 sub-clones, final acquisition 6 strain monoclonal cells, it is respectively designated as MDCK-3D2, MDCK-3D3, MDCK-3D5, MDCK-
3D6, MDCK-3D7 and MDCK-3D9.
The growth characteristics analysis result of 2.2 MDCK clonal cell lines
Growth curve measurement result shows, clonal cell line MDCK-3D6 is compared with parental cells, and fast growth (is schemed
1)；And the growth of other 5 strain cell MDCK-3D2, MDCK-3D3, MDCK-3D5, MDCK-3D7 and MDCK-3D9 and parental cells
Speed is basically identical or the slowest.
The 2.3 MDCK clonal cell lines Adaptability Analysis to bird flu virus
Fowl is flowed by clone cell MDCK-3D2, MDCK-3D3, MDCK-3D5, MDCK-3D6, MDCK-3D7 and MDCK-3D9
The compatibility test of influenza vaccine strain the results are shown in Table 1.
Result shows, clone cell MDCK-3D6 is high to the adaptability of avian influenza vaccine strain, is passaged to 11-20 generation, virus
HA reaches 256；Being passaged to 21-25 generation, virus HA reaches 512；Be significantly higher than clonal cell line MDCK-3D2, MDCK-3D3,
MDCK-3D5, MDCK-3D7 and MDCK-3D9 and parental cells.
Table 1 clone cell compatibility test result to avian influenza vaccine strain
Therefore, the MDCK clonal cell line MDCK-3D6 obtained is submitted to Chinese microorganism strain preservation management to entrust by the present invention
Member can carry out preservation in common micro-organisms center, and its microbial preservation is numbered: CGMCC No.12040.
Embodiment 2 microcarrier cultivates MDCK clonal cell line propagation bird flu virus
Mdck cell described in the present embodiment is the MDCK clonal cell line MDCK-3D6 that embodiment 1 obtains, and its microorganism is protected
Hide numbered: CGMCC No.12040.
1, experimental technique
1.1 microcarriers process
With without Ca2+、Mg2+After the phosphate buffer of ion cleans and soaks microcarrier at least 3h, through 121 DEG C of high steams
Sterilizing 25min, is stored in 4 DEG C of sealings stand-by after cooling.Clean 2 times with the cell culture fluid of 37 DEG C before using.
The bioreactor culture optimization of 1.2 mdck cells
1.2.1 the impact of different initial density cell by cell growths
Different initial density 1.5 × 105Individual cell/ml, 3.0 × 105Individual cell/ml, 4.5 × 105Individual cell/ml's
Mdck cell is inoculated in 5L reactor respectively, and microcarrier consumption is 3g/L, observes the shadow of different initial density cell growth
Ring, determine optimal inoculating cell density.
1.2.2 the impact of mixing speed cell growth
When investigation mixing speed is 50/min, 100r/min and 120r/min respectively, mdck cell is in DMEM culture medium
Growing state, microcarrier concentration is 3g/L.With 3.0 × 105Cells/ml cell density is inoculated in 5L reactor, working volume
For 2L, it is monitored every 12h, sampling counting cell.
1.2.3 the impact of different microcarrier concentration cell growth
The microcarrier concentration of microcarrier supplier GE business recommendations is 2～3g/L, is usually no more than during batch culture cell
5g/L.The present invention has investigated the growing state of cell when microcarrier concentration is 1g/L, 3g/L and 5g/L.
1.2.4 the impact of metabolite cell growth
In mdck cell incubation, the metabolic condition of lactic acid and glucose is the leading indicator of cell growth.Therefore, originally
Glucose, lactic acid metabolism situation in invention detection cell growth process, and perfusion strategy is analyzed.
1.3 microcarriers cultivate bird flu virus condition optimizing
1.3.1 the determination of virus infective dose (MOI)
Cell is cultivated after 72h, respectively with MOI=0.005,0.01,0.1,0.5,1 and 5 virus inoculation, cultivate 48 respectively,
After 72h, detect virus titer.
1.3.2 the determination of TPCK-pancreas enzyme concentration
Growing mdck cell and the impact of virus titer to investigate TPCK-pancreatin, the present invention carefully cultivates at MDCK
During the infection time optimized, virus maintains in liquid adds 1 μ g/mL, 2 μ g/mL, 5 μ g/mL, 7.5 μ g/mL and 10 μ g/mL respectively
TPCK pancreatin, detects virus titer.
1.3.3 virus measures
Viral hemoagglutination value and TCID50The method that being worth provides according to WHO handbook measures.
(1) viral hemoagglutination pH-value determination pH: carry out on " u "-shaped 96 orifice plate, the most each hole adds 50 μ L normal saline；Yu Zuo
Hole, side the 1st adds 50 μ LL virus liquids (allantoic fluid or freeze dried vaccine liquid), after mix homogeneously, inhales 50 μ L to the 2nd holes, and 2 multiple proportions are dilute successively
Releasing to the 11st hole, inhale and abandon 50 μ L, the 12nd hole is erythrocyte comparison；Add 0.5% chicken red blood cell successively to hang from right-to-left to each hole
Liquid 50 μ L, vibrates on the oscillator, observed result after left at room temperature 10min.
(2) sample TCID50Measure: will digest by cultured mdck cell in containing blood serum medium, and be diluted to required
Cell density (0.8～1.0 × 105Cells/mL, i.e. every hole 8000～10000 cells), in 96 orifice plates, every hole adds 100 μ L
Cell suspension；Put in incubator 37 DEG C to hatch 24h and be paved into monolayer about 60% abundance to cell；Take out orifice plate, suck culture fluid, often
Hole interpolation about 100 μ L PBS rock and wash 2 times to remove serum；Virus liquid good for 10 multiple proportions serial dilutions is added to 96 orifice plates
On, every hole 100 μ L, to make 11 gradients from 1 to 11, from A to H, make 8 repetitions, it is (each that last string (A12-H12) does negative control
Add 100 μ L viral dilution liquid), put incubator 37 DEG C and hatch 1h；Absorb virus liquid, add 100 μ L cell maintenance mediums；Put incubator
37 DEG C hatch 72h after observation of cell pathological changes determine TCID50Value.
2, experimental result
The determination of 2.1 initial cell densities
When microcarrier consumption is 3g/L, initial cell density 1.5 × 105Individual cell/ml, 3.0 × 105Individual cell/ml and
4.5×105Individual cell/ml makes mdck cell at grown on microcarriers curve difference (Fig. 2).Low initial density (1.5 ×
105Individual cell/ml) make extend the period of delay of mdck cell, this is due to the hypocellularity of distribution on single microcarrier, also simultaneously
There is higher microcarrier zero load ratio (Fig. 3 A), cell growth lack initial density effect and cause growth phase to slowly,
Maxicell density is minimum, but cell survival rate maintains higher level all the time.4.5×105The initial density of individual cell/ml can
So that mdck cell rapidly enters trophophase, but cell survival rate is relatively low, this be due to inoculation after cell exist agglomerate and
The phenomenon that free cell does not sticks, although on single microcarrier, the number of cells of distribution is a lot, but can not provide and effectively stick
Growing surface, is unfavorable for that cell grows (Fig. 3 C).During so cell is inoculated on microcarrier cultivation, suitable cell initial density
(3×105Individual cell/ml) cell can be uniformly distributed in each micro-carrier surface, promote cell fast-growth, be also thin simultaneously
Abundant growing surface has been reserved in the growth of born of the same parents, the preferable culture effect of final acquisition, maintains higher cell survival rate (Fig. 2
With Fig. 3 B).
The impact of 2.2 mixing speed cell growth
In order to make microcarrier fully suspend in microcarrier incubation, it is necessary to there is sufficiently high mixing speed；But excessively
Stirring can extend the cell attachment time, major injury attached cell, especially bigger to the injury of mitotic cycle cell,
Cell density is caused to reduce.Therefore the present invention has investigated mixing speed when being 50r/min, 100r/min and 120r/min respectively
Mdck cell growing state in DMEM culture medium.Result as shown in Figure 4, when mixing speed is 50r/min, cell density
Slowly rising, high-cell density is only 9.3 × 105Cell/ml, it is too low that this is possibly due to mixing speed, and in rolling bottle, oxygen passes
Matter speed can not meet cell consumption, and therefore cell growth occurs without exponential phase；When mixing speed is 120r/min, the highest
Density is more than the former, but is also significantly less than the high-cell density when mixing speed is 100r/min, illustrates the most stirred
Degree, creates serious shearing injury to cell.When mixing speed is 100r/min, cell rapidly enters exponential phase, 56h
Time cell high density be 1.5 × 106cell/ml.Therefore, present invention determine that 100r/min is optimal mixing speed.
The determination of 2.3 microcarrier concentration
Microcarrier concentration affects the preparation amount of seed cell, high-cell density, cell algebraically, changes liquid frequency and strategy,
Again because microcarrier is expensive, toxigenic capacity is also had a significant impact, so selecting suitable microcarrier concentration on a large scale
Industrialization cell is cultivated significant.The microcarrier concentration of microcarrier supplier GE business recommendations is 2～3g/L.The present invention examines
Having examined the growing state of cell when microcarrier concentration is 1g/L, 3g/L and 5g/L, experimental result is as it is shown in figure 5, microcarrier concentration
During for 1g/L, it is impossible to the surface area providing enough grows for cell, and cell is by more serious contact inhibition, and during 60h, cell is close
Degree only 1.0 × 106cell/ml；And microcarrier concentration is when being 3g/L and 5g/L, before 36h, cell density is without significant difference, 36～
60h the former be slightly above the latter by cell density, high-cell density is respectively 1.5 × 106Cell/ml and 1.3 × 106cell/ml。
In view of use cost, select 3g/L as the use weight of microcarrier.
2.4 bioreactor cells cultivate metabolic analysis and perfusion strategy
During mdck cell enters exponential phase, glucose content quickly falls to minimum 5mmol/L, metabolism
Lactic acid is risen rapidly to 20mmol/L by 0mmol/L, along with total cellular score gradually becomes close to maximum, the glucose consumption of cell
In stable, lactic acid content also drops to a relatively low level (Fig. 6) simultaneously.Owing to the 1 complete metabolism of molecule glucose is lactic acid
In the case of, 2 molecule lactic acid can be generated.At phase of cell growth, glucose utilization is slightly below lactic acid growing amount, and Fructus Vitis viniferae is described
Sugar is not that complete metabolism generates lactic acid, the glucose complete oxidation supply cellular energy less than 50%.Therefore, training is amplified at cell
During Yanging, cell is characterized with the glucose metabolism a large amount of lactic acid of generation in early days, and perfusion cultures base is to control lactic acid concn relatively
For the purpose of low-level, groundwater increment 3～4 volume；Middle and late stage is survived as perfusion purpose with cell, groundwater increment 1～2 volume, by
Step reduces to reach higher culture medium utilization rate.
The condition optimizing of 2.5 avian influenza mdck cells
2.5.1 virus infective dose (MOI) and the determination of receipts poison time
Different infection multiplicities on AIV propagation titer to affect result as shown in table 2.Too high or too low infection multiplicity (5
With 0.005) all cause relatively low AIV to breed titer, illustrate that connecing toxic agent amount directly affects the propagation efficiency of virus.Infection multiplicity is
The highest AIV can be obtained when 0.1 and breed titer, reach 108.9TCID50/ml.Respectively group result of the test all shows, after connecing poison
The virus titer of 48h is above connecing the virus titer of 72h after poison, and illustrating to connect 48h after poison can the most in good time as results virus
Between.
The impact on AIV propagation titer of the table 2 different infection multiplicity (MOI)
2.5.2TPCK-the pancreatin impact on AIV virus multiplication
By MOI=0.1 inoculation AIV virus, TPCK-pancreas enzyme concentration is respectively 1.0,2.0,5.0,7.5 and 10 μ g/ml, and 38
DEG C cultivate, every 12h, results virus, measure HA-HI test (HA), the results are shown in Table 3.
The different TPCK-pancreas enzyme concentration of table 3 connects different time viral hemoagglutination valency (log2) after poison
During from table 3 it is observed that TPCK-pancreas enzyme concentration is 2.0 μ g/ml, AIV virus the highest blood clotting when cultivating 48-72h
Valency brings up to 10log2 from 8log2, is better than other and processes.Therefore, the present invention utilizes the virus of propagation AIV virus to maintain in liquid
TPCK-pancreas enzyme concentration is 2.0 μ g/ml.The TPCK-pancreatin of 7.5 and 10 μ g/ml is relatively big to cytoclasis effect because of excessive concentration,
Cause complete cell death.
2.6 bird flu viruss are the optimum results of proliferation conditions in 5L reactor
To sum up, present invention determine that the optimum condition that H5N1 subtype avian influenza virus is bred in 5L reactor is: the thinnest
Born of the same parents' density 3.0 × 105Cell/ml, mixing speed is 100r/min, and microcarrier concentration is 3g/L；Sick by MOI=0.1 inoculation AIV
Poison；Virus maintains the TPCK-pancreatin simultaneously containing 2.0 μ g/ml in liquid；Cultivate 48-72h for 38 DEG C.
The assessment of embodiment 3 inactivated avian influenza vaccine
1, experimental technique
1.1 bird flu viruss are cultivated
The result optimized according to embodiment 2 utilizes 5L bioreactor to carry out the cultivation of bird flu virus.The present embodiment institute
Stating mdck cell is the MDCK clonal cell line MDCK-3D6 that embodiment 1 obtains, and its microbial preservation is numbered: CGMCC
No.12040；Bird flu virus is H 5 N 1 avian influenza subtype virus strain Re-6.
1.2 results viruses
When CPE (CPE) occurs in the cell of cultivation to more than 80%, virus can be gathered in the crops.
1.3 inactivation of virus with join Seedling
Viral level >=10 to every 0.1ml are adjusted before inactivation7.0EID50, HA titre >=29, meet and join Seedling requirement.
Virus liquid 5000r/min is centrifuged 30min, removes dead cell and fragment, takes supernatant；Supernatant is carried out 10 times of concentrations
After, use formalin inactivation, make the 0.1% of inactivator final concentration of virus liquid supernatant cumulative volume, 37 DEG C of inactivation 16h, permissible
Complete inactivation；After aseptic is checked, inactivation of viruses is mixed with imported oil adjuvant (1:1.5) by volume respectively, breast
For animal immune after change.
1.4 immunization experiment
Contrived experiment, compares the most qualified H5N1 subtype avian influenza virus inactivated vaccine and Kazakhstan medicine that the present invention produces
The H5 subtype avian influenza vaccine of 2 manufacturer production of biovaccine company limited of group and Harbin Wei Ke biotechnology development company
The effect of pre-avian influenza-prevention.
1.4.1 safety examination
Each batch vaccine that the inventive method produces is with 40 age in days SPF chicken 10, and each cervical region is subcutaneous or chest muscle is injected
Vaccine 2 plumage part, observes 14.
1.4.2 immunoprotection
The SPF chicken of 4 week old is randomly divided into A, B and C test group, often group 10；D group is negative control group, SPF chicken 5.
Bird flu virus inactivated vaccine immunity A test group prepared by the present invention；The H5N1 subtype avian influenza epidemic disease of domestic 2 manufacturer production
Seedling immunity B and C test group respectively；Wherein, the inactivated avian influenza vaccine immunity B test of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.
Group, the inactivated avian influenza vaccine immunity C test group of Harbin Wei Ke biotechnology development company；Often group vaccine cervical region subcutaneous vaccination
Test chicken 10, dosage of inoculation 0.3mL/ is only；Matched group is the most immune.
After immunity the 7th, 14,21 days, gather every chicken serum, by existing " Chinese veterinary pharmacopoeia " annex method determination test group
HI antibody titer with negative control group every chicken.
After immunity 3 weeks, with H5N1 subtype avian influenza virus intravenous injection challenge trial group and matched group chicken, after attacking 5 days,
Gather cloacal swabs, carry out virus purification with 10 age in days SPF Embryo Gallus domesticus.It is judged to exempt from not being separated to virus from cloaca
Epidemic disease counteracting toxic substances is protected.
2, experimental result
The safety examination of 2.1 H5N1 subtype avian influenza inactivated vaccines
Safety examination result shows, the chicken inoculating each batch vaccine prepared by 2 plumage part the inventive method is anti-without whole body
Should, injection site absorbs preferably, without observing pathological changes.Vaccine prepared by proof the inventive method is to chicken safety.
2.2 immunization experiment results
3 kinds of vaccination tests chickens are after 3 weeks, and blood sampling detection antibody titer, result shows (table 4), compares chicken geometric average
Titer is below 2log2, and immunity chicken HI antibody geometric mean titer is between 7.5log2～9.4log2.After counteracting toxic substances, the guarantor of A group
The rate of protecting reaches 100%, hence it is evident that be better than B group and the C group of similar-type products immunity.Showing, vaccine prepared by the inventive method can be right
The attack of H5N1 subtype avian influenza virus realizes 100% immunoprotection.
The Immunoprotection test result of 43 groups of vaccines of table
Claims (10)
1. a strain MDCK clonal cell line MDCK-3D6, it is characterised in that its microbial preservation numbering is: CGMCC
No.12040。
2. the application in cultivating virus of the MDCK clonal cell line MDCK-3D6 described in claim 1.
3. according to the application described in claim 2, it is characterised in that described virus includes: influenza virus (Influenza
Virus), rabies virus (Rabies virus) or parainfluenza virus (Parainfluenza Viruses).
4. according to the application described in claim 3, it is characterised in that described influenza virus includes: bird flu virus (Avain
Influenza Virus), human influenza virus (Human Influenza Viruses) or equine influenza virus (Equine
Influenza virus)；It is preferably bird flu virus.
5. the production method of an inactivated avian influenza vaccine, it is characterised in that comprise the following steps: (1) is by described in claim 1
MDCK clonal cell line MDCK-3D6 is inoculated on the microcarrier in bioreactor, cultivates cell；(2) inoculation avian influenza
Poison, breeds described virus, gathers in the crops virus liquid；(3) virus liquid is inactivated, add adjuvant mixing, emulsifying, to obtain final product.
6. according to the production method described in claim 5, it is characterised in that: step (1) described MDCK clonal cell line MDCK-
3D6 is according to 1.5 × 105-4.5×105Individual/ml, preferably 3.0 × 105The initial density of individual/ml is inoculated on microcarrier；
The concentration of described microcarrier is 1-5g/L, preferably 3-5g/L, most preferably 3g/L；
It is furthermore preferred that wherein said microcarrier is Cytodex-1.
7. according to the production method described in claim 5, it is characterised in that: the mixing speed of step (1) described cultivation cell is
50-120r/min, preferably 100r/min；
Preferably, the time of described cultivation cell is 72h.
8. according to the production method described in claim 5, it is characterised in that: the virus of step (2) described inoculation bird flu virus
Infection multiplicity MOI is 0.005-5；Preferably, virus infection multiplicity MOI is 0.1；
During described propagation, virus maintains the concentration of TPCK-pancreatin in liquid to be 1-10 μ g/mL, preferably 1-5 μ g/mL,
It is preferably 2 μ g/mL；
The time of described results virus liquid is 24-96h, preferably 48-72h, most preferably 48h after inoculation bird flu virus；
Preferably, described bird flu virus is H 5 N 1 avian influenza subtype virus strain Re-6.
9. according to the production method described in claim 5, it is characterised in that: step (3) is described to be included virus liquid inactivation: by disease
Venom is centrifuged, and takes supernatant；By supernatant concentration, add inactivator, inactivation；
Wherein, described inactivator is formalin；The 0.1% of the final concentration of virus liquid supernatant cumulative volume of described inactivator；Institute
Stating inactivation is 37 DEG C of inactivation 16h；
The virus liquid of inactivation mixes according to volume ratio 1:1.5 with adjuvant；Described adjuvant is oil adjuvant.
10. the inactivated avian influenza vaccine that production method described in claim 5 to 9 any one produces.
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| CN110520525A (en) * | 2017-07-05 | 2019-11-29 | Sk生物科学株式会社 | It is used to prepare the method for influenza working virus seedling, the method for using identical seedling preparation influenza vaccines and the viral seedling prepared by this method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104338127A (en) * | 2014-10-09 | 2015-02-11 | 江苏省农业科学院 | Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine |
| CN104694458A (en) * | 2015-01-06 | 2015-06-10 | 哈药集团生物疫苗有限公司 | MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof |
-
2016
- 2016-06-14 CN CN201610423180.3A patent/CN106085947B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104338127A (en) * | 2014-10-09 | 2015-02-11 | 江苏省农业科学院 | Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine |
| CN104694458A (en) * | 2015-01-06 | 2015-06-10 | 哈药集团生物疫苗有限公司 | MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 吴培培等: "无血清微载体培养Vero细胞增殖传染性法氏囊病毒", 《江苏农业科学》 * |
| 张启龙: "稳定表达siat7e和st3galI基因MDCK细胞株的建立", 《中国优秀硕士学位论文全文数据库.基础科学辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110520525A (en) * | 2017-07-05 | 2019-11-29 | Sk生物科学株式会社 | It is used to prepare the method for influenza working virus seedling, the method for using identical seedling preparation influenza vaccines and the viral seedling prepared by this method |
| CN110520525B (en) * | 2017-07-05 | 2024-04-16 | Sk生物科学株式会社 | Influenza working virus seedling and preparation, and method for increasing infectivity and preparing vaccine |
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