CN109908337A - The preparation method and products thereof of chicken pox live vaccine - Google Patents
The preparation method and products thereof of chicken pox live vaccine Download PDFInfo
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Abstract
The invention discloses preparation methods of chicken pox live vaccine and products thereof.Chicken embryo fibroblasts cell is cultivated obtained adaptation serum free medium by domestication, in the passage chicken embryo fibroblasts of single-cell suspension growth conditions by the present invention；Invention further provides a kind of methods for preparing chicken pox live vaccine, it is characterised by comprising: the adaptation serum free medium is inoculated into progress cell suspension Multiplying culture in bioreactor in the passage chicken embryo fibroblasts of suspension growth by (1)；(2) harvest virus liquid after carrying out virus multiplication culture to the cell inoculation bird pox virus after suspension Multiplying culture, (3) mix virus liquid and freeze drying protectant, freeze-drying to get.Preparation method of the present invention significantly reduces pollution risk and production cost, effectively shortens the production time of chicken pox live vaccine, is convenient for expanding the scale of production；Chicken pox live vaccine safety prepared by the present invention is good, and immune protection effectiveness is high.
Description
Technical field
The present invention relates to preparation methods of chicken pox live vaccine and products thereof, more particularly to use serum free suspension culture skill
The method of art preparation chicken pox live vaccine and obtained chicken pox live vaccine product, belong to the preparation neck of chicken pox live vaccine live vaccine
Domain.
Background technique
Chicken pox is that one kind as caused by bird pox virus (Fowlpox virus) is acute, contagious disease, main infection
Chicken, turkey and a variety of wild rare birds.Clinically it is mainly shown as 3 kinds of forms.One is skin-type, with skin without
There is acne rash and are characterized in hair and few hair position, and this type of clinic process is milder, and the death rate is lower；Other two is mucous membrane type, custom
Claim " diphtheria " and mixed type, often causes acne rash, and Chang Yin in respiratory tract organ and the mucous membrane of digestive organ due to this type of
It has difficulty in breathing and keeps the death rate higher.The chicken group for infecting the disease can often make the sick fowl of infection raw although the death rate is not high
Long hypoevolutism, egg production decline, hatching rate reduces after the onset of planting fowl, and once secondary animal infectious diease, parasitic disease,
It is easy to cause the death of large quantities of infection poultry, especially chick is even more serious.After disease outburst infection, can not only it bring huge
Economic loss, and some birds in imminent danger can be threatened.Each large-scale commercialization farm is using the weak poison of pigeon avipoxvirus and quail
Change fowl pox attenuated vaccine to prevent chicken pox outburst.
DF-1 origin of cell now has become a kind of continuous cell line of maturation in chicken embryo fibroblasts.DF-1 cell
In vitro culture has very strong proliferative capacity, and the density of cell can be more than CEF4 times or more.DF-1 cell is reverse transcriptase
Negative, non-tumorigenic cell.DF-1 cell line is the global commerce authorized through U.S. Food and Drug Administration (FDA)
Cell line has been widely used in the proliferation of avian viral, the expression of recombinant protein, the research of tumour virus and animal at present
With the production of human vaccination.
The mode that traditional DF-1 culture mostly uses serum adherent greatly, the training method complex process take up a large area,
Need a large amount of manual operations and need to add serum during the cultivation process, increase by pollutions such as exogenous virus, mycoplasmas can
Energy property, and the serum difference between different batches also results in product quality and is difficult to control, while it is pure also to increase downstream separation
The difficulty of change.Compared with serum adhere-wall culture technology, serum free suspension culture technique eliminates and disappears without expensive microcarrier
The operation for changing harvest cell, reduces pollution risk and production cost, shortens the production time.Due to serum free suspension culture
Technology does not need to attach matrix, can save the device space, utilization rate of equipment and installations is improved, convenient for expanding the scale of production.Therefore, no blood
Clear suspension culture techniques are the inexorable trends of cell industrialized production vaccine.
Therefore, DF-1 cell suspend entirely taming to cultivate and obtain adaptation serum free medium and be in suspension growth shape
The cell of state and the preparation for being applied to chicken pox live vaccine, for reducing the production cost and pollution wind of chicken pox live vaccine
Danger, progress large-scale production etc. all have important meaning.
Summary of the invention
An object of the present invention is to provide one plant and adapts to serum free medium and be in the biography of single-cell suspension growth conditions
For chicken embryo fibroblasts cell；
The adaptation serum free medium obtained is tamed the second object of the present invention is to application and in suspension growth state
It passes on chicken embryo fibroblasts cell (DF-1 cell) and prepares chicken pox live vaccine, which is dropped using suspension culture techniques
The production cost and pollution risk of low chicken pox live vaccine, are able to carry out scale industrial production.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
The present invention will pass on chicken embryo fibroblasts (DF-1 cell) through the adhere-wall culture stage low serum domestication process and
Serum free medium suspension domestication process obtain one plant adapt to serum free medium and in suspension growth state passage chicken embryo at
Fibrocyte, the cell are named as DF-1-XF.
The present invention uses the method for gradually reducing serum domestication DF-1 cell to adapt to low concentration serum free culture system first: this
When invention is by DF-1 cell length to the mid log phase for stablizing growth in the MEM culture solution containing 10% newborn bovine serum, replacement
For the MEM culture solution containing 5% newborn bovine serum, when the convergence degree of cell length to 80%~90%, trypsin digestion is thin
Born of the same parents, with cell density for 2.0 × 105Cells/ml passage is in the MEM culture solution containing 5% newborn bovine serum；After number generation,
Survival rate of the DF-1 cell in the MEM culture solution containing 5% newborn bovine serum maintains 90% or more, and keeps very fast growth
Rate further decreases serum domestication culture；Adapt to DF-1 cell gradually containing 1% newborn bovine serum
MEM condition of culture.As serum uses the reduction of concentration, the form of DF-1 cell adherent growth gradually changes, finally
Cell shows single-cell suspension growth conditions after serum-free domestication adapts to.DF-1 cell is dropped to from serum-concentration 10%
5%, cell does not occur macroscopic morphological differences, does not show to be not suitable with.When serum-concentration is reduced to 1%, cell
The speed of growth slows down, and after passage, the nutritional condition that cell adapted serum-concentration is 1%, the speed of growth is restored, but thin
There is the trend being rounded in born of the same parents' form, only the respective cells state that shows slightly suspension growth.Driven suspension culture adapts to, DF-
1 cell shows the form of cell suspension growth, but cell clustering phenomena is serious, and respective cells group is larger.It is cultivated suspending
It tames the later period, preferable single-cell suspension growth conditions are presented in suspension DF-1 cell, and cell is rounded, and clustering phenomena is less,
Cell space size is almost the same, and the speed of growth is normal, illustrates that DF-1 cell has been already adapted to serum free medium and in unicellular outstanding
Floating life long status；The present invention tames process through the low serum in adhere-wall culture stage and serum free medium suspension domestication process obtains
Adaptations serum free medium and be in single-cell suspension growth conditions DF-1 cell be named as DF-1-XF cell.
The DF-1-XF for obtaining and adapting to serum free medium and being in single-cell suspension growth conditions is cultivated in domestication by the present invention
The mechanism that cell is submitted to patent approval carries out preservation, and deposit number is: CGMCC No.16295, classification naming are: adapting to
The passage chicken embryo fibroblasts of full suspension growth；Preservation date is: on September 6th, 2018；Depositary institution is: China Microbiological
Culture presevation administration committee common micro-organisms center；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology, the academy of sciences.
The present invention further adapts to serum free medium and in the DF- of single-cell suspension growth conditions using described
1-XF cell prepares chicken pox live vaccine, comprising:
(1) cell suspension Multiplying culture will be carried out in DF-1-XF cell inoculation to bioreactor or Tissue Culture Flask；
(2) virus liquid is harvested after carrying out virus multiplication culture to the DF-1-XF cell inoculation chicken pox after suspension Multiplying culture, (3) will receive
The virus liquid obtained is uniformly mixed with freeze drying protectant, is freeze-dried to get chicken pox live vaccine.
The present invention is by DF-1-XF cell respectively with initial density for 0.3 × 106cells/ml、 0.5×106cells/ml
With 0.75 × 106Cells/ml, which is inoculated in Tissue Culture Flask, carries out suspension culture, counts every sampling in 24 hours, as a result table
Clear-cells Initial seeding density is 0.75 × 106Cells/ml cultivates 48 hour cell density up to 3.0 × 106cells/ml
More than, it is suitble to mass production demand；Therefore, step (1) is by DF-1-XF cell according to 0.75 × 106Cells/ml is that cell is initial
Inoculum density is inoculated into progress cell suspension Multiplying culture in bioreactor or Tissue Culture Flask；In addition, institute in step (1)
The cell suspension Multiplying culture condition stated further include: the conditions such as pH value, dissolved oxygen, temperature and speed of agitator, these parameters can be with
It is cultivated using the parameter of conventional cell suspension cultures, as a reference, the pH may be controlled to 7.2, oxygen dissolving value can be with
Control (DO) be 50%, temperature may be controlled to 37 DEG C, speed of agitator may be controlled to 50-500r/min.
The present invention is 1%, cell Initial seeding density 0.75 × 10 in serum-concentration6A/ml, pH 7.2, dissolved oxygen
(DO) for 50%, under conditions of 37 DEG C of temperature, by DF-1-XF cell respectively with 60r/min, 80r/min, 100r/min and
Tetra- speeds of agitator of 120r/min, are inoculated in 7L bioreactor and carry out cell suspension cultures, small every 24 in incubation
When sampling meter cell number.As a result, it has been found that cell Proliferation is most fast, and 48 hour cell density are when speed of agitator is 100r/min
3.2×106cells/ml；When therefore carrying out cell suspension Multiplying culture in step (1), used speed of agitator is preferred
For 100r/min.
By DF-1-XF cell with 0.75 × 106The initial density of a/ml is inoculated in bioreactor, respectively at cell
Bird pox virus is inoculated with by 2v/v% within culture 36 hours, 48 hours and 60 hours, sampled in incubation every 24 hours, measurement
Viral level connects toxic dose so that determination is most suitable.Other condition of culture: pH control is 7.2, dissolved oxygen (DO) is 50%, temperature 37
DEG C, speed of agitator be set as 100r/min.
The present invention is it has furthermore been found that connect the malicious time to the DF-1-XF cell inoculation bird pox virus after suspension Multiplying culture
It is larger for the Multiplying culture effect of virus:
The present invention is by DF-1-XF cell with 0.75 × 106The initial density of a/ml is inoculated in bioreactor, respectively
Every milliliter 2 × 10 was pressed in cell culture 24 hours, 36 hours, 48 hours and 60 hours3PFU's connects poison amount inoculation bird pox virus,
Connect 48 hours sampling and measuring viral levels after poison.The result shows that cell culture 36h is followed by kind of a bird pox virus, virus titer is reachable
10.6×106PFU/ml.Therefore, preferably by 36 hours inoculation fowl pos diseases of DF-1-XF cell suspension Multiplying culture in step (2)
Poison.
The present invention is it has furthermore been found that connect toxic dose to the DF-1-XF cell inoculation bird pox virus after suspension Multiplying culture
And the acquisition time of harvest venom also has the Multiplying culture effect of bird pox virus the influence of highly significant:
The present invention is by DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, cell training
After supporting 36 hours, according to volume basis, respectively to be inoculated with bird pox virus with 1%, 3%, 6%, the 10% poison amount that connects, after connecing poison
48 hours sampling and measuring viral levels.The result shows that connecing toxic dose is 6%, 72 hours viral titers reach after connecing poison
107.40EID50/0.2ml；Therefore, the DF-1-XF cell inoculation bird pox virus after the preferably floating Multiplying culture of step (2) is best
Connecing toxic dose is 6%.
The present invention is by DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, using same
Step connects the mode of poison, is inoculated with bird pox virus by 6v/v%, sampled in incubation every 24 hours, measure viral level.As a result
Show that 72 hours viral titers are up to 10 after connecing poison7.50 EID50/0.2ml；Therefore, when the poison of receipts described in step (2) is best
Between to connect after poison 72 hours.
Freeze drying protectant described in step (3) can be the freeze drying protectant of any routine；The present invention is through being sieved
Choosing test discovery, can obtain optimal frozen-dried protective effect using following freeze drying protectants:
Freeze drying protectant is made of A liquid and B liquid, wherein the composition of the A liquid is: gelatin 5g, sucrose 5g, dextrin 5g,
Tryptone 2g is settled to 100ml with deionized water；Preparation method includes: that prepared A liquid is carried out high pressure sterilization, i.e.,
?.
The composition of the B liquid is: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g, deionized water are settled to
100ml；Preparation method includes: by the 0.22 μm of membrane filtration degerming of prepared B liquid, 4 DEG C of preservations.
Freeze drying protectant is obtained in order to reach more preferably to be uniformly mixed protective agent A liquid and B liquid by the volume ratio of 1 ︰ 1；It will be thin
Cellular virus liquid and freeze drying protectant are matched according to the volume ratio of 1 ︰ 1.
The preparation method of chicken pox live vaccine of the present invention uses serum free suspension culture technique, with serum adhere-wall culture technology
It compares, preparation method of the present invention eliminates the operation of digestion harvest cell, significantly reduce pollution without expensive microcarrier
Risk and production cost, effectively shorten the production time；It does not need to attach matrix, the device space can be saved, improve equipment utilization
Rate, convenient for expanding the scale of production.Chicken pox live vaccine safety prepared by preparation method of the present invention is good；Immune protection effectiveness
The experimental results showed that prepared chicken pox live vaccine has good immune protection effectiveness for chicken.
Detailed description of the invention
Cellular morphology variation during the domestication of Fig. 1 DF-1 cell；A: suspension Initial stage of culture；B: suspension late stage of culture.
Specific embodiment
The present invention is further described below in conjunction with specific embodiment, the advantages and features of the present invention will be with describing
And it is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.This field skill
Art personnel should be understood that and can carry out without departing from the spirit and scope of the invention to details of the invention and form
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment 1 adapts to the domestication culture of serum free medium and the DF-1 cell in suspension growth state
Use the method for gradually reducing serum domestication DF-1 cell to adapt to low concentration serum free culture system.10% new life will be contained
When stablizing DF-1 cell length to the mid log phase of growth in the MEM culture solution of cow's serum, it is changed to containing 5% newborn ox blood
Clear MEM culture solution, when the convergence degree of cell length to 80%~90%, trypsin digestion and cell is with cell density
2.0×105Cells/ml passage is in the MEM culture solution containing 5% newborn bovine serum.After number generation, DF-1 cell is containing
Survival rate in the MEM culture solution of 5% newborn bovine serum maintains 90% or more, and keeps very fast growth rate, makees into one
Step reduces serum domestication culture.DF-1 cell is set gradually to adapt to the MEM culture item containing 1% newborn bovine serum in the same way
Part.The DF-1 cell for having adapted to 1% newborn bovine serum condition of culture is carried out suspending in cell triangular flask, and it is suitable to cultivate domestication
It answers.Cell culture fluid is that DF-1 serum free medium is trained in Shanghai source, and cell Initial seeding density is 1.0 × 106Cells/ml,
Revolving speed is set as 160r/min, is placed in 5%CO2Suspension culture is carried out in incubator.
As serum uses the reduction of concentration, the form of DF-1 cell adherent growth gradually changes, finally through no blood
Cell shows single-cell suspension growth conditions after clear domestication adapts to.DF-1 cell drops to 5% from serum-concentration 10%, cell
Do not occur macroscopic morphological differences, does not show to be not suitable with.When serum-concentration is reduced to 1%, vitro growth rates
Slow down, after passage, the nutritional condition that cell adapted serum-concentration is 1%, the speed of growth is restored, but cellular morphology goes out
The trend being now rounded, cell out of the ordinary show the state of suspension growth slightly.Driven suspension culture adapts to, and DF-1 cell is presented
The form of cell suspension growth out, but cell clustering phenomena is serious, and respective cells group is larger.The domestication later period is cultivated suspending, is hanged
Preferable single-cell suspension growth conditions are presented in floating DF-1 cell, and cell is rounded, and clustering phenomena is less, cell space size base
This is consistent, and the speed of growth is normal, illustrates that DF-1 cell has been already adapted to serum free suspension state growth, is named as DF-1-
XF cell.
The preparation of 2 chicken pox live vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106At the beginning of cells/ml is cell
Beginning inoculum density, which is inoculated into 7L bioreactor, carries out cell suspension cultures, and incubation time is 36 hours；Other suspension cultures
Condition is as follows: pH control be 7.2, dissolved oxygen (DO) control be 50%, temperature control be 37 DEG C, speed of agitator control be 100r/
min；
(2) the DF-1-XF cell to suspension Multiplying culture 36 hours is inoculated with chicken pox quail according to the dosage of inoculation of 6% (V/V)
Quailization low virulent strain (F282E4) carries out virus multiplication culture；Inoculation harvested virus liquid after 72 hours；Other condition of suspension culture are such as
Under: pH control be 7.2, dissolved oxygen (DO) control be 50%, temperature control be 37 DEG C, speed of agitator control be 100r/min；Through examining
It surveys, viral titer 107.50EID50/0.2ml。
(3) match seedling and packing
(3.1) preparation of freeze drying protectant
A liquid: gelatin 5g, sucrose 5g, dextrin 5g, tryptone 2g, deionized water are settled to 100ml, and 116 DEG C, high pressure is gone out
Bacterium 30min, 15 DEG C of preservations are no more than 7 days；
B liquid: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g, deionized water are settled to 100ml, 0.22 μm of filter membrane
Filtration sterilization, 4 DEG C of preservations, is no more than 3 days；
(3.2) it dispenses
Protective agent A liquid, B liquid are uniformly mixed by the volume ratio of 1 ︰ 1 and obtain freeze drying protectant, qualified cell will be examined
Virus liquid is added in freeze drying protectant, quantitative separating by 1 ︰, 1 volume ratio；
(3.3) it is lyophilized
Vacuum freezedrying is carried out after packing rapidly, obtains chicken pox live vaccine.
The preparation of 3 chicken pox live vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.3 × 106At the beginning of cells/ml is cell
Beginning inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
Being made as 7.2, dissolved oxygen (DO) to control as the control of 50%, temperature is 37 DEG C, speed of agitator control is 60r/min；
(2) the DF-1-XF cell to suspension Multiplying culture 72 hours is inoculated with chicken pox quail according to the dosage of inoculation of 3% (V/V)
Quailization low virulent strain (F282E4) carries out virus multiplication culture；Inoculation harvested virus liquid after 96 hours；Through detecting, viral titer is
106.05EID50/0.2ml；
(3) match seedling and packing
(3.1) preparation of freeze drying protectant
A liquid: gelatin 5g, sucrose 5g, dextrin 5g, tryptone 2g, deionized water are settled to 100ml, and 116 DEG C, high pressure is gone out
Bacterium 30min, 15 DEG C of preservations are no more than 7 days；
B liquid: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g, deionized water are settled to 100ml, 0.22 μm of filter membrane
Filtration sterilization, 4 DEG C of preservations, is no more than 3 days；
(3.2) it dispenses
Protective agent A liquid, B liquid are uniformly mixed by the volume ratio of 1 ︰ 1 and obtain freeze drying protectant, qualified cell will be examined
Virus liquid is added in freeze drying protectant, quantitative separating by 1 ︰, 1 volume ratio；
(3.3) it is lyophilized
Vacuum freezedrying is carried out after packing rapidly, obtains chicken pox live vaccine.
The preparation of 4 chicken pox live vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106At the beginning of cells/ml is cell
Beginning inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
Being made as 7.2, dissolved oxygen (DO) to control as the control of 50%, temperature is 37 DEG C, speed of agitator control is 120r/min；
(2) the DF-1-XF cell to suspension Multiplying culture 24 hours is inoculated with chicken according to the dosage of inoculation inoculation of 1% (V/V)
Acne quailization low virulent strain (F282E4) carries out virus multiplication culture；Inoculation harvested virus liquid after 120 hours；Through detecting, virus poison
Valence is 105.15EID50/0.2ml；
(3) match seedling and packing
(3.1) preparation of freeze drying protectant
A liquid: gelatin 5g, sucrose 5g, dextrin 5g, tryptone 2g, deionized water are settled to 100ml, and 116 DEG C, high pressure is gone out
Bacterium 30min, 15 DEG C of preservations are no more than 7 days；
B liquid: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g, deionized water are settled to 100ml, 0.22 μm of filter membrane
Filtration sterilization, 4 DEG C of preservations, is no more than 3 days；
(3.2) it dispenses
Protective agent A liquid, B liquid are uniformly mixed by the volume ratio of 1 ︰ 1 and obtain freeze drying protectant, qualified cell will be examined
Virus liquid is added in freeze drying protectant, quantitative separating by 1 ︰, 1 volume ratio；
(3.3) it is lyophilized
Vacuum freezedrying is carried out after packing rapidly, obtains chicken pox live vaccine.
The preparation of 5 chicken pox live vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106At the beginning of cells/ml is cell
Beginning inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
Being made as 7.2, dissolved oxygen (DO) to control as the control of 50%, temperature is 37 DEG C, speed of agitator control is 120r/min；
(2) the DF-1-XF cell to suspension Multiplying culture 48 hours is inoculated with chicken pox according to the dosage of inoculation of 10% (V/V)
Quailization low virulent strain (F282E4) carries out virus multiplication culture；Inoculation harvested virus liquid after 48 hours；Through detecting, viral titer
It is 105.89EID50/0.2ml；
(3) match seedling and packing
(3.1) preparation of freeze drying protectant
A liquid: gelatin 5g, sucrose 5g, dextrin 5g, tryptone 2g, deionized water are settled to 100ml, and 116 DEG C, high pressure is gone out
Bacterium 30min, 15 DEG C of preservations are no more than 7 days；
B liquid: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g, deionized water are settled to 100ml, 0.22 μm of filter membrane
Filtration sterilization, 4 DEG C of preservations, is no more than 3 days；
(3.2) it dispenses
Protective agent A liquid, B liquid are uniformly mixed by the volume ratio of 1 ︰ 1 and obtain freeze drying protectant, qualified cell will be examined
Virus liquid is added in freeze drying protectant, quantitative separating by 1 ︰, 1 volume ratio；
(3.3) it is lyophilized
Vacuum freezedrying is carried out after packing rapidly, obtains chicken pox live vaccine.
The preparation technology parameter Optimum Experiment of 1 chicken pox live vaccine of test example
1. test method
The optimization of 1.1 cell suspension cultures conditions
1.1.1 the optimization of cell Initial seeding density
By DF-1-XF cell respectively with 0.3 × 106A/ml, 0.5 × 106A/ml and 0.75 × 106A/ml tri- is initial
Density, which is inoculated in bioreactor, carries out suspension culture, counts cell number every sampling in 24 hours in incubation.Other suspensions
Condition of culture: pH control is 7.2, dissolved oxygen (DO) is 37 DEG C of 50%, temperature, speed of agitator 100r/min.
1.1.2 the optimization of speed of agitator
By DF-1-XF cell respectively with 60,80,100 and 120r/min, tetra- speeds of agitator, it is inoculated in 7L biological respinse
DF-1-XF cell suspension cultures are carried out in device, every the meter cell number of sampling in 24 hours in incubation.Item is cultivated in other suspensions
Part: serum-concentration 1%, cell Initial seeding density 0.75 × 106A/ml, pH 7.2, dissolved oxygen (DO) are 50%, temperature
37℃。
1.2 viral suspension culture parameters optimizations
1.2.1 the optimization of malicious time is connect
By DF-1-XF cell with 0.75 × 106The initial density of a/ml is inoculated in bioreactor, respectively at cell
Bird pox virus is inoculated with by 3v/v% within culture 0,12,24,36 hour, connects 72 hours sampling and measuring viral levels after poison, to determine most
It is suitable to connect the malicious time.Other condition of culture: pH control is 7.2, dissolved oxygen (DO) is that 37 DEG C of 50%, temperature, speed of agitator are set as
100r/min。
1.2.2 the optimization of toxic dose is connect
By DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, connects poison using synchronizing
Mode it is small to connect after poison 72 respectively to connect poison amount inoculation bird pox virus by 1v/v%, 3v/v%, 6v/v% and 10v/v%
When viral level is measured by sampling, connect toxic dose so that determination is most suitable.Other condition of culture: pH control is 7.2, dissolved oxygen (DO) is
50%, 37 DEG C of temperature, speed of agitator are set as 100r/min.
1.2.3 the optimization of malicious time is received
By DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, connects poison using synchronizing
Mode, be inoculated with bird pox virus by 6v/v%, sampled every 24 hours in incubation, measure viral level, it is most suitable with determination
Receive the malicious time.Other condition of culture: pH control is 7.2, dissolved oxygen (DO) is that 37 DEG C of 50%, temperature, speed of agitator are set as
100r/min。
2. test result
2.1 cell Initial seeding density optimum results
By DF-1-XF cell respectively with initial density for 0.3 × 106cells/ml、0.5×106Cells/ml and 0.75
×106Cells/ml, which is inoculated in Tissue Culture Flask, carries out suspension culture, counts every sampling in 24 hours, the results showed that cell
Initial seeding density is 0.75 × 106Cells/ml, 48 hour cell density of culture can 3.0 × 106Cells/ml or more is suitble to
Mass production demand, the results are shown in Table 1.Therefore selection 0.75 × 106Cells/ml is cell Initial seeding density.
The relationship of table 1 different cell-seeding-densities and suspension cell growth speed
The optimum results of 2.2 speeds of agitator
By DF-1-XF cell respectively with 60,80,100 and 120r/min, tetra- speeds of agitator, it is inoculated in 7L biological respinse
It carries out suspension culture in device, counts cell number every sampling in 24 hours in incubation.The result shows that when speed of agitator is 100r/
When min, cell Proliferation is most fast, and 48 hour cell density are 3.2 × 106Cells/ml the results are shown in Table 2.Therefore selection stirring turns
Fast 100r/min.
The relationship of table 2 different speeds of agitator and suspension cell growth speed
2.3 connect the optimum results of malicious time
By DF-1-XF cell with 0.75 × 106The initial density of a/ml is inoculated in bioreactor, respectively at cell
Bird pox virus is inoculated with by 3v/v% within culture 0 hour, 12 hours, 24 hours, 36 hours, connect 72 hours sampling and measuring virus after poison
Content.The result shows that cell culture 0h, i.e., using synchronizing the method for connecing poison inoculation bird pox virus, virus titer up to 10.6 ×
106PFU/ml the results are shown in Table 3.Therefore select most preferably to connect the malicious time as cell culture 36h.
3 difference of table connects influence of the malicious time to viral level
2.4 connect the optimum results of toxic dose
By DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, connects poison using synchronizing
Mode it is small to connect after poison 72 respectively to connect poison amount inoculation bird pox virus by 1v/v%, 3v/v%, 6v/v% and 10v/v%
When viral level is measured by sampling.The result shows that connecing toxic dose is 6v/v%, 72 hours viral titers are reachable after connecing poison
107.40EID50/ 0.2ml, the results are shown in Table 4.Therefore it selects most preferably to connect toxic dose as 6%.
4 difference of table connects influence of the malicious time to viral level
2.5 receive the optimum results of malicious time
By DF-1-XF cell with 0.75 × 106The density of a/ml is inoculated in 7L bioreactor, connects poison using synchronizing
Mode, be inoculated with bird pox virus by 6v/v%, sampled every 24 hours in incubation, measure viral level.The result shows that
72 hours viral titers are up to 10 after connecing poison7.50 EID50/ 0.2ml, the results are shown in Table 5.Therefore it selects most preferably to receive the malicious time to connect poison
72 hours afterwards.
5 difference of table connects influence of the malicious time to viral level
The safety testing of 2 chicken pox live vaccine of test example
With the susceptible chick of 1-2 week old 10, the live vaccine 0.2ml of 10 times of dosages of every intramuscular injection, 7-10 is observed
Day, 10 chickens are all good for and live.Safety testing the results show that the method for the present invention preparation chicken pox live vaccine have it is good
Safety.
The diagnostic test of 3 chicken pox live vaccine of test example is tested
Chicken pox live vaccine prepared by the embodiment of the present invention is diluted to sterile saline containing 100EID50/ 0.1ml, adds
Enter the anti-bird pox virus specific positive serum of equivalent, in room temperature and 1h is followed by kind of a chicken embryo fibroblasts, observes 120h, does not go out
Existing lesion.
The efficacy test of 4 chicken pox live vaccine of test example is tested
Chicken pox live vaccine normal saline dilution prepared by the embodiment of the present invention 1, allantocherion vaccination 11-12 days
Instar chicken embryo 10, every embryo 0.2ml, 96-120h after inoculation, whole chick chorioallantoic membrane oedema thicken or occur pox spots；Test
The results show that chicken pox live vaccine effect prepared by preparation method of the present invention is qualified.
Claims (10)
1. a kind of method for preparing chicken pox live vaccine characterized by comprising (1) chicken embryo fibroblasts are carried out domestication training
It supports and obtains adapting to serum free medium, the passage chicken embryo fibroblasts in single-cell suspension growth conditions；(2) by step (1)
The passage chicken embryo fibroblasts that domestication culture obtains, which are inoculated into bioreactor or Tissue Culture Flask, carries out cell suspension increasing
Grow culture；(3) it is received after carrying out virus multiplication culture to the passage chicken embryo fibroblasts of suspension Multiplying culture inoculation bird pox virus
Obtain virus liquid；(4) virus liquid of harvest is uniformly mixed with freeze drying protectant, is freeze-dried to get chicken pox live vaccine.
2. according to the method for claim 1, which is characterized in that adaptation serum free medium described in step (1) is in slender
The microbial preservation number of the passage chicken embryo fibroblasts of born of the same parents' suspension growth state is: CGMCC No.16295.
3. according to the method for claim 1, which is characterized in that the passage chicken that will be obtained by domestication culture in step (2)
Embryo fibroblast is 0.3 × 10 according to initial density6cells/ml、0.5×106Cells/ml or 0.75 × 106cells/ml
Inoculum density be inoculated into bioreactor or Tissue Culture Flask and carry out suspension culture；It preferably, will be by taming and dociling in step (2)
Changing the passage chicken embryo fibroblasts cultivated and obtained according to initial density is 0.75 × 106The inoculum density of cells/ml is inoculated into
Suspension culture is carried out in bioreactor or Tissue Culture Flask.
4. according to the method for claim 1, which is characterized in that the adaptation nothing that will be obtained by domestication culture in step (2)
Blood serum medium is inoculated into bioreactor or cell culture in the passage chicken embryo fibroblasts of single-cell suspension growth conditions
Cell suspension Multiplying culture is carried out under agitation in bottle；Wherein, used speed of agitator is 50-500r/min；It is preferred that
For 60-120r/min；Most preferably 100r/min.
5. according to the method for claim 1, which is characterized in that the item of cell suspension Multiplying culture described in step (2)
Part further include: pH value 7.2, oxygen dissolving value 50%, cultivation temperature are 37 DEG C.
6. according to the method for claim 1, which is characterized in that thin to suspension Multiplying culture 24-60 hours in step (3)
Born of the same parents are inoculated with bird pox virus and carry out virus multiplication culture；Preferably, to 36 hours cell inoculations of suspension Multiplying culture in step (3)
Bird pox virus carries out virus multiplication culture.
7. according to the method for claim 1, which is characterized in that in step (3), according to volume basis, according to 1-10%'s
Poison is connect to measure to cell inoculation bird pox virus；Preferably, it measures according to 6% poison that connects to cell inoculation bird pox virus.
8. according to the method for claim 1, which is characterized in that virus multiplication condition of culture described in step (3) also wraps
It includes: pH value 7.2, dissolved oxygen 50%, 37 DEG C of temperature, speed of agitator 100r/min.
9. according to the method for claim 1, which is characterized in that be inoculated with bird pox virus Multiplying culture 24-120 in step (3)
Hour harvest virus liquid；Preferably, step (3) is inoculated with 72 hours harvest virus liquids of bird pox virus Multiplying culture.
10. according to the method for claim 1, which is characterized in that freeze drying protectant described in step (4) is by A liquid and B liquid group
At；Wherein,
The composition of the A liquid is: gelatin 5g, sucrose 5g, dextrin 5g, tryptone 2g；Deionized water is settled to 100ml；
The composition of the B liquid is: vitamin C 0.1g, sorbierite 2g, sodium glutamate 1.5g；Deionized water is settled to 100ml.
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| CN115627259A (en) * | 2022-11-30 | 2023-01-20 | 北京赛尔富森生物科技有限公司 | Adaptation method of virus in chick embryo fibroblast |
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