CN109602900A - The preparation method and products thereof of chook MDV live vaccine - Google Patents
The preparation method and products thereof of chook MDV live vaccine Download PDFInfo
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Abstract
The invention discloses preparation methods of chook MDV 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 Marek's disease viral lived 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) virus liquid is harvested after carrying out virus multiplication culture to the cell inoculation chicken Marek's disease virus after suspension Multiplying culture, virus liquid is centrifuged by (3)；By sedimentation cell cracking, filtering, be freeze-dried to get.Live vaccine safety prepared by preparation method of the present invention is good, has good immune protection effectiveness；Pollution risk and production cost are effectively reduced, the production time of vaccine is shortened, is convenient for expanding the scale of production.
Description
Technical field
The present invention relates to preparation methods of chicken Marek's disease viral lived vaccine and products thereof, more particularly to use serum-free
The chicken Marek's disease viral lived vaccine that suspension culture techniques prepare the method for chicken Marek's disease viral lived vaccine and obtain
Product belongs to the preparation field of chicken Marek's disease viral lived vaccine live vaccine.
Background technique
Marek's disease (MD) is to cause chicken to be drenched by cell-associated (CA) marek's disease virus (MDV) of herpetoviridae
A kind of highly contagious disease of bar hyperblastosis tumour.With the raising of the intensive degree of poultry husbandry, which is brought
Harm also increasingly increasing, prevent and treat it is improper chicken group dead, production performance decline or product quality will be caused to reduce, while can also
Weaken the preventive effect of other vaccines because of its serious immunosuppressive action.Therefore the prevention of Marek's disease becomes poultry husbandry
The most important thing.MD is the currently the only chicken tumor disease that can be prevented with vaccine.MD is prevented and is controlled, the use of vaccine
It is current and unique prevention or controlling soil moist in the future.
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 that the global commerceization through U.S. Food and Drug Administration (FDA) authorization is thin
Born of the same parents system, has been widely used in the proliferation of avian viral, the expression of recombinant protein, the research of tumour virus and animal and people at present
The production of class vaccine.
The mode that traditional DF-1 culture mostly uses serum adherent greatly, the training method complex process take up a large area, need
It a large amount of manual operations and needs to add serum during the cultivation process, increase by the possibility of the pollutions such as exogenous virus, mycoplasma
Property, and the serum difference between different batches also results in product quality and is difficult to control, while also increasing downstream separation purifying
Difficulty.Compared with serum adhere-wall culture technology, serum free suspension culture technique eliminates digestion and receives without expensive microcarrier
The operation for obtaining cell, reduces pollution risk and production cost, shortens the production time.Not due to serum free suspension culture technique
It needs to attach matrix, the device space can be saved, utilization rate of equipment and installations is improved, convenient for expanding the scale of production.Therefore, serum free suspension is trained
Feeding technology is the inexorable trend 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 state
Cell and be applied to the preparation of chicken Marek's disease viral lived vaccine, for reducing chook MDV live vaccine
Production cost and pollution risk, carry out large-scale production etc. and 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
Passage chicken embryo fibroblasts cell (DF-1 cell) prepares chicken Marek's disease viral lived vaccine, and the preparation method is using suspension
Culture technique reduces the production cost and pollution risk of chicken Marek's disease viral lived vaccine, and it is raw to be able to carry out large-scale industrial
It produces.
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: this hair first
When bright DF-1 cell length to the mid log phase that will contain stable growth in the MEM culture solution of 10% newborn bovine serum, it is changed to
MEM culture solution containing 5% newborn bovine serum, when the convergence degree of cell length to 80%~90%, trypsin digestion and cell, with
Cell density is 2.0 × 105Cells/ml passage is in the MEM culture solution containing 5% newborn bovine serum；After number generation, DF-1
Survival rate of the cell in the MEM culture solution containing 5% newborn bovine serum maintains 90% or more, and keeps very fast growth rate,
Further decrease serum domestication culture；Make DF-1 cell gradually adapt to the MEM containing 1% newborn bovine serum in the same way to train
The condition of supporting.As serum uses the reduction of concentration, the form of DF-1 cell adherent growth gradually changes, finally through serum-free
Cell shows single-cell suspension growth conditions after domestication adapts to.DF-1 cell drops to 5% from serum-concentration 10%, and cell does not go out
Existing 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 is rounded
Trend, the only respective cells state that shows slightly suspension growth.Driven suspension culture adapts to, and DF-1 cell shows cell
The form of suspension growth, but cell clustering phenomena is serious, and respective cells group is larger.Domestication later period, suspension DF-1 are cultivated suspending
Preferable single-cell suspension growth conditions are presented in cell, and cell is rounded, and clustering phenomena is less, and cell space size is almost the same,
The speed of growth is normal, illustrates that DF-1 cell has been already adapted to serum free medium and in single-cell suspension growth conditions；The present invention
The adaptation free serum culture that low serum domestication process and serum free medium suspension domestication process through the adhere-wall culture stage obtain
Base and DF-1-XF cell is named as in the DF-1 cells of single-cell suspension growth conditions.
The present invention will domestication culture obtain adapt to serum free medium and in single-cell suspension growth conditions DF-1-XF it is thin
The mechanism that born of the same parents are submitted to patent approval carries out preservation, and deposit number is: CGMCCNo.16295, classification naming are: adapting to complete outstanding
The passage chicken embryo fibroblasts for length of growing floating on water；Preservation date is: on September 6th, 2018；Depositary institution is: Chinese microorganism strain
Preservation administrative center common micro-organisms center；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are micro-
Biological study institute.
The present invention further adapts to serum free medium and in the DF-1- of single-cell suspension growth conditions using described
XF cell prepares chicken Marek's disease viral lived 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 is harvested after carrying out virus multiplication culture to the DF-1-XF cell inoculation chook MDV after suspension Multiplying culture
The virus liquid of harvest is centrifuged, discards supernatant by liquid, (3), is cracked after stabilizer is added to sedimentation cell, filters, and freezing is dry
It is dry to get chook MDV live vaccine.
The present invention is 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 thin
Born of the same parents' Initial seeding density is 0.75 × 106Cells/ml cultivates 48 hour cell density up to 3.0 × 106Cells/ml or more,
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 initial inoculation is close
Degree is inoculated into progress cell suspension Multiplying culture in bioreactor or Tissue Culture Flask；In addition, cell described in step (1)
Suspension Multiplying culture condition further include: the conditions such as pH value, dissolved oxygen, temperature and speed of agitator, these parameters can be using conventional
The parameters of cell suspension cultures cultivated, as a reference, the pH, which may be controlled to 7.2, oxygen dissolving value, can control (DO)
For 50%, cultivation 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)
Under conditions of 37 DEG C of 50%, temperature, by DF-1-XF cell respectively with 60r/min, 80r/min, 100r/min and 120r/min
Four speeds of agitator are inoculated in progress DF-1 cell suspension cultures in 7L bioreactor, took in incubation every 24 hours
Sample meter cell number.As a result, it has been found that cell Proliferation is most fast, and 48 hour cell density are up to 3.2 when speed of agitator is 100r/min
×106cells/ml；When therefore carrying out cell suspension Multiplying culture in step (1), used speed of agitator is preferably
100r/min。
By DF-1-XF cell with 0.75 × 106The initial density of a/ml is inoculated in bioreactor, respectively at cell
Chook MDV is inoculated with by 2v/v% within culture 36 hours, 48 hours and 60 hours, sampled in incubation every 24 hours,
Viral level is measured, 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 connecing to the DF-1-XF cell inoculation chook MDV after suspension Multiplying culture
The malicious time is affected for viral Multiplying culture effect:
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 chicken Marek's
Virus connects 48 hours sampling and measuring viral levels after poison.The result shows that cell culture 36h is followed by kind of a chook MDV, disease
Malicious titre is up to 10.6 × 106PFU/ml.Therefore, preferably DF-1-XF cell suspension Multiplying culture 36 hours are connect in step (2)
Kind chook MDV.
The present invention is it has furthermore been found that connecing to the DF-1-XF cell inoculation chook MDV after suspension Multiplying culture
Toxic dose and time of virus liquid is harvested for the influence also highly significant of the Multiplying culture effect of chook MDV:
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, respectively with every milliliter 2 × 103PFU、5×103PFU、10×103PFU、15×103PFU's connects poison amount inoculation
Chook MDV connects 48 hours sampling and measuring viral levels after poison.The result shows that connect toxic dose be every milliliter 5 ×
103PFU, 48 hours viral titers are up to 12.7 × 10 after connecing poison6PFU/ml.Therefore, after the preferably floating Multiplying culture of step (2)
The toxic dose that most preferably connects of DF-1-XF cell inoculation chook MDV is every milliliter 5 × 103PFU。
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, with every milliliter 5 × 103PFU's connects poison amount inoculation chook MDV, and sampling in each 12 hours is surveyed after connecing poison
Determine viral level.The result shows that connecing after poison 48 hours viral titers up to 12.8 × 106PFU/ml；Therefore, institute in step (2)
The receipts poison Best Times stated are to connect after poison 48 hours.
Centrifugation described in step (3) is preferably centrifuged 10min under conditions of 2000r/min；The cleavage method is excellent
The method that choosing is cracked using ultrasonic treatment device.
The preparation method of chicken Marek's disease viral lived vaccine of the present invention uses serum free suspension culture technique, pastes with serum
Wall culture technique is compared, and preparation method of the present invention eliminates the operation of digestion harvest cell without expensive microcarrier, significant to drop
Low pollution risk and production cost, effectively shorten the production time；It does not need to attach matrix, the device space can be saved, improve
Utilization rate of equipment and installations, convenient for expanding the scale of production.The safety of chicken Marek's disease viral lived vaccine prepared by preparation method of the present invention
Property is good；Immune protection effectiveness examine the experimental results showed that, prepared chicken Marek's disease viral lived vaccine for chicken have
There is good immune protection effectiveness.
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
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description 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.Those skilled in the art
Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or
Replacement, 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 born bovine will be contained
When stablizing DF-1 cell length to the mid log phase of growth in the MEM culture solution of serum, it is changed to containing 5% newborn bovine serum
MEM culture solution, when cell it is long to 80%~90% convergence degree when, trypsin digestion and cell, with cell density be 2.0 ×
105Cells/ml passage is in the MEM culture solution containing 5% newborn bovine serum.After number generation, DF-1 cell is newborn containing 5%
Survival rate in the MEM culture solution of cow's serum maintains 90% or more, and keeps very fast growth rate, further decreases serum
Domestication culture.DF-1 cell is set gradually to adapt to the MEM condition of culture containing 1% newborn bovine serum in the same way.It will adapt to
The DF-1 cell of 1% newborn bovine serum condition of culture carries out the culture domestication adaptation that suspends in cell triangular flask.Cell culture fluid
DF-1 serum free medium is trained for Shanghai source, cell Initial seeding density is 1.0 × 106Cells/ml, revolving speed are 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%, and cell is not
There is macroscopic morphological differences, does not show to be not suitable with.When serum-concentration is reduced to 1%, vitro growth rates subtract
Slowly, after passage, the nutritional condition that cell adapted serum-concentration is 1%, the speed of growth is restored, but cellular morphology becomes
Round trend, cell out of the ordinary show the state of suspension growth slightly.Driven suspension culture adapts to, and DF-1 cell shows cell
The form of suspension growth, but cell clustering phenomena is serious, and respective cells group is larger.Domestication later period, suspension DF-1 are cultivated suspending
Preferable single-cell suspension growth conditions are presented in cell, and cell is rounded, and clustering phenomena is less, and cell space size is almost the same,
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 Marek's disease viral lived vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106Cells/ml is that cell is initial
Inoculum density, which is inoculated into 7L bioreactor, carries out cell suspension cultures, and incubation time is 36 hours；Item is cultivated in other suspensions
Part 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) to suspension Multiplying culture 36 hours DF-1-XF cells according to every milliliter 5 × 103The dosage of inoculation of PFU is inoculated with
The CVI988 plants of progress virus multiplication cultures of chicken Marek's disease virus；Inoculation harvested virus liquid after 48 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 120r/
min；Through detecting, viral titer is 12.8 × 106PFU/ml。
(3) the virus liquid 2000r/min of harvest being centrifuged 10min, discarded supernatant, appropriate stabilizer is added in sedimentation cell,
It is cracked with ultrasonic treatment device, through filtered through gauze, packing, every plumage part answers >=2000PFU；It carries out rapidly freezing after packing true
Sky is dry, obtains chicken Marek's disease viral lived vaccine.
The preparation of 3 chicken Marek's disease viral lived vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.3 × 106Cells/ml is that cell is initial
Inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
For 7.2, dissolved oxygen (DO) control be 50%, temperature control be 37 DEG C, speed of agitator control be 60r/min；
(2) to suspension Multiplying culture 72 hours DF-1-XF cells according to every milliliter 2 × 103The dosage of inoculation of PFU is inoculated with
The CVI988 plants of progress virus multiplication cultures of chicken Marek's disease virus；Inoculation harvested virus liquid after 72 hours；Through detecting, virus poison
Valence is 9.8 × 106PFU/ml；
(3) the culture solution 2000r/min of harvest being centrifuged 10min, discarded supernatant, appropriate stabilizer is added in sedimentation cell,
It is cracked with ultrasonic treatment device, through filtered through gauze, packing, every plumage part answers >=2000PFU；It carries out rapidly freezing after packing true
Sky is dry, obtains chicken Marek's disease viral lived vaccine.
The preparation of 4 chicken Marek's disease viral lived vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106Cells/ml is that cell is initial
Inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
For 7.2, dissolved oxygen (DO) control be 50%, temperature control be 37 DEG C, speed of agitator control be 120r/min；
(2) to suspension Multiplying culture 24 hours DF-1-XF cells according to every milliliter 10 × 103The dosage of inoculation of PFU connects
Kind inoculation CVI988 plants of progress virus multiplication cultures of chicken Marek's disease virus；Inoculation harvested virus liquid after 24 hours；Through detecting,
Viral titer is 8.7 × 106PFU/ml；
(3) the culture solution 2000r/min of harvest being centrifuged 10min, discarded supernatant, appropriate stabilizer is added in sedimentation cell,
It is cracked with ultrasonic treatment device, through filtered through gauze, packing, every plumage part answers >=2000PFU；It carries out rapidly freezing after packing true
Sky is dry, obtains chicken Marek's disease viral lived vaccine.
The preparation of 5 chicken Marek's disease viral lived vaccine of embodiment
(1) embodiment 1 is tamed into the DF-1-XF cell of culture acquisition according to 0.5 × 106Cells/ml is that cell is initial
Inoculum density is inoculated into progress cell suspension Multiplying culture in 7L bioreactor；Other condition of suspension culture are as follows: pH control
For 7.2, dissolved oxygen (DO) control be 50%, temperature control be 37 DEG C, speed of agitator control be 120r/min；
(2) to suspension Multiplying culture 48 hours DF-1-XF cells according to every milliliter 15 × 103The dosage of inoculation of PFU connects
Kind inoculation CVI988 plants of progress virus multiplication cultures of chicken Marek's disease virus；Inoculation harvested virus liquid after 60 hours；Through detecting,
Viral titer is 9.3 × 106PFU/ml；
(3) the culture solution 2000r/min of harvest being centrifuged 10min, discarded supernatant, appropriate stabilizer is added in sedimentation cell,
It is cracked with ultrasonic treatment device, through filtered through gauze, packing, every plumage part answers >=2000PFU；It carries out rapidly freezing after packing true
Sky is dry, obtains chicken Marek's disease viral lived vaccine.
The preparation technology parameter Optimum Experiment of 1 chicken Marek's disease viral lived 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 tetra- speeds of agitator of 60r/min, 80r/min, 100r/min and 120r/min, connect
It plants and carries out DF-1 cell suspension cultures in 7L bioreactor, count cell number every sampling in 24 hours in incubation.It is other
Condition of suspension culture: serum-concentration 1%, cell Initial seeding density 0.75 × 106A/ml, pH 7.2, dissolved oxygen (DO) are
50%, 37 DEG C of temperature.
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
Press every milliliter 2 × 10 within culture 24,36,48 and 60 hours3PFU's connects poison amount inoculation chook MDV, connects after poison 48 hours
Viral level is measured by sampling, connects the malicious time 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.
1.2.2 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, and cell culture 36 is small
Shi Hou, respectively with every milliliter of inoculation 2 × 103PFU、5×103PFU、8×103PFU、10×103PFU's connects poison amount inoculation chicken horse
Li Keshi virus, connects 48 hours sampling and measuring viral levels after poison, connects toxic dose so that determination is most suitable.Other condition of culture: pH control
It is made as 7.2, dissolved oxygen (DO) and is set as 100r/min for 50%, 37 DEG C of temperature, speed of agitator.
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, and cell culture 36 is small
Shi Hou, with every milliliter of inoculation 5 × 103PFU's connects poison amount inoculation chook MDV, takes in incubation every 24 hours
Sample measures viral level, with the determination most suitable receipts malicious time.Other condition of culture: pH control is 7.2, dissolved oxygen (DO) is 50%, temperature
37 DEG C of degree, 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 is initial
Inoculum density is 0.75 × 106Cells/ml, 48 hour cell density of culture can 3.0 × 106Cells/ml or more is suitble to big raw
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 tetra- speeds of agitator of 60r/min, 80r/min, 100r/min and 120r/min, connect
It plants and carries out suspension culture in 7L bioreactor, count cell number every sampling in 24 hours in incubation.The result shows that when stirring
Mix revolving speed be 100r/min when, cell Proliferation is most fast, 48 hour cell density be 3.2 × 106Cells/ml the results are shown in Table 2.Cause
This selection speed of agitator 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
Press every milliliter 2 × 10 within culture 24 hours, 36 hours, 48 hours and 60 hours3PFU's connects poison amount inoculation chook MDV,
Connect 48 hours sampling and measuring viral levels after poison.The result shows that cell culture 36h is followed by kind of a chook MDV, virus drop
Degree reaches as high as 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, and cell culture 36 is small
Shi Hou, respectively with every milliliter 2 × 103PFU、5×103PFU、10×103PFU、15×103PFU's meets poison amount inoculation chicken Ma Li
Kirschner virus, connects 48 hours sampling and measuring viral levels after poison.The result shows that connecing toxic dose is every milliliter 5 × 103PFU connects poison
48 hours viral titers reach as high as 12.7 × 10 afterwards6PFU/ml the results are shown in Table 4.Therefore it selects most preferably to connect toxic dose as every milli
Rise 5 × 103PFU。
4 difference of table connects influence of the toxic dose 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, and cell culture 36 is small
Shi Hou, with every milliliter 5 × 103PFU's connects poison amount inoculation chook MDV, connects each 12 hours sampling and measuring virus after poison
Content.The result shows that connecing 48 hours viral titers after poison reaches as high as 12.8 × 106PFU/ml the results are shown in Table 5.Therefore it selects
It is best to receive the malicious time to connect after poison 48 hours.
The different influences for receiving the malicious time to viral level of table 5
The safety testing of 2 chicken Marek's disease viral lived vaccine of test example
With 1-3 age in days SPF chick 20, it is divided into 2 groups.Immune group 10, each muscle or subcutaneous injection vaccine (embodiment 1
Prepared live vaccine) 0.2ml (contain 10 dosages), control group 10, not virus inoculation, after 14 days immune group with compare
Group is strong to live, and no specificity is dead.Experiment results proved, the tool of chicken Marek's disease viral lived vaccine prepared by the method for the present invention
There is good safety.
Claims (10)
1. a kind of method for preparing chook MDV live vaccine characterized by comprising (1) by chicken embryo fibroblasts
Obtained adaptation serum free medium is cultivated, in the passage chicken embryo fibroblasts of single-cell suspension growth conditions by domestication；
(2) cell inoculation that step (1) domestication culture obtains is subjected to cell suspension proliferation into bioreactor or Tissue Culture Flask
Culture；(3) virus multiplication culture is carried out to the cell inoculation chicken Marek's disease virus after suspension Multiplying culture；Harvest virus
The virus liquid of harvest is centrifuged by liquid, (4)；Sedimentation cell cracking, filtering are taken, freeze-drying is living to get chicken Marek's disease virus
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 in step (2) by cell according to initial density be 0.3 ×
106cells/ml、0.5×106Cells/ml or 0.75 × 106The inoculum density of cells/ml be inoculated in Tissue Culture Flask into
Row, which suspends, to be cultivated；Preferably, in step (1) by cell according to initial density be 0.75 × 106The inoculum density of cells/ml connects
Kind carries out suspension culture in Tissue Culture Flask.
4. 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 inoculated into bioreactor or Tissue Culture Flask carries out cell suspension Multiplying culture under stirring conditions；
Wherein, used speed of agitator is 50-500r/min；Preferably 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 after suspension Multiplying culture 24-60 hours in step (3)
Cell inoculation chook MDV carries out virus multiplication culture；Preferably, to suspension Multiplying culture 36 hours in step (2)
Cell inoculation chook MDV carries out virus multiplication culture.
7. according to the method for claim 1, which is characterized in that in step (3), with every milliliter of inoculation 2 × 103PFU、5×
103PFU、8×103PFU or 10 × 103The poison that connects of PFU is measured to cell inoculation chook MDV；Preferably, it is connect with every milliliter
Kind 5 × 103The poison that connects of PFU is measured to cell inoculation chook MDV.
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 24-72 hours harvest diseases of virus multiplication culture in step (3)
Venom；Preferably, 48 hours harvest virus liquids of virus multiplication culture in step (2).
10. according to the method for claim 1, which is characterized in that centrifugation described in step (4) is 2000r/ in revolving speed
10min is centrifuged under conditions of min；The cracking is cracked using ultrasound.
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| CN112210530A (en) * | 2020-10-15 | 2021-01-12 | 山东信得动物疫苗有限公司 | Cell full-suspension domestication method, DF-1 cell full-suspension domestication method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101194012A (en) * | 2005-04-11 | 2008-06-04 | 维涡里斯公司 | Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines |
| CN107058243A (en) * | 2017-03-23 | 2017-08-18 | 华南农业大学 | A kind of suspension culture method of MDV |
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2018
- 2018-12-04 CN CN201811469417.7A patent/CN109602900A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101194012A (en) * | 2005-04-11 | 2008-06-04 | 维涡里斯公司 | Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines |
| CN107058243A (en) * | 2017-03-23 | 2017-08-18 | 华南农业大学 | A kind of suspension culture method of MDV |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112210530A (en) * | 2020-10-15 | 2021-01-12 | 山东信得动物疫苗有限公司 | Cell full-suspension domestication method, DF-1 cell full-suspension domestication method and application |
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