CN106282098B - A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus - Google Patents
A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus Download PDFInfo
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Abstract
The invention discloses a kind of methods using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus, belong to veterinary biologics technical field, mainly comprise the steps that the passage and culture of S1. preparation cell；S2. cell adapted low blood serum medium is tamed；S3. cell adapted low serum free culture system environment is tamed；S4. the breeding of cell kind poison；S5. the processing of virus liquid is harvested；S6. the TCID50 measurement of virus liquid is harvested.Method provided by the invention mainly utilizes fibronectin (FN) promoting growth of cell, improves adherence rate, after the metaboilic level of enhancing cell makes the culture environment of cell adapted low serum, gradually remove fibronectin (FN) again, it can get the DF1 cell for adapting to low serum free culture system by the method, production cost can be significantly reduced, it is capable of expanding the scale of production for fast and stable, quality is easily achieved equalization stable.
Description
Technical field
The present invention relates to veterinary biologics technical fields, and in particular to a kind of to utilize lower serum content nutrients culture
Method of the DF1 cell to prepare infections chicken cloacal bursa virus.
Background technique
DF1 cell is chick embryo continuous cell line and bioreactor, and in vitro culture is in fibrous attached cell, and the cell is to avian infectious Fa Shi
Capsule virus is more sensitive, can be used for the preparation of infections chicken cloacal bursa virus vaccine.Currently, culture DF1 cell is used to prepare
Mainly using rolling bottle technique, culture medium generally uses DMEM, DMEM/F12 to add 10% serum and is cultivated vaccine.
Currently, the production of infections chicken cloacal bursa virus cell inactivation vaccine mainly uses spinner culture technology, either
Advanced bioreactor suspension culture techniques are all made of high serum content cell culture technology, animal derived components bring
The problems such as vaccine safety, cost, quality, is increasingly significant, and be mainly manifested in: 1) cow's serum dosage is big, and cell is grown to serum
With certain dependence, increase production cost, component has differences between serum batch, directly affects the stability of product quality；
2) serum origin is in animal, it is possible to carry exogenous virus, the cell and vaccine product to growth are likely to bring danger.Cause
The usage amount that serum is reduced in this cell cultivation process is the key that save production cost and raising vaccine quality stability.
Summary of the invention
The present invention is directed to be directed to the technological deficiency of the prior art, provide a kind of utilization lower serum content nutrients culture
Method of the DF1 cell to prepare infections chicken cloacal bursa virus, to solve in the prior art when thin as host using DF1 cell
When born of the same parents prepare infections chicken cloacal bursa virus, the larger technical problem of serum amount needed for the culture to DF1 cell.
To realize the above technical purpose, the invention adopts the following technical scheme:
A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus,
The following steps are included:
1) DF1 cell is taken, the DMEM/F12 culture containing 8% (v/v) newborn bovine serum, 10mg/L fibronectin is inoculated in
Base replaces several subcultures in incubation, and newborn bovine serum content is constant in the culture medium replaced every time and fibronectin
Content is more previous low, tames and dociles until being changed to the DMEM/F12 culture medium containing 8% (v/v) newborn bovine serum to get to the first generation
Change cell；
2) it takes first generation domestication cell, is inoculated in containing 6% (v/v) newborn bovine serum, 20mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 6% (v/v) newborn bovine serum, i.e.,
Obtain second generation domestication cell；
3) it takes second generation domestication cell, is inoculated in containing 4% (v/v) newborn bovine serum, 30mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 4% (v/v) newborn bovine serum, i.e.,
Obtain third generation domestication cell；
4) it takes third generation domestication cell, is inoculated in containing 2% (v/v) newborn bovine serum, 40mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 2% (v/v) newborn bovine serum, i.e.,
Obtain forth generation domestication cell；
5) it takes forth generation described in step 4 to tame cell, is inoculated with infections chicken cloacal bursa virus, to contain 0~2% (v/v)
The DMEM/F12 culture medium culture of newborn bovine serum harvests culture.
Preferably, pre-treatment obtains DF1 cell used in step 1) in the following manner: taking in cell bottle
On cover with the DF1 cell of single layer, digested through EDTA- pancreatin, 10mL cell growth medium be added, cell dispersion is blown and beaten, by DF1 cell
It is placed in 37 DEG C of carbon dioxide incubators of temperature and carries out culture 72h, when good cell monolayer to be formed, expand culture.
Preferably, the cell growth medium is the DMEM/F12 culture solution containing 10% (v/v) newborn bovine serum.
Preferably, the DMEM/F12 described in step 1) containing 8% (v/v) newborn bovine serum, 10mg/L fibronectin
Culture medium is by that will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum and containing 50mg/L fibronectin
DMEM/F12 culture medium is mixed to get with the ratio of 4:1 (v/v).
Preferably, the DMEM/F12 described in step 2) containing 6% (v/v) newborn bovine serum, 20mg/L fibronectin
Culture medium is by that will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum and containing 50mg/L fibronectin
DMEM/F12 culture medium is mixed to get with the ratio of 3:2 (v/v).
Preferably, the DMEM/F12 described in step 3) containing 4% (v/v) newborn bovine serum, 30mg/L fibronectin
Culture medium is by that will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum and containing 50mg/L fibronectin
DMEM/F12 culture medium is mixed to get with the ratio of 2:3 (v/v).
Preferably, the DMEM/F12 described in step 4) containing 2% (v/v) newborn bovine serum, 40mg/L fibronectin
Culture medium is by that will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum and containing 50mg/L fibronectin
DMEM/F12 culture medium is mixed to get with the ratio of 1:4 (v/v).
Preferably, step 5) specifically includes following operation: the DMEM/F12 to contain 0~2% (v/v) newborn bovine serum
The resulting forth generation of culture medium step 4) tames cell, until being passed when DF1 cell covers with single layer with the ratio inoculation chicken of 1% (v/v)
Metachromia bursal disease virus is virulent, and control temperature continues to cultivate at 37 DEG C, and when 80% or more lesion occurs in DF1 cell, harvest is thin
Born of the same parents' culture.
Preferably, the EDTA- pancreatin is the PBS solution containing 0.05~0.25% (w/v) pancreatin.
Preferably, also containing in step 5) the DMEM/F12 culture medium containing 0~2% (v/v) newborn bovine serum
The antibiotic of 100~200IU/mL, the DMEM/F12 medium pH for containing 0~2% (v/v) newborn bovine serum be 7.2~
7.4。
It can also include the processing of step 6) harvest virus liquid: the virus liquid of harvest on the basis of above technical scheme
Multigelation 3 times, the cell venom containing supernatant is obtained, fractionated viral liquid is taken to carry out steriling test and titration, remaining disease
Venom is set -70 DEG C of refrigerators and is saved.
It further, can also include receiving to step 6) product TCID50Measurement: will be received with the cell culture fluid of serum-free
The IBDV venom obtained does 10 times and is serially diluted, and takes 10-5、10-6、10-7、10-84 dilutions are inoculated with DF1 cell, each dilution
8 holes are inoculated with, every hole 0.1mL adsorbs 1h, and 0.1mL maintaining liquid, 37 DEG C of culture 72h are added in every hole.DF1 cytopathy is observed, is calculated
TCID50。
The beneficial effects are mainly reflected as follows following several respects:
1, the present invention can make the culture environment of the low serum of the cell adapted DMEM/F12 of DF1 by adding fibronectin (FN),
After cytotostatic increment it can remove fibronectin step by step, acclimation and screening arrives the high-quality performance that has suitable for low serum free culture system
DF1 cell.
2, the DF1 cell of serum free culture system is reduced in cell Proliferation density and is bred in the ability of infections chicken cloacal bursa virus
It is unaffected, and the serum content in maintaining liquid can be reduced to 0%.
3, due to taming the obtained cell adapted low serum free culture system of DF1 and serum-free virus multiplication, production can be significantly reduced
Cost, and it is capable of expanding the scale of production for fast and stable, quality is easily achieved equalization stable.
Detailed description of the invention
Fig. 1 be control group in the embodiment of the present invention 1 (i.e. serum content be 10% DMEM/F12 culture medium culture DF1
Cell) growing state microscopy figure.
Fig. 2 is that the DF1 cell for the DMEM/F12 culture medium culture for being 8% using serum content in the embodiment of the present invention 1 is raw
Long situation microscopy figure.
Fig. 3 is that the DF1 cell for the DMEM/F12 culture medium culture for being 6% using serum content in the embodiment of the present invention 1 is raw
Long situation microscopy figure.
Fig. 4 is that the DF1 cell for the DMEM/F12 culture medium culture for being 4% using serum content in the embodiment of the present invention 1 is raw
Long situation microscopy figure.
Fig. 5 is that the DF1 cell for the DMEM/F12 culture medium culture for being 2% using serum content in the embodiment of the present invention 1 is raw
Long situation microscopy figure.
Fig. 6 is the DF1 in 1 cell kind poison reproductive process of the embodiment of the present invention, when 80% or more lesion occurs in DF1 cell
Cell growth status microscopy figure.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details, In
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1
A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus,
The following steps are included:
S1. the passage and culture with cell are prepared:
The chick embryo continuous cell line and bioreactor DF1 cell for covering with single layer in T25 cell bottle is taken, through 0.05% EDTA- pancreatin
10mL cell growth medium is added in vitellophag, blows and beats cell dispersion, DF1 cell is placed in 37 DEG C of carbon dioxide incubators of temperature
In carry out culture 72h, when good cell monolayer to be formed, expand culture, above-mentioned cell growth medium is 10% newborn
The DMEM/F12 culture solution of cow's serum content.
S2. cell adapted low blood serum medium is tamed: the following steps are included:
The domestication of S2.1 first generation cell:
The step S1 DF1 cell inoculation for expanding culture is tamed into the low blood serum medium of the first generation, described the
The low blood serum medium of a generation is the DMEM/F12 containing 10% newborn bovine serum and contains the DMEM/ of 50mg/L fibronectin (FN)
The mixed-culture medium that F12 ratio is 4: 1, wherein the content of newborn bovine serum is 8%, and expands culture for fibronectin (FN)
Content carries out 50mg/L, 40mg/L, 30mg/L, 20mg/L, 10mg/L, 0mg/L and successively decreases step by step, keeps DF1 cell adapted containing 8%
The DMEM/F12 culture medium of newborn bovine serum.
The domestication of S2.2 second generation cell:
The first generation DF1 cell inoculation of domestication is tamed into the low blood serum medium of the second generation, the second generation
Low blood serum medium is the DMEM/F12 containing 10% newborn bovine serum and contains the DMEM/F12 of 50mg/L fibronectin (FN)
The mixed-culture medium that ratio is 3: 2, wherein the content of newborn bovine serum is 6%, and expands culture containing fibronectin (FN)
Amount carries out 50mg/L, 40mg/L, 30mg/L, 20mg/L, 10mg/L, 0mg/L and successively decreases step by step, keeps DF1 cell adapted new containing 6%
The DMEM/F12 culture medium of raw cow's serum.
The domestication of S2.3 third generation cell:
The second generation DF1 cell inoculation of domestication is tamed into the low blood serum medium of the third generation, the third generation
Low blood serum medium is the DMEM/F12 containing 10% newborn bovine serum and contains the DMEM/F12 of 50mg/L fibronectin (FN)
The mixed-culture medium that ratio is 2: 3, wherein the content of newborn bovine serum is 4%, and expands culture containing fibronectin (FN)
Amount carries out 50mg/L, 40mg/L, 30mg/L, 20mg/L, 10mg/L, 0mg/L and successively decreases step by step, keeps DF1 cell adapted new containing 4%
The DMEM/F12 culture medium of raw cow's serum.
The domestication of S2.4 forth generation cell:
The third generation DF1 cell inoculation of domestication is tamed into the low blood serum medium of forth generation, the forth generation
Low blood serum medium is the DMEM/F12 containing 10% newborn bovine serum and contains the DMEM/F12 of 50mg/L fibronectin (FN)
The mixed-culture medium that ratio is 1: 4, wherein the content of newborn bovine serum is 2%, and expands culture containing fibronectin (FN)
Amount carries out 50mg/L, 40mg/L, 30mg/L, 20mg/L, 10mg/L, 0mg/L and successively decreases step by step, keeps DF1 cell adapted new containing 2%
The DMEM/F12 culture medium of raw cow's serum.
S3. cell adapted low serum free culture system environment is tamed:
The DF1 cell expansion culture for the reduction serum free culture system that success is tamed simultaneously freeze-stored cell, and preparation production seed
Cell.
S4. the breeding of cell kind poison
The DF1 cell of serum free culture system is reduced when covering with single layer, in the ratio of cell suspension 1% be inoculated with the separation of this laboratory,
The virulent Shandong strain of the infections chicken cloacal bursa virus of identification (SD) controls temperature at 37 DEG C, and maintaining liquid is new containing 0% when connecing poison
The DMEM/F12 of raw cow's serum harvests cell culture when 80% or more lesion occurs in DF1 cell.
S5. the processing of virus liquid is harvested:
The virus liquid multigelation of harvest 3 times obtains the cell venom containing supernatant, and fractionated viral liquid is taken to carry out without bacterial examination
It tests and titration, remaining virus liquid is set -70 DEG C of refrigerators and saved.
S6. the TCID of virus liquid is harvested50Measurement:
The IBDV venom of harvest is done 10 times with the cell culture fluid of serum-free to be serially diluted, takes 10-5、10-6、10-7、10-8
4 dilutions are inoculated with DF1 cell, and each dilution is inoculated with 8 holes, and every hole 0.1mL adsorbs 1h, and 0.1mL maintaining liquid is added in every hole,
37 DEG C of culture 72h.DF1 cytopathy is observed, TCID is calculated50。
Embodiment 2
The present embodiment is used to investigate the domestication effect that embodiment 1 tames DF1 cell
By by the DF1 cell of drop serum free culture system, serum adding proportion is 8%, 6%, 4%, 2%, through reducing fibre step by step
Even for the content of albumen (FN) until when the content of FN is 0,37 DEG C of carbon dioxide incubator cultures, control group is that serum content is
The DF1 cell of 10% DMEM/F12 culture.
Table 1:DMEM/F12 reduces serum and tames DF1 cell effect
As shown in Table 1: except the DF1 cell of 2% serum content be significantly lower than control group point kind of ratio and cell it is average
Harvest yield, point kind of the ratio of the DF1 cell of 8%, 6%, 4% serum content and the cell harvest yield that is averaged are similar to control group.
Embodiment 3
The present embodiment is used to investigate infections chicken cloacal bursa virus potency prepared after the decreased serum of DF1 cell is tamed
It is horizontal.
By the DF1 cell by reducing serum free culture system, serum adding proportion is 8%, 6%, 4%, 2%, is reduced step by step
The content of fibronectin (FN) until when the content of FN is 0, inoculative proportion be the separation of 1% this laboratory, identification it is avian infectious
The virulent Shandong strain of bursal disease virus (SD), maintaining liquid be the DMEM/F12 containing 0% serum content, control group maintaining liquid be containing
The DMEM/F12 of 2% serum.
DF1 cell proliferation infections chicken cloacal bursa virus situation after table 2. is tamed
Embodiment 4
The present embodiment is used to assess the cost of 1~3 preparation process of above embodiments
By taking 100L volume of culture as an example, to original process and after reducing serum domestication, technique carries out Cost comparisons, to culture solution
It is estimated and is compared with the cost of maintaining liquid, concrete outcome is as follows:
Table 3: cost estimation
Embodiment 5
A method of using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus,
The following steps are included:
1) DF1 cell is taken, the DMEM/F12 culture containing 8% (v/v) newborn bovine serum, 10mg/L fibronectin is inoculated in
Base replaces several subcultures in incubation, and newborn bovine serum content is constant in the culture medium replaced every time and fibronectin
Content is more previous low, tames and dociles until being changed to the DMEM/F12 culture medium containing 8% (v/v) newborn bovine serum to get to the first generation
Change cell；
2) it takes first generation domestication cell, is inoculated in containing 6% (v/v) newborn bovine serum, 20mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 6% (v/v) newborn bovine serum, i.e.,
Obtain second generation domestication cell；
3) it takes second generation domestication cell, is inoculated in containing 4% (v/v) newborn bovine serum, 30mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 4% (v/v) newborn bovine serum, i.e.,
Obtain third generation domestication cell；
4) it takes third generation domestication cell, is inoculated in containing 2% (v/v) newborn bovine serum, 40mg/L fibronectin
DMEM/F12 culture medium replaces several subcultures in incubation, and newborn bovine serum content is not in the culture medium replaced every time
Become and fibronectin content is more previous low, until be changed to the DMEM/F12 culture medium containing 2% (v/v) newborn bovine serum, i.e.,
Obtain forth generation domestication cell；
5) it takes forth generation described in step 4 to tame cell, is inoculated with infections chicken cloacal bursa virus, with newborn containing 2% (v/v)
The DMEM/F12 culture medium culture of cow's serum harvests culture.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All any modifications, equivalent replacements, and improvements etc. done in application range of the invention, should all
It is included within protection scope of the present invention.
Claims (10)
1. a kind of method using lower serum content nutrients culture DF1 cell to prepare infections chicken cloacal bursa virus,
Be characterized in that the following steps are included:
1) DF1 cell is taken, the DMEM/F12 culture medium containing 8% (v/v) newborn bovine serum, 10mg/L fibronectin is inoculated in,
Several subcultures are replaced in incubation, newborn bovine serum content is constant in the culture medium replaced every time and fibronectin contains
Amount is more previous low, tames until being changed to the DMEM/F12 culture medium containing 8% (v/v) newborn bovine serum to get to the first generation
Cell；
2) first generation domestication cell is taken, the DMEM/ containing 6% (v/v) newborn bovine serum, 20mg/L fibronectin is inoculated in
F12 culture medium replaces several subcultures in incubation, in the culture medium replaced every time newborn bovine serum content it is constant and
Fibronectin content is more previous low, until being changed to the DMEM/F12 culture medium containing 6% (v/v) newborn bovine serum to get arriving
The second generation tames cell；
3) second generation domestication cell is taken, the DMEM/ containing 4% (v/v) newborn bovine serum, 30mg/L fibronectin is inoculated in
F12 culture medium replaces several subcultures in incubation, in the culture medium replaced every time newborn bovine serum content it is constant and
Fibronectin content is more previous low, until being changed to the DMEM/F12 culture medium containing 4% (v/v) newborn bovine serum to get arriving
The third generation tames cell；
4) third generation domestication cell is taken, the DMEM/ containing 2% (v/v) newborn bovine serum, 40mg/L fibronectin is inoculated in
F12 culture medium replaces several subcultures in incubation, in the culture medium replaced every time newborn bovine serum content it is constant and
Fibronectin content is more previous low, until being changed to the DMEM/F12 culture medium containing 2% (v/v) newborn bovine serum to get arriving
Forth generation tames cell；
5) it takes forth generation described in step 4 to tame cell, is inoculated with infections chicken cloacal bursa virus, with newborn containing 0~2% (v/v)
The DMEM/F12 culture medium culture of cow's serum harvests culture.
2. according to claim 1 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that pre-treatment obtains DF1 cell used in step 1) in the following manner: taking
The DF1 cell for covering with single layer in cell bottle, digests through EDTA- pancreatin, and 10mL cell growth medium is added, and piping and druming dispersion is thin
DF1 cell is placed in 37 DEG C of carbon dioxide incubators of temperature and carries out culture 72h by born of the same parents, when good cell monolayer to be formed, into
Row expands culture.
3. according to claim 2 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that the cell growth medium is the DMEM/F12 containing 10% (v/v) newborn bovine serum
Culture solution.
4. according to claim 3 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that contain 8% (v/v) newborn bovine serum, 10mg/L fibronectin described in step 1)
DMEM/F12 culture medium be by will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum with it is fine containing 50mg/L
Even the DMEM/F12 culture medium of albumen is mixed to get with the ratio of 4:1 (v/v).
5. according to claim 4 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that contain 6% (v/v) newborn bovine serum, 20mg/L fibronectin described in step 2)
DMEM/F12 culture medium be by will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum with it is fine containing 50mg/L
Even the DMEM/F12 culture medium of albumen is mixed to get with the ratio of 3:2 (v/v).
6. according to claim 5 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that contain 4% (v/v) newborn bovine serum, 30mg/L fibronectin described in step 3)
DMEM/F12 culture medium be by will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum with it is fine containing 50mg/L
Even the DMEM/F12 culture medium of albumen is mixed to get with the ratio of 2:3 (v/v).
7. according to claim 6 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that contain 2% (v/v) newborn bovine serum, 40mg/L fibronectin described in step 4)
DMEM/F12 culture medium be by will contain the DMEM/F12 culture medium of 10% (v/v) newborn bovine serum with it is fine containing 50mg/L
Even the DMEM/F12 culture medium of albumen is mixed to get with the ratio of 1:4 (v/v).
8. according to claim 7 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that step 5) specifically includes following operation: to contain 0~2% (v/v) newborn bovine serum
The resulting forth generation of DMEM/F12 culture medium step 4) tame cell, until when DF1 cell covers with single layer, with the ratio of 1% (v/v)
Example inoculation infections chicken cloacal bursa virus is virulent, and control temperature continues to cultivate at 37 DEG C, when 80% or more lesion occurs in DF1 cell
When, harvest cell culture.
9. according to claim 8 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that the EDTA- pancreatin is the PBS solution containing 0.05~0.25% (w/v) pancreatin.
10. according to claim 9 a kind of avian infectious to prepare using lower serum content nutrients culture DF1 cell
The method of bursal disease virus, it is characterised in that the step 5) DMEM/F12 containing 0~2% (v/v) newborn bovine serum is cultivated
Also containing the antibiotic of 100~200IU/mL, the DMEM/F12 culture medium for containing 0~2% (v/v) newborn bovine serum in base
PH is 7.2~7.4.
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