CN106237324A - A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine - Google Patents
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine Download PDFInfo
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Abstract
The present invention relates to a kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine.The technique of the full suspension culture of ST cell that the present invention relates to; its key technology be ST cell entirely suspend tame successfully after can carry out the large-scale culture of transmissible gastro-enteritis virus at bioreactor; have that simple to operate, controllability is strong, culture efficiency and automaticity is high, need not the advantages such as carrier, the large-scale production for cell or vaccine provides a new path.
Description
Technical field
The present invention relates to a kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine, belong to biology for animals
Arts.
Background technology
Transmissible gastro-enteritis virus (Transmissible Gastroenteritis virus of Swine, TGEV)
Belong to coronaviridae coronavirus genus member, may result in transmissible gastroenteritis of swine (Transmissible
Gastroenteritis, TGE).This disease is the one acute high degree in contact sexually transmitted disease (Saifetal., 1992) of pig, clinical
It is characterized as diarrhoea, serious vomiting and dehydration.The pig of all ages and classes and kind is the most susceptible to primary disease, and incubation period is short, propagates fast, 2～3
Swinery can be involved in it.This disease has height fatality rate to newborn piglet, and piglet sickness rate and fatality rate within two week old can
Reach 100%, become the worldwide disease of one of pig.China just has the report of TGE from the sixties, and in recent years, TGE is at me
State some areas happen occasionally and popular, and in the area that swinery is intensive, TGE is one of piglet morbidity and main causes of death,
Carry out harm seriously to industrial belt of raising pigs.Pig farm is once infected TGEV and is difficult to eradicate, and can only rely on vaccine prevention control at present.
ST cell, i.e. Pig testicular cell, belong to fibroblast, external can continuous passage adhere-wall culture, this cell line pair
Multiple virus is sensitive, such as: pig parvoviral, PRV (Pseudorabies virus), swine fever virus, transmissible gastro-enteritis virus etc., be vaccine
One of main raw and auxiliary material of propagative viruses in production.The cultivation of ST cell is the critical process link of virus type production of vaccine, directly
Connect the quality having influence on vaccine, the most stably obtain enough cells particularly important for ensureing production capacity and product quality.
Spinner culture ST cell technology is cell cultivation side most commonly used in current virus type production of vaccine for animals
Method, advantage is that technique is simple, with low cost, but the problems such as production efficiency is low, labor intensity is high, product uniformity difference are the most very
Difficulty be fundamentally addressed (Chen Wenqing, Wang Jianchao, Liu Huajie etc. suspension culture technique is relatively divided with spinner culture technique
Analysis. China's veterinary drug magazine, 2010,44 (10): 37-41).
Drawback based on spinner culture, suspension culture starts to be applied to produce among reality.ST cell suspension cultures technique
Can control a series of cell culture parameters, have cultivation viral level high, need not mix, differences between batches are little, and training method is automatic
Change, need not producers in a large number, cultivate that demand area is little, pollute little, low cost and other advantages, be the cell of first-selection in vaccine preparation
Culture technique.
The mainly microcarrier suspension culture of suspension culture application at present.Full suspension culture is in terms of live vaccine, and taking the lead in should
For the production of foot-and-mouth disease vaccine, suspension culture technique causes certain effect in bio-pharmaceuticals industry afterwards, and production of vaccine is looked forward to
Industry increasingly pays close attention to new technology.
Passage cell of the present invention entirely suspend produce transmissible gastroenteritis of swine vaccine technique.Its key technology is ST
Cell suspends after taming successfully entirely, can be used for the cultivation of transmissible gastro-enteritis virus.Relatively other microcarrier suspension culture system,
This system has that simple to operate, controllability is strong, culture efficiency and automaticity is high, need not the advantages such as carrier, for cell or
The large-scale production of vaccine provides a new path.
Summary of the invention
It is an object of the invention to provide a kind of ST cell entirely to suspend and produce the technique of transmissible gastroenteritis of swine vaccine, thus drop
Low production cost, carries out scale, homogeneity production, the popular situation sick to tackle current domestic transmissible gastroenteritis of swine.
Technical scheme
1. one kind uses the method that full suspension technology produces transmissible gastroenteritis of swine vaccine, it is characterised in that its preparation method
Cultivate including cell, change cell culture fluid and connect poison and results operation, the most first ST cell shaking flask is carried out suspension culture expansion
Access bioreactor after increasing and carry out suspension culture, after cell grows to virus inoculation density, change a part of cell culture fluid
And access virus seed liquor, continue to cultivate in bioreactor and gather in the crops virus liquid after terminating, add suitable frozen-dried protective
Form through vacuum freeze-drying after agent mixing subpackage.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, it is special
Levy and be described replacing cell culture fluid and connect poison operation for using ST cell suspension culture in bioreactor extremely can connect poison
During state, change new cell culture fluid, and Pigs Inoculated Transmissible gastroenteritis virus into by the 1/3 of cell culture fluid.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, it is special
Levy be described method particularly as follows:
(1) by ST cell with centrifugal, with cell culture fluid by cell by centrifugation again after flask suspension culture 48h～72h
With density 3 × 10 after suspension5～3 × 106/ ml accesses bioreactor, carries out the full suspension culture of ST cell；
(2) treat that the full suspension culture of ST cell to density is 6 × 105/ ml～6 × 106/ ml, bioreactor 15 DEG C sedimentation 2
Hour, the original fluid pumping out upper strata 1/3 injects new cell culture fluid, and inoculates 0.1%～0.5% (V/V) pig transmissible
Marcy agent kind poison；
(3) continue to cultivate suspension cell in bioreactor, when there is typical case CPE in the cell until about 80%, results
Virus liquid, after adding suitable heat-resisting lyophilized protecting agent mixing, vaccine is made in subpackage, chilled vacuum drying.
4. a kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine as described in claim 1-3, its
The virus being characterised by described be preserving number be the pig infectious gastroenteritis virus S D/L strain of CGMCC No.6001.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, it is special
Levy the foundation being described ST cell strain, be through the low serum in adhere-wall culture stage by pig testis continuous cell line (ST cell line)
Domestication process and low serum free culture system liquid suspension domestication process and obtain, this strain cell is named as Pig testicular cell system (Swine
Testis) suspension adapted strain (being called for short ST-1 strain), this strain cell delivered the Chaoyang District, Beijing City North Star on 08 23rd, 2016
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers of West Road 1 institute
Preservation, deposit number is CGMCC No.12698.
Cell culture fluid the most of the present invention means that low serum cell culture fluid, its serum content (V/V) are:
0.5%～2%.
The bioreactor used is APPLIKON company 30L～2000L bioreactor.
The technique of ST cell disclosed by the invention full suspension culture transmissible gastroenteritis of swine vaccine, it is simple to operate, controlled
Property strong, culture efficiency and automaticity is high, need not carrier, can in low serum free culture system liquid suspension culture, for cell or
The large-scale production of vaccine provides a new path.
Detailed description of the invention
1. the foundation domestication process of the ST cell strain of full suspension culture
(1) domestication (reduction serum) of ST cell attachment cultivation stage
ST (CVCC, No.CL27, " China's veterinary's strain catalogue " (second edition), p171) attached cell is taken from liquid nitrogen container
Seed, adds the cell growth medium of 10% new-born calf serum in T75 culture bottle, carries out ST cell recovery, and adhere-wall culture, makes
Secondary Culture domestication is carried out with the adhere-wall culture liquid gradually reducing serum.Serum content is from 10%, 8%, 5%, 3%, 2%, 1%
It is down to 0.5%.
(2) the suspension domestication of ST cell (low serum free culture system liquid)
The part results of successful ST cell F39 will be tamed, and frozen, it is designated as ST-1F0.
ST cell domestication process is it can be seen that this cell is down to low serum levels at adherent phase sera content by 10%
(0.5%～2.0%), the quantity of cell and vigor all can reach requirement of experiment.And from adherent to suspension, by the domestication in 39 generations
Cultivating, cell just adapts to suspension culture state, and can stably rise in value to 1.2 × 106/ ml, cell viability reaches more than 90%.
By the process optimization to full condition of suspension culture (pH, rotating speed), it is determined that pH7.0 ± 0.1, rotating speed 120r/min, it is achieved that
ST cell amplification culture from triangle culture bottle to bioreactor, and the dissolved oxygen amount in bioreactor is improved,
Thus complete ST cell from adherent to the complete whole domestication process suspended, and the named pig of suspension cell after taming successfully
Testicular cell system (Swine Testis) suspension adapted strain, is called for short ST-1 strain, and this strain cell was delivered on 08 23rd, 2016
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, deposit number is this CGMCC No.12698.(detailed in Example 1)
2. the preparation of full suspension cell
(1) cell shaking flask amplification culture: with 5 × 10 after cell recovery5The density of/ml is linked into 125ml triangle culture bottle
In, with 120r/min rotating speed in 37 DEG C, 5%CO2 incubator is cultivated.Treat that cell grows to 1.2 × 106During/ml, pass
In generation, proportionally adding fresh culture, making cell initial density is 5 × 105/ml。
(2) cell inoculation: correct dissolved oxygen electrode, logical CO before cell inoculation2Adjust pH to 7.0 ± 0.1, take shaking of requirement
Bottle cell (density about 1.2 × 106/ ml) centrifugal after, with 200ml (200ml/1000ml) culture fluid return outstanding after access biological respinse
Device.
3. the preparation of seedling venom and join Seedling
(1) poison is connect: treat that the full suspension culture of ST-1 cell is to density about 6 × 105/ ml～6 × 106/ ml, bioreactor 15
DEG C sedimentation 2 hours, the original fluid pumping out upper strata 1/3 injects new cell culture fluid, and inoculates 0.2% (V/V) pig transmissible
Marcy agent kind poison SD/L strain (CGMCC No.6001).
(2) results virus liquid, joins Seedling: continue to cultivate suspension cell in bioreactor, treat that the cell of about 80% goes out
During existing typical case CPE, gather in the crops virus liquid, add suitable heat-resisting lyophilized protecting agent and add subpackage, warp after suitable freeze drying protectant mixing
Lyophilisation makes vaccine.
4. the inspection of transmissible gastroenteritis of swine vaccine
(1) character faint yellow Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding diluent.
(2) steriling test by existing " Chinese veterinary pharmacopoeia " (Chinese veterinary pharmacopoeia committee, People's Republic of China's veterinary drug allusion quotation,
2 years versions three, Chinese agriculture publishing house, 2011, hereinafter referred to as " Chinese veterinary pharmacopoeia ") annex is tested, should aseptic life
Long.
(3) mycoplasma inspection is tested by existing " Chinese veterinary pharmacopoeia " annex, should grow without mycoplasma.
(4) exogenous virus inspection by vaccine and transmissible gastroenteritis of swine specific positive serum and after, inoculate Vero thin
Born of the same parents, MDBK cell, ST cell monolayer, test by existing " Chinese veterinary pharmacopoeia " annex, should pollute without exogenous virus.
(5) vaccine is indicated head part according to label and is diluted to 1 part/ml, mix homogeneously by diagnostic test, then is diluted to
200TCID50/ 0.1ml, mixes with equivalent TGEV specific positive serum, after putting 37 DEG C of neutralizations 30 minutes, inoculates respectively and grows up to
The ST cell 24 porocyte culture plate of monolayer, every kind of cell inoculates 12 holes, every hole 0.5ml.Set simultaneously and do not neutralize matched group (general
Vaccine indicates head part according to label and is diluted to 1 part/ml, mix homogeneously, then is diluted to 200TCID50/ 0.1ml, cultivates with equivalent
Liquid mixes) and normal cell controls group, each inoculation 6 holes, every hole 0.5ml.Every hole is added the cell containing 2% new-born calf serum and is cultivated
Liquid 0.5ml.Tissue Culture Plate is put 37 DEG C, containing 5%CO2Incubator is cultivated, observes 96～120 hours.Neutralization group and cell
Matched group should occur without CPE, does not neutralize matched group and CPE should all occurs.
(6) safety verification is with the healthy susceptible piglet 5 of 28～35 ages in days, 10 parts of every intramuscular inoculation vaccine, observes 14
Day, piglet should be all good for and be lived.
(7) the following method of efficacy test is appointed and is selected one.
1. TGEV viral level measures, with the culture fluid containing 2% new-born calf serum, vaccine is diluted to 1 part/ml, makees 10
Times serial dilution, takes 10-3、10-4、10-5、10-64 dilution factors, inoculate that to have grown up to the ST cell 96 hole trace of monolayer thin respectively
Born of the same parents' culture plate, each dilution factor is inoculated 8 holes, every hole 100 μ l, is set simultaneously and do not connect poison comparison 8 holes.Every hole is added containing 2% new born bovine
The cell culture fluid 100 μ l of serum.Put 37 DEG C, containing 5%CO2Incubator is cultivated, observes 96～120 hours, record CPE hole
Number.TCID is calculated by Reed-Muench method50, every part vaccine virus content answers >=105.0TCID50。
2. piglet immunological counteracting toxic substances method is with the 3～5 healthy susceptible piglets 5 of ages in days, 1 part of each intramuscular injection vaccine.Immunity 14
In the future, by 5 immune piglets, together with 5 comparison piglets that condition is identical, porcine Transmissible gastroenteritis virus poison TGEV/Q by force
Strain 4ml is (containing 1000ID50), Continuous Observation 7 days, comparison piglet falls ill entirely, and immunity piglet at least protects 4；Or comparison piglet 4
Morbidity, piglet is all protected in immunity.
(8) vacuum measures and is measured by " Chinese veterinary pharmacopoeia " annex, should meet regulation.
(9) residual moisture measures and is measured by " Chinese veterinary pharmacopoeia " annex, should meet regulation.
The biomaterial resource information that the present invention relates to
The pig infectious gastroenteritis virus S D/L strain that the present invention relates to, is by doubtful of transmissible gastroenteritis of swine (TGE)
The pathological material of disease passage method of sick piglet is separated to a strain TGE virus SD strain and carries out passage and cause weak and obtain, this disease
Poison has been caused weak and has been had good immunogenicity, can be named as transmissible gastro-enteritis virus as vaccine strain
(Transmissible gastroenteritis virus) SD/L strain, this strain delivers Beijing on April 13rd, 2012
North Star West Road, Chaoyang District 1 No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute are common
The preservation of microorganism center, preserving number is CGMCC No.6001；Pig testis subculture cells ST strain, deposit number is CVCC,
No.CL27, purchased from (China Veterinery Drug Inspection Office, " China's veterinary's strain mesh is write by Chinese microorganism strain preservation administrative center
Record " (second edition), Chinese agriculture publishing house, 2002, p171)；(the letter of Pig testicular cell system (Swine Testis) suspension adapted strain
Claim ST-1 strain), this cell strain delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science on 08 23rd, 2016
China Committee for Culture Collection of Microorganisms of institute of microbiology of institute common micro-organisms center CGMCC No.12698.
The positive effect of the present invention
The present invention relates to a kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine.The present invention relates to
The technique of the full suspension culture of ST cell, after its key technology is that ST cell entirely suspends and tames successfully, can enter at bioreactor
The large-scale culture of row transmissible gastro-enteritis virus, has that simple to operate, controllability is strong, culture efficiency and an automaticity
High, need not the advantages such as carrier, the large-scale production for cell or vaccine provides a new path.
Embodiment
The following is specific embodiment of the present invention, so that the present invention is expanded on further, but be not construed as limiting the present invention
Scope.People in the art it should be understood that can be to the technology of the present invention under without departing from the spirit and scope of the present invention
The details of scheme and form are modified or replace, but these amendments or replacement each fall within protection scope of the present invention.
Embodiment 1
The foundation of the ST cell strain of full suspension culture
The domestication (low serum domestication process) of 1.ST cell attachment cultivation stage
Take from liquid nitrogen container ST attached cell seed (pig testis subculture cells ST strain, deposit number is CVCC,
No.CL27, purchased from China Veterinery Drug Inspection Office, " China's veterinary's strain mesh is write by Chinese microorganism strain preservation administrative center
Record " (second edition), Chinese agriculture publishing house, 2002, p171), T75 culture bottle adds 10%MEM cell growth medium, carries out
Cell recovery is cultivated, and inoculative proportion is 1:1, changes liquid after 24h.When cell grows up to fine and close monolayer and growth conditions is good,
0.05% trypsinization 2 times, blows and beats into single-cell suspension liquid gently with pipet, passes on according to the ratio of 1:3 (V/V), carries out
ST cell attachment is cultivated, and continues downwards to be passaged to F3.With 0.05% trypsinization liquid, cell is separated, 500～
1000r/min is centrifuged, and uses culture fluid to re-mix uniformly, with 3 × 105～5 × 105Individual/ml carries out Secondary Culture domestication.Blood
Clear content is down to 0.5% from 10%, 8%, 5%, 3%, 2%, 1%, and each serum content cell state is stablized and follow-up resumed generation
Three generations, then goes to next serum content.The growing state of observation of cell.It is shown in Table 1.
Table 1ST cell attachment cultivates low serum cell culture fluid Domestication tests result
| Serum content | Inoculum density | Adherent situation | Upgrowth situation | Quantity | Vigor |
| 10% | 3.2×105 | ++++ | ++++ | 3.4×106 | 98% |
| 8% | 2.7×105 | ++++ | ++++ | 3.5×106 | 99% |
| 5% | 2.9×105 | +++ | ++++ | 3.0×106 | 96% |
| 3% | 3.0×105 | +++ | ++++ | 3.1×106 | 97% |
| 2% | 2.8×105 | +++ | ++++ | 2.9×106 | 97% |
| 1% | 3.1×105 | +++ | ++++ | 2.8×106 | 96% |
| 0.5% | 3.1×105 | +++ | ++++ | 3.0×106 | 98% |
Through the cell domestication Secondary Culture in slowly (37 generation), the result of the test display training of 10%～5% serum content
Nutrient solution cultivates cell, and about 48h can grow up to fine and close cell monolayer；Cultivate thin from 3% culture fluid being down to 0.5% serum content
Born of the same parents, when passing 1st generation, have a little dead cell, but do not affect cell attachment and growth, can conventionally carry out after passing for 3 to 5 generations
Sub-bottle passes on, and after domestication is stable, about 72h grows up to fine and close cell and cell state is good.In view of production cost problem, will be thin
The serum content of intracellular growth liquid is set to 0.5%～2%.
The suspension domestication of 2.ST cell (containing the cell culture fluid of 0.5%～2% new-born calf serum)
(1) domestication of different generation cell suspension cultures
Densification is grown up to by what the cell culture fluid (cell culture fluid of 0.5%～2% new-born calf serum) tamed was cultivated
After the ST attached cell of monolayer stably passed on for 3 generations, it is designated as ST F0.
1) when cell culture fluid cultivation ST attached cell (F5) tamed grows into fine and close cell monolayer, 0.05% pancreas
Enzymic digestion, blows and beats into single-cell suspension liquid gently, with 5 × 10 with pipet5The density of individual/ml is linked into 125ml triangle and cultivates
In Ping, complement to 40ml with low serum free culture system liquid, with 120r/min rotating speed in 37 DEG C, 5%CO2Incubator is cultivated.Every 72h
It is centrifuged and changes liquid (containing the cell culture fluid of 2% new-born calf serum), and the continuous subculture of method according to this, until 50 generations, cultivated
To often for cell sampling counting observation of cell form in journey.
2) when cell culture fluid cultivation ST attached cell (F8) tamed grows into fine and close cell monolayer, 0.05% pancreas
Enzymic digestion, blows and beats into single-cell suspension liquid gently, with 5 × 10 with pipet5The density of individual/ml is linked into 125ml triangle and cultivates
In Ping, complement to 40ml with low serum free culture system liquid, with 120r/min rotating speed in 37 DEG C, 5%CO2Incubator is cultivated.Every 72h
It is centrifuged and changes liquid (containing the cell culture fluid of 2% new-born calf serum), and the continuous subculture of method according to this, until 50 generations, cultivated
To often for cell sampling counting observation of cell form in journey.
3) when cell culture fluid cultivation ST attached cell (F11) tamed grows into fine and close cell monolayer, 0.05%
Trypsinization, blows and beats into single-cell suspension liquid gently, with 5 × 10 with pipet5The density of individual/ml is linked into the training of 125ml triangle
Support in bottle, complement to 40ml with low serum free culture system liquid, with 120r/min rotating speed in 37 DEG C, 5%CO2Incubator is cultivated.Every
72h is centrifugal changes liquid (containing the cell culture fluid of 2% new-born calf serum), and the continuous subculture of method according to this, until 50 generations, cultivates
During to often for cell sampling counting and observation of cell form.
Result of the test: start attached cell is domesticated for suspension cell from " 2) " F8, it is more just to have started cell death number, warp
Changing liquid pass on after constantly centrifugal, when " 2) " reach F39, full suspension culture 72h, cell density reaches 2.26 × 106/ ml, carefully
Born of the same parents' vigor reaches 94%.1) and 3) all do not tame successfully.The part results of successful ST cell F39 will be tamed, and frozen, note
For ST-1F0.
(2) the culture fluid pH impact on suspended culture cell
Under conditions of other condition of culture are consistent, the pH of cell culture fluid is respectively set to 6.8 ± 0.1,7.0 ±
0.1,7.2 ± 0.1 and 7.4 ± 0.1, after 37 DEG C are cultivated 72h, sampling counting observation of cell form.
When ST cell culture fluid pH is 7.0 ± 0.1, the pH of culture fluid declines very fast, and cell growth is fast, cell density ratio
The same period, other test group were high, and cytoactive is preferable.When pH is in 6.8 ± 0.1 or 7.4 ± 0.1, cultivate cell growth after 48h
Slowly, cell density is substantially less than other test group.So ST cell culture fluid ideal pH is 7.0 ± 0.1.
(3) rotating speed impact on suspended culture cell
Condition of culture, with " (2) ", carries out suspension culture with different rotating speeds, and the 1st kind: 80r/min, the 2nd kind: 120r/min,
After all cultivating 72h at 37 DEG C, sampling counting observation of cell form.
80r/min rotating speed cultivates ST cell, and cell agglomerate is serious, and cell is covered in tank skin and tank bottoms situation is more, and
120r/min rotating speed is used to be more beneficial for the growth of ST cell.
The lowest serum full suspension culture ST-1 cell amplifies in bioreactor
Cell inoculates the previous day, squeezes into 800ml (800ml/1000ml) cell culture fluid (new containing 2% in bioreactor
Raw Ox blood serum), logical saturated air is overnight.Recover from liquid nitrogen the ST-1 suspension cell tamed by conventional method, through 500～
1000r/min is centrifuged, and is joined by cell in 250ml triangular flask, adds 0.5%～2% new-born calf serum cell and cultivates
Liquid, is adjusted to 5 × 10 by cell density5/ ml, is placed in 37 DEG C, and 120rpm cultivates in shaking table.Cell grows into 1.2 ×
106/ more than ml can carry out Secondary Culture, is amplified in this approach cultivating.When cell culture volumes reaches 200ml, can
To go to suspension cell 3L bioreactor is cultivated.According to this cell is proceeded in 10L, 30L bioreactor, grope
The optimum condition of cell growth.PH is respectively set as 7.0 ± 0.1, and dissolved oxygen amount is set as 40%, 80%, observes two kinds of feelings
Low serum suspension cell ST-1 proliferative conditions in bioreactor under condition.When dissolved oxygen amount is 40% and 80%, the highest cell
Density is all higher than 2.0 × 106/ ml, and vigor all can reach more than 90%, the two is more or less the same.Therefore, 40% dissolved oxygen amount is selected
As ST cell suspension cultures ventilation.Result of the test is shown in Table 2.
Table 2ST-1 suspension cell growth test result in each reactor
From ST cell domestication process it can be seen that this cell is down to low serum levels at adherent phase sera content by 10%
(0.5%～2%), the quantity of cell and vigor all can reach requirement of experiment.And from adherent to suspension, trained by the domestication in 39 generations
Supporting, cell just adapts to suspension culture state, and can stably rise in value to 2 × 106/ ml, cell viability reaches more than 90%.Pass through
Process optimization to full condition of suspension culture (pH, rotating speed), it is determined that pH7.0 ± 0.1, rotating speed 120r/min, it is achieved that ST is thin
Born of the same parents' amplification culture from triangle culture bottle to bioreactor, and the dissolved oxygen amount in bioreactor is improved, thus
Complete ST cell from adherent to the complete whole domestication process suspended, and the named pig testis of suspension cell after taming successfully
Cell line (Swine Testis) suspension adapted strain, is called for short ST-1 strain, and this strain cell delivered Beijing on 08 23rd, 2016
North Star West Road, Chaoyang District, city 1 No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute are general
Logical microorganism center preservation, deposit number is CGMCC No.12698.
Embodiment 2
Adapt to the amplification cultivation of the ST cell strain of full suspension culture
By the Pig testicular cell suspension adapted strain ST-1 recovery Secondary Culture of Liquid nitrogen storage, after be inoculated in bioreactor
Suspend amplification culture entirely, obtains ST-1 cell suspending culture solution.Specifically comprise the following steps that
1. take the ST-1 cell strain of liquid nitrogen cryopreservation, be equipped with after rapid fluid resuscitation in the shaking flask of culture fluid, in 36～37 DEG C
Cultivate 60～72h, pass on amplification culture according to the ratio of 1:3～1:5；
2. cell inoculates the previous day, squeezes into 800ml (800ml/1000ml) culture fluid (containing 2% blood in bioreactor
Clearly), logical saturated air is overnight.
3. cell inoculation: correct dissolved oxygen electrode, logical CO before cell inoculation2Adjusting pH to 7.0 ± 0.1, cultivation temperature is 36
～37 DEG C, take the shaking flask cell (density about 1.2 × 10 of requirement6/ ml) centrifugal after, cultivate with 200ml (200ml/1000ml)
Liquid returns to access in bioreactor after hanging and carries out suspension culture.
Embodiment 3
ST cell entirely suspend produce transmissible gastroenteritis of swine vaccine technique
1. prepared by production seed culture of viruses: takes well-grown ST cell monolayer, discards cell growth medium, change with containing 0.1%～
The cell maintenance medium of 0.5% pig infectious gastroenteritis virus S D/L strain seed culture of viruses, puts 36～37 DEG C and continues to cultivate, treat about 80%
There are results during typical case CPE in cell.Quantitative separating, indicates harvest date, seed culture of viruses algebraically etc., freezen protective.
2. the breeding of seedling venom
(1) bioreactor cleaning, sterilizing: bioreactor is cleaned up, 121 DEG C of sterilizing 30min.
(2) cell inoculation: suspension culture amplification ST-1 cell in shaking flask, reaches 1.2 × 106After/ml, inoculate into biology
Reactor, cell culture condition is: reactor working volume 30 liters, corrects dissolved oxygen electrode, logical CO before cell inoculation2Adjust pH extremely
7.0 ± 0.2, cultivation temperature is 36～37 DEG C, and dissolved oxygen amount is 40%, takes the shaking flask cell (density about 1.2 × 10 of requirement6/
Ml), after centrifugal, return with cell culture fluid, after hanging, access bioreactor carries out suspension culture, cultivate 2～3 continuously.
(3) poison and results are connect: treat that the full suspension culture of ST-1 cell is to density about 1.2 × 106/ ml, bioreactor 15 DEG C
Settle 2 hours, pump out upper strata 1/3 archeocyte culture fluid and change the cell culture fluid of 0.5%～2% new-born calf serum, and according to carefully
0.2% Pigs Inoculated infectious gastroenteritis virus S D/L strain of born of the same parents' culture fluid final volume, Virus culture condition is temperature 33～35 DEG C,
PH value is 7.0 ± 0.2, and dissolved oxygen is 40%, and gathering in the crops and removing tank during typical case CPE occur in the cell until about 80%.Produce 3 batches of diseases altogether
Venom.
3. the inspection of semifinished product
(1) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, should equal bacteria growings.
(2) viral level every milliliter viral level is all >=106.5TCID50。
4. join Seedling and subpackage and will check qualified SD/L strain virus liquid, add freeze drying protectant in the ratio of 1:1 and stablize
Agent.Add appropriate antibiotics, be sufficiently mixed, quantitative separating.
5. carry out rapidly lyophilisation after lyophilizing subpackage to form.
Embodiment 4
Transmissible gastroenteritis of swine vaccine product inspection
1. character slightly yellow Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding diluent.
2. steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, equal asepsis growth.
3. mycoplasma inspection is tested by existing " Chinese veterinary pharmacopoeia " annex, all grows without mycoplasma.
4. exogenous virus inspection by vaccine and transmissible gastroenteritis of swine specific positive serum and after, inoculate Vero thin
Born of the same parents, MDBK cell, ST cell monolayer, test by existing " Chinese veterinary pharmacopoeia " annex, all pollutes without exogenous virus.
5. vaccine is indicated head part according to label and is diluted to 1 part/ml, mix homogeneously by diagnostic test, then is diluted to
200TCID50/ 0.1ml, mixes with equivalent transmissible gastroenteritis of swine specific positive serum, after putting 37 DEG C of neutralizations 30 minutes, and inoculation
Having grown up to the ST cell 24 porocyte culture plate of monolayer, every kind of cell inoculates 12 holes, every hole 0.5ml.Set simultaneously do not neutralize right
(vaccine is indicated head part according to label be diluted to 1 part/ml, mix homogeneously according to group, then be diluted to 200TCID50/ 0.1ml, with
Equivalent culture fluid mixes) and normal cell controls group, each inoculation 6 holes, every hole 0.5ml.Every hole is added containing 2% new-born calf serum
Cell culture fluid 0.5ml.Tissue Culture Plate is put 37 DEG C, containing 5%CO2Incubator is cultivated, observes 96～120 hours.Neutralize
There is not CPE in group and cell controls group, do not neutralize matched group and CPE the most all occur.
6. safety verification is with the healthy susceptible piglet 5 of 28～35 ages in days, 10 parts of every intramuscular inoculation vaccine, observes 14
Day, piglet is all strong to live.
7. the following method of efficacy test is appointed and is selected one.
(1) TGEV viral level measures, with the culture fluid containing 2% new-born calf serum, vaccine is diluted to 1 part/ml, makees 10
Times serial dilution, takes 10-3、10-4、10-5、10-64 dilution factors, inoculate that to have grown up to the ST cell 96 hole trace of monolayer thin respectively
Born of the same parents' culture plate, each dilution factor is inoculated 8 holes, every hole 100 μ l, is set simultaneously and do not connect poison comparison 8 holes.Every hole is added containing 2% new born bovine
The cell culture fluid 100 μ l of serum.Put 37 DEG C, containing 5%CO2Incubator is cultivated, observes 96～120 hours, record CPE hole
Number.TCID is calculated by Reed-Muench method50, every part vaccine virus content >=105.0TCID50。
(2) piglet immunological counteracting toxic substances method is with the 3～5 healthy susceptible piglets 5 of ages in days, 1 part of each intramuscular injection vaccine.Immunity 14
In the future, by 5 immune piglets, together with 5 comparison piglets that condition is identical, porcine Transmissible gastroenteritis virus poison TGEV/Q by force
Strain 4ml is (containing 1000ID50), Continuous Observation 7 days, comparison piglet falls ill entirely, and immunity piglet at least protects 4；Or comparison piglet 4
Morbidity, piglet is all protected in immunity
8. vacuum measures and is measured by " Chinese veterinary pharmacopoeia " annex, all meets regulation.
9. residual moisture measures and is measured by " Chinese veterinary pharmacopoeia " annex, all meets regulation.
Claims (5)
1. one kind uses the method that full suspension technology produces transmissible gastroenteritis of swine vaccine, it is characterised in that its preparation method includes
Cell is cultivated, is changed cell culture fluid and connect poison and results operation, after the most first ST cell shaking flask being carried out suspension culture amplification
Access bioreactor and carry out suspension culture, after cell grows to virus inoculation density, change a part of cell culture fluid and connect
Enter virus seed liquor, continue to cultivate in bioreactor and gather in the crops virus liquid after terminating, add suitable freeze drying protectant and mix
Form through vacuum freeze-drying after even subpackage.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, its feature exists
In described replacing cell culture fluid and connect poison operation for use ST cell in bioreactor suspension culture to can connect poison state
Time, new cell culture fluid, and Pigs Inoculated Transmissible gastroenteritis virus is changed into by the 1/3 of cell culture fluid.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, its feature exists
In described method particularly as follows:
(1) by ST cell with centrifugal, with cell culture fluid by cell settling flux by centrifugation after flask suspension culture 48h～72h
After with density 3 × 105～3 × 106/ ml accesses bioreactor, carries out the full suspension culture of ST cell；
(2) treat that the full suspension culture of ST cell to density is 6 × 105/ ml～6 × 106/ ml, bioreactor 15 DEG C settles 2 hours,
The original fluid pumping out upper strata 1/3 injects new cell culture fluid, and inoculates 0.1%～0.5% (V/V) transmissible gastroenteritis of swine
Virus plants poison；
(3) continue to cultivate suspension cell in bioreactor, when there is typical case CPE in the cell until about 80%, results virus
Liquid, after adding suitable freeze drying protectant mixing, vaccine is made in subpackage, chilled vacuum drying.
4. a kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine, its feature as described in claim 1-3
Be described virus be preserving number be the pig infectious gastroenteritis virus S D/L strain of CGMCC No.6001.
A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine the most as claimed in claim 1, its feature exists
In described ST cell strain, be by the pig testis continuous cell line (ST cell line) the low serum through the adhere-wall culture stage tame process and
Low serum free culture system liquid suspension domestication process and obtain, this strain cell be named as Pig testicular cell system (Swine Testis) suspend
Adapted strain (is called for short ST-1 strain), and this strain cell delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 08 23rd, 2016
The center preservation of China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms, deposit number
For CGMCC No.12698.
Cell culture fluid the most of the present invention means that low serum cell culture fluid, its serum content (V/V) are: 0.5%～
2%.
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| CN109402068A (en) * | 2018-11-09 | 2019-03-01 | 四川华神兽用生物制品有限公司 | A method of preparing the remaining porcine pseudorabies virus of serum-free |
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