CN106318899B - The foundation and its application of one plant of bull testis passage cell strain - Google Patents
The foundation and its application of one plant of bull testis passage cell strain Download PDFInfo
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Abstract
The present invention relates to the foundation and its application of one plant of bull testis passage cell strain.This method comprises: (1) prepares primary cell using fresh bull testis and establishes ZJS-1 plants of bull testis cell line；(2) live vaccines of hog cholera is produced using cell monolayer method culture bull testis passage cell；(3) live vaccines of hog cholera is produced using suspension cell method culture bull testis cell.The bull testis cell line that the present invention establishes has simple production process and stabilization, easy to operate for live vaccines of hog cholera production, and viral level is high, and differences between batches are small, easy to control the quality, is remarkably improved vaccine yield and quality.It is good using live vaccines of hog cholera safety produced by the invention, immune efficacy is high, to swine fever strong virus attack have complete immanoprotection action.
Description
Technical field
The present invention relates to the foundation and its application of one plant of bull testis passage cell strain, belong to veterinary biologics field.
Background technique
It is bull testis primary cell and porcine kidney cell that China produces cell used in hog cholera lapinised virus vaccine at present.In order to
The culture process of swine fever virus is improved, bull testis passage cell strain, also known as bull testis cell line are established in special independent research, in this base
Basic bacteria batch and production seed lot are established on plinth, and system calibration is carried out to it.
The live vaccines of hog cholera live vaccine excellent for the safety and effect of China's independent research, is worldwide answered extensively
With for preventing the infection of swine fever virus.Original production is bull testis primary cell with cell, need to acquire fresh bull testis system
It is standby, recently as the increasingly sophisticated of cattle disease epidemic situation, acquire fresh bull testis and prepare Cells for production there are exogenous virus, bacterium
And the risk of mycoplasma contamination, porcine kidney cell as the progress that Cells for production is on the basis of bull testis primary cell,
As pig source cell, other viruses in pig source are also easier to adapt to the cell, and are proliferated on cell, cause pig source virus dirty
Dye.The pollution of these exogenous factors constitutes a serious threat to the safety of live vaccine and effect, to using animal and the environment may
Cause huge loss.
Summary of the invention
In place of improving the shortcomings of the prior art, provide a kind of raw with bull testis passage cell
The method for producing live vaccines of hog cholera.This method has simple production process and stabilization, easy to operate, and viral level is high, and differences between batches are small,
It is easy to control the quality, it is remarkably improved vaccine yield and quality, can be given birth in rolling bottle cell production line and suspension cell production line
It produces.It is good using live vaccines of hog cholera safety produced by the invention, immune efficacy is high, have to swine fever strong virus attack complete immune
Protective effect.
Technical solution of the present invention
1. the foundation of one plant of bull testis passage cell strain, it is characterised in that the cell line is originated from and is prepared by fresh bull testis
Primary cell obtains one plant after passing on and is not only suitable for rolling bottle monolayer cell culture, and is suitable for cell suspension cultures technique, and
The bull testis passage cell strain to swine fever virus high susceptibility is still maintained, it is thin that this plant of passage cell strain is named as bull testis
Born of the same parents are ZJS-1 plants of (Bovine testis cell line), have delivered Chaoyang District, Beijing City North Star west on August 23rd, 2016
The China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, institute 3 of road 1 is protected
Hiding, deposit number are CGMCC No.12699.
2. the application of one plant of bull testis passage cell strain described in claim 1, it is characterised in that this plant of bull testis cell is adopted
The preparation of live vaccines of hog cholera is used for monolayer method and swine fever virus Lapinized strain CVCC No.AV1412.
3. the application of one plant of bull testis passage cell strain as claimed in claim 2, it is characterised in that this plant of bull testis cell
The preparation of live vaccines of hog cholera is used for using cell suspension cultures method and swine fever virus Lapinized strain CVCC No.AV1412.
4. the application of one plant of bull testis passage cell strain as described in claim 2,3, it is characterised in that hog cholera therein
Malicious Lapinized strain CVCC No.AV1412 is that the spleen leaching poison of swine fever virus is inoculated with the bull testis cell of good single layer or the training that suspends
Feeding bull testis cell is added in 3.5% serum maintaining liquid postposition incubator and cultivates, cell state observed daily, after 4~5 days
Harvest virus, according to said method continues passaged virus, and two receipts, three is taken to receive cell culture venom as production seed culture of viruses.
The specific embodiment of the invention
1. the foundation of bull testis continuous cell line
New born healthy calf is chosen, testis is won in operation, and 0.25% trypsin solution is added after being handled with Hanks liquid and disappears
Change, discard trypsin solution, shakes method cell dispersion with bead, stationary culture is at single layer.The bull testis cell of single layer will be grown up to
Dispersed and passed on 0.25% pancreatin, be passaged to F50 and obtain one plant and be not only suitable for rolling bottle monolayer cell culture, but be applicable in cell in
Suspension culture process, and still maintain the bull testis passage cell strain to the high susceptibility of swine fever virus, this plant of passage cell
It is named as ZJS-1 plants of bull testis cell line (Bovine testis cell line), delivered north on 08 23rd, 2016
No. 3 China Committee for Culture Collection of Microorganisms, Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1
Common micro-organisms center preservation, deposit number are CGMCC No.12699.
2. the foundation of bull testis cell microcarrier suspension culture technique
(1) in the bull testis passage cell P11 generation frozen, is taken out in cell monolayer preparation, sets in 37 DEG C of water-baths and thaws, 1000r/
Min is centrifuged 15min, discards supernatant, and inoculation 110cm is resuspended with the DMEM culture medium containing 8% newborn bovine serum2Culture bottle sets 37
DEG C, contain 5%CO2Culture to cell grows up to fine and close single layer in incubator, is digested with 0.25% pancreatin/EDTA, passes by 1:3~1:5
In generation, expands culture.The bull testis passage cell that will be enlarged by culture is inoculated into 3000ml cell spinner bottle, sets 37 DEG C of cultures to cell
Grow up to fine and close single layer.Control revolving speed is 8~12r/h.
(2) bioreactor culture condition
The condition of laboratory microcarrier suspension culture bull testis passage cell is determined as 37 DEG C of temperature, pH value 7.2, dissolved oxygen
50%, microcarrier concentration 5g/L, 40~45r/min of speed of agitator (2L bioreactor), 45~50r/min (10L biological respinse
Device), cell-seeding-density 1.5 × 105A/mg~2.0 × 105A/mg.
3. the preparation of swine fever cell live vaccine
(1) breeding of cell seed culture of viruses
Swine fever virus Lapinized strain (C plants) spleen is drenched into poison and is inoculated with the bull testis passage cell of good single layer or the culture that suspends
Passage cell adds 3.5% serum maintaining liquid, sets in incubator and cultivate, and observes cell state daily, and virus is harvested after 4~5 days,
According to said method continue passaged virus, two receipts, three is taken to receive cell culture venom as production seed culture of viruses；
Cell virus seed identification: cell virus seed identification complies fully with fever virus lapinized Chinese Strain seed culture of viruses standard, to pig safety without pair
Effect, the every 1ml of cell seed culture of viruses contain virus >=500,000 rabbit infective dose.
(2) inoculation cell monolayer prepares live vaccines of hog cholera
The above-mentioned bull testis passage cell culture bottle for having formed good single layer is taken, cell growth medium is discarded, inoculation contains swine fever
The cell maintenance medium of viral Lapinized strain spleen leaching poison, continues to cultivate；Harvest changes liquid to work on the 5th for the first time after connecing poison, later every
It is changed liquid 1 time every harvest on the 5th, the venom of harvest sets -20 DEG C or less preservations；Qualified virus-culturing fluid will be examined, is mixed in same
In container, stabilizer is added, while antibiotic is added, sufficiently shakes up, quantitative separating；Every part venom containing cell is no less than
0.015ml is got product after carrying out vacuum freezedrying rapidly after packing.
(3) live vaccines of hog cholera is prepared by suspension culture process
The bull testis passage cell for having formed good single layer is taken, individual cells is digested to 0.25% pancreatin/EDTA and counts
Number.According to bioreactor culture volume, every liter of volume 5g containing microcarrier, by 1.5 × 105~2.0 × 105A/mg cell density
After accessing cell, the DMEM cell culture fluid for containing 6%~8% newborn bovine serum is added, in 37 DEG C, 30~60r/min, pH value
7.2, it is cultivated under conditions of dissolved oxygen 50%, daily sampling carries out cell observation and digestion counts.
When microcarrier cell is counted not less than 4.0 × 10 in level-one bioreactor6When a/ml, stop stirring, to micro- load
After body cell precipitates completely, growth-promoting media is discarded, well-grown BT cell microcarrier is collected through rewinding pipe to cell dissociation bottle
In, it is cleaned 2 times with PBS (0.01mol/L, pH value 7.2), is counted with being sampled after 0.25% pancreatin/EDTA digestion.It is raw according to second level
Object bioreactor culture volume, by 1.5 × 105~2.0 × 105A/mg cell density access two stage biological reactor amplifies training
It supports, when microcarrier cell is counted not less than 3.0 × 10 in two stage biological reactor6When a/ml, growth-promoting media is discarded, is added and contains 1%
The DMEM cell maintenance medium of~3.5% newborn bovine serum or horse serum is after 0.1~0.5 inoculation produces seed culture of viruses, 37 with MOI
DEG C, cultivate under conditions of 30~60r/min, pH value 7.2, dissolved oxygen 50%, connect and do within 5th harvest for the first time after poison and change liquid (stopping is stirred
Mix, precipitate, harvest supernatant, add the DMEM cell maintenance medium containing 1%~3.5% newborn bovine serum or horse serum), after
It is changed the liquid once every harvest on the 4th, harvesting frequency is no more than 8 times.- 15 DEG C or less preservations are set, are no more than 6 months.It is qualified to examine
Virus-culturing fluid, be mixed in same container, stabilizer be added, while antibiotic is added, sufficiently shakes up, quantitative separating；Often
Head part venom containing cell is no less than 0.015ml, gets product after carrying out vacuum freezedrying rapidly after packing.
Product inspection: by " Chinese veterinary pharmacopoeia " (magnificent people's republic, Chinese veterinary pharmacopoeia committee China veterinary drug allusion quotation 2005
Three Chinese agriculture publishing houses of version, 2006, the present invention hereinafter referred to as " Chinese veterinary pharmacopoeia ") it tests, Ying Fuhe " swine fever epidemic disease living
Seedling (cell source) " set quota, it is without side-effects safely to pig.Every part vaccine contains virus >=7500 rabbit infective doses.
In above-mentioned technical proposal, the cell maintenance medium of the spleen of Lapinized strain containing the swine fever virus leaching poison is containing 0.3% spleen
Drench the maintaining liquid (virus liquid that virus passes through rabbit body passage harvest rabbit spleen and lymph preparation) of seed culture of viruses.
In above-mentioned technical proposal, the formula (V/V) of the growth-promoting media is: 10% calf serum, adds in right amount 90%DMEM liquid
Antibiotic, pH value is adjusted to 7.0.
In above-mentioned technical proposal, the formula (V/V) of the maintaining liquid is: 5% calf serum, adds in right amount 90%DMEM liquid
Antibiotic, pH value is adjusted to 7.2.
The present invention relates to biomaterial resource informations
Passage cell strain of the present invention is named as bull testis cell line (Bovine testis cell line)
ZJS-1 plants, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research was delivered on August 23rd, 2016
The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute, deposit number are CGMCC No.12699；Pig
Pestivirus Lapinized strain, also known as C plant, deposit number are CVCC No.AV1412；Classical swine fever virus Shimen system velogen strain, preservation are compiled
It number is compiled see China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation administrative center for CVCC No.AV1411
It writes, Chinese animal doctor's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, 2002, p145).
Detailed description of the invention
Show that (1-1) first round purifies the single green screened in Fig. 1 swine fever virus Lapinized strain purification result figure
Fluorescent spot；(1-2) is the single green fluorescence spot that the second wheel purifying screens；(1-3) is negative control.
Fig. 2 virus neutralization tests (2-1) as shown in the figure is special to occur in CSFV F7 and negative serum and in the cell cytosol of hole
Anisotropic green fluorescence；(2-2) is to occur specific green fluorescence in CSFV F7 virus control wells cell cytosol；(2-3) is normal
Without specificity fluorescent in cell control well cell cytosol.
Fig. 3 attacks each group experimental animal mean body temperature change curve after poison.
Fig. 4 is preparation flow figure of the invention.
Positive effect of the present invention
The present invention relates to the methods with bull testis continuous cell line production live vaccines of hog cholera.The present invention relates to one plant of bull testis
The foundation and its application of continuous cell line, including: (1) primary cell is prepared using fresh bull testis and establish bull testis biography
Continuous cell line；(2) live vaccines of hog cholera is produced using cell monolayer method culture bull testis passage cell；(3) suspension cell method is used
It cultivates bull testis passage cell and produces live vaccines of hog cholera.The bull testis passage cell that the present invention establishes is produced for live vaccines of hog cholera
With simple production process and stabilization, easy to operate, viral level is high, and differences between batches are small, easy to control the quality, is remarkably improved vaccine production
Amount and quality.It is good using live vaccines of hog cholera safety produced by the invention, immune efficacy is high, have to swine fever strong virus attack complete
Immanoprotection action.
Embodiment
It is next with reference to the accompanying drawings and specific embodiments of the specification that the invention will be further described.
Embodiment 1
--- the seedling preparation of cell primary cell and the foundation of continuous cell line
New born healthy calf is chosen, testis is won in operation, and 0.25% trypsin solution is added after being handled with Hanks liquid and disappears
Change, discard trypsin solution, shakes method cell dispersion with bead, stationary culture is at single layer.The bull testis cell of single layer will be grown up to
Dispersed and passed on 0.25% pancreatin, be passaged to F50, obtained one plant and be not only suitable for rolling bottle monolayer cell culture, and be applicable in cell
In suspension culture process, and still maintain the bull testis continuous cell line to swine fever virus high susceptibility, this plant of passage cell
System is named as ZJS-1 plants of bull testis continuous cell line (Bovine testis cell line), later every 10 generations, packing
Cell cryopreservation tube, freeze-stored cell, for continuing passage or virus inoculation.The cell line delivered Beijing on August 23rd, 2016
No. 3 China Committee for Culture Collection of Microorganisms, Institute of Microorganism, Academia Sinica, institute of city, North Star West Road, Chaoyang District 1 are general
Logical microorganism center preservation, deposit number CGMCC No.12699 thus.
Embodiment 2
--- adaptability of the swine fever virus Lapinized strain to bull testis passage cell
1. viral passages and clone purification
(1) fever virus lapinized Chinese Strain (C plants) spleen is drenched the bull testis passage cell that poison is inoculated with good single layer by viral passages, is added
3.5% serum maintaining liquid sets 37 DEG C, containing 5%CO2It is cultivated in incubator, observes cell state daily, disease is harvested after 4~5 days
Poison according to said method continues passaged virus 2 times.
(2) viral clone purification uses limiting dilution assay purified virus, and concrete operations are as follows: will upload in bull testis cell
Make 10 times for resulting generation in 3rd generation virus F3 to be serially diluted, takes 10-2、10-3、10-4、10-5、10-65 dilutions are inoculated with respectively
96 well culture plate of bull testis cell, each dilution is inoculated with 6 holes, and negative control hole is arranged, and sets 37 DEG C, containing 5%CO2Culture
It is cultivated 3~4 in case.Supernatant is moved in a new 96 sterile, cell-free well culture plate corresponding apertures by hole is corresponding, 4
DEG C save.The culture plate cell monolayer that will be taken off supernatant is fixed with 80% acetone and 20% methyl alcohol mixed liquor, carries out direct fluorescence
The fluorescence antibody 0.1ml of working concentration is added in dyeing, every hole, sets and acts on 45~60min in 37 DEG C of wet box, discards fluorescence antibody,
It is washed 1 time with PBST (PBS containing 0.5 ‰ Tween-20,0.01mol/L, pH7.2), in fluorescence microscopy microscopic observation.To occur
24 well culture plate of BT cell that 70%~80% single layer has been covered in the corresponding supernatant inoculation of the cell hole of single fluorescence carries out the 2nd
Time cloning purifying, is inoculated with 25cm for supernatant2Square vase obtains fever virus lapinized Chinese Strain (C plants) passage cell poison F7 after amplifying
In generation, lot number: 003-013 is shown in Fig. 1 as swine fever virus Lapinized strain swine fever passage cell toxogen beginning seed culture of viruses.
2. viral level measures
(1) minimal infecting dose (MID) of rabbit is made F7 for hog cholera lapinised virus passage cell poison sterile saline respectively
100000 times, 200,000 times, 300,000 times, 500,000 times, 1,000,000 times of dilutions, per generation seed culture of viruses viral suspension are inoculated with rabbit 2, every rabbit ear
It is injected intravenously 1ml, provides to carry out thermometric by existing " live vaccines of hog cholera " standard, determine.Every milliliter of CSFV F7 (lot number: 003-
013) viral level is 200,000 RID.
(2)FAID50F7 is used the DMEM containing 3.5% newborn bovine serum to cultivate by measurement for hog cholera lapinised virus passage cell poison
Base is made 10 times and is serially diluted.With 10-3、10-4、10-5、10-6The virus of 4 dilutions is inoculated with 96 orifice plate bull testis cell monolayers,
Each dilution is inoculated with 6 holes, the hole 0.1ml/.Set 37 DEG C, 5%CO2After cultivating 3~4 in incubator, culture solution is discarded.With
PBST (PBS containing 0.5 ‰ Tween-20,0.01mol/L, pH7.2) is washed 1 time, and 80% acetone and 20% methanol is added in every hole
Mixed liquor 0.1ml, -20 DEG C are fixed 30 minutes.Acetone mixture is abandoned, the swine fever monoclonal fluorescence antibody of working concentration is added in every hole
0.1ml, 37 DEG C of wet box dye 45~60min.It inhales and abandons fluorescence antibody, washed 3 times with PBST, set fluorescence microscopy under the microscope, carefully
Occurring specific green fluorescence in endochylema is virus-positive.Virus FAID is calculated with Spearman-Karber method50.Fluoremetry
It as a result is 105.5FAID50。
3. specific assay
1) F7 is diluted to every 1ml with sterile saline for hog cholera lapinised virus passage cell poison and contained by rabbit body neutralization test
There is 100RID, be sufficiently mixed with the CSFV specific positive serum of equivalent inactivation, sets 10~15 DEG C and neutralize 1 hour, shake 2 therebetween
~3 times.Positive control and negative control are set simultaneously.After neutralization, it is inoculated with rabbit 2 respectively, every rabbit ear vein injects 1ml,
Temp measuring method and body temperature reaction normal imitate detecting method with rabbit.Only there is thermal response to rabbit body neutralization test in positive controls, remaining 2
Group does not cause thermal response in 120 hours after inoculation.
2) fluorescence antibody virus neutralization tests is by F7 for hog cholera lapinised virus passage cell poison serum-free DMEM culture solution
It is diluted to 200FAID50/ 0.1ml, (serum neutralize antibody titers are not with the CSFV specific positive serum of 56 DEG C of inactivations 30 minutes
Lower than 1:8) mixed in equal amounts, it sets after being neutralized 1 hour at 37 DEG C, is inoculated with 96 hole bull testis tissue culture plates, each sample connects 4
Hole, the hole 0.1ml/.Normal cell controls, positive control and negative serum control are set simultaneously.Set 37 DEG C, containing 5%CO2In incubator
Culture, observation 3~4 days.Culture solution is discarded, with PBST (PBS containing 0.5 ‰ Tween-20,0.01mol/L, pH7.2) washing 1
Secondary, 80% acetone and 20% methyl alcohol mixed liquor 0.1ml is added in every hole, and -20 DEG C are fixed 30 minutes.Acetone mixture is abandoned, every hole adds
Enter the 100 μ l of swine fever monoclonal fluorescence antibody of working concentration, 37 DEG C of wet box dye 45~60 minutes.It inhales and abandons fluorescence antibody, use
PBST is washed 3 times, sets fluorescence microscopy under the microscope.In virus neutralization tests, CSFV F7 (lot number: 003-013) and positive serum
With do not occur specificity fluorescent in hole and normal cell controls hole cell cytosol, virus control wells and negative serum control hole are thin
There is specificity fluorescent in born of the same parents' endochylema, sees Fig. 2.
4. pure property
CSFV F7 (lot number: 003-013) is tested by existing " Chinese veterinary pharmacopoeia " annex and carries out steriling test, branch
Substance is examined, exogenous virus inspection result shows virus without bacterium, mould, mycoplasma, exogenous virus pollution (1~table of table 4).
1) steriling test takes seed culture of viruses inoculation TG tubule, each 2, every 0.2ml, 1, the inclined-plane GA to set 37 DEG C of cultures, and 1 is set
25 DEG C of cultures, separately take 0.2ml, are inoculated with 1 GP tubule, set 25 DEG C, cultivate 7.
2) mycoplasma is examined detects seed culture of viruses with two methods of fluid nutrient medium culture, agar solid plate culture simultaneously.
Positive and negative control need to be set simultaneously by checking every time, cultivate observation under the conditions of same, and when detection uses mycoplasma hyorhinis as compareing.
3) as inspection product after seed culture of viruses is used swine fever hyper-immune serum to neutralize by exogenous virus inspection, it is thin that Vero, MDBK, PK15 are inoculated with
Born of the same parents, separately set one bottle of normal cell controls, and the total incubation time of each cell should be no less than 14.Cell culture is answered during culture
At least subculture 1 time is judged to against regulation if there is cytopathy during culture.Such as cell-free lesion, culture terminates
When, cell monolayer carries out following examine.
1. the cell monolayer of fluorescence antibody inspection technique last time subculture is used for fluorescence antibody inspection after should cultivating 5~7.
MDBK cell is for examining bovine viral diarrhea virus (BVDV)；PK15 cell is for examining pig parvoviral (PPV).To every kind
The inspection of specific exogenous virus includes at least 3 groups of cell monolayers: (1) test sample cell culture；(2) in last time subculture
When be inoculated with 100~300FAID50The positive control of specific virus；(3) normal cell controls.Each cell monolayer checks area
6.0cm should be not less than2。
Vero, PK15 cell monolayer cultivated at least 7 days after passage 2. cytopathogenic effect inspection technique is learnt from else's experience are tested.With
Giemsa staining liquid dyes cell monolayer.Observe cell monolayer, check inclusion body, giant cell or other by exogenous virus
The appearance situation of caused CPE.
3. hemadsorption exogenous virus is examined learn from else's experience passage after cultivate at least 7 days Vero, PK15 cell monolayers into
Performing check.With PBS washing cell monolayer 2~3 times, appropriate 0.2% guinea pig red blood cells and the equivalent suspension of chicken red blood cell are added,
It is subject to and covers entire monolayer surface.2 cell monolayers are selected, respectively in 2~8 DEG C and 25 DEG C placement 30min, are washed with PBS, are examined
Look into hemadsorption situation.
1 bacterium of table, mould, mycoplasma are examined
Note: "-" indicates asepsis growth.
2. exogenous virus inspection of table-fluorescent antibody examination result
Note: "-" indicates no specificity fluorescent；There is specificity fluorescent in "+" expression.
3. exogenous virus inspection of table-cytopathogenic effect (CPE) inspection result
Note: "-" indicates no specific C PE.
4. exogenous virus inspection of table-hemadsorption inspection result
Note: "-" indicates no hemadsorption phenomenon.
5. Security test
With health susceptible piglet 5 of hog cholera antibody feminine gender, every muscle passes on through injection F7 for hog cholera lapinised virus thin
Born of the same parents poison virus stock solution used 5ml, (lot number: 003-013) seed culture of viruses stoste 5ml (5 × 105.5FAID50) after the susceptible pig of inoculation, thermometric observation
21 days.The body temperature of tested pig in the normal range, spirit, appetite with injection before compared with no significant change.
6. immunogenicity determining
F7 is made 10 times with sterile saline for hog cholera lapinised virus passage cell malicious (lot number: 003-013) respectively is
Column dilution.With 10-4、10-5、10-6The virus liquid of 3 dilutions distinguishes inoculation test pig 5, every intramuscular injection 1ml, simultaneously
3 controls are set up not to be inoculated with.14 days after inoculation, together with 3 control pigs, every pig muscle injection swine fever crossdrift is virulent blood poison
1ml.After attacking poison, daily morning and afternoon is respectively surveyed body temperature 1 time, and observes that spirit, appetite has 16 days without exception.Protection result after attacking poison
Respectively 5/5,5/5,0/5.Control group 3/3 is fallen ill, and 3/3 is dead.CSFV F7 is 10 to the minimum immune dosage of pig-5/ml.It passes
It conforms to current standards for cytotoxic immunogenicity.
Hog cholera lapinised virus spleen is drenched into poison inoculation bull testis passage cell, passage proliferation, by cell culture using limited
Dilution method carries out two-wheeled clone purification, obtains 1 plant of swine fever virus Lapinized strain passage cell adapted strain, which is passed
In generation, is prepared for the former malicious seed culture of viruses of swine fever production seed culture of viruses, is named as CSFV F7 (lot number: 003-013), which can be in ox
It is grown on testis passage cell.
The research such as specific, pure, safety, immunogenicity, rabbit body neutralization test research knot are carried out to the seed culture of viruses of acquisition
Fruit shows that BT passage seed culture of viruses is hog cholera lapinised virus, and seed culture of viruses is pure, safe, and immunogenicity is good.
This test is used above:
(1) swine fever virus seed culture of viruses and cell are that the spleen of swine fever virus Lapinized strain preparation drenches poison, generation: F480, preservation
Number: CVCC No.AV1412；Bull testis continuous cell line, P50 generation.It is prepared by China Veterinery Drug Inspection Office and is saved.
(2) swine fever standard positive serum and negative serum are prepared and are provided by China Veterinery Drug Inspection Office.
(3) monoclonal fluorescence antibody and red cell suspension are prepared and are protected by Sinovet (Beijing) Biotechnology Co., Ltd.
It deposits.
(4) the test health susceptible piglet negative with 4 week old hog cholera antibody of pig.
Embodiment 3
--- swine fever virus Lapinized strain passage cell adapts to malicious highest generation vaccine virus immunogenicity research
1. observation is immunized first 3 days before immune, daily timing measurement experimental animal rectal temperature, whether there is or not clinical conditions for observation
Shape.Highest generation swine fever virus Lapinized strain passage cell is adapted into poison CSFV F13 (3,000,000 RID/ml, 106.5FAID50/
Ml 400 times first) are done and is diluted to 7500RID/ml (103.9FAID50/ ml), after being done 3000 times of dilutions again later (120 are diluted altogether
Ten thousand times) the 1st group of piglet of inoculation, every pig is inoculated with 1ml, by the 2nd group of experimental animal musculi colli injection PBS, 1ml/ head.After immune
Daily observation experimental animal had no adverse reaction to the 7th day.Poison is attacked within 14th after immune.Experimental animal CSF ELISA is anti-before immune
Body is detected as feminine gender, and clinical manifestation is normal (table 5).After immune, each group experimental animal body temperature is normal.
5. experimental design of table and animal packet information
Note: N/A table is not applicable；It * is immune according to being extrapolated after former times of viral level calculating extension rate of CSFV F13
Dosage.
2. attacking poison to attack before poison 3, daily timing thermometric, whether there is or not clinical abnormal for observation.Attack poison 0, all pig muscle injections
It examines with the former poison of virulent crossdrift blood poison, 1ml/ pigs.Day by day thermometric and observe to 16 days.Count every group of animal morbidity, dead, guarantor
Protect situation.After strong virus attack, immune group experimental animal has 2 (2028,2036) slightly body temperature to react, remaining is normal.Immune group
CSFV clinical symptoms are not shown.3/3 body temperature of negative control group experimental animal increases (being shown in Table 6, Fig. 3).3/3, which shows spirit, withers
It wastes, burnout, chilly, loss of appetite, anorexia, even useless food, vomiting, instability of gait；Alternating constipation and diarrhea；Palpebral edema, eye
The obvious clinical symptoms of the swine fevers such as eyeball flush.Attack protection situation are as follows: immune group 5 have no morbidity, all strong to live, 5/5 protection,
CSF negative control group experimental animal 3/3 is fallen ill, 3/3 dead (being shown in Table 7).
Table 6. attacks each group experimental animal temperature recording table (DEG C) after poison
Note: * BBT indicates basal body temperature."/" table animal dead, no data.
Each group is protected after table 7.CSFV strong virus attack
| Animal group | Size of animal | Morbidity | It is dead | Protection |
| 1st group (immune group) | 5 | 0/5 | 0/5 | 5/5 |
| 2nd group (control group) | 3 | 3/3 | 3/3 | N/A |
Note: N/A (Not Available) is not applicable.
The present embodiment is the result shows that dilute by swine fever virus Lapinized strain passage cell adaptation poison F13 highest generation vaccine virus
It releases to 7500RID (103.9FAID50), then done after 3000 times of dilutions after immunity test animal, swine fever strong virus attack can be produced
Raw preferable protective effect, up to 5/5, control pig whole morbidity is simultaneously all dead for protection, meets existing regulation, it was demonstrated that highest generation
Vaccine virus CSFV F13 immunogenicity is excellent.The passage of basic seed culture of viruses was no more than for 4 generations as production seed, CSFV F13 is determined
It is swine fever virus Lapinized strain (C plants) for cell toxicant highest generation vaccine virus, it is determined that the passage of swine fever virus Lapinized strain
Passage range of the cell adapted basic seed culture of viruses of poison in seedling.
Used in the present embodiment:
Swine fever virus Lapinized strain seed culture of viruses is that swine fever virus Lapinized strain passage cell adapts to poison F13, lot number: 003-
015, viral level is 3,000,000 RID/ml, 106.5FAID50/ ml comes from Sinovet (Beijing) Biotechnology Co., Ltd..
It examines with virulent classical swine fever virus Shimen system (CSFV-SM blood poison), lot number: 003-011,106.0MLD/ml, by China
Veterinary medicament supervision is prepared and saves.
Experimental animal: the negative susceptible piglet of health of 4 week old hog cholera antibodies is examined purchased from the Inner Mongol Huhehaote City left flag of soil
Plain flag dragon seed of forest pig farm.
IDEXX antibody against swine fever virus detection kit is purchased from Beijing Ai Deshi Yuan Heng Biotechnology Co., Ltd.
Embodiment 4
--- cell monolayer method prepares live vaccines of hog cholera
(1) selection of seedling cell: the bull testis continuous cell line for selecting the present invention to establish.
(2) passage and culture of seedling cell: bull testis cell is through 0.25% pancreatin had digestive transfer culture, with cell growth medium
Continue to cultivate in 37 DEG C, when forming good single layer, for continuing passage or virus inoculation.
(3) breeding of cell seed culture of viruses: using cell maintenance medium, and fresh spleen leaching poison is made to 0.3% viral suspension, inoculation life
Long good BT cell line single layer, sets 37 DEG C and continues to cultivate.It is changed the liquid once every harvest on the 5th, two receipts, three is taken to receive cell culture poison
Liquid is as production seed culture of viruses.
Cell virus seed identification: cell virus seed identification is without side-effects safely to pig, and the every 1ml of cell seed culture of viruses contains virus >=500,
000 rabbit infective dose；Identification complies fully with swine fever virus Lapinized strain strain seed culture of viruses standard.
(4) breeding of seedling virus liquid: taking the BT continuous cell line culture bottle for having formed good single layer, and it is raw to discard cell
Long liquid, inoculation contain the cell maintenance medium of 0.3% spleen seed culture of viruses, set 37 DEG C and continue to cultivate, and work on the 5th for the first time change by harvest after connecing poison
Liquid changes liquid 1 time every harvest on the 4th later, and the venom of harvest sets -20 DEG C or less preservations.
The inspection of venom: by " Republic of China Veterinary Pharmacopoeia ", (the People's Republic of China (PRC) is compiled by the Chinese veterinary pharmacopoeia committee
Veterinary drug allusion quotation (version in 2005) three) Chinese agriculture publishing house, 2015, hereinafter referred to as " Chinese veterinary pharmacopoeia " annex page 15,19 progress
It examines, it should be without bacterium, mould, mycoplasma growth.Cell venom is without side-effects safely to pig, and each time virus-culturing fluid of receiving is used respectively
Rabbit measurement, every 1ml contain virus >=500,000 rabbit infective dose.
(5) dispense and be lyophilized: qualified virus-culturing fluid will be examined, be mixed in same container, according to a certain percentage plus
Enter suitable stabilizer, while suitable antibiotic is added, sufficiently shake up, quantitative separating, every part venom containing cell is no less than
0.015ml is got product after carrying out vacuum freezedrying rapidly after packing.
Product inspection: testing by " Republic of China Veterinary Pharmacopoeia " (version in 2005), Ying Fuhe " live vaccines of hog cholera
(cell source) " regulation, it is without side-effects safely to pig.Every part vaccine contains virus >=7500 rabbit infective doses.
The formula of above-mentioned cell growth medium used is: 10% calf serum, 90%DMEM liquid plus suitable antibiotic, pH
Value is adjusted to 7.0.
The formula of above-mentioned cell maintenance medium used is: 5% calf serum, 90%DMEM liquid plus suitable antibiotic, pH value
It is adjusted to 7.2.
Embodiment 5
--- microcarrier suspension culture method prepares swine fever virus live vaccine
The present embodiment will produce seed culture of viruses highest generation CSFV F12 pickup kind microcarrier suspension culture bull testis cell ZJS-1
Strain has carried out the research for most preferably meeting poison amount (MOI) and harvesting frequency, the results showed that, microcarrier is inoculated with according to MOI value 0.1~0.5
Suspend culture bull testis cell, and first time on the 5th harvests and change liquid after inoculation, harvested and changed the liquid once every 4 days later, harvest can
Up to 8 receipts time, every milliliter of viral level reaches 106.5FAID50More than.
1. connecing the determination of poison amount (MOI)
With 2L bioreactor, volume of culture 2L, when density reaches 3.0 × 106When a/ml or more, by MOI value 0.05,
0.1,0.5,0.8 access virus liquid connects and does within 5th harvest for the first time after poison and change liquid (the DMEM culture containing 3.5% newborn bovine serum
Base), it is changed the liquid once (the DMEM culture medium containing 3.5% newborn bovine serum) every harvest on the 4th later, and test sample viral level
(FAID50Measurement), it determines and most preferably connects poison amount (MOI), when cell starts shedding off, stop experiment.
When connecing poison by MOI value 0.05, virus liquid viral level is compared with the virus liquid harvested when connecing poison by MOI value 0.1~0.8
It is low；When connecing poison by MOI value 0.1~0.8, each time virus liquid viral level of receiving is above 106.5FAID50/ ml, but consider to press MOI
Value 0.8 connects poison, and seed culture of viruses dosage is big, therefore selects MOI 0.1~0.5 most preferably to connect poison amount (being shown in Table 8).
8. difference MOI of table connects the relationship of poison amount and viral level
2. the determination of harvesting frequency
With 2L bioreactor, volume of culture 2L, when bull testis cell density reaches 3.0 × 106When a/ml or more, press
MOI value 0.1 accesses virus liquid, connects and does within 5th harvest for the first time after poison and change liquid (the DMEM culture medium containing 3.5% newborn bovine serum),
(the DMEM culture medium containing 3.5% newborn bovine serum), test sample viral level (FAID are changed the liquid once every harvest on the 4th later50
Measurement), when cell falls off, stop experiment, repeats experiment 3 batches, determine best harvesting frequency
Test result shows that first time on the 5th harvests and change liquid after connecing poison, harvested and changed the liquid once every 4 days later, harvests
Secondary, viral level reachable 10 is received up to 86.5FAID50/ ml or more；After harvesting 8 receipts time, cell starts to fall off, because
This, determines that harvesting frequency is no more than 8 times (being shown in Table 9).
9. virus harvest number of table determines test
Note: it is to test with a batch that this, which tests the determination (MOI value 0.1) that first connects poison amount (MOI) with 3.1,.
3.10L bioreactor viral growth process certification test
With 10L bioreactor, volume of culture 10L breeds 4 batches of virus liquids according to determining virus culture process.Inoculation
First time on the 5th harvests and changes liquid afterwards, harvests and changed later liquid, the viral level (FAID of detection harvest sample every 4 days50It surveys
It is fixed).
Indicate: in 10L bioreactor viral growth process certification test, breeding CSFV-2013001 is tieed up when criticizing virus liquid
Holding liquid is the DMEM culture medium containing 3.5% newborn bovine serum；Breed CSFV-2013002, CSFV-2013003, CSFV-
Maintaining liquid is the DMEM culture medium containing 3.5% horse serum when 2013004 batches of virus liquids.
The result shows that after inoculation first time on the 5th harvest and change liquid, later every 4 days harvest and change the liquid once, harvest do not surpass
8 receipts time are crossed, every milliliter of viral level can reach 106.5FAID50(it is shown in Table 10) above.
Table 10.10L bioreactor viral growth process certification
In conclusion it is thin in microcarrier suspension culture bull testis to determine that swine fever virus Lapinized strain passage cell adapts to poison
ZJS-1 plants of optimal culture conditions of born of the same parents system are as follows: MOI value is 0.1~0.5, and first time on the 5th harvests and change liquid after inoculation, later every 4
Day harvests and changes the liquid once, and no more than 8 receipts of harvest are secondary, and every milliliter of viral level can reach 106.5FAID50More than.
It is described above in the present embodiment:
Production seed culture of viruses is CSFV F12 generation, in bull testis passage cell P11 generation, is saved by China Veterinery Drug Inspection Office.
Cell culture medium is DMEM culture medium, is purchased from Hyclone company, batch: AXM51149RC.
Serum is that newborn bovine serum is purchased from Jinan Jing Niu company, batch: 120101；Horse serum is purchased from Hyclone company, batch
It is secondary: AXF42323.
Cell growth medium is the DMEM culture medium containing 8% newborn bovine serum, and maintaining liquid is containing 3.5% newborn bovine serum
DMEM culture medium or DMEM culture medium containing 3.5% horse serum.
Microcarrier Cytodex 1 is purchased from GE Healthcar company, batch: 10021510.
Bioreactor is 310 type bioreactor of BioFlo, and maximum functional volume is respectively 2L and 10L, by the U.S.
The manufacture of NBS bio-engineering corporation.
Claims (4)
1. one plant of bull testis passage cell strain, it is characterised in that the cell strain is originated from the primary cell prepared by fresh bull testis,
One plant is obtained after passing on and is not only suitable for rolling bottle monolayer cell culture, and is suitable for cell suspension cultures technique, and still maintain
To the bull testis passage cell strain of swine fever virus high susceptibility, this plant of passage cell strain is named as bull testis cell line
ZJS-1 plants of (Bovine testis cell line), delivered BeiChen West Road, Chaoyang District, BeiJing City 1 on August 23rd, 2016
No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica preservations of institute are protected
Hiding number is CGMCC No.12699.
2. one plant of bull testis passage cell strain described in claim 1, it is characterised in that this plant of bull testis cell uses cell monolayer
Cultivation and swine fever virus Lapinized strain CVCC No.AV1412 are used for the preparation of live vaccines of hog cholera.
3. one plant of bull testis passage cell strain as claimed in claim 2, it is characterised in that this plant of bull testis cell is outstanding using cell
Floating cultivation and swine fever virus Lapinized strain CVCC No.AV1412 are used for the preparation of live vaccines of hog cholera.
4. one plant of bull testis passage cell strain as described in claim 2,3, it is characterised in that the weak poison of swine fever virus rabbitization therein
Strain CVCC No.AV1412 is that the spleen leaching poison of swine fever virus is inoculated with the bull testis of the bull testis cell of good single layer or the culture that suspends
Cell is added in 3.5% serum maintaining liquid postposition incubator and cultivates, observes cell state daily, and virus is harvested after 4~5 days, is pressed
The method continues passaged virus, and two receipts, three is taken to receive cell culture venom as production seed culture of viruses.
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| CN103083653A (en) * | 2011-10-28 | 2013-05-08 | 辽宁成大动物药业有限公司 | Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology |
| CN105311630A (en) * | 2014-06-20 | 2016-02-10 | 普莱柯生物工程股份有限公司 | Method of preparing vaccine through suspension culture of mammal cells and application of the method |
| CN105582535A (en) * | 2014-11-18 | 2016-05-18 | 广东永顺生物制药股份有限公司 | Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine |
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| CN103083653A (en) * | 2011-10-28 | 2013-05-08 | 辽宁成大动物药业有限公司 | Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology |
| CN105311630A (en) * | 2014-06-20 | 2016-02-10 | 普莱柯生物工程股份有限公司 | Method of preparing vaccine through suspension culture of mammal cells and application of the method |
| CN105582535A (en) * | 2014-11-18 | 2016-05-18 | 广东永顺生物制药股份有限公司 | Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine |
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