CN104587460B - Mink viral enteritis, canine distemper bigeminal live vaccine and its preparation method and application - Google Patents
Mink viral enteritis, canine distemper bigeminal live vaccine and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of mink viral enteritis, canine distemper bigeminal live vaccine and its preparation method and application, a kind of bigeminal live vaccine of the present invention, its effective ingredient includes mink viral enteritis virus antigen and canine distemper virus antigen, wherein said mink viral enteritis virus antigen is mink viral enteritis virus JLM strain, and its culture presevation is numbered: CGMCC No.9904；Described canine distemper virus antigen is canine distemper virus JTM strain, and its culture presevation is numbered: CGMCC No.9905.The present invention is from several respects such as humoral immunization, Immunization level of protection and Pathologic changes; all confirm between MEV JLM strain and CDV JTM strain without immune interference; and on this basis; further study safety and the effect of this bigeminal live vaccine; result shows that a kind of bigeminal live vaccine of the present invention uses safety, and the effect of Combined vaccine is all not less than the effect standard of each single Seedling.
Description
Technical field
The present invention relates to a kind of bigeminal live vaccine and its preparation method and application, particularly to a kind of viral intestinal of mink
Inflammation, canine distemper bigeminal live vaccine and its preparation method and application.The invention belongs to veterinary biologics technical field,
Background technology
Mink, as the fleece animal of a kind of preciousness, worldwide has and cultivates widely.Denmark, China,
The U.S., Canada, Holland, Russia, Deng Doushi mink farming big country of Finland, China's mink farming amount occupies generation
Boundary first.Mink farming has the features such as market stability, business risk are little and good in economic efficiency, has good supporting
Grow prospect.Owing to China's mink farming industry is many based on small-scale family dispersion cultivation, rearing conditions is poor, exempts from
Epidemic disease prevention and control disunity, not in time, the popular of disease causes integral production inefficiency in addition.The China of harm at present water
The viral infectious of ermine aquaculture mainly has mink viral enteritis, mink canine distemper and Aleutian disease etc..
Mink viral enteritis is that a kind of high degree in contact caused by mink viral enteritis virus (MEV) infects
Disease, is widely current in Ge Yangdiao state of the world, one of disease of harm mink farming industry being well recognized as.This disease has relatively
High M & M, the young ermine at 3 to 6 monthly ages easy infection, mortality rate is up to 60%.China is in 1974
There is this disease in year, afterwards at China's many ground Outbreak, sickness rate reaches 83.4%, and mortality rate reaches 15.1% first,
Serious compromises China's mink farming industry.
Mink canine distemper is a kind of acute, highly infective, the high mortality caused by canine distemper virus (CDV)
Disease, is distributed widely in countries in the world.This disease the most all can be fallen ill, and is multiple season, not the same year with Xia Qiu
Age, different cultivars mink the most susceptible, to the young ermine easy infection at 3 monthly ages after ablactation, there is higher sickness rate
And mortality rate.China's reported first in 1973 mink canine distemper, spreads to the whole nation later, supports to China mink
Grow industry and cause huge economic loss.In August, 2002, Fujin City ermine field outburst canine distemper, sickness rate 41.2%,
Mortality rate 97.1%；In JIUYUE, 2002, Linyi City ermine field outburst canine distemper, sickness rate 67.9%, mortality rate 44%；
In April, 2004, ermine field, Weifang, Shandong outburst canine distemper, sickness rate 50%, mortality rate 20%.
At present, prevention mink viral enteritis and mink canine distemper many employings vaccination.The viral intestinal of domestic mink
Scorching vaccine is inactivated vaccine, and canine distemper is live vaccine, does not join Seedling.The most domestic urgent need develops the viral intestinal of mink
Scorching, canine distemper bigeminal live vaccine, prevents that two is sick reaching a pin, improves immune effect, save the purpose of labour force.
Summary of the invention
The problem existed for prior art, it is high, dry without immunity that an object of the present invention is to provide a kind of immunity
The mink viral enteritis disturbed, canine distemper bigeminal live vaccine, reach to prevent mink viral enteritis and mink simultaneously
The purpose of canine distemper.
In order to achieve the above object, present invention employs techniques below means:
A kind of mink viral enteritis of the present invention, canine distemper bigeminal live vaccine, it is characterised in that effective ingredient includes
Mink viral enteritis virus antigen and canine distemper virus antigen, wherein said mink viral enteritis virus resists
Originally being mink viral enteritis virus JLM strain, Classification And Nomenclature is mink viral enteritis virus, is deposited in China micro-
Biological inoculum preservation administration committee's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Ke Yuan institute of microbiology, its culture presevation is numbered: CGMCC No.9904, and the preservation time is 2014 10
The moon 30；Described canine distemper virus antigen is canine distemper virus JTM strain, and Classification And Nomenclature is canine distemper virus,
Being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is at the Chaoyang District, Beijing City North Star
West Road 1 institute of microbiology of the Chinese Academy of Sciences of institute, its culture presevation is numbered: CGMCC No.9905, the preservation time
It it is on October 30th, 2014.
In the present invention, it is preferred to, in mink viral enteritis described in every part, canine distemper bigeminal live vaccine,
Mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50。
The two of the purpose of the present invention are to provide described mink viral enteritis, canine distemper bigeminal live vaccine in preparation
Prevent or treat the application in mink viral enteritis and mink canine distemper.
The three of the purpose of the present invention are to provide and a kind of prepare mink viral enteritis, the side of canine distemper bigeminal live vaccine
Method.
In the present invention, each provide three kinds and prepare mink viral enteritis, the method for canine distemper bigeminal live vaccine,
It is spinner culture method, suspension culture method and microcarrier suspension culture method respectively.
Wherein, a kind of prepare described mink viral enteritis, the spinner culture method of canine distemper bigeminal live vaccine,
It is characterized in that comprising the following steps:
(1) prepared by seedling mink viral enteritis virosis venom
Taking well-grown CRFK cell by MOI is that 0.005-0.5 inoculates mink viral enteritis virus JLM strain
Virus, puts and cultivates at 37 DEG C, gathers in the crops virus liquid after cultivating 4～5, preserves, measure sick after-15 DEG C of freeze thawing 1 time
Poison content to carry out steriling test standby；
(2) prepared by seedling canine distemper virus virus liquid
Taking well-grown Vero or MDCK cell monolayer by MOI is that 0.005-0.5 inoculates canine distemper virus
JTM strain virus, puts and cultivates at 37 DEG C, gathers in the crops virus liquid after cultivating 4～5, preservation after-15 DEG C of freeze thawing 1 time,
Measure viral level and to carry out steriling test standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By viral for mink intestinal
Scorching virus is mixed homogeneously by 4:1 volume ratio with freeze drying protectant with canine distemper virus virus mixed liquor, is vaccine
Stock solution, quantitative separating adding a cover after mixing.
Wherein, a kind of prepare described mink viral enteritis, the suspension culture method of canine distemper bigeminal live vaccine,
It is characterized in that comprising the following steps:
(1) preparation of seedling mink viral enteritis virosis venom: take well-grown CRFK cell and connect
Planting in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell is long
Become individual cells with 0.25% trypsinization during the fine and close monolayer of one-tenth and count, by 0.8 × 105～1.2 × 105Individual/ml cell
Cell is accessed in one-level bioreactor and cultivates 48～72 hours by density, when cell density is not less than 2 × 106Individual/ml
Time, it is transferred in two stage biological reactor cultivate 48～72 hours by cell according to similarity condition, works as cell density
Reach 2 × 106Individual/ml time, with MOI for 0.005-0.5 inoculate mink viral enteritis virus production seed culture of viruses JLM
Strain, cultivates 36～48 hours under conditions of 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50%, results
Virus liquid, puts less than-15 DEG C preservations, measures viral level and to carry out steriling test standby；
(2) preparation of seedling canine distemper virus virus liquid: take well-grown Vero or mdck cell inoculation
In cell spinner bottle, putting 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell grows up to
Become individual cells with 0.25% trypsinization during fine and close monolayer and count, by 0.8 × 105～1.2 × 105Individual/ml cell is close
Cell is accessed in one-level bioreactor and cultivates 48～72 hours by degree, when cell density is not less than 2 × 106Individual/ml
Time, it is transferred in two stage biological reactor cultivate 48～72 hours by cell according to similarity condition, works as cell density
Reach 2 × 106Individual/ml time, with MOI for 0.005-0.5 inoculation canine distemper virus produce seed culture of viruses JTM strain, 37 DEG C,
40～60r/min, cultivate 36～48 hours under conditions of pH value 7.2, dissolved oxygen 50%, gather in the crops virus liquid, put-15 DEG C
Hereinafter preserve, measure viral level and to carry out steriling test standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By MEV and CDV
Virus mixed liquor is mixed homogeneously by 4:1 volume ratio with freeze drying protectant, is vaccinogen liquid, quantitatively divides after mixing
Fill and add a cover.
Wherein, a kind of described mink viral enteritis, the microcarrier suspension culture of canine distemper bigeminal live vaccine are prepared
Method, it is characterised in that comprise the following steps:
(1) preparation of seedling mink viral enteritis virosis venom: take well-grown CRFK cell and connect
Planting in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell is long
Become individual cells with 0.25% trypsinization during the fine and close monolayer of one-tenth and count, by 0.8 × 105～1.2 × 105Individual/ml cell
Cell is accessed in one-level bioreactor by density, every liter of 5g Han microcarrier, cultivates 48～72 hours, works as cell
Density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in two stage biological reactor, every liter contains
Microcarrier 5g, cultivates 48～72 hours, when cell density reaches 2 × 106Individual/ml time, with MOI as 0.005-0.5
Inoculation mink viral enteritis virus production seed culture of viruses JLM strain, at 37 DEG C, 40～60r/min, pH value 7.2, molten
Cultivate 36～48 hours under conditions of oxygen 50%, gather in the crops virus liquid, put less than-15 DEG C preservations, measure viral level
And it is standby to carry out steriling test；
(2) preparation of seedling canine distemper virus virus liquid: take well-grown Vero or mdck cell inoculation
In cell spinner bottle, putting 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell grows up to
Become individual cells with 0.25% trypsinization during fine and close monolayer and count, by 0.8 × 105～1.2 × 105Individual/ml cell is close
Cell is accessed in one-level bioreactor by degree, and every liter of 5g Han microcarrier cultivates 48～72 hours.When cell is close
Degree is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in two stage biological reactor, every liter containing micro-
Carrier 5g, cultivates 48～72 hours, when cell density reaches 2 × 106Individual/ml time, with MOI as 0.005-0.5
Inoculation canine distemper virus produces seed culture of viruses JTM strain, at 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50%
Under the conditions of cultivate 36～48 hours, gather in the crops virus liquid, put less than-15 DEG C preservations, mensuration viral level also carries out nothing
Bacterium inspection is standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By MEV and CDV
Virus mixed liquor is mixed homogeneously by 4:1 volume ratio with freeze drying protectant, is vaccinogen liquid, quantitatively divides after mixing
Fill and add a cover.
Mink viral enteritis, canine distemper bigeminal live vaccine R&D process in, it has been found that the viral intestinal of mink
The JLM strain of scorching virus is respectively provided with excellent safety and immunogenicity, Liang Zhetong with mink canine distemper virus JTM strain
Time immunity mink without immune interference phenomenon.Therefore mink viral enteritis virus JLM strain and canine distemper virus are selected
JTM strain have developed mink viral enteritis, canine distemper bigeminal live vaccine (JLM strain+JTM strain) as seed culture of viruses
(abbreviation Combined vaccine).For further determining that between two seeds culture of viruses without immune interference effect, research carries out multinomial examination
Test, from several respects such as humoral immunization, Immunization level of protection and Pathologic changes, it was demonstrated that MEV JLM strain with
Without immune interference between CDV JTM strain.On this basis, further study safety and the effect of Combined vaccine, knot
Fruit shows that vaccine uses safety, and the effect of Combined vaccine is all not less than the effect standard of each single Seedling.
Accompanying drawing explanation
Fig. 1 is mink viral enteritis, 2008121 batches of goods immune group water of canine distemper bigeminal live vaccine potency test
Ermine counteracting toxic substances rear intestinal (normally)；
Fig. 2 is mink viral enteritis, MEV pair, 2008121 batches of goods of canine distemper bigeminal live vaccine potency test
According to group No.0464 mink counteracting toxic substances rear intestinal (hemorrhage).
Detailed description of the invention
Further describe the present invention, advantages of the present invention and feature below in conjunction with specific embodiment to will be with describing
And it is apparent.But embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, can be to technical solution of the present invention under without departing from the spirit and scope of the present invention
Details and form are modified or replace, but these amendments and replacement each fall within protection scope of the present invention.
The source of embodiment 1 seedling seed culture of viruses and characteristic
1. mink viral enteritis virus JLM strain
Manufacture this product seed culture of viruses system separating obtained from the mink excrement swab that ermine field, Jilin Province gathers, thin through CRFK
Born of the same parents are passed on, and specificity neutralization test, immunofluorescence test, PCR and gene sequencing etc. are identified, it was demonstrated that this poison
Strain is mink viral enteritis virus (MEV).Animal Orthogonal Rotational Regressive Tests shows that this strain only has more weak pathogenicity.
This strain is carried out Attenuation on CRFK cell by us immediately, and virus titer can reach 105.6TCID50/ml
Above.MEV JLM strain virus is carried out the comprehensive identification such as safety, immunogenicity, specificity, pure property,
Show that MEV JLM strain virus has excellent safety, immunogenicity good, aseptic, without mycoplasma, nothing outward
Source virus is polluted.
Described mink viral enteritis virus, named JLM strain, Classification And Nomenclature is mink viral enteritis virus,
Being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is at the Chaoyang District, Beijing City North Star
West Road 1 institute of microbiology of the Chinese Academy of Sciences of institute, its culture presevation is numbered: CGMCC No.9904, the preservation time
It it is on October 30th, 2014
2. mink canine distemper virus JTM strain
Manufacture the morbidity ermine lungs that this product strain system gathers from ermine field, Jiutai City of Jilin Province separating obtained, through Vero
Passage, specificity neutralization test, immunofluorescence test, RT-PCR, gene sequencing and animal return examination
Test etc. and to identify, it was demonstrated that this virus is mink canine distemper virus (CDV).Animal Orthogonal Rotational Regressive Tests, mink tissue of falling ill
After internal organs poison inoculation mink prepared by internal organs, the mink of 2/4 occurs that body temperature raises, eye nasal secretions, loose stool
Etc. symptom.Separation poison is carried out Attenuation on Vero cell by us immediately, and virus titer reaches
105.2TCID50/ more than ml.CDV JTM strain virus is carried out safety, immunogenicity, specificity, pure property
Deng comprehensive identification, show that CDV JTM strain virus safety, immunogenicity are good, aseptic, without mycoplasma, nothing
Exogenous virus pollutes.
Described canine distemper virus, named JTM strain, Classification And Nomenclature is canine distemper virus, is deposited in Chinese micro-life
Thing culture presevation administration committee's common micro-organisms center, address is section in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Institute of microbiology of institute, its culture presevation is numbered: CGMCC No.9905, and the preservation time is in October, 2014
30 days.
The preparation method of embodiment 2 Combined vaccine
1, prepared by seedling virus liquid
Prepared by 1.1 seedling MEV virus liquids
1.1.1 prepare by rolling bottle method
Taking well-grown CRFK cell, synchronizing by MOI is 0.005-0.5 inoculation MEV JLM virus
(CGMCC No.9904), puts and cultivates at 37 DEG C.Virus liquid ,-15 DEG C of freeze thawing 1 time is gathered in the crops after cultivating 4～5
Rear preservation, measures each batch viral level respectively and to carry out steriling test standby.
1.1.2 prepare with suspension culture method
Take well-grown CRFK cell to be inoculated in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours.Control
Rotating speed is 8～12 turns/hour.Become individual cells when cell grows up to fine and close monolayer with 0.25% trypsinization and count,
By 0.8 × 105～1.2 × 105Cell is accessed in 15L bioreactor and cultivates 48～72 hours by individual/ml cell density.
When cell density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in 150L bioreactor
Cultivate 48～72 hours.When cell density reaches 2 × 106Individual/ml time, inoculate MEV with MOI for 0.005-0.5
Produce seed culture of viruses JLM strain, under conditions of 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50% cultivate 36～
48 hours, gather in the crops virus liquid, put less than-15 DEG C preservations, mensuration viral level to carry out steriling test standby.
1.1.3 prepare by microcarrier suspension culture method
Take well-grown CRFK cell to be inoculated in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, control
Rotating speed is 8～12 turns/hour；Become individual cells when cell grows up to fine and close monolayer with 0.25% trypsinization and count,
By 0.8 × 105～1.2 × 105Cell is accessed in one-level bioreactor by individual/ml cell density, and every liter contains microcarrier
5g, cultivates 48～72 hours, when cell density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is turned
Move in two stage biological reactor, every liter of 5g Han microcarrier, cultivates 48～72 hours, when cell density reaches 2 × 106
Individual/ml time, with MOI for 0.005-0.5 inoculate mink viral enteritis virus production seed culture of viruses JLM strain, 37 DEG C,
40～60r/min, cultivate 36～48 hours under conditions of pH value 7.2, dissolved oxygen 50%, gather in the crops virus liquid, put-15 DEG C
Hereinafter preserve, measure viral level and to carry out steriling test standby；
Prepared by 1.2 seedling CDV virus liquids
1.2.1 prepare by rolling bottle method
Taking well-grown Vero cell monolayer by MOI is 0.005-0.5 inoculation CDV JTM virus (CGMCC
No.9905), put and cultivate at 37 DEG C.Gather in the crops virus liquid after cultivating 4～5, preserve after-15 DEG C of freeze thawing 1 time,
Measure each batch viral level respectively and to carry out steriling test standby.
1.2.2 prepare with suspension process
Take well-grown Vero cell to be inoculated in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours.Control to turn
Speed is 8～12 turns/hour.Become individual cells when cell grows up to fine and close monolayer with 0.25% trypsinization and count,
By 0.8 × 105～1.2 × 105Cell is accessed in 15L bioreactor and cultivates 48～72 hours by individual/ml cell density.
When cell density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in 150L bioreactor
Cultivate 48～72 hours.When cell density reaches 2 × 106Individual/ml time, inoculate CDV with MOI for 0.005-0.5
Produce seed culture of viruses JTM strain, under conditions of 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50% cultivate 36～
48 hours, gather in the crops virus liquid, put less than-15 DEG C preservations, mensuration viral level to carry out steriling test standby.
1.1.3 prepare by microcarrier suspension culture method
Take well-grown Vero cell to be inoculated in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, control to turn
Speed is 8～12 turns/hour；Become individual cells when cell grows up to fine and close monolayer with 0.25% trypsinization and count,
By 0.8 × 105～1.2 × 105Cell is accessed in one-level bioreactor by individual/ml cell density, and every liter contains microcarrier
5g, cultivates 48～72 hours.When cell density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is turned
Move in two stage biological reactor, every liter of 5g Han microcarrier, cultivates 48～72 hours, when cell density reaches 2 × 106
Individual/ml time, with MOI for 0.005-0.5 inoculation canine distemper virus produce seed culture of viruses JTM strain, 37 DEG C, 40～
Under conditions of 60r/min, pH value 7.2, dissolved oxygen 50% cultivate 36～48 hours, gather in the crops virus liquid, put-15 DEG C with
Lower preservation, measures viral level and to carry out steriling test standby；
2, viral level measures
2.1MEV viral level measures
The DMEM containing 2% new-born calf serum by seed culture of viruses or vaccine (vaccine first becomes 1 part/ml by diluted)
The vaccine diluted is made 10 times of serial dilutions by culture medium.Take 10-2、10-3、10-4、10-54 dilution factors, connect
Kind cultivating 96 well culture plates of 1～4 hour CRFK cell, each dilution factor inoculates 8 holes, 0.1ml/ hole, with
Time set normal cell controls hole.Put 37 DEG C, containing 5%CO2Incubator is cultivated.Observe 7 ,-15 DEG C of freeze thawing once,
The every hole of Microhemagglutination testing inspection is coagulation, if there is red cell agglutination, is then judged to the positive, uses Spearman-Karber
Formula calculates TCID50。
2.2CDV viral level measures
The DMEM containing 2% new-born calf serum by seed culture of viruses or vaccine (vaccine first becomes 1 part/ml by diluted)
Make 10 times of serial dilutions respectively.Take 10-2、10-3、10-4、10-54 dilution factors, it is thin that monolayer Vero is covered with in inoculation
96 well culture plates of born of the same parents, 8 holes of each dilution factor inoculation, 0.1ml/ hole, set normal cell controls hole simultaneously.Put
37 DEG C, containing 5%CO2Incubator is cultivated 4～5, and observation of cell pathological changes (CPE) uses Spearman-Karber
Method calculates TCID50。
3, steriling test
Steriling test is carried out by " Chinese veterinary pharmacopoeia " (two O mono-O versions) (three) annex.
4, Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid.Two-strain psma ligand ratio is defined as every part vaccine
It is not less than 10 containing MEV2.5TCID50, CDV is not less than 102.0TCID50, calculate two kinds of compositions joins Seedling consumption,
Mix homogeneously, adds required antigen-agent with diluent.MEV with CDV virus mixed liquor is pressed with freeze drying protectant
4:1 volume ratio mix homogeneously, is vaccinogen liquid, quantitative separating adding a cover after mixing.
Being prepared for three batches of mink viral enteritis, canine distemper bigeminal live vaccine the most respectively, lot number is respectively
It is 2008121,2008122 and 2008123.
Embodiment 3 mink viral enteritis, canine distemper bigeminal live vaccine overdose inoculation safety testing
For evaluating mink viral enteritis, canine distemper bigeminal live vaccine inoculates the safety after mink, uses 7～8
Week old mink viral enteritis, canine distemper antigen, antibody equal negative healthy mink 20, be divided into 4 groups, often organize 5
Only.1st group is vaccination group to the 3rd group, and (lot number is: 2008121, to inoculate 3 batches of goods of this vaccine respectively
2008122,2008123), 10 parts/only (normal dose be 1 part/only), the 4th group is matched group, often
Only inoculation 1ml PBS solution, carries out thermometric and clinical observation to experimental animal in after vaccination continuous 21 days, examination
Test all animals of end and cut open inspection.Result shows, after vaccination, each group mink body temperature is normal, the mental status and appetite
Well, injection site and whole body have no untoward reaction.The all animals of off-test are cutd open inspection and observe, and mink is respectively organized dirty
Device is showed no exception.Result shows, mink viral enteritis, canine distemper bigeminal live vaccine (JLM strain+JTM strain)
10 multiple doses are inoculated mink safety.
Embodiment 4, mink viral enteritis, the potency test of canine distemper bigeminal live vaccine
The present embodiment carries out the effect of 3 batches of goods of this vaccine (batch: 2008121,2008122,2008123)
Test.7～10 week old mink viral enteritis, canine distemper antigen, the susceptible water of the equal negative healthy of antibody are selected in test
Ermine 40, test point 4 groups, often group 10.Inoculate 3 batches of these vaccine products respectively for 1st group to the 3rd group, note
Penetrating dosage is 1 part/ml/.4th group is PBS control group.Latter 28 days of immunity, from each immune group and matched group
Each take 5 minks at random, every mink injection MEV ZJ strain poison 1ml by force.After counteracting toxic substances, gather mink excrement every day
Swab carries out Blood coagulation test, and observation mink is spiritual, appetite state, pellet morphology, with or without other symptoms such as diarrhoea,
Test 14 days after counteracting toxic substances and terminate.Remain each immune group and matched group mink, every mink injection CDV DL strain
Attack with strong poison 2ml.After counteracting toxic substances, observe every day mink spirit, appetite state, pellet morphology, with or without diarrhoea,
The symptoms such as eye nose stickiness or purulent secretion, callosity thicken, test 21 days after counteracting toxic substances and terminate.Challenge test is tied
Shu Hou, adds up every treated animal protection situation.Result shows: after vaccination 28 days, and Combined vaccine group attacks MEV ZJ
The protective rate of the most malicious rear 3 batches of vaccine products of strain is respectively 5/5,4/5,5/5；MEV negative control group attacks MEV
ZJ strain poison sequela rate by force is 4/5；Combined vaccine group attacks the protection of the CDV DL strain internal organs rear 3 batches of vaccine products of poison
Rate is respectively 4/5,5/5,5/5；It is 4/5 that CDV negative control group attacks CDV DL strain internal organs poison sequela rate.
Result shows, laboratory products all can provide good protection to MEV and CDV strong virus attack, can effective prevention of water
Ermine viral enteritis and canine distemper.
Fig. 1 is mink viral enteritis, 2008121 batches of goods immune group water of canine distemper bigeminal live vaccine potency test
Ermine counteracting toxic substances rear intestinal photo, display is normal；Fig. 2 is mink viral enteritis, the examination of canine distemper bigeminal live vaccine effect
Test 2008121 batches of goods MEV matched group No.0464 mink counteracting toxic substances rear intestinal photos, show the most hemorrhage
Phenomenon.
Claims (6)
1. a mink viral enteritis, canine distemper bigeminal live vaccine, it is characterised in that effective ingredient includes mink
Viral enteritis virus antigen and canine distemper virus antigen, wherein said mink viral enteritis virus antigen is
Mink viral enteritis virus JLM strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, its culture presevation is numbered: CGMCC No.9904；Described canine distemper virus antigen is canine distemper virus JTM
Strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered:
CGMCC No.9905。
2. mink viral enteritis, canine distemper bigeminal live vaccine as claimed in claim 1, it is characterised in that every
In head part vaccine, mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than
102.0TCID50。
3. mink viral enteritis described in claim 1 or 2, canine distemper bigeminal live vaccine in preparation prevention or
Application in treatment mink viral enteritis and canine distemper medicine.
4. the mink viral enteritis prepared described in claim 1 or 2, the turning of canine distemper bigeminal live vaccine
Bottle cultural method, it is characterised in that comprise the following steps:
(1) prepared by seedling mink viral enteritis virosis venom
Taking well-grown CRFK cell by MOI is that 0.005-0.5 inoculates mink viral enteritis virus JLM strain
Virus, puts and cultivates at 37 DEG C, gathers in the crops virus liquid after cultivating 4～5, preserves, measure sick after-15 DEG C of freeze thawing 1 time
Poison content to carry out steriling test standby；
(2) prepared by seedling canine distemper virus virus liquid
Taking well-grown Vero or MDCK cell monolayer by MOI is that 0.005-0.5 inoculates canine distemper virus
JTM strain virus, puts and cultivates at 37 DEG C, gathers in the crops virus liquid after cultivating 4～5, preservation after-15 DEG C of freeze thawing 1 time,
Measure viral level and to carry out steriling test standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By viral for mink intestinal
Scorching virus is mixed homogeneously by 4:1 volume ratio with freeze drying protectant with canine distemper virus virus mixed liquor, is vaccine
Stock solution, quantitative separating adding a cover after mixing.
5. the mink viral enteritis prepared described in claim 1 or 2, canine distemper bigeminal live vaccine outstanding
Floating cultural method, it is characterised in that comprise the following steps:
(1) preparation of seedling mink viral enteritis virosis venom: take well-grown CRFK cell and connect
Planting in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell is long
Become individual cells with 0.25% trypsinization during the fine and close monolayer of one-tenth and count, by 0.8 × 105～1.2 × 105Individual/ml cell
Cell is accessed in one-level bioreactor and cultivates 48～72 hours by density, when cell density is not less than 2 × 106Individual/ml
Time, it is transferred in two stage biological reactor cultivate 48～72 hours by cell according to similarity condition, works as cell density
Reach 2 × 106Individual/ml time, with MOI for 0.005-0.5 inoculate mink viral enteritis virus production seed culture of viruses JLM
Strain, cultivates 36～48 hours under conditions of 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50%, results
Virus liquid, puts less than-15 DEG C preservations, measures viral level and to carry out steriling test standby；
(2) preparation of seedling canine distemper virus virus liquid: take well-grown Vero or mdck cell inoculation
In cell spinner bottle, putting 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell grows up to
Become individual cells with 0.25% trypsinization during fine and close monolayer and count, by 0.8 × 105～1.2 × 105Individual/ml cell is close
Cell is accessed in one-level bioreactor and cultivates 48～72 hours by degree, when cell density is not less than 2 × 106Individual/ml
Time, it is transferred in two stage biological reactor cultivate 48～72 hours by cell according to similarity condition, works as cell density
Reach 2 × 106Individual/ml time, with MOI for 0.005-0.5 inoculation canine distemper virus produce seed culture of viruses JTM strain, 37
DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50% under conditions of cultivate 36～48 hours, gather in the crops virus liquid,
Put less than-15 DEG C preservations, measure viral level and to carry out steriling test standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By viral for mink intestinal
Scorching virus is mixed homogeneously by 4:1 volume ratio with freeze drying protectant with canine distemper virus virus mixed liquor, is vaccine
Stock solution, quantitative separating adding a cover after mixing.
6. the mink viral enteritis prepared described in claim 1 or 2, canine distemper bigeminal live vaccine micro-
Carrier suspension culture method, it is characterised in that comprise the following steps:
(1) preparation of seedling mink viral enteritis virosis venom: take well-grown CRFK cell and connect
Planting in cell spinner bottle, put 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell is long
Become individual cells with 0.25% trypsinization during the fine and close monolayer of one-tenth and count, by 0.8 × 105～1.2 × 105Individual/ml cell
Cell is accessed in one-level bioreactor by density, every liter of 5g Han microcarrier, cultivates 48～72 hours, works as cell
Density is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in two stage biological reactor, every liter contains
Microcarrier 5g, cultivates 48～72 hours, when cell density reaches 2 × 106Individual/ml time, with MOI as 0.005-0.5
Inoculation mink viral enteritis virus production seed culture of viruses JLM strain, at 37 DEG C, 40～60r/min, pH value 7.2, molten
Cultivate 36～48 hours under conditions of oxygen 50%, gather in the crops virus liquid, put less than-15 DEG C preservations, measure viral level
And it is standby to carry out steriling test；
(2) preparation of seedling canine distemper virus virus liquid: take well-grown Vero or mdck cell inoculation
In cell spinner bottle, putting 37 DEG C and cultivate 48～72 hours, controlling rotating speed is 8～12 turns/hour；When cell grows up to
Become individual cells with 0.25% trypsinization during fine and close monolayer and count, by 0.8 × 105～1.2 × 105Individual/ml cell is close
Cell is accessed in one-level bioreactor by degree, and every liter of 5g Han microcarrier cultivates 48～72 hours, when cell is close
Degree is not less than 2 × 106Individual/ml time, according to similarity condition, cell is transferred in two stage biological reactor, every liter containing micro-
Carrier 5g, cultivates 48～72 hours, when cell density reaches 2 × 106Individual/ml time, with MOI as 0.005-0.5
Inoculation canine distemper virus produces seed culture of viruses JTM strain, at 37 DEG C, 40～60r/min, pH value 7.2, dissolved oxygen 50%
Under the conditions of cultivate 36～48 hours, gather in the crops virus liquid, put less than-15 DEG C preservations, mensuration viral level also carries out nothing
Bacterium inspection is standby；
(3) Seedling and subpackage are joined
Check qualified virus liquid as seedling virus liquid, two-strain psma ligand ratio is defined as every part vaccine
Middle mink viral enteritis viral level is not less than 102.5TCID50, canine distemper virus content is not less than 102.0TCID50,
Calculate two kinds of compositions joins Seedling consumption, mix homogeneously, adds required antigen-agent with diluent；By viral for mink intestinal
Scorching virus is mixed homogeneously by 4:1 volume ratio with freeze drying protectant with canine distemper virus virus mixed liquor, is vaccine
Stock solution, quantitative separating adding a cover after mixing.
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| CN109652344A (en) * | 2016-02-17 | 2019-04-19 | 中国农业科学院特产研究所 | Bacterial strain and its application and vaccine and preparation method thereof |
| CN105816869A (en) * | 2016-04-26 | 2016-08-03 | 中国农业科学院特产研究所 | Preparation method of mink canine distemper virus live vaccine and vaccine prepared by same |
| CN105816872A (en) * | 2016-04-26 | 2016-08-03 | 中国农业科学院特产研究所 | Preparation method of mink parvoviral enteritis inactivated vaccine and vaccine prepared by using same |
| CN105838683A (en) * | 2016-05-18 | 2016-08-10 | 山东省滨州畜牧兽医研究院 | Method for proliferation of mink canine distemper virus by applying novel cell microcarrier |
| CN107794248A (en) * | 2017-10-24 | 2018-03-13 | 广州齐志生物工程设备有限公司 | A kind of method with microcarrier suspension culture CDV |
| CN109280648B (en) * | 2017-12-20 | 2021-11-30 | 吉林特研生物技术有限责任公司 | Method for preparing mink parvoviral enteritis antigen-protein complex, antigen-protein complex and application of antigen-protein complex |
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