CN107115525A - Racoon dog canine distemper live vaccine and its preparation method and application - Google Patents
Racoon dog canine distemper live vaccine and its preparation method and application Download PDFInfo
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- CN107115525A CN107115525A CN201710343618.1A CN201710343618A CN107115525A CN 107115525 A CN107115525 A CN 107115525A CN 201710343618 A CN201710343618 A CN 201710343618A CN 107115525 A CN107115525 A CN 107115525A
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- racoon dog
- cdv
- canine distemper
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10051—Methods of production or purification of viral material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention discloses a kind of racoon dog canine distemper live vaccine and its preparation method and application, belong to veterinary biologicses technical field.A kind of racoon dog canine distemper live vaccine of the present invention, it is aqueous vaccine, wherein active ingredient is racoon dog CDV attenuated vaccine strain, the attenuated vaccine strain is inventor by way of racoon dog body continuous passage, one plant obtained is tamed from canine distemper vaccine strain CDV O2 to racoon dog safety, the canine distemper attenuated vaccine strain that immunogenicity is good, titre is stable, OR12 plants of CDV is named as, its microbial preservation number is CGMCC NO.13793.It is demonstrated experimentally that the vaccine can effectively protect racoon dog from the attack of the strong poison of CDV, protective rate can effectively prevent the generation of racoon dog canine distemper more than 90%, and duration of immunity is 6 months.The proposition of the present invention provides a kind of new technological means for the prevention and control of racoon dog canine distemper.
Description
Technical field
The present invention relates to a kind of racoon dog canine distemper live vaccine and its preparation method and application, the invention belongs to veterinary biologicses
Technical field.
Background technology
Canine distemper is a kind of acute, hot, high degree in contact as caused by paramyxovirus section Morbillivirus CDV
Sexually transmitted disease, young animal is mostly acute, lethal is passed through, and adults can be into chronic, persistent infection, and clinical symptoms are with double
Phase body temperature is raised, and is principal character with catarrhal pneumonia, gastroenteritis, occasionally there is nervous symptoms appearance, other easy secondary bacteriums
The mixed infection of virus and superinfection, the death rate may be up to 80%, be the most serious for endangering China's fur animal farming
One of epidemic disease.
Racoon dog canine distemper annual any season can occur, and especially young racoon dog is before and after ablactation and finishing period is quicker
Sense, with very high morbidity and mortality, aged racoon dog has certain resistance.It is the main of racoon dog canine distemper with malicious animal
The infection sources, by eye and nasal discharge, saliva, urine, excrement discharge virus, mainly through alimentary canal, respiratory tract infection.Due to racoon dog dog
The characteristics of pest have that morbidity is anxious, lack specific treatment method, racoon dog canine distemper still using vaccine inoculation as main preventions, but
Do not have the sale of racoon dog canine distemper vaccine in the market, many raisers are with the canine distemper vaccine of the animals such as dog, mink to pre-
Anti- racoon dog canine distemper, it is impossible to obtain preferable immune protective effect.Therefore, the good racoon dog canine distemper epidemic disease of exploitation safety, immune effect
Seedling, is not only the active demand in industry and market, with wide market application foreground, has for prevention and control racoon dog canine distemper important
Meaning.
The content of the invention
It is an object of the invention to provide a kind of racoon dog canine distemper live vaccine and preparation method thereof.
In order to achieve the above object, present invention employs following technological means：
The present invention is by way of the racoon dog body continuous passage, and domestication's canine distemper vaccine strain CDV-O2 obtains one plant
To racoon dog safety, CDV-OR12 plants of the racoon dog CDV attenuated vaccine strain that immunogenicity is good, titre is stable, and with this strain system
For racoon dog canine distemper live vaccine (aqueous vaccine).
A kind of racoon dog canine distemper live vaccine of the present invention, is aqueous vaccine, and its active ingredient is the weak malicious epidemic disease of racoon dog CDV
Miao Zhu, described racoon dog CDV attenuated vaccine strain, is named as CDV-OR12 plants, Classification And Nomenclature is CDV
(Canine Distemper Virus), the vaccine strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, preservation address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, microbial preservation
Number be CGMCC NO.13793, the preservation time be on 04 21st, 2017.
In racoon dog canine distemper live vaccine of the present invention, it is preferred that contained racoon dog CDV attenuated vaccine strain
TCID50≥104.5/ head part, it is furthermore preferred that the TCID of contained racoon dog CDV attenuated vaccine strain50For 104.5-
105.5/ head part.
Further, the invention also provides a kind of method for preparing described racoon dog canine distemper live vaccine, it is characterised in that：
Comprise the following steps：
(1) CDV-OR12 plants described of racoon dog CDV attenuated vaccine strain is taken to be inoculated with Vero in the way of adsorbing and connect poison thin
Virus amplification culture is carried out on born of the same parents, daily observation cell virus situation passes through multigelation when cytopathy reaches 75-85%
Method harvest virus liquid, and carry out viral level, pure property examine；
(2) after qualified virus liquid will be examined with the MEM nutrient solutions dilution of serum-free, quantitative separating, and it is stored in -20
DEG C, as racoon dog canine distemper live vaccine.
In method of the present invention, it is preferred that racoon dog CDV attenuated vaccine in the virus liquid that step (1) is obtained
The TCID of strain50≥104.5/ml。
In method of the present invention, it is preferred that racoon dog CDV attenuated vaccine in the vaccine finished product prepared
The content TCID of strain50≥104.5/ head part, it is furthermore preferred that the TCID of contained racoon dog CDV attenuated vaccine strain50For
104.5-105.5/ head part.
Further, the invention also provides described racoon dog canine distemper live vaccine is in prevention racoon dog canine distemper medicine is prepared
Purposes.
It is demonstrated experimentally that the vaccine that the present invention is prepared can effectively protect racoon dog from the attack of the strong poison of CDV, exempt from
Epidemic disease 3 weeks and protective rate is 100% after 3 months, protective rate is 90% after being immunized 6 months.Therefore, the vaccine can effectively prevent racoon dog
The generation of canine distemper, duration of immunity is 6 months, and proposition of the invention provides a kind of new technology hand for the prevention and control of racoon dog canine distemper
Section.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.
The CDV vaccine strain CDV-O2 of embodiment 1 domestication and identification
1. CDV vaccine strain CDV-O2 domestication
(1) the CDV vaccine strain CDV-O2 of preservation is inoculated in African green monkey kidney cell in the way of adsorbing and connect poison
(Vero) virus amplification culture, daily observation cell virus situation, by anti-when cytopathy reaches 80% or so are carried out on
The method harvest virus liquid of multiple freeze thawing, and carry out viral level (TCID50≥105.0/ ml), steriling test.Qualified disease will be examined
Venom is dispensed, and is stored in -20 DEG C.
(2) the CDV-O2 virus liquid stostes hindlimb muscle of amplification is inoculated with the healthy racoon dog 3 of negative antibody, every 5ml divides three
Point inoculation, isolated rearing and the every physical signs of the racoon dog of observation inoculation daily.The 3rd after inoculation, cut open respectively within 5 days and kill racoon dog 1, sterile bar
Spleen is gathered under part, and virus purification is carried out using Vero cells, and passes through CDV specific primer and hundstaupe pyreticosis
Malicious positive serum is identified.
(3) CDV being separated to is labeled as CDV-OR3, domestication 1st generation (F1) as in racoon dog body, by CDV-
OR3 is inoculated in progress virus amplification culture on Vero cells in the way of adsorbing and connect poison, and daily observation cell virus situation treats thin
Virus liquid is harvested by the method for multigelation when born of the same parents' lesion reaches 80% or so, and carries out viral level (TCID50≥105.0/
Ml), steriling test.Qualified virus liquid packing will be examined, and be stored in -20 DEG C.
(4) repeat step (2), (3), by CDV, continuous domestication 9 times again in racoon dog body, obtain CDV weak
Toxic vaccine strain, it is the 10th generation (F10) strain tamed in racoon dog body, and the strain is named as into CDV-OR12 plants, in being deposited in
State's Microbiological Culture Collection administration committee common micro-organisms center, preservation address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica, microbial preservation number is CGMCC NO.13793, and the preservation time is 04 month 2017 21
Day.
2. CDV attenuated vaccine strain CDV-OR12 is to racoon dog immunogenicity determining
Go bail for the CDV attenuated vaccine strain CDV-OR12 deposited, and adjustment viral level is 104.5TCID50/ ml, hind leg
Intramuscular inoculation negative antibody health racoon dog 5, every 1ml, inoculation determines CDV neutralize antibody titers in serum after 21 days,
And poison is attacked with strong HBF-1 plants of the poison of CDV, determine protective rate.As a result show, CDV attenuated vaccine strain CDV-
CDV neutralize antibody titers are respectively in OR12 inoculation racoon dog serum：1:256、1:362、1:256、1:181、1:362, attack
Malicious protective rate is 100% (5/5).
3. CDV attenuated vaccine strain CDV-OR12 returns strong experiment to the virulence of racoon dog
Go bail for the continuous 5 generation intramuscular inoculation negative antibody racoon dogs of the CDV attenuated vaccine strain CDV-OR12 deposited, per Dai Lian
Continuous observation 14 days.As a result show and acted normally per physical signs such as pickup kind racoon dog body temperature, appetite, the state of mind, excrement, do not gone out
Existing canine distemper symptom, illustrates that the CDV attenuated vaccine strain CDV-OR12 avirulences return strong phenomenon, available for racoon dog canine distemper
The research of vaccine.
The preparation of the racoon dog canine distemper live vaccine (aqueous vaccine) of embodiment 2
1. the preparation of CDV seedling liquid
CDV-OR12 plants of the CDV attenuated vaccine strain deposited of going bail for is inoculated with the way of adsorbing and connecing poison on Vero cells
Virus amplification culture is carried out, daily observation cell virus situation passes through multigelation when cytopathy reaches 80% or so
Method harvests virus liquid, and carries out racoon dog CDV attenuated vaccine strain in viral level, the inspection of pure property, obtained virus liquid
Content TCID50≥104.5/ml.According to the viral level data measured, qualified virus liquid will be examined to be trained with the MEM of serum-free
After nutrient solution suitably dilution, quantitative separating is stored in -20 DEG C in 90ml vaccines bottle.
2. the measure of CDV content in vaccine finished product
Vaccine finished product carries out viral level measure, CDV content TCID50For 104.5-105.5/ head part.
The safety testing of the racoon dog canine distemper live vaccine of embodiment 3 (CDV-OR12 plants)
Take and examine qualified racoon dog canine distemper live vaccine (prepared by embodiment 2), hindlimb muscle inoculation negative antibody racoon dog, if single dose
Amount (1 part of inoculation, CDV content TCID50=104.5/ head part), single dose repeats and overdose (10 times) immunoprophylaxis
Group, while setting not vaccine inoculation racoon dog as negative control.After vaccine inoculation, body temperature, appetite, the spiritual shape of continuous 14 days observation racoon dogs
The physical signs such as state, excrement, and cut open inspection observation inoculation racoon dog main organs lesion situation.Result of the test shows that racoon dog is through single dose, list
Dosage is repeated with after overdose inoculation, and every physical signs is normal, and any clinical symptoms, cut open inspection observation inoculation racoon dog are not shown
Main organs illustrate racoon dog canine distemper live vaccine to racoon dog safety without lesion.
The effect and immune period test of the racoon dog canine distemper live vaccine of embodiment 4 (CDV-OR12 plants)
Take and examine qualified racoon dog canine distemper live vaccine (prepared by embodiment 2), hindlimb muscle inoculation negative antibody racoon dog, every
1ml (1 part of inoculation, CDV content TCID50=104.5/ head part), while setting inoculation Vero cell culture fluid control groups.
Inoculation 3 weeks, 3 months, 6 months and blood sampling, separation serum after 9 months, determine CDV neutralizing antibody in serum, make simultaneously
With 100 median lethal dose (LD50) hundstaupe hot inspection attacked with strong poison HBF-1, continuous 14 days observation attack malicious racoon dog body temperature,
The physical signs such as appetite, the state of mind, excrement.
As a result show, be inoculated with 3 weeks and after 3 months, CDV neutralize antibody titers are 1 in inoculation racoon dog serum:256
More than, racoon dog malicious protective rate strong to CDV is 100% (10/10)；After inoculation 6 months, racoon dog is strong to CDV
The protective rate of poison is 90% (9/10)；After inoculation 9 months, racoon dog malicious protective rate strong to CDV is 70% (7/10).Cause
This, the duration of immunity of the vaccine is set to 6 months.
The racoon dog canine distemper live vaccine of embodiment 5 (CDV-OR12 plants) is with similar vaccine (CDV3-CL plants) to the immune efficacy of racoon dog
Comparative test
Take and examine qualified racoon dog canine distemper live vaccine (prepared by embodiment 2), and mink canine distemper on the market is lived
Hindlimb muscle is inoculated with negative antibody racoon dog, 1 part (CDV content TCID of every inoculation to (CDV3-CL plants) of vaccine respectively50=
104.5/ head part), while setting the control group of non-vaccine inoculation.Inoculation 3 weeks, 3 months, 6 months and blood sampling, separation blood after 9 months
Clearly, CDV neutralizing antibody in serum is determined, while using 100 median lethal dose (LD50) hundstaupe hot inspection with strong
Malicious HBF-1 is attacked, and the physical signs such as malicious racoon dog body temperature, appetite, the state of mind, excrement are attacked in observation in continuous 14 days.
As a result show, be inoculated with 3 weeks and after 3 months, it is in CDV and anti-in racoon dog canine distemper live vaccine immune group serum
Body potency is 1:256, inoculation racoon dog malicious protective rate strong to CDV is 100% (10/10), mink canine distemper live vaccine
CDV neutralize antibody titers are 1 in immune group serum:More than 128, it is inoculated with racoon dog malicious protection strong to CDV
Rate is 100% (10/10)；After inoculation 6 months, the inoculation racoon dog malicious protection strong to CDV of racoon dog canine distemper live vaccine immune group
Rate is 100% (10/10), and mink canine distemper live vaccine immune group inoculation racoon dog malicious protective rate strong to CDV is 80%
(8/10)；After inoculation 9 months, racoon dog canine distemper live vaccine immune group inoculation racoon dog malicious protective rate strong to CDV is 70%
(7/10), mink canine distemper live vaccine immune group inoculation racoon dog malicious protective rate strong to CDV is only 50% (5/10).
Result above shows：The immune efficacy of racoon dog canine distemper live vaccine (CDV-OR12 plants) prepared by the present invention is better than facing
The mink canine distemper live vaccine applied on bed.
Above test result indicates that the racoon dog canine distemper live vaccine (CDV-OR12 plants) that the present invention is prepared can be protected effectively
Generation of the racoon dog from the attack, effectively prevention racoon dog canine distemper of the strong poison of CDV.
Claims (8)
1. a kind of racoon dog canine distemper live vaccine, is aqueous vaccine, it is characterised in that its active ingredient is the weak malicious epidemic disease of racoon dog CDV
Miao Zhu, described racoon dog CDV attenuated vaccine strain, is named as CDV-OR12 plants, the vaccine strain is deposited in China Microbiological bacterium
Preservation administration committee common micro-organisms center is planted, preservation address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science
Institute of microbiology of institute, microbial preservation number is CGMCC NO.13793, and the preservation time is on 04 21st, 2017.
2. racoon dog canine distemper live vaccine as claimed in claim 1, it is characterised in that contained racoon dog canine distemper attenuated vaccine strain
TCID50≥104.5/ head part.
3. racoon dog canine distemper live vaccine as claimed in claim 1, it is characterised in that contained racoon dog canine distemper attenuated vaccine strain
TCID50For 104.5-105.5/ head part.
4. a kind of method of the racoon dog canine distemper live vaccine prepared described in claim 1, it is characterised in that：Comprise the following steps：
(1) CDV-OR12 plants described of racoon dog CDV attenuated vaccine strain is taken to be inoculated with the way of adsorbing and connect poison on Vero cells
Virus amplification culture is carried out, daily observation cell virus situation passes through the side of multigelation when cytopathy reaches 75-85%
Method harvests virus liquid, and carries out viral level, the inspection of pure property；
(2) after qualified virus liquid will be examined with the MEM nutrient solutions dilution of serum-free, quantitative separating, and be stored in -20 DEG C, i.e.,
For racoon dog canine distemper live vaccine.
5. method as claimed in claim 4, it is characterised in that the weak malicious epidemic disease of racoon dog CDV in the virus liquid that step (1) is obtained
The TCID of seedling strain50≥104.5/ml。
6. method as claimed in claim 4, it is characterised in that the weak malicious epidemic disease of racoon dog CDV in the vaccine finished product prepared
The TCID of seedling strain50≥104.5/ head part.
7. method as claimed in claim 6, it is characterised in that the weak malicious epidemic disease of racoon dog CDV in the vaccine finished product prepared
The TCID of seedling strain50For 104.5-105.5/ head part.
8. purposes of the racoon dog canine distemper live vaccine in prevention racoon dog canine distemper medicine is prepared described in claim any one of 1-3.
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Cited By (1)
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| CN109868262A (en) * | 2018-04-27 | 2019-06-11 | 青岛农业大学 | A kind of Strain 3 of Canine Distemper and its application |
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| CN109868262B (en) * | 2018-04-27 | 2022-03-04 | 青岛农业大学 | Canine distemper attenuated strain and application thereof |
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