CN110302374A - Canine vaccine and its preparation method and application - Google Patents
Canine vaccine and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of Canine vaccines and its preparation method and application, are related to veterinary drug biological product technical field.The Canine vaccine includes the canine distemper virus antigen, the immune Canine Parvovirus antigen measured, the rabies virus antigen that amount is immunized of immune amount, and the canine distemper virus antigen includes the canine distemper velogen strain LN (10) 1 of deactivated form；The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.The effect that the vaccine can be realized by primary immunization while be prevented and treated canine distemper, Canine Parvovirus Enteritis and rabies, the immune time of canid vaccine can be effectively reduced, and then alleviates existing single vaccine and need to repeat repeatedly the problem of immune, caused working strength increases and be easy to cause the stress reaction of canid during canid immunoprophylaxis.
Description
Technical field
The present invention relates to veterinary drug biological product technical field, more particularly, to a kind of Canine vaccine and preparation method thereof and
Using.
Background technique
Canine distemper is by paramyxovirus section (Paramyxoviridae) Morbillivirus (Morbillivirus) canine distemper
Acute, hot, highly contagious disease caused by viral (Canine Distemper Virus, CDV), is mainly characterized by
To encroach on based on mucosal system, there is the raising of two-phase body temperature, with catarrhal pneumonia, gastroenteritis, occasionally there is nervous symptoms, be easy after
Mixed infection and the superinfection of other bacterial viruses are sent out, the death rate may be up to 80%.It is the most dangerous infection sources with poison animal,
By eye and nasal discharge, saliva, urine, excrement discharge virus, mainly through alimentary canal, respiratory tract infection, in recent years, with army
Dog, police dog, experimental dog and increasing considerably for pet dog breeding amount and increasing for strange land exchange, the hair in China dog
Sick rate and lethality have raised trend.
Canine parvoviral enteritis is to belong to (Parvovirus) by Parvoviridae (Parvoviridae) parvovirus
In the highly contagious disease of world's distribution caused by canine parvovirus (Canine Parvovirus, CPV) infection, mainly
Cause dog acute hemorrhagic entertitis or Neonatal Canine acute myocarditis, higher morbidity and mortality are presented.CPV can infect difference
The dog at age and different cultivars, but the neurological susceptibility of purebred puppy is higher.The clinical symptoms of morbidity dog are mainly that body temperature increases, acutely
Vomiting, hemorrhagic enteritis and leucocyte substantially reduce, and rehabilitation dog can still pass through excrement outside toxin expelling for a long time.The height of canine parvovirus
Infection rate and lethality cause huge loss to the cultivation of canine farming and pet.
Rabies are by the rabies viruses of Rhabdoviridae (Rhabdoviridae) Lyssavirus (Lyssavirus)
The Arbo infectious disease of the performance of acute central nervous system caused by (Rabies virus, RV).Rabies belong to nature epidemic disease
Source property disease, case fatality rate are in first of each infectious disease, its lethality is 100% after symptom occurs in infection, mainly pass through breakage
Skin or newborn film invade body, central nervous system is advanced on nerve endings, clinical manifestation is predominantly acute, carries out
Property, almost irreversible encephalomyelitis, be with mad dry uneasiness, behavior abnormality, attack, progressive paralysis and final death
Feature.Rabies viruses is mainly to infect the sick dog of rabies viruses and with malicious animal as major source of infection.In China, rabies are
Caused by sick dog is propagated.Can be with virus in some salivas without obvious clinical symptoms or the dog for seeming health, the malicious rate of band is also very high,
The dangerous infection sources can also be become.
Currently, in canid field of immunology, three kinds of above-mentioned canine distemper, canine parvoviral enteritis and rabies diseases
The health for drastically influencing canid, solving these problems most effective, direct method is exactly vaccine inoculation.But due to
Above-mentioned three kinds of virus uncommon antigenicity each other causes to need during immunoprophylaxis duplicate animal to be immunized
Inoculation, not only increases working strength, also be easy to cause the stress reaction of canid.
Therefore, a kind of safe and effective Canine vaccine is researched and developed, which can be to canine distemper, parvovirus
Enteritis prevents and treats simultaneously with rabies, and then realizes the effect of " anti-three disease of primary immunization ", and existing exempt from is effectively relieved
Duplicate the problem of immunity inoculation is carried out to animal is needed during epidemic disease prevention, becomes very necessary and urgent, will also have
Vast market prospect.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of Canine vaccine, and the Canine vaccine can be realized by primary immunization
The effect prevented and treated simultaneously canine distemper, Canine Parvovirus Enteritis and rabies, can effectively reduce canid vaccine
Immune time, and then alleviate what existing single vaccine needed to repeat repeatedly to be immunized during canid immunoprophylaxis
Problem.
The second object of the present invention is to provide a kind of preparation method of Canine vaccine, and this method has preparation process letter
It is single, easy to operate, the advantages of being suitble to large-scale industrial production.
The third object of the present invention is to provide a kind of application of Canine vaccine, which can be widely applied to make
It is standby to prevent and treat in canid canine distemper, Canine Parvovirus Enteritis and rabic drug.
A kind of Canine vaccine provided by the invention, the Canine vaccine include the canine distemper virus antigen of immune amount, are immunized
The rabies virus antigen of the Canine Parvovirus antigen of amount and immune amount；
The canine distemper virus antigen includes the canine distemper velogen strain LN (10) 1 of deactivated form；
The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；
The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.
Further, in the Canine vaccine, the content that the canine distemper virus antigen of amount is immunized is 105.0~
107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 105.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~107.0TCID50/ head part.
Further, in the Canine vaccine, the content that the canine distemper virus antigen of amount is immunized is 106.0~
107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 106.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~106.0TCID50/ head part.
Further, the canine distemper virus antigen, Canine Parvovirus antigen and rabies virus antigen are deactivated form
Totivirus antigen.
Further, the biological deposits number of the canine distemper velogen strain LN (10) 1 are CGMCC No.17981；
Preferably, the biological deposits number of parvovirus velogen strain JL (18) 1-Beagle are CGMCC No.17983；
Preferably, the biological deposits number of the rabies viruses low virulent strain HEP-Flury are CGMCC No.17982.
Further, the Canine vaccine further includes veterinarily acceptable carrier；
Preferably, the veterinarily acceptable carrier includes freeze drying protectant and adjuvant；
It is furthermore preferred that the adjuvant includes ADJ-801.
A kind of preparation method of above-mentioned Canine vaccine provided by the invention, the preparation method comprises the following steps:
First by the canine distemper virus antigen of immune amount, the rabies viruses of the Canine Parvovirus antigen of immune amount and immune amount
Antigen mixes, and veterinarily acceptable adjuvant is then added, and Canine vaccine is made.
Further, the preparation method further includes the steps that Canine vaccine obtained carrying out freeze-drying preservation；
Preferably, the step of freeze-drying preservation includes that Canine vaccine obtained is added to freeze drying protectant, is then carried out
Lyophil preservation.
Further, the preparation method specifically includes the following steps:
(a), it is proliferated strain: culture proliferation canine distemper velogen strain LN (10) 1, parvovirus velogen strain JL (18) 1- respectively
Beagle and rabies viruses low virulent strain HEP-Flury；
(b), strain inactivates: by canine distemper velogen strain LN (10) 1, the parvovirus velogen strain after step (a) culture proliferation
JL (18) 1-Beagle and rabies viruses low virulent strain HEP-Flury are inactivated, and canine distemper virus, canine parvovirus are respectively obtained
With the totivirus antigen of rabies viruses deactivated form；
(c), vaccine formulation: canine distemper virus antigen, Canine Parvovirus antigen, the rabies virus antigen after inactivation are mixed
And each antigenic content is adjusted, veterinarily acceptable adjuvant is then added, Canine vaccine is made；
(d), preservation is lyophilized: freeze drying protectant is added in Canine vaccine made from step (c), then carries out freeze-drying guarantor
Hiding.
Preferably, the inactivator of step (b) the mesogen strain inactivation is beta-propiolactone.
A kind of above-mentioned Canine vaccine provided by the invention prevents and treats canid canine distemper, parvovirus in preparation
Application in enteritis and rabic drug.
Compared with prior art, the invention has the benefit that
Canine vaccine provided by the invention, the Canine vaccine include the canine distemper virus antigen of immune amount, immune amount
The rabies virus antigen of Canine Parvovirus antigen and immune amount, wherein the canine distemper virus antigen includes the hundstaupe of deactivated form
Hot velogen strain LN (10) 1；The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；
The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.The vaccine passes through primary immunization
It realizes the effect prevented and treated simultaneously canine distemper, Canine Parvovirus Enteritis and rabies, canid epidemic disease can be effectively reduced
The immune time of seedling, and then alleviate existing single vaccine and need to repeat multi-time no during canid immunoprophylaxis
The problem of epidemic disease, caused working strength increases and be easy to cause the stress reaction of canid.
The preparation method of Canine vaccine provided by the invention, the preparation method first resist the canine distemper virus of immune amount
The Canine Parvovirus antigen of former, immune amount and the rabies virus antigen of immune amount mix, and are then added veterinarily acceptable
Canine vaccine is made in adjuvant.The advantages of this method has preparation process simple, easy to operate, is suitble to large-scale industrial production.
Canine vaccine provided by the invention can be widely applied to preparation and prevent and treat canid canine distemper, tiny disease
In viral enteritis and rabic drug.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
According to an aspect of the present invention, a kind of Canine vaccine, the Canine vaccine include the canine distemper virus of immune amount
The rabies virus antigen of antigen, the Canine Parvovirus antigen of immune amount and immune amount；
The canine distemper virus antigen includes the canine distemper velogen strain LN (10) 1 of deactivated form；
The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；
The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.
Canine vaccine provided by the invention, the Canine vaccine include the canine distemper virus antigen of immune amount, immune amount
The rabies virus antigen of Canine Parvovirus antigen, immune amount, wherein the canine distemper virus antigen includes the hundstaupe of deactivated form
Hot velogen strain LN (10) 1；The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；
The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.The vaccine passes through primary immunization
It realizes the effect prevented and treated simultaneously canine distemper, Canine Parvovirus Enteritis and rabies, canid epidemic disease can be effectively reduced
The immune time of seedling, and then alleviate existing single vaccine and need to repeat multi-time no during canid immunoprophylaxis
The problem of epidemic disease, caused working strength increases and be easy to cause the stress reaction of canid.
In the preferred embodiment of the present invention, in the Canine vaccine, the canine distemper virus antigen of amount is immunized
Content is 105.0~107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 105.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~107.0TCID50/ head part.
As a preferred embodiment, the canine distemper virus antigen, canine parvovirus in above-mentioned content range are anti-
After the mixing of former and rabies virus antigen, the mutual immune interference or influence of antigenic component will not be not only generated, is generating guarantor respectively
While the response of shield property, canine distemper virus antigen, Canine Parvovirus antigen and rabies virus antigen also have mutually enhancing immune
The effect of effect.
The typical but non-limiting preferred embodiment of content of above-mentioned canine distemper virus antigen are as follows: 105.0TCID50/ head
Part, 105.5TCID50/ head part, 106.0TCID50/ head part, 106.5TCID50/ head part and 107.0TCID50/ head part；Above-mentioned canine parvovirus
The typical but non-limiting preferred embodiment of the content of malicious antigen are as follows: 105.0TCID50/ head part, 105.5TCID50/ head part,
106.0TCID50/ head part, 106.5TCID50/ head part and 107.0TCID50/ head part；The content of above-mentioned rabies virus antigen is typical but non-
Restrictive preferred embodiment are as follows: 105.67TCID50/ head part, 106.0TCID50/ head part, 106.5TCID50/ head part and
107.0TCID50/ head part.
Further, in the Canine vaccine, the content that the canine distemper virus antigen of amount is immunized is 106.0~
107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 106.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~106.0TCID50/ head part.
Preferably, in the Canine vaccine, the content that the canine distemper virus antigen of amount is immunized is 106.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 106.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67TCID50/ head part.
In the present invention, by the further adjustment and optimization to each viral antigen content ratio, to advanced optimize
The drug effect of Canine vaccine of the present invention.
In the preferred embodiment of the present invention, the canine distemper virus antigen, Canine Parvovirus antigen and mad dog
Viral antigen is the totivirus antigen of deactivated form.
As a preferred embodiment, above-mentioned canine distemper virus antigen, Canine Parvovirus antigen and rabies viruses are anti-
Original is the totivirus antigen of deactivated form, and above-mentioned antigen does not have an infectivity after inactivating, but its as totivirus antigen according to
So maintain all natural conformation of canine distemper virus, canine parvovirus and rabies viruses, i.e., whole immunogenicity, after inoculation
Immune response can be carried out with effective stimulus canine animals body.
In the preferred embodiment of the present invention, the biological deposits number of the canine distemper velogen strain LN (10) 1 are
CGMCC No.17981；The preservation time are as follows: on June 3rd, 2019；Depositary institution: China Committee for Culture Collection of Microorganisms
Common micro-organisms center；Preservation address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute.
In the preferred embodiment of the present invention, the biology of parvovirus velogen strain JL (18) 1-Beagle is protected
Hiding number is CGMCC No.17983；The preservation time are as follows: on June 3rd, 2019；Depositary institution: Chinese microorganism strain preservation management
Committee's common micro-organisms center；Preservation address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute.
In the preferred embodiment of the present invention, the biological deposits number of the rabies viruses low virulent strain HEP-Flury
For CGMCC No.17982；The preservation time are as follows: on June 3rd, 2019；Depositary institution: Chinese microorganism strain preservation conservator
It can common micro-organisms center；Preservation address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute.
In the preferred embodiment of the present invention, the Canine vaccine further includes veterinarily acceptable carrier；
As a preferred embodiment, above-mentioned veterinarily acceptable carrier and above-mentioned canine distemper virus antigen,
After Canine Parvovirus antigen and rabies virus antigen mixing, it can make antigen is more stable to be stored in vaccine, stabilization of vaccines
Potency, and then be conducive to the preservation of vaccine.
In above-mentioned preferred embodiment, the veterinarily acceptable carrier includes freeze drying protectant and adjuvant；
Preferably, the freeze drying protectant includes gelatin, lactoalbumin hydrolysate and sucrose.The freeze drying protectant resists with above-mentioned
After original mixing, it can be effectively ensured in freeze-drying preserving process, canine distemper virus antigen, Canine Parvovirus antigen and rabies viruses
The titer plateaus of antigen.
It is furthermore preferred that the freeze drying protectant the preparation method comprises the following steps: gelatin, lactoalbumin hydrolysate and sucrose are dissolved in water
In, solution is obtained after being matched according to mass percent；Then solution is obtained in 116 DEG C of at a temperature of high pressure sterilization 30min
To freeze drying protectant.
Preferably, the adjuvant includes ADJ-801.ADJ-801 adjuvant is that Import and Export Co., Ltd. produced had trade relations in Henan
Vaccine adjuvant, which, which has, changes antigen physical behavior, delays antigen degradation and exclusion, the time retained in extension body
Advantage, so as to more efficiently stimulation immune system.
According to an aspect of the present invention, a kind of preparation method of above-mentioned Canine vaccine, the preparation method includes following
Step:
First by the canine distemper virus antigen of immune amount, the rabies viruses of the Canine Parvovirus antigen of immune amount and immune amount
Antigen mixes, and veterinarily acceptable adjuvant is then added, and Canine vaccine is made.
The preparation method of Canine vaccine provided by the invention, the preparation method first resist the canine distemper virus of immune amount
The Canine Parvovirus antigen of former, immune amount and the rabies virus antigen of immune amount mix, and are then added veterinarily acceptable
Canine vaccine is made in adjuvant.The advantages of this method has preparation process simple, easy to operate, is suitble to large-scale industrial production.
In the preferred embodiment of the present invention, the preparation method further includes freezing Canine vaccine obtained
The step of dry preservation；
Preferably, the step of freeze-drying preservation includes that Canine vaccine obtained is added to freeze drying protectant, is then carried out
Lyophil preservation.
In the preferred embodiment of the present invention, the preparation method specifically includes the following steps:
(a), it is proliferated strain: culture proliferation canine distemper velogen strain LN (10) 1, parvovirus velogen strain JL (18) 1- respectively
Beagle and rabies viruses low virulent strain HEP-Flury；
(b), strain inactivates: by canine distemper velogen strain LN (10) 1, the parvovirus velogen strain after step (a) culture proliferation
JL (18) 1-Beagle and rabies viruses low virulent strain HEP-Flury are inactivated, and canine distemper virus, canine parvovirus are respectively obtained
With the totivirus antigen of rabies viruses deactivated form；
(c), vaccine formulation: canine distemper virus antigen, Canine Parvovirus antigen, the rabies virus antigen after inactivation are mixed
And each antigenic content is adjusted, veterinarily acceptable adjuvant is then added, Canine vaccine is made；
(d) preservation is lyophilized: freeze drying protectant is added in Canine vaccine made from step (c), then carries out freeze-drying guarantor
Hiding.
As a preferred embodiment, the inactivator of above-mentioned steps (b) mesogen strain inactivation is beta-propiolactone.
According to an aspect of the present invention, a kind of above-mentioned Canine vaccine preparation prevent and treat canid canine distemper,
Application in Canine Parvovirus Enteritis and rabic drug.
Canine vaccine provided by the invention can be widely applied to preparation and prevent and treat canid canine distemper, tiny disease
In viral enteritis and rabic drug.
Technical solution of the present invention is described further below in conjunction with embodiment and comparative example.
The culture of embodiment 1 canine parvovirus velogen strain JL (18) 1-Beagle is proliferated
To T75cm2When feline kidney cells F81 grows up to cell monolayer in Tissue Culture Flask, the canine parvovirus for being inoculated with preservation is strong
Strain 1ml JL (18) 1-Beagle (106.0TCID50/mL), it is placed in 35 DEG C of 5%CO2Middle progress virus amplification culture, often
Day observation cell virus situation, after harvest virus liquid after multigelation 3 times when cytopathy reaches 80% or so, and carries out
Viral level (>=106.0TCID50/mL), steriling test, mycoplasma are examined and exogenous virus is examined, and will examine qualified virus
Liquid is stored in -80 DEG C；
The culture of 2 wild-type canine distemper virus strain LN (10) 1 of embodiment is proliferated
To T75cm2When the African green monkey kidney cell VDS of expression dog SLAM receptor grows up to cell monolayer in Tissue Culture Flask,
It is inoculated with the wild-type canine distemper virus strain 1ml LN (10) 1 (10 of preservation6.0TCID50/mL), it is placed in 35 DEG C of 5%CO2Middle progress disease
Malicious amplification cultivation, it is daily to observe cell virus situation, after be harvested after multigelation 3 times when cytopathy reaches 80% or so
Virus liquid, and carry out viral level (>=106.0TCID50/mL), steriling test, mycoplasma are examined and exogenous virus is examined, will
Qualified virus liquid is examined to be stored in -80 DEG C；
The culture of 3 rabies viruses attenuated vaccine strain HEP-Flury of embodiment is proliferated
To T75cm2When cream hamster kidney cell BHK-21 grows up to cell monolayer in Tissue Culture Flask, it is inoculated with the rabies of preservation
Malicious attenuated vaccine strain 1ml HEP-Flury (105.67TCID50/mL), it is placed in 35 DEG C of 5%CO2Middle progress virus amplification culture,
By harvesting virus liquid after multigelation 3 times after 96h, and carry out viral level (>=105.67TCID50/mL), steriling test,
Mycoplasma is examined and exogenous virus is examined, and qualified virus liquid will be examined to be stored in -80 DEG C；
Embodiment 4 prepares freeze drying protectant
Gelatin, lactoalbumin hydrolysate and sucrose are dissolved in the water, according to mass percent meter, make final concentration of the 2 of gelatin
~7wt%, final concentration of 10~25wt% of lactoalbumin hydrolysate, the final concentration of 15~30wt% of sucrose obtain solution；
Solution is then obtained into freeze drying protectant in 116 DEG C of at a temperature of high pressure sterilization 30min.
The preparation of 5~7 Canine vaccine of embodiment
Firstly, by Examples 1 to 3, canine distemper virus antigen, Canine Parvovirus antigen and rabies viruses obtained resist respectively
Stoste is prepared, and 5~7 vaccine of specific embodiment proportion is as shown in table 1.Then, beta-propiolactone is added in 1:2000 ratio, it is thorough
Bottom mixes, and 4 DEG C inactivate 24 hours, and shaking therebetween mixes twice, hydrolyzes 1 hour for 37 DEG C later, by ADJ-801 and inactivation of viruses liquid
After the mixing of 1:9 ratio, freeze drying protectant is added, in sterile ampulla, capping is placed in pre-freeze in freeze dryer, does quantitative separating
It is dry, obtain Canine vaccine.
Table 1: the specific vaccine of embodiment 5~7 proportion
Comparative example 1~6
Firstly, by Examples 1 to 3, canine distemper virus antigen, Canine Parvovirus antigen and rabies viruses obtained resist respectively
Stoste is prepared, and specific 1~6 vaccine of comparative example proportion is as shown in table 2.Then, beta-propiolactone is added in 1:2000 ratio, it is thorough
Bottom mixes, and 4 DEG C inactivate 24 hours, and shaking therebetween mixes twice, hydrolyzes 1 hour for 37 DEG C later, by ADJ-801 and inactivation of viruses liquid
After the mixing of 1:9 ratio, freeze drying protectant is added, in sterile ampulla, capping is placed in pre-freeze in freeze dryer, does quantitative separating
It is dry, obtain Canine vaccine.
Table 2: the specific vaccine of comparative example 1~6 proportion
1 efficacy test of experimental example
1, method
(1) Canine vaccine that Example 5~7 and comparative example 1~6 are prepared, the nape of the neck inoculate antibody yin
Property 14~21 age in days beasle dogs, every vaccinates 1ml, while setting not vaccine inoculation beasle dog as a control group, for the first time
Each booster immunization is primary when the 21st day, 42 days after inoculation, and vaccine 1ml is subcutaneously injected in every the nape of the neck of test group, the 63rd after inoculation
It when, every beasle dog is taken a blood sample respectively, separates serum, carries out antibody titer and titer determination.
After immunity inoculation 1 month, 6 months and 9 months, tested beasle dog uses canine distemper, Canine Parvovirus Enteritis, mad
Dog disease is intranasal, oral with virulent difference and intramuscular inoculation 1ml, contains 100 LD50.Observation beasle dog body temperature, appetite, spirit daily
The physical signs such as state.
2, result:
After canine distemper virus, canine parvovirus and rabies viruses inactivation triple vaccine are immune, using serum neutralisation, survey
Determining canine parvovirus prevention neutralize antibody titers is 1:50；Using hemagglutination-inhibition test method, anti-dog parvovirus antibody blood is measured
It is solidifying to inhibit potency for 1:128；Using Rabies Q-AB ELISA method, measuring rabies poison neutralizing antibody titers is
6.902EU/mL。
It has been observed that 1 month beasle dog is to the protective rate of canine distemper, parvovirus and rabies viruses after being inoculated with three times
100%, beasle dog items physical signs is normal, and no any clinical symptoms occur；After being inoculated with 6 months three times, beasle dog is to dog
The protective rate of distemper virus, parvovirus and rabies viruses is 95%；After being inoculated with 9 months three times, beasle dog is to hundstaupe pyreticosis
The protective rate of poison, parvovirus and rabies viruses is respectively 80% and 90% and 80%.Therefore, the duration of immunity of vaccine is set as 6
A month.
Inoculation hundstaupe hot inspection shows body temperature with virulent control group beasle dog and increases up to 40 DEG C or more, and body temperature drops to often
2~3d after temperature, the body temperature of sick dog are so recycled again above 40 DEG C, and can generate heat simultaneously continued for several weeks again；Flow clear nasal mucus and purulence
Nose juice, the gum of outflow are purulence, passage of loose stools, it is viscous just or even coal tar oil sample bloody stool.
Inoculation parvovirus, which is examined, shows body temperature raising with virulent control group beasle dog, and violent diarrhea, discharge is mixed with
The water sample loose stools or cast of intestinal mucosa just, and are significantly reduced through inspection blood middle leukocytes.
Inoculation rabies, which are examined, with virulent control group beasle dog shows low-heat, drowsiness, loss of appetite, to the underwater sound, light,
The stimulation such as wind is very sensitive, causes ictal laryngeal muscles, pharyngismus, expiratory dyspnea.
The above result shows that triple inactivated vaccine provided by the present invention can effectively protect beasle dog from hundstaupe pyreticosis
Poison, parvovirus and the virulent attack of rabies viruses, can effectively prevent canine distemper, Canine Parvovirus Enteritis and rabic hair
It is raw.
2 safety verification of experimental example
The Canine vaccine that Example 5~7 is prepared, the nape of the neck inoculates negative antibody beasle dog, if single dose
(canine distemper virus content >=106.0TCID50/ parts, canine parvovirus content >=106.0TCID50/ parts, rabies viruses contain
Amount >=105.67TCID50/ parts), single dose repeat (single dose be inoculated with 3 times, be spaced 21 days) and (10 times) of overdose be immunized
Group, while not vaccine inoculation beasle dog is set as control.After vaccine inoculation, the body temperature of continuous 14 days observation beasle dogs, appetite, essence
The physical signs such as refreshing state, and dissect observation inoculation beasle dog main organs lesion situation.Test result shows beasle dog through list
After dosage, single dose repetition and overdose inoculation, every physical signs is normal, does not show any clinical symptoms, and dissect is seen
Inoculation beasle dog main organs are examined without lesion.Illustrate dog canine distemper, Canine Parvovirus Enteritis and rabies triple inactivated vaccine
To beasle dog safety.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of Canine vaccine, which is characterized in that the Canine vaccine includes the canine distemper virus antigen of immune amount, immune amount
The rabies virus antigen of Canine Parvovirus antigen and immune amount；
The canine distemper virus antigen includes the canine distemper velogen strain LN (10) 1 of deactivated form；
The Canine Parvovirus antigen includes parvovirus velogen strain JL (18) 1-Beagle of deactivated form；
The rabies virus antigen includes the rabies viruses low virulent strain HEP-Flury of deactivated form.
2. Canine vaccine according to claim 1, which is characterized in that in the Canine vaccine,
The content of the canine distemper virus antigen of immune amount is 105.0~107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 105.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~107.0TCID50/ head part.
3. Canine vaccine according to claim 1, which is characterized in that in the Canine vaccine,
The content of the canine distemper virus antigen of immune amount is 106.0~107.0TCID50/ head part；
The content of the Canine Parvovirus antigen of immune amount is 106.0~107.0TCID50/ head part；
The content of the rabies virus antigen of immune amount is 105.67~106.0TCID50/ head part.
4. described in any item Canine vaccines according to claim 1~3, which is characterized in that the canine distemper virus antigen, dog are thin
Small virus antigen and rabies virus antigen are the totivirus antigen of deactivated form.
5. Canine vaccine according to claim 1, which is characterized in that the biology of the canine distemper velogen strain LN (10) 1 is protected
Hiding number is CGMCC No.17981；
Preferably, the biological deposits number of parvovirus velogen strain JL (18) 1-Beagle are CGMCC No.17983；
Preferably, the biological deposits number of the rabies viruses low virulent strain HEP-Flury are CGMCC No.17982.
6. Canine vaccine according to claim 1, which is characterized in that the Canine vaccine further includes veterinarily acceptable
Carrier；
Preferably, the veterinarily acceptable carrier includes freeze drying protectant and adjuvant；
It is furthermore preferred that the adjuvant includes ADJ-801.
7. a kind of preparation method of any one according to claim 1~6 Canine vaccine, which is characterized in that the preparation side
Method the following steps are included:
First by the canine distemper virus antigen of immune amount, the rabies virus antigen of the Canine Parvovirus antigen of immune amount and immune amount
It mixes, veterinarily acceptable adjuvant is then added, Canine vaccine is made.
8. the preparation method of Canine vaccine according to claim 7, which is characterized in that the preparation method further includes that will make
The step of Canine vaccine obtained carries out freeze-drying preservation；
Preferably, the step of freeze-drying preservation includes that freeze drying protectant is added in Canine vaccine obtained, is then freezed
Drying preservation.
9. the preparation method of Canine vaccine according to claim 7, which is characterized in that the preparation method specifically includes following
Step:
(a), it is proliferated strain: culture proliferation canine distemper velogen strain LN (10) 1, parvovirus velogen strain JL (18) 1-Beagle respectively
With rabies viruses low virulent strain HEP-Flury；
(b), strain inactivates: by canine distemper velogen strain LN (10) 1, the parvovirus velogen strain JL after step (a) culture proliferation
(18) 1-Beagle and rabies viruses low virulent strain HEP-Flury are inactivated, respectively obtain canine distemper virus, canine parvovirus and
The totivirus antigen of rabies viruses deactivated form；
(c), vaccine formulation: canine distemper virus antigen, Canine Parvovirus antigen, the rabies virus antigen after inactivation are mixed and adjusted
Each antigenic content is saved, veterinarily acceptable adjuvant is then added, Canine vaccine is made；
(d), preservation is lyophilized: freeze drying protectant is added in Canine vaccine made from step (c), then carries out lyophil preservation；
Preferably, the inactivator of step (b) the mesogen strain inactivation is beta-propiolactone.
10. a kind of Canine vaccines described in any item according to claim 1~6 prevent and treat canid hundstaupe in preparation
Application in heat, Canine Parvovirus Enteritis and rabic drug.
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| CN114470184A (en) * | 2021-12-30 | 2022-05-13 | 孙嘉玮 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
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