CN114470184A - Combined vaccine for tetanus, canine distemper and rabies for livestock - Google Patents
Combined vaccine for tetanus, canine distemper and rabies for livestock Download PDFInfo
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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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- 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
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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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
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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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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- 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
- A61K2039/70—Multivalent 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
- 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
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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
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention discloses a combined vaccine for tetanus, canine distemper and rabies for animals, which comprises an immune amount of inactivated canine distemper virus antigen, an immune amount of inactivated rabies virus antigen and an immune amount of tetanus toxoid antigen. The invention utilizes the function of tetanus toxoid macromolecular protein for promoting the immune response of organisms, combines with canine distemper and rabies virus antigens, improves the neutralizing antibody level of the canine distemper and rabies antigens, can generate antibodies against rabies viruses and canine distemper viruses, and simultaneously generates antibodies against corynebacterium parvum, prevents tetanus and realizes prevention of three diseases by one injection.
Description
Technical Field
The invention belongs to the field of animal vaccines, and particularly relates to a combined vaccine for tetanus, canine distemper and rabies for animals.
Technical Field
Canine Distemper (CDV) is a highly contagious disease caused by canine distemper virus, mainly infects canines, belongs to a second class of animal epidemic diseases, and can be infected by directly contacting with animals, or by air or food. The disease has strong infectivity, the death rate is more than 80 percent, and the disease can be listed as the leaders in the aspects of infection intensity, disease severity and death rate of various diseases suffered by the dogs. Meanwhile, mixed infection of bacteria is mostly caused in the process of canine distemper.
Tetanus is caused by tetanus bacillus, a gram-positive anaerobic bacterium present in dust, soil, human and animal feces, and when subjected to trauma, tetanus bacillus invades through wounds, releases a highly toxic protein, tetanus spastic toxin, which passes through the blood circulation and lymphatic system and binds to serum globulins to the cranial nerves, causing neuropathic symptoms with spastic limb paralysis (topical tetanus); tetanus spasm toxin can further cause systemic excitement of the toxin, such as a large amount of toxin can produce a systemic convulsion (systemic tetanus), which is characterized in that the affected animal has a spasmodic skeletal muscle and has an increased reflex excitement to external stimuli.
Rabies is a natural epidemic disease, is a zoonosis disease which is caused by rabies virus and is susceptible to all warm-blooded animals, and any animal can suffer from rabies. Once the morbidity and mortality rate is 100%, the acute infectious disease with the highest morbidity and mortality rate in human is the highest to date.
At present, attenuated live vaccines are mainly used for canine distemper and rabies, and because the attenuated live vaccines have some unknown potential risks, such as enhanced virulence reversion of viruses, the vaccines originally used for preventing diseases have certain virulence, so that healthy animals are ill, and the health of the animals and groups is influenced. In the case of bacterial diseases such as tetanus, tetanus toxoid vaccine is mainly used for the prevention at present.
The combined vaccine contains two or more organisms or purified antigens, can prevent various diseases simultaneously, is favorable for improving the coverage rate and the inoculation rate of the vaccine, reduces the difficulty and the inoculation cost in vaccine management, can also reduce the dose of an adjuvant necessary in vaccine production, reduces the adverse reaction of the vaccine, and has important practical significance for guaranteeing the animal welfare and the continuous healthy development of animal husbandry.
At present, combined vaccines against canine distemper, canine parvovirus disease, rabies, canine infectious hepatitis, canine parainfluenza virus and the like are available for dogs, and reports of combining the inactivated vaccine with tetanus toxoid are few. The inactivated distemper and rabies viruses are combined with the tetanus toxoid to prepare the combined vaccine capable of preventing the distemper, rabies and tetanus of the canines.
Disclosure of Invention
The invention aims to provide a combined vaccine capable of preventing and treating canine distemper, rabies and tetanus of canines simultaneously.
The above purpose is realized by the following technical scheme:
a combined vaccine for treating tetanus, canine distemper and rabies for animals comprises an immunizing amount of inactivated canine distemper virus antigen, an immunizing amount of inactivated rabies virus antigen and an immunizing amount of tetanus toxoid antigen.
Preferably, the virus content of the inactivated canine distemper virus antigen is not less than 0.5x105TCID50Per ml; the antigen titer of the inactivated rabies virus antigen is not less than 1 IU/ml; the toxin titer of the tetanus toxoid antigen is not lower than 2 Lf/ml.
Further preferably, the virus content of the inactivated canine distemper virus antigen is 0.67x105TCID50Per ml; the antigen titer of the inactivated rabies virus antigen is 1.67 IU/ml; the tetanus toxoid antigen has a toxin titer of 2.33 Lf/ml.
The invention further provides a method for preparing the combination vaccine, which comprises the following steps:
(1) recovering and passaging the Vero cells, respectively inoculating the canine distemper virus and the rabies virus, collecting virus liquid after propagation culture, concentrating and centrifuging, adding beta-propiolactone for inactivation, and purifying by Sepharose column chromatography to obtain inactivated canine distemper virus antigen liquid and inactivated rabies virus antigen liquid;
(2) culturing Clostridium tetani to generate toxin, sterilizing with formaldehyde, refining with ammonium sulfate, detoxicating, sterilizing, and filtering to obtain tetanus toxoid antigen solution;
(3) mixing the three antigen solutions according to a certain volume ratio, adding an adjuvant, and subpackaging to obtain the antigen.
Preferably, in step (1), the virus inoculation and propagation culture method is as follows: and (2) thawing virus seeds by using water at 35 ℃, inoculating Vero cells according to the infection index of 0.01-0.1 MOI, adding 199 nutrient solution containing 0.2-0.5% of bovine serum as a maintenance solution, carrying out virus culture at 33-35 ℃, and adding gentamicin into the culture solution according to 24 IU/ml.
Preferably, in the step (1), the virus liquid is concentrated by using a 100 KD-300 KD ultrafiltration membrane, wherein the concentration multiple is 20-40 times of the volume.
Preferably, in the step (1), newly prepared beta-propiolactone is added to the concentrated and centrifuged virus solution so that the volume ratio of the beta-propiolactone to the concentrated solution is 1: and (4000), shaking for 30 minutes, inactivating in a cold storage at the temperature of 2-8 ℃ for 24 hours, and then standing at the temperature of 37 ℃ for 2 hours for hydrolysis.
Preferably, the volume ratio of the three antigen liquids is 1:1: 1.
according to the method, the tetanus toxoid content is 6-10 Lf/ml, the rabies virus antigen content is 4-6 IU/ml, and the canine distemper virus antigen content is 2-4 x105TCID50Mixing the three stock solutions according to the ratio of 1:1:1, and fully stirring and combining the three stock solutions in a cold storage environment at the temperature of 2-8 ℃ to ensure that the content of tetanus toxoid is not lower than 2Lf per dose, the titer of rabies virus antigen is not lower than 1IU per dose, and the content of virus before inactivation of canine distemper virus antigen is not lower than 0.5x105TCID 50/ml.
The invention has the beneficial effects that:
the invention utilizes the effect of tetanus toxoid macromolecular protein on promoting the immune response of organisms, combines with canine distemper and rabies virus antigens, improves the neutralizing antibody level of the canine distemper and rabies antigens, can generate antibodies against rabies viruses and canine distemper viruses, and also generates antibodies against tetanus toxin, prevents tetanus, prevents three diseases by one injection, and reduces the use of antibiotics in the animal feeding process. The invention can also reduce the possible risk of live virus vaccine, and has the advantages of good safety, convenient storage and transportation, maternal antibody interference resistance and the like.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples.
EXAMPLE 1 preparation of combination vaccine
1. Preparation of inactivated viruses
Reviving Vero cells: taking out the Vero cells from the liquid nitrogen tank, quickly putting the Vero cells into a beaker filled with injection water at the temperature of 37-39 ℃, shaking the Vero cells to quickly melt the Vero cells within 1 minute, wiping the melted Vero cells with a disinfectant for disinfection, and moving the dissolved Vero cells into an operation room. Sucking the Vero cell suspension out, putting the Vero cell suspension into a culture bottle, supplementing a culture solution, and culturing at a constant temperature of 37 ℃, wherein the cell culture solution is 199 culture solution containing 5-10% calf serum.
Passage of Vero cells: pouring out the culture solution in the cell culture bottle, digesting with cell digestive juice, blowing and beating to suspend when the cell wall has a ground glass shape, supplementing the cell suspension to the cell culture solution, and culturing at constant temperature of 37 ℃ for vaccine production.
Virus inoculation: thawing virus seeds by using water at 35 ℃, inoculating cells according to an infection index of 0.01-0.1 MOI, adding 199 nutrient solution containing 0.2-0.5% of bovine serum as a maintenance solution, placing at 33-35 ℃ for virus culture, and adding gentamicin into the culture solution according to 24 IU/ml.
And (3) harvesting viruses: and after 3-5 days of culture, harvesting virus liquid, wherein the virus liquid harvested each time is virus single-time harvest liquid, and the harvest liquid is stored at 2-8 ℃ for not more than 3 months.
Merging, ultrafiltering and concentrating: the virus harvest liquid produced by the same batch of cells can be combined into 1 batch under the strict aseptic operation condition, and concentrated by 20-40 times of volume by a 100 KD-300 KD ultrafiltration membrane, and the concentrated liquid is clarified by centrifugation at 7000rpm for 30 minutes.
Virus inactivation: adding newly prepared beta-propiolactone into the clarified concentrated virus solution to ensure that the volume ratio of the beta-propiolactone to the concentrated solution is 1: and (4000), shaking for 30 minutes, inactivating in a cold storage at the temperature of 2-8 ℃ for 24 hours, and then standing at the temperature of 37 ℃ for 2 hours for hydrolysis.
And (3) virus purification: and concentrating and detecting qualified virus liquid, and purifying by using a Sepharose 4FF gel chromatographic column.
And (3) stock solution verification: and (4) carrying out stock solution verification on the purified inactivated virus to obtain a virus stock solution meeting the quality requirement as an antigen.
2. Preparation of tetanus toxoid stock solution:
clostridium tetani is subjected to deep culture in a large tank by using a tetanus culture medium to generate toxin, and is sterilized by 0.3-0.6% of formaldehyde, refined by ammonium sulfate, detoxified by 0.15% of formaldehyde at 37 ℃ for 15-22 days, sterilized, filtered and refined to obtain tetanus toxoid stock solution.
Obtaining a batch of vaccine antigen stock solution according to the above embodiment, and obtaining the antigen contents of the batch of stock solution before mixing according to the traditional detection methods of different antigens as follows: the content of tetanus toxoid is 7Lf/ml, the titer of rabies virus antigen is 5IU/ml, and the content of canine distemper virus antigen is 2x105TCID50The three stock solutions are mixed according to the proportion of 1:1:1 to obtain the product with the tetanus toxoid content of 2.33Lf/ml, the rabies virus antigen titer of 1.67IU/ml and the canine distemper virus antigen content of 0.67x105TCID50A semi-finished product of the combined vaccine of/ml. Meanwhile, the antigen stock solution of the batch is diluted 3 times each to be used as an experimental control.
Example 2: antibody titers of combination vaccines
The animals are immunized according to the assay protocol for the relevant preparation in the biological preparation assay protocol, and the titer of the combined antigen after immunization with each single antigen is compared.
| Tetanus potency | Hydrophobia titer | Canine distemper titer | |
| Laboratory animal | Mouse | Mouse | Rat |
| 3-fold dilution of a Single antigen | 7.33IU | 1.67IU | 1040IU |
| Combined antigens | 7.67IU | 3.50IU | 1877IU |
The comparison shows that the tetanus titer is not obviously changed after the combined antigen immunization, and the antibody titer after the rabies and canine distemper antigen immunization is obviously higher than that of the corresponding single antigen.
Example 3:
8 puppies of 2 months old who have no related disease antibodies detected by ELISA are purchased from the market, combined antigens and 3 single antigens are respectively injected, serum is collected after one month, and serum antibody detection is carried out by adopting corresponding ELISA antibody detection kits purchased from the market to obtain an average value for comparison.
| Tetanus potency | Hydrophobia titer | Canine distemper titer | |
| 3-fold dilution of a Single antigen | 9.0 | 2.0 | 1549 |
| Combined antigens | 8.67 | 3.83 | 2355 |
Experiments of different animal examples show that the neutralizing antibodies of the canine distemper and rabies antigens of the product are higher than those of the common single vaccine, the effect is obvious, three antigens are injected into an organism at the same time after being fully combined, mutual immune suppression is not generated, the improvement of the antibody generated by the organism is probably the result of the synergic immunity of tetanus toxoid macromolecular protein, the product of the invention technically not only improves the neutralizing antibody level of the same canine distemper and rabies antigen amount, but also generates the immune antibody of tetanus, solves the risk of canine distemper and rabies infected by canine distemper through one-time immunity, meanwhile, the risk that the canine animals are infected with tetanus in the activity of the canine animals is solved, the use of antibiotics after the canine animals are infected with tetanus is avoided, the national macro-policy of restricting the large-scale use of antibiotics is met, and the canine animals play a very important role in preventing and treating animal diseases.
Claims (9)
1. A combined vaccine for tetanus, canine distemper and rabies for animals is characterized in that: the vaccine comprises an immunizing amount of inactivated canine distemper virus antigen, an immunizing amount of inactivated rabies virus antigen and an immunizing amount of tetanus toxoid antigen.
2. The combination vaccine of claim 1, wherein: the virus content of the inactivated canine distemper virus antigen is not less than 0.5x105TCID50Per ml; the antigen titer of the inactivated rabies virus antigen is not less than 1 IU/ml; the toxin titer of the tetanus toxoid antigen is not lower than 2 Lf/ml.
3. The combination vaccine of claim 1, wherein: the virus content of the inactivated canine distemper virus antigen is 0.67x105TCID50Per ml; the antigen titer of the inactivated rabies virus antigen is 1.67 IU/ml; the tetanus toxoid antigen has a toxin titer of 2.33 Lf/ml.
4. A method for preparing a combination vaccine according to any one of claims 1 to 3, comprising the steps of:
(1) recovering and passaging the Vero cells, respectively inoculating the canine distemper virus and the rabies virus, collecting virus liquid after propagation culture, concentrating and centrifuging, adding beta-propiolactone for inactivation, and purifying by Sepharose column chromatography to obtain inactivated canine distemper virus antigen liquid and inactivated rabies virus antigen liquid;
(2) culturing Clostridium tetani to generate toxin, sterilizing with formaldehyde, refining with ammonium sulfate, detoxicating, sterilizing, and filtering to obtain tetanus toxoid antigen solution;
(3) mixing the three antigen solutions according to a certain volume ratio, adding an adjuvant, and subpackaging to obtain the antigen.
5. The method for preparing the combination vaccine according to claim 4, wherein in the step (1), the virus inoculation and propagation culture method comprises the following steps: and (2) thawing virus seeds by using water at 35 ℃, inoculating Vero cells according to the infection index of 0.01-0.1 MOI, adding 199 nutrient solution containing 0.2-0.5% of bovine serum as a maintenance solution, carrying out virus culture at 33-35 ℃, and adding gentamicin into the culture solution according to 24 IU/ml.
6. A method for preparing a combination vaccine according to claim 4, wherein: in the step (1), a 100 KD-300 KD ultrafiltration membrane is used for concentrating virus liquid, and the concentration multiple is 20-40 times of the volume.
7. A method for preparing a combination vaccine according to claim 4, wherein: in the step (1), newly prepared beta-propiolactone is added into the concentrated and centrifuged virus solution, so that the volume ratio of the beta-propiolactone to the concentrated solution is 1: and (4000), shaking for 30 minutes, inactivating the mixture after the mixture is placed in a cold storage for 24 hours at the temperature of 2-8 ℃, and then placing the mixture for 2 hours at the temperature of 37 ℃ for hydrolysis.
8. A method for preparing a combination vaccine according to claim 4, wherein: the volume ratio of the three antigen liquids is 1:1: 1.
9. use of the combination vaccine of any one of claims 1-3 for the preparation of a medicament for the prevention and treatment of canine distemper, rabies and tetanus in canines.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111664620.1A CN114470184A (en) | 2021-12-30 | 2021-12-30 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
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| CN202111664620.1A CN114470184A (en) | 2021-12-30 | 2021-12-30 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
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