CN107789622A - A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine - Google Patents
A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine Download PDFInfo
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- CN107789622A CN107789622A CN201710968232.XA CN201710968232A CN107789622A CN 107789622 A CN107789622 A CN 107789622A CN 201710968232 A CN201710968232 A CN 201710968232A CN 107789622 A CN107789622 A CN 107789622A
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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
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
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Abstract
The present invention relates to animal bacteria field, there is provided a kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine, the vaccine is with C.perfringens β2White-oil adjuvant after toxin toxoid nutrient solution and sterilizing presses 1:1 ratio is added in C.perfringens toxoid nutrient solution and emulsified, and C.perfringens β is made2Toxin toxoid vaccine, the vaccine therapy effect is good, immunocompetence is strong and has no toxic side effect, and available for immune pregnancy phase cow and the calf that has just been born, can prevent the enterotoxemia of calf.
Description
Technical field
The present invention relates to animal bacteria field, and the invention provides a kind of calf enterotoxemia C.perfringens β2Poison
The preparation method of plain toxoid vaccine.
Background technology
C.perfringens is a kind of gram-positive anaerobic bacterium for being widely present in nature, can be produced outside up to 17 kinds
Toxin, wherein main has α, β1、β2, the toxin such as ε, 5 kinds of toxin types of A, B, C, D, E are divided into according to institute's toxin producing.In A, B, C, D
β is found that in type C.perfringens2Toxin, increasing research show：The intestines of the enterotoxemia of ox especially calf
Toxaemia and production β2The C.perfringens of toxin is relevant.
The pathogenesis of Roll toxaemia is as caused by C.perfringens：Production β in Roll road2The aerogenesis of toxin
Capsular clostridium is largely bred so as to produce substantial amounts of β2Toxin, the toxin have strong cell toxicant to small intestinal epithelial cells
Property, intestinal wall is destroyed, causes toxaemia.Therefore preventing the disease should be to control β2Toxin is attached most importance to.
Toxoid vaccine is to retain the vaccine made by the toxin of immunogenicity with toxicity is lost, and it can effective pin
To disease caused by C.perfringens exotoxin.However, there is presently no for C.perfringens β2Class prepared by toxin
Toxin vaccine,
The content of the invention
The present invention is directed to blank existing for prior art, there is provided a kind of toxin of calf enterotoxemia C.perfringens β 2
Toxoid vaccine and preparation method thereof, the vaccine are helped with the white oil after the toxin toxoid nutrient solutions of C.perfringens β 2 and sterilizing
Agent presses 1:1 ratio is added in C.perfringens toxoid nutrient solution and emulsified, and the poison of C.perfringens β 2 is made
Plain toxoid vaccine, the vaccine therapy effect is good, immunocompetence is strong and has no toxic side effect, available for immune pregnancy phase cow with
And the calf that is just born, the enterotoxemia of calf can be prevented.
Used c-type C.perfringens uses existing strain to inventor in the present invention, in particular selected from being preserved in
National Collection of Type Cultures Britain Culture Collection, preserving number：NCTC's 4989
Reference culture, it can be directly obtained by existing channel, so the biomaterial belongs to the material that can be directly obtained in the prior art,
It is not required to carry out single biological deposits.
The concrete technical scheme of the present invention is as follows：Comprise the following steps that：
(1) reference culture, is inoculated with about 2-5 μ L on blood agar base plate culture medium, 41 DEG C of recovery 36h, after recovery
Single bacterium colony on picking blood plate is inoculated in 7mL thioglycollate mediums, 39 DEG C of increasing bacterium Anaerobic culturel 12h, is produced
Gas capsular clostridium bacterium solution；7mL C.perfringens bacterium solutions are added in 100mL Gordon soup toxin producing mediums, anaerobic environment
Lower 43 DEG C of cultures 6h efficiently produces poison, and then 4 DEG C of 8000rpm centrifugation 30min, take supernatant, with 0.22 μm of the seitz filter mistake in aperture
Bacterium is filtered out, obtains containing β2The C.perfringens exotoxin nutrient solution of toxin；
(2), in the β of above-mentioned acquisition2The formaldehyde of volume ratio 0.3% is added in exotoxin nutrient solution, after fully vibration mixes
It is placed in 37 DEG C persistently to inactivate 5 days, obtains C.perfringens β2Toxin toxoid nutrient solution；
(3), by above-mentioned β2Tween-80 is added in toxin toxoid nutrient solution, its final concentration is reached 2wt%, is rocked
It is even, obtain aqueous phase；White-oil adjuvant is sterilized, obtains oil phase；By oil phase by volume 1:1 ratio is added in aqueous phase, then
Emulsified in tissue pulverizer three times, each 10min, C.perfringens β is made2Toxin toxoid vaccine.
Wherein described blood agar base plate culture medium is that 1g glucose and 3.8g beans agar powders are dissolved in 100ml distilled water
After sterilizing, add 5ml Sheep Bloods and obtain；
Described Gordon soup toxin producing mediums are：Per 100mL PBSs dissolving 3g tryptones, 2g dextrin, 2g
Yeast extract and 2.2g L-arginines；
The steriling test reference of vaccine《People's Republic of China's veterinary biologicses quality standard》(version in 2001) annex
Page 301 are carried out, as a result asepsis growth.
The perfringens shuttle of above-mentioned acquisition is each subcutaneously injected with body weight 2kg-3kg rabbit 4 in the safety verification of vaccine
The toxin toxoid vaccine 5mL of bacterium β 2, observe 30 days, inoculation animal does not occur the diseases such as infection, necrosis at health survival and injection
Reason change.
When specifically used, it is subcutaneously injected, is inoculated with first within 2 months before cow childbirth, is inoculated with once within 4 weeks after calf birth, 2 weeks
Booster immunization once, each 5mL of immunizing dose, as a result confirms that antibody titer caused by vaccine reaches as high as 1 again afterwards：3200.
In summary, after the vaccine that the present invention is obtained is with the toxin toxoid nutrient solutions of C.perfringens β 2 and sterilizing
White-oil adjuvant presses 1:1 ratio is added in C.perfringens toxoid nutrient solution and carries out emulsification and is made, the vaccine therapy
Effect is good, immunocompetence is strong and has no toxic side effect, and available for immune pregnancy phase cow and the calf that has just been born, can prevent calf
The enterotoxemia of ox.
Embodiment
Embodiment
A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine, is comprised the following steps that：
(1) reference culture, is inoculated with 2-5 μ L in blood agar base plate culture medium, 41 DEG C of recovery 36h, used after recovery
Thioglycollate medium, 39 DEG C of increasing bacterium Anaerobic culturel 12h, obtains C.perfringens bacterium solution；By 7mL perfringens shuttles
Bacterium bacterium solution is added in 100mL Gordon soup toxin producing mediums, and the lower 43 DEG C of cultures 6h of anaerobic environment efficiently produces poison, then 4 DEG C
8000rpm centrifuges 30min, takes supernatant, with 0.22 μm of the seitz filter filtration sterilization in aperture, obtains containing β2The perfringens of toxin
Clostridium exotoxin nutrient solution；
(2), in the β of above-mentioned acquisition2The formaldehyde of volume ratio 0.3% is added in exotoxin nutrient solution, after fully vibration mixes
It is placed in 37 DEG C persistently to inactivate 5 days, obtains toxoid nutrient solution；
(3), by β2Toxin toxoid nutrient solution adds tween-80, its final concentration is reached 2%, rocks uniformly, obtains
Aqueous phase；White-oil adjuvant is sterilized, obtains oil phase.By oil phase by volume 1:1 ratio is added in aqueous phase, then in tissue powder
Emulsified in broken machine three times, each 10min, C.perfringens β is made2Toxin toxoid vaccine；
Wherein described blood agar base plate culture medium is that 1g glucose and 3.8g beans agar powders are dissolved in 100ml distilled water
After sterilizing, add 5ml Sheep Bloods and obtain；
Described Gordon soup toxin producing mediums are：Per 100mL PBSs dissolving 3g tryptones, 2g dextrin, 2g
Yeast extract and 2.2g L-arginines；
The steriling test reference of vaccine《People's Republic of China's veterinary biologicses quality standard》(version in 2001) annex
The progress of page 301, asepsis growth.
The safety verification of vaccine body weight 2kg-3kg rabbit 4, respective hypodermic injection C.perfringens β2Endotoxin
Toxin vaccine 5mL, observe 30 days, inoculation animal does not occur the pathological changes such as infection, necrosis at health survival and injection.
Experimental example
Vaccine effect inspection with more than the 50kg of Shandong high speed bioengineering cattle farm healthy susceptible calf 10, wherein
C.perfringens β is subcutaneously injected in 8 calves every2Toxin toxoid vaccine 5mL；Entered after 2 weeks with identical approach and dosage
Second of inoculation of row, as vaccine immunity group, with the identical 2 calves hypodermic injection 5mL of feeding management physiological saline
For blank control.10 calves are carried out with jugular vein blood collection week about, detects β in serum2Toxin antibody potency.Vaccine immunity
The antibody titers from serum of group reaches up to 1 in 4th week：3200, the serum of blank control group is not detected by antibody, as a result
It is as shown in the table：
Vaccine immunity group calf serum antibody titer testing result
It can be seen that the vaccine therapy effect that is obtained of the present invention is good, immunocompetence is strong and has no toxic side effect, it is pregnant available for being immunized
It is pregnent phase cow and the calf that is just born, the enterotoxemia of calf can be prevented.
Claims (3)
1. a kind of preparation method of the toxin toxoid vaccines of calf enterotoxemia C.perfringens β 2, it is characterised in that specific
Step is as follows：
(1) reference culture, is inoculated with 2-5 μ L on blood agar base plate culture medium, 41 DEG C of recovery 36h, picking blood after recovery
Single bacterium colony on flat board is inoculated in 7mL thioglycollate mediums, 39 DEG C of increasing bacterium Anaerobic culturel 12h, obtains perfringens
Clostridium bacterium solution；7mL C.perfringens bacterium solutions are added in 100mL Gordon soup toxin producing mediums, lower 43 DEG C of anaerobic environment
Culture 6h efficiently produces poison, and then 4 DEG C of 8000rpm centrifugation 30min, takes supernatant, with 0.22 μm of the seitz filter filtration sterilization in aperture,
Obtain containing β2The C.perfringens exotoxin nutrient solution of toxin；
(2), in the β of above-mentioned acquisition2The formaldehyde of volume ratio 0.3% is added in exotoxin nutrient solution, fully vibration is placed in 37 after mixing
DEG C persistently inactivate 5 days, obtain C.perfringens β2Toxin toxoid nutrient solution；
(3), by above-mentioned β2Tween-80 is added in toxin toxoid nutrient solution, its final concentration is reached 2wt%, rocks uniformly, obtains
To aqueous phase；White-oil adjuvant is sterilized, obtains oil phase；By oil phase by volume 1:1 ratio is added in aqueous phase, is then being organized
Emulsified in pulverizer three times, each 10min, C.perfringens β is made2Toxin toxoid vaccine.
2. preparation method according to claim 1, it is characterised in that：Described blood agar base plate culture medium is 1g grapes
After sugar and 3.8g beans agar powders are dissolved in the sterilizing of 100ml distilled water, add 5ml Sheep Bloods and obtain.
3. preparation method according to claim 1, it is characterised in that：Described Gordon soup toxin producing mediums are：Per 100mL
PBS dissolving 3g tryptones, 2g dextrin, 2g yeast extracts and 2.2g L-arginines.
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| CN201710968232.XA CN107789622A (en) | 2017-10-18 | 2017-10-18 | A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine |
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| CN201710968232.XA CN107789622A (en) | 2017-10-18 | 2017-10-18 | A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB958574A (en) * | 1959-11-23 | 1964-05-21 | Wellcome Found | Clostridium perfringens ª‰-toxoid vaccines |
| CN101541343A (en) * | 2006-11-20 | 2009-09-23 | 英特威国际有限公司 | Use of clostridium perfringens type C bacterium for the manufacture of a vaccine |
| CN103432578A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of C-type clostridium perfringens toxin vaccine of piglet red dysentery |
| CN103432579A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine |
| CN103463632A (en) * | 2013-09-05 | 2013-12-25 | 山东农业大学 | Preparation method for D type clostridium perfringen toxoid vaccine of cattle and sheep enterotoxaemia |
| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
-
2017
- 2017-10-18 CN CN201710968232.XA patent/CN107789622A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB958574A (en) * | 1959-11-23 | 1964-05-21 | Wellcome Found | Clostridium perfringens ª‰-toxoid vaccines |
| CN101541343A (en) * | 2006-11-20 | 2009-09-23 | 英特威国际有限公司 | Use of clostridium perfringens type C bacterium for the manufacture of a vaccine |
| CN103463632A (en) * | 2013-09-05 | 2013-12-25 | 山东农业大学 | Preparation method for D type clostridium perfringen toxoid vaccine of cattle and sheep enterotoxaemia |
| CN103432578A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of C-type clostridium perfringens toxin vaccine of piglet red dysentery |
| CN103432579A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine |
| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 潘卫三主编: "《工业药剂学》", 31 August 2015, 北京：中国医药科技出版社 * |
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