CN104623651B - A kind of preparation method of the A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease - Google Patents
A kind of preparation method of the A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease Download PDFInfo
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Abstract
The present invention relates to animal bacteria field, provide a kind of preparation method of the A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease, the method is with A type bacillus perfringens as original strain, after firsts and seconds culture, final acquisition bacterium solution, this bacterium solution is mixed with white-oil adjuvant A type bacillus perfringens inactivated vaccine is obtained；The vaccine immunity rabbit of the method for the invention preparation, after counteracting toxic substances, its protective rate reaches 100%, serves good immanoprotection action.
Description
Technical field
A kind of the present invention relates to animal bacteria field, the invention provides the A type perfringens of rabbit clostridieum welchii disease
The preparation method of clostridium inactivated vaccine.
Background technology
Clostridieum welchii disease primary disease is also known as bacillus perfringens disease.Clostridieum welchii enteritis is mainly by A type bacillus perfringens
Caused by the alpha toxin producing.It is widely present in soil, feedstuff, vegetable, sewage, in feces.The generation of primary disease no obvious season
Property, in addition to suckling son rabbit, various ages, kind, the rabbit of sex all have susceptibility.The main infection of primary disease is former to be disease rabbit and band
Bacterium rabbit and Excreta.Pathogen self-digestion road or wound invade body, amount reproduction producing on small intestinal and caecum chorion
Strong alpha toxin, changes the permeability of blood capillary, makes toxin enter blood in a large number, cause whole body stercoraemia.
In recent years, the sickness rate of piglet red dysentery in all parts of the country and ruminant necrotic enteritis and enterotoxemia was also bright
The enterotoxemia of aobvious ascendant trend, especially rabbit, newborn lamb and calf and lamb dysentery have then become the animal husbandry of puzzlement China
The a great problem of development, has severely impacted the development of animal husbandry.For the treatment of above-mentioned disease, can use theoretically
Antibiotic and sulfa drugss etc. carry out symptomatic treatment, but due to affected animal morbidity typically all fall ill anxious, dead soon, symptom is not sometimes
Substantially and at all have little time very much to treat；Patient's condition is relatively slow, is treated using these medicines, and effect is also very undesirable will be from
Fundamentally control the propagation of primary disease, be reduced or avoided and conscientiously have it is necessary to take to the huge economic losses that Animal husbandry production brings
The preventive measure of effect.
Research to bacillus perfringens inactivated vaccine has had a lot.Old stannum congruence has carried out " rabbit hemorrhagic disease "
Join the research of Seedling with A type clostridieum welchii；Tong Cheng just etc. had developed the clostridieum welchii list Seedling of rabbit, can produce 82.01% after counteracting toxic substances
Protective rate；Lu Cheng etc. is studied to rabbit hemorrhagic disease and A type clostridieum welchii Combined vaccine, and A type clostridieum welchii produces for 21 days
Raw immunity, protective rate is 82.14%；Yang Ying studies to A type clostridieum welchii aluminium hydroxide inactivated vaccine, vaccine 0.6ml,
2ml, 4ml can protect mice, rabbit and pig to resist the attack of standard A type 1MLD toxin.But the immunity guarantor to animal for these vaccines
Shield rate and safety etc. are not ideal, and therefore we need good immune effect badly, safe and reliable neoplasm preparation, Ran Houying
Control for this epidemic disease.
Content of the invention
For these reasons, the invention provides a kind of A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease
Preparation method, the method, with A type bacillus perfringens as original strain, final after firsts and seconds culture obtains bacterium
Liquid, this bacterium solution is mixed with white-oil adjuvant A type bacillus perfringens inactivated vaccine is obtained；The epidemic disease of the method for the invention preparation
Seedling immunizing rabbit, after counteracting toxic substances, its protective rate reaches 100%, serves good immanoprotection action.
The A type bacillus perfringens that inventor is adopted in the present invention adopt existing strain, in particular selected from standard strain A
Type NCTC528 (is preserved in National Collection of Type Cultures Britain Culture Collection),
Can be directly obtained by existing channel, so this biomaterial belongs to the material that can directly obtain in prior art, be not required to carry out
Individually biological deposits.
The concrete technical scheme of the present invention is as follows：Comprise the following steps that：
(1). the preparation of production strain
Bacillus perfringens standard strain A type NCTC528 is inoculated in blood agar plate, cultivates in anaerobic box for 37 DEG C
36h recovers；Picking colonies typical is inoculated on 7ml nutrient broth medium 39 DEG C of Anaerobic culturel 12h respectively as first order seed
Culture fluid；Afterwards the first order seed culture fluid of 2ml is inoculated in 39 DEG C of Anaerobic culturel 12h in 100ml nutrient broth medium to make
For secondary seed culture fluid；
(2). the preparation of seedling bacterium solution
The secondary seed culture fluid of above-mentioned for 70ml acquisition is accessed in 1L toxin producing medium, 200r/ under 45 DEG C of anaerobic environments
Min stir culture 4h；
(3). the inactivation of seedling bacterium solution
The bacterium solution obtaining after above-mentioned culture is adjusted pH to 7.2 with 1MNaOH, being subsequently adding formalin makes the final matter of formaldehyde
Amount fraction reaches 0.3% and is inactivated, and is sufficiently stirred for mixing, and 37 DEG C inactivate 3 days；
(4). the preparation of vaccine
The white-oil adjuvant of the inactivation qualified bacterium solution of inspection and sterilizing by volume 1：2 ratio is fully emulsified, and according to every
The vaccine of 100 milliliters of volumes adds the thimerosal that the ratio of 0.01 gram of thimerosal adds can obtain target inactivated vaccine；
The 4 DEG C of preservations of prepared inactivated vaccine are to be checked；
Wherein said toxin producing medium obtains by the following method：By 0.29g Na2HPO4·12H2O,
6.02gKH2PO4, 8.0gNaCl, 20g peptone, 10g dextrin, 20g yeast extract, 12gL- arginine is sequentially added into
1000ml deionized water obtains final product.
Described yeast is extracted as being reagent YEAST EXTRACT, market is directly buied.
Above-mentioned toxin producing medium is a kind of brand-new culture medium that the present inventor obtains after long-felt practice,
This culture medium complies fully with bacillus perfringens and excretes poison the needs to nutritional labeling, is become by adjusting phosphate buffer
Point, make pH reach 7.4, aborning simplified culture keynote section pH.And than traditional meat liver stomach membrane digestion soup and kitchen meat culture
Basigamy simple process, saving production cost and good stability, disclosure satisfy that the requirement of industrialized production.
During described Anaerobic culturel, anaerobic environment gas composition is as follows by volume fraction：88%N2, 7%H2, 5%CO2.
Described blood agar is 1g glucose and 3.8g bean agar powder is dissolved in after the sterilizing of 100ml distilled water, adds 5ml sheep
Blood obtains.
Bacillus perfringens standard strain A type NCTC528 adopting in the present invention, containing for rabbit clostridieum welchii disease
Alpha toxin gene, can produce alpha toxin antigen.The vaccine of preparation can stimulate body to produce for A type bacillus perfringens thalline and its product
Two kinds of antibody of raw alpha toxin.
The present invention is explored to fermentation condition in terms of medium nutrient content, temperature, pH, from cost and toxin producing
And three Elements research of bacterium number find, improve temperature in bacterial growth Suitable ranges and advantageously produce poison in antibacterial
Element, but the growth that all can affect antibacterial too high or too low for temperature, reduce toxin producing amount.And during the fermentation in fermentation tank
The paired bacterial growth of anaerobic environment gas group and toxogenic raising play very important effect.Ensure fermentation liquid pH simultaneously
7.4 about, this is to reduce toxin producing amount higher than 7.8 due to pH value to value, and maintenance 7.0-7.4 toxin producing amount highest is therefore full
The optimum condition that poison is produced in bacillus perfringens growth is can reach after sufficient above-mentioned condition.
In order to verify the effect of above-mentioned vaccine, inventor has carried out effect detection to it, and concrete grammar is as follows：
1.5-2.0kg healthy rabbits 10, wherein 5 subcutaneous injection vaccine 2ml, another 5 compare, all quiet after 21 days
Arteries and veins inject lethal dose A type clostridium perfringens toxoid, observe 10 days, matched group is all dead, immune group all survive it was demonstrated that
This inactivated vaccine reaches 100% to the protective rate of rabbit.
In sum, the vaccine that the present invention is obtained, compared with existing vaccine, has the advantages that：(1) body produces
Raw antibody is high, and animal protection rate is high.Contain antibacterial and toxin due in the vaccine prepared using toxin producing culture medium culturing bacterium solution
Two kinds of antigenic components, can produce two kinds of antibody being directed to somatic antigen and Venom antigens by active immunity in body, reach good
Preventive effect.(2) bacillus perfringens inactivated vaccine involved in the present invention is easy to large-scale production, with low cost, operative employee
Skill is simple.
Specific embodiment
Embodiment 1
A kind of preparation method of the A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease, comprises the following steps that：
(1). the preparation of production strain
Bacillus perfringens standard strain A type NCTC528 is inoculated in blood agar plate, cultivates in anaerobic box for 37 DEG C
36h recovers；Picking colonies typical is inoculated on 7ml nutrient broth medium 39 DEG C of Anaerobic culturel 12h respectively as first order seed
Culture fluid；Afterwards the first order seed culture fluid of 2ml is inoculated in 39 DEG C of Anaerobic culturel 12h in 100ml nutrient broth medium to make
For secondary seed culture fluid；
(2). the preparation of seedling bacterium solution
The secondary seed culture fluid of above-mentioned for 70ml acquisition is accessed in 1L toxin producing medium, 200r/ under 45 DEG C of anaerobic environments
Min stir culture 4h；
(3). the inactivation of seedling bacterium solution
The bacterium solution obtaining after above-mentioned culture is adjusted pH to 7.2 with 1MNaOH, being subsequently adding formalin makes the final matter of formaldehyde
Amount fraction reaches 0.3% and is inactivated, and is sufficiently stirred for mixing, and 37 DEG C inactivate 3 days；
(4). the preparation of vaccine
The white-oil adjuvant of the inactivation qualified bacterium solution of inspection and sterilizing by volume 1：2 ratio is fully emulsified, and according to every
The vaccine of 100 milliliters of volumes adds the thimerosal that the ratio of 0.01 gram of thimerosal adds can obtain target inactivated vaccine；
The 4 DEG C of preservations of prepared inactivated vaccine are to be checked；
Wherein said toxin producing medium obtains by the following method：By 0.29g Na2HPO4·12H2O,
6.02gKH2PO4, 8.0gNaCl, 20g peptone, 10g dextrin, 20g yeast extract, 12gL- arginine is sequentially added into
1000ml deionized water obtains final product；Wherein said yeast is extracted as being reagent YEAST EXTRACT, market is directly buied；
During described Anaerobic culturel, anaerobic environment gas composition is as follows by volume fraction：88%N2, 7%H2, 5%CO2；
Described blood agar is 1g glucose and 3.8g bean agar powder is dissolved in after the sterilizing of 100ml distilled water, adds 5ml sheep
Blood obtains.
Embodiment 2
Vaccine potency is checked, and concrete grammar is as follows：
1.5-2.0kg healthy rabbits 10, the vaccine 2ml in wherein 5 subcutaneous injection embodiments 1, another 5 compare,
The A type clostridium perfringens toxoid of whole intravenous injection lethal doses after 21 days, observes 10, matched group is all dead, immune group
All survival is it was demonstrated that this inactivated vaccine reaches 100% to the protective rate of rabbit.
Claims (3)
1. a kind of preparation method of the A type bacillus perfringens inactivated vaccine of rabbit clostridieum welchii disease, comprises the following steps that：
(1). the preparation of production strain
Bacillus perfringens standard strain A type NCTC528 is inoculated in blood agar plate, cultivates 36h in anaerobic box again for 37 DEG C
Soviet Union；Picking colonies typical is inoculated on 7ml nutrient broth medium 39 DEG C of Anaerobic culturel 12h as first order seed culture fluid；It
Afterwards the first order seed culture fluid of 2ml is inoculated in 100ml nutrient broth medium 39 DEG C of Anaerobic culturel 12h as secondary seed
Culture fluid；
(2). the preparation of seedling bacterium solution
The secondary seed culture fluid of above-mentioned for 70ml acquisition is accessed in 1L toxin producing medium, under 45 DEG C of anaerobic environments, 200r/min stirs
Mix culture 4h；
(3). the inactivation of seedling bacterium solution
The bacterium solution obtaining after above-mentioned culture is adjusted pH to 7.2 with 1MNaOH, being subsequently adding formalin makes formaldehyde final mass divide
Number reaches 0.3% and is inactivated, and is sufficiently stirred for mixing, and 37 DEG C inactivate 3 days；
(4). the preparation of vaccine
The white-oil adjuvant of the inactivation qualified bacterium solution of inspection and sterilizing by volume 1：2 ratio is fully emulsified, and according to every 100 millis
The vaccine rising volume adds the thimerosal that the ratio of 0.01 gram of thimerosal adds can obtain target inactivated vaccine；
Wherein said toxin producing medium obtains by the following method：By 0.29g Na2HPO4.12H2O, 6.02gKH2PO4,
8.0gNaCl, 20g peptone, 10g dextrin, 20g yeast extract, 12gL- arginine is sequentially added into 1000ml deionization
Water obtains final product.
2. according to claim 1 preparation method it is characterised in that：During described Anaerobic culturel, anaerobic environment gas composition presses body
Fraction is as follows：88%N2, 7%H2, 5%CO2.
3. according to claim 1 preparation method it is characterised in that：Described blood agar is 1g glucose and 3.8g bean agar
After powder is dissolved in the sterilizing of 100ml distilled water, 5ml Sheep Blood is added to obtain.
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| CN106066398B (en) * | 2016-05-25 | 2018-08-21 | 山东农业大学 | A kind of indirect ELISA detection method of A types clostridium perfringens toxoid antibody |
| CN107400697A (en) * | 2017-07-24 | 2017-11-28 | 内蒙古东达獭兔循环产业研究院 | A kind of clostridieum welchii culture medium and its prepare discrimination method |
| CN109954135B (en) * | 2017-12-25 | 2023-01-31 | 金宇保灵生物药品有限公司 | Inactivated toxoid vaccine of clostridium perfringens type A cattle and preparation method thereof |
| CN111500482B (en) * | 2019-01-31 | 2023-09-05 | 内蒙古金宇保灵生物技术研究院有限公司 | Clostridium perfringens type A strain of sheep, inactivated vaccine and vaccine preparation method |
| CN109943507B (en) * | 2019-03-26 | 2021-08-17 | 中国兽医药品监察所 | Preparation method and application of veterinary A-type clostridium perfringens toxin |
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| 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 |
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| "产气荚膜梭菌外毒素生产发酵工艺及其类毒素油乳剂疫苗的研究";吕静;《山东农业大学硕士学位论文》;20100315;正文第25页1.1，第26页1.4,1.5，第28页2.2，第35-36页2.2,2.3，第39页3.1，第41-43页3.7，第44-45页4.3 * |
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