CN104623652A - Method for preparing two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus of necrotic enteritis - Google Patents
Method for preparing two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus of necrotic enteritis Download PDFInfo
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Abstract
The invention relates to the field of animal bacteriology and provides a method for preparing two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus of necrotic enteritis. The method comprises the following steps: inoculating A type and C type clostridium perfringens enrichment fluid in a toxin-producing culture medium, and shake cultivating in an anaerobic environment of 43 DEG C for 5 hours to produce toxin; and inactivating via formaldehyde, adding an adjuvant to emulsify to obtain two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus. The protection rate of vaccine immune commercial chickens obtained by the method reaches 100% after counteracting toxic substances, which proves that the toxoid and thallus antigen complex vaccine can generate a good immune protection effect on pathogenic infection of bacteria and pathogenicity of bacterial toxins on poultry.
Description
Technical field
The present invention relates to animal bacteria field, the invention provides a kind of A, C type bacillus perfringens toxoid of chicken necrotizing enterocolitis and the preparation method of thalline bivalence two class antigen vaccine.
Background technology
Necrotic enteritis (Necrotic Enteritis is called for short NE) causes primarily of A type or C type bacillus perfringens, but C type is not general.Produce the characteristic pathological changes such as intestinal mucosa necrosis, the fowl that dies of illness has wheat bran sample necrosis region for feature with hepatomegaly, intestinal bleeding and intestinal mucosa.Can be there is this disease in multiple birds, confirmed that necrotic enteritis has generation in broiler, laying hen, turkey and Carnis Coturnicis japonicae.Along with the forbidding to antibiotics growth promoter, there is generation this disease mainly supports fowl countries and regions in the whole world, and global economy loss reaches 2,000,000,000 dollars.
Bacillus perfringens is anaerobism, produces spore, Gram-positive escherichia coli.It is vertebrates intestinal commensalism bacterium, but quantity is lower in normal host intestinal.It is also a kind of conditioned pathogen, if condition is suitable for, and can at intestinal amount reproduction.Bacillus perfringens cause a disease mainly its produce extracellular toxin cause.The foreign protein of at least 17 kinds of toxin or genotoxic potential can be produced, A, B, C, D, E 5 toxin producing types can be divided into according to α, β, ε, ι toxin playing principal causative effect.
Current research; Meat-type Breeding Hen immunity A type and C type toxoid can produce stronger IgY serum antibody response; and can offspring be passed to, although A type antibody response is higher than C type, the broiler protected effect of immune C type toxoid to subclinical necrotic enteritis and hepatitis is more remarkable.Crouch in 2010 etc. use A type toxoid and the light mineral oil emulsifying seedling of NetvaxTM company, intramuscular injection immunity inoculation Female broiler breeder safety, the mental status and fertility performance, without whole body side reaction, obviously can reduce death and the intestinal injury of the Offspring of Broiler caused by NE.2011, Saleh etc. use local epidemic isolates to prepare A type, C type and A+C type toxoid, subcutaneous injection immunity 7 age in days commercial meat birds, 0.5ml/ only, 21 ages in days add with same dosage and approach exempts from once, mixed hello (2L bacillus perfringens broth culture+1kg feedstuff or 1L bacillus perfringens broth culture+2L water) counteracting toxic substances is carried out at 35 ages in days, result proves immune chicken, the anatoxic chicken intestinal damage of immune especially A+C type obviously declines, but what adopt in said method is directly with toxoid immunity broiler, there is more limitation, as duration of immunity is short, need repeatedly the defects such as booster immunization.Therefore how to overcome above-mentioned defect, the development tool for vaccine has very important significance.
Summary of the invention
The invention provides a kind of A, C type bacillus perfringens toxoid of chicken necrotizing enterocolitis and the preparation method of thalline bivalence two class antigen vaccine, the method is inoculated in toxin producing medium with A type, each 4ml of C type bacillus perfringens enrichment liquid, (88%N under 43 DEG C of anaerobic environments
2, 7%H
2, 5%CO
2) shaken cultivation 5h.Through formalin-inactivated, add adjuvant emulsion and obtain A, C type bacillus perfringens toxoid and thalline bivalence two class antigen vaccine.By the vaccine that said method obtains; containing α, β two kinds of primary toxins antigens; and toxoid and bacillus perfringens somatic antigen composition various; necrotic enteritis bacillus perfringens being infected to the poultry caused has splendid immune effect; after immunity commercial chicken counteracting toxic substances, its protective rate reaches 100%; prove this toxoid and somatic antigen combination vaccine; can the Infective effect of directed toward bacteria, also the pathogenic effects of directed toward bacteria toxin can produce good immune protective effect to poultry.
A, C type bacillus perfringens that inventor is adopted in the present invention adopts existing strain, be selected from standard strain A type NCTC528, C type NCTC3180 (being all preserved in National Collection of Type Cultures Britain Culture Collection) especially, directly obtain by existing channel, so this biomaterial belongs to the material that can directly obtain in prior art, do not need to carry out independent biological deposits.
Concrete technical scheme of the present invention is as follows: concrete steps are as follows:
(1). the recovery of strain and increasing bacterium
Bacillus perfringens standard strain A type NCTC528, C type NCTC3180 are inoculated in blood agar plate respectively, in anaerobic box, cultivate 24h recovery for 39 DEG C; Picking colonies typical is inoculated in 39 DEG C of anaerobism Zengjing Granule 12h in 7ml THIOGLYCOLLIC ACID salt broth;
(2). the preparation of bacterium liquid
A type, the C type bacillus perfringens enrichment culture medium of above-mentioned acquisition are all inoculated in 100ml toxin producing medium with 4ml, and in 43 DEG C of anaerobic box, 160r/min shaken cultivation 5h obtains bacterium liquid;
(3). the deactivation of bacterium liquid
The bacterium liquid 1MNaOH that produces is adjusted pH to 7.2, and then add formalin and make formaldehyde mass fraction reach 0.3% to carry out deactivation, fully vibration mixing is placed in 37 DEG C of shaking tables, and after 4 days, deactivation is complete;
(4). the preparation of vaccine
The ratio of the bacterium liquid that deactivation is up to the standards and the white-oil adjuvant of sterilizing 1:2 is by volume fully emulsified, and adds thimerosal according to the ratio that every 100 milliliters of above-mentioned emulsion mixture add 0.01 gram of thimerosal and can obtain vaccine;
Wherein said toxin producing medium, its preparation method is as follows: 2.5 grams of peptones, 2 grams of yeast extracts, 1.5 grams of dextrin, 1.4 grams of L-arginines are dissolved in 100mlPBS buffer, adjusts pH to 7.4 and get final product with 1M HCl;
It is reagent YEAST EXTRACT that described yeast is extracted as, and market is directly buied.
Above-mentioned culture medium is the brand-new culture medium that inventor designs for bacillus perfringens of the present invention, better can meet bacillus perfringens than existing culture medium and cultivate the needs producing poison, there is obvious specific aim and effectiveness, and preparing process easy, save production cost and good stability, the requirement of suitability for industrialized production can be met.
In wherein said anaerobic box, by volume mark is as follows in gas composition: 88%N
2, 7%H
2, 5%CO
2;
Described blood agar is after 1g glucose and 3.8g bean agar powder are dissolved in the sterilizing of 100ml distilled water, adds 5ml Sheep Blood and obtains.
Wherein, in order to obtain the best inoculative proportion of A type and C type bacterium, inventor is when the preparation of step bacterium liquid, be that 2ml:6ml, 4ml:4ml, 6ml:2ml three components has not carried out corresponding culture experiment by A type, C type bacillus perfringens enrichment culture medium inoculation volume score, conventional double sandwich-ELISA kit measurement is utilized respectively to organize toxin producing amount afterwards, when measurement result inoculative proportion is 4ml:4ml, toxin producing amount is maximum, so select this volume ratio.
In order to verify the effect of above-mentioned vaccine, inventor has carried out effect detection to it, and concrete grammar is as follows:
The healthy susceptible chicken 10 of 9 ages in days; wherein 5 above-mentioned vaccine 0.1ml of subcutaneous injection; another 5 chickens do not inoculate and compare; A, C type clostridium perfringens toxoid of each intramuscular injection lethal dose after 21 days; observe 10; matched group is all dead, and immune group is all survived, and shows that this vaccine is 100% to the protective rate of chicken.
In sum, the vaccine that the present invention obtains is compared with existing attenuated vaccine, recombinant vaccine, there is following beneficial effect: (1) vaccine of the present invention contains the alpha toxin Venom antigens relevant to chicken necrotizing enterocolitis with β toxin two kinds, and the inoculative proportion of A type and C type bacterium is measured by double sandwich-ELISA method, result shows that inoculative proportion reaches the highest toxin producing amount when 4ml:4ml; (2) because toxin producing amount is high, the toxoid antigen in vaccine contained by per unit is high, and body corresponding generation t antibody is high, and animal protection rate is high.Disease occur time, body produce t antibody can directly in and bacillus perfringens generation extracellular toxin, reach good immunoprophylaxis effect.
Detailed description of the invention
Embodiment 1
A, C type bacillus perfringens toxoid of chicken necrotizing enterocolitis and a preparation method for thalline bivalence two class antigen vaccine, concrete steps are as follows:
(1). the recovery of strain and increasing bacterium
Bacillus perfringens standard strain A type NCTC528, C type NCTC3180 are inoculated in blood agar plate respectively, in anaerobic box, cultivate 24h recovery for 39 DEG C; Picking colonies typical is inoculated in 39 DEG C of anaerobism Zengjing Granule 12h in 7ml THIOGLYCOLLIC ACID salt broth;
(2). the preparation of bacterium liquid
A type, the C type bacillus perfringens enrichment culture medium of above-mentioned acquisition are all inoculated in 100ml toxin producing medium with 4ml, and in 43 DEG C of anaerobic box, 160r/min shaken cultivation 5h obtains bacterium liquid;
(3). the deactivation of bacterium liquid
The bacterium liquid 1MNaOH that produces is adjusted pH to 7.2, and then add formalin and make formaldehyde mass fraction reach 0.3% to carry out deactivation, fully vibration mixing is placed in 37 DEG C of shaking tables, and after 4 days, deactivation is complete;
(4). the preparation of vaccine
The ratio of the bacterium liquid that deactivation is up to the standards and the white-oil adjuvant of sterilizing 1:2 is by volume fully emulsified, and adds thimerosal according to the ratio that every 100 milliliters of above-mentioned emulsion mixture add 0.01 gram of thimerosal and can obtain vaccine;
Wherein said toxin producing medium, its preparation method is as follows: 2.5 grams of peptones, 2 grams of yeast extracts, 1.5 grams of dextrin, 1.4 grams of L-arginines are dissolved in 100mlPBS buffer, adjusts pH to 7.4 and get final product with 1M HCl;
It is reagent YEAST EXTRACT that described yeast is extracted as, and market is directly buied.
In wherein said anaerobic box, by volume mark is as follows in gas composition: 88%N
2, 7%H
2, 5%CO
2;
Described blood agar is after 1g glucose and 3.8g bean agar powder are dissolved in the sterilizing of 100ml distilled water, adds 5ml Sheep Blood and obtains.
Embodiment 2
Vaccine potency is checked, and concrete grammar is as follows:
The healthy susceptible chicken 10 of 9 ages in days; the vaccine 0.1ml wherein obtained in 5 subcutaneous injection embodiments 1; another 5 chickens do not inoculate and compare; A, C type clostridium perfringens toxoid of each intramuscular injection lethal dose after 21 days; observe 10; matched group is all dead; immune group is all survived; show that this vaccine is 100% to the protective rate of chicken; prove this toxoid and somatic antigen combination vaccine; can the Infective effect of directed toward bacteria, also the pathogenic effects of directed toward bacteria toxin can produce good immune protective effect to poultry.
Claims (3)
1. A, C type bacillus perfringens toxoid of chicken necrotizing enterocolitis and a preparation method for thalline bivalence two class antigen vaccine, concrete steps are as follows:
(1). the recovery of strain and increasing bacterium
Bacillus perfringens standard strain A type NCTC528, C type NCTC3180 are inoculated in blood agar plate respectively, in anaerobic box, cultivate 24h recovery for 39 DEG C; Picking colonies typical is inoculated in 39 DEG C of anaerobism Zengjing Granule 12h in 7ml THIOGLYCOLLIC ACID salt broth;
(2). the preparation of bacterium liquid
A type, the C type bacillus perfringens enrichment culture medium of above-mentioned acquisition are all inoculated in 100ml toxin producing medium with 4ml, and in 43 DEG C of anaerobic box, 160r/min shaken cultivation 5h obtains bacterium liquid;
(3). the deactivation of bacterium liquid
The bacterium liquid 1MNaOH that produces is adjusted pH to 7.2, and then add formalin and make formaldehyde mass fraction reach 0.3% to carry out deactivation, fully vibration mixing is placed in 37 DEG C of shaking tables, and after 4 days, deactivation is complete;
(4). the preparation of vaccine
The ratio of the bacterium liquid that deactivation is up to the standards and the white-oil adjuvant of sterilizing 1:2 is by volume fully emulsified, and adds thimerosal according to the ratio that every 100 milliliters of above-mentioned emulsion mixture add 0.01 gram of thimerosal and can obtain vaccine;
Wherein said toxin producing medium, its preparation method is as follows: 2.5 grams of peptones, 2 grams of yeast extracts, 1.5 grams of dextrin, 1.4 grams of L-arginines are dissolved in 100mlPBS buffer, adjusts pH to 7.4 and get final product with 1M HCl;
Described yeast extract is reagent YEAST EXTRACT.
2. preparation method according to claim 1, is characterized in that: in described anaerobic box, by volume mark is as follows in gas composition: 88%N
2, 7%H
2, 5%CO
2.
3. preparation method according to claim 1, is characterized in that: described blood agar is after 1g glucose and 3.8g bean agar powder are dissolved in the sterilizing of 100ml distilled water, adds 5ml Sheep Blood and obtains.
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| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
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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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| 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 |
| CN103432578B (en) * | 2013-09-12 | 2015-03-25 | 山东农业大学 | Preparation method of C-type clostridium perfringens toxin vaccine of piglet red dysentery |
Non-Patent Citations (2)
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| 曲海波等: "A型产气荚膜梭菌类毒素疫苗与全菌灭活疫苗的安全性和免疫效力的比较研究", 《中国兽药杂志》 * |
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| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
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