CN101766814A - Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof - Google Patents
Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof Download PDFInfo
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- CN101766814A CN101766814A CN200910193900A CN200910193900A CN101766814A CN 101766814 A CN101766814 A CN 101766814A CN 200910193900 A CN200910193900 A CN 200910193900A CN 200910193900 A CN200910193900 A CN 200910193900A CN 101766814 A CN101766814 A CN 101766814A
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Abstract
The invention provides a swine pasteurellosis bivalent inactivated vaccine, which can prevent infection caused by two different capsule serotypes of pasteurella multocida, reaches the effect that one stitch of vaccine can prevent multiple infections, reduces the cost, does not have the hidden trouble of dispersing toxin and is safe and reliable. The invention also provides a preparation method for the swine pasteurellosis bivalent inactivated vaccine.
Description
Technical field
The present invention relates to the veterinary biologics field, particularly a kind of swine pasteurellosis bivalent inactivated vaccine.
Background technology
Bacillus pasteurii disease is one of main bacterial infectious disease of present China pig.
Bacillus pasteurii disease is to infect the bacterial disease that causes by pasteurella multocida, can cause septicemia, pneumonia.Pasteurellosis bacillus divides by pod membrane can be divided into A, B, four kinds of pod membrane serotypes of D, E.Bacillus pasteurii disease all has generation all over the world, and the torrid areas is comparatively serious.Popular at present Bacillus pasteurii disease also has a spot of D group based on A, B group.
Pasteurellosis bacillus can cause the mixed infection of pig body, it usually causes swinery high incidence and high mortality to occur, because mixed infection and abuse of antibiotics, cause sick many types of, sick many diseases, the many sick phenomenons of a disease very general, cause swine diseases increasingly sophisticated, the diagnosis difficulty is increasing, fails to pinpoint a disease in diagnosis with mistaken diagnosis to happen occasionally.Antibiotic medicine residual makes meat animals face an intransitable difficult problem when outlet.So it is more important that the vaccine of swine pasteurellosis just seems.
At present, the vaccine of the control Bacillus pasteurii disease of Shi Yonging has only the live vaccine that prevents pasteurellosis bacillus capsular serotype A group, B group bacterium to infect respectively on the market, and is merely able to prevent separately a kind of bacterial infection.In addition, existing pasteurellosis bacillus vaccine is the bacterial vaccine of living, and as immunogen, has certain diffusing malicious situation with the antibacterial that lives.
Therefore, need a kind of new swine pasteurellosis vaccine of invention to address the above problem.
Summary of the invention
One of main purpose of the present invention is to provide based on the deficiencies in the prior art a kind of swine pasteurellosis bivalent inactivated vaccine that can prevent by pasteurellosis bacillus capsular serotype A group and the caused bacterial infection of B group bacterium, immunity safety.
Another object of the present invention is to provide a kind of method for preparing swine pasteurellosis bivalent inactivated vaccine easy and simple to handle.
The invention provides a kind of swine pasteurellosis bivalent inactivated vaccine, described swine pasteurellosis bivalent inactivated vaccine comprises antigen and adjuvant: the pasteurellosis bacillus pod membrane B group bacterium that described antigen is cultivated concentrated solution and deactivation by the pasteurellosis bacillus capsular serotype A group bacterium of deactivation cultivate concentrated solution with etc. bacterium measure and mix, described antigen is 75～85 volume parts; Described adjuvant is a gel aluminum hydroxide, and described adjuvant is 15～25 volume parts.
Swine pasteurellosis bivalent inactivated vaccine of the present invention, owing to adopt the pasteurella multocida strain preparation bivalent inactivated vaccine of pod membrane serum A, Type B, the infection that can prevent the pasteurella multocida by two kinds of different pod membrane serotypes to cause, reach the effect that a pin is prevented more clinically, reduce cost, and do not have the malicious hidden danger of loosing, safe and reliable.
As the preferred implementation of swine pasteurellosis bivalent inactivated vaccine of the present invention, the bacterial strain of described pasteurellosis bacillus capsular serotype A group bacterium is capsular serotype A group pasteurella multocida P71 strain.
As the preferred implementation of swine pasteurellosis bivalent inactivated vaccine of the present invention, the bacterial strain of described pasteurellosis bacillus pod membrane B group bacterium is pod membrane B group pasteurella multocida C44-1.
The immunogenicity of above-mentioned two bacterial strains is strong, is suitable as the production bacterial strain.
As the preferred implementation of swine pasteurellosis bivalent inactivated vaccine of the present invention, also contain the thimerosal of 0.01% volume ratio in the described swine pasteurellosis bivalent inactivated vaccine.Thimerosal is a kind of chemical substance that can mould fungus inhibition.Prevent to be subjected to mould contamination in production of vaccine and the use.
The present invention also provides a kind of preparation method of swine pasteurellosis bivalent inactivated vaccine, and described preparation method comprises the steps:
(1) respectively with pasteurellosis bacillus capsular serotype A group bacterium and pasteurellosis bacillus pod membrane B group bacterium propagation, obtains pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid;
(2) pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and the deactivation of pasteurellosis bacillus pod membrane B group bacteria culture fluid that step (1) propagation is obtained;
(3) pasteurellosis bacillus capsular serotype A group's bacteria culture fluid after deactivation and pasteurellosis bacillus pod membrane B group bacteria culture fluid add gel aluminum hydroxide respectively and concentrate, in the concentrated solution that obtains respectively, the volume parts of described gel aluminum hydroxide is 15～25, and the volume parts of above-mentioned two kinds of culture fluid is respectively 75～85;
(4) pasteurellosis bacillus capsular serotype A group bacteria culture fluid concentrated solution that step (3) is obtained and pasteurellosis bacillus pod membrane B group bacteria culture fluid concentrated solution mix by equivalent bacterium number, obtain swine pasteurellosis bivalent inactivated vaccine.
The preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, easy and simple to handle, the infection that the product for preparing can prevent the pasteurella multocida by two kinds of different pod membrane serotypes to cause, reach the effect that a pin is prevented more clinically, reduce cost, and do not have the malicious hidden danger of loosing, safe and reliable.
Preferred implementation as the preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, the deactivation mode is in the pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid that obtain in step (1) propagation, add 0.2% formalin respectively by cumulative volume, be positioned over 37 ℃ of deactivations 24 hours, stir therebetween 3～5 times.
As the preferred implementation of the preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, the bacterial strain of described pasteurellosis bacillus capsular serotype A group bacterium is capsular serotype A group pasteurella multocida P71 strain.
As the preferred implementation of the preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, the bacterial strain of described pasteurellosis bacillus pod membrane B group bacterium is pod membrane B group pasteurella multocida C44-1.
The immunogenicity of above-mentioned two bacterial strains is strong, is suitable as the production bacterial strain.
As the preferred implementation of the preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, this preparation method comprises that also step (5) adds the thimerosal of 0.01% volume ratio to described swine pasteurellosis bivalent inactivated vaccine.The effect that adds thimerosal is mould fungus inhibition.
As the preferred implementation of the preparation method of swine pasteurellosis bivalent inactivated vaccine of the present invention, step (2) can also be carried out deactivation check and toxin check to pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid respectively after deactivation.The effect of carrying out the check of deactivation check and toxin is control to the manufacture process vaccine quality.
The specific embodiment
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.Concrete experimental technique of the present invention can be referring to " People's Republic of China's veterinary drug allusion quotation (in 2005 version) " and appendix.Other reagent or raw materials that do not indicate especially all can be obtained according to prior art by those skilled in the art.
Embodiment 1
This product is to select pathogenic pasteurellosis bacillus capsular serotype A group, B group's virulent strain for use, being inoculated in appropriate media cultivates, with culture after the formalin deactivation, the hydro-oxidation aluminium glue concentrates and makes bivalent inactivated vaccine, is used for prevention and infects caused bacterial disease by pasteurellosis bacillus capsular serotype A group and B group bacterium.The strain of making and checking this product to use is virulent strain such as P71 strain of capsular serotype A group pasteurella multocida and pod membrane B group pasteurella multocida C44-1 strain, is China Veterinery Drug Inspection Office and identifies, takes care of and supply.
1 strain
The strain of making and checking this product to use is pasteurellosis bacillus capsular serotype A group, B group's virulent strain, is to obtain the strong strain of immunogenicity, and the lyophilizing after the rejuvenation of responsive pig body of each strain is preserved.
The 2 vaccine manufacturing and the inspections of semifinished product
2.1 produce preparation with seed
2.1.1 the breeding of first order seed
Freeze-drying lactobacillus, with the martin's bouillon dilution, streak inoculation is put 37 ℃ and was cultivated 18～24 hours on the blood plate, chooses well-grown bacterium colony, is inoculated in and contains on the Sanguis caprae seu ovis agar slant, cultivates 18～24 hours for 37 ℃, as first order seed.
2.1.2 the breeding of secondary seed
Wash the blood agar slant culture of first order seed with a small amount of buffering meat soup, be inoculated in the bassoon of buffering meat soup, put 37 ℃ and cultivate and took out in 18～24 hours, through check pure after as secondary seed.
2.2 seedling culture medium
The pasteurellosis bacillus culture medium that adopts the merchant to sell.
2.3 the seedling preparation of bacterium liquid
Add in the culture medium by 5% amount respectively with strain liquid and cultivate respectively 2.3.1 seedling adds serum 2%, 2 strain seedling with the cultivation of bacterium liquid in culture medium, cultivation in rearmounted 37 ℃ of the mixing; When being reduced to 6, bacterium liquid pH stops when following cultivating.
2.3.2 pure check
After cultivate finishing, respectively from the sampling of seedling bacterium liquid, check purely according to the pure method of inspection of antibacterial class live vaccine in " People's Republic of China's veterinary drug allusion quotation (in 2005 version ", bacterium liquid should be pure.
2.3.3 count plate
From the sampling of seedling bacterium liquid, carry out count plate respectively, to determine to cultivate the bacterium number with the lung plague agar plate.
2.3.4 deactivation
In cultured strong toadstool liquid, the formalin by total amount adding 0.2% is positioned over 37 ℃ of deactivations 24 hours, stirs therebetween 3～5 times.
2.3.5 deactivation check
After the deactivation, get inactivated bacterial liquid 5ml and be inoculated in 100ml and contain in the culture medium of 2% serum 37 ℃ and cultivated 24 hours, transplant in above-mentioned culture medium 100ml 37 ℃ again and cultivated 24 hours; Get inactivated bacterial liquid 0.5ml and be inoculated in blood agar plate, cultivated 24 hours for 37 ℃.Above-mentioned two kinds of methods are cultivated all should asepsis growth.
2.3.6 toxin check
Get 5 of the SPF mices of inactivated bacterial liquid intravenous injection 18～22g, every 0.2ml, 3 days planted agent's 5/5 strong living.
2.3.7 precipitation concentrates
Through deactivation and toxin after the assay was approved, add an amount of aluminium hydroxide gel by inactivated bacterial liquid and shake up and precipitate concentratedly, mixing was put the room temperature precipitation 48～72 hours.
2.3.8 detoxification element
Add the aluminium glue of 15-25% in proportion to the good bacterium liquid of deactivation, mixing removes the toxin of antibacterial to greatest extent.
Antibacterial is in cultivating breeding, can produce a large amount of harmful substances, as the secretion of breeding extracellular toxin (based on protein) and the endotoxin (based on lipopolysaccharide) that discharges after the antibacterial apoptosis breaks in breeding in culture medium, and some organic acid and enzyme.These materials are all to the toxic effect of animal body (with anaphylaxis etc. for the most common).Aluminium hydroxide gel is a kind of weak adsorbing colloid that has, and it can adsorb the antibacterial thalline, with it in conjunction with after, form granule, and under action of gravity, fall to below the liquid, but can not adsorb or little material such as utmost point weight absorption bacteriotoxin.Simultaneously, aluminium hydroxide gel is a kind of immunostimulant, nonspecific immune response that can enhancement antigen.Propagation and deactivation are finished in antigen (antibacterial) liquid medium within, and the result of root a tree name count plate adds an amount of sterilization aluminium hydroxide gel in antigen liquid, after stirring, leave standstill.Aluminium hydroxide gel and antigen (antibacterial) conjugate then falls to the bottom, and harmful substances such as bacteriotoxin are in remaining in supernatant.After the supernatant removal, with physiological saline solution that precipitate is resuspended, the vaccine finished product that is mixed with will improve security of products greatly.
3 join Seedling and packing
3.1 mix
The count plate result of root a tree name 2.3.3 counts equal proportion with the residue of 2.3.8 by bacterium and mixes, and each bacterial strain contains suitable bacterium number by per 2 milliliters and joins Seedling, adds 0.01% thimerosal by total amount.
3.2 the inspection of semifinished product
Get the preceding Seedling sample of packing, carry out steriling test, should not have bacterial growth by existing " People's Republic of China's veterinary drug allusion quotation " appendix.
3.3 packing
With the 100ml/ bottle, or the packing of 20ml/ bottle, cover bottle stopper, and the jewelling lid.
In other embodiments, can also adopt other pathogenic pasteurellosis bacillus capsular serotype A group, the B group's virulent strain of system, and not only be confined to two bacterial strains being adopted among the embodiment 1.The mode of deactivation also can adopt other ablation methods such as beta-propiolactone (BPL), gamma-radiation irradiation and heating.Can adopt other mercurous materials to replace as the thimerosal of antiseptic, dimethylmercury for example, perhaps based on the reason of safety, thimerosal can not add yet.
Embodiment 2 effect research
With 20 of healthy susceptible pigs, the vaccine 2ml that each intramuscular injection embodiment 1 makes contains 1 using dosage; After 21 days, carry out strong virus attack with each virulent strain.
Pasteurellosis bacillus P71 strain:
Choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the strong toadstool liquid of injection P71 strain was observed 10 days, the death more than 4/5 of contrast pig, and immune swine is protected more than 4/5.
Pasteurellosis bacillus C44-1 strain:
Choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the strong toadstool liquid of each intravenous injection C44-1 strain was observed 10 days, the death 4/5 or more of contrast pig, and immune swine is protected more than 4/5.
The result:
| Matched group | Experimental group | |
| The P71 strain | 4/5 death | 4/5 protection |
| The C44-1 strain | 4/5 death | 5/5 protection |
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.
Claims (10)
1. a swine pasteurellosis bivalent inactivated vaccine is characterized in that, described swine pasteurellosis bivalent inactivated vaccine comprises antigen and adjuvant:
The pasteurellosis bacillus pod membrane B group bacterium that described antigen is cultivated concentrated solution and deactivation by the pasteurellosis bacillus capsular serotype A group bacterium of deactivation cultivate concentrated solution with etc. bacterium measure and mix, described antigen is 75～85 volume parts;
Described adjuvant is a gel aluminum hydroxide, and described adjuvant is 15～25 volume parts.
2. swine pasteurellosis bivalent inactivated vaccine as claimed in claim 1 is characterized in that, the bacterial strain of described pasteurellosis bacillus capsular serotype A group bacterium is capsular serotype A group pasteurella multocida P71 strain.
3. swine pasteurellosis bivalent inactivated vaccine as claimed in claim 1 is characterized in that, the bacterial strain of described pasteurellosis bacillus pod membrane B group bacterium is pod membrane B group pasteurella multocida C44-1.
4. swine pasteurellosis bivalent inactivated vaccine as claimed in claim 1 is characterized in that, also contains the thimerosal of 0.01% volume ratio in the described swine pasteurellosis bivalent inactivated vaccine.
5. the preparation method of a swine pasteurellosis bivalent inactivated vaccine, described preparation method comprises the steps:
(1) respectively with pasteurellosis bacillus capsular serotype A group bacterium and pasteurellosis bacillus pod membrane B group bacterium propagation, obtains pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid;
(2) pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and the deactivation of pasteurellosis bacillus pod membrane B group bacteria culture fluid that step (1) propagation is obtained;
(3) pasteurellosis bacillus capsular serotype A group's bacteria culture fluid after deactivation and pasteurellosis bacillus pod membrane B group bacteria culture fluid add gel aluminum hydroxide respectively and concentrate, in the concentrated solution that obtains respectively, the volume parts of described gel aluminum hydroxide is 15～25, and the volume parts of above-mentioned two kinds of culture fluid is respectively 75～85;
(4) pasteurellosis bacillus capsular serotype A group bacteria culture fluid concentrated solution that step (3) is obtained and pasteurellosis bacillus pod membrane B group bacteria culture fluid concentrated solution mix by equivalent bacterium number, obtain swine pasteurellosis bivalent inactivated vaccine.
6. the preparation method of swine pasteurellosis bivalent inactivated vaccine as claimed in claim 5, it is characterized in that, the deactivation mode is in the pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid that obtain in step (1) propagation, add 0.2% formalin respectively by cumulative volume, be positioned over 37 ℃ of deactivations 24 hours, stir therebetween 3～5 times.
7. the preparation method of swine pasteurellosis bivalent inactivated vaccine as claimed in claim 5 is characterized in that, the bacterial strain of described pasteurellosis bacillus capsular serotype A group bacterium is capsular serotype A group pasteurella multocida P71 strain.
8. the preparation method of swine pasteurellosis bivalent inactivated vaccine as claimed in claim 5 is characterized in that, the bacterial strain of described pasteurellosis bacillus pod membrane B group bacterium is pod membrane B group pasteurella multocida C44-1.
9. the preparation method of swine pasteurellosis bivalent inactivated vaccine as claimed in claim 5 is characterized in that, comprises that also step (5) adds the thimerosal of 0.01% volume ratio to described swine pasteurellosis bivalent inactivated vaccine.
10. the preparation method of swine pasteurellosis bivalent inactivated vaccine as claimed in claim 5, it is characterized in that step (2) is carried out deactivation check and toxin check to pasteurellosis bacillus capsular serotype A group's bacteria culture fluid and pasteurellosis bacillus pod membrane B group bacteria culture fluid respectively after deactivation.
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| CN200910193900A CN101766814A (en) | 2009-11-13 | 2009-11-13 | Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof |
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| CN200910193900A CN101766814A (en) | 2009-11-13 | 2009-11-13 | Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102895660A (en) * | 2012-10-25 | 2013-01-30 | 中国兽医药品监察所 | Bivalent inactivated vaccine for duck virus hepatitis |
| CN104056263A (en) * | 2013-03-22 | 2014-09-24 | 普莱柯生物工程股份有限公司 | Vaccine composition for preventing and treating respiratory diseases secondary to atrophic rhinitis, and preparation method and application thereof |
| CN106692962A (en) * | 2016-11-11 | 2017-05-24 | 安徽东方帝维生物制品股份有限公司 | Preparation method of pig pasteurella multocida antigen and application |
| CN107312733A (en) * | 2017-08-17 | 2017-11-03 | 天康生物股份有限公司 | Viable count detection method in serum A types, serum D type pasteurella multocida mixed culturing methods and gained mixed bacteria liquid |
| CN109602902A (en) * | 2018-11-27 | 2019-04-12 | 广东渔跃生物技术有限公司 | One boar Pasteurella polysaccharide protein Conjugate vaccines and preparation method thereof |
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2009
- 2009-11-13 CN CN200910193900A patent/CN101766814A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102895660A (en) * | 2012-10-25 | 2013-01-30 | 中国兽医药品监察所 | Bivalent inactivated vaccine for duck virus hepatitis |
| CN104056263A (en) * | 2013-03-22 | 2014-09-24 | 普莱柯生物工程股份有限公司 | Vaccine composition for preventing and treating respiratory diseases secondary to atrophic rhinitis, and preparation method and application thereof |
| CN104056263B (en) * | 2013-03-22 | 2016-05-11 | 普莱柯生物工程股份有限公司 | A kind of prevention and treatment are by vaccine combination and the preparation method and application of the respiratory disease of atrophic rhinitis secondary |
| CN106692962A (en) * | 2016-11-11 | 2017-05-24 | 安徽东方帝维生物制品股份有限公司 | Preparation method of pig pasteurella multocida antigen and application |
| CN107312733A (en) * | 2017-08-17 | 2017-11-03 | 天康生物股份有限公司 | Viable count detection method in serum A types, serum D type pasteurella multocida mixed culturing methods and gained mixed bacteria liquid |
| CN109602902A (en) * | 2018-11-27 | 2019-04-12 | 广东渔跃生物技术有限公司 | One boar Pasteurella polysaccharide protein Conjugate vaccines and preparation method thereof |
| CN109602902B (en) * | 2018-11-27 | 2022-10-28 | 广东渔跃生物技术有限公司 | Swine pasteurella polysaccharide protein coupling vaccine and preparation method thereof |
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Address after: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant after: GUANGDONG WINSUN BIOPHARMACEUTICAL CO., LTD. Address before: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant before: Guangdong Winsun Bio-Pharmaceutical Co., Ltd. |
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Application publication date: 20100707 |