CN102100911B - Preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine - Google Patents
Preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine Download PDFInfo
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- CN102100911B CN102100911B CN 201010290119 CN201010290119A CN102100911B CN 102100911 B CN102100911 B CN 102100911B CN 201010290119 CN201010290119 CN 201010290119 CN 201010290119 A CN201010290119 A CN 201010290119A CN 102100911 B CN102100911 B CN 102100911B
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Abstract
The invention discloses a preparation method of a Cynoglossus semilaevis vibrio anguillarum vaccine, which is characterized in that Cynoglossus semilaevis pathogeny Listonella anguillarum BH1 serves as an immunogen, and Astragalus mongholicus lixivium serves as an adjuvant. The preparation method comprises the following steps: inoculating the Cynoglossus semilaevis pathogeny Listonella anguillarum BH1 bacterial strain on a common nutrient broth culture medium; after the bacterial strain is cultured, inactivating by formaldehyde to obtain inactivated bacteria; after dipping the Astragalus mongholicus with distilled water, decocting, and filtering; condensing until the final concentration is 1g of raw drug per ml, and obtaining the Astragalus mongholicus lixivium; and mixing the inactivatedbacteria with the Astragalus mongholicus lixivium to obtain the Cynoglossus semilaevis Vibrio anguillarum vaccine. When the vaccine prepared with the method disclosed by the invention is used for immunizing Cynoglossus semilaevis, the capability of the Cynoglossus semilaevis on resisting Vibrio anguillarum infection can be obviously improved, Vibrio anguillarum infection during the culture production of the Cynoglossus semilaevis can be effectively prevented, the death rate of the Cynoglossus semilaevis is reduced, the culture yield of the Cynoglossus semilaevis is improved, the Cynoglossus semilaevis culture production is guaranteed to healthily and sustainably develop, and the economic benefit for Cynoglossus semilaevis culture production is improved.
Description
Technical field
The present invention relates to the preparation method of a kind of preparation method of animal vaccine, particularly a kind of Cynoglossus semilaevis vibrio anguillarum vaccine; The invention still further relates to the immune using method of this vaccine.
Background technology
Vibrio anguillarum is the important pathogen of harm aquatic animal, the vibriosis (Vibriosis) that is occured by its caused marine fish have popular extensively, morbidity and mortality rate high, be quite serious to the harm of Cynoglossus semilaevis breeding production especially.Therefore, to the high-efficiency prevention and control of Vibrio anguillarum disease, to reduce to greatest extent its extent of injury, can improve significantly the economic benefit that cynoglossus semilaevis cultivation is produced.Aspect the sick prevention and control of marine fish Vibrio anguillarum, in breeding production, mostly use at present chemical synthetic drug or antibiotics to control the generation of this disease and popular, but in long-term drug use, it is a lot of not enough that we find that Drug therapy exists: the one, and the cause of disease is complicated, there is blindness in a large amount of medications, and a lot of diseases there is no targetedly active drug; The 2nd, some chemicals significant surfaces are narrow, and a little less than the preventive effect, and sick fish often is difficult to picked-up to effective drug dose because of anorexia; The 3rd, medicine is residual in animal body, jeopardizes human health, and long-term prescription brings environmental pollution, and causes resistant micro-organism to occur in a large number; In addition, under medicine " protective effect ", make for a long time the resistance against diseases decline that the weak individuality of premunition that should be eliminated in a large number survives and proportion increases and cause whole colony gradually in population, had a strong impact on the development of aquaculture.Therefore, the generation of applied immunology technical prevention fish bacterial disease has been subject to increasing attention, has become the important content that various countries' researcher is paid close attention to.Also be not applicable to the anti-vibrio anguillarum disease vaccine of Cynoglossus semilaevis in the prior art.Therefore, develop Cynoglossus semilaevis vibrio anguillarum vaccine, for the generation of effective control Cynoglossus semilaevis Vibrio anguillarum disease and popularly have an important application prospect.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and a kind of preparation method that improves Cynoglossus semilaevis immunity, reduces the Cynoglossus semilaevis vibrio anguillarum vaccine of the harm that the Vibrio anguillarum disease causes in the cynoglossus semilaevis cultivation process is provided.
Another technical problem to be solved by this invention provides the using method of several above-described Cynoglossus semilaevis vibrio anguillarum vaccines.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine, is characterized in: it take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarum BH1) as immunogen, take Radix Astragali leachate as adjuvant; Its preparation methods steps is as follows:
(1) with Listonella anguillarum BH1 inoculation on common nutrient broth medium, cultivate 24h for 28-30 ℃, add the volume final concentration and be 0.3% formaldehyde 28-30 ℃ of deactivation 48h, get the deactivation thalline;
(2) get the Radix Astragali and decoct after with distilled water immersion 30-60min, filter, being concentrated into final concentration is former medicine 1g/mL, gets Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make Cynoglossus semilaevis vibrio anguillarum vaccine; The mixed proportion of the two is: the deactivation thalline that adds in every milliliter of Radix Astragali leachate is 8 * 10
10Cells.
The using method of the Cynoglossus semilaevis vibrio anguillarum vaccine that above-described preparation method makes can adopt the method for immersion immunity, to be exempted from first Cynoglossus semilaevis and with mass concentration be 8% NaCl solution soaking 3-7min under venting condition, then 1: 5 by volume dilute with water Cynoglossus semilaevis vibrio anguillarum vaccine gets Cynoglossus semilaevis vibrio anguillarum vaccine solution, to be exempted from again Cynoglossus semilaevis and be put in inflation immersion 1h in the Cynoglossus semilaevis vibrio anguillarum vaccine solution, be pressed afterwards the preceding method booster immunization 1 time in 5-10 days.
The using method of the Cynoglossus semilaevis vibrio anguillarum vaccine that above-described preparation method makes can adopt the method for injecting immune, get body length and carry out the initial injection immunity for the Cynoglossus semilaevis of 15-20cm by the immunizing dose of 0.1mL/ tail, after week about by aforementioned immunizing dose booster immunization 1 time, immunity is 4 times altogether.
The using method of the Cynoglossus semilaevis vibrio anguillarum vaccine that above-described preparation method makes can adopt the oral immunity method, throws something and feeds after hard pellet diet is soaked with Cynoglossus semilaevis vibrio anguillarum vaccine, and the fish body absorption every day inactivated bacteria scale of construction is 8 * 10
6The cells/g body weight is thrown something and fed a week continuously, carries out one week of booster immunization by preceding method again after every other week.
Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 of the present invention (Listonella anguillarum BH1) (also being Vibrio anguillarum bacterial strain BH1) is in open source literature " Cynoglossus semilaevis cause of disease Listonella anguillarum phenotype and characterization of molecules research " disclosed (bacterial strain BH1), above-mentioned open source literature is published in " ocean journal ", 2009,31 (5): 1-11.DNA sequence length and the Genebank accession number of bacterial strain BH1 see the following form:
This bacterial strain BH1 is public material, now is kept at Huaihai Institute of Technology oceanography institute laboratory, rising present patent application day in 20 years, the public if necessary, Huaihai Institute of Technology oceanography institute laboratory can externally be provided.
Preparation method of the present invention take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarumBH1) as immunogen, take Radix Astragali leachate as adjuvant.Use the vaccine of the inventive method preparation and carry out the Cynoglossus semilaevis immunity, can significantly improve the ability that cynoglossus semilaevis anti-vibrio anguillarum infects, can effectively prevent the Vibrio anguillarum in the cynoglossus semilaevis cultivation production to infect, reduce the Cynoglossus semilaevis mortality rate, increase the output of cynoglossus semilaevis cultivation, guarantee health and sustainable development that cynoglossus semilaevis cultivation is produced, improve the economic benefit that cynoglossus semilaevis cultivation is produced.
The specific embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not consist of its Copyright law.
Embodiment 1.A kind of preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine, it take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarum BH1) as immunogen, take Radix Astragali leachate as adjuvant; Its preparation methods steps is as follows:
(1) with Listonella anguillarum BH1 inoculation on common nutrient broth medium, cultivate 24h for 30 ℃, add the volume final concentration and be 30 ℃ of deactivation 48h of formaldehyde of 0.3%, get the deactivation thalline;
(2) get the Radix Astragali and decoct after with distilled water immersion 60min, filter, being concentrated into final concentration is former medicine 1g/mL, gets Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make Cynoglossus semilaevis vibrio anguillarum vaccine; The mixed proportion of the two is: the deactivation thalline that adds in every milliliter of Radix Astragali leachate is 8 * 10
10Cells.
Adopt the method for immersion immunity during use, to be exempted from first Cynoglossus semilaevis and with mass concentration be 8% NaCl solution soaking 3-7min under venting condition, then 1: 5 by volume dilute with water Cynoglossus semilaevis vibrio anguillarum vaccine gets Cynoglossus semilaevis vibrio anguillarum vaccine solution, to be exempted from again Cynoglossus semilaevis and be put in inflation immersion 1h in the Cynoglossus semilaevis vibrio anguillarum vaccine solution, be pressed afterwards the preceding method booster immunization 1 time in 5-10 days.
Embodiment 2.A kind of preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine, it take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarum BH1) as immunogen, take Radix Astragali leachate as adjuvant; Its preparation methods steps is as follows:
(1) with Listonella anguillarum BH1 inoculation on common nutrient broth medium, cultivate 24h for 28 ℃, add the volume final concentration and be 30 ℃ of deactivation 48h of formaldehyde of 0.3%, get the deactivation thalline;
(2) get the Radix Astragali and decoct after with distilled water immersion 30min, filter, being concentrated into final concentration is former medicine 1g/mL, gets Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make Cynoglossus semilaevis vibrio anguillarum vaccine; The mixed proportion of the two is: the deactivation thalline that adds in every milliliter of Radix Astragali leachate is 8 * 10
10Cells.
Adopt the method for injecting immune during use, get body long for the Cynoglossus semilaevis of 15-20cm carries out the initial injection immunity by the immunizing dose of 0.1mL/ tail, after week about by aforementioned immunizing dose booster immunization 1 time, immune 4 times altogether.
Embodiment 3.A kind of preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine, it take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarum BH1) as immunogen, take Radix Astragali leachate as adjuvant; Its preparation methods steps is as follows:
(1) with Listonella anguillarum BH1 inoculation on common nutrient broth medium, cultivate 24h for 29 ℃, add the volume final concentration and be 29 ℃ of deactivation 48h of formaldehyde of 0.3%, get the deactivation thalline;
(2) get the Radix Astragali and decoct after with distilled water immersion 45min, filter, being concentrated into final concentration is former medicine 1g/mL, gets Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make Cynoglossus semilaevis vibrio anguillarum vaccine; The mixed proportion of the two is: the deactivation thalline that adds in every milliliter of Radix Astragali leachate is 8 * 10
10Cells.
Adopt the oral immunity method during use, throw something and feed after hard pellet diet is soaked with Cynoglossus semilaevis vibrio anguillarum vaccine, the fish body absorption every day inactivated bacteria scale of construction is 8 * 10
6The cells/g body weight is thrown something and fed a week continuously, carries out one week of booster immunization by preceding method again after every other week.
Embodiment 4.Immunization experiment and result thereof that the Cynoglossus semilaevis vibrio anguillarum vaccine that makes with embodiment 1,2 and 3 described methods carries out.
Experimental period: 2010 February～Mays in year.
Experiment place: Jiangsu Province's Huaihai Institute of Technology aquatic animal disease laboratory.
Laboratory animal: Cynoglossus semilaevis.
Experimental vaccine and method: Cynoglossus semilaevis vibrio anguillarum vaccine and the using method of using respectively embodiment 1,2 and 3 described methods to make.
After duration of immunity finishes, getting the Cynoglossus semilaevis that carries out immunity by embodiment 1,2 and 3 described vaccines and using method, is 1.44 * 10 with concentration respectively
8The Listonella anguillarum BH1 of CFU/mL (Listonella anguillarum BH1) bacteria suspension infectable infection is exempted from Cynoglossus semilaevis, and infective dose is 0.3mL.
Matched group is for using concentration 1.44 * 10
8The Listonella anguillarum BH1 of CFU/mL (Listonella anguillarum BH1) bacteria suspension infectable infection is without the Cynoglossus semilaevis of immunity, and infective dose is 0.3mL.
Experimental result: Cynoglossus semilaevis is after experimental example 1,2,3 described vaccines and using method are carried out immunity; can both produce certain immune protective efficiency; wherein best with the injection system immune effect; soak, immune protective rate oral and that inject three kinds of modes is respectively 45%, 40%, 90%, see the following form:
| Group | Test angle number | Survival angle number | Survival rate/% | Immune protective efficiency/% |
| 1 group of embodiment | 20 | 9 | 45％ | 45％ |
| 2 groups of embodiment | 20 | 18 | 90％ | 90％ |
| 3 groups of embodiment | 20 | 8 | 40％ | 40％ |
| Matched group | 10 | 0 | 0 |
Claims (1)
1. the preparation method of a Cynoglossus semilaevis vibrio anguillarum vaccine is characterized in that: it is take Cynoglossus semilaevis cause of disease Listonella anguillarum BH1 (Listonella anguillarum BH1) as immunogen, with
Radix Astragali leachate is adjuvant; Its preparation methods steps is as follows:
(1) with Listonella anguillarum (claiming again Vibrio anguillarum) BH1 inoculation on common nutrient broth medium, cultivate 24h for 28-30 ℃, add the volume final concentration and be 0.3% formaldehyde 28-30 ℃ of deactivation 48h, get the deactivation thalline;
(2) get the Radix Astragali and decoct after with distilled water immersion 30-60min, filter, being concentrated into final concentration is former medicine 1g/mL, gets Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make Cynoglossus semilaevis vibrio anguillarum vaccine; The mixed proportion of the two is: the deactivation thalline that adds in every milliliter of Radix Astragali leachate is 8 * 10
10Cells.
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| CN102836428B (en) * | 2012-09-12 | 2014-01-08 | 淮海工学院 | Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease |
| CN103275890A (en) * | 2013-05-03 | 2013-09-04 | 中国水产科学研究院黄海水产研究所 | Vibrio anguillarum (listonella anguillarum) virulent strain and application thereof |
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