CN102139103B - Preparation and application methods of photobacterium damsela vaccines of cynoglossus semilaevis - Google Patents
Preparation and application methods of photobacterium damsela vaccines of cynoglossus semilaevis Download PDFInfo
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- CN102139103B CN102139103B CN201110086569A CN201110086569A CN102139103B CN 102139103 B CN102139103 B CN 102139103B CN 201110086569 A CN201110086569 A CN 201110086569A CN 201110086569 A CN201110086569 A CN 201110086569A CN 102139103 B CN102139103 B CN 102139103B
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- cynoglossus semilaevis
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- photobacterium damsela
- luminous bacillus
- mermaid luminous
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Abstract
The invention provides a preparation method of photobacterium damsela vaccines of cynoglossus semilaevis, which is characterized by comprising the steps of: inoculating ST1 strains of photobacterium damsela on an ordinary nutrient agar bouillon culture medium, cultivating for 24h at the temperature of 28 DEG C, and adding formaldehyde with the final concentration of 0.3% into the culture medium to inactivate the photobacterium damsela for 48h at the temperature of 28 DEG C so as to obtain inactivated bacteria; soaking milkvetch roots with distilled water, and then decocting, filtering and concentrating to obtain milkvetch lixivium; and mixing the inactivated bacteria and the obtained milkvetch lixivium to prepare the photobacterium damsela vaccines of the cynoglossus semilaevis. In the invention, the vaccines have the advantages of enabling the cynoglossus semilaevis to produce good immune response, enhancing the immunity of the cynoglossus semilaevis to the pathogen (photobacterium damsela), and increasing the survival rate of cultivated cynoglossus semilaevis, the output of the cultivated cynoglossus semilaevis, and the economic benefit of the cultivation and production of the cynoglossus semilaevis. The problem that the cynoglossus semilaevis is easy to catch diseases caused by the photobacterium damsela during the cultivation and production process at present is fundamentally solved. The invention also discloses an application method of the photobacterium damsela vaccines of the cynoglossus semilaevis.
Description
Technical field
The present invention relates to a kind of method for preparing of animal disease resistant vaccine, particularly relate to a kind of method for preparing of Cynoglossus semilaevis Mermaid luminous bacillus vaccine.The invention still further relates to the method for using of this vaccine.
Background technology
Mermaid luminescent bacteria widely present in sea water and feed on marine animals, mainly infects cold-blooded animal, especially farmed fish, in addition to infection damselfish fishes, but also infect sharks, turbot, amberjack, Günther, etc., on aquaculture production of harm is very serious in recent years, along with Günther farming scale, the mermaid luminous bacteria cause diseases to Günther aquaculture production caused a great loss, therefore, mermaid luminous bacteria cause disease effective prevention, to minimize the extent of its damage, will be able to significantly improve aquaculture production C.semilaevi economic benefits.
Do not see at present research and the application that the disease-resistant vaccine of Cynoglossus semilaevis is arranged as yet, at present the raiser use chemical synthetic drug or antibiotics control disease mostly generation with popular, a kind of anti-Mermaid luminous bacillus vaccine that is applicable to Cynoglossus semilaevis of shortage on the market.Therefore, develop Cynoglossus semilaevis Mermaid luminous bacillus vaccine, the generation of the disease that causes for effective control Cynoglossus semilaevis Mermaid luminous bacillus and popular has important application prospects.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, provides a kind of and improve Cynoglossus semilaevis immunity, reduces the harm that caused by the cause of disease Mermaid luminous bacillus in the cynoglossus semilaevis cultivation production, with the method for preparing of the Cynoglossus semilaevis Mermaid luminous bacillus vaccine that satisfies the disease-resistant demand that cynoglossus semilaevis cultivation produces.
Another technology to be solved by this invention has provided the immune method for using of several kinds of aforesaid Cynoglossus semilaevis Mermaid luminous bacillus vaccines.
Technical problem to be solved by this invention is to realize through following technical scheme.The present invention is a kind of method for preparing of Cynoglossus semilaevis Mermaid luminous bacillus vaccine, is characterized in: it with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) being immunogen, is adjuvant with Radix Astragali leachate; Its concrete steps are following:
(1) with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) inoculation is cultivated 24h for 28 ℃ on the ordinary nutrient agar broth bouillon, adds 28 ℃ of deactivation 48h of formaldehyde of final concentration 0.3%, gets 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, Radix Astragali leachate;
(3) with the deactivation thalline with Radix Astragali leachate mix, make water aqua type Radix Astragali adjuvant Cynoglossus semilaevis Mermaid luminous bacillus vaccine; Cause of disease Mermaid luminous bacillus immunogen is 1.17 * 10 in the vaccine
11Cells/mL, Radix Astragali leachate are 1g/mL.
The Mermaid luminous bacillus ST1 that relates in the inventive method (
PhotobacteriumdamselaeST1) at " Cynoglossus semilaevis cause of disease Mermaid luminous bacillus phenotype and characterization of molecules " (Phenotypic and molecular characterization of Photobacterium damselae; A pathogen of the cultured tongue sole Cynoglossus semilaevis in China.) open in; The publication of delivering of this open source literature is: New Zealand's " ocean and fresh water research "; 2011; 45 (1): 1-13 (New Zealand Journal of Marine and Freshwater Research, 2011,45 (1): 1-13.).The isolate DNA sequence length of this bacterial strain and accession number such as following table:
Isolate DNA sequence length and accession number
The immune method for using of the prepared Cynoglossus semilaevis Mermaid luminous bacillus of above-described method for preparing vaccine is:
1, it can adopt the method for immersion immunity: earlier Cynoglossus semilaevis being used mass concentration under venting condition is 8% NaCl solution soaking 5min; 1:5 dilute with water Cynoglossus semilaevis Mermaid luminous bacillus vaccine gets Cynoglossus semilaevis Mermaid luminous bacillus vaccine solution by volume then; Again Cynoglossus semilaevis is put in inflation immersion 1h in the Cynoglossus semilaevis Mermaid luminous bacillus vaccine solution, presses the preceding method booster immunization 1 time after the week.
2, it can adopt the method for injecting immune: it is long for the Cynoglossus semilaevis of 15-20cm carries out the initial injection immunity by the immunizing dose of 0.2mL/ tail to get body, after week about by aforementioned immunizing dose booster immunization 1 time, immunity is 4 times altogether.
3, it can adopt the oral immunity method: throw something and feed after hard pellet diet is soaked with Cynoglossus semilaevis Mermaid luminous bacillus vaccine, the Cynoglossus semilaevis absorption every day inactivated bacteria scale of construction is 1.17 * 10
7The 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.
Huaihai Institute of Technology aquatic animal disease laboratory carries out the Cynoglossus semilaevis immunoprotection with the vaccine of preparation of the present invention and immunization method and tests the inventor in Jiangsu Province 2010 February ~ Mays in year.Duration of immunity is exempted from the fish tail vein and is got blood after finishing, and separating immune serum carries out agglutination reaction of serum with the full bacterium antigen of Mermaid luminous bacillus, measures the serum antibody agglutination titer.Experimental result be Mermaid luminous bacillus vaccine of the present invention through soak, oral and inject three kinds of modes immunity Cynoglossus semilaevis; Exempted from fish and all can be produced immunne response; Best with the injection system immune effect; Serum antibody titer is the highest, soaks, the fish serum antibody titer of being exempted from oral and that inject three kinds of modes immunity is respectively 1:16,1:128,1:8192.
Immunity fish serum antibody titer
| Group | Serum antibody titer |
| The immersion immunity group | 1:16 |
| The oral immunity group | 1:128 |
| The injecting immune group | 1:8192 |
Cynoglossus semilaevis Mermaid luminous bacillus vaccine of the present invention and immune method for using thereof; Can make Cynoglossus semilaevis produce good immunne response; Strengthen the immunity of Cynoglossus semilaevis to the cause of disease Mermaid luminous bacillus; Improve the survival rate of cynoglossus semilaevis cultivation, increase the output of cynoglossus semilaevis cultivation, improve the economic benefit that cynoglossus semilaevis cultivation is produced.Fundamentally solved the problem that is prone to take place the Mermaid luminous bacillus associated diseases in the present cynoglossus semilaevis cultivation production process.
The specific embodiment
Below further describe concrete technical scheme of the present invention,, and do not constitute restriction its right so that those skilled in the art understands the present invention further.
Embodiment 1.A kind of method for preparing of Cynoglossus semilaevis Mermaid luminous bacillus vaccine, it with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) being immunogen, is adjuvant with Radix Astragali leachate; Its concrete steps are following:
(1) with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) inoculation is cultivated 24h for 28 ℃ on the ordinary nutrient agar broth bouillon, adds 28 ℃ of deactivation 48h of formaldehyde of final concentration 0.3%, gets 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, Radix Astragali leachate;
(3) with the deactivation thalline with Radix Astragali leachate mix, make water aqua type Radix Astragali adjuvant Cynoglossus semilaevis Mermaid luminous bacillus vaccine; Cause of disease Mermaid luminous bacillus immunogen is 1.17 * 10 in the vaccine
11Cells/mL, Radix Astragali leachate are 1g/mL.
Embodiment 2.A kind of method for using like the prepared Cynoglossus semilaevis Mermaid luminous bacillus of embodiment 1 described method for preparing vaccine.It adopts the method for immersion immunity; Earlier Cynoglossus semilaevis being used mass concentration under venting condition is 8% NaCl solution soaking 5min; 1:5 dilute with water Cynoglossus semilaevis Mermaid luminous bacillus vaccine gets Cynoglossus semilaevis Mermaid luminous bacillus vaccine solution by volume then; Again Cynoglossus semilaevis is put in inflation immersion 1h in the Cynoglossus semilaevis Mermaid luminous bacillus vaccine solution, presses the preceding method booster immunization 1 time after the week.
Embodiment 3.A kind of method for using like the prepared Cynoglossus semilaevis Mermaid luminous bacillus of embodiment 1 described method for preparing vaccine.Its adopts the method for injecting immune, and it is long for the Cynoglossus semilaevis of 15-20cm carries out the initial injection immunity by the immunizing dose of 0.2mL/ tail to get body, after week about by aforementioned immunizing dose booster immunization 1 time, immune 4 times altogether.
Embodiment 4.A kind of method for using like the prepared Cynoglossus semilaevis Mermaid luminous bacillus of embodiment 1 described method for preparing vaccine.It adopts the oral immunity method, throws something and feeds after hard pellet diet is soaked with Cynoglossus semilaevis Mermaid luminous bacillus vaccine, and the Cynoglossus semilaevis absorption every day inactivated bacteria scale of construction is 1.17 * 10
7The 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.
Claims (1)
1. the method for preparing of a Cynoglossus semilaevis Mermaid luminous bacillus vaccine is characterized in that: it with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) being immunogen, is adjuvant with Radix Astragali leachate; Its concrete steps are following:
(1) with Mermaid luminous bacillus ST1 (
PhotobacteriumdamselaeST1) inoculation is cultivated 24h for 28 ℃ on the ordinary nutrient agar broth bouillon, adds 28 ℃ of deactivation 48h of formaldehyde of final concentration 0.3%, gets 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, Radix Astragali leachate;
(3) the deactivation thalline is mixed with Radix Astragali leachate, make water aqua type Radix Astragali adjuvant Cynoglossus semilaevis Mermaid luminous bacillus vaccine; Cause of disease Mermaid luminous bacillus immunogen is 1.17 * 10 in the vaccine
11Cells/mL, Radix Astragali leachate are 1g/mL.
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| CN201110086569A CN102139103B (en) | 2011-04-07 | 2011-04-07 | Preparation and application methods of photobacterium damsela vaccines of cynoglossus semilaevis |
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| CN102430120B (en) * | 2011-12-01 | 2014-08-20 | 中国水产科学研究院黄海水产研究所 | Adjuvant for enhancing fish vaccine immunization effect and application thereof |
| CN109865136A (en) * | 2019-03-18 | 2019-06-11 | 宁波大学 | A kind of silvery pomfret bivalent inactivated vaccine |
| CN109865135A (en) * | 2019-03-18 | 2019-06-11 | 宁波大学 | A kind of silvery pomfret Mermaid luminous bacillus and Vibrio splindidus combine inactivated vaccine |
| CN109880766B (en) * | 2019-03-18 | 2021-05-07 | 宁波大学 | Photobacterium pomfret mermaid strain and inactivated vaccine |
| CN111304131B (en) * | 2020-03-17 | 2021-07-16 | 中国水产科学研究院黄海水产研究所 | Pathogenic mermaid photobacterium mermaid subspecies strain and application thereof |
| CN111388660B (en) * | 2020-03-17 | 2021-05-25 | 中国水产科学研究院黄海水产研究所 | Mermaid photobacterium mermaid subspecies polyvalent inactivated vaccine and preparation method thereof |
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| AU2005203202C1 (en) * | 2004-07-30 | 2009-02-19 | Intervet International B.V. | Novel bacterium and vaccine |
| CN101461940B (en) * | 2009-01-09 | 2011-01-26 | 中国水产科学研究所南海水产研究所 | Photobacterium damsela vaccine as well as preparation method and use thereof |
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