CN102836431A - Method for preparing inactivated vaccine of anti-Listonella-anguillarum egg yolk antibody - Google Patents
Method for preparing inactivated vaccine of anti-Listonella-anguillarum egg yolk antibody Download PDFInfo
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- CN102836431A CN102836431A CN2012103359286A CN201210335928A CN102836431A CN 102836431 A CN102836431 A CN 102836431A CN 2012103359286 A CN2012103359286 A CN 2012103359286A CN 201210335928 A CN201210335928 A CN 201210335928A CN 102836431 A CN102836431 A CN 102836431A
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Abstract
The invention relates to a method for preparing inactivated vaccine of anti-Listonella-anguillarum egg yolk antibody. The method comprises the following steps: inactivating and centrifuging etiology Listonella-anguillarum BH1 so as to obtain inactivated thallus; preparing Astragalus mongholicus leachate; preparing oil phase by white oil, aluminium stearate and span-80; preparing vaccine water phase by the inactivated thallus and the Astragalus mongholicus leachat; finally, mixing the oil phase with the water phase according to the volume ratio of 2:1, emulsifying and milling for 5 minutes so as to obtain the Vibrio parahaemolyticus oil emulsion inactivated vaccine in which Astragalus mongholicus and oil white are used as united adjuvant. By adoption of the vaccine, the anti-Listonella-anguillarum egg yolk antibody can be obtained; by adoption of the anti-Listonella-anguillarum egg yolk antibody, the immunity of marine fishes such as cynoglossus semilaevis to etiology Listonella-anguillarum is improved, so that the survival rate is improved, the yield is improved, and the economic benefit of culture production of marine fishes such as cynoglossus semilaevis is improved. The problem that the Listonella-anguillarum caused diseases frequently occur in the culture production of marine fishes such as cynoglossus semilaevis, is solved.
Description
Technical field
The present invention relates to a kind of method for preparing of inactivated vaccine, particularly relate to a kind of inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum.
Background technology
Listonella anguillarum is identified as the pathogen of aquatic animal already, and this bacterium is separated to from the anguilla japonica that " red epidemic disease " takes place in 1893 by gondola Canestrini the earliest first.Now know can cause steelhead, salmon, turbot, smelt, eel, Pacific salmon, yellowtail fish, horse mackerel, sea bream, sea bass, flounder, cod, mackerel, a variety of tropical fish as well as shrimp, crab, oysters, clams, abalone and other aquaculture animals developed fatal vibriosis.Disease by Listonella anguillarum causes has caused very big loss for the production of Aquatic product animal cultivation; Therefore; Effective prevention of the disease that Listonella anguillarum is caused to reduce its extent of injury to greatest extent, can improve the economic benefit that aquaculture of aquatic animal is produced significantly.
At present the raiser use chemical synthetic drug or antibiotics control aquatic animal vibriosis mostly generation with popular, a kind of immune formulation that is applicable to the anti-Listonella anguillarum easy to use of aquatic animal germling in seedling stage of shortage on the market.Therefore, develop the vaccine of anti-Listonella anguillarum, have important application prospects for the sick generation of effective control aquatic animal Listonella anguillarum and popular.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, provides a kind of and improve aquatic animal immunity, reduces the harm that caused by the cause of disease Listonella anguillarum in the marine fish culture production, to satisfy the inactivated vaccine method for making of yolk antibody that is used to prepare anti-Listonella anguillarum of disease-resistant demand in the aquaculture of aquatic animal production.
Technical problem to be solved by this invention is to realize through following technical scheme.The present invention is a kind of inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum, is characterized in: this method with cause of disease Listonella anguillarum BH1 (
Listonella anguillarumBH1) after the formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine with Radix Astragali leachate; Its concrete steps are following:
(1) with cause of disease Listonella anguillarum BH1 inoculation on the ordinary nutrient agar broth bouillon, 28-30 ℃ of shaking table cultivated 24h, adds 28 ℃ of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal 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) white oil is heated to 50-55 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 1-3:100 g/ml; Continue to be heated to 65-70 ℃, class of adding department 80, class 80 of department is 5-7:100 with the volume ratio of white oil; Until finishing temperature 115-120 ℃; Keep 15-20 min, make oil phase, cool off subsequent use;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; Aqueous phase cause of disease Listonella anguillarum BH1 immunogen is 10
11Cells/mL, Radix Astragali leachate be 1g/mL, and it is 4% Tween 80 that aqueous phase has also added final volume concentration;
(5) with oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, make the Radix Astragali and white oil Listonella anguillarum oil emulsion inactivated vaccine for the associating adjuvant.
Cause of disease Listonella anguillarum of the present invention (
Listonella anguillarumBH1) 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): 112-121.DNA sequence length and the Genebank accession number of bacterial strain BH1 see the following form:
This bacterial strain BH1 is public material, is kept at Huaihai Institute of Technology oceanography institute laboratory at present, rising present patent application day in 20 years, the public if desired, Huaihai Institute of Technology oceanography institute laboratory can externally be provided.
The method for using of the Listonella anguillarum oil emulsion inactivated vaccine that the inventive method makes is: adopt the adult laying hen of method immunity of cervical region subcutaneous injection immunity, the 1st time immunizing dose is 0.5 mL, and the preceding method booster immunization is pressed in all backs; Immunizing dose is 0.5 mL, presses preceding method after the week and continues booster immunization, and immunizing dose is 1 mL; Press preceding method booster immunization again after one week; Immunizing dose is 0.5 mL, and immunity is 4 times altogether, collects by egg that immune chicken produces; Isolate yolk, and measure its antibody titer.The the 1st the thoughtful the 2nd all yolk antibodies were tired and are maintained 2 after immunity finished
13About, it is 2 that the 3rd all yolk antibodies are later on tired
14About.Then the height that is obtained is exempted from egg as raw material, be prepared into the yolk powder of the high immunity yolk antibody that contains anti-Listonella anguillarum, be added on the use of throwing something and feeding in the mixed feed in 5% left and right sides ratio through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h).
The Listonella anguillarum oil emulsion inactivated vaccine that the inventive method makes can obtain the high immunity yolk antibody of anti-Listonella anguillarum; The use of high immunity yolk antibody can strengthen the immunity of seawater fishs such as Cynoglossus semilaevis to the cause of disease Listonella anguillarum; Improve the survival rate of marine fish cultures such as Cynoglossus semilaevis; Increase the output of marine fish cultures such as Cynoglossus semilaevis, improve the economic benefit of marine fish culture productions such as Cynoglossus semilaevis.Fundamentally solved the problem that is prone to take place the Listonella anguillarum associated diseases in the marine fish culture production processes such as present Cynoglossus semilaevis.
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 inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum, this method with cause of disease Listonella anguillarum BH1 (
Listonella anguillarumBH1) after the formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine with Radix Astragali leachate; Its concrete steps are following:
(1) with cause of disease Listonella anguillarum BH1 inoculation on the ordinary nutrient agar broth bouillon, 30 ℃ of shaking tables are cultivated 24h, add 28 ℃ of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal 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, Radix Astragali leachate;
(3) white oil is heated to 50 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 1:100 g/ml, continues to be heated to 65 ℃; Class of adding department 80, class 80 of department is 5:100 with the volume ratio of white oil, until 115 ℃ of finishing temperatures; Keep 15min, make oil phase, cool off subsequent use;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; Aqueous phase cause of disease Listonella anguillarum BH1 immunogen is 10
11Cells/mL, Radix Astragali leachate be 1g/mL, and it is 4% Tween 80 that aqueous phase has also added final volume concentration;
(5) with oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, make the Radix Astragali and white oil Listonella anguillarum oil emulsion inactivated vaccine for the associating adjuvant.
Embodiment 2, a kind of inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum, this method with cause of disease Listonella anguillarum BH1 (
Listonella anguillarumBH1) after the formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine with Radix Astragali leachate; Its concrete steps are following:
(1) with cause of disease Listonella anguillarum BH1 inoculation on the ordinary nutrient agar broth bouillon, 29 ℃ of shaking tables are cultivated 24h, add 28 ℃ of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal 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, Radix Astragali leachate;
(3) white oil is heated to 52 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 3:100 g/ml, continues to be heated to 68 ℃; Class of adding department 80, class 80 of department is 7:100 with the volume ratio of white oil, until 118 ℃ of finishing temperatures; Keep 18 min, make oil phase, cool off subsequent use;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; Aqueous phase cause of disease Listonella anguillarum BH1 immunogen is 10
11Cells/mL, Radix Astragali leachate be 1g/mL, and it is 4% Tween 80 that aqueous phase has also added final volume concentration;
(5) with oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, make the Radix Astragali and white oil Listonella anguillarum oil emulsion inactivated vaccine for the associating adjuvant.
Embodiment 3, a kind of inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum, this method with cause of disease Listonella anguillarum BH1 (
Listonella anguillarumBH1) after the formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine with Radix Astragali leachate; Its concrete steps are following:
(1) with cause of disease Listonella anguillarum BH1 inoculation on the ordinary nutrient agar broth bouillon, 28 ℃ of shaking tables are cultivated 24h, add 28 ℃ of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal 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, Radix Astragali leachate;
(3) white oil is heated to 55 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 2:100 g/ml, continues to be heated to 70 ℃; Class of adding department 80, class 80 of department is 6:100 with the volume ratio of white oil, until 120 ℃ of finishing temperatures; Keep 20 min, make oil phase, cool off subsequent use;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; Aqueous phase cause of disease Listonella anguillarum BH1 immunogen is 10
11Cells/mL, Radix Astragali leachate be 1g/mL, and it is 4% Tween 80 that aqueous phase has also added final volume concentration;
(5) with oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, make the Radix Astragali and white oil Listonella anguillarum oil emulsion inactivated vaccine for the associating adjuvant.
Embodiment 4, get the application experiment that Listonella anguillarum oil emulsion inactivated vaccine that embodiment 3 makes carries out.
1. Listonella anguillarum oil emulsion inactivated vaccine immunization laying hen.
The Listonella anguillarum oil emulsion inactivated vaccine adopts the adult laying hen of method immunity of cervical region subcutaneous injection immunity, and the 1st time immunizing dose is 0.5 mL, and the preceding method booster immunization is pressed in all backs; Immunizing dose is 0.5 mL, presses preceding method after the week and continues booster immunization, and immunizing dose is 1 mL; Press preceding method booster immunization again after one week, immunizing dose is 0.5 mL, and immunity is 4 times altogether; Collection is isolated yolk and is made high immunity yolk antibody by egg that immune chicken produces, and measures its antibody titer.The the 1st the thoughtful the 2nd all yolk antibodies were tired and are maintained 2 after immunity finished
13, it is 2 that the 3rd all yolk antibodies are later on tired
14
2. the immunization experiment and the result thereof that carry out with the high immunity yolk antibody of anti-Listonella anguillarum.
Experimental period: 2011 April in year ~ Mays.
Experiment place: Jiangsu Province's Huaihai Institute of Technology aquatic animal disease laboratory.
Laboratory animal: Cynoglossus semilaevis and turbot.
High immunity yolk antibody and method: the high immunity yolk antibody that uses the anti-Listonella anguillarum that described method makes.Exempt from egg as raw material with the height that is obtained during use, be prepared into the yolk powder of the high immunity yolk antibody that contains anti-Listonella anguillarum, be added on the use of throwing something and feeding in the mixed feed in 5% ratio through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h).
Test is thrown something and fed with Cynoglossus semilaevis and turbot behind the high immunity yolk antibody feedstuff 2d of anti-Listonella anguillarum, and using concentration respectively is 1.56 * 10
8The Listonella anguillarum BH1 of CFU/mL (
Listonella anguillarumBH1) the bacteria suspension infectable infection is exempted from Cynoglossus semilaevis and turbot, and infective dose is 0.3mL.Experimental period is 7d, and the high immunity yolk antibody feedstuff of the anti-Listonella anguillarum that continues to throw something and feed.
Matched group is with concentration 1.56 * 10
8The Listonella anguillarum BH1 of CFU/mL (
Listonella anguillarumBH1) the bacteria suspension infectable infection Cynoglossus semilaevis and the turbot of normal diet of throwing something and feeding, infective dose is 0.3mL.
Experimental result: Cynoglossus semilaevis and turbot are after the high immunity yolk antibody of experimental example 1 described anti-Listonella anguillarum and method for using are carried out immunity, and two kinds of marine fish can both produce certain immune protective efficiency, and its immune protective rate is respectively 85%, 75%.See the following form:
| Group | Test fish number | Survival fish number | Survival rate/% | Immune protective rate/% |
| The Cynoglossus semilaevis experimental group | 20 | 17 | 85% | 85% |
| The Cynoglossus semilaevis matched group | 20 | 4 | 20% | 20% |
| The turbot experimental group | 20 | 15 | 75% | 75% |
| The turbot matched group | 20 | 3 | 15% | 15% |
Claims (1)
1. inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Listonella anguillarum is characterized in that: this method with cause of disease Listonella anguillarum BH1 (
Listonella anguillarumBH1) after the formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine with Radix Astragali leachate; Its concrete steps are following:
(1) with cause of disease Listonella anguillarum BH1 inoculation on the ordinary nutrient agar broth bouillon, 28-30 ℃ of shaking table cultivated 24h, adds 28 ℃ of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal 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) white oil is heated to 50-55 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 1-3:100 g/ml; Continue to be heated to 65-70 ℃, class of adding department 80, class 80 of department is 5-7:100 with the volume ratio of white oil; Until finishing temperature 115-120 ℃; Keep 15-20 min, make oil phase, cool off subsequent use;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; Aqueous phase cause of disease Listonella anguillarum BH1 immunogen is 10
11Cells/mL, Radix Astragali leachate be 1g/mL, and it is 4% Tween 80 that aqueous phase has also added final volume concentration;
(5) with oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, make the Radix Astragali and white oil Listonella anguillarum oil emulsion inactivated vaccine for the associating adjuvant.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103301457A (en) * | 2013-07-03 | 2013-09-18 | 淮海工学院 | Preparation method for egg yolk antibody inactivated vaccine resisting loach pathogenic vibrio cholerae |
| CN104710530A (en) * | 2015-03-17 | 2015-06-17 | 浙江省海洋水产养殖研究所 | Preparation and application of anti-vibrio parahaemolyticus OMPK (outer membrane protein k) egg yolk antibody |
| CN105169391A (en) * | 2015-10-16 | 2015-12-23 | 扬州大学 | Egg yolk antibody microcapsule feed additive for preventing carassius auratus gibelio bacterial septicemia |
| CN110840841A (en) * | 2019-11-01 | 2020-02-28 | 中国水产科学研究院黄海水产研究所 | Turbot oil emulsion vaccine and application thereof |
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| CN101190332A (en) * | 2006-12-01 | 2008-06-04 | 中国科学院海洋研究所 | Vibrio anguilarum oil oral-emulsified bivalent vaccine and preparation thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103301457A (en) * | 2013-07-03 | 2013-09-18 | 淮海工学院 | Preparation method for egg yolk antibody inactivated vaccine resisting loach pathogenic vibrio cholerae |
| CN104710530A (en) * | 2015-03-17 | 2015-06-17 | 浙江省海洋水产养殖研究所 | Preparation and application of anti-vibrio parahaemolyticus OMPK (outer membrane protein k) egg yolk antibody |
| CN105169391A (en) * | 2015-10-16 | 2015-12-23 | 扬州大学 | Egg yolk antibody microcapsule feed additive for preventing carassius auratus gibelio bacterial septicemia |
| CN105169391B (en) * | 2015-10-16 | 2018-08-24 | 扬州大学 | The egg yolk antibody microcapsules feed addictive of anti-hybridized prussian carp bacterial septicemia |
| CN110840841A (en) * | 2019-11-01 | 2020-02-28 | 中国水产科学研究院黄海水产研究所 | Turbot oil emulsion vaccine and application thereof |
| CN110840841B (en) * | 2019-11-01 | 2021-07-02 | 中国水产科学研究院黄海水产研究所 | Turbot oil emulsion vaccine and application thereof |
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