CN102836431B - 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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- CN102836431B CN102836431B CN201210335928.6A CN201210335928A CN102836431B CN 102836431 B CN102836431 B CN 102836431B CN 201210335928 A CN201210335928 A CN 201210335928A CN 102836431 B CN102836431 B CN 102836431B
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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 preparation method of inactivated vaccine, particularly relate to a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum.
Background technology
Listonella anguillarum is identified already as the pathogen of aquatic animal, and this bacterium is separated in 1893 first by gondola Canestrini the earliest from the anguilla japonica of generation " red epidemic disease ".Existing known its can cause the vibriosis of the multiple aquatic animal generation lethal such as rainbow trout (Salmo gairdneri), Oncorhynchi, turbot, fragrant fish, Anguillar japonica, Pacific Ocean salmon, Yellowtail fish, jack mackerel, snapper, Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis), sole, morrhua, mackerel, various tropical fresh water fish and shrimp, Eriocheir sinensis, Concha Ostreae, Conch Meretricis seu Cyclinae, Bao.The disease being caused by Listonella anguillarum is produced and has been caused very large loss to aquaculture of aquatic animal, therefore, effective prevention of the disease that Listonella anguillarum is caused, to reduce to greatest extent its extent of injury, can improve the economic benefit that aquaculture of aquatic animal is produced significantly.
At present raiser uses the generation of chemical synthetic drug or antibiotics control aquatic animal vibriosis and popular mostly, lacks a kind of immune formulation of the anti-Listonella anguillarum easy to use that is applicable to aquatic animal germling in seedling stage on market.Therefore, develop the vaccine of anti-Listonella anguillarum, for the generation of effective control aquatic animal Listonella anguillarum disease and popularly there is important application prospect.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provides a kind of and improve aquatic animal immunity, reduces harm that marine fish culture causes by cause of disease Listonella anguillarum in producing, to meet the inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum of the disease-resistant demand that aquaculture of aquatic animal produces.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum, is characterized in: the method is with cause of disease Listonella anguillarum BH1(
listonella anguillarumbH1) after formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, using Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are as follows:
(1) by cause of disease Listonella anguillarum BH1 inoculation on ordinary nutrient agar broth bouillon, 28-30 DEG C of shaking table cultivated 24h, adds 28 DEG C of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal deactivation thalline;
(2) decoct after getting Radix Astragali distilled water immersion 30-60min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 50-55 DEG C, add aluminium stearate, the w/v of aluminium stearate and white oil is 1-3:100 g/ml, continue to be heated to 65-70 DEG C, add class of department 80, class 80 of department is 5-7:100 with the volume ratio of white oil, until finishing temperature 115-120 DEG C, maintain 15-20 min, make oil phase, cooling for subsequent use;
(4) deactivation thalline is mixed with Radix Astragali leachate, make vaccine water; In water, cause of disease Listonella anguillarum BH1 immunogen is 10
11cells/mL, Radix Astragali leachate is 1g/mL, has also added the Tween 80 that final volume concentration is 4% in water;
(5) by oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, making the Radix Astragali and white oil is the Listonella anguillarum oil emulsion inactivated vaccine of 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 now kept at Huaihai Institute of Technology oceanography institute laboratory, rises in 20 years in present patent application day, if the public needs, Huaihai Institute of Technology oceanography institute laboratory can externally be provided.
The using method of the Listonella anguillarum oil emulsion inactivated vaccine that the inventive method makes is: the adult laying hen of method immunity that adopts the immunity of cervical region subcutaneous injection, the 1st time immunizing dose is 0.5 mL, after one week, press preceding method booster immunization, immunizing dose is 0.5 mL, after one week, press preceding method and continue booster immunization, immunizing dose is 1 mL, after one week, press preceding method booster immunization again, immunizing dose is 0.5 mL, immunity 4 times altogether, collect by egg that immune chicken produces, isolate yolk, and measure its antibody titer.After immunity finishes, the 1st thoughtful the 2nd week yolk antibody tired and maintained 2
13left and right, it is 2 that the 3rd week later yolk antibody tired
14left and right.Then obtained height is exempted to egg as raw material, be prepared into the yolk powder of the high immunity yolk antibody that contains anti-Listonella anguillarum through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h), be added in 5% left and right ratio the use of throwing something and feeding in mixed feed.
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 the seawater fishs such as Cynoglossus semilaevis to cause of disease Listonella anguillarum, improve the survival rate of the marine fish cultures such as Cynoglossus semilaevis, increase the output of the marine fish cultures such as Cynoglossus semilaevis, improve the economic benefit that the marine fish cultures such as Cynoglossus semilaevis are produced.Fundamentally solve the problem that Listonella anguillarum associated diseases easily occurs in the marine fish culture production processes such as current Cynoglossus semilaevis.
Detailed description of the invention
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 form the restriction to its right.
Embodiment 1, a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum, the method is with cause of disease Listonella anguillarum BH1(
listonella anguillarumbH1) after formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, using Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are as follows:
(1) by cause of disease Listonella anguillarum BH1 inoculation on ordinary nutrient agar broth bouillon, 30 DEG C of shaking tables are cultivated 24h, add 28 DEG C of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal deactivation thalline;
(2) decoct after getting Radix Astragali distilled water immersion 60min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 50 DEG C, adds aluminium stearate, the w/v of aluminium stearate and white oil is 1:100 g/ml, continues to be heated to 65 DEG C, add department class 80, class 80 of department is 5:100 with the volume ratio of white oil, until 115 DEG C of finishing temperatures, maintain 15min, make oil phase, cooling for subsequent use;
(4) deactivation thalline is mixed with Radix Astragali leachate, make vaccine water; In water, cause of disease Listonella anguillarum BH1 immunogen is 10
11cells/mL, Radix Astragali leachate is 1g/mL, has also added the Tween 80 that final volume concentration is 4% in water;
(5) by oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, making the Radix Astragali and white oil is the Listonella anguillarum oil emulsion inactivated vaccine of associating adjuvant.
Embodiment 2, a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum, the method is with cause of disease Listonella anguillarum BH1(
listonella anguillarumbH1) after formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, using Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are as follows:
(1) by cause of disease Listonella anguillarum BH1 inoculation on ordinary nutrient agar broth bouillon, 29 DEG C of shaking tables are cultivated 24h, add 28 DEG C of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal deactivation thalline;
(2) decoct after getting Radix Astragali distilled water immersion 60min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 52 DEG C, adds aluminium stearate, the w/v of aluminium stearate and white oil is 3:100 g/ml, continues to be heated to 68 DEG C, add department class 80, class 80 of department is 7:100 with the volume ratio of white oil, until 118 DEG C of finishing temperatures, maintain 18 min, make oil phase, cooling for subsequent use;
(4) deactivation thalline is mixed with Radix Astragali leachate, make vaccine water; In water, cause of disease Listonella anguillarum BH1 immunogen is 10
11cells/mL, Radix Astragali leachate is 1g/mL, has also added the Tween 80 that final volume concentration is 4% in water;
(5) by oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, making the Radix Astragali and white oil is the Listonella anguillarum oil emulsion inactivated vaccine of associating adjuvant.
Embodiment 3, a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Listonella anguillarum, the method is with cause of disease Listonella anguillarum BH1(
listonella anguillarumbH1) after formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, using Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are as follows:
(1) by cause of disease Listonella anguillarum BH1 inoculation on ordinary nutrient agar broth bouillon, 28 DEG C of shaking tables are cultivated 24h, add 28 DEG C of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal deactivation thalline;
(2) decoct after getting Radix Astragali distilled water immersion 45min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 55 DEG C, adds aluminium stearate, the w/v of aluminium stearate and white oil is 2:100 g/ml, continues to be heated to 70 DEG C, add department class 80, class 80 of department is 6:100 with the volume ratio of white oil, until 120 DEG C of finishing temperatures, maintain 20 min, make oil phase, cooling for subsequent use;
(4) deactivation thalline is mixed with Radix Astragali leachate, make vaccine water; In water, cause of disease Listonella anguillarum BH1 immunogen is 10
11cells/mL, Radix Astragali leachate is 1g/mL, has also added the Tween 80 that final volume concentration is 4% in water;
(5) by oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, making the Radix Astragali and white oil is the Listonella anguillarum oil emulsion inactivated vaccine of associating adjuvant.
Embodiment 4, gets 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.
Listonella anguillarum oil emulsion inactivated vaccine adopts the adult laying hen of method immunity of cervical region subcutaneous injection immunity, the 1st time immunizing dose is 0.5 mL, after one week, presses preceding method booster immunization, and immunizing dose is 0.5 mL, after one week, press preceding method and continue booster immunization, immunizing dose is 1 mL, after one week, presses preceding method booster immunization again, and immunizing dose is 0.5 mL, immunity 4 times altogether, collect by egg that immune chicken produces, isolate yolk and make high immunity yolk antibody, and measure its antibody titer.After immunity finishes, the 1st thoughtful the 2nd week yolk antibody tired and maintained 2
13, it is 2 that the 3rd week later yolk antibody tired
14.
2. immunization experiment and the result thereof of carrying out with the high immunity yolk antibody of anti-Listonella anguillarum.
Experimental period: 2011 April ~ Mays in year.
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 of the anti-Listonella anguillarum that the method described in using makes.When use, exempt from egg as raw material using the height being obtained, be prepared into the yolk powder of the high immunity yolk antibody that contains anti-Listonella anguillarum through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h), be added in 5% ratio the use of throwing something and feeding in mixed feed.
Test is thrown something and fed after the high immunity yolk antibody feedstuff 2d of anti-Listonella anguillarum by Cynoglossus semilaevis and turbot, is 1.56 × 10 respectively by concentration
8the Listonella anguillarum BH1(of CFU/mL
listonella anguillarumbH1) 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 for using concentration 1.56 × 10
8the Listonella anguillarum BH1(of CFU/mL
listonella anguillarumbH1) 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 carried out after immunity through high immunity yolk antibody and the using method of the anti-Listonella anguillarum described in experimental example 1, and two kinds of marine fish can 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/% |
| Cynoglossus semilaevis experimental group | 20 | 17 | 85% | 85% |
| Cynoglossus semilaevis matched group | 20 | 4 | 20% | 20% |
| Turbot experimental group | 20 | 15 | 75% | 75% |
| Turbot matched group | 20 | 3 | 15% | 15% |
Claims (1)
1. for the preparation of an inactivated vaccine method for making for the yolk antibody of anti-Listonella anguillarum, it is characterized in that: the method is with cause of disease Listonella anguillarum BH1(
listonella anguillarumbH1) after formalin deactivation as immunogen, make oil emulsion with white oil, department class 80 and aluminium stearate, using Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are as follows:
(1) by cause of disease Listonella anguillarum BH1 inoculation on ordinary nutrient agar broth bouillon, 28-30 DEG C of shaking table cultivated 24h, adds 28 DEG C of deactivation 48h of formaldehyde of volume final concentration 0.3%, centrifugal deactivation thalline;
(2) decoct after getting Radix Astragali distilled water immersion 30-60min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 50-55 DEG C, add aluminium stearate, the w/v of aluminium stearate and white oil is 1-3:100 g/ml, continue to be heated to 65-70 DEG C, add class of department 80, class 80 of department is 5-7:100 with the volume ratio of white oil, until finishing temperature 115-120 DEG C, maintain 15-20 min, make oil phase, cooling for subsequent use;
(4) deactivation thalline is mixed with Radix Astragali leachate, make vaccine water; In water, cause of disease Listonella anguillarum BH1 immunogen is 10
11cells/mL, Radix Astragali leachate is 1g/mL, has also added the Tween 80 that final volume concentration is 4% in water;
(5) by oil phase and water 2:1 mixing and emulsifying grinding by volume 5min, making the Radix Astragali and white oil is the Listonella anguillarum oil emulsion inactivated vaccine of associating adjuvant.
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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 |
| 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 |
| 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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| 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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| 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 |
| CN102100911A (en) * | 2010-09-21 | 2011-06-22 | 淮海工学院 | Preparation method of Cynoglossus semilaevis vibrio anguillarum vaccine |
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