CN102836428B - Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease - Google Patents
Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease Download PDFInfo
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Abstract
The invention provides an inactivated vaccine preparation method for an egg yolk antibody for resisting a litopenaeus vannamei red body disease. The inactivated vaccine preparation method comprises the steps of inactivating etiology vibrio parahaemolyticus JGB080708-1 bacterial strains and centrifuging to obtain inactivation thalli; preparing astragalus mongholicus lixiviums; preparing an oil phase by using white oil, aluminium stearate and span-80 and mixing the inactivation thalli and the astragalus mongholicus lixiviums to prepare a vaccine aqueous phase; and mixing the oil phase and the aqueous phase according to a volume ratio of 2:1 for emulsification and grinding for 5min to prepare vibrio parahaemolyticus oil emu inactivated vaccines with the astragalus mongholicus and the white oil serving as a unite adjuvant. The vibrio parahaemolyticus oil emulsion inactivated vaccines can be used for immunization of adult laying hens, the high egg yolk antibody for resisting the litopenaeus vannamei red body disease is further obtained, the immunity of litopenaeus vannamei etiology vibrio parahaemolyticus is strengthened, the rate of survival of litopenaeus vannamei culture is improved, the yield of the litopenaeus vannamei culture is increased, and economic benefits of litopenaeus vannamei culture and production are improved. A problem of incidental vibrio parahaemolyticus caused diseases in the litopenaeus vannamei culture and production process at present is fundamentally solved.
Description
Technical field
The present invention relates to a kind of preparation method of inactivated vaccine, particularly a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Red Body Disease In Litopenaeus Vannamei.
Background technology
Litopenaeus vannamei (
litopenaeus vannamei) claim again white shrimp, Penaeus vannamei etc., be tropical shrimp species, the advantages such as wide, the anti-dense stocking of suitable salt scope, growth are fast, high-output stress-resistance that it has, after China introduced in 1988, the existing coastal various places large-scale farming in the whole nation.Along with the expansion of Litopenaeus vannamei cultivation scale, the breeding ecological environment goes from bad to worse, and the disease occurrence frequency sharply rises, by the cause of disease vibrio parahaemolytious (
vibrio parahaemolyticus) eperythrozoonosis that causes all is widely current in each foster shrimp area, the whole nation, and propagates soon, can cause the sea-farming Litopenaeus vannamei crushing dead, become the most common in the Litopenaeus vannamei breeding process and be to cause one of disease that economic loss is the most serious.
At present Litopenaeus vannamei sea-farming is difficult to control the generation of the disease that vibrio parahaemolytious causes with popular in producing, the inactivated vaccine of a kind of anti-Red Body Disease In Litopenaeus Vannamei easy to use of shortage on market.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provide a kind of improve Litopenaeus vannamei for the immunity of vibrio parahaemolytious, reduce harm that the Litopenaeus vannamei sea-farming causes by the cause of disease vibrio parahaemolytious in producing, with the inactivated vaccine method for making of the yolk antibody for the preparation of anti-Red Body Disease In Litopenaeus Vannamei that meets the disease-resistant demand on the Litopenaeus vannamei breeding production.
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-Red Body Disease In Litopenaeus Vannamei, is characterized in: it is with the cause of disease vibrio parahaemolytious JGB080708-1(of the Red Body Disease In Litopenaeus Vannamei after the formalin deactivation
vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, class of department 80, aluminium stearate, and 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 vibrio parahaemolytious JGB080708-1 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 with decocting after 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 ℃, 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 ℃, 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 ℃, maintain 15-20 min, make oil phase, cooling standby;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; In water, cause of disease vibrio parahaemolytious JGB080708-1 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, make the vibrio parahaemolytious oil emulsion inactivated vaccine that the Radix Astragali and white oil are the associating adjuvant.
Cause of disease vibrio parahaemolytious JGB080708-1 of the present invention open source literature " Litopenaeus vannamei (
litopenaeus vannamei) the cause of disease vibrio parahaemolytious (
vibrio parahaemolyticus) phenotype and characterization of molecules " in disclosed (bacterial strain JGB080708-1), above-mentioned open source literature is published in " Oceanologia et Limnologia Sinica ", 2009,40(5): 654-661.DNA sequence length and the Genebank accession number of bacterial strain JGB080708-1 see the following form:
This bacterial strain JGB080708-1 is public material, now is kept at Huaihai Institute of Technology oceanography institute laboratory, is rising present patent application day in 20 years, and the public if necessary, can externally provide by Huaihai Institute of Technology oceanography institute laboratory.
The using method of the inactivated vaccine of the anti-Red Body Disease In Litopenaeus Vannamei that preparation method of the present invention makes is as follows: with the vibrio parahaemolytious oil emulsion inactivated vaccine made, adopt the adult laying hen of method immunity of cervical region subcutaneous injection immunity, the 1st time immunizing dose is 0.5 mL, press the preceding method booster immunization after one week, immunizing dose is 0.5 mL, press preceding method after one week and continue booster immunization, immunizing dose is 1 mL, press preceding method booster immunization again after one week, immunizing dose is 0.5 mL, immunity is 4 times altogether, collection is by egg that immune chicken produces, isolate yolk, make yolk antibody.Immunity finishes latter the 4th week, and yolk antibody is tired and can be reached 2
12.Then the height that said method was obtained of usining is exempted from egg as raw material, through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h) or spray drying be prepared into the yolk powder of the high immunity yolk antibody that contains anti-Red Body Disease In Litopenaeus Vannamei, can in the production of growing seedlings, directly throw something and feed, or be added in mixed feed in 5% ratio the use of throwing something and feeding.The high immunity yolk antibody of anti-Red Body Disease In Litopenaeus Vannamei, not only have good resistant effect, and easy to use, has again higher nutritive value simultaneously.
Vibrio parahaemolytious oil emulsion inactivated vaccine of the present invention, can be for the adult laying hen of immunity, and then obtain the high immunity yolk antibody of anti-Red Body Disease In Litopenaeus Vannamei, can strengthen the immunity of Litopenaeus vannamei to the cause of disease vibrio parahaemolytious, improve the survival rate of Litopenaeus vannamei cultivation, increase the output of Litopenaeus vannamei cultivation, improve the economic benefit of Litopenaeus vannamei breeding production.Fundamentally solved the problem that the vibrio parahaemolytious associated diseases easily occurs in current Litopenaeus vannamei breeding production process.
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 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-Red Body Disease In Litopenaeus Vannamei, and it is with the cause of disease vibrio parahaemolytious JGB080708-1(of the Red Body Disease In Litopenaeus Vannamei after the formalin deactivation
vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, class of department 80, aluminium stearate, and 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 vibrio parahaemolytious JGB080708-1 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 with decocting after 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 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 1:100 g/ml, continues to be heated to 65 ℃, add department class 80, class 80 of department is 5:100 with the volume ratio of white oil, until 115 ℃ of finishing temperatures, maintain 15 min, make oil phase, cooling standby;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; In water, cause of disease vibrio parahaemolytious JGB080708-1 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, make the vibrio parahaemolytious oil emulsion inactivated vaccine that the Radix Astragali and white oil are the associating adjuvant.
Embodiment 2, a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Red Body Disease In Litopenaeus Vannamei, and it is with the cause of disease vibrio parahaemolytious JGB080708-1(of the Red Body Disease In Litopenaeus Vannamei after the formalin deactivation
vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, class of department 80, aluminium stearate, and 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 vibrio parahaemolytious JGB080708-1 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 with decocting after distilled water immersion 30min, filter, being concentrated into final concentration is former medicine 1g/mL, obtains Radix Astragali leachate;
(3) white oil is heated to 52 ℃, add aluminium stearate, the w/v of aluminium stearate and white oil is 3:100 g/ml, continue to be heated to 68 ℃, add class of department 80, class 80 of department is 5-7:100 with the volume ratio of white oil, until 118 ℃ of finishing temperatures, maintain 18 min, make oil phase, cooling standby;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; In water, cause of disease vibrio parahaemolytious JGB080708-1 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, make the vibrio parahaemolytious oil emulsion inactivated vaccine that the Radix Astragali and white oil are the associating adjuvant.
Embodiment 3, a kind of inactivated vaccine method for making of the yolk antibody for the preparation of anti-Red Body Disease In Litopenaeus Vannamei, and it is with the cause of disease vibrio parahaemolytious JGB080708-1(of the Red Body Disease In Litopenaeus Vannamei after the formalin deactivation
vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, class of department 80, aluminium stearate, and 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 vibrio parahaemolytious JGB080708-1 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 with decocting after 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 ℃, adds aluminium stearate, the w/v of aluminium stearate and white oil is 2:100 g/ml, continues to be heated to 70 ℃, add department class 80, class 80 of department is 6:100 with the volume ratio of white oil, until 120 ℃ of finishing temperatures, maintain 20 min, make oil phase, cooling standby;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; In water, cause of disease vibrio parahaemolytious JGB080708-1 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, make the vibrio parahaemolytious oil emulsion inactivated vaccine that the Radix Astragali and white oil are the associating adjuvant.
Embodiment 4, the application experiment of the inactivated vaccine of the anti-Red Body Disease In Litopenaeus Vannamei that embodiment 3 makes.
1. vibrio parahaemolytious oil emulsion inactivated vaccine immunization laying hen.
The vibrio parahaemolytious 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 the preceding method booster immunization, and immunizing dose is 0.5 mL, press preceding method after one week 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 is 4 times altogether, collection, by egg that immune chicken produces, is isolated yolk and is made high immunity yolk antibody, and measures its antibody titer.After immunity finishes, the 1st thoughtful the 2nd week yolk antibody tired and maintained 2
11, it is 2 that the 3rd week later yolk antibody tired
12.
2. the immunization experiment that the high immunity yolk antibody of the anti-Red Body Disease In Litopenaeus Vannamei made with said method carries out and result thereof.
Experimental period: 2012 April in year ~ May.
Experiment place: Jiangsu Province's Huaihai Institute of Technology aquatic animal disease laboratory.
Laboratory animal: Litopenaeus vannamei zoea and mysis.
(1) use the high immunity yolk antibody of anti-Red Body Disease In Litopenaeus Vannamei.The height obtained of usining during use is exempted from egg as raw material, through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h), is prepared into the yolk powder that contains anti-Pathogen of Red Body Disease In Litopenaeus Vannamei vibrio parahaemolytious high immunity yolk antibody.
(2) experimental group Litopenaeus vannamei zoea is thrown something and fed with the yolk powder and the commercially available shrimp crack that contain anti-Pathogen of Red Body Disease In Litopenaeus Vannamei vibrio parahaemolytious high immunity yolk antibody, matched group Litopenaeus vannamei zoea throw something and feed commercially available common yolk powder and commercially available shrimp crack.
(3) throw something and feed after 4d, experimental group and matched group Litopenaeus vannamei germling are carried out to the protection check, get respectively germling 100 tails, with vibrio parahaemolytious, carry out immersion infection, the thalline final concentration is 1.8 * 10
6cFU/mL.Duration of test continues to be thrown something and fed by above-mentioned (2), and experimental period is 72h.
(4) experimental result: experimental group Litopenaeus vannamei germling is thrown something and fed after the high immunity yolk antibody of experimental example 1 described anti-Red Body Disease In Litopenaeus Vannamei, can obtain higher anti-vibrio parahaemolytious infection ability, after germling infects 72h, survival rate reaches 78%, and the larvae survival rate is 26%.Experimental result shows that the high immunity yolk antibody of anti-Red Body Disease In Litopenaeus Vannamei can make Litopenaeus vannamei obtain higher immune protective efficiency.
Claims (1)
1. the method for making of the inactivated vaccine for the preparation of the yolk antibody of anti-Red Body Disease In Litopenaeus Vannamei, it is characterized in that: it is with the cause of disease vibrio parahaemolytious JGB080708-1(of the Red Body Disease In Litopenaeus Vannamei after the formalin deactivation
vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, class of department 80, aluminium stearate, and 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 vibrio parahaemolytious JGB080708-1 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 with decocting after 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 ℃, 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 ℃, 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 ℃, maintain 15-20 min, make oil phase, cooling standby;
(4) the deactivation thalline is mixed with Radix Astragali leachate, make the vaccine water; In water, cause of disease vibrio parahaemolytious JGB080708-1 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, make the vibrio parahaemolytious oil emulsion inactivated vaccine that the Radix Astragali and white oil are the 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 |
| CN105166574A (en) * | 2015-10-16 | 2015-12-23 | 扬州大学 | Microencapsulated feed for resisting vibriosis of prawn larvae |
| CN105126101A (en) * | 2015-10-16 | 2015-12-09 | 扬州大学 | Microcapsule feed additive resisting to litopenaeus vannamei pathogenic vice hemolysis vibrio |
| KR102049976B1 (en) * | 2018-07-30 | 2019-11-28 | (주)애드바이오텍 | Antibody for shrimp’s early mortality syndrome and white spot syndrome virus, and applications thereof |
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