CN102836428A - 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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- CN102836428A CN102836428A CN2012103360512A CN201210336051A CN102836428A CN 102836428 A CN102836428 A CN 102836428A CN 2012103360512 A CN2012103360512 A CN 2012103360512A CN 201210336051 A CN201210336051 A CN 201210336051A CN 102836428 A CN102836428 A CN 102836428A
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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 method for preparing of inactivated vaccine, particularly a kind of inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis.
Background technology
Litopenaeus vannamei (
Litopenaeus vannamei) claim white shrimp, Wan Shi prawn etc. again, be tropical shrimp species, advantages such as wide, the anti-dense stocking of right salt scope, growth are fast, high-output stress-resistance that it has, after China introduced in 1988, the coastal various places large-scale farming in the whole nation at present.Along with Litopenaeus vannamei is cultured the expansion of 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 in the Litopenaeus vannamei breeding process the most common and be to cause one of the most serious disease of economic loss.
The generation that is difficult to the disease that the control vibrio parahaemolytious causes during Litopenaeus vannamei sea-farming is at present produced is with popular, the inactivated vaccine of a kind of anti-Litopenaeus vannamei eperythrozoonosis easy to use of shortage on the market.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, provide a kind of improve Litopenaeus vannamei to the immunity of vibrio parahaemolytious, reduce harm that the Litopenaeus vannamei sea-farming caused by the cause of disease vibrio parahaemolytious in producing, to satisfy the inactivated vaccine method for making of yolk antibody that is used to prepare anti-Litopenaeus vannamei eperythrozoonosis of disease-resistant demand on the Litopenaeus vannamei breeding 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-Litopenaeus vannamei eperythrozoonosis, is characterized in: it with the cause of disease vibrio parahaemolytious JGB080708-1 of the Litopenaeus vannamei eperythrozoonosis after the formalin deactivation (
Vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, department class 80, aluminium stearate, and with Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are following:
(1) with 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 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 vibrio parahaemolytious JGB080708-1 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 vibrio parahaemolytious oil emulsion inactivated vaccine for 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, 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 inactivated vaccine of the anti-Litopenaeus vannamei eperythrozoonosis that method for preparing of the present invention makes is following: with the vibrio parahaemolytious oil emulsion inactivated vaccine that makes, 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, presses preceding method after the week and continues booster immunization; Immunizing dose is 1 mL, presses preceding method booster immunization again after the week, and immunizing dose is 0.5 mL; Immunity is 4 times altogether; Collection is isolated yolk by egg that immune chicken produces, and makes yolk antibody.Immunity finishes the 4th week of back, and yolk antibody is tired and can be reached 2
12Exempt from egg as raw material with the height that said method was obtained then; 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-Litopenaeus vannamei eperythrozoonosis; Can in the production of growing seedlings, directly throw something and feed, or be added on the use of throwing something and feeding in the mixed feed in 5% ratio.The high immunity yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis not only has good resistant effect, and easy to use, has higher nutritive value simultaneously again.
Vibrio parahaemolytious oil emulsion inactivated vaccine of the present invention; Can be used for the adult laying hen of immunity; And then obtain the high immunity yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis, and can strengthen the immunity of Litopenaeus vannamei to the cause of disease vibrio parahaemolytious, improve the survival rate that Litopenaeus vannamei is cultured; Increase the output that Litopenaeus vannamei is cultured, improve the economic benefit of Litopenaeus vannamei breeding production.Fundamentally solved the problem that is prone to take place the vibrio parahaemolytious associated diseases in the present Litopenaeus vannamei breeding 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 inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis, it with the cause of disease vibrio parahaemolytious JGB080708-1 of the Litopenaeus vannamei eperythrozoonosis after the formalin deactivation (
Vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, department class 80, aluminium stearate, and with Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are following:
(1) with 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 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 15 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 vibrio parahaemolytious JGB080708-1 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 vibrio parahaemolytious 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-Litopenaeus vannamei eperythrozoonosis, it with the cause of disease vibrio parahaemolytious JGB080708-1 of the Litopenaeus vannamei eperythrozoonosis after the formalin deactivation (
Vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, department class 80, aluminium stearate, and with Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are following:
(1) with 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 and decoct after with distilled water immersion 30min, 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; Continue to be heated to 68 ℃, class of adding department 80, class 80 of department is 5-7:100 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 vibrio parahaemolytious JGB080708-1 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 vibrio parahaemolytious 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-Litopenaeus vannamei eperythrozoonosis, it with the cause of disease vibrio parahaemolytious JGB080708-1 of the Litopenaeus vannamei eperythrozoonosis after the formalin deactivation (
Vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, department class 80, aluminium stearate, and with Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are following:
(1) with 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 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 vibrio parahaemolytious JGB080708-1 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 vibrio parahaemolytious oil emulsion inactivated vaccine for the associating adjuvant.
Embodiment 4, the application experiment of the inactivated vaccine of the anti-Litopenaeus vannamei eperythrozoonosis 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, 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
11, it is 2 that the 3rd all yolk antibodies are later on tired
12
2. immunization experiment and result thereof that the high immunity yolk antibody of the anti-Litopenaeus vannamei eperythrozoonosis that makes with said method carries out.
Experimental period: 2012 April in year ~ Mays.
Experiment place: Jiangsu Province's Huaihai Institute of Technology aquatic animal disease laboratory.
Laboratory animal: Litopenaeus vannamei zoea and mysis.
(1) high immunity yolk antibody of the anti-Litopenaeus vannamei eperythrozoonosis of use.Exempt from egg as raw material with the height that is obtained during use, be prepared into the yolk powder that contains anti-Litopenaeus vannamei eperythrozoonosis cause of disease vibrio parahaemolytious high immunity yolk antibody through lyophilization (egg yolk liquid thickness 6mm, freeze-drying time 10h).
(2) experimental group Litopenaeus vannamei zoea is thrown something and fed with containing the yolk powder and the commercially available shrimp crack of anti-Litopenaeus vannamei eperythrozoonosis cause of disease 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 behind the 4d, experimental group and matched group Litopenaeus vannamei germling are carried out the protection check, get germling 100 tails respectively, carry out immersion infection with vibrio parahaemolytious, the thalline final concentration is 1.8 * 10
6CFU/mL.Duration of test continues to throw something and feed by above-mentioned (2), and experimental period is 72h.
(4) experimental result: experimental group Litopenaeus vannamei germling is thrown something and fed behind the high immunity yolk antibody of experimental example 1 described anti-Litopenaeus vannamei eperythrozoonosis; Can obtain higher anti-vibrio parahaemolytious infection ability; Survival rate reached 78% after germling infected 72h, and the larvae survival rate is 26%.Experimental result shows that the high immunity yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis can make Litopenaeus vannamei obtain higher immune protective efficiency.
Claims (1)
1. inactivated vaccine method for making that is used to prepare the yolk antibody of anti-Litopenaeus vannamei eperythrozoonosis is characterized in that: it with the cause of disease vibrio parahaemolytious JGB080708-1 of the Litopenaeus vannamei eperythrozoonosis after the formalin deactivation (
Vibrio parahaemolyticus) as immunogen, make oil emulsion with white oil, department class 80, aluminium stearate, and with Radix Astragali leachate as immunostimulant, emulsifying is prepared into oil emulsion inactivated vaccine; Its concrete steps are following:
(1) with 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 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 vibrio parahaemolytious JGB080708-1 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 vibrio parahaemolytious oil emulsion inactivated vaccine for the associating adjuvant.
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Cited By (5)
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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 |
| CN105126101A (en) * | 2015-10-16 | 2015-12-09 | 扬州大学 | Microcapsule feed additive resisting to litopenaeus vannamei pathogenic vice hemolysis vibrio |
| CN105166574A (en) * | 2015-10-16 | 2015-12-23 | 扬州大学 | Microencapsulated feed for resisting vibriosis of prawn larvae |
| CN110776565A (en) * | 2018-07-30 | 2020-02-11 | （株）Ad生物技术 | Antibody against early death syndrome and white spot virus of shrimp and application thereof |
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Cited By (5)
| 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 |
| CN105126101A (en) * | 2015-10-16 | 2015-12-09 | 扬州大学 | Microcapsule feed additive resisting to litopenaeus vannamei pathogenic vice hemolysis vibrio |
| CN105166574A (en) * | 2015-10-16 | 2015-12-23 | 扬州大学 | Microencapsulated feed for resisting vibriosis of prawn larvae |
| CN110776565A (en) * | 2018-07-30 | 2020-02-11 | （株）Ad生物技术 | Antibody against early death syndrome and white spot virus of shrimp and application thereof |
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