CN101020050A - Prepn and usage of extracellular product subunit vaccine of seawater fish morbid vibrio - Google Patents
Prepn and usage of extracellular product subunit vaccine of seawater fish morbid vibrio Download PDFInfo
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- CN101020050A CN101020050A CNA2006101243383A CN200610124338A CN101020050A CN 101020050 A CN101020050 A CN 101020050A CN A2006101243383 A CNA2006101243383 A CN A2006101243383A CN 200610124338 A CN200610124338 A CN 200610124338A CN 101020050 A CN101020050 A CN 101020050A
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Abstract
The present invention is preparation and usage of extracellular product subunit morbid vibrio vaccine for seawater fish. The vibrio spawn is two or more of Vibrio alginolyticus, Vibrio Hrveyi, Vibrio vulnificus, Vibrio parahaemolyticus, etc. The vaccine preparing process includes culturing vibrio, extracting extracellular product and preparing vaccine. The vaccine containing partial pathogen and no infecting component has stable immunological specificity and no hidden danger of restoring toxici. The vaccine is used in preventing fishes' skin ulcer, eyeball turbidity and other vibrio caused diseases and may be used widely for immunizing seawater fish.
Description
Technical field:
The present invention relates to a kind of preparation and using method of seawater fish morbid vibrio extracellular product subunit vaccine.
Background technology:
In recent years, along with developing rapidly of mariculture industry, cultivation density strengthens, and cultures water environment and runs down, and the generation of disease is also more and more frequent.That bacterial disease becomes is the most common in the aquaculture, endanger one of severe diseases, often cause the mortality of cultured fishes, wherein vibriosis (Vibrosis) is again a disease the most common in the marine fish culture, that harm is maximum, already causes enormous economic loss for marine fish culture.
For a long time, tradition is prevented and treated method and generally is to use chemicals and antibiotic, uses these medicines that pathogen is developed immunity to drugs in a large number, destroys the microecosystem of breeding water body, also can cause medicine intravital residual, have a strong impact on the quality of cultivated animals at cultivated animals.Therefore, efficient, the safe vaccine of exploitation carries out effective immunity inoculation to Fish and is only the most effective most economical method of bacterial disease of preventing and treating.In existing technology, have only the production and the application of Vibrio anguillarum thalline vaccine and vibrio mimicus coupled subunit vaccine at present.Vibrio anguillarum thalline vaccine because have that virulence rebounds easily, that using dosage is big, immunne response level body is lower etc. is former thereby cause immune effect not good.The vibrio mimicus coupled subunit vaccine has the specificity of more single-minded anti-vibrio mimicus in aquaculture production, relatively poor to the pathogenic microbial disease defense reaction of other vibrios, has very significantly limitation in production application.
Summary of the invention
The objective of the invention is provides a kind of preparation and using method of seawater fish morbid vibrio extracellular product subunit vaccine in order to remedy the deficiency that above-mentioned prior art exists.
The technical scheme taked of the present invention is for achieving the above object:
A kind of preparation method of seawater fish morbid vibrio extracellular product subunit vaccine, can be used as the vaccine that multiple seawater fish improves the anti-vibrio of cultured output, used strain is: the strain of two or more in vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis; This preparation method comprises 3 main steps, is respectively: the preparation of the cultivation of antibacterial, the extraction of extracellular products, vaccine.
The preparation method of described seawater fish morbid vibrio extracellular product subunit vaccine, according to the following steps:
(1) cultivation of antibacterial
Vibrio activates with the tryptone soya peptone fluid medium TSB that contains 2%NaCl and initial pH 7.2, under 28 ℃, and shaken cultivation 18h under the 150r/min; Getting 1mL bacterium liquid then, to coat contain 2%NaCl and the initial pH that are covered with aseptic cellophane be 7.2 tryptone soya peptone Agar Plating TSA, leaves standstill under 28 ℃ and cultivate 18h;
(2) extraction of extracellular products
Add the PBS of 4mL pH 7.2 in every ware, wash bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min, the supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products ECP, puts 4 ℃ of refrigerators and preserves.With the protein concentration in the ultraviolet spectrophotometer working sample;
(3) vaccine preparation
Concentration is the extracellular products of 0.05mg/ml, adds 0.1% formalin, 60 ℃ of deactivation 1h, and 4 ℃ of preservations are standby.
The using method of this seawater fish morbid vibrio subunit vaccine:
(1) infusion method: should carry out immersion immunity during the seedling.Method is put in the container that fills vaccine for the fish that will need immunity, and soak time is one day;
(2) injection: the juvenile fish stage can be carried out the lumbar injection immunity, and injected dose is the 0.1ml/ tail;
(3) oral immunity: the juvenile fish stage also can be carried out oral vaccine.Every double centner fish 0.05ml every day mixes to raise and throws something and feeds, and connects to feed 3 days.
Advantage of the present invention and effect:
1. the immune effect of multicomponent vaccine is better than the one-component vaccine;
2. seawater fish morbid vibrio extracellular product subunit vaccine highly effective and safe, drug residue free pollutes.Simultaneously, infect composition and be removed, relevant residual toxicity or the regressive hidden danger of toxicity no longer exist;
3. a part that contains pathogen in this vibrio vaccine, thereby this vaccine is more definite on chemical property, immunologic opsonin is more stable;
4. this subunit vaccine all has defense reaction to the pathogenic microbial disease of multiple vibrio;
5. seawater fish morbid vibrio extracellular product subunit vaccine wide spectrum is resisted the invasion and attack infection of various seawater fish morbid vibrios, and its applicable object is various marine fishs;
6. this vaccine is easy to use, can the multipath immunity inoculation, can during seedling, carry out immersion immunity, and can carry out injecting immune in the juvenile fish stage again, also can carry out oral immunity.
7. this vaccine immunity protective rate is 86-92%, specifically protects effect to see the following form:
The trial effect of table 1 vaccine immersion immunity in the cage culture cabrilla
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Hainan, Yangjiang | Immune group matched group immune group matched group | 9 9 9 9 | 500 500 500 500 | 47 238 52 228 | 9.4 47.6 10.4 54.4 | 90.6 52.4 89.6 45.6 |
The trial effect of table 2 vaccine injecting immune in the cage culture red snapper
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Zhanjiang, Leizhou | Immune group matched group immune group matched group | 9 9 9 9 | 500 500 500 500 | 67 258 38 229 | 13.4 51.6 7.6 45.8 | 86.6 48.4 92.4 54.2 |
The trial effect of table 3 vaccine oral immunity in the cage culture rock salmon
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| The spy is the Sanya, island | Immune group matched group immune group | 9 9 9 | 500 500 500 | 67 248 52 | 13.4 49.6 10.4 | 86.6 50.4 89.6 |
| Matched group | 9 | 500 | 228 | 54.4 | 45.6 |
The specific embodiment
The present invention further specifies with the following example.
Embodiment 1
Respectively vibrio alginolyticus and vibrio harveyi are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus and vibrio harveyi extracellular products respectively.Method is: the PBS with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.Then prepare vibrio alginolyticus and vibrio harveyi extracellular product subunit vaccine respectively.Method is: is respectively the vibrio alginolyticus of 0.05mg/ml and the extracellular products of vibrio harveyi with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.At last the vibrio alginolyticus and the vibrio harveyi extracellular product subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio extracellular product subunit vaccine.
Embodiment 2
Respectively vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus extracellular products respectively.Method is: the PBS with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.
Then prepare vibrio alginolyticus, vibrio harveyi and Vibrio vulnificus extracellular product subunit vaccine respectively.Method is: is respectively the extracellular products of vibrio alginolyticus, vibrio harveyi and the Vibrio vulnificus of 0.05mg/ml with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.At last the vibrio alginolyticus, vibrio harveyi and the Vibrio vulnificus extracellular product subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio extracellular product subunit vaccine.
Embodiment 3
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum extracellular products respectively.Method is: the PBS with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum extracellular product subunit vaccine respectively.Method is: is respectively the extracellular products of vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and the Vibrio anguillarum of 0.05mg/ml with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus and the Vibrio anguillarum extracellular product subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio extracellular product subunit vaccine.
Embodiment 4
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and vibrio parahaemolytious are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and vibrio parahaemolytious extracellular products respectively.Method is: the PBS. with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and vibrio parahaemolytious extracellular product subunit vaccine respectively.Method is: is respectively the extracellular products of vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and the vibrio parahaemolytious of 0.05mg/ml with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.The broad algae vibrio that will make at last, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum and vibrio parahaemolytious extracellular product subunit vaccine equal proportion are mixed, and obtain the morbid vibrio extracellular product subunit vaccine.
Embodiment 5
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus extracellular products respectively.Method is: the PBS with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and vibrio mimicus extracellular product subunit vaccine respectively.Method is: is respectively the extracellular products of vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and the vibrio mimicus of 0.05mg/ml with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious and the vibrio mimicus extracellular product subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio extracellular product subunit vaccine.
Embodiment 6
Respectively vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis are adopted tryptone soya peptone fluid medium (TSB) activation earlier, under 28 ℃, 150r/min shaken cultivation 18h, getting 1mL bacterium liquid separate application then is 7.2 tryptone soya peptone Agar Plating (TSA) in contain 2%NaCl and the initial pH that are covered with aseptic cellophane, leaves standstill under 28 ℃ and cultivates 18h.Then, extract vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis extracellular products respectively.Method is: the PBS with pH 7.2 washes bacterium liquid, 4 ℃ of centrifugal 30min of following 10000r/min.The supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products (ECP).With the protein concentration in the ultraviolet spectrophotometer working sample.Then prepare vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and vibrio fluvialis extracellular product subunit vaccine respectively.Method is: is respectively the extracellular products of vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis of 0.05mg/ml with concentration, adds 0.1% formalin, and 60 ℃ of deactivation 1h, 4 ℃ of preservations are standby.At last the vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis extracellular product subunit vaccine equal proportion that make are mixed, obtain the morbid vibrio extracellular product subunit vaccine.
Claims (3)
1, a kind of preparation method of seawater fish morbid vibrio extracellular product subunit vaccine, it is characterized in that: can be used as the vaccine that multiple seawater fish improves the anti-vibrio of cultured output, used strain is: the strain of two or more in vibrio alginolyticus, vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, vibrio parahaemolytious, vibrio mimicus and the vibrio fluvialis; This preparation method comprises 3 main steps, is respectively: the preparation of the cultivation of antibacterial, the extraction of extracellular products, vaccine.
2,, it is characterized in that according to the following steps according to the preparation method of the described seawater fish morbid vibrio extracellular product subunit vaccine of claim 1:
(1) cultivation of antibacterial
Vibrio activates with the tryptone soya peptone fluid medium TSB that contains 2%NaCl and initial pH 7.2, under 28 ℃, and shaken cultivation 18h under the 150r/min; Getting 1mL bacterium liquid then, to coat contain 2%NaCl and the initial pH that are covered with aseptic cellophane be 7.2 tryptone soya peptone Agar Plating TSA, leaves standstill under 28 ℃ and cultivate 18h;
(2) extraction of extracellular products
Add the PBS of 4mL pH7.2 in every ware, wash bacterium liquid, in 4 ℃ of centrifugal 30min of following 10000r/min, the supernatant via hole diameter is the filtering with microporous membrane of 0.22 μ m, obtains vibrio extracellular products ECP, puts 4 ℃ of refrigerators and preserves.With the protein concentration in the ultraviolet spectrophotometer working sample;
(3) vaccine preparation
Concentration is the extracellular products of 0.05mg/ml, adds 0.1% formalin, 60 ℃ of deactivation 1h, and 4 ℃ of preservations are standby.
3, according to the using method of the seawater fish morbid vibrio subunit vaccine of claim 1 method preparation, it is characterized in that:
(1) infusion method: should carry out immersion immunity during the seedling.Method is put in the container that fills vaccine for the fish that will need immunity, and soak time is one day;
(2) injection: the juvenile fish stage can be carried out the lumbar injection immunity, and injected dose is the 0.1ml/ tail;
(3) oral immunity: the juvenile fish stage also can be carried out oral vaccine.Every double centner fish 0.05ml every day mixes to raise and throws something and feeds, and connects to feed 3 days.
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| CNA2006101243383A CN101020050A (en) | 2006-12-22 | 2006-12-22 | Prepn and usage of extracellular product subunit vaccine of seawater fish morbid vibrio |
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| CNA2006101243383A CN101020050A (en) | 2006-12-22 | 2006-12-22 | Prepn and usage of extracellular product subunit vaccine of seawater fish morbid vibrio |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101879317A (en) * | 2010-05-21 | 2010-11-10 | 中国海洋大学 | Preparation method and application of Vibro harveyi and Vibrio parahaemolyticus bigeminy DNA vaccine |
| CN102274494A (en) * | 2011-09-01 | 2011-12-14 | 淮海工学院 | Preparation method and use of vibrio parahaemolyticus toxin vaccine |
| WO2020130782A1 (en) * | 2018-12-19 | 2020-06-25 | Centro De Investigaciones Biologicas Del Noroeste, S.C. | Homeopathic compositions based on vibrio parahaemolyticus and vibrio alginolyticus and use thereof as immunostimulants in the culture of aquatic species |
| CN115885900A (en) * | 2022-12-05 | 2023-04-04 | 中国海洋大学 | Method for improving resistance of crassostrea gigas and vibrio larvae of crassostrea gigas |
-
2006
- 2006-12-22 CN CNA2006101243383A patent/CN101020050A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101879317A (en) * | 2010-05-21 | 2010-11-10 | 中国海洋大学 | Preparation method and application of Vibro harveyi and Vibrio parahaemolyticus bigeminy DNA vaccine |
| CN101879317B (en) * | 2010-05-21 | 2012-06-27 | 中国海洋大学 | Preparation method and application of Vibro harveyi and Vibrio parahaemolyticus bigeminy DNA vaccine |
| CN102274494A (en) * | 2011-09-01 | 2011-12-14 | 淮海工学院 | Preparation method and use of vibrio parahaemolyticus toxin vaccine |
| WO2020130782A1 (en) * | 2018-12-19 | 2020-06-25 | Centro De Investigaciones Biologicas Del Noroeste, S.C. | Homeopathic compositions based on vibrio parahaemolyticus and vibrio alginolyticus and use thereof as immunostimulants in the culture of aquatic species |
| CN115885900A (en) * | 2022-12-05 | 2023-04-04 | 中国海洋大学 | Method for improving resistance of crassostrea gigas and vibrio larvae of crassostrea gigas |
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Open date: 20070822 |