CN101461940B - Photobacterium damsela vaccine as well as preparation method and use thereof - Google Patents
Photobacterium damsela vaccine as well as preparation method and use thereof Download PDFInfo
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- CN101461940B CN101461940B CN2009100365298A CN200910036529A CN101461940B CN 101461940 B CN101461940 B CN 101461940B CN 2009100365298 A CN2009100365298 A CN 2009100365298A CN 200910036529 A CN200910036529 A CN 200910036529A CN 101461940 B CN101461940 B CN 101461940B
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- 229920006008 lipopolysaccharide Polymers 0.000 claims abstract description 34
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Abstract
The invention discloses a mermaid photobacterium vaccine, a method for preparing the same and application thereof. The vaccine consists of mermaid photobacterium lipopolysaccharide and an adjuvant. The method comprises: inactivating the mermaid photobacterium first, then extracting the mermaid photobacterium lipopolysaccharide from the inactivated mermaid photobacterium, and mixing the lipopolysaccharide with the adjuvant to obtain the mermaid photobacterium vaccine. The vaccine can be used for preventing diseases of fish, particularly sea fish. The vaccine has the advantages of safety and high efficiency, simple preparation method, low cost and convenient large-scale popularization.
Description
Technical field
The present invention relates to the fish disease prevention and control field, be specifically related to a kind of Mermaid luminous bacillus vaccine and its production and application.
Background technology
Mermaid luminous bacillus (Photobacterium damselae subsp.damselae) is a kind of Gram-negative, can be luminous, have a liking for salt conditionality pathogenic bacterium, it is 1981 at first, from the ulcer wound of the catfish that warm sea water, grows, be separated to, and be categorized into vibrio, and be called the mermaid vibrio, be distributed widely in ocean, inshore and the sea water biology.People and animals' disease that it can cause is similar to vibrio, all can cause human gastroenteritis, wound infection and people and animals' septicemia (CURRENT MICROBIOLOGY Vo1.48 (2004), pp.167-174Cloning and Characterization of the Gene Encoding forOMP-PD Porin:The Major Photobacterium damsela Outer Membrane Protein).
Mermaid luminous bacillus is a kind of antibacterial that causes the tissue necrosis infectious disease of tool extreme damage, external many scholars are referred to as " mermaid suffers from pathogen altogether ", it not only can cause pseudotuberculosis and fish hueppe's disease, cause aquatic products to culture loss more than 50%, can also the people be caused a disease by people's wound or edible underdone marine product, can cause healthy human wound infection as it, the constitutional septicemia, even can also cause fatal necrotizing fasciitis etc., with regard to taking place, a lot of fishermen infected Mermaid luminous bacillus and serious morbific incident in Japan.More fearful is, increasing report shows that Mermaid luminous bacillus has strong lethal, can be simultaneously so that immunodeficiency or healthy factitious host to be arranged, and bring out fast, fulminant infection (Fraser SL, Purcell BK, Delgado B Jr, Baker AE, Whelen AC.Rapidly fatal infection due to Photobacterium (Vibrio) damela.Clin Infect Dis 1997; 25:935-936.).
The present virulence factor that studies confirm that Mermaid luminous bacillus has extracellular hemolysin, mermaid cytolysin, outer membrane protein and neuraminidase etc.Have the scholar that certain marine site sea water of China has been carried out the antibacterial general adjustment and look into, the Mermaid luminous bacillus recall rate is in the 3rd, accounts for 6.9%, be the ocean dominant bacteria, and this has been carried out virulence experiment and wound infection tested, the result shows that it has the invasiveness of organizing, and simultaneously animal is also had stronger appeal.As pathogenic bacterium, it can cause traumatic infection, the bovine pasteurellosis of multiple seawater fishs such as golden pomfret, damselfish, shallow sea turtle, golden head Channa argus, barramundi, turbot, anguilla japonica, even dead, brings enormous economic loss.
Though traditional antibiotic has been brought into play important function in treatment and control seawater fish bacterial disease, but can cause drug-resistance of bacteria with antibiotic for a long time, make the dosage of medication increasing, drug residue is serious, influence the quality of aquatic products, even threaten human beings'health.As seen develop vaccine and very be necessary.
Directed toward bacteria is to adopt the full vaccine of deactivation mostly now, but the full vaccine of deactivation usually can produce complicated microbial metabolic products in preparation process, thereby side effect occurs, makes immune effect unsatisfactory.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of Mermaid luminous bacillus that can effectively prevent to cause a disease, and safe in utilization, the tangible Mermaid luminous bacillus vaccine of immune protective effect.
Another object of the present invention is to provide a kind of method for preparing above-mentioned Mermaid luminous bacillus vaccine, and this preparation method is simple to operate, and is with low cost.
Another object of the present invention is to provide the application of above-mentioned Mermaid luminous bacillus vaccine in the medicine of preparation control Fish especially seawater fish disease.
Above-mentioned purpose of the present invention is achieved by following scheme:
The protective antigen that the present invention is directed to Mermaid luminous bacillus is explored, and has the better protect antigenic characteristic by the lipopolysaccharide (LPS) of relatively finding this bacterium adventitia, can make the invasion and attack that vaccine is resisted Mermaid luminous bacillus.
A kind of Mermaid luminous bacillus vaccine, this vaccine are the Mermaid luminous bacillus lipopolysaccharide is mixed back preparation with adjuvant and to get.Described adjuvant can select any preparation vaccine to use always, as incomplete Freund, Freund's complete adjuvant and aluminum hydroxide adjuvant etc.
In the above-mentioned Mermaid luminous bacillus vaccine, every 1ml vaccine contains Mermaid luminous bacillus lipopolysaccharide 0.25～1mg, and preferred every 1ml vaccine contains Mermaid luminous bacillus lipopolysaccharide 0.5mg.
In the above-mentioned Mermaid luminous bacillus vaccine, the consumption of adjuvant and it goes without doing special the qualification can select during practical operation that the mass ratio of Mermaid luminous bacillus lipopolysaccharide and adjuvant is 2: 3～3: 2 in the vaccine, and preferably both mass ratioes are 1: 1.
The preparation method of Mermaid luminous bacillus vaccine of the present invention, be to cultivate earlier Mermaid luminous bacillus and with after its deactivation, from the thalline of deactivation, extract the Mermaid luminous bacillus lipopolysaccharide, this lipopolysaccharide is mixed obtaining the Mermaid luminous bacillus vaccine then with adjuvant.
In the above-mentioned preparation method, conventional cultural methods such as slant culture, liquid culture are adopted in the cultivation of Mermaid luminous bacillus.
In the above-mentioned preparation method, the conventional method of this area is also adopted in the deactivation of Mermaid luminous bacillus, as available formalin deactivation, formaldehyde etc.
In the above-mentioned preparation method, the conventional extracting method of this area is adopted in the extraction of Mermaid luminous bacillus lipopolysaccharide, as hot phenol water law etc.
Mermaid luminous bacillus vaccine of the present invention, through toxicity test and Fish in vivo test, prove that this vaccine is not only little to fish body toxicity, and all very remarkable in multiple Fish (as silvery pomfret Scad etc.) immune protection effect on one's body, therefore can be used for Fish, the diseases prevention and treatment of especially southern seawater fish.
Compared with prior art, the present invention has following beneficial effect:
1. Mermaid luminous bacillus vaccine of the present invention is safe in utilization, and immune protection is effective, can effectively prevent Mermaid luminous bacillus to cause a disease;
2. vaccine its preparation method of the present invention is simple to operate, with low cost, and vaccine can be used for the especially diseases prevention and treatment of seawater fish of Fish, has very high commercial value.
The specific embodiment
Below the invention will be further described by specific embodiment, but not with any situation restriction the present invention.
The cultivation and the deactivation of embodiment 1 Mermaid luminous bacillus
The inventor adopts conventional separation method to isolate for the Mermaid luminous bacillus that tries from morbidity fish body, and it is inoculated in Zobell 2216E slant medium, cultivate 24h for 28 ℃, transfer in Zobell 2216E fluid medium, 28 ℃, 180rpm shaking table cultivation 18h, add 0.25 volume % formalin deactivation antibacterial, spend the night in 4 ℃.The centrifugal 15min of 7000rpm abandons supernatant, adds sterile saline washing thalline, the centrifugal 15min of 5000rpm, and the precipitation thalline with sterile saline washed twice again, is collected thalline.
In the present embodiment Zobell 2216E slant culture and and the preparation of Zobell 2216E fluid medium all adopt universal method, for those skilled in the art in common knowledge.
The extraction of embodiment 2 Mermaid luminous bacillus lipopolysaccharide (LPS)
The phenol water law is adopted in the extraction of the Mermaid luminous bacillus lipopolysaccharide of present embodiment, and its step is as follows:
(1) get about 20 grams of thalline that embodiment 1 prepares gained, be suspended from the 200ml distilled water, add 90% phenol of equal-volume preheating again, water intaking is preserved mutually.
(2) distilled water that adds preheating in mutually to above-mentioned phenol makes total system recover original volume (about 400ml), vigorous stirring 30min in 65 ℃ of water-baths again dashes with tap water and to drench the glass container outer wall, makes content be cooled to room temperature rapidly, the centrifugal 20min of 10000rpm, water intaking is preserved mutually;
(3) repeating step is (2) 2～3 times, merge each time extracting gained water, put in the bag filter with the tap water dialysis 24h that flows, reuse distill water dialysis 48h changes distilled water therebetween 3～4 times, then, the anti-dialysis of PEG20000 makes solution concentration in the bag filter to the 50ml, and this concentrated solution in the centrifugal 5h of 30000g, is got supernatant, lyophilization obtains purer lipopolysaccharide (LPS) powder.
The preparation of embodiment 3 Mermaid luminous bacillus vaccines
Lipopolysaccharide (LPS) the powder dilution back of embodiment 2 preparation gained is promptly obtained the Mermaid luminous bacillus vaccine with the abundant mixing of incomplete Freund.
Present embodiment prepares 5 parts of Mermaid luminous bacillus vaccines altogether, and the concentration of Mermaid luminous bacillus lipopolysaccharide is respectively and contains Mermaid luminous bacillus lipopolysaccharide 0.25mg, 0.5mg, 1mg, 2mg and 4mg in the 1ml vaccine in its every part vaccine.
The toxicity test of embodiment 4 Mermaid luminous bacillus lipopolysaccharide vaccines
In order to ensure the safety of vaccine of the present invention, 5 parts of vaccines that embodiment 3 is prepared carry out toxicity test.
With golden pomfret 120 tails of health, the about 45g of average weight is divided into 6 groups at random, every group 20 tail.
With Rachycentron canadum 120 tails of health, the about 60g of average weight is divided into 6 groups at random, every group 20 tail.
With 5 parts of vaccines and PBS contrast of embodiment 3 preparation gained, be injected into respectively in the fish body abdominal cavity by the amount of 0.1mg/ tail, experimental result sees Table 1,2.
Table 1 Mermaid luminous bacillus vaccine is to golden pomfret toxicity test result
Table 2 Mermaid luminous bacillus vaccine is to Rachycentron canadum toxicity test result
By table 1,2 as can be seen, when the concentration of Mermaid luminous bacillus lipopolysaccharide in prepared vaccine is 0.25mg/ml, 0.5mg/ml and 1mg/ml, the toxicity of vaccine all a little less than, from fish body toxicity and economic angle are considered that the concentration of the preferred Mermaid luminous bacillus lipopolysaccharide of Mermaid luminous bacillus vaccine is 0.5mg/ml.
Embodiment 5 Mermaid luminous bacillus vaccines are to the influence of golden pomfret growth
In order to understand fully the influence of Mermaid luminous bacillus lipopolysaccharide vaccine of the present invention to the fish bulk-growth, employing embodiment 3 preparation gained Mermaid luminous bacillus lipopolysaccharide concentration are that the vaccine of 0.5mg/ml carries out the test of upgrowth situation to the fish body.
With golden pomfret 90 tails of health, average weight 50.5g, Rachycentron canadum 90 tails, average weight 63.2g, each is divided into 3 groups at random, is respectively vaccine immunity group (0.5mg/ml), the full vaccine (1.8 * 10 of deactivation
8Cfu/ml) immune group and matched group (PBS), every group 30 tail.Lumbar injection 0.1mg/ tail, experimental result sees Table 3,4.
Table 3 Mermaid luminous bacillus vaccine is to the influence of golden pomfret growth
Table 4 Mermaid luminous bacillus vaccine is to the influence of Rachycentron canadum growth
By table 3,4 as can be seen, in 4 time-of-weeks of raising, behind the injection Mermaid luminous bacillus vaccine, the average daily gain in weight of fish is a little more than matched group and the full vaccine of deactivation, and this illustrates that vaccine of the present invention can't influence the growth of fish body.
Embodiment 6 Mermaid luminous bacillus lipopolysaccharide vaccines are to golden pomfret immune protection effect
Adopting embodiment 3 preparation gained Mermaid luminous bacillus lipopolysaccharide concentration is the vaccine of 0.5mg/ml, carries out the immune protection effect test of vaccine to the fish body.
With golden pomfret 90 tails and Rachycentron canadum 90 tails of health, each is divided into 3 groups at random, is respectively vaccine immunity group (0.5mg/ml), the full vaccine (1.8 * 10 of deactivation
8Cfu/ml) immune group and matched group (PBS), lumbar injection 0.1mg/ tail behind the immune 28d, is got 30 tail fishes for every group, carries out counteracting toxic substances with Mermaid luminous bacillus LD50 dosage and infects, and calculates protective rate and relative protective rate (RPS) respectively, and experimental result sees Table 5,6.
Table 5 Mermaid luminous bacillus vaccine is to the immune protective rate of golden pomfret
Table 6 Mermaid luminous bacillus vaccine is to the immune protective rate of Rachycentron canadum
Experimental result data by his-and-hers watches 5,6 is analyzed, and therefore the relative protective rate of vaccine group illustrates that Mermaid luminous bacillus vaccine effect of the present invention is fine far above the full vaccine group of deactivation as can be seen.
Claims (5)
1. a Mermaid luminous bacillus vaccine is mixed and made into by Mermaid luminous bacillus lipopolysaccharide and adjuvant; Every 1ml vaccine contains Mermaid luminous bacillus lipopolysaccharide 0.25～1mg; Described adjuvant is an incomplete Freund.
2. according to the described Mermaid luminous bacillus vaccine of claim 1, it is characterized in that in the described vaccine that every 1ml vaccine contains Mermaid luminous bacillus lipopolysaccharide 0.5mg.
3. method for preparing the described Mermaid luminous bacillus vaccine of claim 1, it is characterized in that this method is first deactivation Mermaid luminous bacillus, therefrom extract the Mermaid luminous bacillus lipopolysaccharide then, this lipopolysaccharide is mixed obtaining the Mermaid luminous bacillus vaccine again with adjuvant.
4. according to the described preparation method of claim 3, the extracting method that it is characterized in that described Mermaid luminous bacillus lipopolysaccharide is the phenol water law.
5. the application of the described Mermaid luminous bacillus vaccine of claim 1 in preparation control fish disease medicine.
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| CN2009100365298A CN101461940B (en) | 2009-01-09 | 2009-01-09 | Photobacterium damsela vaccine as well as preparation method and use thereof |
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| CN2009100365298A CN101461940B (en) | 2009-01-09 | 2009-01-09 | Photobacterium damsela vaccine as well as preparation method and use thereof |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102139103B (en) * | 2011-04-07 | 2012-10-24 | 淮海工学院 | Preparation and application methods of photobacterium damsela vaccines of cynoglossus semilaevis |
| CN108330142B (en) * | 2018-02-09 | 2021-09-17 | 河北科技师范学院 | Mermaid photorhabditis hemolysin Hly with immune protection effectchProtein |
| CN109880766B (en) * | 2019-03-18 | 2021-05-07 | 宁波大学 | Photobacterium pomfret mermaid strain and inactivated vaccine |
| CN109865135A (en) * | 2019-03-18 | 2019-06-11 | 宁波大学 | A kind of silvery pomfret Mermaid luminous bacillus and Vibrio splindidus combine inactivated vaccine |
| CN109865136A (en) * | 2019-03-18 | 2019-06-11 | 宁波大学 | A kind of silvery pomfret bivalent inactivated vaccine |
| CN111304131B (en) * | 2020-03-17 | 2021-07-16 | 中国水产科学研究院黄海水产研究所 | Pathogenic mermaid photobacterium mermaid subspecies strain and application thereof |
| CN111388660B (en) * | 2020-03-17 | 2021-05-25 | 中国水产科学研究院黄海水产研究所 | Mermaid photobacterium mermaid subspecies polyvalent inactivated vaccine and preparation method thereof |
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| CN1306399A (en) * | 1999-03-26 | 2001-08-01 | 杣源一郎 | Additives for crustacean or fish feeds and feeds |
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| CN1306399A (en) * | 1999-03-26 | 2001-08-01 | 杣源一郎 | Additives for crustacean or fish feeds and feeds |
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Correction item: Patentee Correct: South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences|Guangzhou City, Guangdong province Xingang road 510300 South China Sea Fisheries Research Institute No. 231 fishing ward False: South China Sea Fishery Research Institute, Chinese Academy of Fishery Sciences|Guangzhou City, Guangdong province Xingang road 510300 South China Sea Fisheries Research Institute No. 231 fishing ward Number: 04 Volume: 27 |
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Free format text: CORRECT: PATENTEE; FROM: SOUTH CHINA SEA FISHERY RESEARCH INSTITUTE, CHINESE ACADEMY OF FISHERY SCIENCES:510300 FISH DISEASE OFFICE, SOUTH CHINA SEA FISHERIES RESEARCH INSTITUTE, NO. 231, XINGANG WEST ROAD, GUANGZHOU CITY, GUANGDONG PROVINCE TO: NANHAI INST. OF AQUATIC PRODUCTS, CHINESE ACADEMY OF AQUATIC PRODUCTS SCIENCES:510300 FISH DISEASE OFFICE, SOUTH CHINA SEA FISHERIES RESEARCH INSTITUTE, NO. 231, XINGANG WEST ROAD, GUANGZHOU CITY, GUANGDONG PROVINCE |
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