CN109395070A - A kind of Edwardsiella tarda microballoon oral vaccine and preparation method - Google Patents
A kind of Edwardsiella tarda microballoon oral vaccine and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of Edwardsiella tarda microballoon oral vaccine and preparation method, which is that inactivation Edwardsiella tarda is coated with the microballoon that calcium alginate obtains.Vaccine of the invention, the partial size of microballoon are 4.64 μm -28.38 μm, 16.51 μm of average grain diameter, its partial size is smaller, and the clad ratio of the inactivation Edwardsiella tarda of calcium alginate pair in 91.04%-96.5%, stablize in the gastric juice of simulation by microballoon, it is discharged in intestinal juice, shows good slow release；The use of the microspheres vaccine is remarkably improved the serum titer of fish body and the phagocytic activity of macrophage and phagocytic index, and highest immune protective rate is up to 77.33%.The oral vaccine effectively can activate fish body to be immunoreacted, and prevent fish disease.Method and process of the invention is relatively simple, and cost is relatively low, is conducive to industrialized production.
Description
Technical field
The present invention relates to a kind of seawater fish Edwardsiella tarda microballoon oral vaccine and preparation methods, belong to biological skill
Art field.
Background technique
In recent years, seawater industrial culturing scale expands rapidly, and production intensification degree is continuously improved.Disease, which becomes, to be restricted
The bottleneck that seawater industrial culturing industry develops in a healthy way.According to statistics, since the generation of disease is to caused by seawater industrial culturing industry
Annual up to 10,000,000,000 yuan of loss.Edwardsiella tarda is a kind of gram negative pathogenic bacterium, is infected in marine fish
Extremely extensively.It, which is noteworthy characterized by, can survive and breed in different host cells, can hide endocytosis,
It is replicated in cytoplasm.The infection and outburst of Edwardsiella tarda cause biggish economic loss to marine fish.
Aquatic animal disease control is at present mainly based on medical treatment.The fishing medicine used in production it is most of by veterinary drug,
Pesticide transplanting, lacks the fundamental researches such as pharmacokinetics, toxicology and the influence to breeding ecological environment, and abuse is wrong
The phenomenon that with fishing medicine, is very universal.Long-term drug abuse is not only restricted the effect of drug, can also induce the micro- life of cause of disease
Object develops drug resistance, and finally results in " no medicine is available ".Meanwhile drug can also remain and to breeding environment in cultivated animals body
It causes further to pollute.As people are food-safe and the attention of Environmental security, aquatic products medicament residue hidden danger is eliminated
Disease-resistant technical research has been particularly important.Substitute of the vaccine as antibiotic can make fish are high specifically to resist attacking for cause of disease
It hits, reduces the generation of fish diseases, preserve the ecological environment.
Vaccines for fish is divided into according to the difference of immunization ways to be vaccinated, oral vaccine and soaking vaccine.It is developed at present
The vaccine about Edwardsiella tarda be to vaccinate.Since the aquatic feature of fish and Population are big, it is difficult picture
Livestock products animal equally uses injecting method vaccine inoculation.Immersion way is big to the demand of vaccine, and immune protective rate is low.Fish
Oral vaccine has easy to use, and safety is good, and the shoal of fish stress be small, is suitable for scale and repeats the features such as immune.Therefore in recent years
Come, oral vaccine becomes the first choice of vaccines for fish vaccination ways.But compared with vaccinating, fish oral vaccine is easy
By the destruction of fish stomach acidity environment and enteral digestive ferment, so that vaccine is largely degraded before reaching intestinal mucosa, to exempt from
Epidemic disease effect is poor.
In recent years, domestic and foreign scholars are successively conducted a research using various polymer as fish oral vaccine transfer medium.
2008, Sun Jinsheng etc. using spraying technology using the polymer wrappeds such as polyvinyl alcohol vaccine preparation at vaccine pellet as oral epidemic disease
Seedling uses, and 2014, using spraying technology, by the package such as acrylic resin vaccine oral vaccine, to prepare vaccine micro- for Zhang Zhen state etc.
Ball.These oral vaccines can pass through the acidic environment of fish body stomach under the protection of polymer, and protection antigen completely arrives
Up to enteron aisle, but the oral vaccine is easy to appear phenomenon of burst release in enteron aisle, cannot reach good immune effect.
Sodium alginate (C6H7O8Na)nIt is to be relied on by α-L- mannuronic acid (M unit) and β-D- guluronic acid (G unit)
Linear copolymer made of 1,4 glucosides key connections.Sodium alginate powder is met water and is got wet, and can be slowly dissolved.Alginate can pass through
The sodium ion and bigeminy cation (Ca of its golonic acid2+) exchange and transient gel chemical conversion ball.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide one kind can be used for sea-farming by slow Edward
The Edwardsiella tarda microballoon oral vaccine of the immunoprophylaxis of disease caused by Salmonella.
A second object of the present invention is to provide a kind of preparation methods of Edwardsiella tarda microballoon oral vaccine.
Technical solution of the present invention is summarized as follows:
A kind of Edwardsiella tarda microballoon oral vaccine is that inactivation Edwardsiella tarda is coated with calcium alginate and obtains
Microballoon.
Edwardsiella tarda is preferably Edwardsiella tarda (Edwardsiella tarda) GX-1, and deposit number is
CCTCC NO:M2018379.
The clad ratio of calcium alginate is 91.04%-96.5%.
A kind of preparation method of Edwardsiella tarda microballoon oral vaccine, includes the following steps:
(1) the Edwardsiella tarda bacterium solution of inactivation is added in sodium alginate aqueous solution, is uniformly mixed, must mixes
Liquid；
(2) mixed liquor is added in vegetable oil and obtains water oil mixture, emulsifier is added；Stir to get water oil cream
Agent；
(3) under stirring, by water emulsifier and CaCl2Aqueous solution mixing, stirring form water-in-oil emulsion；
(4) centrifugation removal upper oil phase and water phase, ultrapure water are resuspended sediment, obtain suspension；
(5) freeze drying protectant is added, vacuum freeze drying is stored refrigerated.
Step (1) is preferred are as follows: by the concentration of inactivation is 1~3 × 1010Cfu/ml Edwardsiella tarda bacterium solution is added to
Volume, mass concentration are to be uniformly mixed in 3% sodium alginate aqueous solution, obtain mixed liquor.
Edwardsiella tarda is preferably Edwardsiella tarda (Edwardsiella tarda) GX-1, and deposit number is
CCTCC NO:M2018379.
Step (2) is preferred are as follows: is by volume the ratio of 1:2, mixed liquor is added in vegetable oil and obtains the mixing of water oil
The emulsifier span 80 of water oil mixture volume 1% is added in object；Water emulsifier is obtained in 1200rpm stirring 15min；It is described
Vegetable oil is soybean oil, corn oil, at least one of peanut oil and rapeseed oil.
Step (3) is preferred are as follows: under 1200rpm stirring, by water emulsifier and isometric mass concentration 4%CaCl2
Aqueous solution mixing, 1200rpm stir 15min, and 800rpm continues to stir 1h, form water-in-oil emulsion.
Step (4) is preferred are as follows: 3000rpm is centrifuged 4min removal upper oil phase and water phase, by the water phase and oily phase with removal
The ratio that 1 times of total volume is added ultrapure water and sediment is resuspended, and obtains suspension；
Step (5) is preferably the ratio of 1:1 by volume, and freeze drying protectant, the freeze-drying is added in Xiang Suoshu suspension
Protective agent is that mass volume ratio is 2% Osmitrol, and vacuum freeze drying is stored refrigerated.
Advantages of the present invention:
Edwardsiella tarda microballoon oral vaccine of the invention, the partial size of microballoon are 4.64 μm -28.38 μm, average grain
16.51 μm of diameter, partial size is smaller, the clad ratio of the inactivation Edwardsiella tarda of calcium alginate pair in 91.04%-96.5%,
Microballoon is stablized in the gastric juice of simulation, discharges in intestinal juice, shows good slow release；The use of the microspheres vaccine can be shown
The phagocytic activity and phagocytic index of the serum titer and macrophage that improve fish body are write, highest immune protective rate is up to 77.33%.
The oral vaccine effectively can activate fish body to be immunoreacted, and prevent fish disease.Method and process of the invention is relatively simple, at
This is lower, is conducive to industrialized production.
Detailed description of the invention
Fig. 1 is Edwardsiella tarda microballoon oral vaccine form；
Fig. 2 is the release rate of Edwardsiella tarda microballoon oral vaccine under the conditions of simulated intestinal fluid；
Fig. 3 is Bastard halibut serum antibody titer variation diagram after oral Edwardsiella tarda microballoon oral vaccine；
Fig. 4 is Bastard halibut leukocytes phagocytic percentage variation diagram after oral Edwardsiella tarda microballoon oral vaccine；
Fig. 5 is Bastard halibut leukocytes phagocytic index variation figure after oral Edwardsiella tarda microballoon oral vaccine.
Specific embodiment
Combined with specific embodiments below, the present invention is further illustrated.
Embodiment 1
A kind of preparation method of Edwardsiella tarda microballoon oral vaccine, includes the following steps:
It (1) is 2 × 10 by the concentration of inactivation10Cfu/ml Edwardsiella tarda bacterium solution is added to isometric, mass concentration
To be uniformly mixed, obtaining mixed liquor in 3% sodium alginate aqueous solution.
(2) it is by volume the ratio of 1:2, the mixed liquor is added in soybean oil and obtains water oil mixture, is added
The emulsifier span 80 of water oil mixture volume 1%；Water emulsifier is obtained in 1200rpm stirring 15min；
(3) under 1200rpm stirring, by water emulsifier and isometric mass concentration 4%CaCl2Aqueous solution mixing,
1200rpm stirs 15min, and 800rpm continues to stir 1h, forms water-in-oil emulsion.
(4) 3000rpm is centrifuged 4min removal upper oil phase and water phase, by the water phase and 1 times of total volume of phase of oil with removal
Ratio is added ultrapure water and sediment is resuspended, and obtains suspension；
(5) freeze drying protectant is added in Xiang Suoshu suspension in the ratio of 1:1 by volume, and the freeze drying protectant is matter
Amount volume ratio is 2% Osmitrol, and vacuum freeze drying is stored refrigerated.
The clad ratio of calcium alginate is 96.5%.
The vaccine strain Edwardsiella tarda that various embodiments of the present invention use is from Tianjin seawater industrial culturing Bastard halibut
It separates and obtains in kidney, belong to the Edwardsiella (Edwardsiella) of enterobacteriaceae.
Edwardsiella tarda is Gram-negative brevibacterium, and amphitrichous can move, no pod membrane, amphimicrobian.4-10℃
It can grow, 25-32 DEG C of optimum growth temperature, 42 DEG C or more do not grow, and can grow in the range of saliferous 0-4%, can be
It is cultivated on nutrient agar.The bacterium causes disease, such as common eel, Bastard halibut in a variety of fish, and turbot etc. is given
Aquaculture causes huge loss.
This laboratory separates and saves, and is signed as Edwardsiella tarda (Edwardsiella tarda) GX-1 bacterium
Strain is preserved in China typical culture collection center (CCTCC) and survives, and deposit number is CCTCC NO:M2018379, when preservation
Between be on June 19th, 2018.Address is the Wuhan Wuhan University, China postcode 430072.
It is all the Edwardsiella tarda of CCTCC NO:M2018379 used by various embodiments of the present invention
(Edwardsiella tarda)GX-1。
Inactivate the preparation of full bacterium bacterium solution
The Edwardsiella tarda bacterial strain taking-up that 80 DEG C of ﹣ save is immediately placed in 37 DEG C of water-baths, battalion is inoculated in after defrosting
Agar plate is supported, 28 DEG C of cultures for 24 hours, add sterile saline to rinse lawn and bacteria suspension is made.It is inoculated in nutrient broth, shaking table
It cultivates for 24 hours, 28 DEG C, 150rpm.0.5% (v:v) formalin is added, for 24 hours, nutrient agar panel is detected without viable bacteria for 4 DEG C of inactivations,
It is centrifuged (5000rpm, 5min) to collect, sterile saline is resuspended, and is collected by centrifugation, is repeated 3 times.Adjust bacterial concentration to 1~3 ×
1010Cfu/ml, 4 DEG C save backup.
Embodiment 2
A kind of preparation method of Edwardsiella tarda microballoon oral vaccine, includes the following steps:
It (1) is 1 × 10 by the concentration of inactivation10Cfu/ml Edwardsiella tarda bacterium solution is added to isometric, mass concentration
To be uniformly mixed, obtaining mixed liquor in 3% sodium alginate aqueous solution.
(2) it is by volume the ratio of 1:2, the mixed liquor is added in peanut oil and obtains water oil mixture, is added
The emulsifier span 80 of water oil mixture volume 1%；Water emulsifier is obtained in 1200rpm stirring 15min；
(3), (3), (4) and (5) of (4) and (5) with embodiment 1.
A kind of Edwardsiella tarda microballoon oral vaccine that the present embodiment obtains, immune effect and embodiment 1 obtain
A kind of Edwardsiella tarda microballoon oral vaccine immune effect it is similar.
The clad ratio of calcium alginate is 93.22%
Embodiment 3
A kind of preparation method of Edwardsiella tarda microballoon oral vaccine, includes the following steps:
It (1) is 3 × 10 by the concentration of inactivation10Cfu/ml Edwardsiella tarda bacterium solution is added to isometric, mass concentration
To be uniformly mixed, obtaining mixed liquor in 3% sodium alginate aqueous solution.
(2) by volume be 1:2 ratio, by the mixed liquor be added to vegetable oil (volume ratio be 1:1 corn oil and
Rapeseed oil) in obtain water oil mixture, the emulsifier span 80 of water oil mixture volume 1% is added；It is stirred in 1200rpm
15min obtains water emulsifier；
(3), (3), (4) and (5) of (4) and (5) with embodiment 1.
A kind of Edwardsiella tarda microballoon oral vaccine that the present embodiment obtains, immune effect and embodiment 1 obtain
A kind of Edwardsiella tarda microballoon oral vaccine immune effect it is similar.
The clad ratio of calcium alginate is 91.04%.
Embodiment 4
According to the orthogonal design scheme of Tables 1 and 2,5 factors, 4 levels are set up, using clad ratio as index, are designed orthogonal
Test.
A kind of optimum preparation condition of Edwardsiella tarda microballoon oral vaccine (hereinafter referred to as microspheres vaccine) can by table 2
See, can be seen that emulsifier concentration according to very poor size is to influence the vaccine clad ratio maximum factor, followed by mixing speed,
It is CaCl again2Concentration, followed by sodium alginate concentration are finally water-oil factors.
It is A2B1C1D1E2, i.e. sodium alginate concentration 3%, CaCl that analysis data, which obtain preferred plan,2Concentration 4%, water oil
Than 1:2, emulsifier concentration 1%, mixing speed 1200rpm.
Thus it establishes following optimal preparation process: seeing embodiment 1.
1 five factor of table, four horizontal quadrature designs table
The orthogonal design result of 2 Edwardsiella tarda microballoon oral vaccine preparation technology parameter of table
Embodiment 5
The usage and dosage of Edwardsiella tarda microballoon oral vaccine
The Edwardsiella tarda microballoon oral vaccine for taking 1mg embodiment 1 to prepare adds 100mL water, mixes, is uniformly sprayed onto
In the feed that 10 kilograms of Bastard halibuts are ingested daily, then 1 ‰ sodium alginate aqueous solution of 100mL mass concentration is sprayed, stirred evenly, yin
It is fed after dry.
Sooner or later it respectively feeds primary.After continuously feeding three days, then change throwing normal diet four days.Then it is repeated by the program immune
Twice.
Embodiment 6
The in-vitro evaluation of Edwardsiella tarda microballoon oral vaccine
The form of Edwardsiella tarda microballoon oral vaccine prepared by embodiment 1, size, clad ratio carry bacterium amount, manually
The main features such as gastric juice intestinal juice release characteristic are determined evaluation.
(1) morphological observation
A little microspheres vaccine is taken, using desk-top microscopically observation microballoon form, and acquires image.It will be seen from figure 1 that
Microballoon is substantially rounded, and partial size is relatively uniform.50 microballoon measurement partial sizes are randomly selected, average value is 16.51 μm.
(2) clad ratio measures
Take a certain amount of Edwardsiella tarda microspheres vaccine prepared, be added to solution cyst fluid (0.2M sodium bicarbonate,
0.06M sodium citrate, surplus are water) in, it 28 DEG C, is sampled after acting on 16h under the conditions of 150rpm, dyes, be filled into through DAPI
0.22 μm of black nuclear pore filter film counts bacterial number under the ultraviolet session of epifluorescence microscope.
Clad ratio=(bacterial population encapsulated in microballoon/total number of bacteria added when preparing microspheres vaccine) × 100%.
Edwardsiella tarda microballoon oral vaccine clad ratio according to the preparation of this scheme is 91.04%, and clad ratio is higher,
Antigen losses are few.
(3) simulated gastric fluid, intestinal juice simulate release test
The Edwardsiella tarda microballoon oral vaccine for taking a certain amount of embodiment 1 to prepare, is added separately to simulated gastric fluid
(2.0g sodium chloride and 3.2g pepsin, water 1000ml, salt acid for adjusting pH to 1.2) and simulated intestinal fluid (trypsase 10.0g,
Potassium dihydrogen phosphate 6.8g, water 1000ml, sodium hydroxide adjust pH in 7.5).28 DEG C, after 150rpm acts on 2h, 4h and 16h,
DAPI is dyed, and bacterial number is counted under the ultraviolet session of epifluorescence microscope, calculates the percentage of coating bacterium.The results show that
After acting on 16h in simulated gastric fluid, the disintegration rate of Edwardsiella tarda microspheres vaccine is only 0.8%, and in simulated intestinal fluid,
After acting on 2h, disintegration rate reaches when the disintegration rate when disintegration rate of microspheres vaccine reaches 78.5%, 4h reaches 96.2%, 16h
98.1%.(see Fig. 2)
Embodiment 7
Edwardsiella tarda microballoon oral vaccine immunoprotective effec
Using the Bastard halibut of a length of 10-15cm of body as animal pattern, raise in the glass jar of 100L.It is converted originally with brine
Water prepares seawater, salinity 26 ‰, water temperature 16 ± 0.5-18 ± 0.5 DEG C, for 24 hours recirculated water and aeration.It temporarily supports and opens after a week in laboratory
Begin to test.
(1) immunization ways
In order to guarantee that every tail fish obtains the slow Edwardsiella vaccine of equivalent, this test fills embodiment 1 using mouth and makes
The mode of standby microballoon oral vaccine is immunized.Test setting vaccine group and control group, every group of 20 tail fishes, each group setting
One parallel, and every daily mouth of tail fish fills 0.5ml Edwardsiella tarda microspheres vaccine, wherein including full bacterium about 109Cfu, control
Group mouth fill without coating Edwardsiella tarda empty microballoon (in embodiment 1, water consumption substitution step (1) obtain mixed liquor,
Its step (2), (3), (4), (5) are obtained with (2) of embodiment 1, (3), (4), (5)).Continuous mouth fills three days, stops four days
Afterwards, then by identical program it repeats immune primary.Commercialization feed is normally fed during entire experiment.
(2) serum antibody titer measures
Blood is taken within the 14th, 21,28 day after initial immunity, serum antibody titer is measured.After the completion of being immunized twice, blood is taken.It is anti-
The measuring method of body potency is micro-agglutination method.On 96 orifice plates, 100 μ l sterile salines of every hole addition, the 1 of each sample
Number hole adds the supernatant of 100 μ l samples to be tested, takes turns doing twice of gradient dilution to No. 9 holes.No. 10 holes are set as blank control.So
Each hole adds 100 μ l antigens afterwards, and antigen is the Edwardsiella tarda of inactivation, and concentration is 3 × 108CFU/ml.Each hole mixes
37 DEG C of incubation 18-24h afterwards, microscopically observation read antibody titer.As seen from Figure 3, the 14th, 21,28 day serum after being immunized
Antibody titer oral vaccine group is above control group, and after illustrating that oral vaccine is immune, fish body produces the immune response of specificity.
(3) phagocyte phagocytic activity measures
It takes blood within the 14th, 21,28 day after initial immunity, measures the phagocytic activity of phagocyte.After the completion of being immunized twice, often
Tail fish takes 200 μ l of blood from tail portion again with the 1ml disposable syringe of the heparin of >=6 μ l (30U can anticoagulant 1ml blood) solution-wet,
It is put into the 1.5ml centrifuge tube for having added 199 culture solution of 200 μ l sterile salines and 200 μ l in advance, mixes gently, be used for blood
The analysis of cell phagocytic activity.In the 1.5ml centrifuge tube for taking 200 μ l to one of anticoagulation new, it is 1.0 that 100 μ l concentration, which are then added,
×108The staphylococcus aureus suspension of cfu/ml inactivation, mixes, is placed in 28 ° of water-baths and is incubated for 30min, the 10th, 20min
It shakes up respectively primary；Blood-bacterium mixed liquor the smear being incubated for is taken, blood plasma is drawn with liquid-transfering gun (pipette tips point is cut) and red blood cell is handed over
The liquid layer 0.05ml that place is rich in leucocyte is met, each sample smear 3 is opened.30s, then plus Liu Shi B liquid dyeing are dyed with Liu Shi A liquid
90-120s is washed, dry, microscopy.Red blood cell takes on a red color, and center is slightly weaker and slightly butterfly, leucocytes system clearly easily divides, carefully
After birth is clearly in black, the nucleus coloring aubergine different in the depth, endochylema light red, and the granulomere in endochylema is shown clearly；
The number of the number of cells (percentage phagocytosis PP) that phagocytosis is participated in 100 cells and each cell phagocytosis bacterium is recorded respectively
(phagocytic index PI).
Percentage phagocytosis (PP)=(cell number/100 of phagocytosis are participated in 100 cells) × 100%；
Bacterial population/phagocytosis bacterium the cell number for phagocytic index (PI)=swallowed.
Haemocyte phagocytosis is the important component of body immune system, can directly swallow and kill the micro- life of a variety of cause of diseases
Object.From fig. 4, it can be seen that the haemocyte phagocytic rate of oral vaccine group is higher than control group, show non-specificity occur after mouth fills vaccine
Immune response.As seen from Figure 5, haemocyte phagocytic index, the 14th day oral vaccine group is slightly below control group after just exempting from, and the
It is above control group within 21 days and the 28th day.
(4) immune protective rate measures
The 28th day development challenge test after initial immunity, every tail fish intraperitoneal injection 0.1ml concentration is 1 × 107cfu/
The Edwardsiella tarda of ml, observes and records death condition daily.
By formula immune protective rate (RPS)=[(the control group death rate ﹣ immune group death rate)/control group death rate] ×
100% calculating adaptive immune protective rate is 77.33%.
Develop it is a kind of in fish digestive system with the microballoon antigen delivery systems of enteric slow release performance, and by the microballoon
Antigen delivery systems are applied to Edwardsiella tarda, develop Edwardsiella tarda microballoon oral vaccine, and the vaccine is available
In the immunoprophylaxis of sea-farming flounder flounder class disease as caused by Edwardsiella tarda.
Present patent application obtains Tianjin science and technology supporting project project (14ZCZDNC00007), Tianjin fishery industry skill
Art system innovation team (ITTFRS2017007), the subsidy of state natural sciences fund general project (31472299).
Claims (10)
1. a kind of Edwardsiella tarda microballoon oral vaccine, it is characterized in that inactivation Edwardsiella tarda is coated with calcium alginate
Obtained microballoon.
2. vaccine according to claim 1, it is characterized in that the Edwardsiella tarda is Edwardsiella tarda
(Edwardsiella tarda) GX-1, deposit number are CCTCC NO:M2018379.
3. vaccine according to claim 1 or 2, it is characterized in that the clad ratio of calcium alginate is 91.04%-96.5%.
4. the preparation method of a kind of Edwardsiella tarda microballoon oral vaccine of claim 1, it is characterized in that including following step
It is rapid:
(1) the Edwardsiella tarda bacterium solution of inactivation is added in sodium alginate aqueous solution, is uniformly mixed, obtains mixed liquor；
(2) mixed liquor is added in vegetable oil and obtains water oil mixture, emulsifier is added；Stir to get the emulsification of water oil
Agent；
(3) under stirring, by water emulsifier and CaCl2Aqueous solution mixing, stirring form water-in-oil emulsion；
(4) it is centrifuged, removes upper oil phase and water phase, ultrapure water is resuspended sediment, obtains suspension；
(5) freeze drying protectant is added, vacuum freeze drying is stored refrigerated.
5. according to the method described in claim 4, it is characterized in that the step (1) are as follows: by the concentration of inactivation be 1~3 ×
1010It is in 3% sodium alginate aqueous solution that cfu/ml Edwardsiella tarda bacterium solution, which is added to isometric, mass concentration, and mixing is equal
It is even, obtain mixed liquor.
6. method according to claim 4 or 5, it is characterized in that the Edwardsiella tarda is Edwardsiella tarda
(Edwardsiella tarda) GX-1, deposit number are CCTCC NO:M2018379.
7. according to the method described in claim 4, it is characterized in that the step (2) are as follows: be by volume the ratio of 1:2, by institute
It states mixed liquor and is added in vegetable oil and obtain water oil mixture, the emulsifier span 80 of water oil mixture volume 1% is added；?
1200rpm stirring 15min obtains water emulsifier；The vegetable oil is soybean oil, corn oil, in peanut oil and rapeseed oil at least
It is a kind of.
8. according to the method described in claim 4, it is characterized in that the step (3) are as follows: under 1200rpm stirring, by water oil cream
Agent and isometric mass concentration 4%CaCl2Aqueous solution mixing, 1200rpm stir 15min, and 800rpm continues to stir 1h, shape
At water-in-oil emulsion.
9. according to the method described in claim 4, it is characterized in that the step (4) are as follows: 3000rpm is centrifuged 4min, removes upper layer
Oily phase and water phase are added ultrapure water in the ratio of water phase and oily 1 times of total volume of phase with removal and sediment are resuspended, obtain suspension.
10. according to the method described in claim 4, it is characterized in that the step (5) is the ratio of 1:1 by volume, Xiang Suoshu
Freeze drying protectant is added in suspension, the freeze drying protectant is that mass volume ratio is 2% Osmitrol, and vacuum refrigeration is dry
It is dry, it is stored refrigerated.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115074288A (en) * | 2022-07-04 | 2022-09-20 | 福建益昕葆生物制药有限公司 | Anti-diarrhea egg yolk antibody coated lactobacillus preparation, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178942A (en) * | 2011-04-02 | 2011-09-14 | 中国科学院海洋研究所 | High-efficiency non-injection type transmission method for live vaccine |
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
| CN106620683A (en) * | 2015-10-29 | 2017-05-10 | 四川农业大学 | Bicombinant oral microsphere vaccine used for fish and purpose thereof |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178942A (en) * | 2011-04-02 | 2011-09-14 | 中国科学院海洋研究所 | High-efficiency non-injection type transmission method for live vaccine |
| CN106620683A (en) * | 2015-10-29 | 2017-05-10 | 四川农业大学 | Bicombinant oral microsphere vaccine used for fish and purpose thereof |
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
Non-Patent Citations (2)
| Title |
|---|
| SHUANG CHENG等: "Analysis of the vaccine potential of a natural avirulent Edwardsiella tarda isolate", 《VACCINE》 * |
| 魏俊利等: "鱼类迟缓爱德华氏菌肠溶口服疫苗的制备及免疫保护效应研究", 《中国水产学会.2017年中国水产学会学术年会论文摘要集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115074288A (en) * | 2022-07-04 | 2022-09-20 | 福建益昕葆生物制药有限公司 | Anti-diarrhea egg yolk antibody coated lactobacillus preparation, preparation method and application thereof |
| CN115074288B (en) * | 2022-07-04 | 2024-05-14 | 福建益昕葆生物制药有限公司 | Anti-diarrhea egg yolk antibody coated lactobacillus preparation and preparation method and application thereof |
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