CN105920593A - Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product - Google Patents
Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product Download PDFInfo
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Abstract
The invention relates to an edwardsiella tarda subunit oral microencapsule vaccine for an aquatic product. The edwardsiella tarda subunit oral microencapsule vaccine comprises a calcium alginate-chitosan release-controlled microcapsule and three recombinant protein-lipopolysaccharide conjugates which are encapsulated in the microcapsule, wherein the three recombinant protein-lipopolysaccharide conjugates include edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenases, outer membrane protein and flagellin which are mixed with astragalus polysaccharide. The oral microencapsule is characterized in that the encapsulation efficiency is 78%, the protein load is 102mg/g, and the grain size is 150+/-10 microns. The prepared oral microencapsule vaccine is convenient to use, proper in grain size, high in slow release performance and high in antigen protection performance, and can effectively irritate a fish intestinal immune system to perform immune response so as to enable a fish to gain the specific immunity protection capacity for edwardsiella tarda; and as a result, the edwardsiella tarda subunit oral microencapsule vaccine is applicable to prevention and treatment of fish edwardsiella tarda disease.
Description
Technical field
The invention belongs to aquaculture technical field of vaccines, be specifically related to a kind of Aquatic product Edwardsiella tarda
Subunit oral microencapsule vaccine.
Background technology
Recently as the fast development of culture fishery, aquatic animal disease problem also becomes increasingly conspicuous,
Wherein bacterial disease the most worldwide causes huge loss to fishery economic.Blunt Edward
Salmonella (Edwardsiella tarda) is the pathogenic bacterium that a kind of aquatic animal is important, can infect include light
Water and the multiple Fish of sea-farming, and mortality can be caused in a short time, to China's culture fishery
Bring tremendous economic loss.The use of vaccine can improve cultured fishes specific immunity level, makes fish body
Produce the problems such as the immunity that opposing specific pathogen microorganism is infected, free from environmental pollution, drug residue free,
It it is the mainstream development direction of fish disease prevention and control from now on.Current aquaculture vaccines both domestic and external mostly with injection or
It is main that dipping bath mode is inoculated, the rare report of oral immunity.Large-scale intensive culture pattern uses note
The mode vaccination of penetrating needs to expend substantial amounts of manpower and time, and fish body is often made by injection inoculation simultaneously
Upper physiological damage, and small dimension fish body is difficult to carry out by injection inoculation.Then grasped by oral immunization
Make simplicity, safe and practical, do not limited by fish body size, and the usage amount of vaccine is than dipping bath immunity more
Save, the most there is not the potential pollution problem caused to water body because of dipping bath immunity simultaneously.Therefore, oral epidemic disease
The development of Seedling and application have been increasingly becoming the focus of current vaccines for fish research.
Summary of the invention
It is an object of the invention to exploitation one keep relative stability in fish body digestive system, particle diameter closes
Suitable, there is slow-releasing and Protection of antigen, and immune answering can be produced by effective stimulus fish body intestinal tract immune system
Answer, make the paired Edwardsiella tarda of the fish bodily form have the oral microcapsule epidemic disease of specific immunity protection
Seedling.
Present invention firstly provides a kind of vaccine, wherein antigen protein includes aminoacid sequence is SEQ ID
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of NO:1, aminoacid sequence are outside SEQ ID NO:2
Memebrane protein (ompW) and aminoacid sequence are SEQ ID NO:3 flagellin (fliC)；
Wherein flagellin (fliC) all or part of aminoacid sequence is the flagellum of SEQ ID NO:4
Albumen (fliC) replaces；
Above-mentioned albumen lipopolysaccharide has carried out coupling；
Described lipopolysaccharide is from the Edwardsiella tarda that deposit number is CGMCC No.11270
ET-1008 bacterial strain separates；Edwardsiella tarda (Edwardsiella tarda) ET-1008 strain,
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 24th, 2015
(being positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.11270.
Another aspect of the present invention provides a kind of microcapsule vaccine；It is with yellow by the albumen of 3 kinds of LPS couplings
Astragalus polysaccharides mixes with 3:1 (W/W) ratio, is then mixed with sodium alginate by antigen mixture, with steaming
Distilled water is configured to emulsion, wherein sodium alginate final concentration of 2%, wraps under the stirring action of vortex mixer
Bury 30min；This emulsion is utilized atomizing orifice coagulating bath method, is injected directly into through vortex mixer agitation
Containing 3%CaCl2Solution in, formed calcium alginate microcapsule, after spraying continue stirring 20min；
Centrifugal collection calcium alginate microcapsule, is then distributed to microcapsule in the chitosan solution of 0.2%, fills
Divide stirring 30min, be centrifuged and collect Ah-ACMS microcapsule, distilled water wash 3 times, then will receipts
The microcapsule of collection is dispersed in the mannitol solution of 8%, freezing after mixing, freeze-dried after, capping
Packaging, is microcapsule vaccine.
The oral microencapsule vaccine of the present invention can be used as feed additive and uses.
There is advantages below in the present invention:
1) processing technology is simple, and material therefor is natural macromolecular material, and wide material sources are cheap,
And have good immunoenhancement result.
2) easy to operate, good immune effect.Oral immunity stress be little to the fish body that individuality is less, and manpower becomes
This is low, it is simple to large-scale promotion is applied.This vaccine free from extraneous odour, good palatability, fish body is easily searched for food.
3) oral microcapsule shape is ideal, and size is suitable, can show preferable slow-releasing,
Immunogen can be protected again to suffer, and fish body gastrointestinal destroys.In microcapsule, add astragalus polysaccharides simultaneously,
The non-specific immunity of fish body can be strengthened.
4) storing conveniently, this vaccine preserves the immunity that can keep good in more than 8 months at ambient temperature
Originality.
Accompanying drawing explanation
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Fig. 1: Edwardsiella tarda bacterial strain ET-1008,
The purified product electrophoretogram of the RT-PCR expressing protein of outer membrane protein ompW and flagellin fliC；
The apparent characteristic figure (× 200) of Fig. 2: Edwardsiella tarda subunit oral microencapsule vaccine；
Fig. 3: the Edwardsiella tarda subunit oral microencapsule vaccine immune protective effect figure to Paralichthys olivaceus；
The fliC albumen of Fig. 4: the present invention and the fliC that aminoacid sequence is SEQ ID NO:3 in NCBI
The gene comparision figure of gene.
Detailed description of the invention
The present invention is described in detail with detailed description of the invention below in conjunction with the accompanying drawings.
Embodiment 1: the separation of Edwardsiella tarda bacterial strain and virulence property analysis thereof
(1) ill Paralichthys olivaceus is taken from Shandong Huang Island and is breeded fish factory, and sick fish average weight be (200 ± 5) g, puts down
All body length (20 ± 5) cm, cardinal symptom is that ascites, anus redness be prominent, liver and kidney enlargement.
(2) the typical dying sick fish of ascites symptom is taken, from liver, kidney, ascites etc. under aseptic condition
Affected area picking portion of tissue, lines on General nutrition agar plate, cultivates 24h, picking for 28 DEG C
The dominant colony that form is consistent is purified cultivation, until obtaining pure culture bacterium.Ordinary nutrient agar is cultivated
Based formulas is: yeast extract 3g, peptone 10g, sodium chloride 15g, distilled water 1L, pH 7.6-7.8,
Agar 20g.
(3) separation is obtained each bacterial strain pure culture, General nutrition agar plate is enlarged training
Support, after 28 DEG C are cultivated 24h, with 0.9% physiological saline solution eluting bacterium colony, it is thus achieved that bacteria suspension, utilize
The bacterial concentration of each bacterial strain is set to 1 × 10 by turbidimetry6CFU/mL, for infectable infection.
(4) 1 group of every 20 tail of experiment healthy Paralichthys olivaceus, size specification is identical with ill Paralichthys olivaceus, and sets life
Reason saline control group.Temperature of cultivation is 20 ± DEG C, whole day is inflated, and changes water 50% every day.After supporting 7d temporarily,
The bacteria suspension of lumbar injection 0.2mL different strains, 0.9% sterile physiological salt of matched group injection same volume
Water.Regularly observed and recorded, the focus and the internal organs that take dying fish carry out antibacterial and separate, are separated excellent
Gesture bacterial strain infects Paralichthys olivaceus, observed result again.
(5) infection experiment result shows, only bacterial strain ET-1008 demonstrates have healthy Paralichthys olivaceus relatively
High virulence, can cause 90% Paralichthys olivaceus dead, and the symptom identical with ill Paralichthys olivaceus occurs in dead Paralichthys olivaceus.
Therefore, it was demonstrated that bacterial strain ET-1008 is the cause of disease of Paralichthys olivaceus ascites symptoms.And can in infecting ill Paralichthys olivaceus body
Again it is separated to this bacterial strain.
(6) contrast experiment shows, with the Edwardsiella tarda vaccine sold in the market to Paralichthys olivaceus
After seedling carries out immunity, then carry out challenge viral dosage with the ET-1008 strain of present invention screening；Result shows mesh
The immune effect of the vaccine sold on front market is far below the inactivated vaccine made with ET-1008 strain of the present invention
Effect.It is presumably due to certain Disease-causing gene of ET-1008 strain morph and cause exempting from of current vaccine
Epidemic disease effect is bad.
(7) customary physiological biochemical identification and 6SrRNA and HSP60 gene sequencing is utilized to show,
Bacterial strain ET-1008 is Edwardsiella tarda (Edwardsiella tarda), and by this bacterial strain in 2015
On August 24, in is deposited in the China Microbiological bacterium being positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Planting preservation administration committee common micro-organisms center, deposit number is: CGMCC No.11270.
Embodiment 2: the GAPDH of Edwardsiella tarda bacterial strain ET-1008, outer membrane protein ompW
And the RT-PCR expression of flagellin fliC
(1) sequence amplification of purpose expressing protein: according to Genbank blunt Ai Dehuashi GAPDH,
The gene order of ompW and fliC, separately designs its specific primer (being shown in Table 1).Utilize bacterial gene
Group extraction agent box, to extract bacterial genomes DNA as template, is respectively cooperating with the forward and reverse of each gene
Primer carries out PCR amplification, PCR reaction system and reaction condition: 50 μ l reaction systems, comprises 37
μl H2O, 5 μ l 10 × Taq buffer, 4 μ l dNTP (2.5mM), each 1 μ l of positive anti-primer, 1 μ l Taq
DNA Polymerase and 1 μ l DNA profiling；PCR response procedures is 94 DEG C of degeneration 10min；94℃
Degeneration, 1min, 50 DEG C of annealing 1min, 72 DEG C extend 1min, carry out 30 circulations, last 72 DEG C
Extend 8min.PCR primer detects through 1% agarose gel electrophoresis, and gel imaging system is taken pictures observation,
Result display GAPDH, ompW and fliC gene amplification success, and amplified production size and purpose base
Because of consistent (Fig. 1).
Table 1 Edwardsiella tarda flagellin flic, the primer of outer membrane protein ompW and GAPDH
To amplified production reclaim after check order, result shows, it is thus achieved that glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) aminoacid sequence is SEQ ID NO:1, the aminoacid sequence of outer membrane protein (ompW)
It is classified as SEQ ID NO:2；NCBI comparison result shows that above two albumen is published with NCBI
Sequence, does not change always.But the sequence of fliC gene exists with published sequence in NCBI
Significantly difference；The present invention obtain the fliC albumen that aminoacid sequence is SEQ ID NO:4 compared to
Aminoacid sequence in NCBI is the district of fliC gene more than 10 amino of existence of SEQ ID NO:3
Not (Fig. 4).
And immune effect shows, the vaccine made with the fliC albumen that aminoacid sequence is SEQ ID NO:4
The immune effect of ET-1008 strain is significantly better than the fliC albumen system that aminoacid sequence is SEQ ID NO:3
The vaccine become.
(2) structure of GAPDH, ompW and fliC expression vector: by the blunt love moral of amplification
Fahrenheit bacterium GAPDH, ompW and fliC gene outcome is cut glue recovery and is purified, purified product warp
After BamH I and Xhol I double digestion, it is connected to prokaryotic expression carrier pET32a, builds it respectively and express
Carrier pET32a-GAPDH, pET32a-ompW and pET32a-fliC, and expression vector is converted extremely
E. coli bl21 (DE3), cultivates on the LB flat board containing ampicillin, after growing bacterium colony,
Utilizing PCR method screening positive clone to express bacterium, bacterial strain PCR test positive cloned is further
Order-checking is utilized to confirm.
(3) expression and purification of recombiant protein: after insertion sequence is correct in order-checking confirms carrier, will
Edwardsiella tarda GAPDH, ompW and the fliC positive colony that screen are expressed bacterium and are inoculated respectively
In the LB culture medium containing ampicillin, when shaking table being cultivated to exponential phase of growth, add the denseest
Degree is the IPTG abduction delivering 6h of 1mM, centrifugal collects thalline, after ultrasonication, with Ni from
Recombiant protein is purified by the affinity column (HisTrap HP 1ml, GE) of son chelating, and eluting produces
Thing, after urea concentration gradients is dialysed, is finally dialysed with PBS, is carried out cold to sample after having dialysed
Lyophilizing is dry with concentrating sample, the recombiant protein of SDS-PAGE detection purification.Edwardsiella tarda fliC
Recombiant protein molecular weight is 63kDa, this genes of interest 416 aminoacid of coding, the protein fragments of expression
Theoretical molecular is 43kDa, and the label protein size of plasmid pET32a is then about about 20kDa, knot
Fruit is consistent with theoretical size；GAPDH recombiant protein molecular weight is 55kDa, and this genes of interest encodes
331 aminoacid, the protein fragments theoretical molecular of expression is 35kDa, the label of plasmid pET32a
Albumen size is then about about 20kDa, and result is consistent with theoretical size；OmpW recombiant protein molecule
Amount is 43kDa, and this genes of interest 214 aminoacid of coding, the protein fragments theoretical molecular of expression is
The label protein size of 23kDa, plasmid pET32a is then about about 20kDa, result and theoretical size
It is consistent.
Embodiment 3: the extracting of Edwardsiella tarda lipopolysaccharide (LPS)
(1) yeast culture: the ET-1008 bacterium colony preserved on a small quantity with aseptic inoculation ring picking lines general
Logical nutrient agar panel, washes down well-grown lawn with fluid medium, transfers and trains into equipped with 40ml
Support in the 100ml conical flask of base, 28 DEG C of shaken cultivation overnight (160rpm).In 10ml centrifuge tube
Adding 7ml bacterium solution, under the conditions of 4 DEG C, 5000r/min is centrifuged 10min and collects antibacterial, washes with normal saline
Wash 3 times, with 7ml 50mmol/L phosphate buffer (pH 7.0, includes 5mmol/L EDTA, 20
mmol/L MgCl2) making bacterial suspension, concentration is 1 × 108CFU/ml。
(2) hot phenol-water method prepares LPS: carry out ultrasonic disruption under condition of ice bath, set the time as
5min, wherein 4s opens, and 4s closes；Bacterium solution is transferred in 250ml beaker, add lysozyme to the denseest
Degree is 200 μ g/ml, and 37 DEG C of stirring incubation 1h, 4 DEG C are stirred overnight；Add E.C. 3.4.21.64 to final concentration
Being 100 μ g/ml, 1h is hatched in 65 DEG C of stirrings, adds RNase and DNase to final concentration of 40
μ g/ml, 37 DEG C of incubation 1h, be placed in the water-bath of 65-70 DEG C.After balance, add the pre-of same volume
90% phenol of heat to 65-70 DEG C, limit edged is stirred vigorously 30min, quickly cools down 15min with ice-water bath,
Being transferred in 1.5ml centrifuge tube, 4 DEG C 10,000r/min is centrifuged 15min.Discard phenol phase, take upper water
To dialyzer, stand dialysis 3d with distilled water, change water every day 3 times.Sodium acetate is added in aqueous phase
To final concentration of 0.5M and 95% ethanol of 10 times of volumes ,-20 DEG C of overnight precipitation LPS, 2000g 4 DEG C
Centrifugal 10min, 1ml distilled water is resuspended, and lyophilization obtains high-purity lipopolysaccharide dry powder and is used for and albumen
Coupling.
Embodiment 4: Edwardsiella tarda recombinant expression protein and the coupling of LPS
(1) recombiant protein and the coupling of LPS: 110mg lipopolysaccharide dry powder is dissolved in 10ml distillation
In water, in this LPS aqueous solution, add 400mg solid NaIO4, mixing is hatched 2min, is then added
Enter 220 μ l ethanolamine and terminate oxidation reaction, product is added in the bag filter that aperture is 3K size,
Being placed in the PBS of 0.01mol/L, dialysed overnight under the conditions of 4 DEG C, period changes twice dialysis solution.With
Time, weigh 390mg carbodiimides (EDC), be dissolved in 5ml distilled water, be subsequently adding 90mg
GAPDH recombiant protein after purification, utilizes 1M HCl reaction system pH to be adjusted to 5.4, room temperature
2h is hatched in concussion.The LPS after oxidation reaction and the restructuring GAPDH albumen containing EDC is completed by above-mentioned
Dissolve mixing, under the conditions of 22 DEG C, hatch 1h, finally place reaction liquid in PBS and dialysed
At night, period changes dialysis solution, finally by the solution after dialysis centrifugal 30min, gained under 8000g
Supernatant is the rough thing of coupling of GAPDH Yu LPS.FliC and ompW is as the coupling of LPS
Carry out with reference to above method.
(2) purification of recombiant protein-LPS conjugate: the rough thing of coupling of each recombiant protein, utilizes
The isolated and purified each recombiant protein-LPS conjugate of Sephacryl S-300 gel filtration chromatography.Particularly as follows: first
By crude coupled product with the membrane filtration that aperture is 0.22 μm, then toward column volume be 120mL's
The rough thing of loading 10ml coupling in Sephacryl S-300 chromatographic column, using PBS as balance with
Elution buffer, is uniformly 2ml/min by flow speed control, collects sample according to uv absorption ripple, according to
Molecular sieve principle, first the coupled product that molecular weight is bigger will separate acquisition.The coupling collected is produced
Thing is dialysed with ultra-pure water, then carries out lyophilization, and-20 DEG C save backup.
Embodiment 5: the preparation of Edwardsiella tarda subunit oral microencapsule vaccine
(1) preparation of calcium alginate-chitosan microcapsule: by GAPDH, ompW of above-mentioned preparation
And after 3 kinds of recombiant protein-LPS coupled products of fliC press 3:3:1 (W/W) mixing, then with total amount be
The ratio of 3:1 (W/W) mixes with astragalus polysaccharides, by antigen mixture with sodium alginate with 1:4 (W/W)
Ratio mixes, and is configured to sodium alginate-antigen emulsion with distilled water, and wherein sodium alginate is final concentration of
2%, under the stirring action of vortex mixer, embed 30min.This emulsion is utilized atomizing orifice coagulating bath
Method, is injected directly into the 3%CaCl through vortex mixer agitation2In solution, form calcium alginate microcapsule, milkiness
Liquid and CaCl2The volume ratio of solution is 1:1, continues stirring 20min after spraying.Centrifugal collection Sargassum
Acid calcium microcapsule, is then distributed to microcapsule in the chitosan solution of 0.2%, is sufficiently stirred for 30min,
Centrifugal Ah-ACMS microcapsule of collecting, distilled water wash 3 times and centrifugal after, then by collection
Microcapsule is dispersed in the mannitol solution of 8%, freezing after mixing, freeze-dried after, capping packaging,
It is Edwardsiella tarda subunit oral microencapsule vaccine of the present invention.Utilize microscope to system
Standby microcapsule carries out apparent characteristic observation, and result is as shown in Figure 2, it is seen that microcapsule granule size is uniform,
Average diameter is 150 ± 10 μm.
(2) oral microencapsulated rate and the mensuration of protein load: measure alginic acid with Brad-ford method
After the solidification of calcium microcapsule, CaCl2Protein concentration in solution, then according to CaCl2The stereometer of solution
Calculate the protein content in the recombiant protein-LPS coupled product of loss in solution.According to formula: envelop rate %=
Total amount-the CaCl of coupling protein2The total amount of the content/coupling protein of the coupling protein of loss in solution
× 100%, calculate the envelop rate of microcapsule；According to formula: the total amount × encapsulating of protein load=coupling protein
Rate/microcapsule dry weight, calculates the protein load of microcapsule.Final result shows, prepared oral micro-glue
Capsule envelop rate is 78%, and protein load is 102mg/g.
Embodiment 6: the immunoprotection of Paralichthys olivaceus is imitated by Edwardsiella tarda subunit oral microencapsule vaccine
Really
Preparing healthy Paralichthys olivaceus totally 80 tail, average weight is 50g ± 5g, utilizes controllable temperature recirculation system
Supporting Paralichthys olivaceus temporarily 7 days, it is 21 DEG C that water temperature controls, and period normally throws something and feeds commercialization pellet.Experiment
Before, fish is equally divided into 2 groups, often organizes 40 tails, after to for examination fish body fasting 24h, to immune group
Fish body throw something and feed the present invention develop oral microencapsule vaccine, feeding method: by oral microencapsule vaccine and fish
Feedstuff mixing puts into water body, and for fish body free choice feeding, using dosage is: every kg body weight fish body is thrown in micro-
Capsule vaccine 0.5g, throws something and feeds 5 days continuously.Throw something and feed first latter 15 days, again carry out secondary immunity, use
Method and consumption are with the most identical.Matched group fish body is thrown something and fed with the sodium alginate without immunogen Yu astragalus polysaccharides
Microcapsule granule prepared by solution, feeding method is identical with immune group with feeding volume, after throwing something and feeding first 35
My god, utilizing normal saline Edwardsiella tarda viable bacteria suspension, concentration is 1 × 107CFU/ml, adopts
With muscle injection mode, experiment fish body being carried out counteracting toxic substances, every endnote penetrates 100 μ l.After counteracting toxic substances, Continuous Observation is also
Record fish body morbidity and death state, calculate Edwardsiella tarda subunit oral microencapsule vaccine to tooth
The relative immunity protective rate of Flounder.Result, as it is shown on figure 3, matched group fish body cumulative mortality is 95%, is exempted from
Epidemic disease group fish body cumulative mortality of 20 days after counteracting toxic substances is 17.5%, is learnt by calculating, and the present invention grinds
The relative immunity protective rate of Paralichthys olivaceus is by the Edwardsiella tarda subunit oral microencapsule vaccine of system
81.6%.
In sum, the Edwardsiella tarda subunit oral microencapsule vaccine that the present invention develops, use
Convenient, particle diameter is suitable, has good slow-releasing and Protection of antigen, and can effective stimulus fish body intestinal
Immune system produces immunne response, makes fish body obtain the specific immunity to Edwardsiella tarda and protects
Power, has good development prospect.
Claims (6)
1. an Edwardsiella tarda subunit vaccine, it is characterised in that the antigen protein of described vaccine
Include aminoacid sequence be the glyceraldehyde-3-phosphate dehydrogenase of SEQ ID NO:1, aminoacid sequence be SEQ
The outer membrane protein of ID NO:2 and aminoacid sequence are SEQ ID NO:3 flagellin.
2. subunit vaccine as claimed in claim 1, it is characterised in that described flagellin all or
Part aminoacid sequence is that the flagellin of SEQ ID NO:4 replaces.
3. subunit vaccine as claimed in claim 1 or 2, it is characterised in that described albumen fat is many
Sugar carries out coupling.
4. subunit vaccine as claimed in claim 3, it is characterised in that described lipopolysaccharide is from preservation
The Edwardsiella tarda ET-1008 bacterial strain of numbered CGMCC No.11270 separates.
5. a microcapsule vaccine, it is characterised in that described microcapsule vaccine is by claim 1 or 2
The described vaccine protein in subunit vaccine mixes with astragalus polysaccharides, is then mixed with sodium alginate by mixture
Conjunction is configured to emulsion, adds CaCl2Solution forms calcium alginate microcapsule, is then distributed to by microcapsule
In chitosan solution, after being sufficiently stirred for, centrifugal collection Ah-ACMS microcapsule, is dispersed in sweet after washing
In dew alcoholic solution, mixing postlyophilization makes microcapsule vaccine.
6. the microcapsule vaccine described in claim 5 is as the application of feed additive.
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| CN201610406736.8A CN105920593A (en) | 2016-06-08 | 2016-06-08 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
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| CN201610406736.8A CN105920593A (en) | 2016-06-08 | 2016-06-08 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
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Cited By (4)
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| CN106727715A (en) * | 2016-12-08 | 2017-05-31 | 重庆文洪水产品养殖有限公司 | Antibacterial modified cellulose aerogels base aquatic immune reinforcing agent and preparation method thereof |
| CN108285904A (en) * | 2018-02-09 | 2018-07-17 | 河北科技师范学院 | A kind of Wdwardsiella tarda flagellin FlgJ with immanoprotection action |
| CN108330143A (en) * | 2018-02-09 | 2018-07-27 | 河北科技师范学院 | A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action |
| CN109395070A (en) * | 2018-10-12 | 2019-03-01 | 天津市水生动物疫病预防控制中心 | A kind of Edwardsiella tarda microballoon oral vaccine and preparation method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106727715A (en) * | 2016-12-08 | 2017-05-31 | 重庆文洪水产品养殖有限公司 | Antibacterial modified cellulose aerogels base aquatic immune reinforcing agent and preparation method thereof |
| CN108285904A (en) * | 2018-02-09 | 2018-07-17 | 河北科技师范学院 | A kind of Wdwardsiella tarda flagellin FlgJ with immanoprotection action |
| CN108330143A (en) * | 2018-02-09 | 2018-07-27 | 河北科技师范学院 | A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action |
| CN108285904B (en) * | 2018-02-09 | 2021-07-06 | 河北科技师范学院 | Edwardsiella tarda flagellin FlgJ with immune protection effect |
| CN108330143B (en) * | 2018-02-09 | 2021-07-09 | 河北科技师范学院 | Edwardsiella tarda flagellin FliC with immune protection effect |
| CN109395070A (en) * | 2018-10-12 | 2019-03-01 | 天津市水生动物疫病预防控制中心 | A kind of Edwardsiella tarda microballoon oral vaccine and preparation method |
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