CN107858317A - A kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage - Google Patents
A kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage Download PDFInfo
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- CN107858317A CN107858317A CN201711138001.2A CN201711138001A CN107858317A CN 107858317 A CN107858317 A CN 107858317A CN 201711138001 A CN201711138001 A CN 201711138001A CN 107858317 A CN107858317 A CN 107858317A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Abstract
The invention discloses a kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage.The invention provides a kind of recombinant bacterium, is that the aerolysin gene in Aeromonas veronii is knocked out into what is obtained.The recombinant bacterium can be used for preparing aquatic livestock Aeromonas veronii vaccine and/or Evaluation of Aeromon As Hydrophila Vaccine and/or Aeromonas vaccine and/or bleeding disease vaccine.The oral or dipping bath attenuated live vaccine that the present invention knocks out crucial virulence factor technique construction using antibiotic-free mark can reduce the pathogenic of pathogen while challeng is retained; immune protective effect is notable; strengthen natural immune system reaction and cellular immunity; the infection ability of other pathogens is resisted in enhancing, while greatly reduces immunization amount.
Description
Technical field
The present invention relates to a kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage.
Background technology
China is aquaculture production country maximum in the world, accounts for more than the 60% of world aquaculture yield.With
Increasingly intensive and commercialization, outbreak of disease of the aquaculture during production practices have become the main of fish farming industry
Problem.Particularly fish hemorrhage caused by Aeromonas has protruded very much as one in culture fishery evolution
Problem.It is estimated that fish disease outburst causes the economy of about multi-million dollar in global culture fishery every year.For example,
The outburst of motile Aeromonas septicemia (MAS), typically results in the whole world as caused by Aeromonas (Aeromonas spp.)
The high mortality of aquaculture Mesichthyes simultaneously causes serious economic loss.In the past few decades, antibiotic is as aquatic products
Cultivate conventional measures of the improvement of Mesichthyes disease with controlling and promoting fish growth.However, the drug resistance problems of pathogen
And the residue problem of antibiotic has become the problem of global people are of interest in animal product, antibiotic is in culture fishery
In disabling it is imperative.At present in addition to using antibiotic, inactivated vaccine is the main of the hemorrhage caused by Aeromonas
Prevention method, but destruction of the inactivated vaccine to antigen is to causing immune response is unstable, lacks immunogenicity action time not hold
Long.Therefore exploitation cause of disease bacteria vaccine will be one of important nonreactive prevention and controls of preventing and treating Aeromonas hemorrhage.
At present the vaccine control of bacterial septicemia is caused mainly to concentrate on intestines due to failing to understand pathogenesis early stage
In development in terms of the bacterial infection Evaluation of Aeromon As Hydrophila Vaccine availed oneself of the opportunity to get in after road/gill barrier breakdown, and on destroying host
The exploitation report of the crucial bacterium Aeromonas veronii vaccine of physical barriers is seldom, mainly going out including traditional supper toxic strain
Live vaccine, it has further been reported that the Aeromonas veronii vaccine of Aeromonas veronii ghost and introducing SPECIFIC APTAMER peptide, and
Majority is immunized in a manner of injection, following defect be present：1) inactivation process, which causes partially or completely to lose protectiveness, resists
Original, unstable or low SI immune response can only be caused, or even correct immune response can not be excited and triggered secondary
Effect etc.；2) Aeromonas veronii ghost has that production yields is low, and lysis genes have the shortcomings that toxicity；3) SPECIFIC APTAMER peptide
Aeromonas veronii vaccine the shortcomings that itself virulence gene expression can not be avoided to cause a disease, 4) workload be present in fish injecting immune
The shortcomings that big.
The content of the invention
It is an object of the invention to provide a kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage.
Present invention firstly provides a kind of recombinant bacterium, is that the aerolysin gene in Aeromonas veronii is knocked out into what is obtained.
The aerolysin gene is the gene of coding gas lysin albumen.
The gas lysin albumen is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 6；
(2) by the amino acid sequence shown in sequence in sequence table 6 by one or several amino acid residues substitution and/or
Missing and/or addition and the protein as derived from sequence 6 with identical function.
The aerolysin gene is following (a1) or (a2) or (a3) or (a4)：
(a1) DNA molecular shown in the sequence 5 of sequence table；
(a2) DNA molecular of the code area as shown in the sequence 5 of sequence table；
(a3) albumen of DNA sequence dna hybridization and coding with identical function limited under strict conditions with (a1) or (a2)
The DNA molecular of matter；
(a4) with more than 90% homology and encoded with the DNA sequence dna that (a1) or (a2) or (a3) is limited with identical work(
The protein DNA molecule of energy.
It is described to knock out to knock out whole ORFs or knockout portion gene section.
It is described knockout concretely by the sequence 5 of sequence table in Aeromonas veronii genomic DNA from 5 ' end the 91st to
1381 knockouts.
The knockout is realized by homologous recombination.
The homologous recombination is to realize specific DNA fragment importing Aeromonas veronii；The specific DNA fragment includes
The upper homology arm of aerolysin gene and lower homology arm；The sequence 3 of the upper homology arm such as sequence table holds 3092-4276 positions from 5 '
Shown in nucleotides；The lower homology arm is as shown in the sequence 3 from 5 ' end 4277-5408 positions nucleotides of sequence table.It is described special
DNA fragmentation is as shown in the sequence 3 from 5 ' end 3092-5408 positions nucleotides of sequence table.
The homologous recombination is that the recombinant vector containing the specific DNA fragment is imported into Aeromonas veronii to realize；
Linearized plasmid vector, fragment first are connected to obtain by the recombinant vector with fragment second；The fragment first is such as sequence table
Shown in sequence 1；The fragment second is as shown in the sequence 2 of sequence table.The connection is realized by seamless clone.The plasmid
Carrier is plasmid pRE112.The recombinant vector is specially shown in the sequence 3 of sequence table.
The present invention also protects a kind of recombinant bacterium, is after specific DNA fragment is imported into Aeromonas veronii progress homologous recombination
The obtained recombinant bacterium with the specific DNA fragment；The specific DNA fragment includes the upper homology arm of aerolysin gene with
Homology arm；The upper homology arm is as shown in the sequence 3 from 5 ' end 3092-4276 positions nucleotides of sequence table；The lower homology arm
As shown in the sequence 3 from 5 ' end 4277-5408 positions nucleotides of sequence table.
The homologous recombination is that the recombinant vector containing the specific DNA fragment is imported into Aeromonas veronii to realize；
Linearized plasmid vector, fragment first are connected to obtain by the recombinant vector with fragment second；The fragment first is such as sequence table
Shown in sequence 1；The fragment second is as shown in the sequence 2 of sequence table.The connection is realized by seamless clone.The plasmid
Carrier is plasmid pRE112.The recombinant vector is specially shown in the sequence 3 of sequence table.
The concretely Aeromonas veronii A.veronii Hm091 of Aeromonas veronii described in any of the above.
Concretely Aeromonas veronii (Aeromonas veronii) the Hm091 △ aer of recombinant bacterium described in any of the above.
Aeromonas veronii (Aeromonas veronii) Hm091 △ aer, on October 09th, 2017 is preserved in
State Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC；Address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number is CGMCC No.14776.
The present invention also protects application of the recombinant bacterium in vaccine is prepared described in any of the above；The vaccine be following (b1)-
Any of (b4)：
(b1) aquatic livestock Aeromonas veronii vaccine；
(b2) aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine；
(b3) aquatic livestock Aeromonas vaccine；
(b4) aquatic livestock bleeding disease vaccine.
The present invention also protects a kind of method for preparing vaccine, comprises the following steps：Using recombinant bacterium described in any of the above as
The active component of vaccine is packed；The vaccine is any of following (b1)-(b4)：
(b1) aquatic livestock Aeromonas veronii vaccine；
(b2) aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine；
(b3) aquatic livestock Aeromonas vaccine；
(b4) aquatic livestock bleeding disease vaccine.
The present invention also protects application of the recombinant bacterium in product is prepared described in any of the above；The purposes of the product is increase
The immunity of aquatic livestock.
The present invention also protects application of the recombinant bacterium in product is prepared described in any of the above；The purposes of the product is as follows
(c1) at least one of-(c4)：
(c1) prevent and/or treat the infection of aquatic livestock Aeromonas veronii；
(c2) prevent and/or treat aquatic livestock infected by Aeromonas hydrophila；
(c3) prevent and/or treat aquatic livestock Aeromonas infection；
(c4) prevent and/or treat aquatic livestock hemorrhage.
The present invention also protects aquatic livestock Aeromonas veronii vaccine, and its active component is recombinant bacterium described in any of the above.
The present invention also protects aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine, and its active component is recombinant bacterium described in any of the above.
The present invention also protects aquatic livestock Aeromonas vaccine, and its active component is recombinant bacterium described in any of the above.
The present invention also protects aquatic livestock bleeding disease vaccine, and its active component is recombinant bacterium described in any of the above.
The present invention also protects a kind of product, and active component is recombinant bacterium described in any of the above；The purposes of the product is such as
Under at least one of (d1)-(d5)：
(d1) prevent and/or treat the infection of aquatic livestock Aeromonas veronii；
(d2) prevent and/or treat aquatic livestock infected by Aeromonas hydrophila；
(d3) prevent and/or treat aquatic livestock Aeromonas infection；
(d4) prevent and/or treat aquatic livestock hemorrhage；
(d5) immunity of aquatic livestock is increased.
The concretely oral or dipping bath of the occupation mode of vaccine described in any of the above.
The concretely aquatic animal feed additive of product described in any of the above.
Aeromonas described in any of the above concretely Aeromonas veronii or Aeromonas hydrophila.
The concretely Aeromonas veronii A.veronii Hm091 of Aeromonas veronii described in any of the above.
The concretely Aeromonas hydrophila A.hydrophila NJ-1 of Aeromonas hydrophila described in any of the above.
Aquatic livestock described in any of the above concretely zebra fish, grass carp, crucian carp, flathead, silver carp, mandarin fish, carp, black carp, Tilapia mossambica, eel
The fish such as eel, white crucian carp, pond crucian carp, sturgeon, yellow tail silver xenocypris, bream, channel catfish, goldfish, swamp eel, lefteye flounder or the frog, giant salamander, shrimp, crab, soft-shelled turtle,
The aquatic animals such as tortoise.
The present inventor has found Vickers gas to South China hemorrhage Burst mechanism research during 2009-2014
Monad is occupied an leading position, and the mixed infection of Aeromonas veronii and Aeromonas veronii and Aeromonas hydrophila is South China
The main reason for causing fish septicemia to break out, Aeromonas veronii is the precondition for causing mixed infection, is further passed through
Transposons knocks out screening and specify that the gas lysin (Aerolysin) of Aeromonas veronii II types excretory system secretion is key pathogenetic
The factor, during mixed infection, the gas lysin of Aeromonas veronii destroys the enteron aisle of host/gill barrier first, can first cause
Around fish head portion oral cavity and serious damage is caused at enteron aisle position, then can make Aeromonas veronii, Aeromonas hydrophila
And other bacteriums are entered in zebra fish body by damage location, the systemic intrusion to host, finally zebra fish is caused to lose
Mass formed by blood stasis occurs and death.
The present invention has advantages below：
1) the gas lysin of the invention based on Aeromonas veronii causes the damage of zebra fish infection site further to promote related
It is main cause this mechanism of causing a disease of disease outburst of causing bleeding that bacterium, which enters in fish body, from triggering the Aeromonas veronii that infects
The vaccine of preventing and treating hemorrhage is developed, is avoided after causing attention to cause because Aeromonas hemorrhage pathogenesis is not clear for a long time
Phase bacterial infection and the exploitation for ignoring the crucial bacterial vaccine for triggering damage, cause the vaccine of bacterial septicemia for a long time
This present situation of prevention effect difference；
2) present invention causes gene to inactivate using homologous recombination principle fixed point deletion specific gene fragment, makes acquisition
Gene deletion strains have preferable genetic stability, avoid using the labile gene engineering methods such as transposons structure attenuation bacterium
The risk for the Genomic instability that strain is brought.The attenuated live vaccine of the present invention marks without any antibiotic, in the absence of to environment
Discharge the potential hazard brought of resistant strain or antibiotics resistance gene；
3) gas lysin (Aerolysin) crucial virulence factor is knocked out so as to obtain using the method for gene knockout in the present invention
Attenuated strain, virulence significantly reduce compared with parent strain, while preferably avoid the security of the not thorough band of inactivated vaccine inactivation
The side effect that problem and inactivation reagent strip are come；
4) attenuated live vaccine in the present invention has certain cross-protection to the Aeromonas of separate sources, has wide
Spectrality；
5) attenuated live vaccine in the present invention has certain immune protective efficiency in zebra fish, illustrates the attenuation of the present invention
Live vaccine can effectively activate the immune response of zebra fish.
6) vaccine provided by the invention is used, the immunity of aquatic livestock can be significantly increased, improve disease-resistant phenotype.
The present invention knocks out the oral or dipping bath attenuated live vaccine of crucial virulence factor technique construction using antibiotic-free mark
The pathogenic of pathogen can be reduced while challeng is retained, immune protective effect is notable, enhancing innate immunity system
The infection ability of other pathogens is resisted in system reaction and cellular immunity, enhancing, while greatly reduces immunization amount.
Brief description of the drawings
Fig. 1 is the structure schematic flow sheet that embodiment 1 knocks out plasmid.
Fig. 2 is that embodiment 1 knocks out dientification of bacteria result.
Fig. 3 is the fluorescence localization result of embodiment 2.
Fig. 4 is the safety detection result of embodiment 3.
Fig. 5 is the immune protective effect statistical result of embodiment 4.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
The genomic DNA reference sequences of Aeromonas veronii aerolysin gene are as shown in the sequence 5 of sequence table.The institute of sequence 5
The protein shown in DNA encoding sequence 6 shown.
Plasmid pRE112：BioVector plasmid vector bacterium cell gene collections, article No.：3573410.
Aeromonas veronii A.veronii Hm091：Bibliography：Zhang Defeng, Liu Lihui, Li Ningqiu, wait south Chinas
The regional different types of epidemiological features of source of fish Aeromonas [J] aquatic sciences, 2015,34 (11):673-682.；The public can be with
Obtained from Institute of Feeds,China Academy of Agriculture Sciences.
Aeromonas hydrophila A.hydrophila NJ-1：Bibliography：Li J,Ni X D,Liu Y J,et
al.Detection of three virulence genes alt,ahp and aerA in Aeromonas
hydrophila and their relationship with actual virulence to zebrafish[J]
.Journal of Applied Microbiology,2011,110(3):823-30.；The public can be from the Chinese Academy of Agricultural Sciences
Feed research institute obtains.
E.coli MC1061：BioVector NTCC Type Tissue Collections, numbering：MC1061.
E.coli S17-1(λpair)：BioVector NTCC Type Tissue Collections, numbering：s17-1.
MC1061 competent cells：E.coli MC1061 single bacterium colonies are inoculated in 50ml LB fluid nutrient mediums, 37 DEG C,
200rpm overnight incubations, it is inoculated into by 5% inoculum concentration in the 1l triangular flasks containing 500ml LB fluid nutrient mediums, 37 DEG C,
250rpm is cultivated, and works as OD600nmBe worth for 0.35~0.4 when, by triangular flask ice bath 15-30min, (period rocks triangular flask, makes
Bacterium solution uniformly cools down), then bacterium solution is transferred in the centrifuge tube of ice bath precooling in advance, 1000g, 4 DEG C of centrifugation 15min, abandoned
Supernatant, thalline is resuspended with the deionized water of 500ml ice bath precoolings；1000g, 4 DEG C of centrifugation 20min, remove supernatant, collect thalline simultaneously
Be resuspended in 10% (referring to percentage) glycerine water solution of 250ml ice bath precoolings, 1000g, 4 DEG C centrifugation 20min, remove on
Clearly, thalline is resuspended with the GYT fluid nutrient mediums of 1ml ice bath precoolings；Bacterium solution is dispensed into the sterile EP pipes of 1.5ml of ice bath precooling
In, often the μ l of pipe 50, after being cooled down rapidly with liquid nitrogen, put -80 DEG C of refrigerators and preserve.
S17 (λ) competent cell：E.coli S17-1 (λ pair) single bacterium colony is inoculated in 50ml LB fluid nutrient mediums
In, 37 DEG C, 200rpm overnight incubations, it is inoculated into by 5% inoculum concentration in the 1l triangular flasks containing 500ml LB fluid nutrient mediums,
37 DEG C, 250rpm cultures, work as OD600nmBe worth for 0.35~0.4 when, by triangular flask ice bath 15-30min, (period rocks triangle
Bottle, makes bacterium solution uniformly cool down), then bacterium solution is transferred in the centrifuge tube of ice bath precooling in advance, 1000g, 4 DEG C of centrifugations
15min, supernatant is abandoned, thalline is resuspended with the deionized water of 500ml ice bath precoolings；1000g, 4 DEG C of centrifugation 20min, remove supernatant, receive
Collection thalline is simultaneously resuspended in 10% (referring to percentage) glycerine water solution of 250ml ice bath precoolings, 1000g, 4 DEG C of centrifugation 20min,
Remove supernatant, thalline is resuspended with the GYT fluid nutrient mediums of 1ml ice bath precoolings；The 1.5ml that bacterium solution is dispensed into ice bath precooling is sterile
In EP pipes, often the μ l of pipe 50, after being cooled down rapidly with liquid nitrogen, put -80 DEG C of refrigerators and preserve.
Zebra fish：The Tu strain zebra fish that Peking University's zebra fish water body laboratory provides.
Embodiment 1, Aeromonas veronii knock out the structure of bacterium
First, the structure of plasmid is knocked out
The structure schematic flow sheet for knocking out plasmid is shown in Fig. 1, comprises the following steps that：
1st, with PCR method (response procedures：98 DEG C, 5min, 32 × [98 DEG C, 20s；58 DEG C, 20s；72 DEG C, 1min], 72
DEG C, 5min) plasmid pRE112 is linearized, obtain linearization plasmid fragment.
2nd, using Aeromonas veronii A.veronii Hm091 genomic DNA as template, using primer Aer-up-F and draw
The primer pair of thing Aer-up-R compositions enters performing PCR amplification, and obtaining pcr amplification product (sequence 1 of sequence table, has upstream homologous
Arm).
Aer-up-F：5'-TGAATTCCCGGGAGAATGATCTCGGCGGTACCTGG-3'；
Aer-up-R：5'-GATCCACACCGGTAAATCAGGGTAGACAGGTTCAG-3'.
3rd, using Aeromonas veronii A.veronii Hm091 genomic DNA as template, using primer Aer-down-F and
The primer pair of primer Aer-down-R compositions enters performing PCR amplification, and obtaining pcr amplification product (sequence 2 of sequence table, has downstream
Homology arm).
Aer-down-F：5'-CCTGTCTACCCTGATTTACCGGTGTGGATCTGGAC-3'；
Aer-down-R：5'-GCTTCTTCTAGAGGTTGAGTGAAGGTGGAGCTGAG-3'.
4th, useHiFi DNA Assembly Master Mix (NEB, article No.：E2621L) by step 1
The pcr amplification product that the pcr amplification product and step 3 that obtained linearization plasmid, step 2 obtain obtain carries out homologous recombination company
(method is with reference to kit specification) is connect, obtaining knockout plasmid, (cyclic plasmid shown in the sequence 3 of sequence table, has been sequenced and has tested
Card).
2nd, structure and the identification of bacterium are knocked out
1st, the knockout plasmid for obtaining step 1 converts MC1061 competent cells, and 25uF, 2.5kV, 200ohm carry out electricity
Turn.After electricity turns, immediately plus 1ml LB fluid nutrient mediums, 37 DEG C, after shaking table 200r/min cultures 1-1.5h, centrifugation, remove 900 μ
L, stay 100 μ l to be resuspended, be then all coated onto on the LB solid plates of chloramphenicol containing 30ug/ml (Cm), 37 DEG C of overnight incubations.To mistake
The single bacterium colony grown after night filters out positive bacterium colony by bacterium colony PCR detections and (uses primer Aer-up-F and primer Aer-up-R
Enter performing PCR to detect to obtain the band of 2348bp sizes).
The 2nd, single bacterium colony that step 1 is accredited as to the positive is seeded to the 30ml LB Liquid Cultures of chloramphenicol containing 30ug/ml (Cm)
In base, 37 DEG C, shaking table 200r/min culture 12h, picking single bacterium colony extraction plasmid, carried out using Hind III restriction enzymes
Digestion identifies, obtain 8029bp sizes band for positive plasmid.
3rd, step 2 is identified that correct positive plasmid converts S17 (λ) competent cell, 25uF, 2.5kV, 200ohm are carried out
Electricity turns.After electricity turns, immediately plus 1ml LB fluid nutrient mediums, 37 DEG C, after shaking table 200r/min cultures 1-1.5h, centrifugation, remove 900
μ l, stay 100 μ l to be resuspended, be then all coated onto on the LB solid plates of chloramphenicol containing 30ug/ml (Cm), 37 DEG C of overnight incubations.It is right
The single bacterium colony grown after overnight filters out positive bacterium colony by bacterium colony PCR detections and (uses primer Aer-up-F and primer Aer-up-
R enters performing PCR and detects to obtain the band of 2348bp sizes).
The 4th, single bacterium colony that step 3 is accredited as to the positive is scoring to oese chloramphenicol containing 30ug/ml (Cm) LB again
On solid plate, 37 DEG C of culture 24h.
The 5th, Aeromonas veronii A.veronii Hm091 inoculations are scoring to the LB solids of chloramphenicol containing 30ug/ml (Cm)
On flat board, 37 DEG C of culture 24h.
6th, the bacterium colony that the bacterium colony and step 5 that scraping step 4 obtains respectively obtain is combined (scraping step 4 acquisition bacterium colony
In the flat lining out of step 5), 37 DEG C combine 8h.
7th, after completing step 6, bacterium colony is scraped, is applied to after being diluted with LB fluid nutrient mediums containing 30 μ g/ml chloramphenicol
(Cm) on the LB solid plates of+100 μ g/ml ampicillins (Amp), 37 DEG C of culture 24h.
8th, after completing step 7, picking single bacterium colony, it is inoculated into containing the μ g/ml ampicillins of 30 μ g/ml chloramphenicol (Cm)+100
(Amp) on LB solid plates, 37 DEG C of culture 24h.
9th, after completing step 8, picking single bacterium colony, 37 DEG C of culture 24h on the LB solid plates for do not contain antibiotic are inoculated into.
10th, after completing step 9, it is scoring to the LB containing 15% (mass percent) sucrose with the appropriate bacterium colony of oese scraping and consolidates
On body flat board, 15-25 DEG C of Low- temperature culture 3 days, plasmid loss bacterial strain is screened.
11st, after completing step 10,50-80 monoclonal of picking distinguish respective score line to contain 30 μ g/ml chloramphenicol (Cm)+
On the LB solid plates of 100 μ g/ml ampicillins (Amp), 37 DEG C of culture 24h.
12nd, after completing step 11, picking single bacterium colony filters out knockout mutations bacterial strain by bacterium colony PCR detections.
Using Aeromonas veronii A.veronii Hm091 (WT) as control.Primer Aer-up-F is respectively adopted and draws
The primer pair that primer pair, primer Aer-confirm-F and the primer Aer-confirm-R of thing Aer-down-R compositions are formed is carried out
Identification.Primer sequence and expected product sheet segment length are shown in Table 1.
The primer information of table 1 and expected results
As a result it is as shown in Figure 2.Primer Aer-confirm-F and primer Aer-confirm-R designs for Aer genes
Purpose fragment 1722bp is arrived in primer, wild mushroom (WT) amplification, is knocked out bacterium (Hm091 △ aer) and is not expanded to purpose fragment；Primer
Aer-up-F and primer Aer-down-R is to be directed to the primer that Aer gene upstream sequences and downstream sequence design, wild mushroom (WT)
The sequence of amplified fragments is about 4000bp, and the sequence for knocking out bacterium (Hm091 △ aer) amplified fragments is about 2300bp, is just cand be compared to
The about 1700bp of the few Aer genetic fragment sizes of wild mushroom.
Wild mushroom and knockout bacterium are sequenced, the results showed that, it is by the DNA pieces shown in sequence in sequence table 4 to knock out bacterium
What the respective segments that section instead of in wild mushroom genome aerolysin gene obtained.
The wherein one plant all knockout bacterium obtained by the above method are named as Aeromonas veronii (Aeromonas
Veronii) Hm091 △ aer, Aeromonas veronii (Aeromonas veronii) Hm091 △ aer are referred to as Vickers gas unit cell
Bacterium A.veronii Hm091 △ aer, will wherein one plant progress preservation.
3rd, Aeromonas veronii (Aeromonas veronii) Hm091 △ aer preservation
Aeromonas veronii (Aeromonas veronii) Hm091 △ aer, on October 09th, 2017 is preserved in
State Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC；Address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number is CGMCC No.14776.
The positioning of embodiment 2, Aeromonas veronii A.veronii Hm091 △ aer in zebra fish
1st, Aeromonas veronii A.veronii Hm091 are seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shaking table
200r/min cultivates 18h；2ml bacterium solutions are taken, 5000r/mim centrifugation 10min, removes supernatant, is washed twice, centrifuged on removing with PBS
Clearly, add 1ml 0.1M sodium bicarbonate buffer liquid that thalline is resuspended, obtain A.veronii Hm091 sodium bicarbonate buffer liquid.
2nd, Aeromonas hydrophila A.hydrophila NJ-1 are seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shaking table
200r/min cultivates 18h；2ml bacterium solutions are taken, 5000r/mim centrifugation 10min, removes supernatant, is washed twice, centrifuged on removing with PBS
Clearly, add 1ml 0.1M sodium bicarbonate buffer liquid that thalline is resuspended, obtain A.hydrophila NJ-1 sodium bicarbonate buffer liquid.
3rd, Aeromonas veronii A.veronii Hm091 △ aer are seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shake
Bed 200r/min cultures 18h；2ml bacterium solutions are taken, 5000r/mim centrifugation 10min, removes supernatant, is washed twice with PBS, centrifugation is removed
Supernatant, add 1ml 0.1M sodium bicarbonate buffer liquid that thalline is resuspended, obtain A.veronii Hm091 △ aer sodium bicarbonate buffers
Liquid.
4th, by red fluorescence dyestuff (Texas) (the silent winged generation of match your scientific and technological T7471) added by 10mg/ml concentration
Add DMSO to be dissolved, obtain red fluorescence dyestuff solution.By blue fluorescent dyes (Pacific BlueTM) (Invitrogen
P10163) dissolved by 10mg/ml concentration addition DMSO, obtain blue fluorescent dyes solution.
5th, the red fluorescence dyestuff solution for taking 50 μ l steps 4 to obtain adds to the A.hydrophila NJ-1 carbon that step 2 obtains
In sour hydrogen sodium buffer solution, room temperature lucifuge slight wobble is incubated 1.5h, is then centrifuged for taking supernatant, is washed 2 times with PBS, centrifuges on removing
Clearly, finally it is resuspended with 1ml PBS, obtains A.hydrophila NJ-1 red fluorescence label solutions.
6th, the red fluorescence dyestuff solution that 50 μ l steps 4 obtain is added into the A.veronii Hm091 carbonic acid that step 1 obtains
In hydrogen sodium buffer solution, room temperature lucifuge slight wobble is incubated 1.5h, is then centrifuged for taking supernatant, is washed 2 times with PBS, and supernatant is removed in centrifugation,
Finally it is resuspended with 1ml PBS, obtains A.veronii Hm091 red fluorescence label solutions.
7th, the blue fluorescent dyes solution that 50 μ l steps 4 obtain is added into the A.veronii Hm091 carbonic acid that step 1 obtains
In hydrogen sodium buffer solution, room temperature lucifuge slight wobble is incubated 1.5h, is then centrifuged for taking supernatant, is washed 2 times with PBS, and supernatant is removed in centrifugation,
Finally it is resuspended with 1ml PBS, obtains A.veronii Hm091 blue-fluorescence label solutions.
8th, the red fluorescence dyestuff solution that 50 μ l steps 4 obtain is added into the A.veronii Hm091 △ that step 3 obtains
In aer sodium bicarbonate buffer liquid, room temperature lucifuge slight wobble is incubated 1.5h, is then centrifuged for taking supernatant, is washed 2 times with PBS, centrifugation is gone
Fall supernatant, be finally resuspended with 1ml PBS, obtain A.veronii Hm091 △ aer red fluorescence label solutions.
9th, Aeromonas hydrophila A.hydrophila NJ-1 are seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shaking table
200r/min cultivates 18h；2ml bacterium solutions are taken, 5000r/mim centrifugation 10min, removes supernatant, is washed twice, centrifuged on removing with PBS
Clearly, add PBS that thalline is resuspended, obtain A.hydrophila NJ-1 solution.
10th, Aeromonas veronii A.veronii Hm091 △ aer are seeded in 30ml LB fluid nutrient mediums, 37 DEG C,
Shaking table 200r/min cultivates 18h；2ml bacterium solutions are taken, 5000r/mim centrifugation 10min, removes supernatant, is washed twice with PBS, centrifugation is gone
Fall supernatant, add PBS that thalline is resuspended, obtain A.veronii Hm091 △ aer solution.
11st, the zebra fish of 2 days after hatching is randomly divided into following 5 groups (every group of fish to be dispensed into 6 orifice plates, per hole 10ml
Water, 15 tail fishes)：
Group A (control group)：Zebra fish is infected using PBS；
Group B (A.hydrophila NJ-1 red fluorescences mark infected group)：It is glimmering using A.hydrophila NJ-1 red
Signal solution infects zebra fish, and concentration of the A.hydrophila NJ-1 in hole is infected is 4 × 107CFU/ml；
Group C (A.veronii Hm091 red fluorescences mark infected group)：Using A.veronii Hm091 red fluorescence marks
Remember solution infection zebra fish, concentration of the A.veronii Hm091 in hole is infected is 4 × 107CFU/ml；
Group D (A.hydrophila NJ-1 red fluorescences mark+unmarked A.veronii Hm091 infected groups)：Using
A.hydrophila NJ-1 red fluorescences label solutions and A.veronii Hm091 solution infection zebra fish, A.hydrophila
Concentration of the NJ-1 in hole is infected is 4 × 107Concentration of CFU/ml, the A.veronii Hm091 in hole is infected is 4 × 107CFU/
ml；
Group E (A.hydrophila NJ-1 red fluorescence mark+A.veronii Hm091 blue-fluorescences mark)：Using
A.hydrophila NJ-1 red fluorescences label solutions and A.veronii Hm091 blue-fluorescences marking fluid infection zebra fish,
Concentration of the A.hydrophila NJ-1 in hole is infected is 4 × 107CFU/ml, A.veronii Hm091 are dense in hole is infected
Spend for 4 × 107CFU/ml；
Group F (A.veronii Hm091 △ aer red fluorescences mark infected group)：Using A.veronii Hm091 △ aer
Red fluorescence label solution infects zebra fish, and concentration of the A.veronii Hm091 △ aer in hole is infected is 4 × 107CFU/
ml；
Group G (A.hydrophila NJ-1 red fluorescences mark+unmarked A.veronii Hm091 △ aer infected groups)：
Zebra fish is infected using A.hydrophila NJ-1 red fluorescences label solutions and A.veronii Hm091 △ aer solution,
Concentration of the A.hydrophila NJ-1 in hole is infected is 4 × 107CFU/ml, A.veronii Hm091 △ aer are in infection hole
In concentration be 4 × 107CFU/ml。
Small fish is fixed respectively with 4% paraformaldehyde after above-mentioned each group infection 4h；The small fish fixed is in laser
Taken pictures under Laser Scanning Confocal Microscope positioning of the observation bacterium in zebra fish.
As a result it is as shown in Figure 3.In control group zebra fish small fish, any fluorescence signal (A) is not observed；In red
The Aeromonas hydrophila A.hydrophila NJ-1 infected groups (B) of fluorescence labeling, the Aeromonas veronii of red fluorescence mark
A.veronii Hm091 △ aer infected groups (F), the Aeromonas hydrophila A.hydrophila NJ-1+ dimensions of red fluorescence mark
In family name's Aeromonas A.veronii Hm091 △ aer infected groups (G), it is observed that red in the enteron aisle of zebra fish small fish
Fluorescence signal, but be limited only in enteric cavity.However, in red fluorescence mark or the Aeromonas veronii of blue-fluorescence mark
In A.veronii Hm091 infected groups, very strong fluorescence signal is not only observed that in the enteron aisle of zebra fish, and in intestines
Around road and head may detect that fluorescence signal (C and E)；It is interesting that in the thermophilic aqueous vapor list of red fluorescence mark
Born of the same parents bacterium NJ-1+ is whether there is in the Aeromonas veronii A.veronii Hm091 infected groups of blue-fluorescence mark, NJ-1 fluorescence signal
Equally can be around zebra fish enteron aisle and head can detect (D and E).These as shown by data are by Aeromonas veronii point
The gas lysin secreted can destroy the gut barrier of zebra fish, promote itself and other Aeromonas to be particularly thermophilic aqueous vapor unit cell
Bacterium is entered inside fish, causes haemolysis and the death of fish.And Aeromonas veronii A.veronii Hm091 △ aer do not have then
The ability of the standby gut barrier for destroying zebra fish.
The security of embodiment 3, Aeromonas veronii A.veronii Hm091 △ aer vaccines
1st, Aeromonas veronii A.veronii Hm091 are seeded on LB solid mediums, 37 DEG C of culture 12h, then
Picking monoclonal is seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shaking table 200r/min cultures 18h.
2nd, Aeromonas veronii A.veronii Hm091 △ aer are seeded on LB solid mediums, 37 DEG C of culture 12h,
Then picking monoclonal is seeded in 30ml LB fluid nutrient mediums, 37 DEG C, shaking table 200r/min cultures 18h.
3rd, the Aeromonas veronii A.veronii Hm091 and Aeromonas veronii for respectively cultivating step 1 and step 2
A.veronii Hm091 △ aer are with different concentration (2.5 × 108CFU/ml、1.0×108CFU/ml、5.0×107CFU/ml、
3.33×107CFU/ml、2.5×107CFU/ml and 1.67 × 107CFU/ml the zebra fish (dipping bath) of 5 days after) infection is hatched, system
Count the death condition of zebra fish in 96h.
As a result it is as shown in Figure 4.Aeromonas veronii A.veronii Hm091 △ aer are respectively 3.3 × 107CFU/ml、
2.5×107CFU/ml、1.67×107There is no zebra fish to occur under CFU/ml dipping bath concentration dead, and after infecting wild strain,
The death rate of zebra fish 100% can be caused in 24 hours.As a result show that knocking out Aer substantially reduces the virulence of Aeromonas veronii,
Aeromonas veronii A.veronii Hm091 △ aer are in infection concentration≤3.3 × 107Dipping bath is safe under CFU/ml.
Embodiment 4, the research of Aeromonas veronii A.veronii Hm091 △ aer vaccine protectives
First, shadow of the Aeromonas veronii A.veronii Hm091 △ aer vaccines to zebra fish Immunoglobulins in Serum
Ring
The zebra fish at 3 monthly ages is randomly divided into following three groups：
1 (control group) of group：Normal raising zebra fish 2 weeks, is satiated with food feeds basal feed twice daily；
2 (feeding immune groups) of group：Normal raising zebra fish 2 weeks, is satiated with food feeds containing Aeromonas veronii twice daily
A.veronii Hm091△aer(2×107CFU/g basal feed)；
3 (dipping bath immune groups) of group：Normally raise zebra fish 2 weeks, weekly dipping bath A.veronii containing Aeromonas veronii
Hm091 △ aer water once (2 × 107CFU/ml, dipping bath time 12h), it is satiated with food feed basal feed twice daily.
After each group is handled, every group of zebra fish (builds up biology by fish IGM kits through tail vein blood purchased from Nanjing
Graduate School of Engineering) specification to carry out zebra fish immunoglobulin Indexs measure.
Logistic curves are determined according to kit protocol, draw immunoglobulin equation y=(A-D)/[1+ (x/C)B]+
D is (wherein：A=5.61378；B=0.36217；C=0.14147；D=-0.12176；r2=0.99394).After measured, three groups
Zebra fish immunoglobulin concentrations average value is as follows：Control group:7.304175ng/ml；Feed immune group:13.646945ng/
ml；Dipping bath immune group:16.00466ng/ml.As a result show, zebra fish Immunoglobulins in Serum has after vaccine immunity
Certain lifting.
2nd, the serum antibody agglutination titer of Aeromonas veronii A.veronii Hm091 △ aer vaccine zebra fish
The zebra fish at 3 monthly ages is randomly divided into following three groups：
1 (control group) of group：Normal raising zebra fish 2 weeks, is satiated with food feeds basal feed twice daily；
2 (feeding immune groups) of group：Normal raising zebra fish 2 weeks, is satiated with food feeds containing Aeromonas veronii twice daily
A.veronii Hm091△aer(2×107CFU/g basal feed)；
3 (dipping bath immune groups) of group：Normally raise zebra fish 2 weeks, weekly dipping bath A.veronii containing Aeromonas veronii
Hm091 △ aer water once (4 × 107CFU/L, dipping bath time 12h), it is satiated with food feed basal feed twice daily.
After each group is handled, every group of zebra fish takes serum to be used to determine serum antibody titer through tail vein blood.Using
96 hole blood-coagulation-board methods are carried out.Normal is used after Aeromonas veronii A.veronii Hm091 nutrient solutions 12000rpm is centrifuged
Salt solution is resuspended, and obtaining bacteria suspension, (concentration is 4 × 107CFU/ml).80 μ l physiology salts are added in l holes with micropipettor
Water, remaining each hole add 50 μ l；20 μ l test serum is added into the 1st hole, pressure-vaccum takes 50 μ l to add the 2nd hole (1 after mixing:2
Dilution), then take 50 μ l to add the 3rd hole (1 from the 2nd hole after mixing:4 dilutions), the rest may be inferred to the 9th hole (1：256 dilutions), discard
50 μ l, the 10th hole is as control；50 μ l Aeromonas veronii suspensions are added into each hole, pressure-vaccum mixes, and is placed in 37 DEG C of cultures
1h is incubated in case, is detected overnight under the conditions of being placed on 4 DEG C.
Experiment is repeated twice (first group and second group), as a result as shown in table 2.Aeromonas veronii A.veronii
Adult Zebrafish is immunized by feeding and dipping bath two ways for Hm091 △ aer vaccines, control group, feeding group and dipping bath
The serum antibody agglutination titer of group is respectively 23~24、24~25And 24, feeding group antibody level of serum is slightly above control group serum
Antibody level.
The antibody titer testing result of table 2
3rd, dipping bath and oral Aeromonas veronii A.veronii Hm091 △ aer vaccines are protected to the relative immunity of zebra fish
Shield rate
1 (control group, CK) of group：Normal raising zebra fish 2 weeks, is satiated with food feeds basal feed twice daily；
Group 2 (feeding immune group, T1)：Normal raising zebra fish 2 weeks, is satiated with food feeds containing Aeromonas veronii twice daily
A.veronii Hm091△aer(2×107CFU/g basal feed)；
3 (dipping bath immune group, T2) of group：Normally raise zebra fish 2 weeks, weekly dipping bath A.veronii containing Aeromonas veronii
Hm091 △ aer water once (3 × 107CFU/ml, dipping bath time 12h), it is satiated with food feed basal feed twice daily.
After each group is handled, every group of zebra fish is randomly divided into following three groups：
Group A (A.hydrophila NJ-1+ ammonium chlorides)：Using 200mg/L ammonium chlorides infection zebra fish (dipping bath), after 12h
Add 2 × 107CFU/ml Aeromonas veronii A.veronii Hm091.
Group B (A.veronii Hm091)：Aeromonas veronii A.veronii Hm091 are with 2 × 107CFU/ml concentration
Infect zebra fish (dipping bath).
Group C (A.veronii Hm091+A.hydrophila NJ-1)：Aeromonas veronii A.veronii Hm091 and
Aeromonas hydrophila A.hydrophila NJ-1 are with every kind of bacterium 2 × 107CFU/ml concentration infection zebra fish (dipping bath).
The death condition of zebra fish in 96h is counted, and according to formula Computation immunity protective rate (RPS), (1- is immunized RPS=
Group death toll/control death toll) × 100%
As a result as shown in Fig. 5 and table 3.
The vaccine protective rate statistical result of table 3
Fig. 5 results show that the dipping bath immune group zebra fish death time extends, but without obvious protecting effect.And feed
Immunization wayses have obvious immanoprotection action to NJ-1+ ammonium chlorides, Hm091, Hm091+NJ-1 dipping bath work(poison, and zebra fish is deposited
Motility rate is obviously improved.
The result of table 3 shows that dipping bath immune group immune protective rate is relatively low, does not play significant protective effect.And feed and exempt from
Epidemic disease group reaches 33% to NJ-1+HM091 immune protective rates, there is certain protective role；To NJ-1+ ammonium chloride groups, HM091 is immune to be protected
Shield rate reaches 80%, there is very strong immanoprotection action.
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>A kind of attenuated live vaccine of prevention and control aquiculture animal Aeromonas hemorrhage
<160> 6
<210> 1
<211> 1200
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
tgaattcccg ggagaatgat ctcggcggta cctgggtcga gatgaaaggg gtggtggaga 60
acggcgtgcg ctatgtacgc aaggtcgaag ccctcgacga agatgacaag gacgagatgg 120
atctggaagg gccggtcgaa ggggggcgga tctggggcta caccacctcg gacaacagtc 180
tggcgccgtt cgaagggcgt tggctcgagc tggagtgcaa gtttgacggc atgcgcctga 240
gccgctgtcg cgaggatgac tgatccgatc tctgcgttcc gattagaaat gctcccgaat 300
gaagcccatg cttgtcctgc atgggctttt tgttcaggga cgaacgaaga tggaacaatc 360
gagaaagtgg tgtgcatgga tgctgggggt gccgctctgg gcggcgcagg cgggagccga 420
cgaattgtgg ctggtggagc tggagcacag cgacggcatc cgcctgcaat ttcagggggc 480
cgaggtggag cagggcagtg ccgccatctg gcggcagggg gagatttggc cgcagccact 540
caaacccggc gaccagctcg ctctgctggt ggccgatggc gttgccaccc ggattgagct 600
gctggccgcc agagccccgc tggccaatca gccctggcag cgggcgcaag atcgcctgca 660
atccttcgat gaccggcagc tcacgttggc caccctggac actgtgccac tcaatccgca 720
ggtgcgctgg gtcaacggct cggccgccga tctgcatgcc gggagcgagc tggtattgat 780
ccgttcggcg gatggaatat tgcagggaat tgaagtgatc aatccggaag agtaattcag 840
catcttctta tttaatataa cttcaccata ttgtggcagc cattaaataa tgactgtcac 900
aatatttatg atttattcct caataaagtc atcgctctaa ttattttcct gtatcaccat 960
tattttgttt gtcatggatg ttttttggtg gtttatttta tttgtgtcag gttttgtcgg 1020
agtccatacc tgtcaaatga aaaaggatat ttctacacaa taacaattat cgtatgtctg 1080
aaagagggat aaatgatgaa tagaataatt accgccaatc tggcgttttt ggcgtcttca 1140
ttgatgctcg ctcaggtgca ggcggctgaa cctgtctacc ctgatttacc ggtgtggatc 1200
<210> 2
<211> 1148
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
cctgtctacc ctgatttacc ggtgtggatc tggacagcga ggcccttgcc aacgaagggt 60
ttggcaacgt atccctgacc atagttccgg ttcaataacc tccctcacag gcccctttca 120
taggggcctg tttctgttct gcgcaagacg ctgctcttgc tctgtgtccc tcgactttcc 180
ctctttttct cctcttctcc ctgcatgatc acagcgggaa cctgtggcgt gccaagttga 240
tgccaacgca ggtttgacct gtgcttgggg tggccccgtg agcgaggcat cgttcactca 300
tttgttgaaa gaggcgtgtt ggccagattg ttaacggctt gtgctttaaa ttttccccct 360
tatactggcg ccatcttgtc ggagtgctga ccgtcgaacg acgcgaagct gagaccgtta 420
attcgggatc cgtggaacct gatcaggtta aaacctgcga agggaacaag ggtaacttgc 480
gggttaccgc gccggaggag ggacaagcct cttccgctca tcgaagggat cacccttgat 540
gggtcagggc gcacaggagg gagtctgtcc cgtccggttg tccagcacgt ggggcgcctt 600
gcccaaagca tgcagagagc gggaagcggc agaacagcag cgcggagatc catcccgcgc 660
gcactccctc ccgagataaa caccccgccg caagttcatc ttctcctcga actccagaca 720
agcaaatacc cataacttca accagttaat gtgagttttg ctatgtctac ttctgtatct 780
gataccgcca aatccagccg ccgcgagcag cgctccagcg ctcaggcctt tatcgacagc 840
ctcaaaggga tggctcatcc caactcccgc cgcatcttta tcgaaggctc ccgcgccgat 900
atccgggtgc ccctgcggga gatccagctg gccgatacct tcgtcggtgg caccaaagaa 960
gatcccaagt tcgaacccaa cgaaccgatc ccggtctatg acacctcggg ccgttacggg 1020
gaggaggggg tcgccatcga cgtgcgcctc ggcctgccgc gcctgcggga aaactgggtg 1080
ctggagcggg atgacaccga cgaactgccg gggctcagct ccaccttcac tcaacctcta 1140
gaagaagc 1148
<210> 3
<211> 8029
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
cgcgtttccg agaaccgcgc gaacgacatg gagcggcacg cgggcgtgga aagcctggtc 60
ggctggatcg gcacgatgcg tccggcgtag aggatctgaa gatccagcag ttcaacctgt 120
tgatagtacg tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa 180
gctctcatgt ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg 240
tactaagctc tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa 300
atcagcaact taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa 360
ggcttttaaa gcttttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact 420
gagaagccct tagagcctct caaagcaatt ttcagtgaca caggaacact taacggctga 480
catgggaatt ctgatccttt ttaacccatc acatatacct gccgttcact attatttagt 540
gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc 600
aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta 660
ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca 720
gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt 780
tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca 840
ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag 900
gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa 960
catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg 1020
aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg 1080
catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa 1140
atatcaagtt cctgagttcg attcgtccac aattaaaaat atctcttctg caaaaggcct 1200
ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg 1260
ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta 1320
catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt 1380
ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga 1440
atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt 1500
ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc 1560
agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg 1620
aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa 1680
ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga 1740
agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata 1800
ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa 1860
aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta 1920
cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga 1980
acgcgcgaac gtctttaaaa tgaacggcaa atggtatctg ttcactgact cccgcggatc 2040
aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa 2100
ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct 2160
tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa 2220
caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac 2280
gtttgcgccc agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag 2340
catccttgaa caaggacaat taacagttaa caaataaaaa cgcaaaagaa aatgccgata 2400
tcctattggc attttctttt atttcttatc aacataaagg tgaatcccat atgaactata 2460
taaaagcagg caaatggcta accgtattcc taaccttttg gtaatgactc caacttattg 2520
atagtgtttt atgttcagat aatgcccgat gactttgtca tgcagctcca ccgattttga 2580
gaacgacagc gacttccgtc ccagccgtgc caggtgctgc ctcagattca ggttatgccg 2640
ctcaattcgc tgcgtatatc gcttgctgat tacgtgcagc tttcccttca ggcgggattc 2700
atacagcggc cagccatccg tcatccatat caccacgtca aagggtgaca gcaggctcat 2760
aagacgcccc agcgtcgcca tagtgcgttc accgaatacg tgcgcaacaa ccgtcttccg 2820
gagactgtca tacgcgtaaa acagccagcg ctggcgcgat ttagccccga catagcccca 2880
ctgttcgtcc atttccgcgc agacgatgac gtcactgccc ggctgtatgc gcgaggttac 2940
cgactgcggc ctgagttttt taagtgacgt aaaatcgtgt tgaggccaac gcccataatg 3000
cgggctgttg cccggcatcc aacgccattc atggccatat caatgatttt ctggtgcgta 3060
ccgggttgag aagcggtgta agtgaactgc atgaattccc gggagaatga tctcggcggt 3120
acctgggtcg agatgaaagg ggtggtggag aacggcgtgc gctatgtacg caaggtcgaa 3180
gccctcgacg aagatgacaa ggacgagatg gatctggaag ggccggtcga aggggggcgg 3240
atctggggct acaccacctc ggacaacagt ctggcgccgt tcgaagggcg ttggctcgag 3300
ctggagtgca agtttgacgg catgcgcctg agccgctgtc gcgaggatga ctgatccgat 3360
ctctgcgttc cgattagaaa tgctcccgaa tgaagcccat gcttgtcctg catgggcttt 3420
ttgttcaggg acgaacgaag atggaacaat cgagaaagtg gtgtgcatgg atgctggggg 3480
tgccgctctg ggcggcgcag gcgggagccg acgaattgtg gctggtggag ctggagcaca 3540
gcgacggcat ccgcctgcaa tttcaggggg ccgaggtgga gcagggcagt gccgccatct 3600
ggcggcaggg ggagatttgg ccgcagccac tcaaacccgg cgaccagctc gctctgctgg 3660
tggccgatgg cgttgccacc cggattgagc tgctggccgc cagagccccg ctggccaatc 3720
agccctggca gcgggcgcaa gatcgcctgc aatccttcga tgaccggcag ctcacgttgg 3780
ccaccctgga cactgtgcca ctcaatccgc aggtgcgctg ggtcaacggc tcggccgccg 3840
atctgcatgc cgggagcgag ctggtattga tccgttcggc ggatggaata ttgcagggaa 3900
ttgaagtgat caatccggaa gagtaattca gcatcttctt atttaatata acttcaccat 3960
attgtggcag ccattaaata atgactgtca caatatttat gatttattcc tcaataaagt 4020
catcgctcta attattttcc tgtatcacca ttattttgtt tgtcatggat gttttttggt 4080
ggtttatttt atttgtgtca ggttttgtcg gagtccatac ctgtcaaatg aaaaaggata 4140
tttctacaca ataacaatta tcgtatgtct gaaagaggga taaatgatga atagaataat 4200
taccgccaat ctggcgtttt tggcgtcttc attgatgctc gctcaggtgc aggcggctga 4260
acctgtctac cctgatttac cggtgtggat ctggacagcg aggcccttgc caacgaaggg 4320
tttggcaacg tatccctgac catagttccg gttcaataac ctccctcaca ggcccctttc 4380
ataggggcct gtttctgttc tgcgcaagac gctgctcttg ctctgtgtcc ctcgactttc 4440
cctctttttc tcctcttctc cctgcatgat cacagcggga acctgtggcg tgccaagttg 4500
atgccaacgc aggtttgacc tgtgcttggg gtggccccgt gagcgaggca tcgttcactc 4560
atttgttgaa agaggcgtgt tggccagatt gttaacggct tgtgctttaa attttccccc 4620
ttatactggc gccatcttgt cggagtgctg accgtcgaac gacgcgaagc tgagaccgtt 4680
aattcgggat ccgtggaacc tgatcaggtt aaaacctgcg aagggaacaa gggtaacttg 4740
cgggttaccg cgccggagga gggacaagcc tcttccgctc atcgaaggga tcacccttga 4800
tgggtcaggg cgcacaggag ggagtctgtc ccgtccggtt gtccagcacg tggggcgcct 4860
tgcccaaagc atgcagagag cgggaagcgg cagaacagca gcgcggagat ccatcccgcg 4920
cgcactccct cccgagataa acaccccgcc gcaagttcat cttctcctcg aactccagac 4980
aagcaaatac ccataacttc aaccagttaa tgtgagtttt gctatgtcta cttctgtatc 5040
tgataccgcc aaatccagcc gccgcgagca gcgctccagc gctcaggcct ttatcgacag 5100
cctcaaaggg atggctcatc ccaactcccg ccgcatcttt atcgaaggct cccgcgccga 5160
tatccgggtg cccctgcggg agatccagct ggccgatacc ttcgtcggtg gcaccaaaga 5220
agatcccaag ttcgaaccca acgaaccgat cccggtctat gacacctcgg gccgttacgg 5280
ggaggagggg gtcgccatcg acgtgcgcct cggcctgccg cgcctgcggg aaaactgggt 5340
gctggagcgg gatgacaccg acgaactgcc ggggctcagc tccaccttca ctcaacctct 5400
agaagaagct tgggatcggg ccctatcact tattcaggcg tagcaaccag gcgtttaagg 5460
gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg 5520
taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat 5580
cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg 5640
ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg 5700
attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc 5760
accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta 5820
ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg 5880
aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc 5940
attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt 6000
tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc 6060
aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt 6120
atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc 6180
aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg 6240
ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac 6300
accaggattt atttattctg cgaagtgatc ttccgtcaca ggtattaggg cccgatcctt 6360
tttgtccggt gttgggttga aggtgaagcc ggtcggggcc gcagcggggg ccggcttttc 6420
agccttgccc ccctgcttcg gccgccgtgg ctccggcgtc ttgggtgccg gcgcgggttc 6480
cgcagccttg gcctgcggtg cgggcacatc ggcgggcttg gccttgatgt gccgcctggc 6540
gtgcgagcgg aacgtctcgt aggagaactt gaccttcccc gtttcccgca tgtgctccca 6600
aatggtgacg agcgcatagc cggacgctaa cgccgcctcg acatccgccc tcaccgccag 6660
gaacgcaacc gcagcctcat cacgccggcg cttcttggcc gcgcgggatt caacccactc 6720
ggccagctcg tcggtgtagc tctttggcat cgtctctcgc ctgtcccctc agttcagtaa 6780
tttcctgcat ttgcctgttt ccagtcggta gatattccac aaaacagcag ggaagcagcg 6840
cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc 6900
ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat 6960
ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt 7020
cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg 7080
gccggctacc gccggcgtaa cagatgaggg caagcggatg gctgatgaaa ccaagccaac 7140
caggaagggc agcccaccta tcaaggtgta ctgccttcca gacgaacgaa gagcgattga 7200
ggaaaaggcg gcggcggccg gcatgagcct gtcggcctac ctgctggccg tcggccaggg 7260
ctacaaaatc acgggcgtcg tggactatga gcacgtccgc gagctggccc gcatcaatgg 7320
cgacctgggc cgcctgggcg gcctgctgaa actctggctc accgacgacc cgcgcacggc 7380
gcggttcggt gatgccacga tcctcgccct gctggcgaag atcgaagaga agcaggacga 7440
gcttggcaag gtcatgatgg gcgtggtccg cccgagggca gagccatgac ttttttagcc 7500
gctaaaacgg ccggggggtg cgcgtgattg ccaagcacgt ccccatgcgc tccatcaaga 7560
agagcgactt cgcggagctg gtgaagtaca tcaccgacga gcaaggcaag accgagcgcc 7620
tgggtcacgt gcgcgtcacg aactgcgagg caaacaccct gcccgctgtc atggccgagg 7680
tgatggcgac ccagcacggc aacacccgtt ccgaggccga caagacctat cacctgctgg 7740
ttagcttccg cgcgggagag aagcccgacg cggagacgtt gcgcgcgatt gaggaccgca 7800
tctgcgctgg gcttggcttc gccgagcatc agcgcgtcag tgccgtgcat cacgacaccg 7860
acaacctgca catccatatc gccatcaaca agattcaccc gacccgaaac accatccatg 7920
agccgtatcg ggcctaccgc gccctcgctg acctctgcgc gacgctcgaa cgggactacg 7980
ggcttgagcg tgacaatcac gaaacgcggc agcgcgtttc cgagaaccg 8029
<210> 4
<211> 173
<212> DNA
<213>Artificial sequence
<400> 4
atgaatagaa taattaccgc caatctggcg tttttggcgt cttcattgat gctcgctcag 60
gtgcaggcgg ctgaacctgt ctaccctgat ttaccggtgt ggatctggac agcgaggccc 120
ttgccaacga agggtttggc aacgtatccc tgaccatagt tccggttcaa taa 173
<210> 5
<211> 1464
<212> DNA
<213>Aeromonas veronii
<400> 5
atgaatagaa taattaccgc caatctggcg tttttggcgt cttcattgat gctcgctcag 60
gtgcaggcgg ctgaacctgt ctaccctgat caggtcaagt gggcgggtct tgggacgggc 120
gtctgtgcca gtggttatcg tcctctgacc cgcgatgaag ccatgagtat caagggtaat 180
ctggtcagcc gcatggggca gtggcagatc accgggctag cggatcgctg ggtgattatg 240
gggccgggat ataatggcga aatcaaacag ggcactgccg gagaaacctg gtgttatccc 300
aattcacctg tttccggaga aataccaaca ctctctgact ggaatattcc ggcgggtgat 360
gaggtcgatg tgcaatggcg tcttgtgcac gacaatgatt attttatcaa accagtcagc 420
tatctcgccc attatttggg atacgcctgg gtgggtggaa atcatagccc ctatgtcggg 480
gaagatatgg atgttacccg ggttggcgat ggctggttga taaggggaaa taatgacggc 540
ggctgctccg gatatcgttg cggggagaag agctccatca aggtaagcaa cttctcatat 600
actctggaac ccgactcctt cagccatggt caggtcaccg agagcggcaa gcagctggtc 660
aagaccatca cggccaatgc gaccaactac accgacctgc cccagcaggt ggtggtgacc 720
ctgaagtacg acaaggccac caactggtcg aaaaccgata cctacagcct gagcgagaag 780
gtgaccacca agaacaagtt tcagtggccg ttggtaggtg aaaccgaact ggccatcgag 840
atcgcagcga gccagagctg ggcttcccaa cacgggggct cgaccaccga gactgtctcg 900
gttgaagcgc gccccacggt gccacctcac tccagcctgc cggtgcgggt tgccctctac 960
aagtccaata tctcataccc ctatgagttc aaggccgagg tcaattatga cctgaccatg 1020
aagggcttcc tgcgttgggg cggcaatgcc tggtataccc atcctgataa tcgtccgacc 1080
tgggagcaca cctttgccgt cggcccgttc cgcgacaagg cgagcagcat tcgctaccag 1140
tgggacaagc gttatattcc gggtgaagtg aagtggtggg actggaactg gaccatcagc 1200
gagtatggtt tgtcgaccat gcagaataac ctgggtcgtg tgctgcgccc gatccgttcg 1260
gcggtgaccg gtgacttcta tgccgagagc cagtttgccg gcgatatcga aatcggtcag 1320
ccccagaccc gttccgcgaa ggctgcccag ctgcgcagtg cctctgccga tgaggtggcg 1380
cttaccggtg tggatctgga cagcgaggcc cttgccaacg aagggtttgg caacgtatcc 1440
ctgaccatag ttccggttca ataa 1464
<210> 6
<211> 487
<212> PRT
<213>Aeromonas veronii
<400> 6
Met Asn Arg Ile Ile Thr Ala Asn Leu Ala Phe Leu Ala Ser Ser Leu
1 5 10 15
Met Leu Ala Gln Val Gln Ala Ala Glu Pro Val Tyr Pro Asp Gln Val
20 25 30
Lys Trp Ala Gly Leu Gly Thr Gly Val Cys Ala Ser Gly Tyr Arg Pro
35 40 45
Leu Thr Arg Asp Glu Ala Met Ser Ile Lys Gly Asn Leu Val Ser Arg
50 55 60
Met Gly Gln Trp Gln Ile Thr Gly Leu Ala Asp Arg Trp Val Ile Met
65 70 75 80
Gly Pro Gly Tyr Asn Gly Glu Ile Lys Gln Gly Thr Ala Gly Glu Thr
85 90 95
Trp Cys Tyr Pro Asn Ser Pro Val Ser Gly Glu Ile Pro Thr Leu Ser
100 105 110
Asp Trp Asn Ile Pro Ala Gly Asp Glu Val Asp Val Gln Trp Arg Leu
115 120 125
Val His Asp Asn Asp Tyr Phe Ile Lys Pro Val Ser Tyr Leu Ala His
130 135 140
Tyr Leu Gly Tyr Ala Trp Val Gly Gly Asn His Ser Pro Tyr Val Gly
145 150 155 160
Glu Asp Met Asp Val Thr Arg Val Gly Asp Gly Trp Leu Ile Arg Gly
165 170 175
Asn Asn Asp Gly Gly Cys Ser Gly Tyr Arg Cys Gly Glu Lys Ser Ser
180 185 190
Ile Lys Val Ser Asn Phe Ser Tyr Thr Leu Glu Pro Asp Ser Phe Ser
195 200 205
His Gly Gln Val Thr Glu Ser Gly Lys Gln Leu Val Lys Thr Ile Thr
210 215 220
Ala Asn Ala Thr Asn Tyr Thr Asp Leu Pro Gln Gln Val Val Val Thr
225 230 235 240
Leu Lys Tyr Asp Lys Ala Thr Asn Trp Ser Lys Thr Asp Thr Tyr Ser
245 250 255
Leu Ser Glu Lys Val Thr Thr Lys Asn Lys Phe Gln Trp Pro Leu Val
260 265 270
Gly Glu Thr Glu Leu Ala Ile Glu Ile Ala Ala Ser Gln Ser Trp Ala
275 280 285
Ser Gln His Gly Gly Ser Thr Thr Glu Thr Val Ser Val Glu Ala Arg
290 295 300
Pro Thr Val Pro Pro His Ser Ser Leu Pro Val Arg Val Ala Leu Tyr
305 310 315 320
Lys Ser Asn Ile Ser Tyr Pro Tyr Glu Phe Lys Ala Glu Val Asn Tyr
325 330 335
Asp Leu Thr Met Lys Gly Phe Leu Arg Trp Gly Gly Asn Ala Trp Tyr
340 345 350
Thr His Pro Asp Asn Arg Pro Thr Trp Glu His Thr Phe Ala Val Gly
355 360 365
Pro Phe Arg Asp Lys Ala Ser Ser Ile Arg Tyr Gln Trp Asp Lys Arg
370 375 380
Tyr Ile Pro Gly Glu Val Lys Trp Trp Asp Trp Asn Trp Thr Ile Ser
385 390 395 400
Glu Tyr Gly Leu Ser Thr Met Gln Asn Asn Leu Gly Arg Val Leu Arg
405 410 415
Pro Ile Arg Ser Ala Val Thr Gly Asp Phe Tyr Ala Glu Ser Gln Phe
420 425 430
Ala Gly Asp Ile Glu Ile Gly Gln Pro Gln Thr Arg Ser Ala Lys Ala
435 440 445
Ala Gln Leu Arg Ser Ala Ser Ala Asp Glu Val Ala Leu Thr Gly Val
450 455 460
Asp Leu Asp Ser Glu Ala Leu Ala Asn Glu Gly Phe Gly Asn Val Ser
465 470 475 480
Leu Thr Ile Val Pro Val Gln
485
Claims (10)
1. recombinant bacterium, it is that the aerolysin gene in Aeromonas veronii is knocked out what is obtained.
2. recombinant bacterium as claimed in claim 1, it is characterised in that：It is described to knock out to knock out whole ORFs or knockout portion
Divide constant gene segment C.
3. recombinant bacterium, it is to be obtained after specific DNA fragment importing Aeromonas veronii progress homologous recombination with described special
The recombinant bacterium of DNA fragmentation；The specific DNA fragment includes the upper homology arm of aerolysin gene and lower homology arm；It is described homologous
Arm is as shown in the sequence 3 from 5 ' end 3092-4276 positions nucleotides of sequence table；The sequence 3 of the lower homology arm such as sequence table is certainly
Shown in 5 ' end 4277-5408 positions nucleotides.
Aeromonas veronii 4. (Aeromonas veronii) Hm091 △ aer, deposit number is CGMCC No.14776.
5. any described recombinant bacteriums of claim 1-3, or, the Aeromonas veronii (A.veronii) described in claim 4
Applications of the Hm091 △ aer in vaccine is prepared；The vaccine is any of following (b1)-(b4)：
(b1) aquatic livestock Aeromonas veronii vaccine；
(b2) aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine；
(b3) aquatic livestock Aeromonas vaccine；
(b4) aquatic livestock bleeding disease vaccine.
6. a kind of method for preparing vaccine, comprises the following steps：By any described recombinant bacteriums of claim 1-3, or, right will
Aeromonas veronii (Aeromonas veronii) Hm091 △ aer described in 4 are asked to be packed as the active component of vaccine；
The vaccine is any of following (b1)-(b4)：
(b1) aquatic livestock Aeromonas veronii vaccine；
(b2) aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine；
(b3) aquatic livestock Aeromonas vaccine；
(b4) aquatic livestock bleeding disease vaccine.
7. any described recombinant bacteriums of claim 1-3, or, the Aeromonas veronii (Aeromonas described in claim 4
Veronii) applications of the Hm091 △ aer in product is prepared；The purposes of the product is the immunity of increase aquatic livestock.
8. any described recombinant bacteriums of claim 1-3, or, the Aeromonas veronii (Aeromonas described in claim 4
Veronii) applications of the Hm091 △ aer in product is prepared；The purposes of the product is at least one in following (c1)-(c4)
Kind：
(c1) prevent and/or treat the infection of aquatic livestock Aeromonas veronii；
(c2) prevent and/or treat aquatic livestock infected by Aeromonas hydrophila；
(c3) prevent and/or treat aquatic livestock Aeromonas infection；
(c4) prevent and/or treat aquatic livestock hemorrhage.
9. aquatic livestock Aeromonas veronii vaccine or aquatic livestock Evaluation of Aeromon As Hydrophila Vaccine or aquatic livestock Aeromonas epidemic disease
Seedling or aquatic livestock bleeding disease vaccine, its active component are any described recombinant bacteriums of claim 1-3, or, claim 4 institute
Aeromonas veronii (Aeromonas veronii) the Hm091 △ aer stated.
10. a kind of product, active component is any described recombinant bacteriums of claim 1-3, or, the Vickers described in claim 4
Aeromonas (Aeromonas veronii) Hm091 △ aer；The purposes of the product is at least one in following (d1)-(d5)
Kind：
(d1) prevent and/or treat the infection of aquatic livestock Aeromonas veronii；
(d2) prevent and/or treat aquatic livestock infected by Aeromonas hydrophila；
(d3) prevent and/or treat aquatic livestock Aeromonas infection；
(d4) prevent and/or treat aquatic livestock hemorrhage；
(d5) immunity of aquatic livestock is increased.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711138001.2A CN107858317B (en) | 2017-11-16 | 2017-11-16 | Attenuated live vaccine for preventing and controlling aeromonas hemorrhagic disease of aquaculture animals |
| PCT/CN2018/075580 WO2019095566A1 (en) | 2017-11-16 | 2018-02-07 | Live attenuated vaccine for preventing or controlling hemorrhagic disease caused by aeromonas in aquaculture animal |
| JP2020502746A JP6971378B2 (en) | 2017-11-16 | 2018-02-07 | Live attenuated vaccine for the prevention and control of Aeromonas hemorrhagic disease in aquaculture animals |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711138001.2A CN107858317B (en) | 2017-11-16 | 2017-11-16 | Attenuated live vaccine for preventing and controlling aeromonas hemorrhagic disease of aquaculture animals |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107858317A true CN107858317A (en) | 2018-03-30 |
| CN107858317B CN107858317B (en) | 2020-05-26 |
Family
ID=61703014
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| Application Number | Title | Priority Date | Filing Date |
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| CN201711138001.2A Active CN107858317B (en) | 2017-11-16 | 2017-11-16 | Attenuated live vaccine for preventing and controlling aeromonas hemorrhagic disease of aquaculture animals |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP6971378B2 (en) |
| CN (1) | CN107858317B (en) |
| WO (1) | WO2019095566A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019095566A1 (en) * | 2017-11-16 | 2019-05-23 | 中国农业科学院饲料研究所 | Live attenuated vaccine for preventing or controlling hemorrhagic disease caused by aeromonas in aquaculture animal |
| CN110106268A (en) * | 2019-05-28 | 2019-08-09 | 电子科技大学中山学院 | A kind of the LAMP detection primer composition and kit of Aeromonas veronii |
| CN110283767A (en) * | 2019-06-21 | 2019-09-27 | 武汉生物工程学院 | It is a kind of become oxygen acceptor gene aer function lose coli strain and preparation method |
| CN114015711A (en) * | 2021-10-25 | 2022-02-08 | 中国农业科学院饲料研究所 | Recombinant protein for inhibiting Aeromonas veronii infection and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235287B (en) * | 2020-03-06 | 2022-08-16 | 集美大学 | Primer group for detecting aeromonas containing Aer gene based on LAMP method and application thereof |
| CN111635914A (en) * | 2020-06-05 | 2020-09-08 | 电子科技大学中山学院 | Plasmid for knocking out aerolysin gene of aeromonas hydrophila and construction method thereof |
| CN112154946B (en) * | 2020-11-05 | 2022-03-08 | 华中农业大学 | Method for cultivating indoor controllable inactivated bait fish with initial fish fries |
| CN115040506A (en) * | 2022-06-23 | 2022-09-13 | 四川农业大学 | Application of 7-hydroxycoumarin in preparation of medicament for preventing and treating aeromonas hydrophila infection of grass carps |
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| CN107858317B (en) * | 2017-11-16 | 2020-05-26 | 中国农业科学院饲料研究所 | Attenuated live vaccine for preventing and controlling aeromonas hemorrhagic disease of aquaculture animals |
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| CN110283767A (en) * | 2019-06-21 | 2019-09-27 | 武汉生物工程学院 | It is a kind of become oxygen acceptor gene aer function lose coli strain and preparation method |
| CN114015711A (en) * | 2021-10-25 | 2022-02-08 | 中国农业科学院饲料研究所 | Recombinant protein for inhibiting Aeromonas veronii infection and preparation method and application thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| JP2020512835A (en) | 2020-04-30 |
| CN107858317B (en) | 2020-05-26 |
| WO2019095566A1 (en) | 2019-05-23 |
| JP6971378B2 (en) | 2021-11-24 |
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