CN107099509A - Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application - Google Patents
Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application Download PDFInfo
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Abstract
Bactericidal composition the present invention relates to aeromonas salmonicida bacteriophage and comprising the bacteriophage and application.The bacteriophage can quickly suppress the growth of Atlantic Ocean aeromonas salmonicida in a short time, and suppress the generation of the pathogen phage resistance.In addition, the bactericidal composition of the bacteriophage is safely, effectively, and high specificity, production cost is low, and the deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
Description
Technical field
The present invention relates to biological technical field, the sterilization group in particular to aeromonas salmonicida bacteriophage, comprising it
Compound and its application.
Background technology
Atlantic Ocean aeromonas salmonicida (Aeromonas salmonicida) belongs to the leather of Aeromonas under Proteobacteria
Blue formula negative bacillus, is distributed extremely wide in water environment, is to cause Salmons especially in the enteron aisle of aquatic animal
The Main Pathogenic Bacteria of furunculosis and routed enteropathy.The host range of aeromonas salmonicida is wide, in some cases, can trigger allow people with
Multi-infection disease inside and outside animal intestinal tract.Under conditions of high-density breeding, because aeromonas salmonicida is with very high
Lethal and the incidence of disease, such pathogenic bacteria are considered as to cause one of explosive main causes of death of a variety of aquatic animals, often
Year causes extremely serious loss to Salmons.In addition, such pathogenic bacteria can infect non-salmon fishes, brought to aquaculture
Huge economic loss.
Clinically because the cause of disease to the fish that falls ill does not recognize clearly, cause the abuse of antibiotic, both cause raiser
Economic loss, also have impact on the quality safety of aquatic products.The furunculosis occurred for Salmons in aquaculture and routed enteropathy,
The main prevention and control mode used is a large amount of antibiotic that come into operation.Although the use of antibiotic being considered as that Current therapeutic is thin
Bacterium infects most effective way, but huge antibiotics production and consumption patterns have also greatly encouraged the formation of drug-fast bacteria simultaneously,
And make it that the drug resistance problems of pathogenic bacteria are increasingly serious.Report, has found that one plant of wide spectrum is resistance in the animal body for suffer from disease at present
Property of medicine aeromonas salmonicida, up to more than 9 kinds of its drug resistance scope.
Bacteriophage is the virus of a class energy bacterial infection, is widely present in the environment.Bacteriophage has high host special
The opposite sex, self growth and breeding are realized by infecting specific targeting host, and ultimately result in the cracking death of Host Strains, and to it
His flora and human body are without influence.Phagotherapy refers to kill pathogen to treat pathogen sense using the single-minded cracking performance of bacteriophage
The treatment means of disease caused by dye.The theory of pathogenic bacteria is controlled using bacteriophage, the i.e. quilt soon after bacteriophage is found
Propose, and be used successfully in the treatment case of bacterial infection disease.However, due to the discovery of antibiotic, weakening bacteriophage
Application in this respect, but be continued for so far as the application of antiseptic in East European countries bacteriophage.
Nearly ten years, because abuse of antibiotics causes a large amount of drug-fast bacterias even appearance of superbacteria, directed toward bacteria infection
The phagotherapy of property disease returns the sight of people again, and is gradually highly valued.Currently, in most of Eastern Europe state
Family and the U.S., bacteriophage are widely used to during field, the especially food processing and productions such as environment, industry and agricultural to food
The prevention and control of source property disease.In addition, current scientists from all over the world are directed to by building scale-model investigation Phage Infection Host Strains
Mechanism characteristic, and attempt bacteriophage products application in the clinically research of prevention and control epidemiology.As should resist
One of important weapon of raw element resistance, the clinical practice of bacteriophage has a high potential.But with regard to answering for its current single phagotherapy
For, it engenders some potential shortcomings, such as cleavable host range narrow range in application process, cracks a certain amount of place
The dosage of main needs is high, and resistance of bacteriophage etc. is evolved in host's short time.
In view of this, it is special to propose the present invention.
The content of the invention
The first aspect of the present invention is provided in addition to selectivity cracking Aeromonas such as aeromonas salmonicida, also to intestines
Bacteriaceae such as Salmonella, vibrios and Escherichia coli have the bacteriophage of certain cracking performance.
One aspect of the present invention is related to aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), preservation
Entitled AS-ZJ, is preserved in China typical culture collection center, and deposit number is： CCTCC NO:M 2017091；During preservation
Between be：On March 6th, 2017.
The bacteriophage is preserved in China typical culture collection center (CCTCC), and preservation address is：Wuhan City, Hubei Province
Wuchang District Bayi Road Luo Jia Shan, Wuhan University's China typical culture collection center；The preservation time is：On March 6th, 2017.Through
Collection was detected as survival strains on March 16th, 2017.
The bacteriophage of the present invention can not only crack Aeromonas such as aeromonas salmonicida, and can also crack enterobacteria
Section such as Salmonella, vibrios and Escherichia coli.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the phage selection flow chart in the embodiment of the present invention；
Fig. 2 is the plaque morphology of aeromonas salmonicida bacteriophage；
Fig. 3 is the adsorption curve of aeromonas salmonicida bacteriophage；
Fig. 4 is the one step growth curve of aeromonas salmonicida bacteriophage；
Fig. 5 is the infection curve map that host mixes with individual plant aeromonas salmonicida bacteriophage；A：MOI=0.1；B：MOI=
1；
Fig. 6 is the infection curve map that host mixes with aeromonas salmonicida bacteriophage cocktail.
Aeromonas salmonicida bacteriophage AS-YJ (Aeromonas salmonicida phage AS-YJ) provided by the present invention,
Preserving number is CCTCC NO:M 2017095；
Aeromonas salmonicida bacteriophage AS-GZ (Aeromonas salmonicida phage AS-GZ) provided by the present invention,
Preserving number is CCTCC NO:M 2017094；
Aeromonas salmonicida bacteriophage AS-SW (Aeromonas salmonicida phage AS-SW) provided by the present invention,
Preserving number is CCTCC NO:M 2017093；
Aeromonas salmonicida bacteriophage AS-SZW (Aeromonas salmonicida phage AS- provided by the present invention
SZW), preserving number is CCTCC NO:M 2017092；
Aeromonas salmonicida bacteriophage AS-ZJ (Aeromonas salmonicida phage AS-ZJ) provided by the present invention,
Preserving number is CCTCC NO:M 2017091；
The preservation address of above-mentioned bacterial strains is：Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan, Wuhan University's Chinese Typical Representative training
Support thing collection；The preservation time is：On March 6th, 2017.Survival is detected as on March 16th, 2017 through collection
Bacterial strain.
Embodiment
The first aspect of the present invention is provided in addition to selectivity cracking aeromonas salmonicida category such as aeromonas salmonicida, also
To the bacteriophage of enterobacteriaceae such as Salmonella, vibrios and E. coli lytic.
One aspect of the present invention is related to aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), preservation
Entitled AS-ZJ, is preserved in China typical culture collection center, and deposit number is：CCTCC NO:M 2017091；The preservation time
For：On March 6th, 2017.
Bactericidal composition containing bacteriophage as described above.
The present invention establishes a kind of feasible method, and efficiently quickly salmon gas is killed in the separating broad spectrum resistance Atlantic Ocean from environment
The bacteriophage of monad.In one embodiment, by recognizing single bacteriophage for infecting the characteristic of host, assess single
The infection ability of bacteriophage, bacteriophage cocktail is built using certain mixed method, is determined finally by specific indexes optimal
Cocktail composition, the bacteriophage cocktail both have bacteriophage selectivity feature, single phagotherapy is compensate for again easy
Cause the deficiency of Host Strains resistance, application prospect is extensive.
The bacteriophage bactericidal composition that the present invention is provided can quickly suppress Atlantic Ocean aeromonas salmonicida in a short time
Growth, and suppress the generation of the pathogen phage resistance.In addition, the bacteriophage cocktail is safely, effectively, and high specificity,
Production cost is low, and the deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
The invention further relates to a kind of bactericidal composition containing bacteriophage as described above and/or its mutant, for treating
And/or prevention animal such as fish furunculosis or canker.
The invention further relates to a kind of bactericidal composition containing bacteriophage as described above and/or its mutant, for killing
And/or prevention aeromonas salmonicida, Salmonella, vibrios and Escherichia coli.
It is preferred that, the bactericidal composition also includes aeromonas salmonicida bacteriophage AS-GZ, and preserving number is：CCTCC NO:
M 2017094；
Aeromonas salmonicida bacteriophage AS-SW, preserving number is CCTCC NO:M 2017093；
Aeromonas salmonicida bacteriophage AS-YJ, preserving number is CCTCC NO:M 2017095；And
Aeromonas salmonicida bacteriophage AS-SZW, preserving number is CCTCC NO:One or more in M 2017092；
Above-mentioned bacteriophage is preserved in China typical culture collection center, and the preservation time is：On March 6th, 2017.Through
Collection was detected as survival strains on March 11st, 2017.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ and AS-GZ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ and AS-SW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ, AS-SW and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ, AS-SW and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ, AS-YJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-GZ, AS-SW, AS-SZW and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ and AS-SW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-SW and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-SW and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-YJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly aeromonas salmonicida
Bacteriophage AS-ZJ, AS-SW, AS-YJ and AS-SZW.
In certain embodiments of the present invention, the bactericidal composition is also including AS-GZ mutant, AS-SZW
One or more in mutant, AS-YJ mutant, AS-ZJ mutant and AS-SW mutant
It is preferred that, the mutant sequence at least 90% is identical with the native sequences of corresponding bacteriophage.
Because virus is very easy to undergo mutation in a replication process, thus preferred, the mutant of above-mentioned bacteriophage
In the range of the application is claimed.AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW mutant at least 90% and institute
The native sequences for stating bacteriophage are identical；And the mutant has the work(of killing pathogen roughly the same with initial bacteriophage
Energy.It is furthermore preferred that mutant has the native sequences phase of 92%, 94%, 96%, 98% or 99% and each self-corresponding bacteriophage
Together.
Bacteriophage AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW mutant can be point mutation, deletion mutation or addition
Mutation, relative to initial bacteriophage sequences, there is 1,2,3,4,5,6,7,8,9,10 or more
Base can change.To those skilled in the art, the phage selection provided according to the present invention goes out and its character phase
As mutant and would not require any inventive effort.
As described above, bacteriophage can exist to induce bacteriolysis with sufficiently high concentration, when mixture resulting mixture,
It is preferred that, bactericidal composition as described above, content >=10 of every kind of aeromonas salmonicida bacteriophage in the composition6PFU/
mL；
It is preferred that, bactericidal composition as described above, the content of every kind of aeromonas salmonicida bacteriophage in the composition
For 106PFU/mL~1010PFU/mL, more preferably 106PFU/mL~109PFU/mL, more preferably 106PFU/mL~108PFU/
ML, more preferably 106PFU/mL~107PFU/mL, can also take 2 × 106PFU/mL, 3 × 106PFU/mL, 4 × 106PFU/mL,
5×106PFU/mL, 6 × 106PFU/mL, 7 × 106PFU/mL, 8 × 106PFU/mL, 9 × 106PFU/mL, 5 × 107PFU/
ML, 5 × 108PFU/mL, 5 × 109The intermediate values such as PFU/mL.
It is preferred that, as AS-ZJ and AS-GZ, AS-SW, AS-YJ, when one kind in AS-SZW or many plants carry out mixture, by sense
The mode of the ratios such as dye plural number is mixed.
It is preferred that, bactericidal composition as described above, the bactericidal composition also includes auxiliary material；
The auxiliary material is the one or more in SM buffer solutions, sodium alginate, sucrose, maltodextrin, glucose；
The compound method of SM buffer solutions is conventional method, for example：NaCl 5.8g, MgSO4·7H2O 2g, 1mol/L's
TrisHCl 50mL (pH=7.0), 5mL 2%gelatin, add pure water and complement to 1000mL.
It is preferred that, the bacteriophage of the bactericidal composition also specific pathogen bacterium including variety classes bacterium.
Above-mentioned bactericidal composition can be used as virus formulation, and formulation used can be various common dosage forms, such as pulvis, water
Agent, freeze-dried, gel, creme, paste etc..
It is preferred that, bactericidal composition as described above is killing aeromonas salmonicida, Salmonella, vibrios and large intestine bar
Application in bacterium；The application is therapeutical uses or non-therapeutic use.
It is preferred that, aeromonas salmonicida bacteriophage as described above or bactericidal composition are being prepared for preventing and treating animal furuncle
Application in the medicine of sore disease or canker；
It is preferred that, application as described above, the animal includes：The cold-blooded property animal of warm-blooded animal and part
It is preferred that, application as described above, the animal is fish；
It is preferred that, application as described above, the fish are salmon fishes；
It is furthermore preferred that application as described above, the salmon fishes include cherry salmon, humpbacked salmon, big
Fiber crops breathe out fish, Ishikawa taimen, taimen, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Coregonus ussuriensis,
Card reaches whitefish, Atlantic salmon, Pacific Ocean salmon, silverside, rainbow trout, grayling, golden trout.
A kind of method for preventing and treating fish furunculosis or canker, bactericidal composition as described above is added to as medicine
In fish meal, or fish body surface is sprayed, or gavaged to fish, or give fish injection, or the bactericidal composition is dissolved in water
In contacted again with fish.
It is preferred that, method as described above, the fish are salmon fishes；
It is furthermore preferred that method as described above, the salmon fishes include cherry salmon, humpbacked salmon, big
Fiber crops breathe out fish, Ishikawa taimen, taimen, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Coregonus ussuriensis,
Card reaches whitefish, Atlantic salmon, Pacific Ocean salmon, silverside, rainbow trout, grayling, golden trout.
In some embodiments, the present invention is directed to causes aquatic products Salmons to occur by Atlantic Ocean aeromonas salmonicida
The problem of furunculosis and routed enteropathy, occur and the application of single bacteriophage in aeromonas salmonicida drug resistance caused by abuse of antibiotics
On the basis of defect, pathogenic salmon gas is killed with one plant isolated from the Atlantic salmon muscle of exanthemv, liver, nephrosis stove
Monad is host, using double-layer agar technique, isolated 10 plants of lytic phages from water environment, by recognizing single bite
The infectivity of thalline, so as to assess its infection ability to Host Strains, is finally built and is determined using certain mixed method
Optimal cocktail composition.The bacteriophage cocktail can quickly suppress the growth of Atlantic Ocean aeromonas salmonicida in a short time,
And suppress the generation of the pathogen phage resistance.Safely, effectively, and high specificity, production cost is low for the bacteriophage cocktail,
The deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
It is used to efficiently crack the research of Atlantic Ocean aeromonas salmonicida bacteriophage cocktail agent the invention discloses one kind.With
Pathogenic aeromonas salmonicida from the Atlantic salmon muscle of exanthemv, liver, one plant of wide spectrum resistance of kidney separation is host, from
Isolated 10 plants of fragility bacteriophages in water environment, cracking diameter is all higher than 0.3mm, is evaluated with individual phage lysis efficiency
It is 0.1 to optimal infection proportion MOI, with reference to indexs such as the adsorption curve of individual phage, one step growth curve and highest potency
The individual phage that screening can add bacteriophage cocktail is AS-SZW, AS-YJ, AS-ZJ, AS-SW, AS-GZ.Pass through arrangement
Combination, the burst size and pyrolysis time of Host Strains obtain high in one group of short time as foundation after being trained altogether using Host Strains and bacteriophage
The bacteriophage cocktail thereof of effect cracking Atlantic Ocean aeromonas salmonicida.In addition, further to analyze the bacteriophage cocktail
Environmental resistance is analyzed, it is determined that the pH tolerances scope of two groups of bacteriophage cocktail thereofs is 3~11,70 DEG C of temperature more than
Under still have certain activity, and in seawater cultivate 7d potency slightly have reduction, about 80%.The side mixed using conventional liq
Formula, the bacteriophage cocktail can quickly suppress the growth of Atlantic Ocean aeromonas salmonicida, the growth curve OD600 in the range of 80h
It is worth for less than 0.05.The above five plants of bacteriophages AS-SZW, AS-YJ, AS-ZJ, AS-SW, AS-GZ are preserved in Chinese Typical Representative training
Support thing collection, preservation date on March 6th, 2017, deposit number is respectively CCTCC NO:M 2017092、CCTCC NO:M
2017095、CCTCC NO:M 2017091、CCTCC NO:M 2017093、CCTCC NO:M 2017094.Bacteriophage of the present invention
Screening process figure it is as shown in Figure 1.Specifically, the present invention is based on common antibiotics species on the market, using diffusion method (K-
B methods) detect the pathogenic bacteria to chloramphenicol, Florfenicol, Enrofloxacin, tetracycline, CTX, Doxycycline, pipemidic acid, promise
Flucloxacillin, Ofloxacin, Ceftizoxime, cephazoline, gentamicin, SMZco, rifampin, vancomycin, erythromycin,
Streptomysin, neomycin, card receive the medicines of 24 kinds of medicines such as mycin, TOB, penicillin, neomycin, furazolidone, furantoin
Quick experiment, confirms that the pathogenic aeromonas salmonicida has very strong drug resistance to preceding 11 kinds of antibiotic.
More than walk in the aeromonas salmonicida that confirms be host, using double-layer agar technique and combine sample characteristic from seawater sample
Bacteriophage is separated in product, speculum is determined by many subinfections, the single plaque of picking is isolated and purified, most finally -80 DEG C
Glycerine (20%) is preserved.During this separation and subsequent analysis used medium are LB meat soups/agar medium, double-layer plate,
Bottom is 1.5%LB agar mediums, and upper strata is 0.7% agar medium.
The genome of each bacteriophage is extracted using phage genome extraction agent box (Aidlab, DN22), after use Miseq
Platform carries out genome sequencing, and splicing and the annotation of sequence are carried out to sequencing result, and sequence determines AS- after being compared through Blastn
Five plants of bacteriophages of SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ are aeromonas salmonicida bacteriophage, wherein preceding 4 plants of bacteriophages
It is respectively 97%, 97%, 96%, 96% with the Aeromanas phage CC2 whole genome sequence similitudes reported,
AS-GZ and the aeromonas salmonicida bacteriophage phiAS4 reported similitude are 97%.
The evaluation of the single morphology of phages and microbiology level：1) morphologic observation：Single phage-infect Host Strains
Afterwards, spread after double-layer plate, 30 DEG C of culture 12h, determine original speculum size with slide measure, obtain bacteriophage AS-SZW, AS-
YJ, AS-ZJ, AS-SW, AS-GZ size are respectively 0.83mm, 0.48mm, 0.5mm, 0.5mm, 1mm.Plaque morphology such as Fig. 2
It is shown.2) measure of adsorption capacity：The Host Strains (aeromonas salmonicida) of bacteriophage and logarithmic phase are mixed with MOI=0.005,
Mixed-culture medium is taken according to time point (0,1,2,3,4,5,6,10,20,30min) is pre-seted, after 16000g centrifugations, using double
Layer flat band method determines free state bacteriophage, and AS-SZW, AS-YJ, AS-ZJ, AS-SW, AS-GZ adsorption times point are obtained according to subtraction
Wei not 5min, 10min, 10min, 5min, 20min；The adsorption curve of bacteriophage is as shown in Figure 3.3) bacteriophage one step growth is bent
The measure of line, MOI is 0.01 (host OD600=0.5), and Host Strains at advance 30 DEG C of bacteriophage with being incubated 5min, then
12000r/min centrifuges 30s, removes clear liquid, adds 5ml LB and suspends, is cultivated at last 30 DEG C, every 10min samplings, altogether training
Support 90min (AS-SW is 120min).The potency of determination sample, obtains AS-SZW, and AS-YJ, AS-ZJ, AS-SW, AS-GZ's splits
The solution cycle is respectively 80min, 40min, 40min, 100min and 60min, and burst size is respectively 145,98,86,86 and 135；Bite
The one step growth curve of thalline is as shown in Figure 4.4) measure of host range, and according to speculum shape on double-layer plate after infection culture
Into feature, evaluate sensitiveness (table 1 shown in) of the bacteriophage to 40 plants of selected indicator bacterias, obtain 5 plants of bacteriophage decapacitation of the above
Outside aeromonas salmonicida under selectivity cracking Aeromonas, to the part Salmonella under enterobacteriaceae, vibrios and big
Enterobacteria has faint cracking performance.In addition, determining the optimal bacteriophage chicken determined in above-mentioned experimental result using universal method
Tail wine AS-cocktail2-2,5 host's spectral limit obtains the superposition that host range is individual phage AS-YJ and AS-GZ.
5 plants of bacteriophages and the host range of optimal cocktail thereof that the present invention of table 1 is provided
Note："-" represents that without infectivity, "+" represents infectious, forms transparent circle, but do not form speculum, " ++ "
Strong infectivity is indicated, speculum can be formed；" * " is expressed as the Host Strains of this patent content bacteriophage
The combined method of bacteriophage cocktail：With four kinds of infection multiplicities (MOI=0.01,0.1,1 or 10) mixing individual plant bite
Thalline and Host Strains, the growth curve (Fig. 5) after bacteriophage is infected according to Host Strains and determines (OD600 values) evaluation phage splitting
Efficiency, it is determined that optimal MOI values, and according to the phage-infect of optimal MOI progress Host Strains, will be preselected with permutation and combination method
Bacteriophage is mixed, and is respectively combined according to 1:1,1:1:1,1:1:1:1 or 1:1:1:1:After 1 is mixed, by the place of logarithmic phase
Main bacterium carries out mixing co-cultivation with bacteriophage.
The determination of optimal bacteriophage cocktail：Bacteriophage is mixed with Host Strains after co-cultivation, by determining nutrient solution OD600
The change of value, draws the growth curve (Fig. 6) of Host Strains, it is determined that optimal bacteriophage cocktail thereof；Label 1 wherein in figure, 2,
3rd, 4,5 five plants of bacteriophages of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ are represented successively.A length of 80h during measure.Obtain most
Good bacteriophage cocktail thereof is Cocktail2-2,5 (i.e. AS-YJ and AS-GZ), its growth curve OD600 in the range of 80h
It is worth for less than 0.01
The evaluation of optimal bacteriophage cocktail envirment factor tolerance：Determine optimal cocktail thereof in above-mentioned experimental result
PH, temperature and tolerance in the seawater.Wherein pH, temperature tolerance are determined as：The bacteriophage of a certain amount of potency
(100ul, 108PFU/mL in the EP pipes for) being added to the 1.5mL of liquid containing 900ul (LB fluid nutrient mediums), in different pH (pH
=2,4,6,8,10,12；Adjusted with 0.1mol HCl or NaOH to specified pH) and temperature (4 DEG C, 20 DEG C, 37 DEG C, 60 DEG C, 80
DEG C) under cultivate, rear sampling dilution suitable multiple simultaneously spreads double-layer plate and determines potency at once.Wherein pH minute is 3h, temperature
It is, every 15min samplings, 45min to be determined altogether to spend the setting time.Tolerance in briny environment determines consistent with temperature measuring, and one
Bacteriophage (100ul, 10 of quantitative potency8PFU/mL) being added to seawater containing 900uL, (it is big that seawater sample is derived from Bay in Shenzhen, Shenzhen
Mei Sha, Huizhou Xun Liaowan and Zhuhai Port, 8000r/min centrifugation 10min, 0.22 μm filtering) 1.5mL EP pipes in, in 20
Cultivated at DEG C, every 1d samplings, cultivate one week (7d), determine bacteriophage activity, determine bacteriophage activity.Obtain the bacteriophage chicken
Tail wine is still active between pH 3~11, and its optimum pH is 8, and 30min still keeps certain cracking at a temperature of 60 DEG C
Activity, about 70%, to its activity influence less, 30min is inactivated at 80 DEG C for 37 DEG C and temperature below, and its lytic activity is less than
The potency in 7d is cultivated in detection line, seawater without significantly reducing, and indifference between each seawater sample.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), the entitled AS-ZJ of preservation, in being preserved in
State's Type Tissue Collection, deposit number is：CCTCC NO:M 2017091；The preservation time is：On March 6th, 2017.
2. the bactericidal composition containing the aeromonas salmonicida bacteriophage described in claim 1.
3. bactericidal composition according to claim 2, it is characterised in that the bactericidal composition also includes killing salmon gas unit cell
Bacterium bacteriophage AS-GZ, preserving number is：CCTCC NO:M 2017094；
Aeromonas salmonicida bacteriophage AS-SW, preserving number is CCTCC NO:M 2017093；
Aeromonas salmonicida bacteriophage AS-YJ, preserving number is CCTCC NO:M 2017095；And
Aeromonas salmonicida bacteriophage AS-SZW, preserving number is CCTCC NO:One or more in M 2017092；
Above-mentioned bacteriophage is preserved in China typical culture collection center, and the preservation time is：On March 6th, 2017.
4. bactericidal composition according to claim 3, it is characterised in that the bactericidal composition kills salmon gas list including described
Born of the same parents bacterium bacteriophage AS-YJ, the aeromonas salmonicida bacteriophage AS-ZJ and the aeromonas salmonicida bacteriophage AS-GZ；
Or, the bactericidal composition includes the aeromonas salmonicida bacteriophage AS-SZW, the aeromonas salmonicida bacteriophage
AS-YJ, the aeromonas salmonicida bacteriophage AS-ZJ and the aeromonas salmonicida bacteriophage AS-GZ；
Or, the bactericidal composition includes the aeromonas salmonicida bacteriophage AS-ZJ and the aeromonas salmonicida phagocytosis
Body AS-GZ.
5. the bactericidal composition according to any one of claim 2~4, it is characterised in that also wrapped in the bactericidal composition
Include the one or more in each bacteriophage mutants.
It is preferred that, the mutant at least 90% is identical with the native sequences of corresponding aeromonas salmonicida bacteriophage.
6. the bactericidal composition according to any one of claim 2~4, it is characterised in that every kind of in the bactericidal composition
Content >=10 of aeromonas salmonicida bacteriophage6PFU/mL；
It is preferred that, the content of every kind of aeromonas salmonicida bacteriophage is 10 in the composition6PFU/mL~107PFU/mL。
7. the bactericidal composition according to any one of claim 2~4, it is characterised in that the bactericidal composition also includes
Auxiliary material；
It is preferred that, the auxiliary material is the one or more in SM buffer solutions, sodium alginate, sucrose, maltodextrin, glucose；
It is preferred that, the bacteriophage of the bactericidal composition also specific pathogen bacterium including variety classes bacterium.
8. the bactericidal composition according to any one of claim 2~4, it is characterised in that the formulation of the bactericidal composition
For pulvis, aqua, freeze-dried, gel, creme, paste.
9. the bactericidal composition described in aeromonas salmonicida bacteriophage or any one of claim 2~8 described in claim 1
Application in killing and/or preventing aeromonas salmonicida, Salmonella, vibrios and Escherichia coli.
10. the bactericidal composition described in aeromonas salmonicida bacteriophage or any one of claim 2~8 described in claim 1
Preparing the application in being used to treat and/or prevent animal such as fish furunculosis or the medicine of canker；
It is preferred that, the fish are salmon fishes；
It is furthermore preferred that the salmon fishes include cherry salmon, humpbacked salmon, chum salmon, Ishikawa taimen, wise man
Sieve fish, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Coregonus ussuriensis, card reach whitefish, Atlantic salmon, too
Flat ocean salmon, silverside, rainbow trout, grayling, golden trout.
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| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
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| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
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| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119024A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119025A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107022529B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107119025B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107119024B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN108148815A (en) * | 2017-12-11 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | The bacteriophage of one plant of fish bacteria and its application |
| CN113528461A (en) * | 2021-07-15 | 2021-10-22 | 华东理工大学 | Isolated Aeromonas salmonicida phage, compositions and uses thereof |
| CN113528461B (en) * | 2021-07-15 | 2023-10-24 | 华东理工大学 | Isolated aeromonas salmonicida phage, compositions and uses thereof |
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