CN108048412A - Fragility Vibriophage ValLY-4 and the bactericidal composition comprising the bacteriophage and its application - Google Patents
Fragility Vibriophage ValLY-4 and the bactericidal composition comprising the bacteriophage and its application Download PDFInfo
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Abstract
The present invention relates to fragility Vibriophage ValLY 4 and the bactericidal composition comprising the bacteriophage and its applications.The bacteriophage can quickly inhibit the growth of vibrios in a short time, and inhibitory action is held time length.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 compensated for during the control of pathogenic bacteria.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of fragility Vibriophage ValLY-4 and comprising
The bactericidal composition of the bacteriophage and its application.
Background technology
China is aquaculture big country.In recent years, the culture fishery in China is grown rapidly.As sea-farming is advised
The continuous popularization being growing with intensive breeding way of mould, the frequent of fish disease occur to seawater fish large-scale cultivation
Sound development cause serious threat.The pathogen of fish mainly includes bacterium, virus, parasite etc., wherein bacillary
Disease is a kind of disease more serious to the fish extent of injury.
Vibrios is movable gemma rod-short bacterium, is distributed widely in inshore, mouth of sea area seawater and organism,
It is one of most common bacterial groups in ocean, while is also to cause the most important pathogen of mariculture fish shrimp bacterial disease
One of.The pathogenicity of vibrios is related with the composite factors such as the physiological status of host and aquatic environment condition, belongs to conditionity cause
Germ mainly by mouth or wound infection, generates toxin, causes the serious disease of the suppuration of wound muscle ulceration and internal organs
Become the death so as to cause fish and shrimp.Disease is also known as vibriosis as caused by vibrios, with " popular area is wide, and incidence is high, causes
Dead property is strong " the features such as, it is the principal disease of aquiculture animal, while is also determined as being the weight for hindering culture fishery development
Restrictive factor is wanted, huge harm is caused to aquaculture.Wherein, with Vibrio anguillarum (Vibrio anguillarum), molten algae
Vibrios (Vibrio alginolyticus), Vibrio harveyi (Vibrio harveyi), vibrio parahaemolytious (Vibrio
Parahaemolyticus) and vibriosis caused by the vibrios such as Vibrio salmonicida (Vibrio salmonicida) is considered as fishes and shrimps
One of disease the most serious in class cultivation.At present, the prevention and control method for aquaculture relevant animal disease are main
With chemobiotic method and immune vaccine method, wherein based on chemobiotic use.As current control aquatic animal disease
Main means, chemobiotic method has many advantages, such as easy to use, quick and good effect.But high-volume frequently uses
Antibiotic is very big to have encouraged the drug resistance of pathogen while pathogen is controlled, and accelerates the generation of wide spectrum drug-fast bacteria.
This causes the drug resistance problems getting worse of pathogenic bacteria.In addition, chemicals residue is also that mankind's food origin disease is caused to generate
One of the major reasons, human health is caused to seriously endanger.
Bacteriophage is also known as bacterial virus, is the virus of a kind of specificity cracking bacterium, widely distributed in the environment, species
It is various, it is simple in structure, there is very strong host specificity.The most important, the mechanism of phage splitting bacterium is from bacterium
Own genetic material by Receptor recognition so as to adsorb on specific host bacterium surface, is injected host by the influence of drug resistance
Interior progress self-replacation assembling, and filial generation is discharged so as to fulfill the growth and breeding of self eventually by cracking host strain.Using biting
Thalline controls aquatic pathogenic bacteria just to have application before bacteriophage is found, but due to being not enough to the research of bacteriophage,
And antibiotic had broad-spectrum antibacterial effect at that time, and good effect is quick, this causes bacteriophage application to be weakened severely.In recent years
Come, a series of drug-fast bacteria problems triggered due to abuse of antibiotics so that bacteriophage returns the visual field of people again, using phagocytosis
The research of body control pathogenic bacteria also causes broad interest in scientific circles.One of important weapon as reply antibiotic resistance,
The clinical practice of bacteriophage has a high potential, but for the application of its current single phagotherapy, in application process still
There are some it is potential the shortcomings that, such as crack host range it is narrow, the cracking ability duration is short, and host strain is also easy to produce resistance etc., wherein
The major defect that resistance applies as individual phage is also easy to produce using host strain.
For deficiency existing for above single phagotherapy, the cocktail therapy that a variety of bacteriophages are used in mixed way is met the tendency of
And it gives birth to.It had both expanded host's spectral limit by the compounding between a variety of bacteriophages, while effectively inhibited host strain resistance
It generates, there is splitting action rapidly and efficiently, obtained preferable effect in the research of clinical practice at present.In addition, phagocytosis
Body cocktail reduces the additive capacity of the individual phage under the conditions of equal by the synergistic effect between bacteriophage, plays
Cost-effective effect.
In view of this, it is special to propose the present invention.
The content of the invention
The present invention is on the problem of fish and shrimp disease is propagated without restraint in aquaculture as caused by vibrios, in antibiotic mistake
On the basis of degree use causes the generation of vibrios endurance strain, kinds of pathogenic vibrio is separated from diseased fishes and shrimps lesion, and is passed through existing
Experiment determines the pathogenic of the vibrios pathogen, using the kinds of pathogenic vibrio as host, from water environment isolated 7 plants it is new
Fragility Vibriophage, pass through morphologic observation, it is determined that the classification position of the Vibriophage, and pass through genome sequencing
The genotype of 7 plants of bacteriophages is determined.In addition, by tablet drop method, according to the presence or absence of speculum, determine vibrios and bite
Host range of the thalline under 32 plants of different genera bacterial strains.By recognizing infectivity of the single bacteriophage to host strain, further
Bacteriostasis of the individual phage under different gradient infection multiplicities (MOI) is had evaluated, and then according to each bacteriophage most cause of disease
Property vibrios cracking ability be combined with 2 bacteriophage cocktail thereofs, by under the conditions of individual phage determine MOI so as to
This 2 combinations are further had evaluated to the growth inhibition effects of vibrio pathogen, thus we determine optimal vibrios phagocytosis
Body cocktail thereof.We are controlling the aquatic livestock pathogenic bacteria as caused by vibrios with certain potentiality, application by the bacteriophage
Space is extensive.
One aspect of the present invention is related to a kind of fragility Vibriophage ValLY-4, and Chinese allusion quotation is preserved within 25th in September in 2017
Type culture collection, deposit number are：CCTCC NO:M 2017553, Classification And Nomenclature are Vibrio phage.
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 September 25th, 2017.Through
Collection was detected as survival strains on October 10th, 2017.
Fragility Vibriophage ValLY-4 provided by the present invention has many excellent in vibrio infection disease is treated
Gesture, it can be to drug-fast bacteria also with good control effect first；Secondly as the fungicidal spectrum of bacteriophage is relatively narrow, complicated thin
When bactericidal effect occurs for --- such as animal intestinal tract --- in the environment of bacterium, probiotics will not be damaged；3rd, potency
Higher, control effect is good.
However bacterium can develop immunity to drugs to antibiotic by Natural Selection or artificial screening, it similarly, also can be to single
Bacteriophage be gradually resistant to.In view of this, for the present invention by recognizing single bacteriophage for infecting the characteristic of host, assessment is single
The infection ability of bacteriophage builds bacteriophage cocktail using certain mixed method, is determined most preferably finally by specific indexes
Cocktail composition, which had not only had the specificity feature of bacteriophage, but also to compensate for single phagotherapy easy
Cause the deficiency of host strain resistance, application prospect is extensive.
According to an aspect of the present invention, the invention further relates to contain fragility Vibriophage ValLY-4's as described above
Bactericidal composition.
In certain embodiments of the present invention, the bactericidal composition contains the fragility Vibriophage ValLY-4
And at least one of other Vibriophages；Preferably fragility Vibriophage.
Since route of infection, the molecular basis between viral (bacteriophage) of the same race are respectively provided with very big similitude, work as
It may promote efficiency of infection during bacteriophage co-infection host of the same race to each other, play synergistic effect.
In certain embodiments of the present invention, the bactericidal composition further includes fragility Vibriophage VpaJT-1,
Preserving number is：CCTCC NO:M 2017547；
Fragility Vibriophage ValSw4-1, preserving number are CCTCC NO:M 2017552；
Fragility Vibriophage VspSw-1, preserving number are CCTCC NO:M 2017551；
Fragility Vibriophage ValLY-2, preserving number are CCTCC NO:M 2017554；
Fragility Vibriophage ValLY-3, preserving number are CCTCC NO:M 2017548；
And fragility Vibriophage ValDsh-1, preserving number are CCTCC NO:One or more in M 2017549
Above-mentioned bacteriophage was preserved in China typical culture collection center on 25th in September in 2017, and Classification And Nomenclature is
Vibrio phage。
Bacteriophage bactericidal composition provided by the invention can quickly inhibit the growth of vibrios in a short time, and inhibit the disease
The generation of opportunistic pathogen phage resistance.In addition, the bacteriophage cocktail is safely, effectively, and high specificity, production cost is low, is causing
The deficiency of single phagotherapy is compensated for during the control of germ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4 and VpaJT-1, ValSw4-1, VspSw-1, ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1 and ValSw4-1, VspSw-1, ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValSw4-1 and VspSw-1, ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VspSw-1 and ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, ValSw4-1 and VspSw-1, ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, VspSw-1 and ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValSw4-1, VspSw-1 and ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValSw4-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、ValSw4-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VspSw-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VspSw-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, ValSw4-1, VspSw-1 and ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, ValSw4-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValSw4-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, VspSw-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、VspSw-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValSw4-1, VspSw-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、ValSw4-1、VspSw-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, ValSw4-1, ValLY-2, ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VspSw-1、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
Any one of ValLY-4, VpaJT-1, ValSw4-1, VspSw-1, ValLY-2 and ValLY-3, ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValSw4-1、VspSw-1、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValSw4-1、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、VspSw-1、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、ValSw4-1、VspSw-1、ValLY-2、ValLY-3、ValDsh-1；
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly Vibriophage
ValLY-4、VpaJT-1、ValSw4-1、VspSw-1、ValLY-2、ValLY-3、ValDsh-1。
In certain embodiments of the present invention, the bactericidal composition further includes the mutant of VpaJT-1, ValLY-4
Mutant, the mutant of ValSw4-1, the mutant of VspSw-1, the mutant of ValLY-2, ValLY-3 mutant with
And the one or more in the mutant of ValDsh-1.
Preferably, the mutant sequence at least 90% is identical with the native sequences of corresponding bacteriophage.
Since 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.Homology can suitably be measured by computer program commonly known in the art,
Such as provided in GCG program bags notch program (Wisconsin software package procedure manual, the 8th edition, in August, 1994, science of heredity meter
Suan Ji groups, 575 science roads, Madison, the state of Wisconsin, the U.S. 53711) (Needleman, S.B. and Wunsch,
C.D., (1970)《Molecular biology impurity》48,443-453).The following setting compared by being used for DNA sequence dna lacks to use
Mouth program：Gap opening penalty 5.0, gap extension penalty 0.3.VpaJT-1、 ValLY-4、ValSw4-1、VspSw-1、
The mutant of ValLY-2, ValLY-3 and ValDsh-1 at least 90% is identical with the native sequences of the bacteriophage；It is and described
Mutant have the function of it is roughly the same with initial bacteriophage kill pathogen, vibrios phagocytosis is fallen on taxology
Body.It is furthermore preferred that mutant has the day of 92%, 94%, 95%, 96%, 97%, 98% or 99% and corresponding bacteriophage
Right sequence is identical.Wherein, VpaJT-1, ValLY-4, ValSw4-1, VspSw-1, ValLY-2, ValLY-3 and ValDsh-1
Sequence can the biomaterial of preservation according to the present invention be sequenced to obtain by well known method.
Bacteriophage VpaJT-1, ValLY-4, ValSw4-1, VspSw-1, ValLY-2, ValLY-3 and ValDsh-1's
Mutant can be point mutation, deletion mutation or addition mutation, compared with initial bacteriophage sequences, have 1,2,3,4,
5,6,7,8,9,10 or more bases can change.To those skilled in the art, according to this hair
The phage selection of bright offer goes out the mutant similar to its character and would not require any inventive effort.
As described above, bacteriophage can exist with sufficiently high concentration to induce bacteriolysis, when mixture resulting mixture,
Preferably, bactericidal composition as described above, content >=10 of each fragility Vibriophage in the composition6PFU/mL；
Preferably, bactericidal composition as described above, the content of each fragility Vibriophage is in the composition
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.
Preferably, as VpaJT-1, ValLY-4, ValSw4-1, VspSw-1, ValLY-2, ValLY-3 and ValDsh-
When one kind in 1 or more plants carry out mixture, mixed in the way of the ratios such as infection multiplicity.
Preferably, bactericidal composition as described above, the bactericidal composition further include auxiliary material；
The auxiliary material is SM buffer solutions, the one or more in sodium alginate, sucrose, maltodextrin, glucose；
The preparation method of SM buffer solutions is conventional method, such as：NaCl 5.8g, MgSO4·7H2O 2g, 1mol/L's
TrisHCl 50mL (pH=7.0), 5mL 2%gelatin add in pure water and complement to 1000mL.
Preferably, the bactericidal composition further includes the bacteriophage of the specific pathogen bacterium of variety classes bacterium.
Above-mentioned bactericidal composition can be used as virus formulation, and dosage form used can be various common dosage forms, such as pulvis, water
Agent, freeze-dried, gelling agent, creme, paste etc..
Preferably, application of the bactericidal composition as described above in killing and/or preventing vibrios microorganism belonging to genus；It is described to answer
With for therapeutical uses or non-therapeutic use；
It is furthermore preferred that the vibrios microorganism belonging to genus includes vibrio alginolyticus V.alginolyticus, Vibrio anguillarum V.
Anguillarum, comma bacillus V.cholerae, non-Nonther conserved quantity non-O1V.cholerae, Fermi operator
V.fischeri, vibrio fluvialis V.fluvialis, Vibrio harveyi V.harveyi, sick Vibrio damsela V.ordalii, secondary haemolysis
Vibrios V.parahaemolyticus, Vibrio salmonicida V.salmonicida, vibrio mimicus V.mimicus, Vibrio splindidus
V.splendidus, Vibrio aureus V.azureus, Vibrio gazogenes V.gazogenes, Vibrio vulnificus V.vulnificus, bamboo
Pod vibrio piscium V.trachuri, damselfish vibrios V.damsela, floating vibrios V.natriegen, clam worm vibrios V.nereis, fish
Enteron aisle vibrios V. ichthyoenteri, shark vibrio V.carchariae, Vibrio campbellii V.campbellii and kill prawn
One or more in vibrios V.penaeicida；
It is furthermore preferred that the vibrios microorganism belonging to genus includes vibrio parahaemolytious V.parahaemolyticus, vibrio alginolyticus
One or more in V.alginolyticus, Vibrio aureus V.azureus.
Preferably, fragility Vibriophage or bactericidal composition as described above are moved in preparation for treating and/or preventing
Application in the drug of object vibriosis；
It is furthermore preferred that the drug is used to treat vibrio alginolyticus V.alginolyticus, Vibrio anguillarum V.
Anguillarum, comma bacillus V.cholerae, non-Nonther conserved quantity non-O1V.cholerae, Fermi operator
V.fischeri, vibrio fluvialis V.fluvialis, Vibrio harveyi V.harveyi, sick Vibrio damsela V.ordalii, secondary haemolysis
Vibrios V.parahaemolyticus, Vibrio salmonicida V.salmonicida, vibrio mimicus V.mimicus, Vibrio splindidus
V.splendidus, Vibrio aureus V.azureus, Vibrio gazogenes V.gazogenes, Vibrio vulnificus V.vulnificus, bamboo
Pod vibrio piscium V.trachuri, damselfish vibrios V.damsela, floating vibrios V.natriegen, clam worm vibrios V.nereis, fish
Enteron aisle vibrios V. ichthyoenteri, shark vibrio V.carchariae, Vibrio campbellii V.campbellii and kill prawn
The vibriosis caused by one or more in vibrios V.penaeicida；
It is furthermore preferred that the drug is used to treat vibrio parahaemolytious V.parahaemolyticus, vibrio alginolyticus
The vibriosis caused by one or more in V.alginolyticus, Vibrio aureus V.azureus；
Preferably, application as described above, the animal include：The cold-blooded property animal of warm-blooded animal and part；
Preferably, application as described above, the animal behaviour, fish, shrimp or mollusk (such as shellfish).
A kind of method for preventing animal vibriosis, is added to animal feed using bactericidal composition as described above as drug
In or spray to animal body surface gavage to animal or injected to animal or by the bactericidal composition it is soluble in water again with
Animal contact.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, can also be obtained according to these attached drawings other attached drawings.
Fig. 1 is the phage selection flow chart in the embodiment of the present invention；
Fig. 2 is the host strain chadogram based on 16s DNA；
3- vibrio parahaemolytious (Vibrio.parahaemolyticus)；18- vibrio alginolyticus (Vibrio.
alginolyticus)；F1- Vibrio aureus (Vibrio.azureus)；F3- vibrio alginolyticus (Vibrio.
alginolyticus)；F4- vibrios (Vibrio.sp)；F10- vibrio parahaemolytious (Vibrio. parahaemolyticus)；
Fig. 3 be as MOI=1 different bacteriophages cocktail thereof to the action diagram of 6 pathogen strain bacterium growth curves；
A-vibrio parahaemolytious (Vibrio.parahaemolyticus)；B-vibrio alginolyticus (Vibrio.
alginolyticus)；C-Vibrio aureus (Vibrio.azureus)；D-vibrio alginolyticus (Vibrio.
alginolyticus)；E-vibrios (Vibrio.sp)；f:- vibrio parahaemolytious (Vibrio. parahaemolyticus)；
Cocktail A：VpaJT-1,ValLY-4,ValSw4-1,VspSw-1,ValLY-2；cock-3： Cocktail
B：ValLY-3,VpaJT-1,VpaSw-1,ValSw4-1,ValDsh-1；Control：Bacterium solution+culture medium (is not added with bacteriophage)；
Blank：Culture medium.
Fragility Vibriophage VpaJT-1 (Vibrio phage VpaJT-1) provided by the present invention, preserving number is
CCTCC NO:M 2017547；
Fragility Vibriophage ValLY-4 (Vibrio phage ValLY-4) provided by the present invention, preserving number is
CCTCC NO:M 2017553；
Fragility Vibriophage ValSw4-1 (Vibrio phage ValSw4-1) provided by the present invention, preserving number is
CCTCC NO:M 2017552；
Fragility Vibriophage VspSw-1 (Vibrio phage VspSw-1) provided by the present invention, preserving number is
CCTCC NO:M 2017551；
Fragility Vibriophage ValLY-2 (Vibrio phage ValLY-2) provided by the present invention, preserving number is
CCTCC NO:M 2017554；
Fragility Vibriophage ValLY-3 (Vibrio phage ValLY-3) provided by the present invention, preserving number is
CCTCC NO:M 2017548；
Fragility Vibriophage ValDsh-1 (Vibrio phage ValDsh-1) provided by the present invention, preserving number is
CCTCC NO:M 2017549；
The preservation address of above-mentioned bacterial strains is：Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan, China of Wuhan University allusion quotation
Type culture collection；The preservation time is：On September 25th, 2017.Detected through collection on October 10th, 2017
For survival strains.
Specific 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 invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment
The present invention is by fishes and shrimps sample environmentally friendly in gathering Shanwei prawn culturing water sample, isolated 7 from its lesion
Strain kinds of pathogenic vibrio, the screening process figure of bacteriophage of the present invention are as shown in Figure 1.The cause of separation vibrios is confirmed by field experiment
Characteristic of disease, it is determined that wherein 5 plants pathogenic extremely notable, LD50<104CFU/mL, action time<100h.
By tablet drop method, according to the presence or absence of speculum, the Vibriophage is determined in 32 plants of different genera bacterial strains
Under host range (table 1), draw the bacteriophage have very strong host specificity, be vibrio specificity fragility bacteriophage.
1 host range of table
Remarks："+" expression can crack, and "-" expression cannot crack；More than bacterial strain from aquaculture fish and shrimp animal body and
It is isolated in cultivation water；ValLY-2, ValLY-3, ValLY-4, ValDsh-1, VspSw-1, VpaJT-1 and ValSw4-1
Represent Vibriophage number；Host strain chadogram is as shown in Figure 2.
By recognizing infectivity of the single bacteriophage to host strain, so as to have evaluated 3 plants of most wide bacteriophages of host range
Individual phage under different gradient infection multiplicities (MOI=10,1,0.1) draws single plant item to the bacteriostasis of host strain
Under part as MOI=10, the inhibitory action of vibrio pathogen significantly and is continued most to grow, OD600 is less than control in 15h
Group, as MOI≤1, inhibition is not notable, has thereby determined that the application conditions MOI=10 of the Vibriophage.It is bitten according to 6 plants
Thalline to the splitting action of kinds of pathogenic vibrio and host's spectral property, it is determined that 2 bacteriophage cocktail thereofs： A(VpaJT-
1,ValLY-4,ValSw4-1,VspSw-1,ValLY-2)；B(ValLY-3, VpaJT-1,VpaSw-1,ValSw4-1,
ValDsh-1), with 1 between each combination:1 ratio is added.And above-mentioned 2 combinations are evaluated under the conditions of MOI=10 to pathogenic arc
The growth inhibition effect of bacterium.Consider inhibition of each bacteriophage cocktail thereof to 7 plants of kinds of pathogenic vibrio, it is determined that group
It is optimum combination to close B (ValLY-3, VpaJT-1, VpaSw-1, ValSw4-1, ValDsh-1), compared with the control group, to corresponding
The concentration (OD600) of pathogenic bacteria plays inhibitory action.Thus, it is believed that the bacteriophage chicken being made of above 7 plants of bacteriophages
Tail wine has certain potentiality in control aquatic livestock pathogenic bacteria as caused by vibrios, and application space is extensive.
Specifically, fragility Vibriophage provided by the present invention and bactericidal composition screen to obtain by the following method：
The screening of kinds of pathogenic vibrio：The fish and shrimp sample of aquaculture factory illness is gathered, using selective medium from hair
It isolates and purifies to obtain 7 plants of kinds of pathogenic vibrio, RNA isolation kit (Omega DNA of bacteria extracts kit) extraction in the fishes and shrimps lesion of disease
The genomic DNA of single plant bacterial strain, is sequenced by 16S, is built chadogram with maximum similar sequences, and then is determined 7 plants of vibrios
Classification position.
The pathogenic detection of vibrios：By field experiment, using mid-term prawn as experimental subjects, 8 plants of vibrios are had rated to prawn
Lethal ability.30 prawns of every group of setting per observing for 24 hours once, are observed 1 week altogether.
Phage selection：Using the 6 plants of kinds of pathogenic vibrio confirmed in steps 1 and 2 as host, using double-layer agar technique and combine
Sample characteristic separates bacteriophage from water environment, by repeatedly infecting definite speculum, after isolating and purifying, individual phage
It is preserved in -80 DEG C of glycerine (20%).
Host range measures：Using drop method, nothing is formed with by observing speculum on host's tablet, it is determined that it is to the bacterium
The cracking performance of strain.
Infection ability is evaluated：Bacteriophage and logarithmic phase host strain are mixed with different gradient MOI (MOI=10,1,0.1)
(OD600=0.2), using the OD of host strain in growth measurement instrument detection 18h600Variation, every 30min records once.
Vibriophage cocktail thereof：According to 7 plants of bacteriophages to the fragmentation pattern of kinds of pathogenic vibrio, 2 combination (figures are obtained
3), it is respectively：A(VpaJT-1,ValLY-4,ValSw4-1,VspSw-1, ValLY-2)；B(ValLY-3,VpaJT-1,
VpaSw-1,ValSw4-1,ValDsh-1).Not homophyletic under af, bd belong to of the same race in Fig. 3, to the pathogenic difference of animal.
The host range respectively combined can cover the kinds of pathogenic vibrio that screening is drawn, the ratio in combination between each bacteriophage is 1:1.
The screening of optimal bacteriophage cocktail thereof：By the MOI determined under the conditions of individual phage, have evaluated each
Combine to the growth inhibition effect of vibrio pathogen, to inhibit vibrios growth in the short time, the inhibition duration it is most long with
And vibrios resistance time of occurrence is the latest requirement, draws optimal Vibriophage cocktail thereof.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution recorded in foregoing embodiments either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is not made to depart from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. fragility Vibriophage ValLY-4 was preserved in China typical culture collection center, preservation on 25th in September in 2017
Number is：CCTCC NO:M 2017553, Classification And Nomenclature are Vibrio phage.
2. the bactericidal composition containing fragility Vibriophage ValLY-4 described in claim 1；
Preferably, the bactericidal composition contains in the fragility Vibriophage ValLY-4 and other Vibriophages
It is at least one.
3. bactericidal composition according to claim 2, which is characterized in that the bactericidal composition further includes fragility vibrios and bites
Thalline VpaJT-1, preserving number are：CCTCC NO:M 2017547；
Fragility Vibriophage ValSw4-1, preserving number are CCTCC NO:M 2017552；
Fragility Vibriophage VspSw-1, preserving number are CCTCC NO:M 2017551；
Fragility Vibriophage ValLY-2, preserving number are CCTCC NO:M 2017554；
Fragility Vibriophage ValLY-3, preserving number are CCTCC NO:M 2017548；
And fragility Vibriophage ValDsh-1, preserving number are CCTCC NO:One or more in M 2017549；
Above-mentioned bacteriophage was preserved in China typical culture collection center on 25th in September in 2017, and Classification And Nomenclature is
Vibrio phage。
4. bactericidal composition according to claim 3, which is characterized in that the bactericidal composition includes the fragility vibrios
Bacteriophage ValLY-4, the fragility Vibriophage VpaJT-1, the fragility Vibriophage ValSw4-1, the fragility arc
The bacterium bacteriophage VspSw-1 and fragility Vibriophage ValLY-2.
5. according to claim 2~4 any one of them bactericidal composition, which is characterized in that also wrapped in the bactericidal composition
Include the one or more in each fragility Vibriophage mutant；
Preferably, the mutant at least 90% is identical with the native sequences of corresponding fragility Vibriophage.
6. according to claim 2~4 any one of them bactericidal composition, which is characterized in that each in the bactericidal composition
Content >=10 of fragility Vibriophage6PFU/mL；
Preferably, the content of each fragility Vibriophage is 10 in the composition6PFU/mL~107PFU/mL。
7. according to claim 2~4 any one of them bactericidal composition, which is characterized in that the bactericidal composition further includes
Auxiliary material；
Preferably, the auxiliary material is the one or more in SM buffer solutions, sodium alginate, sucrose, maltodextrin, glucose；
Preferably, the bactericidal composition further includes the bacteriophage of the specific pathogen bacterium of variety classes bacterium.
8. according to claim 2~4 any one of them bactericidal composition, which is characterized in that the dosage form of the bactericidal composition
For pulvis, aqua, freeze-dried, gelling agent, creme, paste.
9. fragility Vibriophage described in claim 1 or claim 2~8 any one of them bactericidal composition are killing
It goes out and/or prevents the application in vibrios microorganism belonging to genus；
The vibrios microorganism belonging to genus includes vibrio alginolyticus V.alginolyticus, Vibrio anguillarum V.anguillarum, comma bacillus
V.cholerae, non-Nonther conserved quantity non-O1V.cholerae, Fermi operator V.fischeri, vibrio fluvialis
V.fluvialis, Vibrio harveyi V.harveyi, sick Vibrio damsela V.ordalii, vibrio parahaemolytious
V.parahaemolyticus, Vibrio salmonicida V.salmonicida, vibrio mimicus V.mimicus, Vibrio splindidus
V.splendidus, Vibrio aureus V.azureus, Vibrio gazogenes V.gazogenes, Vibrio vulnificus V.vulnificus, bamboo
Pod vibrio piscium V.trachuri, damselfish vibrios V.damsela, floating vibrios V.natriegen, clam worm vibrios V.nereis, fish
Enteron aisle vibrios V.ichthyoenteri, shark vibrio V.carchariae, Vibrio campbellii V.campbellii and kill prawn arc
One or more in bacterium V.penaeicida.
10. fragility Vibriophage described in claim 1 or claim 2~8 any one of them bactericidal composition are being made
The application being ready for use in the drug for the treatment of and/or prevention animal vibriosis.
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Cited By (3)
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| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112391356A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong2 and application thereof |
| CN112646786A (en) * | 2021-01-21 | 2021-04-13 | 海南海壹水产种苗有限公司 | Rapid preliminary separation method for vibrio kammaticus phage |
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| WO2017173229A1 (en) * | 2016-04-01 | 2017-10-05 | Tufts University | Methods and compositions for preventing infection by a vibrio species |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112391356A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong2 and application thereof |
| CN112391355B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio harveyi efficient lytic phage vB_VhaS-yong3 and application thereof |
| CN112646786A (en) * | 2021-01-21 | 2021-04-13 | 海南海壹水产种苗有限公司 | Rapid preliminary separation method for vibrio kammaticus phage |
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