CN110093321A - Bacteriophage AH10-Phage-QY01 is in preparation treatment or the purposes of prevention and control aquaculture bacterial disease drug - Google Patents
Bacteriophage AH10-Phage-QY01 is in preparation treatment or the purposes of prevention and control aquaculture bacterial disease drug Download PDFInfo
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- CN110093321A CN110093321A CN201910361627.2A CN201910361627A CN110093321A CN 110093321 A CN110093321 A CN 110093321A CN 201910361627 A CN201910361627 A CN 201910361627A CN 110093321 A CN110093321 A CN 110093321A
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Abstract
The invention discloses Aeromonas hydrophila bacteriophage AH10-Phage-QY01 in preparation treatment or the purposes of prevention and control aquaculture bacterial disease drug, belongs to aquaculture bacterial disease Prevention Technique field.Bacteriophage AH10-Phage-QY01 is preserved in China typical culture collection center, deposit number is CCTCC NO:M2019109, further include purposes of the bacteriophage AH10-Phage-QY01 in preparation bacterial septicemia disease as caused by Aeromonas hydrophila, can be used alone or it is combined with antibiotic.The present invention utilizes generality and the high specific to host strain existing for bacteriophage, filter out Aeromonas hydrophila bacteriophage AH10-Phage-QY01 for the first time from natural environment for pathogenic bacteria, for inhibiting, cracking pathogenic bacteria, treatment or prevention and control are by bacterial diseases of fishes such as bacterial septicemias caused by the pathogenic bacteria.
Description
Technical field
The invention belongs to aquaculture bacterial disease Prevention Technique fields, and in particular to Aeromonas hydrophila bacteriophage
AH10-Phage-QY01 and its preparation treatment or prevention and control aquaculture bacterial disease drug purposes.
Background technique
The bacterial diseases of fishes are the Major Diseases of fresh water culture fishery, not only cause breed variety and fish-egg big during popular
Area is dead, also cultured product is made to lose commodity value, causes serious economic loss.The bacterial diseases of fishes are easy to get refractory and easy production
Raw drug resistance, at present due to largely more and more stronger, the bacillary fish using the drug resistance of various aquatic pathogenic bacterias of antibiotic medicine
The prevention and control difficulty of disease is also increasing.
Septicemia caused by Aeromonas hydrophila (Aeromonas Hydrophila) infects is a kind of high cause of carp, crucian
Characteristic of disease bacterial disease, cause of disease are Aeromonas hydrophila (Aeromonas hydrophila, AH), and bacterial septicemia takes place frequently, and gives
Crucian cultivation industry causes damaging influence.Aeromonas (Aeromonas) belongs to brevibacterium, and single flagellum, movement is extremely
Active amphimicrobian type Gram-negative bacteria, growth temperature is relatively wide (0~45 degree), and nutritional requirement is not high, ordinary flat, micro- white
The translucent bacterium colony of color, forms greyish white, smooth, wet, raised bacterium colony on blood agar, and majority has β haemolysis, enteron aisle selection training
It supports on base, most of bacterial strains form lactose azymic bacterium colony, undergrowth on TCBS agar.Infected by Aeromonas hydrophila crucian can
Cause its viscera tissue, the gill and body surface extensive bleeding, general patient's condition is more violent, mostly pernicious infectious disease, and the death rate is high.
The conventional antimicrobial drug such as Thiamphenicol, gentamicin, terramycin, radonil etc. of existing Aeromonas hydrophila,
Main component is mostly antibiotic or sulfa drugs.Due to a large amount of uses of the people to antibiotic for a long time, bacterium is to antibiosis
The drug resistance of element enhances year by year, and the fungistatic effect of antibiotic declines year by year, to reach the antibiotic agent used needed for therapeutic effect
It measures increasing, goes down for a long time, drug-fast bacteria strong for Aeromonas hydrophila etc. will face the available situation of no medicine.
Bacteriophage (Phage) is the virus for invading bacterium, it must have stringent host specificity in viable bacteria endoparasitism,
Its molecular structure and complementarity for depending on phage adsorption organ and recipient bacterium surface receptor.Bacteriophage is the most general in virus
All over the group most wide with distribution.Usually in some places full of bacterial community, as soil, animal enteron aisle in, can look for
To bacteriophage.It, can be this for Aeromonas hydrophila using generality existing for bacteriophage and to the high specific of host strain
Pathogenic bacteria filter out its corresponding bacteriophage and for inhibiting, killing the pathogenic bacteria.Use bacteriophage or bacteriophage and antibiotic
Associated with method replace the simple antibiotherapy can to effectively reduce the drug resistance of bacterial antibiotic.Meanwhile it because biting
Thallus is generally existing in the environment, can't be influenced too much to environment using phagotherapy, green, safe and reliable.
Summary of the invention
Using generality existing for bacteriophage, (i.e. safety, itself is widely present in nature the present invention, will not be to ring
Border has an adverse effect) and to the high specific of host strain, filter out thermophilic aqueous vapor list for the first time from natural environment for pathogenic bacteria
Born of the same parents bacterium bacteriophage AH10-Phage-QY01, can be used for inhibiting, crack pathogenic bacteria, caused by treatment or prevention and control are by the pathogenic bacteria
The bacterial diseases of fishes such as bacterial septicemia.
Highly pathogenic bacterial disease Aeromonas hydrophila (Aeromonas is especially for the current bacterial diseases of fishes
Hydrophila septicemia caused by) infecting is easy to get the problem that refractory, drug resistance is strong and prevention and control difficulty is big, the purpose of the present invention
It is to provide Aeromonas hydrophila bacteriophage AH10-Phage-QY01 in preparation treatment or prevention and control aquaculture bacterial disease drug
Purposes.
The present invention solve above-mentioned technical problem the technical solution adopted is as follows:
Aeromonas hydrophila bacteriophage AH10-Phage-QY01 (Aeromonas hydrophila bacteriophages
AH10-Phage-QY01), on 2 25th, the 2019 Chinese allusion quotations being deposited in positioned at Wuhan City, Hubei Province Wuhan University in the school
Type culture collection, deposit number are CCTCC NO:M2019109.
The Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is in preparation treatment or prevention and control aquaculture bacterial disease
The purposes of drug.
Further, the drug is also wrapped using the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 as active constituent
Include pharmaceutically acceptable carrier；The dosage of the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is not less than 1
×107pfu/mL.Further, bacteriophage or its lysate are dropped in breeding water body, make the bacteriophage in breeding water body
Quantity is minimum to reach 1 × 107Pfu/mL~9 × 107(the prophage drop degree being typically prepared is about 10 when pfu/mL12pfu/
ML, i.e. dosage are diluted stoste by 1:100000), preferable control can be played to the Aeromonas hydrophila in water body and made
With.
Further, the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is caused in preparation by Aeromonas hydrophila
Bacterial septicemia disease medicament purposes.
Further, the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is used alone or it joins with antibiotic
With.
Further, the dosage that the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is used alone not less than 1 ×
107pfu/mL；Dose ratio associated with the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 and antibiotic is 1:1, and institute
The dosage of Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is stated not less than 1 × 107pfu/mL。 AH10-phage-
QY01 can be used as antibiotic auxiliary therapeutical agent, play the role of prevention and control to the pathogenic bacteria with drug resistance with antibiotic combination, with
Dosage associated with antibiotic with its be used alone dosage it is consistent, with antibiotic associated with dose ratio be 1:1.
The beneficial effects of the present invention are:
1. the medicinal treatment traditional compared to aquatic pathogenic bacteria, the present invention is using generality existing for bacteriophage and to host
The high specific of bacterium filters out Aeromonas hydrophila bacteriophage AH10-Phage- for pathogenic bacteria for the first time from natural environment
QY01, for inhibiting, cracking pathogenic bacteria, treatment or prevention and control are by bacillary fishes such as bacterial septicemias caused by the pathogenic bacteria
Disease.
2. the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 that the present invention screens is to Aeromonas hydrophila fungistatic effect
It is very strong, and the duration is very long, using the bacteriophage substitute antibiotics or with antibiotic combination can be strong to drug resistance thermophilic water
The good fungistatic effect of Aeromonas reduces the usage amount of antibiotic for preventing and treating aquaculture aeromonas hydrophila disease.
3. the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 that the present invention screens has very high specificity, only right
Aeromonas hydrophila has growth of the good inhibitory effect without influencing other bacterium colonies, green, safe and efficient.
Detailed description of the invention
Fig. 1 is fungistatic effect of the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 to Aeromonas hydrophila；Wherein,
Infection multiplicity is 0.1,37 degree of culture 8h.
When Fig. 2 is that Aeromonas hydrophila bacteriophage AH10-Phage-QY01 continues the fungistatic effect of Aeromonas hydrophila
Between；Wherein, infection multiplicity is 0.1, and from left to right successively are as follows: 37 degree of culture 12h, 37 degree culture for 24 hours, 37 degree of culture 48h.
Preservation information is as follows:
Aeromonas hydrophila bacteriophage AH10-Phage-QY01 (Aeromonas hydrophila bacteriophages
AH10-Phage-QY01), on 2 25th, the 2019 Chinese allusion quotations being deposited in positioned at Wuhan City, Hubei Province Wuhan University in the school
Type culture collection, deposit number are CCTCC NO:M2019109.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
Embodiment: screening, separation and the purification of Aeromonas hydrophila bacteriophage AH10-Phage-QY01
Using generality existing for bacteriophage and to the high specific of host strain, in following instance, supported for all kinds of aquatic products
Pathogenic bacteria are grown to filter out its corresponding bacteriophage from natural environment and be used to treat aquaculture caused by the pathogenic bacteria thin
Bacterium disease.Specific steps are as follows:
Sample 50ml, 4000rpm/min is taken to be centrifuged 30min.
10ml supernatant is drawn, with 0.22um filter filtration sterilization.
The LB liquid medium that 3 times of 25ml are concentrated is added in 200ml conical flask, is cooled to room temperature, filtrate is added
Into conical flask, and host strain 1ml is added, while 0.5M calcium chloride is added to final concentration 0.1mM, 28 degree of 12~14h of culture.
It takes out culture solution 3ml to be added in the sterile test tube of 5ml, chloroform is added to final concentration of 3%, shakes 2min, room temperature
Stand 2h.
1ml supernatant 0.22um filter filtration sterilization is taken, by 10 times of doubling dilutions of filtrate to 10-5。
It takes the dilution 100ul and 200ul host strain of each gradient to be mixed, has melted after 28 degree of incubation 15min with 42 degree of 5ml
The upper layer LB (0.4% agar) of change is mixed, and is cast on bottom LB plate, is solidified after being stored at room temperature 15min to top-layer agar
Afterwards, 28 degree of incubator culture 8h are inverted in.
The plate of observation culture 8h, picking list spot are added the fresh host strain of 200ul in 5ml LB liquid, chlorination are added
Calcium to final concentration 0.1mM, 28 degree of stationary cultures are stayed overnight.
It takes out, 4000rpm/min is centrifuged 30min, 10 times of multiple proportions gradient dilutions of filtrate after 0.22um membrane filtration.
6,7,8 step 3~5 time are repeated, until obtaining shape, size, property stablizes plaque.
It chooses purified plaque and is put into the 1.5ml centrifuge tube containing 1ml host strain, 37 degree of 6~12h of culture.
15000g is centrifuged 10min, and a new 1.5ml centrifuge tube is added after taking supernatant 0.22um membrane filtration, is added
200ul PEG8000 solution.
It is mixed by inversion, places 15min.
15000g centrifugation 20min obtains phage particle.It is resuspended with 200ul TE buffer, obtains the bacteriophage of high titre
Stoste, can be used for conservation or DNA sample extracts material.
Effect example 1: the fungistatic effect of bacteriophage is verified
By taking Aeromonas hydrophila as an example, it will purify good in embodiment and carry out the Aeromonas hydrophila obtained after enrichment culture
(titre is 10 for bacteriophage AH10-Phage-QY01 stoste-10~10-12Puf/ml) with infection multiplicity for 0.1 (20ul bacteriophage
Stoste and the mixing of 200ul Aeromonas hydrophila) ratio mixed with Aeromonas hydrophila, in 37 DEG C of incubators placement 10 minutes
Afterwards fall double-layer plate, seal with sealing film plate and be placed in visible obvious plaque after 37 DEG C of incubator culture 8h, as shown in Figure 1.
Effect example 2: the fungistatic effect duration of bacteriophage is verified
As shown in Fig. 2, Aeromonas hydrophila bacteriophage AH10-Phage-QY01 can quickly and efficiently crack thermophilic aqueous vapor
Monad simultaneously inhibits its bacterium colony to grow, and the plate of plaque occurs, and plaque increases as time increases, until all
Plaque connect together and all nibbled the lawn on plate.
Claims (7)
1. Aeromonas hydrophila bacteriophage AH10-Phage-QY01, is preserved in China typical culture collection center, deposit number is
CCTCC NO:M2019109。
2. Aeromonas hydrophila bacteriophage AH10-Phage-QY01 described in claim 1 is in preparation treatment or prevention and control aquaculture
The purposes of bacterial disease drug.
3. purposes according to claim 2, which is characterized in that the drug is with the Aeromonas hydrophila bacteriophage
AH10-Phage-QY01 is active constituent, further includes pharmaceutically acceptable carrier.
4. purposes according to claim 2 or 3, which is characterized in that the Aeromonas hydrophila bacteriophage AH10-Phage-
The dosage of QY01 is not less than 1 × 107pfu/mL。
5. purposes according to claim 2, which is characterized in that the Aeromonas hydrophila bacteriophage AH10-Phage-
Purposes of the QY01 in preparation bacterial septicemia disease medicament as caused by Aeromonas hydrophila.
6. purposes according to claim 2 or 5, which is characterized in that the Aeromonas hydrophila bacteriophage AH10-Phage-
QY01 is used alone or it is combined with antibiotic.
7. purposes according to claim 6, which is characterized in that
The dosage that the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 is used alone is not less than 1 × 107pfu/mL；
Dose ratio associated with the Aeromonas hydrophila bacteriophage AH10-Phage-QY01 and antibiotic is 1:1, and the thermophilic water
The dosage of Aeromonas bacteriophage AH10-Phage-QY01 is not less than 1 × 107pfu/mL。
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| CN201910361627.2A CN110093321B (en) | 2019-04-30 | 2019-04-30 | Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases |
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| CN201910361627.2A CN110093321B (en) | 2019-04-30 | 2019-04-30 | Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484513A (en) * | 2019-08-23 | 2019-11-22 | 中国水产科学研究院黑龙江水产研究所 | Bacteriophage pAhMJG and its application in the fish disease that treatment Aeromonas hydrophila causes |
| CN110699330A (en) * | 2019-11-11 | 2020-01-17 | 云南大学 | Bacteriophage and application thereof |
| CN113444696A (en) * | 2021-07-13 | 2021-09-28 | 吉林农业大学 | Aeromonas hydrophila phage and application thereof |
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| US20130323209A1 (en) * | 2012-06-04 | 2013-12-05 | Ctc Bio, Inc. | Novel bacteriophage and its use for preventing proliferation of pathogenic bacteria |
| CN104073469A (en) * | 2014-05-20 | 2014-10-01 | 天津科技大学 | Separating and screening method for lytic aquatic pathogenic bacterium phage |
| CN104450629A (en) * | 2014-09-28 | 2015-03-25 | 浙江省淡水水产研究所 | Chinese soft-shell turtle aeromonas phage and application thereof |
| CN108085423A (en) * | 2018-02-08 | 2018-05-29 | 集美大学 | A kind of method and kit using Aeromonas hydrophila bacteriophage Rapid identification Aeromonas hydrophila |
| CN108103031A (en) * | 2018-01-10 | 2018-06-01 | 浙江省淡水水产研究所 | A kind of wide range phage preparation used for aquiculture and preparation method thereof |
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2019
- 2019-04-30 CN CN201910361627.2A patent/CN110093321B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130323209A1 (en) * | 2012-06-04 | 2013-12-05 | Ctc Bio, Inc. | Novel bacteriophage and its use for preventing proliferation of pathogenic bacteria |
| CN104073469A (en) * | 2014-05-20 | 2014-10-01 | 天津科技大学 | Separating and screening method for lytic aquatic pathogenic bacterium phage |
| CN104450629A (en) * | 2014-09-28 | 2015-03-25 | 浙江省淡水水产研究所 | Chinese soft-shell turtle aeromonas phage and application thereof |
| CN108103031A (en) * | 2018-01-10 | 2018-06-01 | 浙江省淡水水产研究所 | A kind of wide range phage preparation used for aquiculture and preparation method thereof |
| CN108085423A (en) * | 2018-02-08 | 2018-05-29 | 集美大学 | A kind of method and kit using Aeromonas hydrophila bacteriophage Rapid identification Aeromonas hydrophila |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484513A (en) * | 2019-08-23 | 2019-11-22 | 中国水产科学研究院黑龙江水产研究所 | Bacteriophage pAhMJG and its application in the fish disease that treatment Aeromonas hydrophila causes |
| CN110699330A (en) * | 2019-11-11 | 2020-01-17 | 云南大学 | Bacteriophage and application thereof |
| CN113444696A (en) * | 2021-07-13 | 2021-09-28 | 吉林农业大学 | Aeromonas hydrophila phage and application thereof |
| CN113444696B (en) * | 2021-07-13 | 2023-09-01 | 吉林农业大学 | Aeromonas hydrophila phage and application thereof |
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