CN108103031A - A kind of wide range phage preparation used for aquiculture and preparation method thereof - Google Patents

A kind of wide range phage preparation used for aquiculture and preparation method thereof Download PDF

Info

Publication number
CN108103031A
CN108103031A CN201810024877.2A CN201810024877A CN108103031A CN 108103031 A CN108103031 A CN 108103031A CN 201810024877 A CN201810024877 A CN 201810024877A CN 108103031 A CN108103031 A CN 108103031A
Authority
CN
China
Prior art keywords
preparation
wide range
bacteriophage
culture
fermented
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810024877.2A
Other languages
Chinese (zh)
Other versions
CN108103031B (en
Inventor
刘莉
卢淑娟
吕孙建
曹铮
林锋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Institute of Freshwater Fisheries
Original Assignee
Zhejiang Institute of Freshwater Fisheries
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Institute of Freshwater Fisheries filed Critical Zhejiang Institute of Freshwater Fisheries
Priority to CN201810024877.2A priority Critical patent/CN108103031B/en
Publication of CN108103031A publication Critical patent/CN108103031A/en
Application granted granted Critical
Publication of CN108103031B publication Critical patent/CN108103031B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The present invention relates to aquaculture fields, disclose a kind of wide range phage preparation used for aquiculture and preparation method thereof, which is fermented preparation by the bacteriophage strain NTHP01 that preserving number is CGMCC No.9623 by host strain of gene engineering colibacillus DH5 α.Preparation method：1）Bacillus coli DH 5 alpha is added to fermented and cultured in culture medium, obtains host's fermented liquid；2）By bacteriophage and MgCl2Mother liquor is put into host's fermented liquid, is stood after mixing, Multiplying culture, obtains fermentation mixed liquor；3）Mixed liquor will be fermented after centrifugation, obtain phage preparation.The present invention wide range phage preparation have splitting action to a variety of pathogenic bacterias, can individually or with other probiotics compounding uses.And its host strain, using genetic engineering bacterium, preparation method is easy, safe to use, without side-effects, can be applied to the bacterial disease prevention of aquaculture.

Description

A kind of wide range phage preparation used for aquiculture and preparation method thereof
Technical field
The present invention relates to aquaculture field more particularly to a kind of wide range phage preparation used for aquiculture and its preparation sides Method.
Background technology
China's aquaculture in recent years is quickly grown, but disease is also serious year by year.According to incompletely statistics, aquaculture at present Disease up to more than 300 is planted, and the annual cultured area that disease occurs accounts for the 1/10 of total cultured area, year loss amount account for cultivation total yield The 15%~30% of amount, year economic loss up to tens billion of members, the sustainable development of serious threat culture fishery.
From nineteen twenty-eight Fleming find penicillin since, antibiotic is widely used in a variety of applications and develops, hereafter the mankind into The high-speed development period of antibiotic is entered.Sensu lato antibiotic is the chemotherapeutant for suppressing or eliminating microorganism growth.Due to Aquaculture has the characteristics that bacterium infection risk is high, for prevention and treatment purpose, in the breeding process using antibiotic into For the common practice of marine industry.Amir Sapkota et al. researchs find grades 13 of the 1990-2007 including China In aquaculture country, the national ratio of terramycin, chloramphenicol and Oxolinic Acid was used to account for 92%, 69% and respectively 69%.Antibiotic was widely used in past more than 60 years, and bacterial drug resistance genes is caused to accelerate diffusion, drug-resistant type bacterial species It is continuously increased.In addition, abuse of antibiotics also results in aquatic products medicament residue, destroys water body environment, endangers beneficial microbe etc. Some arrange serious consequence.With people to antibiotic and its residual to the understanding of Safety of Aquatic Products and Environmental security harmfulness, And trade globalization trend, China has carried out stringent limitation to aquatic products with Antibiotics and use, indiscriminate in aquaculture Preferable control has also been obtained with antibiotic phenomenon.But since the long period, antibiotic is in aquaculture as disease The important means of evil prevention, antibody-resistant bacterium generally existing.According to national flounder flounder class industry's technology system the study found that China flounder All there are different degrees of antibiotic resistances for the 90 various bacterial cause of diseases found in the cultivation of flounder class.Therefore, the limit of antibiotic System using and antibody-resistant bacterium a large amount of appearance, this contradiction often result in production on without medicine can with the phenomenon that occur.Antibiotic-free water Production cultivation is a trend of industry development from now on.
Bacteriophage (phage, bacteriophage) is to parasitize bacterium, Mycoplasma, conveyor screw, actinomyces and indigo plant carefully A viroid in bacterium etc., also known as bacterial virus.Wherein virulent phage in sensitive bacteria cellular proliferative and can be allowed to crack, and be " natural killer " of bacterium.Therefore, bacteriophage is also one of controlling way of bacterial disease.Compared with antibiotic, bacteriophage Also have that high specificity, Small side effects, multiplication capacity are strong, be not easy to produce resistance, pathogen dependence and the spies such as easy to use Point is a kind of bio-control method of green.Existing document report finds bacteriophage to pseudomonas aeruginosa (Pseudomonas aeruginosa), detection of Salmonella (Salmonella), vibrio parahaemolytious (Vibrio ) etc. parahaemolyticus bacteriums have preferable inhibition.
Since bacteriophage has specificity to host strain bactericidal effect, most of bacteriophages have centainly the prevention of pathogen Limitation, i.e., a kind of bacteriophage only have bactericidal effect to a kind or several pathogens.In addition, its specificity also causes bacteriophage Preparation is often prepared as host strain in itself using pathogenic bacteria, higher to the purification requirements in later stage, and is brought to production application latent Risk.
The content of the invention
In order to solve the above technical problem, the present invention provides a kind of wide range phage preparation used for aquiculture and its preparations Method.The present invention wide range phage preparation have splitting action to a variety of pathogenic bacterias, can individually or and other Probiotics compounding use.And its host strain, using genetic engineering bacterium, preparation method is easy, safe to use, without side-effects, can It is applied to the bacterial disease prevention of aquaculture.
The specific technical solution of the present invention is：A kind of wide range phage preparation used for aquiculture is CGMCC by preserving number The bacteriophage strain NTHP01 of No.9623 is fermented using gene engineering colibacillus DH5 α as host strain to be prepared.
The present inventor is in first patent (201410506817.6, one plant of Trionyx Sinensis In Aeromonas bacteriophage and its application) After being found that bacteriophage strain NTHP01, subsequent research has been carried out, it is found that it not only has Aeromonas splitting action, and And also there is wide range.
In addition, in the prior art, being typically due to the specificity of bacteriophage causes phage preparation often with harmful pathogenic bacteria It itself is prepared as host strain, it is higher to the purification requirements in later stage, and bring potential risks to production application.And this hair It is bright using genetic engineering by the use of auxotrophic E. coli DH5 α as host strain, the harmless or less toxic evil of the host strain itself, Therefore later-period purification requirement is low, safe.And the present invention also has good phagocytosis effect to host strain.
In short, the present invention provides the higher host strain of security for follow-up large-scale production, purifies and separates work is simplified Skill.Phage preparation preparation method of the present invention is easy, and safe to use, without side-effects, wide range phage preparation obtained is to a variety of Pathogenic bacteria has splitting action, can individually or with other probiotics compounding uses, water can be applied to The bacterial disease prevention of production cultivation.
The preparation method of above-mentioned wide range phage preparation used for aquiculture, comprises the following steps：
1) the fresh bacterium solution of bacillus coli DH 5 alpha is added to fermented and cultured in culture medium, obtains host's fermented liquid.
2) by preserving number be CGMCC No.9623, preservation date in August, the 2014 bacteriophage strain NTHP01 of 28 days and MgCl2Mother liquor is put into host's fermented liquid, is stood after mixing, then carries out Multiplying culture, obtains fermentation mixed liquor；Its In, Multiplying culture condition is：The activation of bacillus coli DH 5 alpha is to OD when bacteriophage strain NTHP01 is added600For 0.3-0.5, sense Dye plural number is 10-1-10-2；30-37 DEG C of culture more than 5h of temperature.
3) mixed liquor will be fermented after centrifugation, bacteriophage is made to be separated with host strain, obtains wide range phage preparation.
The first patent (201410506817.6, one plant of Trionyx Sinensis In Aeromonas bacteriophage and its application) of team of the present invention Disclose preserving number be CGMCC No.9623 bacteriophage strain NTHP01, and disclose it can be to the thermophilic aqueous vapor unit cell of Shelled Turtle Trionyx Sinensis Bacterium forms plaque, has splitting action to Pathogenic Aeromonas, and life is used available for the disease caused by Aeromonas Object therapy treats and prevents, and especially has significant therapeutic effect to Shelled Turtle Trionyx Sinensis rotten neck disease and ichthyophthirius etc..In the patent skill In art scheme, only disclose bacteriophage strain NTHP01 has splitting action to Aeromonas, does not disclose it with wide range： To other bacterium (Edwardsiella tarda, vibrio parahemolyticus, vibrio alginolyticus, comma bacillus, Lattice Topologies in addition to Aeromonas The common bacterias such as bacterium, citrobacter freundii, enterobacteria) also there is splitting action.Therefore those skilled in the art are according to special The record of profit 201410506817.6 can not simultaneously know that bacteriophage strain NTHP01 has wide range.
Also, in practice remaining those skilled in the art in addition to team of the present invention to the research of the bacteriophage not It is more, therefore common those skilled in the art also and are unaware of the physiological characteristic of bacteriophage NTHP01.For this purpose, the present invention is by long-term A large amount of physiological characteristics for probing into wide range bacteriophage NTHP01, such as tolerable temperature, to the stability of condition of different pH, rule of proliferation, Infection multiplicity, magnesium chloride (MgCl2) the definite proliferation conditions such as concentration are added, and pass through amplification culture and determine preparation condition, it is made The product that can be preserved at 4 DEG C.
The bacteriophage has splitting action to a variety of aquaculture encountered pathogenic bacterias；It is analyzed from electron microscopic morphology, belongs to flesh tail phagocytosis Body section, is named as NTHP01, can survive under the conditions of pH4-10.Room temperature preserve 6 months it is activity stabilized；Add 30% glycerine guarantor Protect agent, 4 DEG C preserve 1 year after it is activity stabilized.
The wide range phage preparation of the present invention can be applied in the prevention of aquaculture bacterial disease, which can For inhibiting aquaculture encountered pathogenic bacteria (including Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, slow love Moral Fahrenheit bacterium, vibrio parahemolyticus, vibrio alginolyticus, comma bacillus, Lattice Topology bacterium, citrobacter freundii, enterobacteria etc. are normal See bacterium) growth.
Preferably, the culture medium is nutrient broth medium；The fresh bacterium solution of bacillus coli DH 5 alpha is added to culture In 37 DEG C of fermented and cultured 3-4h in base.
Preferably, in step 2), MgCl is added into the culture medium2Mother liquor makes MgCl in culture medium2Concentration is 4- 30mM stands 20-40min after mixing.
Preferably, in step 2), Multiplying culture condition is：Infection multiplicity is 10-2, MgCl2Final concentration of 4mM, temperature 37 DEG C, culture 5h makes OD600For 1.9.
Preferably, in step 3), in small-scale production, centrifugal condition is：5000-6000rpm/min, 10- 20min。
Preferably, in step 2), after obtaining fermentation mixed liquor, before entering step 3), also ferment by two-stage tandem Processing：
It will fermentation mixed liquor and MgCl2Mother liquor is added separately in extensive host's fermented liquid, makes MgCl2It is final concentration of 4mM, 20-40min is stood after mixing, secondary fermentation culture is carried out, obtains secondary fermentation mixed liquor；Subsequently into step 3)；Its In, secondary fermentation condition of culture is：37 DEG C, infection multiplicity 10-2, host strain is activated to OD when bacteriophage is added600For 0.3- 0.5, cultivate 5h.
This provides a kind of large-scale method for producing of phage preparation to the present invention, excellent first under the conditions of laboratory shake flask Change the proliferation conditions such as incubation time, temperature, infection multiplicity, measure the influence of addition various concentration ion pair multiplication, it is final to determine The optimal proliferation conditions of bacteriophage.Two-stage tandem fermentation process is designed on this basis, is successfully obtained using 2 tons of fermentation tanks 109The phagocytosis body fluid of pfu/ml.
Preferably, the preparation method of extensive host's fermented liquid is as follows：By the fresh bacterium of bacillus coli DH 5 alpha Liquid is added in nutrient broth medium in 37 DEG C of fermented and cultured 20-24h；It is then transferred in the culture medium of 45-55 times of volume Continue fermented and cultured 3-4h in 37 DEG C.
Preferably, in step 3), in large-scale production, secondary fermentation mixed liquor is passed through into the centrifugation point of butterfly centrifugal machine From host strain, gained liquid is wide range phage preparation.
Application of the wide range phage preparation used for aquiculture of the present invention in the prevention of aquaculture bacterial disease, it is described Wide range phage preparation grows for inhibiting aquaculture encountered pathogenic bacteria；The pathogen includes Aeromonas hydrophila, mild Aeromonas, Aeromonas caviae, Edwardsiella tarda, vibrio parahemolyticus, vibrio alginolyticus, comma bacillus, Lattice Topology are thin The common bacterias such as bacterium, citrobacter freundii, enterobacteria.
It is compared with the prior art, the beneficial effects of the invention are as follows：
The wide range phage preparation of the present invention has splitting action to a variety of pathogenic bacterias, can be individually or micro- with other Ecological agent compounding use.And its host strain, using genetic engineering bacterium, preparation method is easy, safe to use, without side-effects, can push away The wide bacterial disease applied to aquaculture is prevented.
The present invention is by probing into the physiological characteristic of wide range bacteriophage NTHP01, such as tolerable temperature, to the steady of condition of different pH It is qualitative, rule of proliferation, infection multiplicity, magnesium chloride (MgCl2) the definite proliferation conditions such as concentration are added, and pass through amplification culture and determine Preparation condition, the product of preparation can be in 4 DEG C of preservations.
Description of the drawings
Fig. 1 is the zymotechnique flow chart of prepare with scale phage preparation of the present invention；
Fig. 2 is electron microscopic observation bacteriophage NTHP01 aspect graphs (arrow meaning is bacteriophage)；
Fig. 3 is the one step growth curve figure of bacteriophage NTHP01；
Fig. 4 is influence figures of the different pH to bacteriophage NTHP01；
Fig. 5 is influence figure of the different temperatures to bacteriophage NTHP01；
Fig. 6 is the influence figure that different infection multiplicities are proliferated bacteriophage NTHP01；
Fig. 7 is the influence figure that different density of magnesium chloride promote bacteriophage NTHP01 infection；
Fig. 8 is that (A double-layer agar techniques crack thermophilic bacteriostasis figures of the bacteriophage NTHP01 to the common pathogenic bacteria in part Hydrophila, B and C are respectively the Aeromonas hydrophila and vibrio parahaemolytious of drip cracking, and arrow show plaque).
Specific embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1
A kind of wide range phage preparation used for aquiculture, by preserving number be CGMCC No.9623 bacteriophage strain NTHP01 with Gene engineering colibacillus DH5 α are prepared for host strain fermentation.
The preparation method of the wide range phage preparation used for aquiculture, comprises the following steps：
1) the fresh bacterium solution of bacillus coli DH 5 alpha is added in nutrient broth medium in 37 DEG C of fermented and cultured 3.5h, obtains place Main fermented liquid.
2) it is bacteriophage the strain NTHP01 and MgCl of CGMCC No.9623 by preserving number2Mother liquor puts into host strain and sends out In zymotic fluid, make MgCl2Final concentration of 4mM stands 30min after mixing, then carries out Multiplying culture, obtains fermentation mixed liquor；Its In, Multiplying culture condition is：The activation of bacillus coli DH 5 alpha is to OD when bacteriophage strain NTHP01 is added600For 0.3, infection is multiple Number is 10-2；37 DEG C of culture 5h of temperature make OD600For 1.9.
3) mixed liquor will be fermented after centrifuging (5500rpm/min, 15min), bacteriophage is made to be separated with host strain, takes supernatant Liquid obtains wide range phage preparation.
Embodiment 2
A kind of wide range phage preparation used for aquiculture, by preserving number be CGMCC No.9623 bacteriophage strain NTHP01 with Gene engineering colibacillus DH5 α are prepared for host strain fermentation.
The preparation method of the wide range phage preparation used for aquiculture, comprises the following steps：
1) the fresh bacterium solution of bacillus coli DH 5 alpha is added in nutrient broth medium in 37 DEG C of fermented and cultured 3.5h, obtains place Main fermented liquid.
2) it is bacteriophage the strain NTHP01 and MgCl of CGMCC No.9623 by preserving number2Mother liquor puts into host strain and sends out In zymotic fluid, make MgCl2Final concentration of 4mM stands 30min after mixing, then carries out Multiplying culture, obtains fermentation mixed liquor；Its In, Multiplying culture condition is：The activation of bacillus coli DH 5 alpha is to OD when bacteriophage strain NTHP01 is added600For 0.3, infection is multiple Number is 10-2；37 DEG C of culture 5h of temperature make OD600For 1.9.
In step 1) and 2) simultaneously, extensive host's fermented liquid is prepared：The fresh bacterium solution of bacillus coli DH 5 alpha is added Into nutrient broth medium in 37 DEG C of fermented and cultured 22h；It is then transferred in the culture medium of 50 times of volumes in 37 DEG C after supervention Ferment culture 3.5h.
3) two-stage tandem fermentation process：It will fermentation mixed liquor and MgCl2Mother liquor is added separately to extensive host strain fermentation In liquid, make MgCl2Final concentration of 4mM stands 30min after mixing, carries out secondary fermentation culture, obtains secondary fermentation mixed liquor； Wherein, secondary fermentation condition of culture is：37 DEG C, infection multiplicity 10-2, host strain is activated to OD when bacteriophage is added600For 0.3, Cultivate 5h.
4) secondary fermentation mixed liquor is centrifuged into host strain by butterfly centrifugal machine, gained liquid is wide range phagocytosis system Agent.
Embodiment 3
A kind of wide range phage preparation used for aquiculture, by preserving number be CGMCC No.9623 bacteriophage strain NTHP01 with Gene engineering colibacillus DH5 α are prepared for host strain fermentation.
The preparation method of the wide range phage preparation used for aquiculture, comprises the following steps：
1) the fresh bacterium solution of bacillus coli DH 5 alpha is added in nutrient broth medium in 37 DEG C of fermented and cultured 3h, obtains host Fermented liquid.
2) it is bacteriophage the strain NTHP01 and MgCl of CGMCC No.9623 by preserving number2Mother liquor puts into host strain and sends out In zymotic fluid, make MgCl2Final concentration of 30mM stands 30min after mixing, then carries out Multiplying culture, obtains fermentation mixed liquor；Its In, Multiplying culture condition is：The activation of bacillus coli DH 5 alpha is to OD when bacteriophage strain NTHP01 is added600For 0.4, infection is multiple Number is 10-1；37 DEG C of culture 5h of temperature make OD600For 1.9.
3) mixed liquor will be fermented after centrifuging (5000rpm/min, 20min), bacteriophage is made to be separated with host strain, takes supernatant Liquid obtains wide range phage preparation.
Embodiment 4
A kind of wide range phage preparation used for aquiculture, by preserving number be CGMCC No.9623 bacteriophage strain NTHP01 with Gene engineering colibacillus DH5 α are prepared for host strain fermentation.
The preparation method of the wide range phage preparation used for aquiculture, comprises the following steps：
1) the fresh bacterium solution of bacillus coli DH 5 alpha is added in nutrient broth medium in 37 DEG C of fermented and cultured 4h, obtains host Fermented liquid.
2) it is bacteriophage the strain NTHP01 and MgCl of CGMCC No.9623 by preserving number2Mother liquor puts into host strain and sends out In zymotic fluid, make MgCl2Final concentration of 15mM stands 30min after mixing, then carries out Multiplying culture, obtains fermentation mixed liquor；Its In, Multiplying culture condition is：The activation of bacillus coli DH 5 alpha is to OD when bacteriophage strain NTHP01 is added600For 0.5, infection is multiple Number is 10-2；37 DEG C of culture 5h of temperature make OD600For 1.9.
In step 1) and 2) simultaneously, extensive host's fermented liquid is prepared：The fresh bacterium solution of bacillus coli DH 5 alpha is added Into nutrient broth medium in 37 DEG C of fermented and cultureds for 24 hours；It is then transferred in the culture medium of 50 times of volumes in 37 DEG C after supervention Ferment culture 4h.
3) two-stage tandem fermentation process：It will fermentation mixed liquor and MgCl2Mother liquor is added separately to extensive host strain fermentation In liquid, make MgCl2Final concentration of 15mM stands 30min after mixing, carries out secondary fermentation culture, obtains secondary fermentation mixed liquor； Wherein, secondary fermentation condition of culture is：37 DEG C, infection multiplicity 10-2, host strain is activated to OD when bacteriophage is added600For 0.5, Cultivate 5h.
4) secondary fermentation mixed liquor is centrifuged into host strain by butterfly centrifugal machine, gained liquid is wide range phagocytosis system Agent.
Research process embodiment
First, bacteriophage NTHP01 is separately cultured
It isolates and purifies
1) separation of bacteriophage NTHP01 uses tryptose soya agar culture medium, thermophilic to be isolated in Shelled Turtle Trionyx Sinensis culture pond Hydrophila is host strain, using double-layer plate partition method separating and cracking bacteriophage.Preparing for double-layer plate is as follows：First Prepare 2% Tryptose soy agar medium and 0.7% semisolid Tryptose soy agar peptone culture medium.
2) 2% Tryptose soy agar medium after thawing is poured into culture dish bottom, is bottom culture medium after solidification.
3) 0.7% semisolid culturemedium is melted after 46 DEG C of heat preservations, a certain amount of bacteriophage is mixed with host strain, inhaled It after attached 20-30min, then mixes with semisolid culturemedium, pours on bottom culture medium, after 28 DEG C of culture 6-8h, observe phagocytosis Spot.Bacteriophage chooses spot liquid trypticase soybean broth culture after going out spot, repeats above procedure until purifying.
2nd, the amplification cultivation and harvest of bacteriophage NTHP01
Fresh host strain bacterium solution is cultivated to OD600MgCl is added in for 0.32It is allowed to final concentration and reaches 4mM, standing adsorption 20-30min, 150rpm/min is harvested after shaking 5 hours.Culture solution is moved into centrifuge tube, is centrifuged 10 minutes with 5,000g, to precipitate host Bacterium；Supernatant is moved in new pipe, with the remaining thalline of biofilter removal in 0.22 μm of aperture to get without host strain it Bacteriophage stoste.Double-layer agar technique measures its potency, and 4 DEG C save backup.
2nd, the electron microscopic observation of bacteriophage NTHP01
By the bacteriophage stoste of above-mentioned preparation after the super filter tube concentration of 30KD, after being washed with suitable PBS buffer solution, using phosphorus After wolframic acid negative staining, copper mesh, the transmission electron microscope observing morphology of phages are dripped.As shown in Fig. 2, the results show bacteriophage head is in twenty face Body, about 60-145nm, and with tail (80-455nm) different in size.With reference to《Virus taxis-world virus taxis committee member It can the 9th report》, NTHP01 bacteriophages be cauda-bactivirus mesh, Myoviridae bacteriophage.
3rd, one step growth curve measures
One step growth curve can be by the way that bacteriophage be calculated the information such as incubation period, outbreak period and outburst amount.By bacteriophage It is mixed by most suitable MOI with each 3mL of host strain, after 28 DEG C are incubated to a large amount of absorption, 13000g centrifugation 1min abandon supernatant, and precipitation is used The fluid nutrient medium of 28 DEG C of 10mL preheating is resuspended, as 28 DEG C, 120rpm/min shaken cultivations.It is taken every 5min within first 25 minutes Sample, samples afterwards every 15min, and continuous sampling 3.5h takes 500 μ L, 0.22 μm of membrane filtration, using double-deck agar culture every time Method measures filtrate pnagus medius potency.Using incubation time as abscissa, phage titre is ordinate, and it is bent to draw one step growth Line.As shown in Figure 3, the results showed that, the incubation period and outbreak period of bacteriophage NTHP01 multiplication are respectively 150min, 60min.
4th, the pH stability of bacteriophage NTHP01
Using culture solution as medium, it is 4,5,6,7,8,9,10,11 to adjust pH value.It is 1 × 10 to take titre8The prophage of PFU/mL 100 μ L of liquid are added in the TSB culture solutions of 900 μ L difference pH value, and the titre of each pipe bacteriophage is measured after 28 DEG C of water-bath 2h, and Calculate the pH tolerance ranges of bacteriophage.As shown in figure 4, the results show that bacteriophage NTHP01 has wider pH adaptability. Under conditions of pH7 to pH10, titre is 107PFU/mL is basically identical with initial titer, i.e., bacteriophage this pH section keep compared with High bioactivity.In pH7 activity highests.PH is less than 7 or during more than 10, as acid or alkalescence enhances, bacteriophage activity by Gradually decline, but it is overall all with certain activity.
5th, the thermal stability of bacteriophage NTHP01
The bacteriophage stoste of above-mentioned preparation is diluted to 1x107PFU/mL is simultaneously sub-packed in 4 5mL centrifuge tubes, often manages each 2mL, Centrifuge tube is respectively placed in 30 DEG C, 40 DEG C, 50 DEG C and 60 DEG C of thermostat water bath, phagocytosis in each pipe is measured every 20min The titre of body, until 100min.After equalized temperature, its potency is measured.As shown in figure 5, the results show that the bacteriophage is 30 In DEG C -40 DEG C of sections, phage titre is 106More than PFU/mL, compare original titer 107PFU/mL is not substantially reduced, Survival rate is high on the whole.Temperature reach 50 DEG C and more than when, bacteriophage rapid deactivation in 20min, titre only has during 20min 105PFU/mL, when the time is in 100min, 50 DEG C of phage titres are only 104PFU/mL, it is most of dead, show the bacteriophage Extremely sensitive to 50 DEG C or more of temperature, activity is relatively low.
6th, the most suitable infection multiplicity (MOI) of bacteriophage NTHP01
Host strain is shaken to 1 × 108CFU/mL, while prepare 1 × 108The bacteriophage stoste of PFU/mL, the two carry out 10 respectively Times gradient dilution is to 1 × 105PFU/mL is respectively taken by table 1 with 7 difference MOI (1000,100,10,1,0.1,0.01,0.001) 100 μ L are mixed in 1.5mL Eppendorf pipes, and add in 800 μ L fresh liquid culture mediums, 28 DEG C of culture 3.5h.After taking-up Each pipe pnagus medius potency is measured respectively, the as most suitable infection multiplicities of the MOI corresponding to 3.5h titre highests.As shown in fig. 6, knot Fruit show the phage-infect plural number for 1,10,100,1000 and 0.001 when, it is worst to host strain infectious effect, in 0.1 He There is preferable infectious effect when 0.01 to host strain, most suitable infection multiplicity is 0.01.
7th, the influence that different density of magnesium chloride are proliferated bacteriophage NTHP01
It is equipped at 6 in the sterilized 50mL centrifuge tubes of 10ml culture mediums and respectively adds in 100 μ L host strains 1 × 108CFU/mL, 37 DEG C Shaking table culture 3h.It is each to add in 1 × 108The 100 μ L of bacteriophage stoste of PFU/mL and different final concentration magnesium chlorides (0,2mM, 4mM, 8mM, 16mM, 32mM), effect 30min is placed on 37 DEG C of shaking table culture 4h, measures each pipe pnagus medius potency after taking-up respectively. As shown in fig. 7, the results show that the phage titre of addition magnesium chloride apparently higher than being not added with, it is and final concentration of in magnesium chloride 4mM promotes optimal to the infection effect of bacteriophage.
8th, plural serial stage fermentation-scaleization prepares bacteriophage
On the basis of the experimental result of above-mentioned bacteriophage multiplication condition, plural serial stage fermentation process is designed, as shown in Figure 1.Specifically Implement as follows：
1) 30L bacteriophages prepare with host strain mixed liquor.The fresh host's bacterium solutions of 600ml are prepared first, add in 30L culture mediums (50L Fermentation tank) in, 37 DEG C of culture 3-4h make bacterium solution OD600Up to 0.3 or so, bacteriophage stoste 600ml and MgCl are added2Mother liquor 300ml (final concentration of 4mM) stands 30min after mixing, afterwards 37 DEG C of culture 5h, terminates fermentation to get 30L bacteriophages and place Main bacterium mixed liquor, for subsequent fermentation.
2) preparation of 1.5 tons of phagocytosis body fluid.The fresh host's bacterium solutions of 600ml are added in 30L culture mediums (50L fermentation tanks), 37 DEG C culture 20-24h；It adds in afterwards in 1.5 tons of fermentation mediums (2 tons of fermentation tanks), 37 DEG C of culture 3-4h make bacterium solution OD600It reaches 0.3 or so, the 30L bacteriophages of above-mentioned preparation and host strain mixed liquor are added in, while adds in MgCl2Mother liquor 1.5L is (final concentration of 4mM), 30min is stood after mixing, 37 DEG C of culture 5h, terminate fermentation afterwards.Tunning using disk centrifugal separator is centrifuged, is received Collect liquid, packing.Phage titer, potency 2x10 are measured using double-layer agar technique9pfu/ml。
9th, bacteriophage is to the fungistatic effect of various pathogenic bacteria
Using double-layer agar technique and the phagocytosis effect to aquaculture encountered pathogenic bacteria of drip observation bacteriophage.It is tested Pathogen has：It is isolated from the Aeromonas hydrophila of Shelled Turtle Trionyx Sinensis, Aeromonas sobria, Aeromonas caviae, Edwardsiella tarda； It is isolated from Aeromonas hydrophila, vibrio parahemolyticus, vibrio alginolyticus, comma bacillus, the Lattice Topology bacterium of Penaeus Vannmei；Separation From the enterobacteria of Macrobrachium rosenbergii；It is isolated from the citrobacter freundii of red claw crayfish；It is isolated from the Aeromonas hydrophila of grouper And Aeromonas sobria of perch etc..Double-layer agar technique is to prepare 2%LB agar mediums and 0.7% semisolid LB trainings first Support base.2%LB culture mediums after thawing are poured into culture dish bottom, are bottom culture medium after solidification.By 0.7% Semi-solid cell culture Base melt after 46 DEG C heat preservation, a certain amount of bacteriophage mix respectively with pathogen to be measured, adsorb 20-30min after, then with partly Solid medium mixes, and pours on bottom culture medium, after 28 DEG C of culture 6-8h, observes plaque.Drip is first with painting Host strain is filled this tablet by cloth method, after planar surface air-dries 3-5min, then 5 μ L phagocytosis body fluid is added dropwise on tablet, and 28 DEG C culture 8-12h after, observe phagocytosis effect.As shown in Figure 8, the results showed that, visible apparent plaque in double-layer plate instils Method tests above-mentioned pathogen, can substantially observe phagocytosis effect, show the bacteriophage to above-mentioned aquaculture encountered pathogenic bacteria There is preferable lytic effect.
Meanwhile using genetic engineering by the use of auxotrophic E. coli DH5 α as host strain, it may have good phagocytosis Effect.The higher host strain of security is provided for follow-up large-scale production, simplifies purifies and separates technique.
1. bacteriophage of table is to the phagocytosis of part aquaculture common bacteria
(note：+ represent that bacterium is weaker to phage-sensitive degree, ++ represent that bacterium is medium to the susceptibility of bacteriophage strong, +++ it represents Bacterium is strong to the susceptibility of bacteriophage)
Raw materials used in the present invention, equipment is the common raw material, equipment of this field unless otherwise noted；It is used in the present invention Method is the conventional method of this field unless otherwise noted.
The above is only presently preferred embodiments of the present invention, not the present invention imposed any restrictions, it is every according to the present invention Any simple modification, change and the equivalent transformation that technical spirit makees above example, still fall within the technology of the present invention side The protection domain of case.

Claims (10)

1. a kind of wide range phage preparation used for aquiculture, it is characterised in that：By the bacteriophage that preserving number is CGMCC No.9623 Strain NTHP01 is fermented using gene engineering colibacillus DH5 α as host strain to be prepared.
2. a kind of preparation method of wide range phage preparation used for aquiculture as described in claim 1, it is characterised in that including Following steps：
1）The fresh bacterium solution of bacillus coli DH 5 alpha is added to fermented and cultured in culture medium, obtains host's fermented liquid；
2）By bacteriophage the strain NTHP01 and MgCl that preserving number is CGMCC No.96232Mother liquor is put into host's fermented liquid In, it is stood after mixing, then carries out Multiplying culture, obtain fermentation mixed liquor；Wherein, Multiplying culture condition is：Bacteriophage strain The activation of bacillus coli DH 5 alpha is to OD when NTHP01 is added600For 0.3-0.5, infection multiplicity 10-1-10-2；30-37 DEG C of temperature Cultivate more than 5h；
3）Mixed liquor will be fermented after centrifugation, bacteriophage is made to be separated with host strain, obtains wide range phage preparation.
3. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 2, which is characterized in that the culture Base is nutrient broth medium；The fresh bacterium solution of bacillus coli DH 5 alpha is added in culture medium in 37 DEG C of fermented and cultured 3-4h.
4. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 2, which is characterized in that step 2） In, add MgCl into the culture medium2Mother liquor makes MgCl in culture medium2Concentration is 4-30mM, and 20-40min is stood after mixing.
5. the preparation method of the wide range phage preparation used for aquiculture as described in claim 2 or 4, which is characterized in that step 2）In, Multiplying culture condition is：Infection multiplicity is 10-2, MgCl2Final concentration of 4mM, 37 DEG C of temperature, culture 5h make OD600For 1.9。
6. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 2, which is characterized in that step 3） In, centrifugal condition is：5000-6000rpm/min, 10-20min.
7. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 2, which is characterized in that step 2） In, after obtaining fermentation mixed liquor, entering step 3）Before, also by two-stage tandem fermentation process：
It will fermentation mixed liquor and MgCl2Mother liquor is added separately in extensive host's fermented liquid, makes MgCl2Final concentration of 4mM, 20-40min is stood after mixing, secondary fermentation culture is carried out, obtains secondary fermentation mixed liquor；Subsequently into step 3）；Wherein, two Secondary fermentation culture conditions are：37 DEG C, infection multiplicity 10-2, host strain is activated to OD when bacteriophage is added600For 0.3-0.5, training Support 5h.
8. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 7, which is characterized in that the big rule The preparation method of mould host's fermented liquid is as follows：By the fresh bacterium solution of bacillus coli DH 5 alpha be added to nutrient broth medium in 37 DEG C of fermented and cultured 20-24h；It is then transferred in the culture medium of 45-55 times of volume and continues fermented and cultured 3-4h in 37 DEG C.
9. the preparation method of wide range phage preparation used for aquiculture as claimed in claim 7 or 8, which is characterized in that step 3）In, secondary fermentation mixed liquor is centrifuged into host strain by butterfly centrifugal machine, gained liquid is wide range phage preparation.
10. wide range phage preparation used for aquiculture as described in claim 1 is in the prevention of aquaculture bacterial disease Using, which is characterized in that the wide range phage preparation grows for inhibiting pathogenic bacteria；The pathogen includes thermophilic Hydrophila, Aeromonas sobria, Aeromonas caviae, Edwardsiella tarda, vibrio parahemolyticus, vibrio alginolyticus, suddenly Random vibrios, Lattice Topology bacterium, citrobacter freundii, enterobacteria.
CN201810024877.2A 2018-01-10 2018-01-10 Wide-spectrum phage preparation for aquaculture and preparation method thereof Active CN108103031B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810024877.2A CN108103031B (en) 2018-01-10 2018-01-10 Wide-spectrum phage preparation for aquaculture and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810024877.2A CN108103031B (en) 2018-01-10 2018-01-10 Wide-spectrum phage preparation for aquaculture and preparation method thereof

Publications (2)

Publication Number Publication Date
CN108103031A true CN108103031A (en) 2018-06-01
CN108103031B CN108103031B (en) 2021-09-07

Family

ID=62218543

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810024877.2A Active CN108103031B (en) 2018-01-10 2018-01-10 Wide-spectrum phage preparation for aquaculture and preparation method thereof

Country Status (1)

Country Link
CN (1) CN108103031B (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109136195A (en) * 2018-08-16 2019-01-04 广东海大畜牧兽医研究院有限公司 A kind of vibrio parahaemolyticus phage and its application
CN109385406A (en) * 2018-11-12 2019-02-26 中国海洋大学 One plant of vibrio parahaemolyticus phage and its application in terms of enhancing aquatic livestock immunity
CN109652383A (en) * 2019-01-08 2019-04-19 集美大学 A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage
CN110093321A (en) * 2019-04-30 2019-08-06 上海海洋大学 Bacteriophage AH10-Phage-QY01 is in preparation treatment or the purposes of prevention and control aquaculture bacterial disease drug
CN110468110A (en) * 2019-09-11 2019-11-19 大连理工大学 A kind of vibrio parahaemolyticus phage and its application in stichopus japonicus disease prevention
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
CN112391357A (en) * 2019-08-14 2021-02-23 宁波大学 Aeromonas sobria efficient lytic phage vB _ AsoP-yong and application thereof
CN112574959A (en) * 2020-11-17 2021-03-30 厦门昶科生物工程有限公司 Bacteriophage for preventing and treating aeromonas disease of aquatic animals and application thereof
CN114763539A (en) * 2022-05-20 2022-07-19 青岛诺安百特生物技术有限公司 Citrobacter freundii bacteriophage, bacteriophage composition and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009138752A3 (en) * 2008-05-15 2010-03-18 Big Dna Ltd Fish vaccine
US20140105866A1 (en) * 2012-10-17 2014-04-17 Universidad De Chile Bacteriophages useful for the prophylaxis and therapy of vibrio anguillarum
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
CN107012109A (en) * 2017-04-01 2017-08-04 昆明理工大学 Applications of the signaling molecule C6 AHL in Phage Infection Escherichia coli are suppressed

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009138752A3 (en) * 2008-05-15 2010-03-18 Big Dna Ltd Fish vaccine
US20140105866A1 (en) * 2012-10-17 2014-04-17 Universidad De Chile Bacteriophages useful for the prophylaxis and therapy of vibrio anguillarum
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
CN107012109A (en) * 2017-04-01 2017-08-04 昆明理工大学 Applications of the signaling molecule C6 AHL in Phage Infection Escherichia coli are suppressed

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈海阳: "大肠杆菌噬菌体的分离纯化及其在海水养殖中的应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109136195A (en) * 2018-08-16 2019-01-04 广东海大畜牧兽医研究院有限公司 A kind of vibrio parahaemolyticus phage and its application
CN109385406A (en) * 2018-11-12 2019-02-26 中国海洋大学 One plant of vibrio parahaemolyticus phage and its application in terms of enhancing aquatic livestock immunity
CN109652383A (en) * 2019-01-08 2019-04-19 集美大学 A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage
CN110093321B (en) * 2019-04-30 2021-07-13 上海海洋大学 Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases
CN110093321A (en) * 2019-04-30 2019-08-06 上海海洋大学 Bacteriophage AH10-Phage-QY01 is in preparation treatment or the purposes of prevention and control aquaculture bacterial disease drug
CN112391357A (en) * 2019-08-14 2021-02-23 宁波大学 Aeromonas sobria efficient lytic phage vB _ AsoP-yong and application thereof
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
CN110468110A (en) * 2019-09-11 2019-11-19 大连理工大学 A kind of vibrio parahaemolyticus phage and its application in stichopus japonicus disease prevention
CN110699330A (en) * 2019-11-11 2020-01-17 云南大学 Bacteriophage and application thereof
CN112574959A (en) * 2020-11-17 2021-03-30 厦门昶科生物工程有限公司 Bacteriophage for preventing and treating aeromonas disease of aquatic animals and application thereof
CN112574959B (en) * 2020-11-17 2022-05-24 厦门昶科生物工程有限公司 Bacteriophage for preventing and treating aeromonas disease of aquatic animals and microecological preparation
CN114763539A (en) * 2022-05-20 2022-07-19 青岛诺安百特生物技术有限公司 Citrobacter freundii bacteriophage, bacteriophage composition and application thereof
CN114763539B (en) * 2022-05-20 2023-06-27 青岛诺安百特生物技术有限公司 Citrobacter freundii phage, phage composition and application thereof

Also Published As

Publication number Publication date
CN108103031B (en) 2021-09-07

Similar Documents

Publication Publication Date Title
CN108103031A (en) A kind of wide range phage preparation used for aquiculture and preparation method thereof
CN109735471B (en) Microbacterium and application thereof as probiotic in aquatic products
AU2020100853A4 (en) Proteus mirabilis phage rdp-sa-16033 and industrial production process thereof
CN113198011B (en) Duck adenovirus type 3 strain inactivated vaccine and application thereof
CN108651522A (en) A kind of vibrio alginolyticus phage preparation, preparation method and applications
Li et al. Virulence properties of Vibrio vulnificus isolated from diseased zoea of freshness shrimp Macrobrachium rosenbergii
CN110004096A (en) One lactobacillus plantarum and its application
CN111575243B (en) Vibrio campylobacter bacteriophage and application thereof
CN117050954A (en) Broad-spectrum salmonella phage vB-SenS-S1 and composition containing phage
CN115197919B (en) Vibrio phage composition and preparation method and application thereof
CN113444695B (en) Escherichia coli bacteriophage with high fermentation efficiency and good clinical effect and application thereof
CN113201506B (en) High-efficiency-cracking carbapenem drug-resistant Klebsiella pneumoniae phage and application thereof
Ebnetorab et al. Isolation, biochemical and molecular detection of Bacillus subtilis and Bacillus licheniformis from the digestive system of rainbow trout (Oncorhynchus mykiss) and its inhibitory effect on Aeromonas hydrophila
CN114231499A (en) Bacteriophage and application thereof
CN112592851A (en) Lactobacillus acidophilus with broad-spectrum antagonistic effect on aquatic pathogenic bacteria and application thereof
CN110408573A (en) A kind of Lactobacillus rhamnosus YFI-6 and its application in anti-giant salamander irido virus
CN114921419B (en) Richter duck plague bacillus phage
CN114958778B (en) Vibrio parahaemolyticus lytic phage GVP-P20 and application thereof in preventing and treating vibrio parahaemolyticus in eriocheir sinensis
CN114196637B (en) Salmonella phage Jnwz02 and application thereof
CN115418355B (en) Staphylococcus lentus phage and separation method and application thereof
CN116606818A (en) Salmonella phage salmonella phage vB-SalP-JXAU-W3 and application thereof
CN117070472A (en) Vibrio parahaemolyticus phage aiming at highly pathogenic vibrio and drug-resistant vibrio and application thereof
CN117070471A (en) Multivalent salmonella phage capable of entering blood orally and application thereof
CN115747171A (en) Aeromonas veronii phage pAEv1818 and application thereof
CN116751753A (en) Edwardsiella tarda phage with high lytic property and composition and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant