WO2013105781A1 - Nouveau bactériophage isolé et composition antibactérienne comprenant celui-ci - Google Patents

Nouveau bactériophage isolé et composition antibactérienne comprenant celui-ci Download PDF

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WO2013105781A1
WO2013105781A1 PCT/KR2013/000166 KR2013000166W WO2013105781A1 WO 2013105781 A1 WO2013105781 A1 WO 2013105781A1 KR 2013000166 W KR2013000166 W KR 2013000166W WO 2013105781 A1 WO2013105781 A1 WO 2013105781A1
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bacteriophage
vibrio
present
active ingredient
composition
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Korean (ko)
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양시용
김재원
임현정
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씨제이제일제당 (주)
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/153—Nucleic acids; Hydrolysis products or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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

Definitions

  • the present invention relates to a novel isolated bacteriophage and an antimicrobial composition comprising the same. More specifically, the present invention relates to a novel bacteriophage having Vibrio harveyi- specific killing ability and vibrio containing the bacteriophage as an active ingredient.
  • a composition for preventing or treating a Vietnamesei infectious disease, an antibiotic comprising the bacteriophage as an active ingredient, a feed additive comprising the bacteriophage as an active ingredient, a disinfectant or cleaning agent containing the bacteriophage as an active ingredient, and vibrio using the bacteriophage A method for preventing or treating an infectious disease caused by Havey.
  • Vibrio Harvey known as Vibrio bacteria that cause increases in shrimp (penaeid shrimp) (V. harveyi) is alive everywhere in the marine environment, the luminance of Gram-negative bacteria, bacillus. Vibrio Habei has been reported to be an important pathogen for normal marine flora of marine organisms, but several species of marine fish, shellfish and shrimp (Karunasagar I et al., Aquaculture 128: 203-209, 1994, Zhang XH and Austin B, J. Fish Dis). 23: 93-103, 2000).
  • Vibrio Havey The virulence mechanism of Vibrio Havey is not yet clear and depends on the host and species of the fungus.
  • Pathogenic components of Vibrio Havey include extracellular products (ECP), including proteases, hemolysins, lipases, lipopolysaccarides and bacteriocin analogs. -like substances) (Teo JWP et al., Gene 312: 181-188, 2003).
  • Bacteriophage is a bacteria-specific virus that controls the growth of bacteria by infecting only certain bacteria, sometimes called abbreviated phages, and without the bacterial host, self-proliferation is impossible.
  • Bacteriophage is a single or double-stranded DNA or RNA that consists of nucleic acid as a genetic material, and the nucleic acid is a simple structure in which the protein envelope is wrapped, with the tail on the icosahedron head, the tail on the icosahedron head, and It is divided into three basic types of filament type.
  • octahedral head-tailed bacteriophages are Myoviridae with contractile tails, Siphoviridae with long, non-contracting tails, and grapes with short tails, depending on the tail characteristics. Can be subdivided into Podoviridae. Bacteriophages without tails in icosahedral heads are subdivided according to the shape of the head, the composition of the head, and the presence or absence of the sheath. Finally, filamentous bacteriophages are subdivided according to size, shape, sheath and filament composition (HW Ackermann, Frequency of morphological phage descriptions in the year 2000; Arch. Virol. 146: 843-857, 2001; Elizabeth Kutter et al. , Bacteriophages Biology and Application, CRC press).
  • AGP antimicrobial growth promoter
  • Clostridium sporogenes phage NCIMB 30008 and Clostridium tyrobutiricum phage NCIMB 30008 were registered as feed preservatives in Europe in 2003 and 2005, respectively. It was developed as a product for the purpose of controlling the contaminated Clostridium sp. These studies show that the use of bacteriophage to control the control of bacteria that are difficult to treat antibiotics, or to control common infectious bacteria contaminated with livestock products at the food level is ongoing.
  • Vibrio Havey which produces vibriosis in aquaculture fish and shrimp, in order to solve problems of a wide range of host ranges such as the occurrence of antibiotic resistant bacteria due to misuse and abuse of antibiotics, the retention of antibiotics in food and past antibiotics.
  • the bacteriophage infected with the virus was newly isolated from nature, and their morphological, biochemical and genetic properties together with the ability to selectively kill only Vibrio Havey bacteria were completed and the present invention was completed.
  • Another object of the present invention is to provide a composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage as an active ingredient.
  • Still another object of the present invention is to provide an antibiotic comprising the bacteriophage as an active ingredient.
  • Still another object of the present invention is to provide a fish or shellfish feed additive containing the bacteriophage as an active ingredient.
  • Another object of the present invention to provide a disinfectant or cleaning agent containing the bacteriophage as an active ingredient.
  • Still another object of the present invention is to provide a method for preventing or treating an infectious disease caused by Vibrio habay using the bacteriophage or composition.
  • the present invention provides novel bacteriophages with specific killing ability to Vibrio harveyi .
  • the present inventors isolate bacteriophages that selectively lyse Vibrio harveyi BB120, VH-BB120, a host cell, from a sample collected from seawater. Morphologically observed by electron microscopy, the bacteriophage has species specificity that selectively infects Vibrio havey among Vibrio bacteria (see Table 1); Can be infected with various wild isolates Vibrio Havey (see Table 3); Exhibits specific killing ability to Vibrio havey; Morphological type B1 consisting of isometric capsid and long non-contractile tail is identified as belonging to Siphoviridae (see FIG. 1). In addition, the bacteriophage has a total genome size of about 75 kbp (see FIG. 2); As a result of analyzing the protein pattern, it was confirmed that the protein having 50 kDa, 44 kDa, and 36 kDa size as the main structural protein (see FIG. 3).
  • nucleic acid molecules of SEQ ID NOS: 1 to 5 are included as part of the entire genome, and similarity between different species is compared based on SEQ ID NOs: 1 to 5
  • the fragments show 20-45% similarity but all fragments are novel bacteriophages without 100% matching bacteriophages (see Table 2).
  • a primer set specific for the bacteriophage specifically, SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, SEQ ID NO: : PCR was performed with primer sets of 12 and 13, and SEQ ID NOs: 14 and 15.
  • PCR products of 600 bp, 400 bp, 900 bp, 500 bp and 1000 bp were amplified respectively (FIGS. 4A to 4D). Reference).
  • the bacteriophage has acid resistance stably surviving in the range of pH 4 to 11 (see FIG. 5), and stably survives in the range of 43 ° C. to 60 ° C. Confirm that it has a heat resistance that can be (see Fig. 6).
  • the specificity, acid resistance and heat resistance of Vibrio doni, the bacteriophage according to the present invention in a variety of products, including the composition for the prevention and treatment of Vibrio Habei infectious diseases of cultured seafood and shrimp, various products including the bacteriophage as an active ingredient, etc. And applications in the pH range.
  • the present inventors collected a sample from the sea water, named as a bacteriophage "Bacteriophage ⁇ CJ17" having a specific killing ability for Vibrio Habei, and has the characteristics described above, and the Korean microorganisms on January 2, 2012 under the Budapest Treaty It was deposited in the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under accession number KCCM11247P.
  • the present invention provides a composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage as an active ingredient.
  • the bacteriophage of the present invention has an antimicrobial activity that can specifically kill Vibrio Vietnamesei, it can be used for the purpose of preventing or treating diseases caused by its infection.
  • the infectious disease caused by Vibrio Havey may include, but is not limited to, Vibriosis.
  • Vibriosis refers to a disease caused by the infection of Vibrio Havey, and infects various marine animals such as seawater fish, shellfish, and shrimps. In particular, in shrimps, lethargy, tissue necrosis, Delayed growth, delayed transformation of larvae, malformation, luminescence, muddiness, pigmentation, etc., causing massive mortality.
  • prevention refers to any action that inhibits or delays the onset of the disease by administration of the composition
  • treatment refers to any action that improves or advantageously changes the condition of the subject disease by administration of the composition. It means.
  • the composition comprises 5 ⁇ 10 2 to 5 ⁇ 10 12 PFU / mL, preferably 1 ⁇ 10 6 to 1 ⁇ 10 10 PFU / mL of the bacteriophage according to the present invention as an active ingredient.
  • the prophylactic or therapeutic composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injection solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and these
  • One or more of the components may be used in combination, and other conventional additives such as antioxidants, buffers, bacteriostatics may be added as necessary.
  • diluents may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • the prophylactic or therapeutic composition of the present invention can be administered through oral or parenteral administration, and a method of applying or dipping to a diseased area or an individual can also be used.
  • parenteral administration administration may be by intravenous administration, intraperitoneal administration, intramuscular administration, or topical administration.
  • Suitable applications, sprays, and dosages of the compositions of the present invention may include formulation methods, modes of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion, and attenuation of the response of the animals and patients. It can vary depending on factors such as performance, and a skilled practitioner or veterinarian can easily determine and prescribe a dosage that is effective for the desired treatment.
  • Formulations for oral administration comprising a composition of the invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do.
  • lactose For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • Formulations for parenteral administration comprising the composition of the present invention as an active ingredient include, for example, injectable forms such as subcutaneous injection, intravenous injection or intramuscular injection, aerosols for inhalation through suppository infusion or respiratory system, and the like. It can be formulated for spraying.
  • injectable formulations the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials.
  • a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
  • the present invention provides an antibiotic comprising the bacteriophage as an active ingredient.
  • antibiotic refers to an agent that can be given to animals in the form of a medicament to kill germs, and generically refers to preservatives, fungicides and antimicrobials.
  • the animal includes fish and shellfish, shrimp and the like, and includes both natural and cultured.
  • the bacteriophage of the present invention has a very high specificity for Vibrio habay compared to conventional antibiotics, it can be used specifically for the prevention or treatment of diseases caused by Vibrio habay, and does not induce drug resistance, compared to conventional antibiotics. Life cycling can be used as a new novel antibiotic.
  • the present invention provides a fish or shellfish feed additive comprising the bacteriophage as an active ingredient.
  • Feedstock antibiotics used in livestock and fisheries are used for prophylactic purposes.
  • Antibiotics for prophylaxis are a problem because they increase the likelihood of developing resistant bacteria and can deliver antibiotics in fish and shellfish or livestock to humans. If antibiotics are absorbed into the body through meat or shellfish, they can lead to antibiotic resistance, which can lead to the spread of disease.
  • the bacteriophage of the present invention as a new feed additive antibiotic to solve the problems caused by the use of conventional antibiotics more natural friendly It is available.
  • the feed additives for aquaculture or shrimp farming of the present invention may be separately prepared in the form of a bacteriophage as a feed additive, or may be directly added to the feed, or directly mixed in a hatchery or aquaculture tank.
  • Bacteriophages in the feed and in the tank of the present invention may be liquid or dried, preferably in the form of dried powder. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying.
  • Feed additives comprising the bacteriophage of the present invention may be mixed in the form of powder in a component ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed.
  • the feed for fish and shellfish or shrimp farming may further include conventional additives to increase the shelf life of the feed in addition to the bacteriophage of the present invention.
  • the feed additive of the present invention may further be added to other non-pathogenic microorganisms.
  • Microorganisms that may be added include Lactobacillus, which has physiological activity and organic degradability under anaerobic conditions such as Bacillus subtilis , Bacillus subtilis , which can produce proteases, lipolytic enzymes and sugar converting enzymes, and bovine stomach. strain, increasing the weight of livestock, such as increasing the milk yield of milk (Aspergillus oryzae) on Aspergillus duck characters showing the effect of increasing the absorption of digested food fungi (J. Animal Sci 43 (Lactobacillus sp .):. 910- 926, 1976) and yeast (J. Anim. Sci . 56: 735-739, 1983) such as Saccharomyces cerevisiae .
  • the feed additive according to the present invention may include cereals, root fruits, food processing by-products, algae, fiber oil, pharmaceutical by-products, oils, starches, gourds, grain by-products, etc. Minerals, fats, oils, minerals, fats, unicellular proteins, zooplankton, leftovers and the like may be included, but is not particularly limited thereto.
  • the feed additive according to the present invention may further include a binder, an emulsifier, a preservative, and the like, which are added to prevent quality deterioration, and are added to the feed to increase the efficacy of amino acids, vitamins, enzymes, probiotics, flavors, Non-protein nitrogen compounds, silicates, buffers, colorants, extractants, oligosaccharides and the like may be further included, in addition to the feed may include a mixture, but is not particularly limited. It is also possible to continue to reduce the number of Vibrio Habays in the farms by mixing and supplying them to the hatchery and the fish farms, and to find the Vibrio Habay clean farms.
  • the present invention provides a disinfectant or cleaning agent comprising the bacteriophage as an active ingredient.
  • the disinfectant containing the bacteriophage as an active ingredient is also used for spraying to remove Vibrio Vietnamesei, which is used in the active area of fish or shrimp in the farm, hatchery, farmed fish or shrimp mortality area, cultured seafood or shrimp in the cooking place and cooking equipment. And places are not limited to this.
  • the cleaning agent containing the bacteriophage as an active ingredient may be used to remove vibrio Vietnamesei infectious to the aquarium of the hatchery or farm.
  • the present invention provides a method for preventing or treating an infectious disease caused by Vibrio habay using the bacteriophage or the composition.
  • the method includes administering the bacteriophage or the composition in a pharmaceutically effective amount to an individual suffering from an infectious disease caused by Vibrio Havey.
  • the bacteriophage or composition according to the present invention may be administered to a shellfish or shrimp in the form of a pharmaceutical preparation, or may be administered through a method of feeding the shellfish or shrimp into a feed or hatchery and aquaculture tank, and feeding it, preferably a feed additive. It can be administered in the form of a mixture in the feed.
  • the route of administration of the bacteriophage or composition according to the present invention can be administered via various routes, oral or parenteral, as long as the target tissue can be reached.
  • oral, topical, intravenous, intraperitoneal, intramuscular, intraarterial It may be administered in a conventional manner through, transdermal, nasal, inhalation, and the like.
  • suitable total daily usage of the bacteriophage or composition according to the invention administered by the method can be determined by the practitioner within the correct medical judgment.
  • the specific therapeutically effective amount for a particular individual may be determined in conjunction with the type and severity of the response to be achieved, the age, body weight, general health, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and specific composition of the individual. It is desirable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.
  • the novel bacteriophage of the present invention exhibits specific killing ability to Vibrio havey and has excellent acid and heat resistance properties. Therefore, the bacteriophage according to the present invention can be usefully used for the prevention or treatment of Vibrio havey infectious disease Vibriosis, and can be widely used as a feed additive composition, a disinfectant and a cleaning agent.
  • 1 is an electron micrograph of the bacteriophage ⁇ CJ17 isolated in the present invention.
  • FIG. 2 is a photograph showing the result of pulsed field gel electrophoresis (PFGE) of the bacteriophage ⁇ CJ17 isolated in the present invention.
  • PFGE pulsed field gel electrophoresis
  • Figure 3 is a photograph showing the SDS-PAGE results of the bacteriophage ⁇ CJ17 isolated in the present invention.
  • Figure 4a is a photograph showing the PCR results using the primer set of SEQ ID NO: 6 to 9 as a template of the bacteriophage ⁇ CJ17 genomic DNA isolated in the present invention.
  • Figure 4b is a photograph showing the PCR results of the primer set of SEQ ID NO: 10 and 11 with the bacteriophage ⁇ CJ17 genomic DNA isolated in the present invention as a template.
  • Figure 4c is a photograph showing the PCR results using the primer set of SEQ ID NO: 12 and 13 as a template of the bacteriophage ⁇ CJ17 genomic DNA isolated in the present invention.
  • Figure 4d is a photograph showing the PCR results using the primer set of SEQ ID NO: 14 and 15 as a template of the bacteriophage ⁇ CJ17 genomic DNA isolated in the present invention.
  • Figure 5 is a graph showing the acid resistance test results of the bacteriophage ⁇ CJ17 isolated in the present invention.
  • Figure 6 is a graph showing the heat resistance test results of the bacteriophage ⁇ CJ17 isolated in the present invention.
  • Vibrio harveyi BB120, VH-BB120, ATCC BAA-1116TM shake culture (OD 600 2) and 10 ⁇ LB medium (tryptone 10 g / l, yeast extract 5 g / l and 18 ml of the sample solution was added to a mixture of 2 ml NaCl 10 g / L), and the mixture was incubated at 30 ° C. for 18 hours. Subsequently, the culture solution was centrifuged at 4,000 rpm for 10 minutes to separate the supernatant, and the separated supernatant was filtered with a 0.45 ⁇ m filter.
  • a single lysate plaque was obtained by sequentially diluting the supernatant from the selected sample solution and applying it to the soft agar overlay assay at a series of concentration levels. Since one lysate consists of one bacteriophage, one lysate was taken and 400 ⁇ l of SM solution (NaCl 5.8 g / l, MgSO 4 ⁇ 7H 2 O 2 g / l, 1 M Tris-HCl (pH) 7.5) Soak in 50 mL / L), and allowed to stand at room temperature for 4 hours to purely isolate the bacteriophage.
  • SM solution NaCl 5.8 g / l, MgSO 4 ⁇ 7H 2 O 2 g / l, 1 M Tris-HCl (pH) 7.5
  • Example ⁇ 1-1> The bacteriophage selected in Example ⁇ 1-1> was cultured in large quantities using VH-BB120, and the bacteriophage was purified therefrom.
  • VH-BB120 was shaken and cultured to 1.0 ⁇ 10 10 cfu (colony forming unit), centrifuged at 4,000 rpm for 10 minutes, and then resuspended in 4 ml of SM solution.
  • PFU plaque forming unit
  • DNase I and RNase A were added to a final concentration of 1 ⁇ g / ml and 30 ° C. at 30 ° C. Let stand for a minute. Subsequently, NaCl and PEG (polyethylene glycol) were added so that the final concentrations were 1 M and 10% (w / v), respectively, and further left at 4 ° C. for 3 hours. This was centrifuged at 12,000 rpm for 20 minutes at 4 °C, the supernatant was removed and the precipitate was separated. The separated precipitate was suspended in 5 ml of SM solution and allowed to stand at room temperature for 20 minutes.
  • NaCl and PEG polyethylene glycol
  • the purified bacteriophage was named "Bacteriophage ⁇ CJ17" and was deposited with the accession no. It was.
  • Vibrio parahaemolyticus V. parahaemolyticus
  • Vibrio campbelli V. campbelli
  • two Vibrio Havey V. harveyi
  • Bacteriophage ⁇ CJ17 isolated and purified in Example ⁇ 1-2> was diluted in 0.01% (w / v) gelatin solution and fixed with 2.5% glutaraldehyde solution. It was added dropwise onto a carbon-coated mica plate (approximately 2.5 ⁇ 2.5 mm) and acclimated for 10 minutes, then washed with sterile distilled water. Carbon film was placed on a copper grid, dyed in 4% uranyl acetate for 30 to 60 seconds, dried, and then passed through a transmission electron microscope (JEM-1011 transmission electron microscope, 80 kV, magnification ⁇ 120,000 to x 200,000).
  • JEM-1011 transmission electron microscope 80 kV, magnification ⁇ 120,000 to x 200,000
  • the bacteriophage ⁇ CJ17 of the present invention is a morphotype B1 consisting of a morphological icosahedron (isometric capsid) and a long non-contractile tail (Sipovir) It was confirmed that it belongs to Siphoviridae.
  • Genomic DNA was extracted from the bacteriophage ⁇ CJ17 of the present invention. Specifically, EDTA (ethylene diamine tetraacetic acid, pH 8.0), protease K (proteinase K) and SDS (sodium dodecyl sulfate) in the culture of purified bacteriophage ⁇ CJ17, respectively, the final concentration of 20 mM, 50 ⁇ g / ml and 0.5% (w / v) was added and allowed to stand at 55 ° C for 1 hour. An equal volume of phenol (pH 8.0) was added thereto, stirred, and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant.
  • EDTA ethylene diamine tetraacetic acid, pH 8.0
  • protease K proteinase K
  • SDS sodium dodecyl sulfate
  • the supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature.
  • 3 M sodium acetate was added to 10% (v / v) of the total volume, mixed, and 2 volumes of cold 95% ethanol were added thereto at -20 ° C. It was left for 1 hour. Thereafter, the supernatant was completely removed by centrifugation at 12,000 rpm for 10 minutes at 0 ° C to obtain precipitated genomic DNA.
  • TE buffer Tris-EDTA, pH 8.0
  • PFGE pulse-field gel electrophoresis
  • the bacteriophage ⁇ CJ17 of the present invention was confirmed to have a total genomic DNA size of about 75 kbp.
  • the bacteriophage ⁇ CJ17 of the present invention was observed a pattern consisting of proteins consisting of major structural proteins of 50 kDa, 44 kDa and 36 kDa size.
  • genomic DNA of bacteriophage ⁇ CJ17 was co-treated with restriction enzymes EcoRV, ScaI and NruI and co-treatment with restriction enzymes PvuII, HincII and StuI.
  • a pCL1920 vector Promega was digested with restriction enzyme SmaI, and a vector treated with CIP (calf intestinal alkaline phosphatase) was prepared. Reaction conditions were mixed so that the amount of the cut genomic DNA and the vector was 3: 1, and the ligation reaction was performed at 16 ° C. for 5 hours to obtain an expression vector containing each genomic DNA fragment. Expression vectors obtained therefrom were introduced into E.
  • Each transformant prepared was inoculated and cultured in LB plate medium containing ampicillin and X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), and then Two colonies were selected by the blue-white colony screening method. Each of the selected colonies was shaken for 16 hours in a medium containing ampicillin, thereby obtaining respective cells. Plasmid purification kit (Solgent) was extracted from each cell obtained.
  • the extracted plasmid was cloned by PCR using M13 forward and reverse (SEQ ID NOs: 16 and 17) primer sets. Among them, the size of the inserted genomic DNA was 1 kb or more, and the similarity of the nucleotide sequences was analyzed using the primer set. The results are shown in Table 2 below.
  • Example 7 PCR analysis using bacteriophage ⁇ CJ17 specific primers
  • primer sets of SEQ ID NOs: 6 and 7 were prepared based on the nucleotide sequences of SEQ ID NO: 1
  • primer sets of SEQ ID NOs: 8 and 9 were prepared based on the nucleotide sequences of SEQ ID NO: 2.
  • primer sets of SEQ ID NO: 10 and 11, SEQ ID NO: 12 and 13, and SEQ ID NO: 14 and 15 were prepared based on the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.
  • PCR was performed using each primer set thus prepared and using genomic DNA of bacteriophage ⁇ CJ17 as a template.
  • each primer was added to a pre-mix (Preone, Bioneer) so as to have a total genome DAN of 0.5 ⁇ g of bacteriophage ⁇ CJ17 and adjusted to a final volume of 20 ⁇ l.
  • PCR was performed by repeating 30 seconds of denaturation at 94 ° C, annealing at 30 ° C for 30 seconds, and polymerization conditions of 1 minute at 72 ° C.
  • Figure 4a shows the PCR results using the primer set of SEQ ID NO: 6 and 7, and 8 and 9
  • Figure 4B shows the PCR results using the primer set of SEQ ID NO: 10 and 11
  • Figure 4C is SEQ ID NO: PCR results using the primer sets 12 and 13 are shown
  • FIG. 4D shows the PCR results using the primer sets SEQ ID NOs: 14 and 15.
  • the PCR products of 400 bp, 900 bp, 500 bp and about 1000 bp were amplified.
  • the bacteriophage ⁇ CJ17 of the present invention was confirmed to be stable without losing activity in the range from pH 4.0 to pH 11.0.
  • bacteriophage ⁇ CJ17 When bacteriophage ⁇ CJ17 was used as a feed additive, the following experiment was performed to confirm the stability of heat generated during the formulation process. Specifically, 100 ⁇ l of a solution of bacteriophage ⁇ CJ17 with a 3.0 ⁇ 10 11 PFU / mL titer was respectively 0, 30, 60, and 120 at 37 ° C., 45 ° C., 53 ° C., 60 ° C., and 70 ° C. Let stand for a minute. The treated experimental culture was diluted in steps, and 10 ⁇ l of the diluted solution of each step was added dropwise according to the soft agar overlay method, followed by incubation at 30 ° C. for 18 hours to determine the titer through lysis. At this time, relative stability according to temperature and exposure time was compared based on the titer at 0 ° C and 37 ° C.
  • the bacteriophage ⁇ CJ17 of the present invention maintained the activity even when exposed to 60 hours at 60 °C, it can be seen that the activity decreases over time when exposed to more than 10 minutes at 70 °C .
  • bacteriophage ⁇ CJ17 showed bactericidal activity against wild isolates other than the specific Vibrio Havey strain VH-BB120 used in the experiment.
  • the bacteriophage ⁇ CJ17 of the present invention was confirmed to exhibit the ability to infect not only VH-BB120, but also all kinds of Vibrio Habei.
  • the bacteriophage ⁇ CJ17 of the present invention can be seen that when applied to the site, such as fish farms, can exhibit an excellent control effect for Vibrio Habei.
  • novel bacteriophage of the present invention exhibits specific killing ability, excellent acid resistance and heat resistance to Vibrio Havey, it can be usefully used for the prevention or treatment of Vibrio's Vibrio infectious disease Vibrioosis, as well as a composition for feed addition, It can be widely used as a disinfectant and cleaning agent.

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Abstract

La présente invention concerne un nouveau bactériophage ayant une activité bactéricide spécifique de V. harveyi, une composition comprenant le bactériophage comme principe actif pour la prévention ou le traitement de maladies infectieuses provoquées par V. harveyi, et une méthode de prévention ou de traitement de la maladie infectieuse provoquée par V. harveyi par l'utilisation d'un antibiotique, d'un additif alimentaire, d'un désinfectant ou d'un détergent qui comprend le bactériophage comme principe actif, et le bactériophage.
PCT/KR2013/000166 2012-01-09 2013-01-09 Nouveau bactériophage isolé et composition antibactérienne comprenant celui-ci WO2013105781A1 (fr)

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CN108699533A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-1及其用于抑制副溶血弧菌增殖的用途
CN108699532A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN110709510A (zh) * 2017-05-10 2020-01-17 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-7及其用于抑制副溶血弧菌细菌增殖的用途
CN112063595A (zh) * 2020-09-24 2020-12-11 瑞科盟(青岛)生物工程有限公司 一株裂解性哈维弧菌噬菌体rdp-vp-19012及其应用
CN112442487A (zh) * 2019-08-14 2021-03-05 宁波大学 哈维氏弧菌高效裂解性噬菌体vB-VhaS-yong1及其应用
CN113046326A (zh) * 2021-02-05 2021-06-29 广东省科学院微生物研究所(广东省微生物分析检测中心) 一株烈性副溶血性弧菌噬菌体vB_VpP_FE11的分离及应用
CN113337474A (zh) * 2021-02-05 2021-09-03 华南农业大学 一株副溶血性弧菌裂解性噬菌体vB_VpP_DE17及其应用

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CN110616197B (zh) * 2019-07-02 2023-05-26 上海海洋大学 副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型生物杀菌制剂
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CN115181731B (zh) * 2021-04-02 2023-12-15 青岛诺安百特生物技术有限公司 一株坎贝氏弧菌噬菌体、制备方法及其应用
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CN108699533A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-1及其用于抑制副溶血弧菌增殖的用途
CN108699532A (zh) * 2015-12-21 2018-10-23 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN108699533B (zh) * 2015-12-21 2022-08-23 尹特荣生物科技株式会社 副溶血弧菌噬菌体Vib-PAP-1及其用于抑制副溶血弧菌增殖的用途
CN108699532B (zh) * 2015-12-21 2022-08-23 尹特荣生物科技株式会社 副溶血弧菌噬菌体Vib-PAP-2及其用于抑制副溶血弧菌增殖的用途
CN110709510A (zh) * 2017-05-10 2020-01-17 尹特荣生物科技株式会社 新型副溶血弧菌噬菌体Vib-PAP-7及其用于抑制副溶血弧菌细菌增殖的用途
CN112442487A (zh) * 2019-08-14 2021-03-05 宁波大学 哈维氏弧菌高效裂解性噬菌体vB-VhaS-yong1及其应用
CN112063595A (zh) * 2020-09-24 2020-12-11 瑞科盟(青岛)生物工程有限公司 一株裂解性哈维弧菌噬菌体rdp-vp-19012及其应用
CN113046326A (zh) * 2021-02-05 2021-06-29 广东省科学院微生物研究所(广东省微生物分析检测中心) 一株烈性副溶血性弧菌噬菌体vB_VpP_FE11的分离及应用
CN113337474A (zh) * 2021-02-05 2021-09-03 华南农业大学 一株副溶血性弧菌裂解性噬菌体vB_VpP_DE17及其应用
CN113046326B (zh) * 2021-02-05 2022-08-16 广东省科学院微生物研究所(广东省微生物分析检测中心) 一株烈性副溶血性弧菌噬菌体vB_VpP_FE11的分离及应用
CN113337474B (zh) * 2021-02-05 2022-09-27 华南农业大学 一株副溶血性弧菌裂解性噬菌体vB_VpP_DE17及其应用

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