WO2013105781A1 - Novel isolated bacteriophage and antibacterial composition comprising same - Google Patents

Novel isolated bacteriophage and antibacterial composition comprising same 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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PCT/KR2013/000166
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French (fr)
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

The present invention relates to a novel bacteriophage having bactericidal activity specific to V.harveyi, to a composition comprising the bacteriophage as an active ingredient for the prevention or treatment of infectious diseases caused by V.harveyi, and to a method for preventing or treating the infectious disease caused by V.harveyi by using an antibiotic, a feed additive, a disinfectant, or a cleaner which include the bacteriophage as an active ingredient, and the bacteriophage.

Description

신규 분리된 박테리오파지 및 이를 포함하는 항균 조성물Newly Isolated Bacteriophage and Antimicrobial Compositions Comprising the Same
본 발명은 신규 분리된 박테리오파지(bacteriophage) 및 이를 포함하는 항균 조성물에 관한 것으로, 보다 구체적으로 본 발명은 비브리오 하베이(Vibrio harveyi) 특이적 사멸능을 갖는 신규 박테리오파지, 상기 박테리오파지를 유효성분으로 포함하는 비브리오 하베이 감염성 질병의 예방 또는 치료용 조성물, 상기 박테리오파지를 유효성분으로 포함하는 항생제, 상기 박테리오파지를 유효성분으로 포함하는 사료첨가제, 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제, 및 상기 박테리오파지를 이용하여 비브리오 하베이에 의한 감염성 질환을 예방 또는 치료하는 방법에 관한 것이다. 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 habei 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.
양식 새우(penaeid shrimp)에서 비브리오증을 일으키는 세균으로 알려진 비브리오 하베이(V. harveyi)는 그람 음성, 간균의 발광성 세균으로 해양 환경의 도처에서 생존한다. 비브리오 하베이는 해양생물의 정상 세균총이나, 몇몇 종이 해양 어류, 패류, 새우의 중요한 병원체로 보고되었다(Karunasagar I et al., Aquaculture 128: 203-209, 1994, Zhang XH and Austin B, J. Fish Dis. 23: 93-103, 2000). 1990년부터 비브리오 하베이 감염은 유충(larva)기의 새우 Penaeus monodon과 Penaeus japonicas의 폐사를 100% 일으키므로, 새우 양식장의 심각한 경제적 손실을 초래한다(Liu PC et al., Lett. Appl. Microbiol. 22: 413-416). 이와 같은, 새우류에서 비브리오 하베이에 대한 감염을 새우류 비브리오증, 새우류 세균성 패혈증, 발광성 비브리오증 또는 붉은다리(red-leg disease) 병이라고 명명한다. 비브리오증의 임상 증상은 무기력, 조직 괴사, 성장지연, 유충의 변태 지연과 기형, 발광성(어둠 속에서 새우가 빛을 발함), 근 혼탁, 색소 침착 등이 있다(Robertson, PAW et al., J. Microbiol. Methods 34: 31-39, 1998). 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). Since 1990, Vibrio habei infection has caused 100% mortality of larva shrimp Penaeus monodon and Penaeus japonicas , resulting in severe economic losses to shrimp farms (Liu PC et al., Lett. Appl. Microbiol. 22 413-416). Such infections of vibrio havey in shrimp are termed shrimp vibriosis, shrimp bacterial sepsis, luminescent vibriosis or red-leg disease. Clinical symptoms of vibriosis include lethargy, tissue necrosis, delay in growth, metamorphosis and malformation of larvae, luminescence (shrimp glows in the dark), muddiness and pigmentation (Robertson, PAW et al., J) . Microbiol.Method 34: 31-39, 1998).
비브리오 하베이의 병원성 메커니즘은 아직 명확히 밝혀지지 않았으며 숙주 및 균의 종에 따라 달라진다. 비브리오 하베이의 병원성 요소로 세포외물질(extracellular products, ECP)이 있으며, 이에는 프로테아제(protease), 헤모리신(hemolysins), 리파제(lipases), 리포폴리사카라이드(lipopolysaccaride)와 박테리오신 유사물질(bacteriocin-like substances)이 포함된다(Teo JWP et al., Gene 312: 181-188, 2003). 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).
많은 양식 새우 부화장에서 비브리오 하베이의 감염을 막기 위한 예방책으로 항생제를 사용하고 있다. 그러나 항생제 내성균과 항생제 잔류는 국제적인 문제이며, 병원균에 대한 포괄적인 치료방법이 시급한 상황이다. Many farmed shrimp hatcheries use antibiotics as a preventive measure against Vibrio Habei's infection. However, antibiotic-resistant bacteria and antibiotic residues are an international problem and there is an urgent need for comprehensive treatment for pathogens.
한편, 박테리오파지는 특정 세균에만 감염하여 세균의 성장을 통제하는 세균-특이적 바이러스로, 보통 파지라고 줄여서 부르기도 하며, 세균숙주 없이는 자가 증식이 불가능하다. 박테리오파지는 단일 혹은 이중 사슬의 DNA 또는 RNA가 유전물질로서 핵산을 구성하고 있으며, 이 핵산을 단백질 외피가 싸고 있는 단순한 구조로 20면체 머리에 꼬리가 있는 형태, 20면체 머리에 꼬리가 없는 형태, 그리고 필라멘트형의 3가지 기본형 구조로 나뉜다. 가장 흔한 형태인 20면체 머리에 꼬리가 있는 형태의 박테리오파지는 꼬리 특성에 따라 수축성 꼬리를 가지는 미오오비리데(Myoviridae), 길고 수축성이 없는 꼬리를 갖는 시포비리데(Siphoviridae), 및 짧은 꼬리를 갖는 포도비리데(Podoviridae)로 세분화될 수 있다. 20면체 머리에 꼬리가 없는 박테리오파지는 머리의 형태, 머리의 구성성분 및 외피의 유무에 따라 세분화된다. 마지막으로, 필라멘트 형태의 박테리오파지는 크기, 모양, 외피 및 필라멘트 구성성분에 따라 세분화된다(H.W. Ackermann, Frequency of morphological phage descriptions in the year 2000; Arch. Virol. 146: 843-857, 2001; Elizabeth Kutter 등, Bacteriophages Biology and Application, CRC press). Bacteriophage, on the other hand, 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. The most common forms of 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).
박테리오파지의 발견 이후 이들을 감염 질병의 치료제로 이용하기 위한 연구가 진행되었지만, 광범위한 숙주범위(broad target spectrum)를 갖는 항생제의 특성에 비해 숙주특이성(specific target spectrum)을 갖는 박테리오파지가 경쟁에서 밀려나 관심을 받지 못했다. 그러나, 최근 들어 동물의 성장촉진을 위해 사료에 첨가하는 항생제(antimicrobial growth promoter, AGP)가 항생제 내성 유발의 주요 원인임이 밝혀짐에 따라 AGP의 사용을 금지하는 정책들이 입안되었고, 한국은 2011년 7월부터 AGP 사용이 전면 금지되었다.Since the discovery of bacteriophages, studies have been conducted to use them as therapeutic agents for infectious diseases, but bacteriophages with specific target spectrums are attracted attention due to the characteristics of antibiotics with broad target spectrum. I couldn't. However, recently, antimicrobial growth promoter (AGP) added to feeds to promote animal growth has been found to be a major cause of antibiotic resistance, and policies to ban the use of AGP have been enacted. The use of AGP has been banned since March.
이러한 흐름을 바탕으로 박테리오파지의 연구가 다시 관심을 모으고 있다. 예를 들어, 클로스트리디움 스포로진 파아지(Clostridium sporogenes phage) NCIMB 30008과 클로스트리디움 트리로부티리쿰 파아지(Clostridium tyrobutiricum phage) NCIMB 30008은 각각 2003년과 2005년에 유럽에서 사료보존제로 등록되어 사료 내 오염된 클로스트리디움 균주(Clostridium sp.)의 통제를 목적하는 제품으로 개발되었다. 이러한 연구는 박테리오파지를 이용해 항생제 치료가 어려운 세균의 통제나 축산물 등에 오염된 인수공통 전염균 등을 식품 단계에서 통제할 수 있는 연구가 지속적으로 이루어지고 있음을 보여준다.Based on this trend, the bacteriophage research is attracting attention again. For example, 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.
이러한 배경하에서, 본 발명자들은 항생제의 오·남용으로 인한 항생제 내성균의 발생, 식품 내 항생제의 잔류 및 과거 항생제와 같이 광범위한 숙주범위의 문제점을 해결하기 위해, 양식 어류와 새우류에서 비브리오증을 일으키는 비브리오 하베이에 감염하는 박테리오파지를 자연계에서 신규 분리하고, 이들의 형태적, 생화학적 및 유전적 특성과 함께 비브리오 하베이균만을 선택적으로 사멸시킬 수 있는 능력을 확인하고 본 발명을 완성하였다. Against this background, we have developed 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.
본 발명의 목적은 비브리오 하베이에 대하여 특이적 사멸능을 갖는 신규 박테리오파지를 제공하는 것이다.It is an object of the present invention to provide novel bacteriophages having specific killing capacity for Vibrio havey.
본 발명의 다른 목적은 상기 박테리오파지를 유효성분으로 포함하는 비브리오 하베이에 의한 감염성 질환의 예방 또는 치료용 조성물을 제공하는 것이다.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.
본 발명의 또 다른 목적은 상기 박테리오파지를 유효성분으로 포함하는 항생제를 제공하는 것이다.Still another object of the present invention is to provide an antibiotic comprising the bacteriophage as an active ingredient.
본 발명의 또 다른 목적은 상기 박테리오파지를 유효성분으로 포함하는 어패류 또는 새우 양식용 사료첨가제를 제공하는 것이다.Still another object of the present invention is to provide a fish or shellfish feed additive containing the bacteriophage as an active ingredient.
본 발명의 또 다른 목적은 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제를 제공하는 것이다.Another object of the present invention to provide a disinfectant or cleaning agent containing the bacteriophage as an active ingredient.
본 발명의 또 다른 목적은 상기 박테리오파지 또는 조성물을 이용하여 비브리오 하베이에 의한 감염성 질환을 예방 또는 치료하는 방법을 제공하는 것이다.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.
본 발명은 비브리오 하베이(Vibrio harveyi)에 특이적 사멸능을 갖는 신규 박테리오파지를 제공한다.The present invention provides novel bacteriophages with specific killing ability to Vibrio harveyi .
본 발명자들은 바닷물로부터 채취된 시료에서 숙주세포인 비브리오 하베이 균주(Vibrio harveyi BB120, VH-BB120)를 선택적으로 용균시키는 박테리오파지를 분리한다. 전자현미경을 통해 형태학적으로 관찰한 결과, 상기 박테리오파지는 비브리오균들 중 비브리오 하베이를 선택적으로 감염시키는 종 특이성을 가지며(표 1 참조); 다양한 야생 분리주 비브리오 하베이에 감염될 수 있고(표 3 참조); 비브리오 하베이에 특이적 사멸능을 나타내며; 형태학상 정이십면체의 머리(isometric capsid)와 수축성이 없는 꼬리(long non-contractile tail)로 구성된 형태형(morphotype) B1, 시포비리데(Siphoviridae)에 속하는 것을 확인한다(도 1 참조). 또한, 상기 박테리오파지는 전체 게놈 크기가 약 75 kbp이며(도 2 참조); 이의 단백질 패턴을 분석한 결과, 50 kDa, 44 kDa, 및 36 kDa 크기의 단백질을 주요 구조단백질로 갖는 것을 확인한다(도 3 참조). 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).
아울러, 이들의 유전적 특성을 분석한 결과, 서열번호: 1 내지 5의 핵산 분자를 전체 게놈의 일부로서 포함하고, 상기 서열번호: 1 내지 5를 바탕으로 타종간 유사성을 비교한 결과, 핵산 분자 단편에 따라 20 내지 45%의 유사성을 보이나 모든 단편이 100% 일치하는 박테리오파지가 없는 신규한 박테리오파지임을 확인한다(표 2 참조). 유전적 특성을 좀 더 구체적으로 분석하기 위해, 상기 박테리오파지에 특이적인 프라이머 세트(primer set), 구체적으로, 서열번호: 6 및 7, 서열번호: 8 및 9, 서열번호: 10 및 11, 서열번호: 12 및 13, 및 서열번호: 14 및 15의 프라이머 세트로 PCR을 수행한 결과, 각각 600 bp, 400 bp, 900 bp, 500 bp 및 1000 bp의 PCR 산물이 증폭됨을 확인한다(도 4a 내지 4d 참조).In addition, as a result of analyzing their genetic characteristics, 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). To analyze the genetic characteristics more specifically, 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. As a result, PCR products of 600 bp, 400 bp, 900 bp, 500 bp and 1000 bp were amplified respectively (FIGS. 4A to 4D). Reference).
한편, 상기 박테리오파지를 비브리오 하베이에 감염시키면 용균반(phage plaque로 soft agar에서 하나의 박테리오파지에 의해 숙주세포가 용균되어 형성되는 clear zone)이 형성되고, 상기 용균반의 크기와 탁도 등이 동일한 것으로 확인된다. 이러한 결과는 상기 박테리오파지가 비브리오 하베이를 용균시켜서 비브리오 하베이의 성장을 억제함을 나타내는 것이다. On the other hand, when the bacteriophage is infected with Vibrio habay, a clear zone in which host cells are lysed by one bacteriophage in a soft agar with phage plaque is formed, and the size and turbidity of the lysate are confirmed to be the same. . These results indicate that the bacteriophage lysates Vibrio habay to inhibit the growth of Vibrio habay.
상기 박테리오파지의 안정성을 다양한 pH 및 온도 범위에서 조사한 결과, pH 4 내지 11의 범위에서 안정하게 생존하는 내산성을 가지며(도 5 참조), 43℃ 내지 60℃의 범위, 즉, 높은 온도에서도 안정하게 생존할 수 있는 내열성을 가짐을 확인한다(도 6 참조). As a result of investigating the stability of the bacteriophage at various pH and temperature ranges, 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).
따라서, 비브리오 하베이에 대한 특이성, 내산성 및 내열성은 본 발명에 따른 박테리오파지를 양식 어패류 및 새우류의 비브리오 하베이 감염성 질환의 예방 및 치료용 조성물, 박테리오파지를 유효성분으로 포함하는 다양한 제품 등에 적용함에 있어, 광범위한 온도 및 pH 범위에의 적용을 가능하게 한다.Therefore, the specificity, acid resistance and heat resistance of Vibrio habei, 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.
이에, 본 발명자들은 바닷물로부터 시료를 채취하여, 비브리오 하베이에 대한 특이적 사멸능을 갖고 상기와 같은 특징을 갖는 박테리오파지를 "Bacteriophage ΦCJ17"로 명명하고, 부다페스트 조약 하에 2012년 1월 2일자로 한국미생물 보존센터(Korean Culture Center of Microorganisms, 서울시 서대문구 홍제1동 361-221)에 기탁번호 제KCCM11247P호로 기탁하였다.Accordingly, the present inventors collected a sample from the sea water, named as a bacteriophage "Bacteriophage Φ 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.
또 하나의 양태로서, 본 발명은 상기 박테리오파지를 유효성분으로 포함하는 비브리오 하베이에 의하여 유발된 감염성 질환의 예방 또는 치료용 조성물을 제공한다. As another aspect, 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.
본 발명의 박테리오파지는 비브리오 하베이를 특이적으로 사멸시킬 수 있는 항균 활성을 가지므로, 이의 감염에 의해 유발되는 질병을 예방하거나 치료하기 위한 목적으로 이용될 수 있다. 바람직하게, 비브리오 하베이에 의하여 유발되는 감염성 질환은 비브리오증(Vibriosis)을 들 수 있지만, 이에 제한되지는 않는다.Since the bacteriophage of the present invention has an antimicrobial activity that can specifically kill Vibrio habei, it can be used for the purpose of preventing or treating diseases caused by its infection. Preferably, the infectious disease caused by Vibrio Havey may include, but is not limited to, Vibriosis.
본 명세서에 사용된 용어 "비브리오증(Vibriosis)"은 비브리오 하베이균의 감염에 의하여 일어나며, 해수 양식어류, 패류, 새우류 등 다양한 해양동물에 감염되는 질병을 의미하는데, 특히 새우류에서 무기력, 조직괴사, 성장지연, 유충의 변태 지연, 기형, 발광, 근혼탁, 색소 침착 등을 일으켜 대량 폐사를 야기한다. As used herein, the term "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.
본 명세서에 사용된 용어 "예방"이란 조성물의 투여로 질병을 억제하거나 발병을 지연시키는 모든 행위를 의미하는 것이며, "치료"란 조성물의 투여로 대상 질병의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하는 것이다.The term "prevention" as used herein refers to any action that inhibits or delays the onset of the disease by administration of the composition, and "treatment" refers to any action that improves or advantageously changes the condition of the subject disease by administration of the composition. It means.
상기 조성물은 5×102 내지 5×1012 PFU/㎖, 바람직하게는 1×106 내지 1×1010 PFU/㎖의 본 발명에 따른 박테리오파지를 유효성분으로 포함한다.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.
본 발명의 예방 또는 치료용 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있다.The prophylactic or therapeutic composition of the present invention may further comprise a pharmaceutically acceptable carrier.
본 명세서에 사용된 용어, "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 하나 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.As used herein, 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. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 예방 또는 치료용 조성물은 경구 투여 또는 비경구 투여를 통해 투여할 수 있으며, 질환부위 또는 개체에 도포 또는 침지하는 방법도 이용할 수 있다. 비경구 투여의 경우 정맥 내 투여, 복강 내 투여, 근육 내 투여, 또는 국부 투여를 이용하여 투여할 수도 있다. 본 발명의 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 대상이 되는 동물 및 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여시간, 투여 경로, 배설 속도 및 반응 감음성과 같은 요인들에 의해 달라질 수 있으며, 통상 숙련된 의사나 수의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.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. For 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.
본 발명의 조성물을 유효성분으로 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성 현탁액, 조제 분말 또는 과립, 에멀젼, 경질 또는 연질 캡슐, 시럽 또는 엘릭시르제로 제제화할 수 있다. 정제 및 캡슐 등의 제형으로 제제화하기 위해, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마스네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐 제형의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체담체를 더 함유할 수 있다.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. 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.
본 발명의 조성물을 유효성분으로 포함하는 비경구 투여용 제형으로는, 예를 들어 피하주사, 정맥주사 또는 근육 내 주사 등의 주사용 형태, 좌제 주입 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화할 수 있다. 주사용 제형으로 제제화하기 위해서는 본 발명의 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제화할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.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. To formulate 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. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
또 다른 양태로서, 본 발명은 상기 박테리오파지를 유효성분으로 포함하는 항생제를 제공한다.In another aspect, the present invention provides an antibiotic comprising the bacteriophage as an active ingredient.
본 명세서에 사용된 용어 "항생제"는 약제 형태로 동물에게 제공되어 균을 사멸시킬 수 있는 제제를 의미하며, 방부제, 살균제 및 항균제를 총칭하는 것이다. 상기 동물은 어패류, 새우 등을 포함하고, 천연 또는 양식된 것을 모두 포함한다. As used herein, the term "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.
본 발명의 박테리오파지는 기존 항생제에 비하여 비브리오 하베이에 대한 특이성이 매우 높으므로, 비브리오 하베이에 의하여 유발되는 질환의 예방 또는 치료에 특이적으로 사용될 수 있고, 약물 내성을 유도하지 않아 기존의 항생 물질에 비하여 제품수명(life cycling)이 긴 신규 항생제로서 이용될 수 있다.Since 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.
또 다른 양태로서, 본 발명은 상기 박테리오파지를 유효성분으로 포함하는 어패류 또는 새우 양식용 사료첨가제를 제공한다.In still another aspect, the present invention provides a fish or shellfish feed additive comprising the bacteriophage as an active ingredient.
축산, 수산업에서 사용되는 사료 첨가용 항생제는 예방 목적으로 사용되고 있는데, 예방 목적의 항생제 투여는 내성균 발생 가능성을 높이고 어패류 또는 가축에 잔류하는 항생제가 사람에게 전달될 수 있어서 문제이다. 항생제가 육류 또는 어패류를 통해 인체에 흡수되면 항생제 내성을 유발해 질병의 확산을 초래할 수도 있다. 또한, 사료에 섞여 먹이는 항생제의 종류가 많고 이는 다제 내성균의 발생 확률을 상승시키는 문제점이 있기 때문에 좀더 자연 친화적이면서도 기존의 항생제 사용으로 인한 문제를 해결할 새로운 사료첨가용 항생물질로서 본 발명의 상기 박테리오파지를 이용할 수 있다.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. In addition, there are many kinds of antibiotics mixed in the feed, which has a problem of increasing the probability of occurrence of multi-drug-resistant bacteria, 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.
본 발명의 어패류 또는 새우 양식용 사료첨가제는 박테리오파지를 사료첨가제 형태로 따로 제조하여 사료에 혼합시키거나, 사료제조시 직접 첨가시켜 사용할 수 있으며, 부화장이나 양식장 수조에서 직접 혼합하여 사용할 수 있다. 본 발명의 사료 내 및 수 조내 박테리오파지는 액상 또는 건조 상태일 수 있으며, 바람직하게는 건조된 분말 형태이다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다. 본 발명의 박테리오파지를 포함하는 사료첨가제는 분말 형태로 사료 중량의 0.05 내지 10 중량%, 바람직하게는 0.1 내지 2중량%의 성분비로 혼합될 수 있다. 또한, 상기 어패류 또는 새우 양식용 사료는 본 발명의 박테리오파지 외에 사료의 보존성을 높일 수 있는 통상의 첨가제들을 추가로 포함할 수 있다.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. In addition, 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.
본 발명의 사료첨가제에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다. 첨가될 수 있는 미생물로는 단백질 분해효소, 지질 분해효소 및 당 전환 효소를 생산할 수 있는 바실러스 서브틸리스(Bacillus subtilis)와 같은 고초균, 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능이 있는 락토바실러스 균주(Lactobacillus sp.), 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 아스퍼질러스 오리자에(Aspergillus oryzae)와 같은 사상균(J. Animal Sci. 43: 910-926, 1976) 및 사카로미세스 세레비지에(Saccharomyces cerevisiae)와 같은 효모(J. Anim. Sci. 56: 735-739, 1983)로 구성된 군으로부터 선택될 수 있다.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 .
본 발명에 따른 사료첨가제에는 식물성으로 곡물류, 근과류, 식품가공 부산물류, 조류, 섬유질유, 제약 부산물류, 유지류, 전분류, 박류, 곡물 부산물류 등이 포함될 수 있으며, 동물성으로 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질, 동물성 플랑크톤류, 남은 음식물 등이 포함될 수 있으나, 특별히 이에 제한되지는 않는다. 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.
본 발명에 따른 사료첨가제에는 품질 저하를 방지하기 위하여 첨가하는 결착제, 유화제, 보존제 등을 추가로 포함할 수 있고, 효용 증대를 위하여 사료에 첨가하는 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태 질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등을 추가로 포함할 수도 있으며, 그 외에도 사료 혼합제 등을 추가로 포함할 수 있으나, 특별히 이에 제한되지는 않는다. 또한 부화장 및 양식장 수조에 혼합하여 공급함으로써 지속적으로 양식장 내 비브리오 하베이의 숫자를 감소시킬 수 있으며, 비브리오 하베이 청정 양식을 모색할 수 있다.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.
또 다른 양태로서, 본 발명은 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제를 제공한다.In another aspect, the present invention provides a disinfectant or cleaning agent comprising the bacteriophage as an active ingredient.
상기 박테리오파지를 유효성분으로 포함하는 소독제는 비브리오 하베이 제거를 위한 살포에도 활용되어 양식장 내 어패류 또는 새우의 활동 영역, 부화장, 양식 어패류 또는 새우의 폐사 지역, 양식 어패류 또는 새우의 조리장소 및 조리설비에 사용될 수 있으며, 장소는 이에 국한되지 않는다. 또한, 상기 박테리오파지를 유효성분으로 포함하는 세척제는 부화장 또는 양식장의 수조에 감염 가능한 비브리오 하베이를 제거하는데 사용될 수 있다. The disinfectant containing the bacteriophage as an active ingredient is also used for spraying to remove Vibrio habei, 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. In addition, the cleaning agent containing the bacteriophage as an active ingredient may be used to remove vibrio habei infectious to the aquarium of the hatchery or farm.
또 다른 양태로서, 본 발명은 상기 박테리오파지 또는 상기 조성물을 이용하여 비브리오 하베이에 의한 감염성 질환을 예방 또는 치료하는 방법을 제공한다.In another aspect, the present invention provides a method for preventing or treating an infectious disease caused by Vibrio habay using the bacteriophage or the composition.
구체적으로, 본 발명의 방법은 비브리오 하베이에 의한 감염성 질환이 발병된 개체에 상기 박테리오파지 또는 상기 조성물을 약학적 유효량으로 투여하는 단계를 포함한다. 본 발명에 따른 박테리오파지 또는 조성물은 약학적 제제의 형태로 어패류 또는 새우에게 투여되거나, 어패류 또는 새우의 사료 또는 부화장 및 양식장 수조에 혼합하여 이를 섭식시키는 방법을 통해 투여될 수 있으며, 바람직하게는 사료첨가제의 형태로 사료에 혼합되어 투여될 수 있다. Specifically, 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.
본 발명에 따른 박테리오파지 또는 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있으며, 구체적으로, 구강, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비측내, 흡입 등을 통해 통상적인 방식으로 투여될 수 있다. 상기 방법에 의해 투여되는 본 발명에 따른 박테리오파지 또는 조성물의 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다. 특정 개체에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 개체의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.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. Specifically, oral, topical, intravenous, intraperitoneal, intramuscular, intraarterial It may be administered in a conventional manner through, transdermal, nasal, inhalation, and the like. It will be apparent to those skilled in the art that the 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.
본 발명의 신규 박테리오파지는 비브리오 하베이에 대하여 특이적 사멸능을 나타내며 내산성 및 내열성이 뛰어난 특성을 갖는다. 따라서, 본 발명에 따른 박테리오파지는 비브리오 하베이 감염성 질환인 비브리오증의 예방 또는 치료에 유용하게 사용될 수 있을 뿐만 아니라, 사료 첨가용 조성물, 소독제 및 세척제 등으로 광범위하게 이용될 수 있다.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.
도 1은 본 발명에서 분리된 박테리오파지 ΦCJ17의 전자현미경 사진이다.1 is an electron micrograph of the bacteriophage Φ CJ17 isolated in the present invention.
도 2는 본 발명에서 분리된 박테리오파지 ΦCJ17의 PFGE(pulsed field gel electrophoresis) 결과를 나타내는 사진이다.Figure 2 is a photograph showing the result of pulsed field gel electrophoresis (PFGE) of the bacteriophage Φ CJ17 isolated in the present invention.
도 3은 본 발명에서 분리된 박테리오파지 ΦCJ17의 SDS-PAGE 결과를 나타내는 사진이다.Figure 3 is a photograph showing the SDS-PAGE results of the bacteriophage Φ CJ17 isolated in the present invention.
도 4a는 본 발명에서 분리된 박테리오파지 ΦCJ17 게놈 DNA를 주형으로 하고 서열번호: 6 내지 9의 프라이머 세트를 이용한 PCR 결과를 나타내는 사진이다.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 ΦCJ17 genomic DNA isolated in the present invention.
도 4b는 본 발명에서 분리된 박테리오파지 ΦCJ17 게놈 DNA를 주형으로 하고 서열번호: 10 및 11의 프라이머 세트를 PCR 결과를 나타내는 사진이다.Figure 4b is a photograph showing the PCR results of the primer set of SEQ ID NO: 10 and 11 with the bacteriophage ΦCJ17 genomic DNA isolated in the present invention as a template.
도 4c는 본 발명에서 분리된 박테리오파지 ΦCJ17 게놈 DNA를 주형으로 하고 서열번호: 12 및 13의 프라이머 세트를 이용한 PCR 결과를 나타내는 사진이다.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 ΦCJ17 genomic DNA isolated in the present invention.
도 4d는 본 발명에서 분리된 박테리오파지 ΦCJ17 게놈 DNA를 주형으로 하고 서열번호: 14 및 15의 프라이머 세트를 이용한 PCR 결과를 나타내는 사진이다.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 ΦCJ17 genomic DNA isolated in the present invention.
도 5는 본 발명에서 분리된 박테리오파지 ΦCJ17의 내산성 실험 결과를 나타내는 그래프이다.Figure 5 is a graph showing the acid resistance test results of the bacteriophage Φ CJ17 isolated in the present invention.
도 6은 본 발명에서 분리된 박테리오파지 ΦCJ17의 내열성 실험 결과를 나타내는 그래프이다.Figure 6 is a graph showing the heat resistance test results of the bacteriophage Φ CJ17 isolated in the present invention.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 비브리오 하베이 사멸효과를 나타내는 박테리오파지의 분리Example 1 Isolation of Bacteriophage with Vibrio Habay Killing Effect
<1-1> 박테리오파지 스크리닝 및 단일 박테리오파지 순수 분리<1-1> Bacteriophage Screening and Single Bacteriophage Separation
인천 중구 을왕동 바다에서 수득한 바닷물 50 ㎖를 4,000 rpm에서 10분간 원심분리한 후, 상등액을 0.45 ㎛ 필터로 여과하여 시료액을 준비하고, 이를 이용하여 소프트 아가 오버레이(soft agar overlay) 방법을 수행하였다.50 ml of seawater obtained from Eulwang-dong, Jung-gu, Incheon was centrifuged at 4,000 rpm for 10 minutes, and then the supernatant was filtered through a 0.45 μm filter to prepare a sample solution, which was then subjected to a soft agar overlay method. .
구체적으로, 비브리오 하베이 균주(Vibrio harveyi BB120, VH-BB120, ATCC® BAA-1116TM) 진탕 배양액(OD600=2) 150 ㎕와 10× LB 배지(tryptone 10 g/ℓ, yeast extract 5 g/ℓ 및 NaCl 10 g/ℓ) 2 ㎖의 혼합물에 상기 시료액 18 ㎖을 가하여 혼합하고, 30℃에서 18시간 동안 배양하였다. 이어서 배양액을 4,000 rpm에서 10분간 원심분리하여 상등액을 분리한 후 분리된 상등액을 0.45 ㎛ 필터로 여과하였다. LB 평판 배지 위에 2% NaCl을 혼합한 0.6 %(w/v) 한천 4 ㎖과 VH-BB120 진탕 배양액(OD600=2) 150 ㎕의 혼합액을 부어 고형화시킨 후, 그 위에 상기에서 여과된 상등액 10 ㎕를 적가하고, 30℃에서 18시간 동안 배양하여 용균반이 형성된 시료액을 선발하였다. Specifically, 150 μl of 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 μm filter. 4 ml of 0.6% (w / v) agar mixed with 2% NaCl and 150 µl of VH-BB120 shake culture medium (OD 600 = 2) were poured onto the LB plate medium and solidified, followed by filtering the supernatant 10 above. ㎕ was added dropwise and incubated at 30 ° C. for 18 hours to select a sample solution in which lysate plaques were formed.
상기 선발된 시료액으로부터 유래한 상등액을 순차적으로 희석하여 일련의 농도 수준으로 소프트 아가 오버레이 분석법에 적용함으로써 단일 용균반을 획득하였다. 하나의 용균반은 하나의 박테리오파지로 구성된다고 보기 때문에, 하나의 용균반을 취해서 400 ㎕의 SM 용액(NaCl 5.8 g/ℓ, MgSO4·7H2O 2 g/ℓ, 1 M Tris-HCl(pH 7.5) 50 ㎖/ℓ)에 침지하고, 4시간 동안 실온에 정치하여 박테리오파지를 순수하게 분리하였다. 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 μl 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.
순수 분리된 박테리오파지를 대량으로 확보하기 위하여, 상기 용균반을 침지하고 정치시킨 용액의 상등액 100 ㎕, 2 %(w/v) NaCl, 0.6 %(w/v) 한천 12 ㎖ 및 VH-BB120 진탕 배양액 500 ㎕을 혼합한 후, 150 mm 지름의 LB 평판 배지에서 소프트 아가 오버레이를 수행하였다. 완전히 용균이 일어난 평판 배지에 15 ㎖의 SM 용액을 가하고, 실온에서 4시간 동안 저속으로 진탕시켜서 용균반에 포함된 박테리오파지를 용출시켰다. 상기 용출된 박테리오파지를 포함하는 SM 용액을 회수하고, 이를 0.45 ㎛ 필터로 여과하여 냉장 보관하였다. To secure large amounts of purely isolated bacteriophages, 100 μl of the supernatant of the solution in which the lysate was immersed and allowed to stand, 12 ml of agar (0.6% (w / v) agar) and 12 ml of VH-BB120 shake culture After mixing 500 μl, soft agar overlay was performed on 150 mm diameter LB plate media. 15 ml of SM solution was added to a completely lysed plate medium and shaken at low temperature for 4 hours at room temperature to elute the bacteriophage contained in the lysate plaque. The SM solution containing the eluted bacteriophage was recovered and stored in a refrigerator by filtration with a 0.45 μm filter.
<1-2> 박테리오파지의 대량배양 및 정제 <1-2> Mass Culture and Purification of Bacteriophage
상기 실시예 <1-1>에서 선발된 박테리오파지를 VH-BB120을 이용하여 대량으로 배양하고, 이로부터 박테리오파지를 정제하였다The bacteriophage selected in Example <1-1> was cultured in large quantities using VH-BB120, and the bacteriophage was purified therefrom.
구체적으로, VH-BB120를 진탕 배양하여 1.0×1010cfu(colony forming unit)가 되도록 분주하여 4,000 rpm에서 10분간 원심분리한 후, 이를 4 ㎖의 SM 용액에 재부유시켰다. 여기에 박테리오파지를 1.0×106 PFU(plaque forming unit)로 접종하고, MOI(multiplicity of infection)=0.001로 적정한 다음, 30℃에서 20분간 정치시켰다. 이어, 150 ㎖의 LB 배지에 접종하고, 30℃에서 5시간 동안 배양하였다. 배양이 종료된 후, 최종 부피의 1 %(v/v)가 되도록 클로로포름을 첨가하고 20분간 교반하였으며, 제한효소 DNase I과 RNase A를 각각 최종 농도 1 ㎍/㎖이 되도록 첨가하고 30℃에서 30분간 정치시켰다. 이어서, 최종 농도가 각각 1 M과 10 %(w/v)가 되도록 NaCl과 PEG(polyethylene glycol)를 첨가하고, 4℃에서 3시간 동안 추가 정치시켰다. 이를 4℃에서 12,000 rpm으로 20분간 원심분리한 후 상등액을 제거하고 침전물을 분리하였다. 분리한 침전물을 5 ㎖의 SM 용액에 현탁하고, 20분간 실온에서 정치시켰다. 여기에 4 ㎖의 클로로포름을 첨가한 후 교반하고 4℃에서 4,000 rpm으로 20분간 원심분리하여 상등액을 수득하였다. 상등액을 0.45 ㎛ 필터로 여과한 후, 글리세롤 밀도 구배법(밀도: 40%, 5% 글리세롤)을 이용한 초원심분리(35,000 rpm, 1시간, 4℃)를 수행하여 박테리오파지를 정제하였다. 상기 정제한 박테리오파지를 500 ㎕의 SM 용액에 현탁한 후 역가(titer)를 측정하였다.Specifically, 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. The bacteriophage was inoculated with 1.0 × 10 6 plaque forming unit (PFU), titrated with multiplicity of infection (= 0.001), and then left standing at 30 ° C. for 20 minutes. It was then inoculated in 150 ml of LB medium and incubated at 30 ° C. for 5 hours. After the incubation was completed, chloroform was added to 1% (v / v) of the final volume and stirred for 20 minutes. Restriction enzymes DNase I and RNase A were added to a final concentration of 1 μg / 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 ℃, 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. 4 ml of chloroform was added thereto, stirred, and centrifuged at 4,000 rpm for 20 minutes at 4 ° C to obtain a supernatant. The supernatant was filtered with a 0.45 μm filter, followed by ultracentrifugation (35,000 rpm, 1 hour, 4 ° C.) using glycerol density gradient (density: 40%, 5% glycerol) to purify the bacteriophage. The purified bacteriophage was suspended in 500 μl of SM solution and titer was measured.
상기 정제된 박테리오파지를 "Bacteriophage ΦCJ17"로 명명하고, 부다페스트 조약 하에 2012년 1월 2일자로 한국미생물 보존센터(Korean Culture Center of Microorganisms, 서울시 서대문구 홍제1동 361-221)에 기탁번호 제KCCM11247P호로 기탁하였다.The purified bacteriophage was named "Bacteriophage ΦCJ17" and was deposited with the accession no. It was.
실시예 2: 박테리오파지 ΦCJ17의 비브리오 균주 감염성 비교Example 2: Vibrio strain infectivity comparison of bacteriophage ΦCJ17
상기 실시예 <1-2>에서 분리, 정제한 박테리오파지 ΦCJ17이 VH-BB120 이외의 다른 종의 비브리오균에 대하여도 용균 활성을 나타내는지 여부를 확인하기 위해 교차감염을 수행하였다.Cross-infection was performed to confirm whether bacteriophage ΦCJ17 isolated and purified in Example <1-2> showed lytic activity against Vibrio bacteria of other species other than VH-BB120.
구체적으로, 5종의 비브리오 파라하에몰리티쿠스(V. parahaemolyticus), 5종의 비브리오 캠프벨리(V. campbelli) 및 2종의 비브리오 하베이(V. harveyi)를 각각 배양하여 배양액을 수득하고, 각각의 배양액과 상기 박테리오파지 ΦCJ17을 이용한 소프트 아가 오버레이 방법을 수행하여 용균반의 형성 여부를 확인하였다(표 1). Specifically, five Vibrio parahaemolyticus ( V. parahaemolyticus ), five Vibrio campbelli ( V. campbelli ) and two Vibrio Havey ( V. harveyi ) were cultured to obtain a culture solution, respectively A soft agar overlay method using the culture medium and the bacteriophage Φ CJ17 was performed to confirm the formation of lysate plaques (Table 1).
표 1 ΦCJ17의 병원성 비브리오균 감염 여부
균주 명칭 용균반 형성 여부
V.harveyi BB120V.harveyiV.parahaemolyticus(MEBiC06047)V.campbellii(GMD258)V.parahaemolyticus(MEBiC06069)V.campbellii(WDC577)V.parahaemolyticus(MEBiC06080)V.campbellii(MEBiC05073)V.parahaemolyticus(MEBiC06081)V.campbellii(MEBiC05077)V.parahaemolyticus(MEBiC06254)V.campbellii(MEBiC05078) OOXXXXXXXXXX
Table 1 Whether ΦCJ17 is infected with pathogenic Vibrio
Strain name Formation of lytic plaque
V.harveyi BB 120 V. harveyi V. parahaemolyticus (MEBiC06047) V. campbellii (GMD258) V. parahaemolyticus (MEBiC06069) V. campbellii (WDC577) (MEBiC05077) V. parahaemolyticus (MEBiC06254) V.campbellii (MEBiC05078) OOXXXXXXXXXX
* 'MEBiC' source: 한국해양연구원, 해양국한생물지원 BANK* 'MEBiC' source: Korea Maritime Research Institute, Korea Marine Biology Support BANK
상기 표 1에서 보듯이, 본 발명에 따른 박테리오파지 ΦCJ17은 비브리오 하베이 이외의 비브리오균에 대해서는 용균활성을 나타내지 않음을 확인하였고, 이러한 결과는 상기 박테리오파지가 비브리오 하베이에 특이적인 사멸능을 가짐을 나타내는 것이다.As shown in Table 1, it was confirmed that bacteriophage Φ CJ17 according to the present invention does not show the lytic activity against Vibrio bacteria other than Vibrio habei, these results indicate that the bacteriophage has a specific killing ability in Vibrio habei.
실시예 3: 박테리오파지 ΦCJ17의 형태학적 관찰Example 3: Morphological Observation of Bacteriophage Φ CJ17
실시예 <1-2>에서 분리, 정제한 박테리오파지 ΦCJ17을 0.01 %(w/v) 젤라틴 용액에 희석하고, 2.5% 글루타르알데하이드(glutaraldehyde) 용액으로 고정하였다. 이를 탄소-코팅 운모 판(carbon-coated mica plate, 대략 2.5×2.5 mm) 위에 적가하고 10분간 적응시킨 후, 멸균 증류수로 세척하였다. 탄소 필름(Carbon film)을 구리 격자(copper grid)에 끼워 4% 우라닐 아세테이트(uranyl acetate)에서 30 내지 60초간 염색하고 건조한 다음, 투과전자현미경(JEM-1011 transmission electron microscope, 80 kV, 배율 ×120,000 내지 ×200,000)으로 검경하였다. Bacteriophage Φ 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).
그 결과, 도 1에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 형태학상 정이십면체의 머리(isometric capsid)와 수축성이 없는 꼬리(long non-contractile tail)로 구성된 형태형(morphotype) B1, 시포비리데(Siphoviridae)에 속하는 것을 확인하였다.As a result, as shown in Figure 1, the bacteriophage Φ 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.
실시예 4: 박테리오파지 ΦCJ17의 전체 게놈 DNA 크기 분석Example 4 Whole Genomic DNA Size Analysis of Bacteriophage ΦCJ17
본 발명의 박테리오파지 ΦCJ17로부터 게놈 DNA를 추출하였다. 구체적으로, 정제된 박테리오파지 ΦCJ17의 배양액에 EDTA(ethylene diamine tetraacetic acid, pH 8.0), 단백분해효소 K(proteinase K) 및 SDS(sodium dodecyl sulfate)를 각각의 최종 농도가 20 mM, 50 ㎍/㎖ 및 0.5 %(w/v)가 되도록 첨가하고, 55℃에서 1시간 동안 정치하였다. 여기에 동일 부피의 페놀(pH 8.0)을 가하여 교반한 다음, 실온에서 12,000 rpm으로 10분간 원심분리하여 상등액을 수득하였다. 수득된 상등액을 동일 부피의 PC(phenol:chloroform=1:1)와 혼합하고, 실온에서 12,000 rpm으로 10분간 원심분리하여 상등액을 수득하였다. 상기 상등액을 동일 부피의 클로로포름과 혼합하고, 실온에서 12,000 rpm으로 10분간 원심분리를 수행하였다. 이로부터 수득된 상등액에 3 M 초산나트륨(sodium acetate)을 전체 부피의 10 %(v/v)가 되도록 가하여 혼합하고, 2배 부피의 차가운 95% 에탄올(ethanol)을 첨가한 후 -20℃에서 1시간 동안 정치시켰다. 그 후, 0℃에서 12,000 rpm으로 10분간 원심분리하여 상등액을 완전히 제거한 후 침전된 게놈 DNA를 수득하였다. 여기에 50 ㎕의 TE 완충액(Tris-EDTA, pH 8.0)을 가하여 게놈 DNA를 용해시키고, 이의 농도를 측정하였다. 이어서, 1 ㎍의 게놈 DNA를 1% PFGE(pulse-field gel electrophoresis) 아가로즈 겔에 로딩하고, BIORAD PFGE 시스템 7번 프로그램(size range 25-100 kb; switch time ramp 0.4-2.0 seconds, linear shape; forward voltage 180 V; reverse voltage 120 V)을 이용하여 상온에서 20시간 동안 전개하였다. Genomic DNA was extracted from the bacteriophage Φ 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 ΦCJ17, respectively, the final concentration of 20 mM, 50 ㎍ / ㎖ 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. The obtained supernatant was mixed with the same volume of PC (phenol: chloroform = 1: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature. To the supernatant obtained from this, 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. 50 μl of TE buffer (Tris-EDTA, pH 8.0) was added thereto to dissolve genomic DNA and its concentration was measured. Subsequently, 1 μg of genomic DNA was loaded onto a 1% pulse-field gel electrophoresis (PFGE) agarose gel and the BIORAD PFGE System No. 7 program (size range 25-100 kb; switch time ramp 0.4-2.0 seconds, linear shape; Forward voltage 180 V; reverse voltage 120 V) was used for 20 hours at room temperature.
그 결과, 도 2에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 약 75 kbp의 전체 게놈 DNA 크기를 갖는 것으로 확인되었다.As a result, as shown in Figure 2, the bacteriophage Φ CJ17 of the present invention was confirmed to have a total genomic DNA size of about 75 kbp.
실시예 5: 박테리오파지 ΦCJ17의 단백질 패턴 분석Example 5: Protein pattern analysis of bacteriophage ΦCJ17
1010 PFU/㎖ 역가의 정제된 박테리오파지 ΦCJ17 용액 15 ㎕와 5× SDS 시료 용액 5 ㎕을 혼합하고 5분간 가열하였다. 이를 15% SDS-PAGE 겔에 로우딩하여 박테리오파지 ΦCJ17의 전체 단백질을 전개하였다. 겔을 쿠마시블루(coomassie blue)로 1시간 동안 상온에서 염색하였다. 15 μl of purified bacteriophage ΦCJ17 solution with 10 10 PFU / ml titer and 5 μl of 5 × SDS sample solution were mixed and heated for 5 minutes. This was loaded onto a 15% SDS-PAGE gel to develop the total protein of bacteriophage ΦCJ17. Gels were stained with coomassie blue for 1 hour at room temperature.
그 결과, 도 3에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 50 kDa, 44 kDa 및 36 kDa 크기의 주요 구조단백질로 이루어진 단백질을 패턴이 관찰되었다. As a result, as shown in Figure 3, the bacteriophage Φ 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.
실시예 6: 박테리오파지 ΦCJ17의 유전적 특성 분석Example 6: Genetic Characterization of Bacteriophage Φ CJ17
분리된 박테리오파지 ΦCJ17의 유전적 특성을 분석하기 위해서, 박테리오파지 ΦCJ17의 게놈 DNA 5 ㎍을 제한효소 EcoRV, ScaI 및 NruI으로 동시 처리하고, 제한효소 PvuII, HincII 및 StuI로 동시 처리하였다. 또한, pCL1920 벡터(Promega)를 제한효소 SmaI로 절단하고, CIP(calf intestinal alkaline phosphatase)로 처리한 벡터를 준비하였다. 절단된 게놈 DNA와 벡터의 양이 3:1이 되도록 반응조건을 맞추어 혼합하고, 16℃에서 5시간 동안 연결반응을 수행하여 각각의 게놈 DNA 절편을 포함하는 발현벡터를 수득하였다. 이로부터 수득된 발현벡터를 대장균 DH5α 세포에 도입하여 각각의 형질전환체를 제조하였다. 제조된 각각의 형질전환체를 암피실린(ampicillin)과 X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)이 포함된 LB 평판 배지에 접종하고 배양한 후, 통상의 청백 콜로니 선별법을 통해 2개의 콜로니를 선발하였다. 선발된 각각의 콜로니를 암피실린이 포함된 배지에서 16시간 동안 진탕 배양하고, 이로부터 각각의 균체를 수득하였다. 수득한 각 균체로부터 플라스미드 정제 키트(Solgent)를 플라스미드를 추출하였다.To analyze the genetic characteristics of the isolated bacteriophage ΦCJ17, 5 μg of genomic DNA of bacteriophage ΦCJ17 was co-treated with restriction enzymes EcoRV, ScaI and NruI and co-treatment with restriction enzymes PvuII, HincII and StuI. In addition, 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. coli DH5α cells to prepare respective transformants. 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.
추출된 플라스미드를 M13 정방향 및 역방향(서열번호: 16 및 17) 프라이머 세트를 이용하여 PCR로 클로닝 여부를 확인하였다. 이들 중 삽입된 게놈 DNA의 크기가 1 kb 이상인 것을 선별한 후 상기 프라이머 세트를 이용하여 염기서열의 유사성을 분석하였고, 그 결과를 하기 표 2에 나타내었다. 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.
표 2 박테리오파지 ΦCJ17의 염기서열과 다른 박테리오파지의 해독 염기서열의 상동성 비교
서열번호 유기체 단백질 Quary Identity e-value
1 Desulfovibrio vulgaris subsp. vulgaris DP4 hypothetical protein 286-450 26/58(45%) 3.00E-10
2 Bacillus phage SPO1 gp21.9 8-385 26/128(20%) 0.21
Lactobacillus phage Lb338-1 gp027 168-440 31/98(32%) 0.4
3 Pseudomonas phage 73 hypothetical protein ORF034 11-403 42/136(31%) 1.00E-08
4 Pseudomonas phage M6 hypothetical protein ORF038 2-439 39/146(27%) 9.00E-07
Cellvibrio gilvus ATCC 13127 flagellin domain protein 401-595 27/73(37%) 7.5
6 Pseudomonas aeruginosa NCGM2.S1 hypothetical bacteriophage protein 85-660 69/192(36%) 5.00E-25
TABLE 2 Homology Comparison of Bacteriophage ΦCJ17 Sequence and Other Bacteriophage Translational Sequences
SEQ ID NO: organism protein Quary Identity e-value
One Desulfovibrio vulgaris subsp. vulgaris dp4 hypothetical protein 286-450 26/58 (45%) 3.00E-10
2 Bacillus phage SPO1 gp21.9 8-385 26/128 (20%) 0.21
Lactobacillus phage Lb338-1 gp027 168-440 31/98 (32%) 0.4
3 Pseudomonas phage 73 hypothetical protein ORF034 11-403 42/136 (31%) 1.00E-08
4 Pseudomonas phage M6 hypothetical protein ORF038 2-439 39/146 (27%) 9.00E-07
Cellvibrio gilvus ATCC 13127 flagellin domain protein 401-595 27/73 (37%) 7.5
6 Pseudomonas aeruginosa NCGM2.S1 hypothetical bacteriophage protein 85-660 69/192 (36%) 5.00E-25
상기 표 2에 나타난 바와 같이, 핵산 분자 단편에 따라 20 내지 45%의 염기서열 유사성을 보이나 모든 단편이 100% 일치하는 박테리오파지는 존재하지 않으므로, 본 발명에 따른 박테리오파지 ΦCJ17은 신규한 것임을 확인하였다.As shown in Table 2, 20-45% nucleotide sequence similarity according to the nucleic acid molecule fragments, but all fragments are 100% identical bacteriophage because there is no bacteriophage Φ CJ17 according to the present invention was confirmed to be novel.
실시예 7: 박테리오파지 ΦCJ17 특이적 프라이머를 이용한 PCR 분석Example 7: PCR analysis using bacteriophage ΦCJ17 specific primers
분리된 박테리오파지 ΦCJ17을 동정하기 위하여 이에 특이적인 프라이머를 제작하였다. 구체적으로, 서열번호: 1의 염기서열을 바탕으로 서열번호: 6 및 7의 프라이머 세트와, 서열번호: 2의 염기서열을 바탕으로 서열번호: 8 및 9의 프라이머 세트를 제작하였다. 또한 서열번호: 3, 서열번호: 4 및 서열번호: 5의 염기서열을 바탕으로 각각 서열번호: 10 및 11, 서열번호: 12 및 13, 및 서열번호: 14 및 15의 프라이머 세트를 제작하였다. 이와 같이 제작된 각각의 프라이머 세트를 사용하고 박테리오파지 ΦCJ17의 게놈 DNA를 주형으로 하여 PCR을 수행하였다.In order to identify the isolated bacteriophage Φ CJ17, a specific primer was prepared. Specifically, primer sets of SEQ ID NOs: 6 and 7 were prepared based on the nucleotide sequences of SEQ ID NO: 1, and primer sets of SEQ ID NOs: 8 and 9 were prepared based on the nucleotide sequences of SEQ ID NO: 2. In addition, 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 Φ CJ17 as a template.
먼저, 0.1 ㎍의 박테리오파지 ΦCJ17의 전체 게놈 DAN와 0.5 pmol이 되도록 각각의 프라이머를 프리-믹스(pre-mix, Bioneer)에 첨가하고 최종 부피가 20 ㎕가 되도록 맞추었다. 이를 94℃에서 30초의 변성, 60℃에서 30초의 어닐링 및 72℃에서 1분의 중합 조건을 30회 반복하여 PCR을 수행하였다. First, each primer was added to a pre-mix (Preone, Bioneer) so as to have a total genome DAN of 0.5 μg of bacteriophage Φ CJ17 and adjusted to a final volume of 20 μl. 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.
도 4a는 서열번호: 6 및 7과 8 및 9의 프라이머 세트를 이용한 PCR 결과를 나타내는 것이고, 도 4b는 서열번호: 10 및 11의 프라이머 세트를 이용한 PCR 결과를 나타내는 것이며, 도 4c는 서열번호: 12 및 13의 프라이머 세트를 이용한 PCR 결과를 나타내는 것이고, 도 4d는 서열번호: 14 및 15의 프라이머 세트를 이용한 PCR 결과를 나타내는 것이다. 도 4a 내지 4d에 나타난 바와 같이, 서열번호: 6 및 7, 서열번호: 8 및 9, 서열번호: 10 및 11과, 서열번호: 12 및 13의 프라이머 세트를 이용한 PCR 결과, 각각 약 600 bp, 400 bp, 900 bp, 500 bp 및 1000 bp 정도의 PCR 산물을 증폭됨을 확인하였다.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, and FIG. 4D shows the PCR results using the primer sets SEQ ID NOs: 14 and 15. As shown in Figures 4a to 4d, PCR results using a primer set of SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, and SEQ ID NOs: 12 and 13, respectively, about 600 bp, It was confirmed that the PCR products of 400 bp, 900 bp, 500 bp and about 1000 bp were amplified.
실시예 8: 박테리오파지 ΦCJ17의 pH에 대한 안정성 조사Example 8 Investigation of Stability of Bacteriophage ΦCJ17 to pH
박테리오파지 ΦCJ17이 어류 위장 내 낮은 pH에서 안정성을 보유할 수 있는지 여부를 확인하기 위하여 다양한 pH 범위(pH 2.0, 2.5, 3.0, 3.5, 4.0, 5.5, 6.5, 7.5, 8.5, 9.5, 10.0, 및 11.0)에서 안정성 조사 실험을 수행하였다. 다양한 pH 용액으로 아세트산나트륨 완충액(Sodium acetate buffer; pH 4.0, 및 5.5), 시트르산나트륨 완충액(Sodium citrate buffer; pH 2.0, 2.5, 3.0, 및 3.5), 인산타트륨 완충액(Sodium phosphate buffer; pH 6.5, 및 7.5), 및 트리스 용액(Tris-HCl; pH 8.5, 9.5, 10.0, 및 11.0)을 각각 0.2 M로 준비하였다. 180 ㎕의 pH 용액과 20 ㎕의 2.0×1011 PFU/㎖ 역가의 박테리오파지 ΦCJ17 용액을 혼합하고, 2시간 동안 상온에서 정치하였다. 이들을 단계 희석하고, 소프트 아가 오버레이 방법에 따라 각 단계의 희석액을 10 ㎕씩 적가한 후 30℃에서 18시간 동안 배양하여 용균 여부를 통해 역가를 측정하였다. 이때, 0시간째의 벡테리오파지 ΦCJ17의 역가를 기준으로 pH 변화에 따른 역가 변화를 비교하여 상대적 안정성을 확인하였다. Various pH ranges (pH 2.0, 2.5, 3.0, 3.5, 4.0, 5.5, 6.5, 7.5, 8.5, 9.5, 10.0, and 11.0) to determine whether bacteriophage ΦCJ17 can maintain stability at low pH in fish stomachs Stability investigation experiments were performed at. Sodium acetate buffer (pH 4.0, and 5.5), sodium citrate buffer (pH 2.0, 2.5, 3.0, and 3.5), sodium phosphate buffer (pH 6.5, And 7.5), and Tris solution (Tris-HCl; pH 8.5, 9.5, 10.0, and 11.0) were prepared at 0.2 M, respectively. 180 μl of pH solution and 20 μl of 2.0 × 10 11 PFU / ml titer of bacteriophage ΦCJ17 solution were mixed and allowed to stand at room temperature for 2 hours. These were diluted in steps, and 10 μl 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 was confirmed by comparing the titer of the pH change based on the titer of the bacteriophage Φ CJ17 at 0 hours.
도 5에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 pH 4.0부터 pH 11.0까지의 범위에서 활성을 잃지 않고 안정적임을 확인하였다.As shown in Figure 5, the bacteriophage Φ CJ17 of the present invention was confirmed to be stable without losing activity in the range from pH 4.0 to pH 11.0.
실시예 9: 박테리오파지 ΦCJ17의 온도에 대한 안정성 조사Example 9 Investigation of Stability of Bacteriophage ΦCJ17 with Temperature
박테리오파지 ΦCJ17을 사료첨가제로 이용할 경우 제형화 과정에서 발생하는 열에 대한 안정성을 확인하기 위하여 하기 실험을 수행하였다. 구체적으로, 3.0×1011 PFU/㎖ 역가의 박테리오파지 ΦCJ17의 용액 100 ㎕를 37℃, 45℃, 53℃, 60℃, 및 70℃의 온도 조건 하에서 각각 0분, 30분, 60분, 및 120분간 정치시켰다. 처리한 실험 배양액을 단계 희석하고, 소프트 아가 오버레이 방법에 따라 각 단계의 희석액 10 ㎕씩을 적가한 다음, 30℃에서 18시간 동안 배양하여 용균 여부를 통해 역가를 측정하였다. 이때, 0분, 37℃에서의 역가를 기준으로 온도 및 노출시간에 따른 상대적 안정성을 비교하였다. When bacteriophage Φ 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 μl of a solution of bacteriophage ΦCJ17 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 µl 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.
그 결과, 도 6에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 60℃에서 2시간까지 노출되어도 활성을 유지하였으나, 70℃에서는 10분 이상 노출되면 시간의 경과에 따라 활성이 감소함을 알 수 있다.As a result, as shown in Figure 6, the bacteriophage Φ CJ17 of the present invention maintained the activity even when exposed to 60 hours at 60 ℃, it can be seen that the activity decreases over time when exposed to more than 10 minutes at 70 ℃ .
실시예 10: 박테리오파지 ΦCJ17의 야생 분리주 비브리오 하베이에 대한 감염범위 조사Example 10 Investigation of Infection Range of Bacteriophage ΦCJ17 in Wild Strain Vibrio Habay
박테리오파지 ΦCJ17이 실험에 사용된 특정 비브리오 하베이 균주인 VH-BB120 이외 다른 야생 분리주에 대해서도 살균 활성을 나타내는지 여부를 조사하였다. 본 실험에 사용된 야생 분리주는 한국 해양연구원 해양국한생물지원 뱅크에서 분리한 비브리오 하베이 1종, ATCC에서 구매한 비브리오 하베이 1종, 및 실험실에서 자체적으로 분리한 비브리오 하베이 1종을 포함한 총 3종의 비브리오 하베이이다. 각 균주의 진탕 배양액(OD600=2) 150 ㎕을 LB 평판 배지에 도말하고 소프트 아가 오버레이 방법에 따라 108 PFU/㎖ 역가의 박테리오파지 ΦCJ17의 용액 10 ㎕씩을 적가한 다음, 30℃에서 18시간 동안 배양하여 용균반 형성 유무를 관찰하였다. It was examined whether bacteriophage ΦCJ17 showed bactericidal activity against wild isolates other than the specific Vibrio Havey strain VH-BB120 used in the experiment. The wild isolates used in this experiment were a total of three species, including one Vibrio habei from the Korea Maritime Research Institute for Marine Biotechnology, one Vibrio habay purchased from ATCC, and one vibrio habay isolated from the laboratory. Vibrio Harvey. Stain 150 μl of shake culture (OD 600 = 2) of each strain onto LB plate medium, add 10 μl of a solution of bacteriophage ΦCJ17 with 10 8 PFU / ml titer according to the soft agar overlay method, and then 18 hours at 30 ° C. The culture was observed for the formation of lytic plaques.
표 3 야생 분리주 비브리오 하베이에 대한 용균 형성 여부 조사
균주명 ΦCJ13 용균반 형성 유무
V. harveyi(MEBiC05675)V. harveyi(LAB)V. harveyi(BB170) OOO
TABLE 3 Investigation of lysate formation in wild isolate Vibrio Habay
Strain name ΦCJ13 lytic plaque formation
V. harveyi (MEBiC05675) V. harveyi (LAB) V. harveyi (BB170) OOO
* 'MEBiC' source: 한국해양연구원, 해양국한생물지원 뱅크* 'MEBiC' source: Korea Maritime Research Institute, Marine Korea Biotechnology Bank
* 'LAB' source: 수산시장 물 샘플에서 분리한 실험실 자체 분리균주* 'LAB' source: Laboratory self-separating strain isolated from fish market water sample
* 'BB170' source: ATCC® BAA-1117TM* 'BB170' source: ATCC® BAA-1117TM
상기 표 3에 나타난 바와 같이, 본 발명의 박테리오파지 ΦCJ17은 VH-BB120 뿐만 아니라, 모든 종류의 비브리오 하베이에 대해 감염능을 나타냄을 확인하였다.As shown in Table 3, the bacteriophage Φ CJ17 of the present invention was confirmed to exhibit the ability to infect not only VH-BB120, but also all kinds of Vibrio Habei.
따라서, 본 발명의 박테리오파지 ΦCJ17은 어류 양식장과 같은 현장에 적용할 경우, 비브리오 하베이에 대한 우수한 방제효과를 나타낼 수 있음을 알 수 있다.Therefore, the bacteriophage Φ 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.
본 발명의 신규 박테리오파지는 비브리오 하베이에 대하여 특이적 사멸능과, 우수한 내산성 및 내열성을 나타내기 때문에, 비브리오 하베이 감염성 질환인 비브리오증의 예방 또는 치료에 유용하게 사용될 수 있을 뿐만 아니라, 사료 첨가용 조성물, 소독제 및 세척제 등으로 광범위하게 이용될 수 있다.Since the 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.
Figure PCTKR2013000166-appb-I000001
Figure PCTKR2013000166-appb-I000001

Claims (8)

  1. 비브리오 하베이(Vibrio harveyi)에 특이적 사멸능을 갖는 기탁번호 KCCM11247P인 박테리오파지.Bacteriophage with accession number KCCM11247P with specific killing ability to Vibrio harveyi.
  2. 제1항의 박테리오파지를 유효성분으로 포함하는 비브리오 하베이에 의한 감염성 질환의 예방 또는 치료용 조성물.A composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage of claim 1 as an active ingredient.
  3. 제2항에 있어서, 상기 비브리오 하베이에 의한 감염성 질환이 비브리오증인 것인 조성물.3. The composition of claim 2, wherein the infectious disease caused by Vibrio Havey is Vibrioosis.
  4. 제1항의 박테리오파지를 유효성분으로 포함하는 항생제.An antibiotic comprising the bacteriophage of claim 1 as an active ingredient.
  5. 제1항의 박테리오파지를 유효성분으로 포함하는 어패류 또는 새우 양식용 사료첨가제.Feed additives for aquaculture or shrimp farming comprising the bacteriophage of claim 1 as an active ingredient.
  6. 제1항의 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제.Disinfectant or cleaning agent comprising the bacteriophage of claim 1 as an active ingredient.
  7. 제1항의 박테리오파지 또는, 제2항 또는 제3항의 조성물을 양식용 어패류 또는 새우에 투여하는 단계를 포함하는, 비브리오 하베이에 의한 감염성 질환을 예방 또는 치료하는 방법.A method for preventing or treating an infectious disease caused by Vibrio havey, comprising administering the bacteriophage of claim 1 or the composition of claim 2 or 3 to aquaculture seafood or shrimp.
  8. 제7항에 있어서, 상기 비브리오 하베이에 의한 감염성 질환이 비브리오증인 것인 방법.8. The method of claim 7, wherein the infectious disease caused by Vibrio Havey is Vibrioosis.
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CN110709510A (en) * 2017-05-10 2020-01-17 尹特荣生物科技株式会社 Novel Vibrio parahaemolyticus bacteriophage Vib-PAP-7 and use thereof for inhibiting proliferation of Vibrio parahaemolyticus bacteria
CN112442487A (en) * 2019-08-14 2021-03-05 宁波大学 Vibrio harveyi high-efficiency lytic phage vB-Vhas-yong1 and application thereof
CN112063595A (en) * 2020-09-24 2020-12-11 瑞科盟(青岛)生物工程有限公司 Lytic vibrio harveyi phage RDP-VP-19012 and application thereof
CN113046326A (en) * 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) Separation and application of virulent vibrio parahaemolyticus phage vB _ VpP _ FE11
CN113337474A (en) * 2021-02-05 2021-09-03 华南农业大学 Vibrio parahaemolyticus lytic phage vB _ VpP _ DE17 and application thereof
CN113046326B (en) * 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) Separation and application of virulent vibrio parahaemolyticus phage vB _ VpP _ FE11
CN113337474B (en) * 2021-02-05 2022-09-27 华南农业大学 Vibrio parahaemolyticus lytic phage vB _ VpP _ DE17 and application thereof

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