WO2013157813A1 - Novel bacteriophage, and antibacterial composition containing same - Google Patents

Novel bacteriophage, and antibacterial composition containing same Download PDF

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WO2013157813A1
WO2013157813A1 PCT/KR2013/003180 KR2013003180W WO2013157813A1 WO 2013157813 A1 WO2013157813 A1 WO 2013157813A1 KR 2013003180 W KR2013003180 W KR 2013003180W WO 2013157813 A1 WO2013157813 A1 WO 2013157813A1
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bacteriophage
coli
φcj18
kccm11272p
active ingredient
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PCT/KR2013/003180
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French (fr)
Korean (ko)
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서효실
김재원
조영욱
양시용
신은미
이현정
임현정
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씨제이제일제당(주)
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Publication of WO2013157813A1 publication Critical patent/WO2013157813A1/en

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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
    • 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
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/381—Microorganisms
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • Avian Pathogenic E. coli is known to infiltrate the body through the respiratory mucosa as E. coli, which is infected through the respiratory system of birds, for example, chickens, ducks, turkeys.
  • the avian pathogenic Escherichia coli causes a variety of diseases such as sepsis, granulomas, cystitis, ovitis, arthritis, etc. in birds, and causes serious economic damage to the poultry industry, especially in poultry with respect to respiratory diseases, which is a problem. .
  • bacteriophage refers to a bacterial specific virus that inhibits and inhibits the growth of bacteria by infecting certain bacteria.
  • Bacteriophage is not only stronger in host specificity than antibiotics, but has recently become increasingly interested in its application as the problem of the emergence of resistant bacteria against antibiotic use has increased (Cislo, M et al. Arch Immunol. Ther.Exp. 2: 175 -183, 1987; Kim Sung Hoon et al., Bacteriophage, a new alternative antibiotic, BioWave Vol. 7 No.15, 2005, BRIC).
  • the inventors of the present invention have repeatedly studied to solve the emergence of resistant bacteria and antibiotic residues in meat, and to effectively prevent and treat infectious diseases of birds, as a result of APEC Escherichia coli causing poultry respiratory diseases.
  • the new bacteriophage ⁇ CJ18 (KCCM11272P), which has the ability to kill enemies, has been separated from the natural world.
  • An object of the present invention is to provide a novel bacteriophage ⁇ CJ18 (KCCM11272P) having a specific killing ability for avian pathogenic E. coli.
  • an object of the present invention is to provide a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli, comprising the bacteriophage ⁇ CJ18 (KCCM11272P) as an active ingredient.
  • an object of the present invention is to provide an antibiotic, poultry feed or drinking water additives, and a disinfectant or cleaning agent containing the bacteriophage ⁇ CJ18 (KCCM11272P) as an active ingredient.
  • an object of the present invention is to provide a method for preventing or treating infectious diseases caused by avian pathogenic E. coli using the bacteriophage ⁇ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.
  • an antibiotic comprising the bacteriophage ⁇ CJ18 (KCCM11272P) as an active ingredient.
  • a feed or drinking water additives for poultry comprising the bacteriophage ⁇ CJ18 (KCCM11272P) as an active ingredient.
  • a bacteriophage ⁇ CJ18 comprising an active ingredient, disinfectant or cleaning agent is provided.
  • the bacteriophage ⁇ CJ18 (KCCM11272P) Or it provides a method for preventing or treating an infectious disease caused by avian pathogenic E. coli, comprising administering to a poultry a composition comprising the same as an active ingredient.
  • Bacteriophage ⁇ CJ18 (KCCM11272P) of the present invention has the effect of specifically killing avian pathogenic E. coli.
  • the bacteriophage ⁇ CJ18 (KCCM11272P) of the present invention is excellent in acid resistance, heat resistance and dry resistance, can be used as a material for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli at various temperature and pH range, as well as antibiotics, Poultry feed or drinking water has an effect that can be utilized as additives, disinfectants or cleaning agents.
  • the present invention has the effect of preventing or treating infectious diseases caused by avian pathogenic E. coli of poultry by administering to the poultry the bacteriophage ⁇ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.
  • 1 is an electron micrograph of a novel bacteriophage ⁇ CJ18 (KCCM11272P).
  • Figure 2 is a graph showing the PFGE results of the novel bacteriophage ⁇ CJ18.
  • FIG. 3 is a graph showing the SDS-PAGE results of the novel bacteriophage ⁇ CJ18.
  • Figure 5 is a graph showing the heat resistance test results of the novel bacteriophage ⁇ CJ18.
  • Figure 6 is a graph showing the results of the dry resistance test of novel bacteriophage ⁇ CJ18.
  • Figure 7 shows part of the DNA base sequence of novel bacteriophage ⁇ CJ18.
  • ⁇ CJ18 Avian Pathogenic Esherichia new bacteriophage ⁇ CJ18 (KCCM11272P) (hereinafter referred to as ⁇ CJ18) To provide.
  • Avian Escherichia coli is an Escherichia coli that infects birds through the respiratory tract of birds such as chickens, ducks, and turkeys, and enters the body of birds through the respiratory mucosa and causes various diseases such as sepsis, granulomas, cystitis, meningitis and arthritis. .
  • the algal pathogenic Escherichia coli is a gram-negative bacillus like the common E. coli and has a main hair flagella, which is motility, and is an aerobic or breathable anaerobic bacterium that decomposes lactose and fructose to produce acids and gases.
  • the avian pathogenic Escherichia coli grows well in a normal medium, and the developable temperature is about 7 to 48 °C and the optimum culture temperature is 35 to 37 °C, especially the expression of the pathogenic factor is effective at about 42 °C close to the body temperature of algae.
  • the developable pH range is about pH 4.5 to 9.0.
  • Bacteriophage is a bacterial specific virus that infects a particular bacterium and inhibits and inhibits the growth of the bacterium.
  • the bacteriophage refers to a virus including a single or double chain DNA or RNA as a genetic material.
  • ⁇ CJ18 of the present invention is a bacteriophage having species specificity that selectively infects avian pathogenic E. coli, and has a morphological grape structure having an isometric capsid and a tail not observed (FIG. 1). It is a bacteriophage belonging to Poriviridae.
  • the ⁇ CJ18 is a bacteriophage newly isolated by the present inventors, and was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under the accession number KCCM11272P.
  • composition for preventing or treating infectious diseases caused by avian pathogenic E. coli comprising ⁇ CJ18 as an active ingredient.
  • the infectious disease caused by the avian pathogenic E. coli is not particularly limited, but may preferably be avian coliosis (Colibacillosis).
  • prevention means any action that provides a composition comprising the ⁇ CJ18 and / or the ⁇ CJ18 as an active ingredient to inhibit the disease or delay the onset of the disease.
  • treatment refers to any action that provides a composition comprising the ⁇ CJ18 and / or the ⁇ CJ18 as an active ingredient to improve the pathological condition of the disease affected by pre-infection.
  • composition for preventing or treating an infectious disease caused by the avian pathogenic E. coli of the present invention may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • the carrier is a carrier which can be used in the composition for the prevention or treatment of infectious diseases caused by the avian pathogenic E. coli, which is formulated as a liquid solution, and is suitable for sterilization and living body, including saline, sterile water, Ringer's solution, buffered saline, and albumin injection solution. , Dextrose solution, maltodextrin solution, glycerol or ethanol and the like. These can be used individually or in mixture of 2 or more types, and can add other conventional additives, such as antioxidant, buffer, and / or bacteriostatic agent, as needed.
  • Diluents, dispersants, surfactants, binders and / or lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention can be administered to birds via nasal spray, oral or parenteral administration, and can be used by application or spraying to a diseased site.
  • ⁇ CJ18 may be diluted in water to be absorbed into the nasal cavity through a sprayer or a spray system.
  • nasal spray or respiratory formulation for nasal spray include aerosols and the like.
  • the nasal spray or respiratory formulation may further comprise a propellant and / or other additives to disperse the dispersed dispersion or the wet powder.
  • intravenous administration In the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration may be used.
  • Non-limiting examples of the formulation for parenteral administration include injection formulations such as subcutaneous injection, intravenous injection or intramuscular injection, and the parenteral administration formulation is used for infectious diseases caused by the avian pathogenic E. coli of the present invention.
  • Prophylactic or therapeutic compositions can be prepared in the form of solutions or suspensions by mixing in water with stabilizers and / or buffers.
  • lactose For formulation into such tablets or 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 Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the like, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • binders such as cellulose or gelatin
  • excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch
  • stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the like
  • the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned
  • Suitable application, spraying or dosage of a composition for the prophylaxis or treatment of an infectious disease caused by the avian pathogenic E. coli of the present invention, as well as the formulation method, mode of administration, time of administration and / or route of administration of the composition is subject to administration It may vary depending on factors such as the age, weight, sex of the animal, the extent of the disease symptoms, the foods eaten, the rate of excretion, etc. The skilled veterinarian will normally determine and prescribe the effective dosage for the desired treatment. Can be.
  • an antibiotic comprising the ⁇ CJ18 as an active ingredient.
  • the animal means mammals and birds including humans.
  • a feed or drinking water additive for poultry comprising the ⁇ CJ18 as an active ingredient.
  • oultry used in the present invention is a concept that collectively leads to animals belonging to birds in livestock.
  • the poultry is not particularly limited, but may preferably include one or more selected from the group consisting of chicken, duck, and turkey.
  • the feed or drinking water additives for poultry of the present invention may be prepared by separately preparing ⁇ CJ18 in the form of feed or drinking water additives and mixing the feed or drinking water, or directly adding the same when preparing feed or drinking water.
  • composition comprising the ⁇ CJ18 and / or the ⁇ CJ18 used as the feed or drinking water additive of the present invention may be in a liquid or dry state, preferably in the form of a dry powder.
  • the drying method for preparing the feed or drinking water additive of the present invention in the form of a dried powder is not particularly limited, and a method commonly used in the art may be used.
  • Non-limiting examples of the drying method may be ventilation drying, natural drying, spray drying or freeze drying, and these may be carried out by using a single method or a combination of two or more methods.
  • the feed or drinking water additive of the present invention may further be added to other non-pathogenic microorganisms.
  • the poultry feed used in the present invention is not particularly limited and may be used a feed commonly used in the art.
  • Non-limiting examples of the feed for poultry include, but are not limited to, vegetable feeds such as cereals, fruit fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These can be used individually or in mixture of 2 or more types.
  • Non-limiting examples of the usable additives include binders, emulsifiers, preservatives, amino acids, vitamins, enzymes, probiotics, flavors, nonprotein nitrogen compounds, silicates, buffers, colorants, extractants or oligosaccharides, and the like. In addition, it may further include a feed mixture and the like. These may be added alone or in combination of two or more.
  • the feed additive of the present invention may be contained in an amount of preferably 0.05 to 10 parts by weight, more preferably 0.1 to 2 parts by weight based on 100 parts by weight of feed.
  • a disinfectant or cleaning agent comprising the ⁇ CJ18 as an active ingredient.
  • the formulation of the disinfectant or cleaning agent is not particularly limited and may be prepared and used in a formulation known in the art.
  • the disinfectant may be used in, but not limited to, an active area of poultry, a slaughterhouse, a dead zone, a cooking place, or a cooking facility.
  • the cleaning agent may be used for cleaning the surface of the poultry or each part of the body, which is exposed to or may be exposed to avian pathogenic E. coli, but is not limited thereto.
  • a method for preventing or treating an infectious disease caused by avian pathogenic E. coli is provided by using the composition comprising ⁇ CJ18 or ⁇ CJ18 as an active ingredient.
  • the prophylactic or therapeutic method of the present invention specifically includes administering to the individual infected with or at risk of being infected with avian pathogenic E. coli, the ⁇ CJ18 or a composition comprising the ⁇ CJ18 as an active ingredient in a pharmaceutically effective amount.
  • the route of administration of the composition comprising ⁇ CJ18 or ⁇ CJ18 of the present invention as an active ingredient is not particularly limited as long as it can reach the target tissue of interest, and may be administered through various routes, for example, nasal, oral or parenteral. have.
  • a suitable daily dose of the composition comprising the ⁇ CJ18 or the ⁇ CJ18 of the present invention as an active ingredient administered to the prophylactic or therapeutic method may be determined by the treating physician within the scope of correct medical judgment.
  • the specific therapeutically effective amount of the composition comprising ⁇ CJ18 or ⁇ CJ18 as an active ingredient for a particular poultry may vary depending on the type and extent of the reaction to be achieved, the age, weight, general state of health, sex or diet of the subject. It may be determined in consideration of the administration time of ⁇ CJ18 or the composition, the route of administration and the ratio of the composition, the duration of treatment, and the like, and may be appropriately applied in an effective amount depending on various factors including the components of the drug or the composition to be used simultaneously or simultaneously. It is preferable.
  • the culture solution was centrifuged at 4,000 rpm for 10 minutes and the supernatant was filtered with a 0.45 ⁇ m filter.
  • 3 ml of 0.7% (w / v) agar and APEC (E10-5) shake culture (OD) on top of the LB plate medium 600 2) 150 ⁇ l of the mixed solution was poured and hardened.
  • 10 ⁇ l of the sample solution was added dropwise thereto and incubated at 30 ° C. for 18 hours to confirm that lysate plaques were formed.
  • SM solution NaCl 5.8 g / L; MgSO 4 7H 2 O 2 g / L; 1M Tris-Cl (pH 7.5) 50 mL
  • a solution was obtained.
  • APEC E10-5 shake culture
  • the solution was recovered, 1% (v / v) chloroform was added, mixed for 10 minutes and centrifuged at 4,000 rpm for 10 minutes to obtain the supernatant, which was filtered through a 0.45 ⁇ m filter to obtain a final sample.
  • Example 1-1 The bacteriophage obtained in Example 1-1 was incubated in large quantities in APEC (E10-5) and purified from the bacteriophage.
  • Example 1-1 APEC (E10-5) was shaken and cultured to 1.5 X 10 10 cfu, centrifuged at 4,000 rpm for 10 minutes, and then resuspended in 4 ml SM solution.
  • MOI multiplicity of infection
  • the obtained precipitate was suspended in 5 ml SM solution and left to stand at room temperature for 20 minutes. Then, 4 ml chloroform was added thereto, stirred, and centrifuged at 4,000 rpm for 20 minutes to obtain the supernatant. The supernatant was filtered with a 0.45 ⁇ m filter, followed by ultracentrifugation (35,000 rpm, 1 hour, 4 ° C.) using glycerol density gradient (density: 40%, 5% glycerol) to purify the bacteriophage.
  • glycerol density gradient density: 40%, 5% glycerol
  • the present inventors took a sample from the slaughtered meal, named the bacteriophage having a specific killing ability to APEC as "Bacteriophage ⁇ CJ18," and the Korean Culture Center of Microorganisms, Seodaemun-gu, Seoul, March 22, 2012. The deposit was made with Accession No. KCCM11272P to Hongje-dong 361-221).
  • Example 1 ⁇ CJ18 purified in Example 1 was diluted in 0.01% gelatin solution and fixed with 2.5% glutaraldehyde solution. This was added dropwise to a carbon-coated mica plate (ca. 2.5 X 2.5 mm) and then adapted for 10 minutes and then washed with sterile distilled water.
  • Figure 1 shows an electron micrograph of ⁇ CJ18, which was found to have a icosahedron head of about 40 nm size and a tailless morphotype.
  • Genomic DNA was extracted from ⁇ CJ18 purified in Example 1 above.
  • the supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant.
  • add 3 M sodium acetate to the supernatant to be 10% (v / v) of the total volume, mix, add 2 volumes of cold 95% ethanol, mix, and then mix at 1 at -20 ° C. Allowed to stand for hours.
  • the precipitate was obtained by centrifugation at 12,000 rpm for 10 minutes at 0 ° C., to which 50 ⁇ l TE buffer (Tris-EDTA, pH 8.0) was added to dissolve and the concentration thereof was measured.
  • 50 ⁇ l TE buffer Tris-EDTA, pH 8.0
  • 1 ⁇ g of DNA was loaded onto a 1% pulse-field gel electrophoresis (PFGE) agarose gel, and the BioORAD PFGE System No. 7 program (size range 25 to 100 kb; switch time ramp). ) 0.4-2.0 seconds, linear shape; forward voltage 180 V; reverse voltage 120 V) for 20 hours at room temperature (FIG. 2).
  • Figure 2 is an electrophoresis picture of genomic DNA of ⁇ CJ18, genomic DNA of ⁇ CJ18 was confirmed that the size of about 47 kbp.
  • Figure 3 is an electrophoresis picture showing the SDS-PAGE results performed on ⁇ CJ18, the major proteins of 45 kDa, 36 kDa, 35 kDa and 13 kDa size can be confirmed.
  • ⁇ CJ18 purified in Example 1
  • 5 ⁇ g of genomic DNA of ⁇ CJ18 was treated with EcoR V, a restriction enzyme, and pCL1920 (Promega) was cut with Sma I restriction enzyme as a vector.
  • CIP calf intestinal alkaline phosphatase
  • the fragmented genomic DNA was mixed with the reaction conditions such that the amount of the vector is 3: 1, and then ligation was performed at 16 ° C. for 5 minutes. It was then introduced into DH5 ⁇ cells, a type of E. coli.
  • the plasmids were cloned by PCR using M13 forward primer and M13 reverse primer set, and the insertion fragment size was 1 kb or more, and the M13 forward primer and M13 reverse primer set were used. Similarity of base sequences was analyzed (Table 2).
  • the base sequence of the bacteriophage showed various similarities to the base sequence of the previously reported bacteriophage, but it was confirmed that there was no bacteriophage in which all fragments were 100% identical. As a result, the bacteriophages could be identified as newly isolated bacteriophages.
  • ⁇ CJ18 In order to identify ⁇ CJ18, a ⁇ CJ18 specific primer was prepared.
  • primer sets of SEQ ID NOs: 7 and 8 based on SEQ ID NO: 1 and primer sets of SEQ ID NOs: 9 and 10 were prepared based on SEQ ID NO: 2.
  • Primers were added to Pre-mix (Bioneer) to 0.1 ⁇ g of bacteriophage whole genomic DNA and 0.5 pmol and adjusted to a final volume of 20 ⁇ l. This is then denatured; 94 ° C., 30 seconds, annealing; 60 ° C., 30 seconds, polymerization; PCR was carried out for 30 cycles under conditions of 72 ° C. and 1 minute.
  • pH solutions sodium acetate buffer solution (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer solution (pH 2.5, pH 3.0 and pH 3.5), sodium phosphate buffer solution (pH 6.9 and pH 7.4), Tris-HCl solution (pH 8.2, pH 9.0, pH 9.8 and pH 11.0) was prepared at 0.2 M each.
  • each pH solution 90 ⁇ l of each pH solution and 10 ⁇ l of bacteriophage solution of 1.0 ⁇ 10 11 PFU / ml titer were mixed to make the concentration of each pH solution 1M, and then allowed to stand at room temperature for 2 hours. Then, these were diluted in stages, and 10 ⁇ l of the diluted solution of each stage was dropped using a soft agar overlay method, followed by incubation at 37 ° C. for 18 hours to determine titer through lysis (FIG. 4).
  • Figure 4 shows the acid resistance test results of the bacteriophage ⁇ CJ18. As shown in FIG. 4, ⁇ CJ18 was stable without losing activity from pH 5.5 to pH 11.0 as compared to the control group.
  • Figure 5 shows the heat resistance test results of bacteriophage ⁇ CJ18. As shown in FIG. 5, ⁇ CJ18 was decreased by 1 log when exposed to 60 ° C. for 1 hour, and decreased by about 2 log after 2 hours of exposure.
  • Figure 6 shows the results of the drying resistance test of bacteriophage ⁇ CJ18. As shown in FIG. 6, ⁇ CJ18 decreased by 1 log when dried at 60 ° C. for 1 hour, and decreased by about 3 logs after 2 hours of exposure.
  • the O-78 serotype is generally known to be the most frequently appearing strain of avian pathogenic E. coli isolated from poultry farms.

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Abstract

The present invention relates to a novel bacteriophage, ΦCJ18 (KCCM11272P). In addition, the present invention relates to a composition for preventing or treating infectious diseases caused by avian pathogenic Escherichia coli, a feed or drinking water additive for domestic fowls, and an antiseptic or a detergent, containing said ΦCJ18 (KCCM11272P) as an active ingredient. In addition, the present invention relates to method for preventing or treating infectious diseases of domestic fowls caused by avian pathogenic Escherichia coli by using said ΦCJ18 (KCCM11272P) or a composition containing the same as an active ingredient.

Description

신규 박테리오파지 및 이를 포함하는 항균 조성물New Bacteriophage and Antimicrobial Compositions Comprising the Same
본원 발명은 조류 병원성 대장균에 특이적 사멸능을 나타내는 신규 박테리오파지 및 이를 포함하는 항균 조성물과, 이를 활용하여 가금류의 질병을 예방 또는 치료하는 방법에 관한 것이다.The present invention relates to a novel bacteriophage that exhibits specific killing ability against avian pathogenic E. coli, and an antimicrobial composition comprising the same, and a method for preventing or treating diseases of poultry using the same.
대장균은 장내세균과(Enterobacteriaceae), 에스케리키아(Escherichia) 속에 속하는 그람 음성의 단간균으로, 포유동물을 비롯한 다양한 동물의 장관 내에 존재하는 정상 세균총 중의 하나이다. 대장균은 대부분 비병원성으로 기회 감염을 유발할 수 있으나, 일부 고병원성 균주는 사람을 비롯한 동물 내에서 다양한 장 질환 및 패혈증 등을 유발하는 것으로 알려져 있다.Escherichia coli is a Gram-negative simple bacillus belonging to the genus Enterobacteriaceae and Escherichia, and is one of the normal flora in the intestines of various animals including mammals. E. coli is mostly non-pathogenic and can cause opportunistic infections. However, some highly pathogenic strains are known to cause various intestinal diseases and sepsis in animals including humans.
이러한 대장균들 중 특히 조류 병원성 대장균(Avian Pathogenic E. coli, APEC)은 조류, 예를 들어 닭, 오리, 칠면조 등의 호흡기를 통해 감염하는 대장균으로서 호흡기 점막을 통해 체내로 침투하는 것으로 알려져 있다.Among these Escherichia coli, Avian Pathogenic E. coli (APEC) is known to infiltrate the body through the respiratory mucosa as E. coli, which is infected through the respiratory system of birds, for example, chickens, ducks, turkeys.
상기 조류 병원성 대장균은 조류 내에 패혈증, 육아종, 기낭염, 수란관염, 관절염 등의 다양한 질병을 유발하고, 특히 호흡기 질병과 관련해서 가금류에 주로 발병시키는 등 가금 산업에 막대한 경제적 피해를 초래하여 문제가 된다.The avian pathogenic Escherichia coli causes a variety of diseases such as sepsis, granulomas, cystitis, ovitis, arthritis, etc. in birds, and causes serious economic damage to the poultry industry, especially in poultry with respect to respiratory diseases, which is a problem. .
한편, 박테리오파지(bacteriophage)는 특정 세균에 감염하여 세균의 성장을 억제하고 저해하는 세균 특이적 바이러스를 의미한다. 박테리오파지는 항생제에 비하여 숙주 특이성이 강할 뿐만 아니라, 최근 들어 항생제 사용에 대한 내성균 출현의 문제가 심각해짐에 따라 그 활용에 관심이 높아지고 있다(Cislo, M 등. Arch Immunol. Ther. Exp. 2:175-183, 1987; 김성훈 외. 박테리오파지, 새로운 대체 항생제. BioWave Vol.7 No.15, 2005, BRIC).On the other hand, bacteriophage refers to a bacterial specific virus that inhibits and inhibits the growth of bacteria by infecting certain bacteria. Bacteriophage is not only stronger in host specificity than antibiotics, but has recently become increasingly interested in its application as the problem of the emergence of resistant bacteria against antibiotic use has increased (Cislo, M et al. Arch Immunol. Ther.Exp. 2: 175 -183, 1987; Kim Sung Hoon et al., Bacteriophage, a new alternative antibiotic, BioWave Vol. 7 No.15, 2005, BRIC).
이에 세계 여러 나라에서 박테리오파지에 대한 연구가 활발히 진행되고 있으며, 박테리오파지에 대한 특허 출원 및 등록은 물론, 이를 활용한 조성물에 대해 미국 식품의약국(Food and Drug Administration, FDA)의 승인 등을 취득하려는 움직임이 증가되고 있는 추세이다.Accordingly, research on bacteriophage is actively being conducted in various countries around the world, and the patent application and registration of bacteriophage, as well as the movement to obtain approval of the US Food and Drug Administration (FDA) for the composition using the bacteriophage This is an increasing trend.
박테리오파지에 관한 선행 기술로는, 미국 등록 특허 제6,485,902호에서 E.coli 0157:H 균을 통제하기 위한 7 종의 박테리오파지에 대해 개시하고 있으며, 대한민국 등록 특허 제10-0910961호에서 황색 포도상 구균에 특이적인 사멸능을 갖는 박테리오파지에 대해 개시하고 있다. 또한, 대한민국 공개 특허 제10-2009-0021475호에서 세균 세포막의 펩티도글리칸 구조물을 특이적으로 파괴시키는 박테리오파지 유래 용균 단백질 및 이에 의한 세균 파쇄물에 관해 개시하고 있다.Prior arts related to bacteriophages are disclosed in US Patent No. 6,485,902 for seven bacteriophages for controlling E. coli 0157: H, and specific for Staphylococcus aureus in Korean Patent No. 10-0910961. Disclosed is a bacteriophage having an aggressive killing ability. In addition, the Republic of Korea Patent Publication No. 10-2009-0021475 discloses a bacteriophage-derived lysate protein that specifically destroys the peptidoglycan structure of the bacterial cell membrane and thereby bacterial lysate.
본 발명자들은 항생제 사용에 의한 내성균의 출현 및 항생제의 육류 내 잔류 문제 등을 해결하고 조류의 감염성 질병을 효율적으로 예방 및 치료하기 위하여 연구를 거듭한 결과, 가금류의 호흡기성 질병을 일으키는 APEC 대장균에 특이적 사멸능을 갖는 신규 박테리오파지 ΦCJ18(KCCM11272P)을 자연계에서 분리해 내기에 이르렀다.The inventors of the present invention have repeatedly studied to solve the emergence of resistant bacteria and antibiotic residues in meat, and to effectively prevent and treat infectious diseases of birds, as a result of APEC Escherichia coli causing poultry respiratory diseases. The new bacteriophage ΦCJ18 (KCCM11272P), which has the ability to kill enemies, has been separated from the natural world.
또한, 상기 신규 박테리오파지의 형태적, 생화학적, 유전적 특성을 동정하고 상기 박테리오파지가 내산성, 내건성 및 내열성 등이 뛰어남을 확인하여, 이를 활용한 항생제, 소독제, 사료 첨가제, 기타 조성물을 개발하였고, 조류에 발병하는 감염성 질병의 예방 또는 치료를 위한 조성물 및 이를 이용한 질병의 예방 또는 치료 방법을 개발하였다.In addition, by identifying the morphological, biochemical and genetic characteristics of the new bacteriophage and confirming that the bacteriophage is excellent in acid resistance, dry resistance and heat resistance, antibiotics, disinfectants, feed additives, and other compositions using the same were developed. A composition for preventing or treating an infectious disease that develops and a method for preventing or treating a disease using the same have been developed.
본원 발명은 조류 병원성 대장균에 특이적인 사멸능을 갖는 신규 박테리오파지 ΦCJ18(KCCM11272P)을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a novel bacteriophage Φ CJ18 (KCCM11272P) having a specific killing ability for avian pathogenic E. coli.
또한, 본원 발명은 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli, comprising the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.
또한, 본원 발명은 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는 항생제, 가금류용 사료 또는 음용수 첨가제, 및 소독제 또는 세척제를 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide an antibiotic, poultry feed or drinking water additives, and a disinfectant or cleaning agent containing the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.
또한, 본원 발명은 상기 박테리오파지 ΦCJ18(KCCM11272P) 또는 이를 유효 성분으로 포함하는 조성물을 이용하여, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a method for preventing or treating infectious diseases caused by avian pathogenic E. coli using the bacteriophage Φ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.
본원 발명의 일 양태는, 조류 병원성 대장균(Avian Pathogenic Escherichia coli)에 특이적인 사멸능을 갖는 신규 박테리오파지 ΦCJ18(KCCM11272P)을 제공한다.One aspect of the invention, Avian PathogenicEsherichia                      new bacteriophage ΦCJ18 (KCCM11272P) with killing ability specific to coli).
본원 발명의 또 다른 일 양태에 따르면, 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, the bacteriophage ΦCJ18 (KCCM11272P) comprising an active ingredient, provides a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli.
본원 발명의 또 다른 일 양태에 따르면, 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는 항생제를 제공한다.According to another aspect of the present invention, there is provided an antibiotic comprising the bacteriophage ΦCJ18 (KCCM11272P) as an active ingredient.
본원 발명의 또 다른 일 양태에 따르면, 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 가금류용 사료 또는 음용수 첨가제를 제공한다.According to another aspect of the present invention, it provides a feed or drinking water additives for poultry comprising the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.
본원 발명의 또 다른 일 양태에 따르면, 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 소독제 또는 세척제를 제공한다.According to another aspect of the present invention, a bacteriophage ΦCJ18 (KCCM11272P) comprising an active ingredient, disinfectant or cleaning agent is provided.
본원 발명의 또 다른 일 양태에 따르면, 상기 박테리오파지 ΦCJ18(KCCM11272P) 또는 이를 유효 성분으로 포함하는 조성물을 가금류에 투여하는 단계를 포함하는, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법을 제공한다.According to another aspect of the present invention, the bacteriophage Φ CJ18 (KCCM11272P) Or it provides a method for preventing or treating an infectious disease caused by avian pathogenic E. coli, comprising administering to a poultry a composition comprising the same as an active ingredient.
본원 발명의 박테리오파지 ΦCJ18(KCCM11272P)은 조류 병원성 대장균을 특이적으로 사멸시키는 효과를 갖는다.Bacteriophage ΦCJ18 (KCCM11272P) of the present invention has the effect of specifically killing avian pathogenic E. coli.
또한, 본원 발명의 상기 박테리오파지 ΦCJ18(KCCM11272P)은 내산성, 내열성 및 내건성이 우수하여, 다양한 온도 및 pH 범위에서 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 물질로 활용될 수 있을 뿐만 아니라, 항생제, 가금류용 사료 또는 음용수 첨가제, 소독제 또는 세척제 등으로 활용될 수 있는 효과를 갖는다.In addition, the bacteriophage ΦCJ18 (KCCM11272P) of the present invention is excellent in acid resistance, heat resistance and dry resistance, can be used as a material for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli at various temperature and pH range, as well as antibiotics, Poultry feed or drinking water has an effect that can be utilized as additives, disinfectants or cleaning agents.
또한, 본원 발명은 상기 박테리오파지 ΦCJ18(KCCM11272P) 또는 이를 유효 성분으로 포함하는 조성물을 가금류에 투여함으로써, 가금류의 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료할 수 있는 효과를 갖는다.In addition, the present invention has the effect of preventing or treating infectious diseases caused by avian pathogenic E. coli of poultry by administering to the poultry the bacteriophage Φ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.
도 1은 신규 박테리오파지 ΦCJ18(KCCM11272P)의 전자 현미경 사진이다.1 is an electron micrograph of a novel bacteriophage Φ CJ18 (KCCM11272P).
도 2는 신규 박테리오파지 ΦCJ18의 PFGE 결과를 나타낸 그래프이다.Figure 2 is a graph showing the PFGE results of the novel bacteriophage Φ CJ18.
도 3은 신규 박테리오파지 ΦCJ18의 SDS-PAGE 결과를 나타낸 그래프이다.3 is a graph showing the SDS-PAGE results of the novel bacteriophage Φ CJ18.
도 4는 신규 박테리오파지 ΦCJ18의 내산성 실험 결과를 나타낸 그래프이다.Figure 4 is a graph showing the acid resistance test results of the novel bacteriophage Φ CJ18.
도 5는 신규 박테리오파지 ΦCJ18의 내열성 실험 결과를 나타낸 그래프이다.Figure 5 is a graph showing the heat resistance test results of the novel bacteriophage Φ CJ18.
도 6은 신규 박테리오파지 ΦCJ18의 내건성 실험 결과를 나타낸 그래프이다.Figure 6 is a graph showing the results of the dry resistance test of novel bacteriophage Φ CJ18.
도 7은 신규 박테리오파지 ΦCJ18의 DNA 염기 서열의 일부를 나타낸 것이다.Figure 7 shows part of the DNA base sequence of novel bacteriophage Φ CJ18.
도 8은 실시예 7에서 사용한 프라이머의 염기 서열을 나타낸 것이다.Figure 8 shows the nucleotide sequence of the primer used in Example 7.
이하, 본원 발명에 대하여 보다 상세히 설명한다. 본원 명세서에 기재되지 않은 내용은 본원 발명의 기술 분야 또는 유사 분야에서 숙련된 자이면 충분히 인식하고 유추할 수 있는 것이므로 그 설명을 생략한다.Hereinafter, the present invention will be described in more detail. The contents not described in the present specification may be sufficiently recognized and inferred by those skilled in the art or similar fields of the present invention, and thus description thereof is omitted.
본원 발명의 일 양태는, 조류 병원성 대장균(Avian Pathogenic Escherichia coli, APEC)에 특이적인 사멸능을 갖는 신규 박테리오파지 ΦCJ18(KCCM11272P)(이하, 'ΦCJ18') 을 제공한다.One aspect of the invention, Avian PathogenicEsherichia                  new bacteriophage ΦCJ18 (KCCM11272P) (hereinafter referred to as ΦCJ18) To provide.
조류 병원성 대장균은 닭, 오리, 칠면조 등과 같은 조류의 호흡기를 통해 감염하는 대장균으로서 호흡기 점막을 통해 조류의 체내로 침투하여 패혈증, 육아종, 기낭염, 수란관염, 관절염 등의 다양한 질병을 유발하는 대장균이다.Avian Escherichia coli is an Escherichia coli that infects birds through the respiratory tract of birds such as chickens, ducks, and turkeys, and enters the body of birds through the respiratory mucosa and causes various diseases such as sepsis, granulomas, cystitis, meningitis and arthritis. .
상기 조류 병원성 대장균은 일반 대장균과 같이 그람 음성의 간균이며 주모성 편모가 있어서 운동성이 있고, 락토스(Lactose), 프럭토스(Fructose)를 분해하여 산과 가스를 생성하는 호기성 또는 통성 혐기성균이다.The algal pathogenic Escherichia coli is a gram-negative bacillus like the common E. coli and has a main hair flagella, which is motility, and is an aerobic or breathable anaerobic bacterium that decomposes lactose and fructose to produce acids and gases.
상기 조류 병원성 대장균은 보통의 배지에서 잘 자라며, 발육 가능한 온도는 약 7 내지 48℃이고 배양 최적 온도는 35 내지 37℃이며, 특히 조류의 체온과 가까운 약 42℃에서 병원성 인자의 발현이 효과적으로 이루어진다. 발육 가능한 pH 범위는 약 pH 4.5 내지 9.0이다.The avian pathogenic Escherichia coli grows well in a normal medium, and the developable temperature is about 7 to 48 ℃ and the optimum culture temperature is 35 to 37 ℃, especially the expression of the pathogenic factor is effective at about 42 ℃ close to the body temperature of algae. The developable pH range is about pH 4.5 to 9.0.
박테리오파지(bacteriophage)는 특정 세균에 감염하여 당해 세균의 성장을 억제하고 저해하는 세균 특이적 바이러스로, 단일 혹은 이중 사슬의 DNA 또는 RNA를 유전 물질로 포함하는 바이러스를 의미한다.Bacteriophage is a bacterial specific virus that infects a particular bacterium and inhibits and inhibits the growth of the bacterium. The bacteriophage refers to a virus including a single or double chain DNA or RNA as a genetic material.
본원 발명의 ΦCJ18은 조류 병원성 대장균을 선택적으로 감염시키는 종 특이성을 갖는 박테리오파지로서, 정이십면체의 머리(isometric capsid)를 가지며 꼬리(tail)가 관찰되지 않는 구조(도 1)를 갖는, 형태학상 포도비리데(Podoviridae)에 속하는 박테리오파지이다.ΦCJ18 of the present invention is a bacteriophage having species specificity that selectively infects avian pathogenic E. coli, and has a morphological grape structure having an isometric capsid and a tail not observed (FIG. 1). It is a bacteriophage belonging to Poriviridae.
상기 박테리오파지를 APEC에 감염시켰을 때, 용균반(파지 플라크(phage plaque)로 연한천(soft agar)에서 하나의 박테리오파지에 의해 숙주 세포가 용균되어 형성되는 클리어 존)이 형성될 뿐만 아니라, 상기 용균반의 크기와 탁도가 동일하므로, 상기 박테리오파지가 APEC을 용균시켜서 APEC의 성장을 억제시킴을 확인하였다.When the bacteriophage is infected with APEC, not only lytic plaques (clear zones formed by lysis of host cells by one bacteriophage in soft agar with phage plaques) are formed. Since the size and turbidity are the same, it was confirmed that the bacteriophage lysed APEC to inhibit the growth of APEC.
또한, 상기 박테리오파지를 다양한 pH 범위 및 온도에서 안정성을 조사한 결과, pH 5.5 내지 11 범위의 넓은 범위에서 안정하게 생존할 수 있는 특성을 나타냄을 확인하였고(도 4), 특히 높은 온도에서 내열성을 가지는 것을 확인하였다(도 5). 또한 박테리오파지의 제품 제형 중 사료 첨가제로 이용할 경우 박테리오파지의 제형 과정에서 발생하는 건조에 대한 안정성이 높은 것을 확인하였다(도 6).In addition, the stability of the bacteriophage in a variety of pH range and temperature, as a result, it was confirmed that exhibits a stable survival characteristics in a wide range of pH 5.5 to 11 (Fig. 4), especially those having heat resistance at high temperatures It was confirmed (FIG. 5). In addition, when used as a feed additive in the product formulation of the bacteriophage, it was confirmed that the stability to the drying occurring during the formulation process of the bacteriophage was high (Fig. 6).
이러한 APEC 특이적 용균활성, 내산성, 내열성 및 내건성은 본원 발명의 박테리오파지를 조류의 APEC 유래 질병의 예방 및 치료용 조성물, 및 박테리오파지를 유효 성분으로 포함하는 다양한 제품에 적용함에 있어, 다양한 온도 및 pH 범위의 적용이 가능하게 한다.Such APEC-specific lytic activity, acid resistance, heat resistance and dryness resistance, in applying the bacteriophage of the present invention to a composition for the prevention and treatment of diseases of APEC-derived diseases of birds, and various products including the bacteriophage as an active ingredient, a variety of temperature and pH range The application of is made possible.
상기 ΦCJ18은 본 발명자들이 신규 분리한 박테리오파지로서, 2012년 3월 22일자로 한국 미생물 보존 센터(Korean Culture Center of Microorganisms, 서울시 서대문구 홍제1동 361-221)에 기탁번호 KCCM11272P으로 기탁되었다.The ΦCJ18 is a bacteriophage newly isolated by the present inventors, and was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under the accession number KCCM11272P.
본원 발명의 또 다른 일 양태에 따르면, 상기 ΦCJ18을 유효 성분으로 포함하는, 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, a composition for preventing or treating infectious diseases caused by avian pathogenic E. coli, comprising Φ CJ18 as an active ingredient.
상기 조류 병원성 대장균에 의한 감염성 질병은 특별히 제한되지 아니하나, 바람직하게는 조류 대장균증(Colibacillosis)일 수 있다.The infectious disease caused by the avian pathogenic E. coli is not particularly limited, but may preferably be avian coliosis (Colibacillosis).
상기 조류 대장균증이란, 조류의 호흡기 등이 병원성 대장균에 감염되어 발생하는 질병으로서, 그 증상으로 기낭염(airsacculitis), 간포막염(perihepatitis), 복막염(peritonitis), 심장주위염(pericarditis), 수란관염(salpingitis), 제대염(omphalitis), 골수염(osteomyelitis) 또는 패혈증(septicemia) 등 다양한 병변을 나타내어, 감염된 조류의 성장 저하 및 폐사를 유발하는 질병을 말한다.The avian coliform disease is a disease caused by infecting the pathogen Escherichia coli with the bird, such as airsacculitis, perihepatitis, peritonitis, pericarditis, pericarditis, salpingitis ), A disease that causes various lesions such as oomphalitis, osteomyelitis, or septicemia, causing growth and death of infected birds.
본원 발명에서 사용되는 용어 "예방"이란, 상기 ΦCJ18 및/또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물을 제공하여 해당 질병을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that provides a composition comprising the Φ CJ18 and / or the Φ CJ18 as an active ingredient to inhibit the disease or delay the onset of the disease.
본원 발명에서 사용되는 용어 "치료"란, 상기 ΦCJ18 및/또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물을 제공하여 기감염된 해당 질병의 병적 상태를 개선시키는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any action that provides a composition comprising the Φ CJ18 and / or the Φ CJ18 as an active ingredient to improve the pathological condition of the disease affected by pre-infection.
본원 발명의 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물은 바람직하게는 상기 ΦCJ18을 5 x 102 내지 5 x 1012 pfu/ml로 함유할 수 있으며, 보다 바람직하게는 상기 ΦCJ18을 1 x 106 내지 1 x 1010 pfu/ml로 함유할 수 있다.The composition for the prevention or treatment of infectious diseases caused by the avian pathogenic E. coli of the present invention may preferably contain the Φ CJ18 5 x 10 2 to 5 x 10 12 pfu / ml, more preferably the Φ CJ18 1 x 10 6 to 1 x 10 10 pfu / ml.
본원 발명의 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물은 약학적으로 허용 가능한 담체를 추가로 포함할 수 있다.The composition for preventing or treating an infectious disease caused by the avian pathogenic E. coli of the present invention may further include a pharmaceutically acceptable carrier.
상기 "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다.By "pharmaceutically acceptable carrier" is meant a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
상기 담체에 있어서, 액상 용액으로 제제화되는 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물에 사용 가능한 담체로 멸균 및 생체에 적합한 것으로는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤 또는 에탄올 등을 들 수 있다. 이들은 단독 또는 2종 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 및/또는 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The carrier is a carrier which can be used in the composition for the prevention or treatment of infectious diseases caused by the avian pathogenic E. coli, which is formulated as a liquid solution, and is suitable for sterilization and living body, including saline, sterile water, Ringer's solution, buffered saline, and albumin injection solution. , Dextrose solution, maltodextrin solution, glycerol or ethanol and the like. These can be used individually or in mixture of 2 or more types, and can add other conventional additives, such as antioxidant, buffer, and / or bacteriostatic agent, as needed.
또한 희석제, 분산제, 계면활성제, 결합제 및/또는 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.Diluents, dispersants, surfactants, binders and / or lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본원 발명의 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물의 투여 방식은 특별히 제한되지 아니하며, 본원 발명의 기술 분야에서 통상적으로 사용하는 방식에 따를 수 있다.The mode of administration of the composition for the prevention or treatment of infectious diseases by the avian pathogenic E. coli of the present invention is not particularly limited, and may be in accordance with the method commonly used in the art.
예를 들어, 본원 발명의 상기 조성물은 비강 분무, 경구 투여 또는 비경구 투여를 통해 조류에 투여될 수 있으며, 질환 부위에의 도포 또는 분무하는 방식에 의해 사용될 수 있다.For example, the composition of the present invention can be administered to birds via nasal spray, oral or parenteral administration, and can be used by application or spraying to a diseased site.
상기 비강 분무의 경우 일정량의 ΦCJ18을 물에 희석하여 분무기나 분무 시스템을 통해 비강으로 흡수 되도록 할 수 있다. 상기 비강 분무를 위한 비강 분무제 또는 호흡기용 제형으로는 에어로졸제 등을 들 수 있다.In the case of nasal spraying, a certain amount of Φ CJ18 may be diluted in water to be absorbed into the nasal cavity through a sprayer or a spray system. Examples of the nasal spray or respiratory formulation for nasal spray include aerosols and the like.
상기 비강 분무제 또는 호흡기용 제형은, 수분산된 농축물 또는 습윤 분말을 분산시키기 위한 추진제 및/또는 기타 첨가제를 추가로 포함할 수 있다.The nasal spray or respiratory formulation may further comprise a propellant and / or other additives to disperse the dispersed dispersion or the wet powder.
상기 비경구 투여의 경우 정맥 내 투여, 복강 내 투여, 근육 내 투여, 피하 투여 또는 국부 투여 등을 이용할 수 있다.In the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration may be used.
상기 비경구 투여용 제형의 비제한적인 예로는 피하주사, 정맥주사 또는 근육 내 주사 등의 주사용 제형을 들 수 있으며, 상기 비경구 투여용 제형은 본원 발명의 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물을 안정제 및/또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액 형태로 제조할 수 있다.Non-limiting examples of the formulation for parenteral administration include injection formulations such as subcutaneous injection, intravenous injection or intramuscular injection, and the parenteral administration formulation is used for infectious diseases caused by the avian pathogenic E. coli of the present invention. Prophylactic or therapeutic compositions can be prepared in the form of solutions or suspensions by mixing in water with stabilizers and / or buffers.
상기 경구 투여용 제형의 비제한적인 예로는, 정제, 수용성 또는 유성 현탁액, 조제 분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제 등을 들 수 있다.Non-limiting examples of such oral dosage forms include tablets, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like.
상기 정제 또는 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 윤활유 등을 포함할 수 있으며, 캡슐 제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 추가로 함유할 수 있다.For formulation into such tablets or 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 Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the like, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
본원 발명의 상기 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물의 적합한 도포, 분무 또는 투여량은, 상기 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로는 물론, 투여 대상이 되는 동물의 나이, 체중, 성별, 질병 증상의 정도, 섭취하는 음식, 배설 속도 등과 같은 요인들에 의해 다양해 질 수 있으며, 보통으로 숙련된 수의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다.Suitable application, spraying or dosage of a composition for the prophylaxis or treatment of an infectious disease caused by the avian pathogenic E. coli of the present invention, as well as the formulation method, mode of administration, time of administration and / or route of administration of the composition is subject to administration It may vary depending on factors such as the age, weight, sex of the animal, the extent of the disease symptoms, the foods eaten, the rate of excretion, etc. The skilled veterinarian will normally determine and prescribe the effective dosage for the desired treatment. Can be.
본원 발명의 또 다른 일 양태에 따르면, 상기 ΦCJ18을 유효 성분으로 포함하는 항생제를 제공한다.According to another aspect of the present invention, there is provided an antibiotic comprising the Φ CJ18 as an active ingredient.
본원 발명에 사용되는 용어 "항생제"란, 약제 형태로 동물에게 제공되어 균을 사멸시킬 수 있는 제제를 의미하며, 방부제, 살균제 및 항균제를 총칭하는 개념에 해당한다.As used herein, the term "antibiotic" refers to an agent that is provided to an animal in the form of a drug and can kill bacteria, and corresponds to the concept of preservatives, fungicides and antimicrobials.
상기 동물은 인간을 포함한 포유동물 및 조류를 의미한다.The animal means mammals and birds including humans.
본원 발명의 또 다른 일 양태에 따르면, 상기 ΦCJ18을 유효 성분으로 포함하는, 가금류용 사료 또는 음용수 첨가제를 제공한다.According to still another aspect of the present invention, there is provided a feed or drinking water additive for poultry comprising the Φ CJ18 as an active ingredient.
본원 발명에서 사용되는 용어 "가금류"란, 가축 중 조류에 속하는 동물을 총칭하여 이르는 개념이다.The term "poultry" used in the present invention is a concept that collectively leads to animals belonging to birds in livestock.
상기 가금류는 특별히 제한되지 아니하나, 바람직하게는 닭, 오리 및 칠면조로 이루어진 군에서 선택되는 1종 이상을 포함할 수 있다.The poultry is not particularly limited, but may preferably include one or more selected from the group consisting of chicken, duck, and turkey.
본원 발명의 상기 가금류용 사료 또는 음용수 첨가제는 ΦCJ18을 사료 또는 음용수 첨가제 형태로 따로 제조하여 사료 또는 음용수에 혼합시키거나, 사료 또는 음용수 제조 시 이를 직접 첨가하는 방식으로 사용할 수 있다.The feed or drinking water additives for poultry of the present invention may be prepared by separately preparing Φ CJ18 in the form of feed or drinking water additives and mixing the feed or drinking water, or directly adding the same when preparing feed or drinking water.
본원 발명의 상기 사료 또는 음용수 첨가제로 사용되는 상기 ΦCJ18 및/또는 상기 ΦCJ18을 포함하는 조성물은 액상 또는 건조상태일 수 있으며, 바람직하게는 건조된 분말형태일 수 있다.The composition comprising the Φ CJ18 and / or the Φ CJ18 used as the feed or drinking water additive of the present invention may be in a liquid or dry state, preferably in the form of a dry powder.
본원 발명의 상기 사료 또는 음용수 첨가제를 건조된 분말 형태로 제조하기 위한 건조 방법은 특별히 제한되지 아니하며, 본원 발명의 기술 분야에서 통상적으로 사용하는 방법을 사용할 수 있다.The drying method for preparing the feed or drinking water additive of the present invention in the form of a dried powder is not particularly limited, and a method commonly used in the art may be used.
상기 건조 방법의 비제한적인 예로는, 통풍 건조, 자연 건조, 분무 건조 또는 동결 건조 등을 들 수 있으며, 이들은 단독 또는 2 가지 이상의 방법을 함께 이용하는 방식으로 수행될 수 있다.Non-limiting examples of the drying method may be ventilation drying, natural drying, spray drying or freeze drying, and these may be carried out by using a single method or a combination of two or more methods.
본원 발명의 상기 사료 또는 음용수 첨가제에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다.The feed or drinking water additive of the present invention may further be added to other non-pathogenic microorganisms.
상기 첨가될 수 있는 미생물의 비제한적인 예로는, 단백질 분해 효소, 지질 분해 효소 및/또는 당 전환 효소를 생산할 수 있는 바실러스 서브틸리스(Bacillus subtilis)와 같은 고초균, 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능을 갖는 락토바실러스 균주(Lactobacillus sp.), 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 갖는 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 이루어진 군에서 선택될 수 있다. 이들은 단독 또는 2 종 이상을 혼합하여 사용할 수 있다.Non-limiting examples of the microorganisms that can be added include physiological conditions under anaerobic conditions such as Bacillus subtilis, Bacillus subtilis, cattle stomach, which can produce proteolytic enzymes, lipolytic enzymes and / or sugar converting enzymes. Lactobacillus strains having active and organic matter resolution (Lactobacillus sp.), Aspergillus ducka, which increases the weight of livestock, increases milk yield and improves digestive absorption of feed.Aspergillus oryzae(J Animal Sci. 43: 910-926, 1976) and Saccharomyces cerevisiae (Saccharomyces                                  cerevisiaeYeast (J Anim Sci 56: 735-739, 1983). These can be used individually or in mixture of 2 or more types.
본원 발명에서 사용되는 가금류용 사료는 특별히 제한되지 아니하며, 본원 발명의 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다.The poultry feed used in the present invention is not particularly limited and may be used a feed commonly used in the art.
상기 가금류용 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독 또는 2 종 이상 혼합하여 사용할 수 있다.Non-limiting examples of the feed for poultry include, but are not limited to, vegetable feeds such as cereals, fruit fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These can be used individually or in mixture of 2 or more types.
본원 발명에서 사용되는 가금류용 음용수는 특별히 제한되지 아니하며, 본원 발명의 기술 분야에서 통상적으로 사용되는 음용수를 사용할 수 있다.Drinking water for poultry used in the present invention is not particularly limited, it is possible to use drinking water commonly used in the art.
본 발명의 상기 ΦCJ18을 유효 성분으로 포함하는 사료 또는 음용수 첨가제는 필요에 따라 기타 첨가제를 추가로 포함할 수 있다.Feed or drinking water additive containing the Φ CJ18 of the present invention as an active ingredient may further include other additives as necessary.
상기 사용 가능한 첨가제의 비제한적인 예로는, 결착제, 유화제, 보존제, 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백질태질소화합물, 규산염제, 완충제, 착색제, 추출제 또는 올리고당 등이 있으며, 그 외에 사료 혼합제 등을 추가로 포함할 수 있다. 이들은 단독 또는 2 종 이상이 함께 첨가될 수 있다.Non-limiting examples of the usable additives include binders, emulsifiers, preservatives, amino acids, vitamins, enzymes, probiotics, flavors, nonprotein nitrogen compounds, silicates, buffers, colorants, extractants or oligosaccharides, and the like. In addition, it may further include a feed mixture and the like. These may be added alone or in combination of two or more.
본원 발명의 상기 사료 첨가제는 사료 100 중량부에 대하여, 바람직하게는 0.05 내지 10 중량부로 함유될 수 있으며, 보다 바람직하게는 0.1 내지 2 중량부로 함유될 수 있다.The feed additive of the present invention may be contained in an amount of preferably 0.05 to 10 parts by weight, more preferably 0.1 to 2 parts by weight based on 100 parts by weight of feed.
본원 발명의 상기 음용수 첨가제는 음용수 100 중량부에 대하여, 바람직하게는 0.0001 내지 0.01 중량부로 함유될 수 있으며, 보다 바람직하게는 0.001 내지 0.005 중량부로 함유될 수 있다.The drinking water additive of the present invention may be contained with respect to 100 parts by weight of drinking water, preferably 0.0001 to 0.01 parts by weight, more preferably 0.001 to 0.005 parts by weight.
본원 발명의 또 다른 일 양태에 따르면, 상기 ΦCJ18을 유효 성분으로 포함하는 소독제 또는 세척제를 제공한다.According to another aspect of the present invention, there is provided a disinfectant or cleaning agent comprising the Φ CJ18 as an active ingredient.
상기 소독제 또는 세척제의 제형은 특별히 제한되지 아니하며, 본원 발명의 기술 분야에서 알려진 제형으로 제조되어 사용할 수 있다.The formulation of the disinfectant or cleaning agent is not particularly limited and may be prepared and used in a formulation known in the art.
상기 소독제는 가금류의 활동 영역, 도축장, 폐사 지역, 조리 장소 또는 조리 설비 등에 사용될 수 있으나 이에 제한되지 아니한다.The disinfectant may be used in, but not limited to, an active area of poultry, a slaughterhouse, a dead zone, a cooking place, or a cooking facility.
상기 세척제는 조류 병원성 대장균에 노출되었거나 노출될 가능성이 있는 가금류의 피부 표면 또는 신체 각 부위 등을 세척하는 용도로 사용될 수 있으나 이에 제한되지 아니한다.The cleaning agent may be used for cleaning the surface of the poultry or each part of the body, which is exposed to or may be exposed to avian pathogenic E. coli, but is not limited thereto.
본원 발명의 또 다른 일 양태에 따르면, 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물을 이용하여, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법을 제공한다.According to another aspect of the present invention, a method for preventing or treating an infectious disease caused by avian pathogenic E. coli is provided by using the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient.
본원 발명의 상기 예방 또는 치료 방법은 구체적으로, 조류 병원성 대장균에 의해 감염되었거나 감염될 위험이 있는 개체에 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물을 약학적 유효량으로 투여하는 단계를 포함한다.The prophylactic or therapeutic method of the present invention specifically includes administering to the individual infected with or at risk of being infected with avian pathogenic E. coli, the ΦCJ18 or a composition comprising the ΦCJ18 as an active ingredient in a pharmaceutically effective amount.
본원 발명의 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물은 약학적 제제의 형태로 가금류에 분무식 비강 투여되거나, 가금류의 사료 또는 음용수에 직접 첨가되어 이를 섭식시키는 방법을 통해 투여될 수 있으며, 사료 또는 음용수 첨가제의 형태로 사료 또는 음용수에 혼합되어 투여될 수 있다.The composition comprising the Φ CJ18 or the Φ CJ18 of the present invention as an active ingredient may be administered by spraying nasal to poultry in the form of a pharmaceutical preparation, or directly added to feed or drinking water of poultry to be administered by a method of feeding it. It can be administered in admixture with feed or drinking water in the form of feed or drinking water additives.
본원 발명의 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물의 투여 경로는 해당 목적 조직에 도달할 수 있는 한 특별히 제한되지 아니하며, 예를 들어, 비강, 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.The route of administration of the composition comprising ΦCJ18 or ΦCJ18 of the present invention as an active ingredient is not particularly limited as long as it can reach the target tissue of interest, and may be administered through various routes, for example, nasal, oral or parenteral. have.
상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물의 투여 경로의 비제한적인 예로는, 구강, 직장, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비측내 또는 흡입 등을 통해 투여되는 방식을 들 수 있다.Non-limiting examples of the route of administration of the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal or inhaled The way it works.
상기 예방 또는 치료 방법에 투여되는 본원 발명의 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물의 적합한 1일 사용량은, 올바른 의학적 판단 범위 내에서 처치의에 의해 결정될 수 있다.A suitable daily dose of the composition comprising the Φ CJ18 or the Φ CJ18 of the present invention as an active ingredient administered to the prophylactic or therapeutic method may be determined by the treating physician within the scope of correct medical judgment.
특정 가금류에 대한 상기 ΦCJ18 또는 상기 ΦCJ18을 유효 성분으로 포함하는 조성물의 구체적인 치료적 유효량은, 달성하고자 하는 반응의 종류와 정도, 해당 개체의 연령, 체중, 일반적인 건강 상태, 성별 또는 식이는 물론, 상기 ΦCJ18 또는 조성물의 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간 등을 고려하여 결정될 수 있으며, 동시 또는 이시에 함께 사용되는 약물 기타 조성물의 성분 등을 비롯한 다양한 인자에 따라 적합하나 유효량으로 적용되는 것이 바람직하다.The specific therapeutically effective amount of the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient for a particular poultry may vary depending on the type and extent of the reaction to be achieved, the age, weight, general state of health, sex or diet of the subject. It may be determined in consideration of the administration time of ΦCJ18 or the composition, the route of administration and the ratio of the composition, the duration of treatment, and the like, and may be appropriately applied in an effective amount depending on various factors including the components of the drug or the composition to be used simultaneously or simultaneously. It is preferable.
이하, 실시예를 기술함으로써 본원 발명을 보다 상세히 설명한다. 다만, 하기의 실시예는 본원 발명의 일 예시에 불과하며, 본원 발명의 내용이 이에 한정되는 것으로 해석되어서는 아니된다.Hereinafter, the present invention will be described in more detail by describing examples. However, the following examples are merely examples of the present invention, and the contents of the present invention should not be construed as being limited thereto.
실시예EXAMPLE 1 - 조류 병원성 대장균에 감염하는 박테리오파지의 분리 Isolation of Bacteriophage Infected with Avian Pathogenic E. Coli
실시예EXAMPLE 1-1 1-1
박테리오파지 스크리닝 및 단일 박테리오파지 분리Bacteriophage Screening and Single Bacteriophage Separation
전라북도 남원시 소재 오리 농장으로부터 농장 지역의 도계 분변 및 환경 샘플로부터 수득한 시료 50 ㎖를 4,000 rpm에서 10분간 원심 분리한 후, 그 상등액을 0.45 ㎛ 필터로 여과하여 시료액을 준비하고, 이를 이용하여 소프트 아가 오버레이(soft agar overlay) 방법을 수행하였다.After centrifugation of 50 ml of the sample obtained from the farming fecal and environmental samples from a duck farm in Namwon, Jeollabuk-do for 10 minutes at 4,000 rpm, the supernatant was filtered with a 0.45 μm filter to prepare a sample solution. A soft agar overlay method was performed.
구체적으로, APEC(E10-5) 진탕 배양액(OD600 = 2) 150 ㎕ 와 10 X LB배지(tryptone 10 g/ℓ; yeast extract 5 g/ℓ; NaCl 10 g/ℓ) 2 ㎖에 상기 시료 여과액 18 ㎖를 혼합하고 30℃에서 18시간 동안 배양한 다음, 상기 배양액을 4,000 rpm에서 10분간 원심 분리하여 그 상등액을 0.45 ㎛ 필터로 여과하였다. 그 다음, LB 평판 배지의 위에 0.7%(w/v) 한천 3 ㎖와 APEC(E10-5) 진탕 배양액(OD600 = 2) 150 ㎕의 혼합액을 부어 굳힌 후, 그 위에 상기 시료액 10 ㎕를 적가하여 30℃에서 18시간 동안 배양하여 용균반이 형성됨을 확인하였다.Specifically, APEC (E10-5) shake culture (OD600                  = 2) 18 ml of the sample filtrate was mixed with 150 µl and 2 ml of 10 × LB medium (tryptone 10 g / l; yeast extract 5 g / l; NaCl 10 g / l) and incubated at 30 ° C. for 18 hours. Next, the culture solution was centrifuged at 4,000 rpm for 10 minutes and the supernatant was filtered with a 0.45 μm filter. Then, 3 ml of 0.7% (w / v) agar and APEC (E10-5) shake culture (OD) on top of the LB plate medium600                  = 2) 150 μl of the mixed solution was poured and hardened. Then, 10 μl of the sample solution was added dropwise thereto and incubated at 30 ° C. for 18 hours to confirm that lysate plaques were formed.
하나의 용균반에는 한 종류의 박테리오파지가 존재한다고 알려져 있기 때문에, 상기 형성된 용균반으로부터 단일 박테리오파지를 분리하고자 하였다. 구체적으로, 상기 용균반을 400 ㎕의 SM용액(NaCl 5.8 g/ℓ; MgSO47H2O 2 g/ℓ; 1M Tris-Cl(pH 7.5) 50 ㎖)에 가하고 실온에서 4시간 동안 방치하여 박테리오파지 용액을 수득하였다.Since one type of bacteriophage is known to exist in one lysate, an attempt was made to separate a single bacteriophage from the formed lysate. Specifically, the lysate was added to 400 μl of SM solution (NaCl 5.8 g / L; MgSO 4 7H 2 O 2 g / L; 1M Tris-Cl (pH 7.5) 50 mL) and left at room temperature for 4 hours to bacteriophage. A solution was obtained.
그 다음, 상기 박테리오파지 용액 100 ㎕를 0.7%(w/v) 한천 12 ㎖ 및 APEC(E10-5) 진탕 배양액 (OD600 = 2) 500 ㎕와 혼합하고, 150 mm 지름의 LB 평판 배지를 이용한 소프트 아가 오버레이 방법을 수행하였으며, 완전히 용균될 때까지 배양하였다. 배양이 종료된 후, 상기 LB 평판 배지에 15 ㎖의 SM 용액을 가하고 실온에서 4시간 동안 방치하여 박테리오파지 용액을 수득하였다.Next, 100 µl of the bacteriophage solution was added to 12 ml of 0.7% (w / v) agar and the APEC (E10-5) shake culture (OD600                  = 2) mixed with 500 μl, followed by a soft agar overlay method using 150 mm diameter LB plate medium and incubated until complete lysis. After the incubation was completed, 15 ml of SM solution was added to the LB plate medium and left at room temperature for 4 hours to obtain a bacteriophage solution.
상기 용액을 회수하고 1%(v/v) 클로로포름을 가한 다음, 10분간 혼합하고 4,000 rpm으로 10분간 원심 분리하여 그 상등액을 수득하였으며, 상기 상등액을 0.45 ㎛ 필터로 여과하여 최종 시료를 수득하였다.The solution was recovered, 1% (v / v) chloroform was added, mixed for 10 minutes and centrifuged at 4,000 rpm for 10 minutes to obtain the supernatant, which was filtered through a 0.45 μm filter to obtain a final sample.
실시예EXAMPLE 1-2 1-2
박테리오파지의 대량 배양 및 정제Mass Cultivation and Purification of Bacteriophage
상기 실시예 1-1에서 수득한 박테리오파지를 APEC(E10-5)에 대량으로 배양하고 이로부터 박테리오파지를 정제하였다.The bacteriophage obtained in Example 1-1 was incubated in large quantities in APEC (E10-5) and purified from the bacteriophage.
구체적으로, APEC(E10-5)를 진탕 배양하여 1.5 X 1010 cfu가 되도록 분주하여 4,000 rpm에서 10분간 원심 분리한 후, 이를 4 ㎖ SM 용액에 재부유시켰다. 여기에 상기 실시예 1-1에서 수득한 박테리오파지를 1.5 X 106 pfu로 접종하고 MOI(multiplicity of infection) = 0.0001로 적정한 다음, 상온에서 20 분간 정치시켰다.Specifically, APEC (E10-5) was shaken and cultured to 1.5 X 10 10 cfu, centrifuged at 4,000 rpm for 10 minutes, and then resuspended in 4 ml SM solution. Here, the bacteriophage obtained in Example 1-1 was inoculated with 1.5 X 10 6 pfu, titrated with MOI (multiplicity of infection) = 0.0001, and left to stand at room temperature for 20 minutes.
그 다음, 150 ㎖ LB 배지에 접종하고 30℃에서 6시간 동안 배양하였다. 배양이 종료된 후, 최종 부피의 1%(v/v)가 되도록 클로로포름을 첨가하고 20분간 교반하였으며, 제한 효소인 DNase I과 RNase A를 각각 최종 농도 1 ㎍/㎖가 되도록 첨가하고 30℃에서 30분 동안 정치시켰다. 그 다음, 최종 농도가 각각 1M과 10%(w/v)가 되도록 NaCl과 PEG(polyethylene glycol)를 가하고 4℃에서 3 시간 동안 추가 정치시켰으며 4℃에서 12,000 rpm으로 20분간 원심 분리하여 침전물을 수득하였다.It was then inoculated in 150 ml LB medium and incubated at 30 ° C. for 6 hours. After the incubation was completed, chloroform was added to 1% (v / v) of the final volume and stirred for 20 minutes. The restriction enzymes DNase I and RNase A were respectively added to a final concentration of 1 μg / ml and the mixture was heated at 30 ° C. Allow to stand for 30 minutes. Then, NaCl and PEG (polyethylene glycol) were added to the final concentrations of 1 M and 10% (w / v), respectively, and further left at 4 ° C. for 3 hours, followed by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. Obtained.
상기 수득한 침전물을 5 ㎖ SM용액에 현탁시키고 20분간 실온에서 정치시킨 다음, 4 ㎖ 클로로포름을 가하여 교반하고 4℃에서 4,000 rpm으로 20분간 원심 분리하여 그 상등액을 수득하였다. 상기 상등액을 0.45 ㎛ 필터로 여과한 다음, 글리세롤 밀도 구배법(밀도: 40%, 5% 글리세롤)을 이용한 초원심 분리(35,000 rpm, 1시간, 4℃)를 수행하여 박테리오파지를 정제하였다.The obtained precipitate was suspended in 5 ml SM solution and left to stand at room temperature for 20 minutes. Then, 4 ml chloroform was added thereto, stirred, and centrifuged at 4,000 rpm for 20 minutes to obtain the 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.
본 발명자는 도계분으로부터 시료를 채취하여, APEC에 대한 특이적 사멸능을 갖는 상기 박테리오파지를 "Bacteriophage ΦCJ18"으로 명명하고, 2012년 3월 22일 한국 미생물 보존 센터(Korean Culture Center of Microorganisms, 서울시 서대문구 홍제1동 361-221)에 기탁번호 제KCCM11272P호로 기탁하였다.The present inventors took a sample from the slaughtered meal, named the bacteriophage having a specific killing ability to APEC as "Bacteriophage Φ CJ18," and the Korean Culture Center of Microorganisms, Seodaemun-gu, Seoul, March 22, 2012. The deposit was made with Accession No. KCCM11272P to Hongje-dong 361-221).
실시예EXAMPLE 2 2
ΦΦ CJ18CJ18 의 조류 병원성 대장균 감염성 비교Comparison of Avian Pathogenic E. Coli Infection
상기 실시예 1에서 정제한 ΦCJ18이 APEC(E10-5) 이외에 다른 대장균에 대하여도 용균 활성을 나타내는지의 여부를 확인하기 위해 교차 감염을 수행하였다.Cross infection was performed to confirm whether Φ CJ18 purified in Example 1 exhibits lytic activity against E. coli in addition to APEC (E10-5).
구체적으로, 2 종의 APEC(E10-5) 및 APEC(E09-35)와 6 종의 비병원성 대장균(E09-1, E09-10, E09-13, E09-14, E09-15, E09-16)을 각각 배양하여 그 배양액을 수득한 후, 상기 각각의 배양액과 상기 정제한 ΦCJ18을 이용한 소프트 아가 오버레이 방법을 수행하여 용균반의 형성 여부를 확인하였다.Specifically, two types of APEC (E10-5) and APEC (E09-35) and six types of non-pathogenic E. coli (E09-1, E09-10, E09-13, E09-14, E09-15, E09-16) After culturing each of them to obtain a culture solution, the formation of lysate plaques was confirmed by performing a soft agar overlay method using the respective culture solution and the purified Φ CJ18.
상기 결과는 표 1에 나타내었다.The results are shown in Table 1.
표 1
균주 명칭 용균반 형성 여부
APEC(E10-5) O
E.coli(E09-35) O
E.coli(E09-1) X
E.coli(E09-10) X
E.coli(E09-13) X
E.coli(E09-14) X
E.coli(E09-15) X
E.coli(E09-16) X
Table 1
Strain name Formation of lytic plaque
APEC (E10-5) O
E. coli (E09-35) O
E. coli (E09-1) X
E. coli (E09-10) X
E. coli (E09-13) X
E. coli (E09-14) X
E. coli (E09-15) X
E. coli (E09-16) X
상기 표 1에 나타난 바와 같이, 실시예 1에서 정제한 ΦCJ18은 비병원성 대장균에 대하여는 용균 활성을 나타내지 않음을 확인할 수 있었다.As shown in Table 1, ΦCJ18 purified in Example 1 did not show the lytic activity against non-pathogenic E. coli.
실시예EXAMPLE 3 3
ΦΦ CJ18CJ18 의 형태 관찰Form observation
상기 실시예 1에서 정제한 ΦCJ18을 0.01 % 젤라틴 용액에 희석하고, 2.5% 글루타르알데하이드(glutaraldehyde) 용액으로 고정하였다. 이를 탄소 코팅된 운모판(carbon-coated mica plate)(ca. 2.5 X 2.5 mm)에 적가하여 10분간 적응시킨 다음, 멸균 증류수로 세척하였다.ΦCJ18 purified in Example 1 was diluted in 0.01% gelatin solution and fixed with 2.5% glutaraldehyde solution. This was added dropwise to a carbon-coated mica plate (ca. 2.5 X 2.5 mm) and then adapted for 10 minutes and then washed with sterile distilled water.
그 다음, 탄소 필름(Carbon film)을 구리 그리드(Copper grid)에 끼워 4 % 우라닐 아세테이트(uranyl acetate)에서 30 내지 60 초간 염색하고 건조한 다음, 투과 전자 현미경(JEM-1011 transmission electron microscope, 80 kV, 배율 X 120,000 내지 X 200,000)으로 검경하였다(도 1).Next, a carbon film was placed on a copper grid, dyed in 4% uranyl acetate for 30 to 60 seconds and dried, followed by a JEM-1011 transmission electron microscope (80 kV). And magnification X 120,000 to X 200,000) (FIG. 1).
도 1은 ΦCJ18의 전자 현미경 사진을 나타낸 것으로, 살피건대 약 40 nm 크기의 정이십면체의 머리를 가지며 꼬리가 없는 형태형(morphotype)을 나타내는 것으로 보아, 형태학상 포도비리데에 속하는 것으로 판단되었다.Figure 1 shows an electron micrograph of ΦCJ18, which was found to have a icosahedron head of about 40 nm size and a tailless morphotype.
실시예EXAMPLE 4 4
ΦΦ CJ18CJ18 의 전체 게놈 Whole genome DNADNA 의 크기 분석Size analysis
상기 실시예 1에서 정제한 ΦCJ18로부터 게놈 DNA를 추출하였다.Genomic DNA was extracted from ΦCJ18 purified in Example 1 above.
구체적으로, 정제된 ΦCJ18 배양액에 20 mM EDTA, 50 ㎍/㎖ 단백질 분해 효소(proteinase) K 및 0.5 %(w/v) SDS를 가하고, 50℃에서 1시간 동안 정치하였으며, 동일 부피의 페놀(pH 8.0)을 가하여 교반한 다음 실온에서 12,000 rpm으로 10 분간 원심 분리하여 그 상등액을 수득하였다.Specifically, 20 mM EDTA, 50 μg / ml proteinase K and 0.5% (w / v) SDS were added to the purified ΦCJ18 culture, and allowed to stand at 50 ° C. for 1 hour, and the same volume of phenol (pH 8.0) was added and stirred, followed by centrifugation at 12,000 rpm for 10 minutes at room temperature to obtain the supernatant.
그 다음, 상기 상등액을 동일 부피의 PC(페놀 : 클로로포름 = 1 : 1)와 혼합하고, 실온에서 12,000 rpm으로 10분간 원심 분리하여 그 상등액을 수득하였다. 상기 상등액을 동일 부피의 클로로포름과 혼합하고, 실온에서 12,000 rpm으로 10분간 원심 분리하여 상등액을 수득하였다. 그 다음, 상기 상등액에 3 M 초산 나트륨(sodium acetate)을 전체 부피의 10 %(v/v)가 되도록 가하여 혼합하고, 2배 부피의 차가운 95 % 에탄올을 가하여 혼합한 후, -20℃에서 1시간 동안 정치시켰다.The supernatant was then mixed with an equal volume of PC (phenol: chloroform = 1: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain the supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant. Then, add 3 M sodium acetate to the supernatant to be 10% (v / v) of the total volume, mix, add 2 volumes of cold 95% ethanol, mix, and then mix at 1 at -20 ° C. Allowed to stand for hours.
그 다음, 0 ℃에서 10분간 12,000 rpm으로 원심 분리하여 그 침전물을 수득하고, 이에 50 ㎕ TE 완충액(Tris-EDTA, pH 8.0)을 가하여 용해시키고 그 농도를 측정하였다. 그 다음, 1 ㎍의 DNA를 1 % PFGE(pulse-field gel electrophoresis) 아가로즈 겔에 로딩하고, 바이오래드(BIORAD) PFGE 시스템 7번 프로그램(크기 범위 25 내지 100 kb; 스위치 타임 램프(switch time ramp) 0.4 내지 2.0 초, 선형(linear shape); 순방향 전압(forward voltage) 180 V; 역방향 전압(reverse voltage) 120 V)을 이용하여 상온에서 20시간 동안 전개하였다(도 2).Then, the precipitate was obtained by centrifugation at 12,000 rpm for 10 minutes at 0 ° C., to which 50 µl TE buffer (Tris-EDTA, pH 8.0) was added to dissolve and the concentration thereof was measured. Next, 1 μg of DNA was loaded onto a 1% pulse-field gel electrophoresis (PFGE) agarose gel, and the BioORAD PFGE System No. 7 program (size range 25 to 100 kb; switch time ramp). ) 0.4-2.0 seconds, linear shape; forward voltage 180 V; reverse voltage 120 V) for 20 hours at room temperature (FIG. 2).
도 2는 ΦCJ18의 게놈 DNA의 전기 영동 사진으로서, ΦCJ18의 게놈 DNA는 약 47 kbp의 크기를 나타냄을 확인할 수 있었다.Figure 2 is an electrophoresis picture of genomic DNA of ΦCJ18, genomic DNA of ΦCJ18 was confirmed that the size of about 47 kbp.
실시예EXAMPLE 5 5
ΦΦ CJ18CJ18 의 단백질 패턴 분석Protein pattern analysis
1010 pfu/㎖ 타이터(titer)의 정제된 ΦCJ18 용액 15 ㎕와 5X SDS 시료 용액 3 ㎕를 혼합하고 5분간 끓인 다음, 15 % SDS-PAGE를 수행하였다(도 3).15 μl of 10 10 pfu / ml titer purified ΦCJ18 solution and 3 μl of 5X SDS sample solution were mixed and boiled for 5 minutes, followed by 15% SDS-PAGE (FIG. 3).
도 3은 ΦCJ18을 대상으로 수행한 SDS-PAGE 결과를 나타내는 전기 영동 사진으로서, 45 kDa, 36 kDa, 35 kDa 및 13 kDa 크기의 주요 단백질을 확인할 수 있었다.Figure 3 is an electrophoresis picture showing the SDS-PAGE results performed on ΦCJ18, the major proteins of 45 kDa, 36 kDa, 35 kDa and 13 kDa size can be confirmed.
실시예EXAMPLE 6 6
ΦΦ CJ18CJ18 의 유전적 특성 분석Genetic characterization of
상기 실시예 1에서 정제한 ΦCJ18의 유전자적 특성을 알아보기 위하여, ΦCJ18의 게놈 DNA 5 ㎍을 제한 효소인 EcoR V로 처리하고, 벡터로는 pCL1920(프로메가 사)을 Sma I 제한 효소로 자른 후 CIP(calf intestinal alkaline phosphatase) 처리한 것을 사용하였다. 상기 절편된 게놈 DNA와 벡터의 양이 3 : 1이 되도록 반응 조건을 맞추어 섞은 후 16℃에서 5분 동안 라이게이션(ligation)을 진행하였다. 그 다음, 이를 대장균의 일종인 DH5α 세포에 도입시켰다.In order to determine the genetic properties of ΦCJ18 purified in Example 1, 5 μg of genomic DNA of ΦCJ18 was treated with EcoR V, a restriction enzyme, and pCL1920 (Promega) was cut with Sma I restriction enzyme as a vector. CIP (calf intestinal alkaline phosphatase) was used. The fragmented genomic DNA was mixed with the reaction conditions such that the amount of the vector is 3: 1, and then ligation was performed at 16 ° C. for 5 minutes. It was then introduced into DH5α cells, a type of E. coli.
상기 형질 전환된 전환체를 스펙티노마이신(spectinomycin)과 X-갈(5-브로모-4-클로로-3-인돌일-베타-D-갈락토피라노시드(X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside))가 포함된 LB 평판 배지에서 통상의 청백 콜로니 선별법을 통해 2개의 콜로니를 선별하였다. 상기 선별된 콜로니를 엠피실린이 포함된 배양 배지에서 16시간 동안 진탕 배양하였다. 여기서 플라스미드 정제 키트(솔젠트 사)를 이용하여 플라스미드를 추출하였다.The transformant was converted into spectinomycin and X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal (5-bromo-)). Two colonies were selected by conventional blue-white colony selection in LB plate medium containing 4-chloro-3-indolyl-beta-D-galactopyranoside). Shake incubation for hours where plasmids were extracted using plasmid purification kit (Solgent).
상기 플라스미드들을, M13 순방향(forward) 프라이머와 M13 역방향(reverse) 프라이머 세트를 이용하여 PCR로 클로닝 여부를 확인하였으며, 삽입 조각 크기가 1 kb 이상 되는 것을 골라 M13 순방향 프라이머와 M13 역방향 프라이머 세트를 이용하여 염기 서열의 유사성을 분석하였다(표 2).The plasmids were cloned by PCR using M13 forward primer and M13 reverse primer set, and the insertion fragment size was 1 kb or more, and the M13 forward primer and M13 reverse primer set were used. Similarity of base sequences was analyzed (Table 2).
상기 박테리오파지의 염기 서열은 기존에 보고된 박테리오파지의 염기 서열과 다양한 유사성을 보였으나, 모든 단편이 100% 일치하는 박테리오파지는 없는 것으로 확인되었다. 이에 상기 박테리오파지는 신규 분리된 박테리오파지임을 확인할 수 있었다.The base sequence of the bacteriophage showed various similarities to the base sequence of the previously reported bacteriophage, but it was confirmed that there was no bacteriophage in which all fragments were 100% identical. As a result, the bacteriophages could be identified as newly isolated bacteriophages.
하기 표 2는 ΦCJ18의 염기 서열과 다른 박테리오파지의 해독 염기 서열의 상동성을 비교한 결과를 나타낸 것이다.Table 2 below shows the result of comparing the homology of the base sequence of Φ CJ18 and the translation base sequence of other bacteriophages.
표 2
서열번호 organism protein query Identity(아미노산) e-value
1 Escherichia coli O157:H7 str. EC4024 Cell division protein FtsZ 66-383 103/106 (97%) 7e-67
Salmonella enterica subsp. enterica serovar Alachua str. R6-377 Cell division protein FtsZ 66-383 100/106 (94%) 2e-64
2 Enterobacteria phage K1-5 gp30 1-306 101/102(99%) 1.3E-67
Erwinia phage phiEa1H putative N-acetyltransferase 16-303 64/96(67%) 6.0E-26
3 Enterobacteria phage K1-5 gp 31 13-216 67/68(98%) 4.2E-39
Klebsiella phage K11 Tail assembly protein (gp 7.3) 19-186 32/61 (52%) 0.16
4 Enterobacteria phage K1-5 gp32 1-501 165/167(99%) 1.3E-67
Enterobacteria phage K1E Haed to tail connector (gp29) 1-501 165/167(99%) 6.1E-66
5 Enterobacteria phage K1E putative internal virion protein 3-1144 341/390(87%) 3e-158
Enterobacteria phage K1-5 gp36 3-1144 341/390(87%) 2e-157
6 Escherichia coli ETEC H10407 hypothetical protein ETEC_2613C 47-895 159/285 (56%) 8e-107
Escherichia phage vB_EcoM_CBA120 tailspike protein 23-853 165/278 (59%) 1e-104
TABLE 2
SEQ ID NO: organism protein query Identity (amino acid) e-value
One Escherichia coli 0157: H7 str. EC4024 Cell division protein FtsZ 66-383 103/106 (97%) 7e-67
Salmonella enterica subsp. enterica serovar Alachua str. R6-377 Cell division protein FtsZ 66-383 100/106 (94%) 2e-64
2 Enterobacteria phage K1-5 gp30 1-306 101/102 (99%) 1.3E-67
Erwinia phage phiEa1H putative N-acetyltransferase 16-303 64/96 (67%) 6.0E-26
3 Enterobacteria phage K1-5 gp 31 13-216 67/68 (98%) 4.2E-39
Klebsiella phage K11 Tail assembly protein (gp 7.3) 19-186 32/61 (52%) 0.16
4 Enterobacteria phage K1-5 gp32 1-501 165/167 (99%) 1.3E-67
Enterobacteria phage K1E Haed to tail connector (gp29) 1-501 165/167 (99%) 6.1E-66
5 Enterobacteria phage K1E putative internal virion protein 3-1144 341/390 (87%) 3e-158
Enterobacteria phage K1-5 gp36 3-1144 341/390 (87%) 2e-157
6 Escherichia coli ETEC H10407 hypothetical protein ETEC_2613C 47-895 159/285 (56%) 8e-107
Escherichia phage vB_EcoM_CBA120 tailspike protein 23-853 165/278 (59%) 1e-104
실시예EXAMPLE 7 7
ΦΦ CJ18CJ18 특이적  Specific 프라이머primer 염기 서열의 제작 Construction of the base sequence
ΦCJ18을 동정하기 위하여 ΦCJ18 특이적인 프라이머를 제작하였다.In order to identify ΦCJ18, a ΦCJ18 specific primer was prepared.
구체적으로, 서열번호 1을 바탕으로 서열번호 7과 8의 프라이머, 및 서열번호 2를 바탕으로 서열번호 9와 10의 프라이머 세트를 제작하였다. 또한 서열번호 3, 서열번호 4를 바탕으로 각각 서열번호 11과 12, 및 서열번호 13과 14 프라이머 세트를 제작하고, 서열번호 5, 서열번호 6을 바탕으로 각각 서열번호 15와 16, 및 서열번호 17과 18 프라이머 세트를 제작하여 PCR을 진행하였다.Specifically, primer sets of SEQ ID NOs: 7 and 8 based on SEQ ID NO: 1, and primer sets of SEQ ID NOs: 9 and 10 were prepared based on SEQ ID NO: 2. In addition, primer sets SEQ ID NO: 11 and 12, and SEQ ID NO: 13 and 14, respectively, based on SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15, 16, and SEQ ID NO: 15 based on SEQ ID NO: 5, SEQ ID NO: 6 PCR was performed by preparing 17 and 18 primer sets.
0.1 ㎍의 박테리오파지 전체 게놈 DNA와 0.5 pmol이 되도록 프라이머를 Pre-mix(바이오니아(Bioneer) 사)에 첨가하고 최종 부피가 20 ㎕가 되도록 맞추었다. 그 다음, 이를 변성(denaturation); 94℃, 30 초, 어닐링(annealing); 60℃, 30 초, 중합(polymerization); 72℃, 1 분의 조건으로 하여 PCR을 30 사이클 진행하였다.Primers were added to Pre-mix (Bioneer) to 0.1 μg of bacteriophage whole genomic DNA and 0.5 pmol and adjusted to a final volume of 20 μl. This is then denatured; 94 ° C., 30 seconds, annealing; 60 ° C., 30 seconds, polymerization; PCR was carried out for 30 cycles under conditions of 72 ° C. and 1 minute.
그 결과, 서열번호 7과 8, 서열번호 9와 10, 서열번호 11과 12, 서열번호 13과 14, 서열번호 15와 16, 서열번호 17과 18의 프라이머 세트를 이용했을 경우 각각 약 213 bp, 306 bp, 220 bp, 500 bp, 1146 bp 및 897 bp의 PCR 산물을 수득하였다.As a result, when using the primer sets SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, respectively, about 213 bp, PCR products of 306 bp, 220 bp, 500 bp, 1146 bp and 897 bp were obtained.
실시예EXAMPLE 8 8
pHpH 에 따른 ΦΦ according to CJ18CJ18 의 안정성 조사Investigation of stability
ΦCJ18이 위장 내 낮은 pH에서 안정성을 보유할 수 있는지 확인하기 위하여 다양한 pH 범위(pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 7.5, 8.3, 9.2 및 11.0)에서 안정성 조사 실험을 실시하였다.Stability investigation experiments were conducted at various pH ranges (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 7.5, 8.3, 9.2 and 11.0) to ensure that ΦCJ18 can retain stability at low pH in the stomach. .
다양한 pH 용액(아세트산 나트륨 완충 용액(pH 2.1, pH 4.0, pH 5.5, 및 pH 6.4), 시트르산 나트륨 완충 용액(pH 2.5, pH 3.0 및 pH 3.5), 인산 나트륨 완충 용액(pH 6.9 및 pH 7.4), 트리스-HCl 용액(pH 8.2, pH 9.0, pH 9.8 및 pH 11.0)을 각각 0.2 M로 제작하였다.Various pH solutions (sodium acetate buffer solution (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer solution (pH 2.5, pH 3.0 and pH 3.5), sodium phosphate buffer solution (pH 6.9 and pH 7.4), Tris-HCl solution (pH 8.2, pH 9.0, pH 9.8 and pH 11.0) was prepared at 0.2 M each.
상기 각 pH 용액 90 ㎕와 1.0 X 1011 PFU/㎖ 타이터의 박테리오파지용액 10 ㎕를 섞어 각 pH 용액의 농도가 1M이 되게 한 후, 2시간 동안 상온에서 정치하였다. 그 다음, 이들을 단계 희석하고, 소프트 아가 오버레이 방법을 이용하여 각 단계의 희석액을 10 ㎕씩 떨어뜨린 뒤 37℃에서 18시간 동안 배양하여 용균 여부를 통해 타이터를 측정하였다(도 4).90 μl of each pH solution and 10 μl of bacteriophage solution of 1.0 × 10 11 PFU / ml titer were mixed to make the concentration of each pH solution 1M, and then allowed to stand at room temperature for 2 hours. Then, these were diluted in stages, and 10 μl of the diluted solution of each stage was dropped using a soft agar overlay method, followed by incubation at 37 ° C. for 18 hours to determine titer through lysis (FIG. 4).
도 4는 박테리오파지 ΦCJ18의 내산성 실험 결과를 나타낸 것이다. 도 4에 나타난 바와 같이, 대조군과 비교하여 ΦCJ18은 pH 5.5부터 pH 11.0까지 활성을 잃지 않고 안정적임을 확인할 수 있었다.Figure 4 shows the acid resistance test results of the bacteriophage Φ CJ18. As shown in FIG. 4, ΦCJ18 was stable without losing activity from pH 5.5 to pH 11.0 as compared to the control group.
실시예EXAMPLE 9 9
온도에 따른 ΦΦ according to temperature CJ18CJ18 의 안정성 조사Investigation of stability
박테리오파지의 제품 제형 중 사료 첨가제로 이용할 경우 박테리오파지의 제형 과정에서 발생하는 열에 대한 안정성을 확인하기 위한 실험을 수행하였다.When used as a feed additive in the formulation of the bacteriophage experiment was conducted to confirm the stability of the heat generated during the formulation of the bacteriophage.
구체적으로, 4.0 X 108 PFU/㎖ 농도의 ΦCJ18의 용액 100 ㎕를 60℃에서 각각 0분, 10분, 30분, 60분 및 120분 동안 정치시킨 다음, 실험 배양액을 단계 희석하여 소프트 아가 오버레이 방법으로 각 단계의 희석액 10 ㎕씩 떨어뜨린 뒤 30℃에서 18시간 동안 배양하여 용균 여부를 통해 타이터를 측정하였다(도 5).Specifically, 100 μl of a solution of ΦCJ18 at a concentration of 4.0 × 10 8 PFU / ml was allowed to stand at 60 ° C. for 0, 10, 30, 60 and 120 minutes, respectively, and then the experimental culture was diluted in steps to soft agar overlay 10 μl of the diluent of each step was dropped by the method, followed by incubation at 30 ° C. for 18 hours to determine titer through lysis (FIG. 5).
도 5는 박테리오파지 ΦCJ18의 내열성 실험 결과를 나타낸 것이다. 도 5에 나타난 바와 같이, 60℃에서 1시간 노출 시 ΦCJ18은 1 로그 감소되었고, 2시간 노출 시 약 2 로그 감소하였다.Figure 5 shows the heat resistance test results of bacteriophage Φ CJ18. As shown in FIG. 5, ΦCJ18 was decreased by 1 log when exposed to 60 ° C. for 1 hour, and decreased by about 2 log after 2 hours of exposure.
실시예EXAMPLE 10 10
건조에 따른 ΦΦ due to drying CJ18CJ18 의 안정성 조사Investigation of stability
박테리오파지의 제품 제형 중 사료 첨가제로 이용할 경우 박테리오파지의 제형 과정에서 발생하는 건조에 대한 안정성을 확인하기 위한 실험을 수행하였다.When used as a feed additive in the formulation of the bacteriophage experiment was carried out to confirm the stability of the drying occurs during the formulation of the bacteriophage.
구체적으로, 6.0 X 108 PFU/㎖ 농도의 ΦCJ18의 용액 100 ㎕를 60℃에서 각각 0분, 60분 및 120분 동안 정치시키고, 실험 배양액을 단계 희석하여 소프트 아가 오버레이 방법으로 각 단계의 희석액 10 ㎕씩 떨어뜨린 뒤 30℃에서 18시간 동안 배양하여 용균 여부를 통해 타이터를 측정하였다(도 6).Specifically, 100 μl of a solution of ΦCJ18 at a concentration of 6.0 × 10 8 PFU / mL was allowed to stand at 60 ° C. for 0 minutes, 60 minutes, and 120 minutes, respectively. After dropping the ㎕ by incubation at 30 ℃ for 18 hours to determine the titer through the lysis (Fig. 6).
도 6은 박테리오파지 ΦCJ18의 내건성 실험 결과를 나타낸 것이다. 도 6에 나타난 바와 같이, 60℃에서 1시간 건조 시 ΦCJ18은 1 로그 감소되었고, 2시간 노출 시 약 3 로그 감소하였다.Figure 6 shows the results of the drying resistance test of bacteriophage Φ CJ18. As shown in FIG. 6, ΦCJ18 decreased by 1 log when dried at 60 ° C. for 1 hour, and decreased by about 3 logs after 2 hours of exposure.
실시예EXAMPLE 11 11
야생 wild 분리주Segregation APECAPEC 에 대한 ΦΦ for CJ18CJ18 의 감염 범위 조사The scope of infection
ΦCJ18이 실험에 사용된 APEC(E10-5) 이외에 건국대학교 수의과대학에서 분리한 야생 분리주 APEC 6주에 대하여 용균 활성이 있는지 여부를 확인하였다.In addition to the APEC (E10-5) used for the experiment, ΦCJ18 was confirmed to have lytic activity against APEC 6 strains isolated from Konkuk University.
구체적으로, 각 균주의 진탕 배양액 (OD600 = 2) 150 ㎕를 섞어 소프트 아가 오버레이 방법을 진행하여 108 pfu/㎖ 타이터의 ΦCJ18의 용액 10 ㎕씩 떨어뜨린 뒤 30℃에서 18 시간 동안 배양하여 용균반 형성 유무를 관찰하였다(표 3).Specifically, 150 μl of shake culture (OD 600 = 2) of each strain was mixed, followed by a soft agar overlay method, followed by 10 μl of a solution of ΦCJ18 of 10 8 pfu / ml titer, followed by incubation at 30 ° C. for 18 hours. The formation of lytic plaques was observed (Table 3).
상기 결과는 표 3에 나타내었다.The results are shown in Table 3.
표 3
균주명 ΦCJ18 용균반 형성 여부
APEC(E09-6) O
APEC(E09-11) O
APEC(E09-35) O
APEC(E10-03) O
APEC(E10-04) O
APEC(E10-05) O
TABLE 3
Strain name ΦCJ18 lytic plaque formation
APEC (E09-6) O
APEC (E09-11) O
APEC (E09-35) O
APEC (E10-03) O
APEC (E10-04) O
APEC (E10-05) O
상기 표 3에 나타난 바와 같이, 일반 양계 농가에서 조류 대장균증의 원인균인 APEC(O-78 혈청형 포함)에 대해 효과적인 감염능을 보이는 것을 확인할 수 있었다.As shown in Table 3, it was confirmed that an effective infectious ability against APEC (including O-78 serotype) which is the causative agent of avian E. coli in a general poultry farm.
한편, 상기 O-78 혈청형은 일반적으로 양계 농장에서 분리되는 조류 병원성 대장균 중 가장 빈번하게 나타나 균주로 알려져 있다.On the other hand, the O-78 serotype is generally known to be the most frequently appearing strain of avian pathogenic E. coli isolated from poultry farms.
Figure PCTKR2013003180-appb-I000001
Figure PCTKR2013003180-appb-I000001

Claims (10)

  1. 조류 병원성 대장균(Avian Pathogenic Escherichia coli)에 특이적인 사멸능을 갖는 신규 박테리오파지 ΦCJ18(KCCM11272P).Avian PathogenicEsherichia                                  coliNovel bacteriophage ΦCJ18 (KCCM11272P) with specific killing ability.
  2. 제1항에 기재된 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물.A composition for preventing or treating infectious diseases caused by avian pathogenic E. coli, comprising the bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 as an active ingredient.
  3. 제2항에 있어서, 상기 감염성 질병은 조류 대장균증(Colibacillosis)인, 조류 병원성 대장균에 의한 감염성 질병의 예방 또는 치료용 조성물.The composition for preventing or treating infectious diseases caused by avian pathogenic E. coli according to claim 2, wherein the infectious disease is avian coliform (Colibacillosis).
  4. 제1항에 기재된 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는 항생제.An antibiotic comprising the bacteriophage ΦCJ18 (KCCM11272P) according to claim 1 as an active ingredient.
  5. 제1항에 기재된 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 가금류용 사료 또는 음용수 첨가제.A feed or drinking water additive for poultry comprising the bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 as an active ingredient.
  6. 제5항에 있어서, 상기 가금류는 닭, 오리 및 칠면조로 이루어진 군에서 선택되는 1 종 이상을 포함하는, 가금류용 사료 또는 음용수 첨가제.The feed or drinking water additive for poultry according to claim 5, wherein the poultry comprises at least one member selected from the group consisting of chicken, duck, and turkey.
  7. 제1항에 기재된 상기 박테리오파지 ΦCJ18(KCCM11272P)을 유효 성분으로 포함하는, 소독제 또는 세척제.A disinfectant or cleaning agent comprising the bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 as an active ingredient.
  8. 제1항에 기재된 상기 박테리오파지 ΦCJ18(KCCM11272P) 또는 제2항에 기재된 조성물을 가금류에 투여하는 단계를 포함하는, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법.The bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 Or administering the composition of claim 2 to poultry, the method for preventing or treating an infectious disease caused by avian pathogenic E. coli.
  9. 제8항에 있어서, 상기 감염성 질병은 조류 대장균증인, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법.The method of claim 8, wherein the infectious disease is avian coliform bacterium.
  10. 제8항에 있어서, 상기 가금류는 닭, 오리 및 칠면조로 이루어진 군에서 선택되는 1 종 이상을 포함하는, 조류 병원성 대장균에 의한 감염성 질병을 예방 또는 치료하는 방법.The method of claim 8, wherein the poultry comprises at least one member selected from the group consisting of chicken, duck, and turkey.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015160164A1 (en) * 2014-04-15 2015-10-22 씨제이제일제당(주) Novel bacteriophage and composition comprising same
EP3130669A4 (en) * 2014-04-10 2017-12-13 Cj Cheiljedang Corporation Novel bacteriophage and composition containing same
EP3133152A4 (en) * 2014-04-15 2017-12-27 Cj Cheiljedang Corporation Novel bacteriophage, and composition containing same
US10704027B2 (en) * 2013-02-27 2020-07-07 Cj Cheiljedang Corporation Bacteriophage and antibacterial composition comprising the same
CN111868236A (en) * 2017-09-15 2020-10-30 辛特生物实验室公司 Bacteriophage compositions and methods for preventing bacterial infection in livestock
CN115960844A (en) * 2016-01-18 2023-04-14 美国控股实验室公司 Method and system for rapid detection of microorganisms using infectious agents

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101591795B1 (en) * 2014-04-15 2016-02-04 씨제이제일제당(주) Novel bacteriophage and composition comprising the same
KR20190070457A (en) 2017-12-13 2019-06-21 주식회사 오투파워 Composition for preventing Chicken Colibacillosis comprising chlorine dioxide as supplement for drinking water
US11701398B2 (en) * 2020-06-04 2023-07-18 Intron Biotechnology, Inc. Compositions and methods for inhibiting the proliferation of pathogenic Escherichia coli

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6942858B1 (en) * 1996-04-15 2005-09-13 Nymox Corporation Compositions containing bacteriophages and methods of using bacteriophages to treat infections
KR20100116289A (en) * 2009-04-22 2010-11-01 경북대학교 산학협력단 A bacteriophage killing pathogenic enteric bacteria
US20110300528A1 (en) * 2008-12-03 2011-12-08 Sabah Abdel Amir Jassim Methods for Bacteriophage Design
KR101101376B1 (en) * 2009-10-16 2012-01-02 경희대학교 산학협력단 Bacteriophage having killing activity specific to Escherichia coli or Shigella

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101023995B1 (en) * 2008-06-10 2011-03-28 씨제이제일제당 (주) Escherichia coil specific bactriophage and method of preventing or controlling E. coli contamination using the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6942858B1 (en) * 1996-04-15 2005-09-13 Nymox Corporation Compositions containing bacteriophages and methods of using bacteriophages to treat infections
US20110300528A1 (en) * 2008-12-03 2011-12-08 Sabah Abdel Amir Jassim Methods for Bacteriophage Design
KR20100116289A (en) * 2009-04-22 2010-11-01 경북대학교 산학협력단 A bacteriophage killing pathogenic enteric bacteria
KR101101376B1 (en) * 2009-10-16 2012-01-02 경희대학교 산학협력단 Bacteriophage having killing activity specific to Escherichia coli or Shigella

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OLIVEIRA, A. ET AL.: "Isolation and characterization of bacteriophages for avian pathogenic E. coli strains", JOURNAL OF APPLIED MICROBIOLOGY, vol. 106, no. 6, 23 February 2009 (2009-02-23), pages 1919 - 1927 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10704027B2 (en) * 2013-02-27 2020-07-07 Cj Cheiljedang Corporation Bacteriophage and antibacterial composition comprising the same
EP3130669A4 (en) * 2014-04-10 2017-12-13 Cj Cheiljedang Corporation Novel bacteriophage and composition containing same
US10577590B2 (en) 2014-04-10 2020-03-03 Cj Cheiljedang Corporation Bacteriophage and composition comprising same
US11785948B2 (en) 2014-04-10 2023-10-17 Cj Cheiljedang Corporation Bacteriophage and composition comprising same
WO2015160164A1 (en) * 2014-04-15 2015-10-22 씨제이제일제당(주) Novel bacteriophage and composition comprising same
EP3133152A4 (en) * 2014-04-15 2017-12-27 Cj Cheiljedang Corporation Novel bacteriophage, and composition containing same
US9950018B2 (en) 2014-04-15 2018-04-24 Cj Cheiljedang Corporation Bacteriophage and composition comprising same
US10286020B2 (en) 2014-04-15 2019-05-14 Cj Cheiljedang Corporation Bacteriophage and composition comprising same
US10925909B2 (en) 2014-04-15 2021-02-23 Cj Cheiljedang Corporation Bacteriophage and composition comprising same
CN115960844A (en) * 2016-01-18 2023-04-14 美国控股实验室公司 Method and system for rapid detection of microorganisms using infectious agents
CN111868236A (en) * 2017-09-15 2020-10-30 辛特生物实验室公司 Bacteriophage compositions and methods for preventing bacterial infection in livestock

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