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

Abstract

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

Description

Newly Isolated Bacteriophage and Antimicrobial Compositions Comprising the Same

The present invention relates to a novel isolated bacteriophage and an antimicrobial composition comprising the same. More specifically, the present invention relates to a novel bacteriophage having Vibrio harveyi- specific killing ability and vibrio containing the bacteriophage as an active ingredient. A composition for preventing or treating a habei infectious disease, an antibiotic comprising the bacteriophage as an active ingredient, a feed additive comprising the bacteriophage as an active ingredient, a disinfectant or cleaning agent containing the bacteriophage as an active ingredient, and vibrio using the bacteriophage A method for preventing or treating an infectious disease caused by Havey.

Vibrio Harvey, known as Vibrio bacteria that cause increases in shrimp (penaeid shrimp) (V. harveyi) is alive everywhere in the marine environment, the luminance of Gram-negative bacteria, bacillus. Vibrio Habei has been reported to be an important pathogen for normal marine flora of marine organisms, but several species of marine fish, shellfish and shrimp (Karunasagar I et al., Aquaculture 128: 203-209, 1994, Zhang XH and Austin B, J. Fish Dis). 23: 93-103, 2000). Since 1990, Vibrio habei infection has caused 100% mortality of larva shrimp Penaeus monodon and Penaeus japonicas , resulting in severe economic losses to shrimp farms (Liu PC et al., Lett. Appl. Microbiol. 22 413-416). Such infections of vibrio havey in shrimp are termed shrimp vibriosis, shrimp bacterial sepsis, luminescent vibriosis or red-leg disease. Clinical symptoms of vibriosis include lethargy, tissue necrosis, delay in growth, metamorphosis and malformation of larvae, luminescence (shrimp glows in the dark), muddiness and pigmentation (Robertson, PAW et al., J) . Microbiol.Method 34: 31-39, 1998).

The virulence mechanism of Vibrio Havey is not yet clear and depends on the host and species of the fungus. Pathogenic components of Vibrio Havey include extracellular products (ECP), including proteases, hemolysins, lipases, lipopolysaccarides and bacteriocin analogs. -like substances) (Teo JWP et al., Gene 312: 181-188, 2003).

Many farmed shrimp hatcheries use antibiotics as a preventive measure against Vibrio Habei's infection. However, antibiotic-resistant bacteria and antibiotic residues are an international problem and there is an urgent need for comprehensive treatment for pathogens.

Bacteriophage, on the other hand, is a bacteria-specific virus that controls the growth of bacteria by infecting only certain bacteria, sometimes called abbreviated phages, and without the bacterial host, self-proliferation is impossible. Bacteriophage is a single or double-stranded DNA or RNA that consists of nucleic acid as a genetic material, and the nucleic acid is a simple structure in which the protein envelope is wrapped, with the tail on the icosahedron head, the tail on the icosahedron head, and It is divided into three basic types of filament type. The most common forms of octahedral head-tailed bacteriophages are Myoviridae with contractile tails, Siphoviridae with long, non-contracting tails, and grapes with short tails, depending on the tail characteristics. Can be subdivided into Podoviridae. Bacteriophages without tails in icosahedral heads are subdivided according to the shape of the head, the composition of the head, and the presence or absence of the sheath. Finally, filamentous bacteriophages are subdivided according to size, shape, sheath and filament composition (HW Ackermann, Frequency of morphological phage descriptions in the year 2000; Arch. Virol. 146: 843-857, 2001; Elizabeth Kutter et al. , Bacteriophages Biology and Application, CRC press).

Since the discovery of bacteriophages, studies have been conducted to use them as therapeutic agents for infectious diseases, but bacteriophages with specific target spectrums are attracted attention due to the characteristics of antibiotics with broad target spectrum. I couldn't. However, recently, antimicrobial growth promoter (AGP) added to feeds to promote animal growth has been found to be a major cause of antibiotic resistance, and policies to ban the use of AGP have been enacted. The use of AGP has been banned since March.

Based on this trend, the bacteriophage research is attracting attention again. For example, Clostridium sporogenes phage NCIMB 30008 and Clostridium tyrobutiricum phage NCIMB 30008 were registered as feed preservatives in Europe in 2003 and 2005, respectively. It was developed as a product for the purpose of controlling the contaminated Clostridium sp. These studies show that the use of bacteriophage to control the control of bacteria that are difficult to treat antibiotics, or to control common infectious bacteria contaminated with livestock products at the food level is ongoing.

Against this background, we have developed Vibrio Havey, which produces vibriosis in aquaculture fish and shrimp, in order to solve problems of a wide range of host ranges such as the occurrence of antibiotic resistant bacteria due to misuse and abuse of antibiotics, the retention of antibiotics in food and past antibiotics. The bacteriophage infected with the virus was newly isolated from nature, and their morphological, biochemical and genetic properties together with the ability to selectively kill only Vibrio Havey bacteria were completed and the present invention was completed.

It is an object of the present invention to provide novel bacteriophages having specific killing capacity for Vibrio havey.

Another object of the present invention is to provide a composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage as an active ingredient.

Still another object of the present invention is to provide an antibiotic comprising the bacteriophage as an active ingredient.

Still another object of the present invention is to provide a fish or shellfish feed additive containing the bacteriophage as an active ingredient.

Another object of the present invention to provide a disinfectant or cleaning agent containing the bacteriophage as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating an infectious disease caused by Vibrio habay using the bacteriophage or composition.

The present invention provides novel bacteriophages with specific killing ability to Vibrio harveyi .

The present inventors isolate bacteriophages that selectively lyse Vibrio harveyi BB120, VH-BB120, a host cell, from a sample collected from seawater. Morphologically observed by electron microscopy, the bacteriophage has species specificity that selectively infects Vibrio havey among Vibrio bacteria (see Table 1); Can be infected with various wild isolates Vibrio Havey (see Table 3); Exhibits specific killing ability to Vibrio havey; Morphological type B1 consisting of isometric capsid and long non-contractile tail is identified as belonging to Siphoviridae (see FIG. 1). In addition, the bacteriophage has a total genome size of about 75 kbp (see FIG. 2); As a result of analyzing the protein pattern, it was confirmed that the protein having 50 kDa, 44 kDa, and 36 kDa size as the main structural protein (see FIG. 3).

In addition, as a result of analyzing their genetic characteristics, nucleic acid molecules of SEQ ID NOS: 1 to 5 are included as part of the entire genome, and similarity between different species is compared based on SEQ ID NOs: 1 to 5 The fragments show 20-45% similarity but all fragments are novel bacteriophages without 100% matching bacteriophages (see Table 2). To analyze the genetic characteristics more specifically, a primer set specific for the bacteriophage, specifically, SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, SEQ ID NO: : PCR was performed with primer sets of 12 and 13, and SEQ ID NOs: 14 and 15. As a result, PCR products of 600 bp, 400 bp, 900 bp, 500 bp and 1000 bp were amplified respectively (FIGS. 4A to 4D). Reference).

On the other hand, when the bacteriophage is infected with Vibrio habay, a clear zone in which host cells are lysed by one bacteriophage in a soft agar with phage plaque is formed, and the size and turbidity of the lysate are confirmed to be the same. . These results indicate that the bacteriophage lysates Vibrio habay to inhibit the growth of Vibrio habay.

As a result of investigating the stability of the bacteriophage at various pH and temperature ranges, the bacteriophage has acid resistance stably surviving in the range of pH 4 to 11 (see FIG. 5), and stably survives in the range of 43 ° C. to 60 ° C. Confirm that it has a heat resistance that can be (see Fig. 6).

Therefore, the specificity, acid resistance and heat resistance of Vibrio habei, the bacteriophage according to the present invention in a variety of products, including the composition for the prevention and treatment of Vibrio Habei infectious diseases of cultured seafood and shrimp, various products including the bacteriophage as an active ingredient, etc. And applications in the pH range.

Accordingly, the present inventors collected a sample from the sea water, named as a bacteriophage "Bacteriophage Φ CJ17" having a specific killing ability for Vibrio Habei, and has the characteristics described above, and the Korean microorganisms on January 2, 2012 under the Budapest Treaty It was deposited in the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under accession number KCCM11247P.

As another aspect, the present invention provides a composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage as an active ingredient.

Since the bacteriophage of the present invention has an antimicrobial activity that can specifically kill Vibrio habei, it can be used for the purpose of preventing or treating diseases caused by its infection. Preferably, the infectious disease caused by Vibrio Havey may include, but is not limited to, Vibriosis.

As used herein, the term "Vibriosis" refers to a disease caused by the infection of Vibrio Havey, and infects various marine animals such as seawater fish, shellfish, and shrimps. In particular, in shrimps, lethargy, tissue necrosis, Delayed growth, delayed transformation of larvae, malformation, luminescence, muddiness, pigmentation, etc., causing massive mortality.

The term "prevention" as used herein refers to any action that inhibits or delays the onset of the disease by administration of the composition, and "treatment" refers to any action that improves or advantageously changes the condition of the subject disease by administration of the composition. It means.

The composition comprises 5 × 10 ² to 5 × 10 ¹² PFU / mL, preferably 1 × 10 ⁶ to 1 × 10 ¹⁰ PFU / mL of the bacteriophage according to the present invention as an active ingredient.

The prophylactic or therapeutic composition of the present invention may further comprise a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injection solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and these One or more of the components may be used in combination, and other conventional additives such as antioxidants, buffers, bacteriostatics may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The prophylactic or therapeutic composition of the present invention can be administered through oral or parenteral administration, and a method of applying or dipping to a diseased area or an individual can also be used. For parenteral administration, administration may be by intravenous administration, intraperitoneal administration, intramuscular administration, or topical administration. Suitable applications, sprays, and dosages of the compositions of the present invention may include formulation methods, modes of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion, and attenuation of the response of the animals and patients. It can vary depending on factors such as performance, and a skilled practitioner or veterinarian can easily determine and prescribe a dosage that is effective for the desired treatment.

Formulations for oral administration comprising a composition of the invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient include, for example, injectable forms such as subcutaneous injection, intravenous injection or intramuscular injection, aerosols for inhalation through suppository infusion or respiratory system, and the like. It can be formulated for spraying. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

In another aspect, the present invention provides an antibiotic comprising the bacteriophage as an active ingredient.

As used herein, the term "antibiotic" refers to an agent that can be given to animals in the form of a medicament to kill germs, and generically refers to preservatives, fungicides and antimicrobials. The animal includes fish and shellfish, shrimp and the like, and includes both natural and cultured.

Since the bacteriophage of the present invention has a very high specificity for Vibrio habay compared to conventional antibiotics, it can be used specifically for the prevention or treatment of diseases caused by Vibrio habay, and does not induce drug resistance, compared to conventional antibiotics. Life cycling can be used as a new novel antibiotic.

In still another aspect, the present invention provides a fish or shellfish feed additive comprising the bacteriophage as an active ingredient.

Feedstock antibiotics used in livestock and fisheries are used for prophylactic purposes. Antibiotics for prophylaxis are a problem because they increase the likelihood of developing resistant bacteria and can deliver antibiotics in fish and shellfish or livestock to humans. If antibiotics are absorbed into the body through meat or shellfish, they can lead to antibiotic resistance, which can lead to the spread of disease. In addition, there are many kinds of antibiotics mixed in the feed, which has a problem of increasing the probability of occurrence of multi-drug-resistant bacteria, the bacteriophage of the present invention as a new feed additive antibiotic to solve the problems caused by the use of conventional antibiotics more natural friendly It is available.

The feed additives for aquaculture or shrimp farming of the present invention may be separately prepared in the form of a bacteriophage as a feed additive, or may be directly added to the feed, or directly mixed in a hatchery or aquaculture tank. Bacteriophages in the feed and in the tank of the present invention may be liquid or dried, preferably in the form of dried powder. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying. Feed additives comprising the bacteriophage of the present invention may be mixed in the form of powder in a component ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed. In addition, the feed for fish and shellfish or shrimp farming may further include conventional additives to increase the shelf life of the feed in addition to the bacteriophage of the present invention.

The feed additive of the present invention may further be added to other non-pathogenic microorganisms. Microorganisms that may be added include Lactobacillus, which has physiological activity and organic degradability under anaerobic conditions such as Bacillus subtilis , Bacillus subtilis , which can produce proteases, lipolytic enzymes and sugar converting enzymes, and bovine stomach. strain, increasing the weight of livestock, such as increasing the milk yield of milk (Aspergillus oryzae) on Aspergillus duck characters showing the effect of increasing the absorption of digested food fungi (J. Animal Sci 43 (Lactobacillus sp .):. 910- 926, 1976) and yeast (J. Anim. Sci . 56: 735-739, 1983) such as Saccharomyces cerevisiae .

The feed additive according to the present invention may include cereals, root fruits, food processing by-products, algae, fiber oil, pharmaceutical by-products, oils, starches, gourds, grain by-products, etc. Minerals, fats, oils, minerals, fats, unicellular proteins, zooplankton, leftovers and the like may be included, but is not particularly limited thereto.

The feed additive according to the present invention may further include a binder, an emulsifier, a preservative, and the like, which are added to prevent quality deterioration, and are added to the feed to increase the efficacy of amino acids, vitamins, enzymes, probiotics, flavors, Non-protein nitrogen compounds, silicates, buffers, colorants, extractants, oligosaccharides and the like may be further included, in addition to the feed may include a mixture, but is not particularly limited. It is also possible to continue to reduce the number of Vibrio Habays in the farms by mixing and supplying them to the hatchery and the fish farms, and to find the Vibrio Habay clean farms.

In another aspect, the present invention provides a disinfectant or cleaning agent comprising the bacteriophage as an active ingredient.

The disinfectant containing the bacteriophage as an active ingredient is also used for spraying to remove Vibrio habei, which is used in the active area of fish or shrimp in the farm, hatchery, farmed fish or shrimp mortality area, cultured seafood or shrimp in the cooking place and cooking equipment. And places are not limited to this. In addition, the cleaning agent containing the bacteriophage as an active ingredient may be used to remove vibrio habei infectious to the aquarium of the hatchery or farm.

In another aspect, the present invention provides a method for preventing or treating an infectious disease caused by Vibrio habay using the bacteriophage or the composition.

Specifically, the method includes administering the bacteriophage or the composition in a pharmaceutically effective amount to an individual suffering from an infectious disease caused by Vibrio Havey. The bacteriophage or composition according to the present invention may be administered to a shellfish or shrimp in the form of a pharmaceutical preparation, or may be administered through a method of feeding the shellfish or shrimp into a feed or hatchery and aquaculture tank, and feeding it, preferably a feed additive. It can be administered in the form of a mixture in the feed.

The route of administration of the bacteriophage or composition according to the present invention can be administered via various routes, oral or parenteral, as long as the target tissue can be reached. Specifically, oral, topical, intravenous, intraperitoneal, intramuscular, intraarterial It may be administered in a conventional manner through, transdermal, nasal, inhalation, and the like. It will be apparent to those skilled in the art that the suitable total daily usage of the bacteriophage or composition according to the invention administered by the method can be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular individual may be determined in conjunction with the type and severity of the response to be achieved, the age, body weight, general health, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and specific composition of the individual. It is desirable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.

The novel bacteriophage of the present invention exhibits specific killing ability to Vibrio havey and has excellent acid and heat resistance properties. Therefore, the bacteriophage according to the present invention can be usefully used for the prevention or treatment of Vibrio havey infectious disease Vibriosis, and can be widely used as a feed additive composition, a disinfectant and a cleaning agent.

1 is an electron micrograph of the bacteriophage Φ CJ17 isolated in the present invention.

Figure 2 is a photograph showing the result of pulsed field gel electrophoresis (PFGE) of the bacteriophage Φ CJ17 isolated in the present invention.

Figure 3 is a photograph showing the SDS-PAGE results of the bacteriophage Φ CJ17 isolated in the present invention.

Figure 4a is a photograph showing the PCR results using the primer set of SEQ ID NO: 6 to 9 as a template of the bacteriophage ΦCJ17 genomic DNA isolated in the present invention.

Figure 4b is a photograph showing the PCR results of the primer set of SEQ ID NO: 10 and 11 with the bacteriophage ΦCJ17 genomic DNA isolated in the present invention as a template.

Figure 4c is a photograph showing the PCR results using the primer set of SEQ ID NO: 12 and 13 as a template of the bacteriophage ΦCJ17 genomic DNA isolated in the present invention.

Figure 4d is a photograph showing the PCR results using the primer set of SEQ ID NO: 14 and 15 as a template of the bacteriophage ΦCJ17 genomic DNA isolated in the present invention.

Figure 5 is a graph showing the acid resistance test results of the bacteriophage Φ CJ17 isolated in the present invention.

Figure 6 is a graph showing the heat resistance test results of the bacteriophage Φ CJ17 isolated in the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Isolation of Bacteriophage with Vibrio Habay Killing Effect

<1-1> Bacteriophage Screening and Single Bacteriophage Separation

50 ml of seawater obtained from Eulwang-dong, Jung-gu, Incheon was centrifuged at 4,000 rpm for 10 minutes, and then the supernatant was filtered through a 0.45 μm filter to prepare a sample solution, which was then subjected to a soft agar overlay method. .

Specifically, 150 μl of Vibrio harveyi BB120, VH-BB120, ATCC BAA-1116TM shake culture (OD ₆₀₀ = 2) and 10 × LB medium (tryptone 10 g / l, yeast extract 5 g / l and 18 ml of the sample solution was added to a mixture of 2 ml NaCl 10 g / L), and the mixture was incubated at 30 ° C. for 18 hours. Subsequently, the culture solution was centrifuged at 4,000 rpm for 10 minutes to separate the supernatant, and the separated supernatant was filtered with a 0.45 μm filter. 4 ml of 0.6% (w / v) agar mixed with 2% NaCl and 150 µl of VH-BB120 shake culture medium (OD ₆₀₀ = 2) were poured onto the LB plate medium and solidified, followed by filtering the supernatant 10 above. ㎕ was added dropwise and incubated at 30 ° C. for 18 hours to select a sample solution in which lysate plaques were formed.

A single lysate plaque was obtained by sequentially diluting the supernatant from the selected sample solution and applying it to the soft agar overlay assay at a series of concentration levels. Since one lysate consists of one bacteriophage, one lysate was taken and 400 μl of SM solution (NaCl 5.8 g / l, MgSO ₄ · 7H ₂ O 2 g / l, 1 M Tris-HCl (pH) 7.5) Soak in 50 mL / L), and allowed to stand at room temperature for 4 hours to purely isolate the bacteriophage.

To secure large amounts of purely isolated bacteriophages, 100 μl of the supernatant of the solution in which the lysate was immersed and allowed to stand, 12 ml of agar (0.6% (w / v) agar) and 12 ml of VH-BB120 shake culture After mixing 500 μl, soft agar overlay was performed on 150 mm diameter LB plate media. 15 ml of SM solution was added to a completely lysed plate medium and shaken at low temperature for 4 hours at room temperature to elute the bacteriophage contained in the lysate plaque. The SM solution containing the eluted bacteriophage was recovered and stored in a refrigerator by filtration with a 0.45 μm filter.

<1-2> Mass Culture and Purification of Bacteriophage

The bacteriophage selected in Example <1-1> was cultured in large quantities using VH-BB120, and the bacteriophage was purified therefrom.

Specifically, VH-BB120 was shaken and cultured to 1.0 × 10 ¹⁰ cfu (colony forming unit), centrifuged at 4,000 rpm for 10 minutes, and then resuspended in 4 ml of SM solution. The bacteriophage was inoculated with 1.0 × 10 ⁶ plaque forming unit (PFU), titrated with multiplicity of infection (= 0.001), and then left standing at 30 ° C. for 20 minutes. It was then inoculated in 150 ml of LB medium and incubated at 30 ° C. for 5 hours. After the incubation was completed, chloroform was added to 1% (v / v) of the final volume and stirred for 20 minutes. Restriction enzymes DNase I and RNase A were added to a final concentration of 1 μg / ml and 30 ° C. at 30 ° C. Let stand for a minute. Subsequently, NaCl and PEG (polyethylene glycol) were added so that the final concentrations were 1 M and 10% (w / v), respectively, and further left at 4 ° C. for 3 hours. This was centrifuged at 12,000 rpm for 20 minutes at 4 ℃, the supernatant was removed and the precipitate was separated. The separated precipitate was suspended in 5 ml of SM solution and allowed to stand at room temperature for 20 minutes. 4 ml of chloroform was added thereto, stirred, and centrifuged at 4,000 rpm for 20 minutes at 4 ° C to obtain a supernatant. The supernatant was filtered with a 0.45 μm filter, followed by ultracentrifugation (35,000 rpm, 1 hour, 4 ° C.) using glycerol density gradient (density: 40%, 5% glycerol) to purify the bacteriophage. The purified bacteriophage was suspended in 500 μl of SM solution and titer was measured.

The purified bacteriophage was named "Bacteriophage ΦCJ17" and was deposited with the accession no. It was.

Example 2: Vibrio strain infectivity comparison of bacteriophage ΦCJ17

Cross-infection was performed to confirm whether bacteriophage ΦCJ17 isolated and purified in Example <1-2> showed lytic activity against Vibrio bacteria of other species other than VH-BB120.

Specifically, five Vibrio parahaemolyticus ( V. parahaemolyticus ), five Vibrio campbelli ( V. campbelli ) and two Vibrio Havey ( V. harveyi ) were cultured to obtain a culture solution, respectively A soft agar overlay method using the culture medium and the bacteriophage Φ CJ17 was performed to confirm the formation of lysate plaques (Table 1).

Table 1 Whether ΦCJ17 is infected with pathogenic Vibrio

Strain name	Formation of lytic plaque
V.harveyi BB 120 V. harveyi V. parahaemolyticus (MEBiC06047) V. campbellii (GMD258) V. parahaemolyticus (MEBiC06069) V. campbellii (WDC577) (MEBiC05077) V. parahaemolyticus (MEBiC06254) V.campbellii (MEBiC05078)	OOXXXXXXXXXX

* 'MEBiC' source: Korea Maritime Research Institute, Korea Marine Biology Support BANK

As shown in Table 1, it was confirmed that bacteriophage Φ CJ17 according to the present invention does not show the lytic activity against Vibrio bacteria other than Vibrio habei, these results indicate that the bacteriophage has a specific killing ability in Vibrio habei.

Example 3: Morphological Observation of Bacteriophage Φ CJ17

Bacteriophage Φ CJ17 isolated and purified in Example <1-2> was diluted in 0.01% (w / v) gelatin solution and fixed with 2.5% glutaraldehyde solution. It was added dropwise onto a carbon-coated mica plate (approximately 2.5 × 2.5 mm) and acclimated for 10 minutes, then washed with sterile distilled water. Carbon film was placed on a copper grid, dyed in 4% uranyl acetate for 30 to 60 seconds, dried, and then passed through a transmission electron microscope (JEM-1011 transmission electron microscope, 80 kV, magnification × 120,000 to x 200,000).

As a result, as shown in Figure 1, the bacteriophage Φ CJ17 of the present invention is a morphotype B1 consisting of a morphological icosahedron (isometric capsid) and a long non-contractile tail (Sipovir) It was confirmed that it belongs to Siphoviridae.

Example 4 Whole Genomic DNA Size Analysis of Bacteriophage ΦCJ17

Genomic DNA was extracted from the bacteriophage Φ CJ17 of the present invention. Specifically, EDTA (ethylene diamine tetraacetic acid, pH 8.0), protease K (proteinase K) and SDS (sodium dodecyl sulfate) in the culture of purified bacteriophage ΦCJ17, respectively, the final concentration of 20 mM, 50 ㎍ / ㎖ and 0.5% (w / v) was added and allowed to stand at 55 ° C for 1 hour. An equal volume of phenol (pH 8.0) was added thereto, stirred, and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant. The obtained supernatant was mixed with the same volume of PC (phenol: chloroform = 1: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature. To the supernatant obtained from this, 3 M sodium acetate was added to 10% (v / v) of the total volume, mixed, and 2 volumes of cold 95% ethanol were added thereto at -20 ° C. It was left for 1 hour. Thereafter, the supernatant was completely removed by centrifugation at 12,000 rpm for 10 minutes at 0 ° C to obtain precipitated genomic DNA. 50 μl of TE buffer (Tris-EDTA, pH 8.0) was added thereto to dissolve genomic DNA and its concentration was measured. Subsequently, 1 μg of genomic DNA was loaded onto a 1% pulse-field gel electrophoresis (PFGE) agarose gel and the BIORAD PFGE System No. 7 program (size range 25-100 kb; switch time ramp 0.4-2.0 seconds, linear shape; Forward voltage 180 V; reverse voltage 120 V) was used for 20 hours at room temperature.

As a result, as shown in Figure 2, the bacteriophage Φ CJ17 of the present invention was confirmed to have a total genomic DNA size of about 75 kbp.

Example 5: Protein pattern analysis of bacteriophage ΦCJ17

15 μl of purified bacteriophage ΦCJ17 solution with 10 ¹⁰ PFU / ml titer and 5 μl of 5 × SDS sample solution were mixed and heated for 5 minutes. This was loaded onto a 15% SDS-PAGE gel to develop the total protein of bacteriophage ΦCJ17. Gels were stained with coomassie blue for 1 hour at room temperature.

As a result, as shown in Figure 3, the bacteriophage Φ CJ17 of the present invention was observed a pattern consisting of proteins consisting of major structural proteins of 50 kDa, 44 kDa and 36 kDa size.

Example 6: Genetic Characterization of Bacteriophage Φ CJ17

To analyze the genetic characteristics of the isolated bacteriophage ΦCJ17, 5 μg of genomic DNA of bacteriophage ΦCJ17 was co-treated with restriction enzymes EcoRV, ScaI and NruI and co-treatment with restriction enzymes PvuII, HincII and StuI. In addition, a pCL1920 vector (Promega) was digested with restriction enzyme SmaI, and a vector treated with CIP (calf intestinal alkaline phosphatase) was prepared. Reaction conditions were mixed so that the amount of the cut genomic DNA and the vector was 3: 1, and the ligation reaction was performed at 16 ° C. for 5 hours to obtain an expression vector containing each genomic DNA fragment. Expression vectors obtained therefrom were introduced into E. coli DH5α cells to prepare respective transformants. Each transformant prepared was inoculated and cultured in LB plate medium containing ampicillin and X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), and then Two colonies were selected by the blue-white colony screening method. Each of the selected colonies was shaken for 16 hours in a medium containing ampicillin, thereby obtaining respective cells. Plasmid purification kit (Solgent) was extracted from each cell obtained.

The extracted plasmid was cloned by PCR using M13 forward and reverse (SEQ ID NOs: 16 and 17) primer sets. Among them, the size of the inserted genomic DNA was 1 kb or more, and the similarity of the nucleotide sequences was analyzed using the primer set. The results are shown in Table 2 below.

TABLE 2 Homology Comparison of Bacteriophage ΦCJ17 Sequence and Other Bacteriophage Translational Sequences

SEQ ID NO:	organism	protein	Quary	Identity	e-value
One	Desulfovibrio vulgaris subsp. vulgaris dp4	hypothetical protein	286-450	26/58 (45%)	3.00E-10
2	Bacillus phage SPO1	gp21.9	8-385	26/128 (20%)	0.21
	Lactobacillus phage Lb338-1	gp027	168-440	31/98 (32%)	0.4
3	Pseudomonas phage 73	hypothetical protein ORF034	11-403	42/136 (31%)	1.00E-08
4	Pseudomonas phage M6	hypothetical protein ORF038	2-439	39/146 (27%)	9.00E-07
	Cellvibrio gilvus ATCC 13127	flagellin domain protein	401-595	27/73 (37%)	7.5
6	Pseudomonas aeruginosa NCGM2.S1	hypothetical bacteriophage protein	85-660	69/192 (36%)	5.00E-25

As shown in Table 2, 20-45% nucleotide sequence similarity according to the nucleic acid molecule fragments, but all fragments are 100% identical bacteriophage because there is no bacteriophage Φ CJ17 according to the present invention was confirmed to be novel.

Example 7: PCR analysis using bacteriophage ΦCJ17 specific primers

In order to identify the isolated bacteriophage Φ CJ17, a specific primer was prepared. Specifically, primer sets of SEQ ID NOs: 6 and 7 were prepared based on the nucleotide sequences of SEQ ID NO: 1, and primer sets of SEQ ID NOs: 8 and 9 were prepared based on the nucleotide sequences of SEQ ID NO: 2. In addition, primer sets of SEQ ID NO: 10 and 11, SEQ ID NO: 12 and 13, and SEQ ID NO: 14 and 15 were prepared based on the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. PCR was performed using each primer set thus prepared and using genomic DNA of bacteriophage Φ CJ17 as a template.

First, each primer was added to a pre-mix (Preone, Bioneer) so as to have a total genome DAN of 0.5 μg of bacteriophage Φ CJ17 and adjusted to a final volume of 20 μl. PCR was performed by repeating 30 seconds of denaturation at 94 ° C, annealing at 30 ° C for 30 seconds, and polymerization conditions of 1 minute at 72 ° C.

Figure 4a shows the PCR results using the primer set of SEQ ID NO: 6 and 7, and 8 and 9, Figure 4B shows the PCR results using the primer set of SEQ ID NO: 10 and 11, Figure 4C is SEQ ID NO: PCR results using the primer sets 12 and 13 are shown, and FIG. 4D shows the PCR results using the primer sets SEQ ID NOs: 14 and 15. As shown in Figures 4a to 4d, PCR results using a primer set of SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, and SEQ ID NOs: 12 and 13, respectively, about 600 bp, It was confirmed that the PCR products of 400 bp, 900 bp, 500 bp and about 1000 bp were amplified.

Example 8 Investigation of Stability of Bacteriophage ΦCJ17 to pH

Various pH ranges (pH 2.0, 2.5, 3.0, 3.5, 4.0, 5.5, 6.5, 7.5, 8.5, 9.5, 10.0, and 11.0) to determine whether bacteriophage ΦCJ17 can maintain stability at low pH in fish stomachs Stability investigation experiments were performed at. Sodium acetate buffer (pH 4.0, and 5.5), sodium citrate buffer (pH 2.0, 2.5, 3.0, and 3.5), sodium phosphate buffer (pH 6.5, And 7.5), and Tris solution (Tris-HCl; pH 8.5, 9.5, 10.0, and 11.0) were prepared at 0.2 M, respectively. 180 μl of pH solution and 20 μl of 2.0 × 10 ¹¹ PFU / ml titer of bacteriophage ΦCJ17 solution were mixed and allowed to stand at room temperature for 2 hours. These were diluted in steps, and 10 μl of the diluted solution of each step was added dropwise according to the soft agar overlay method, followed by incubation at 30 ° C. for 18 hours to determine the titer through lysis. At this time, relative stability was confirmed by comparing the titer of the pH change based on the titer of the bacteriophage Φ CJ17 at 0 hours.

As shown in Figure 5, the bacteriophage Φ CJ17 of the present invention was confirmed to be stable without losing activity in the range from pH 4.0 to pH 11.0.

Example 9 Investigation of Stability of Bacteriophage ΦCJ17 with Temperature

When bacteriophage Φ CJ17 was used as a feed additive, the following experiment was performed to confirm the stability of heat generated during the formulation process. Specifically, 100 μl of a solution of bacteriophage ΦCJ17 with a 3.0 × 10 ¹¹ PFU / mL titer was respectively 0, 30, 60, and 120 at 37 ° C., 45 ° C., 53 ° C., 60 ° C., and 70 ° C. Let stand for a minute. The treated experimental culture was diluted in steps, and 10 µl of the diluted solution of each step was added dropwise according to the soft agar overlay method, followed by incubation at 30 ° C. for 18 hours to determine the titer through lysis. At this time, relative stability according to temperature and exposure time was compared based on the titer at 0 ° C and 37 ° C.

As a result, as shown in Figure 6, the bacteriophage Φ CJ17 of the present invention maintained the activity even when exposed to 60 hours at 60 ℃, it can be seen that the activity decreases over time when exposed to more than 10 minutes at 70 ℃ .

Example 10 Investigation of Infection Range of Bacteriophage ΦCJ17 in Wild Strain Vibrio Habay

It was examined whether bacteriophage ΦCJ17 showed bactericidal activity against wild isolates other than the specific Vibrio Havey strain VH-BB120 used in the experiment. The wild isolates used in this experiment were a total of three species, including one Vibrio habei from the Korea Maritime Research Institute for Marine Biotechnology, one Vibrio habay purchased from ATCC, and one vibrio habay isolated from the laboratory. Vibrio Harvey. Stain 150 μl of shake culture (OD ₆₀₀ = 2) of each strain onto LB plate medium, add 10 μl of a solution of bacteriophage ΦCJ17 with 10 ⁸ PFU / ml titer according to the soft agar overlay method, and then 18 hours at 30 ° C. The culture was observed for the formation of lytic plaques.

TABLE 3 Investigation of lysate formation in wild isolate Vibrio Habay

Strain name	ΦCJ13 lytic plaque formation
V. harveyi (MEBiC05675) V. harveyi (LAB) V. harveyi (BB170)	OOO

* 'MEBiC' source: Korea Maritime Research Institute, Marine Korea Biotechnology Bank

* 'LAB' source: Laboratory self-separating strain isolated from fish market water sample

* 'BB170' source: ATCC® BAA-1117TM

As shown in Table 3, the bacteriophage Φ CJ17 of the present invention was confirmed to exhibit the ability to infect not only VH-BB120, but also all kinds of Vibrio Habei.

Therefore, the bacteriophage Φ CJ17 of the present invention can be seen that when applied to the site, such as fish farms, can exhibit an excellent control effect for Vibrio Habei.

Since the novel bacteriophage of the present invention exhibits specific killing ability, excellent acid resistance and heat resistance to Vibrio Havey, it can be usefully used for the prevention or treatment of Vibrio's Vibrio infectious disease Vibrioosis, as well as a composition for feed addition, It can be widely used as a disinfectant and cleaning agent.

Claims (8)

1. Bacteriophage with accession number KCCM11247P with specific killing ability to Vibrio harveyi.
2. A composition for the prevention or treatment of infectious diseases caused by Vibrio habay comprising the bacteriophage of claim 1 as an active ingredient.
3. 3. The composition of claim 2, wherein the infectious disease caused by Vibrio Havey is Vibrioosis.
4. An antibiotic comprising the bacteriophage of claim 1 as an active ingredient.
5. Feed additives for aquaculture or shrimp farming comprising the bacteriophage of claim 1 as an active ingredient.
6. Disinfectant or cleaning agent comprising the bacteriophage of claim 1 as an active ingredient.
7. A method for preventing or treating an infectious disease caused by Vibrio havey, comprising administering the bacteriophage of claim 1 or the composition of claim 2 or 3 to aquaculture seafood or shrimp.
8. 8. The method of claim 7, wherein the infectious disease caused by Vibrio Havey is Vibrioosis.

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