Specifications

Title of the invention: new phage and the containing composition

Technical areas

[1]

This invention is Admiral plasticity represents a mortal to the e. coli specific new phage, the containing composition and use of the above new Bacteriophage or composition to prevent or cure infectious diseases of animals on the way.

Background technology

[2]

E. coli (Escherichia coli,E.coli, ' ') is an intestinal bacteria (Enterobacteriaceae), Keisuke Lycian genus (Escherichia) belongs to the bacillus of gram-negative, Secretary of a variety of animals, including mammals exists within the normal germs is one of a total of. Escherichia coli is the most opportunistic infections can lead to chronic non-hospital, but some pathogenic bacteria within the animal, including the person who gives a variety of causes, including bowel disease and septicemia have been known to.

[3]

The chapter on the kind of e. coli-poison plastic Escherichia coli (Enterotoxigenic E.coli, ETEC), pathogenic e. coli (Enteropathogenic E.coli, EPEC), hemorrhagic coli (Enterohemorrhagic E.coli, EHEC), Jean coherent Escherichia coli ( E.coli, Enteroaggregative EAEC), Jean invasive Escherichia coli (EIEC E.coli, Enteroinvacive), a minor Star Escherichia coli ( E.coli, NTEC Necrotoxigenic), and of these, in particular, the amount of money in Escherichia coli e. coli Admiral plastic is infectious diseases have been known to cause.

[4]

At present, the amount of money the industry's large-scale mass breeding of the pigs in accordance with the trend of Escherichia coli is the most common and the most amount of money is another matter: sickness in chapter two. Recently, even in our country increased occurrence of e. coli symptoms of swine diarrhea caused by the growth of a young pig breeding farms with delays and loss of our great cause and economically.

[5]

These swine e. coli symptoms, prevention and treatment in conventional pigs to several antibiotics, antibiotics, using them to abuse or resistant to antibiotics in swine if they're likely to be present in the body, a pig residual antibiotics administered to many restrictions worldwide.

[6]

In the meantime, the phage (bacteriophage) infected with certain bacteria inhibits the growth of bacteria infected and inhibited germ-specific virus. Bacteriophage host specificity compared to strong antibiotics, as well as the recent emergence of resistant bacteria for antibiotic use problems and serious residual problems, and so the meat of my antibiotics as he takes advantage of the growing interest in.

[7]

In many countries around the world in a study on phage and Phage for patent application, of course, the United States regarding the composition of the food and Drug Administration (Food and Drug Administration, FDA), such as the approval of the movement you want to acquire a growing trend.

[8]

However, the good money is an important problem in the industry, including axis, and Admiral in the plasticity of Escherichia coli infectious diseases prevention and/or treatment related to Phage for technology is still lacking, and therefore these phage and the development of technology relevant to this request.

A detailed description of the invention

Technical challenges

[9]

This is one of those who invented the emergence of resistant bacteria to antibiotic use and antibiotic meats my residual issues are addressed and Escherichia coli infectious diseases in order to effectively prevent and cure as a result of his research, Admiral firing against the specific e. coli having to kill new phage Φ C J 27 (KCCM11465P) led to separation from the natural world.

[10]

Also, as mentioned above in the form of a new phage, biochemical, genetic characteristics, compassionate and above, including my acid and heat-resistant phage that takes advantage of this by checking one of the antibiotics, disinfectants, animal feed additives, other composition was developed, which in turn, such as Escherichia coli prevention of infectious diseases, or composition for the treatment and prevention of disease or treatment method was developed.

[11]

This invention is an Admiral firing having to kill the e. coli specific new phage Φ C J 27 (KCCM11465P) aims to provide.

[12]

In addition, this invention is a reminder that phage Φ J 27 C (KCCM11465P), which includes the active ingredient, Admiral firing due to e. coli infectious diseases prevention and/or treatment for the purpose of providing water.

[13]

In addition, this invention is a reminder that phage Φ C J 27 (KCCM11465P) as the active ingredient, which contains antibiotics, feed additives, drinking water additives, feed, drinking water, disinfectants or cleansers aims to provide.

[14]

In addition, this invention is a reminder that phage Φ C J 27 (KCCM11465P) is included as the active ingredient or a composition, with the exception of humans, animals, the Chief of infectious diseases prevention and the e. coli toxin plasticity and/or therapy aims to provide a way to.

Challenges in the Sudan

[15]

The days of this invention may, Admiral plastic having to kill the e. coli specific new phage Φ C J 27 (KCCM11465P).

[16]

According to the other, of the invention dimension, seen above, phage Φ J 27 C (KCCM11465P), which includes the active ingredient, Admiral firing due to e. coli prevention of infectious diseases or treatment for.

[17]

According to another, this invention dimension, the aforementioned phage Φ C J 27 (KCCM11465P) as the active ingredient, including antibiotics, feed additives, drinking water additives, feed, drinking water, disinfectants or cleansers.

[18]

According to another, this invention dimension, the aforementioned phage Φ C J 27 (KCCM11465P) as the active ingredient or a composition which includes steps to administering to animals except humans, Admiral firing to prevent infectious diseases caused by e. coli or how to cure.

The effect of the invention

[19]

This invention of Bacteriophage Φ C J 27 (KCCM11465P;-gun) is a quirky ectopic Escherichia coli as an enemy Admiral deaden the effect to.

[20]

In addition, this invention reminded of Bacteriophage Φ C J 27 (KCCM11465P) is the acid resistance and excellent resistance to a wide range of temperature and pH range-in-Chief of the infectious diseases plastic poison prevention of e. coli or could be utilized for the treatment materials, as well as the aforementioned phage Φ J 27 C (KCCM11465P), which include antibiotics, as feed additives, drinking water additives, feed, drinking water, disinfectants or cleansers, such as the effect that can be utilized.

[21]

In addition, this invention is a reminder that phage Φ J 27 C (KCCM11465P) or active ingredients include antibiotics, compared to conventional antibiotics Pathogenic Escherichia coli the ripe fungus is very high specificity for certain pathogens without killing only a mortal can be resistant to conventional antibiotics, drugs do not induce compared to the effect that extend product life.

[22]

In addition, this invention is a reminder that phage Φ C J 27 (KCCM11465P) is included as the active ingredient or a composition by administering to animals except humans, Admiral firing to prevent infectious diseases caused by e. coli or the effect that can be treated.

Drawing, brief description

[23]

Figure 1 is a new Bacteriophage Φ C J 27 (KCCM11465P) ("Φ J C 27 ') of the electron microscope photographs.

[24]

Two new phage Φ C J PFGE results of 27.

[25]

Three new phage Φ C J SDS-PAGE of 27 results.

[26]

4 new phage Φ C J 27 shows the graph of my acid test results.

[27]

5 new phage Φ C J 27 shows the experimental results of thermal resistance at 60 ° c the graph.

The form for the implementation of the invention

[28]

This, explains in detail than this invention. For information not listed in this specification shall be trained in the field of technical field or similar characters, fully aware that the explanation is omitted because it can be inferred.

[29]

[30]

The days of this invention may, Admiral plasticity of Escherichia coli (Enterotoxigenic Escherichia coli, ETEC) having to kill specific new phage Φ C J 27 (KCCM11465P) ("Φ J C 27 ').

[31]

Admiral firing Escherichia coli is gram-negative Bacillus as of lactose (Lactose), SAT (Fructose), meantime, to dissolve acid and gas produced is aerobic or anaerobic bacteria tub sex. Admiral plastic badge of e. coli is usually about 7 ° c ~ grows well in 48 developing and developed at the optimum temperature is approximately 35 ° c to 37 ° c. In addition, Chapter 4.5 to 9 pH pH the e. coli toxin plasticity is developed from a range of possible.

[32]

Admiral firing Escherichia coli is cholera bacteria (Vibrio cholerae) and produces toxins similar to the Admiral because plastic is infected with cholera-infected e. coli case and similar diseases will be able to represent. The toxin is produced significantly above this recessive toxins (heat-labile enterotoxin, LT) and heat-resistant toxins (heat-stable, ST enterotoxin) are distinguished by two. Approx. 60 ° c above the toxin this zeal in heat for about 10 minutes, losing the active toxin and heat-resistant toxins above 100 ° c in approximately 30 minutes by heating does not lose immunity represents the active toxin.

[33]

In the upper part of the small intestine e. coli multiply Admiral plasticity, and Admiral firing units of volume concentration of e. coli serum (1 ml) per approx. 107 cfu (colony formation unit) 108 cfu if it reaches the e. coli symptoms, including e. coli can cause infectious diseases.

[34]

Bacteriophage infects bacteria (bacteriophage) that takes the cake by a specific bacteria inhibits the growth of bacteria that inhibit specific viral and, with single or double chains of DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid), genetic material or contains a virus.

[35]

Specifically, this invention of Bacteriophage Φ C J Hamm's work 27 Admiral plasticity to selectively infect species Pathogenic Escherichia coli having specificity as a Bacteriophage, long hair (elongated head) and a tail (contractile tail) contraction is having the structure observed morphological Mai o irregularities (Myoviridae) Bacteriophage (see Figure 1).

[36]

Enterobacteria phage Φ J 27 C of the sequence and the other enterobacteria phage Detox batching homology of the column base [table 1]. Bacteriophage Φ C when it comes to my acid pH of 2.0 J 27 pH from 5.0 active without losing the stable (4), heat-resistant when it comes to 60 ℃ 1 hour until I have been exposed to in the log indicated the active reduction of less than (5). Enterobacteria phage Φ C DNA sequence of 27 J is the column number of the column in the list as on the 1.

[37]

The aforementioned phage Φ C J remove these inventors are new 27 phage, dated 25 October 2013, South Korea microbial Conservation Center (Korean Culture Center of Microorganisms, 1-Dong, Seodaemun-Ku, Seoul hongje 361-221) were deposited with the depositary on the number KCCM11465P.

[38]

[39]

According to the other, of the invention dimension, seen above, phage Φ C J as the active ingredient, including 27 Admiral firing prevention of infectious diseases caused by e. coli or treatment for.

[40]

The aforementioned phage Φ C J 27 Admiral deaden as ectopic Escherichia coli specific antimicrobial activity that can indicate the Admiral because of plasticity induced by e. coli infection and prevent or cure their disease can be used for the purpose. The aforementioned phage Φ C J 27 Admiral plasticity using Escherichia coli infectious diseases, for example Escherichia coli (Colibacillosis) can hear, specifically swine Escherichia coli (Colibacillosis in pigs), but is not limited to this.

[41]

The above "e. coli symptoms," Iran, the animal has been infected with pathogenic e. coli on the disease and its symptoms as sepsis, diarrhea (new machine after diarrhea diarrhea and reason), blood (cerebrospinal fluid vascular edema disease and symptoms), etc. Of these, sepsis is not within 3 days after the 2nd newborn in the predecessor of the acute infectious disease that occurs primarily as our rate is significantly higher. Diarrhea is 1 week to 2 weeks major the reason, within mammals and a bunch of Ministers immediately after the infectious causes of death or to develop the disorder. After 8 weeks the reason to poison blood 12 weeks old now and the onset of the edema and the money mainly from the neurological symptoms is sudden death, sometimes causing the indicated hourly.

[42]

This invention, the term "prevention" is used in Iran, the aforementioned phage Φ Φ J 27 C and/or C J 27 with active ingredient above as enterobacteria phage containing the disease by offering animal composition to suppress or delay its onset means all acts.

[43]

This term is used in the invention "cure" refers to above, phage Φ Φ J 27 C and/or C J 27 with active ingredient above as enterobacteria phage containing composition provides the animals already infected the disease symptoms get better or it means all acts to improve.

[44]

This may remind you of an Admiral firing prevention of infectious diseases caused by e. coli or therapeutic composition for water above phage Φ J 27 C 5 × 102 to 5 × 1012 pfu/㎖ may contain and, specifically, to the aforementioned phage Φ J 27 C 1 × 106 to 1 × 1010 pfu/㎖ can be high.

[45]

This may remind you of an Admiral firing prevention of infectious diseases caused by e. coli or composition for treating water to further acceptable about biological substrate can include, along with the aforementioned substrate formulation is food, medicines, feed additives or drinking water additives, such as can be provided. This term is used in the invention "about geopolitical acceptable substrate" refers to organisms biologically active compounds administered without irritating and does not inhibit substrate characteristics, or paint thinner.

[46]

This is the kind of above dimension available substrate does not specifically limit the relevant technology is being used in the field usually is allowed to fence about forensic Chera if either can be used. For example, the aforementioned substrate of nonrestrictive, saline, sterile water, saline solution, large ring, demilitarized albumin injection solution, dextro, saying Windows solution sat dextrin solution, glycerol, ethanol, etc. They are used alone or mixed with two or more can be used.

[47]

In addition, if necessary, antioxidant, buffering, and/or add other usual biocide additives that can be used with paint thinner, dispersant, surfactant, and/or lubricants, etc., in addition, by adding aqueous solution, suspension, suspension injectable formulations, such as pills, capsules, granules or tablets, such as the formulation can be used by.

[48]

This may remind you of an Admiral firing prevention of infectious diseases caused by e. coli or therapeutic doses of water does not specifically limit for the approach, the technology used in the field usually can follow the way. For example, the above doses administered or nonrestrictive way parenteral doses of oral composition in a manner that can be administered.

[49]

The above examples of oral dose formulation for nonrestrictive, Loki (troches), Rosen (lozenge), refining, water soluble oil-based suspension, suspension, and dispensation of powder, granule, emulsion, hard capsules, soft capsules, syrup or Elixir (elixirs).

[50]

The composition of the water, this may include tablets or capsules of formulation to formulation, as lactose, Caro (Saccharose), sorbitol (Sorbitol), Nishi-Tolkien (Mannitol), starch, amylopectin (Amylopectin), cellulose (Cellulose) or gelatin (Gelatin), such as a combined article; (Dicalcium phosphate) calcium-d-force-as part of the brothers; Corn starch or potato starch breakdown, such as; Stearic acid magnesium (magnesium stearate), stearic acid calcium (calcium stearate), te reels fumaric acid sodium (sodium stearyl fumarate) or polyethylene glycol wax (polyethylene glycol wax) and as such can include such things as the lubricant, in the case of the above mentioned substances capsule formulations, in addition to the local oil, such as liquid may contain additional carriers, etc.

[51]

The composition of the water, this parenteral doses of Hamm in a way that, for example, intravenous dose, administered within the abdominal cavity, subcutaneous doses of intramuscular doses, or you can take advantage of the local dose above apply or spray on the composition of water disease and how you can take advantage of these also are limited.

[52]

The above formulation for parenteral doses, for example, Hypodermic, intravenous or intramuscular injections such as injectable form; Left my injection method; Or is it possible to have inhaled through respiratory tract aerosol, spray can be the formulation for this shall not be limited. The above formulation as an injectable formulation composition of water stabilizer in order to have seen Hamm or mixed in water with a buffer solution or suspension, as manufacturing and ampoules (ampoule) or vial (vial), unit dose formulation for can be. The above aerosol, spray formulation for sign language can be distributed, if the concentrate or moist powder dispersion, such as additives and propellant so that it can be the formulation together.

[53]

This may remind you of an Admiral firing prevention of infectious diseases caused by e. coli or therapeutic composition for water suitable for coating, spray or dose is administered, how the composition of the water, the above formulation was administered, and time, and sign language and/or route, of course, the age of the animals which are the subject of a dose, weight, gender, degree of disease symptoms, such as food, intake of excretion rate can be varied by a factor, usually skilled vet care for the purpose of effective dose that facilitates the decision and can prescribe.

[54]

[55]

According to another, this invention dimension, the aforementioned phage Φ C J 27 active ingredients include antibiotics.

[56]

This is a term used in the invention "antibiotic" means, pharmaceutical form is provided to animals including humans, bacteria can kill, and formulation with the efficacy of the preservative, disinfectant and antibacterial umbrella concept.

[57]

This may remind you of Bacteriophage Φ C J 27 active ingredients include antibiotics as compared to the conventional antibiotics Admiral firing very high specificity for e. coli in the ripe fungus is only a particular pathogens without killing death-inducing drug tolerance, and not conventional antibiotics compared to extended product life new has the advantage that can be used with antibiotics.

[58]

[59]

According to another, this invention dimension, the aforementioned phage Φ C J 27 active ingredients containing feed additives or drinking water additives.

[60]

This may remind you of the feed additives or drinking water additives are reminded of Bacteriophage Φ C J 27/composition containing feed additives or drinking water additives in the form of a separately manufactured feed or drinking water, or mixed in a way that reminded of Bacteriophage Φ C J 27 or the containing composition feed or drinking water directly in the manufacture of the additive can be used in a manner that.

[61]

[62]

This invention of the above feed additives or drinking water additive that is used as the aforementioned phage Φ Φ C J J C 27 or above, phage containing the composition of water is liquid or 27 dry can be, for example, can be in the form of dry powder.

[63]

For example, the invention of Bacteriophage Φ C J 27 is in powder form feed additives of 0.1 0.05 to 10 weight percent, specifically through 2 weight% can be blended into seongbunbi.

[64]

This may remind you of the feed additives or drinking water additives to manufacture in the form of dry powder dry method does not specifically limit in the technology field, normally you can use how to use. The above examples of how dry the nonrestrictive, spray drying, ventilation drying, natural drying, freeze drying, etc. They are used alone or with two or more methods can be carried out in such a way that.

[65]

This may remind you of the feed additives or drinking water additives and other non-hospital in the province can be further additions to the microorganisms. The above examples of micro-organisms which can be superimposed nonrestrictive, protease and protease, geology and/or can produce the enzyme conversion per b. serve til Lys (Bacillus subtilis), and second, such as sterilization; Miracle of the anaerobic conditions of the above physiological active and having a resolution of organic materials, lactobacillus b. strain (Lactobacillus sp.) Such as lactic acid bacteria; Increases the weight of cattle milk, increase the rate of digestion and absorption of feed flow rate of the mountain, having the effect of the height of the Asperger's barns-près-duck party (Aspergillus oryzae), such as molds; And Mac and Mrs. levy in the age of (Saccharomyces cerevisiae) is made up of yeast, such as in the military can be chosen. They are used alone or mixed with two or more can be used.

[66]

This may remind you of Bacteriophage Φ C J 27 active ingredients containing feed additives or drinking water additives to add on an as-needed basis, other additives can contain. The above examples of available additives nonrestrictive, feed or drinking water in order to prevent the loss of quality of the lodge added to paints, emulsifiers, preservatives, etc.; Feed or drinking water additive to increase the benefits of amino acids, vitamins, enzymes, flavours, fragrances, non-protein in Bacillus Tae nitrogen compounds, silicate, buffering agents, colorants, such as my or oligosaccharide extract and, in addition, you can include additional fodder mixes, etc. They are used alone or can be added with two species.

[67]

This invention of the above feed additives feed 100 weight Division, containing 0.05 to 10 weight-part can be, specifically, contains 0.1 to 2 weight Division. This invention of the above drinking water additives are about 100 drinking water weight bag, O.0001 or as containing 0.01 weight can be 0.001 to 0.005, specifically in the weight Department can be high. Admiral firing within the scope of the above e. coli Bacteriophage Φ about C J 27 has the advantage of being active enough to demonstrate.

[68]

[69]

According to another, this invention dimension, the aforementioned phage Φ C J 27 active ingredients containing feed additives or feed additives, drinking water or drinking water or added to, the aforementioned phage Φ C J 27 feed or drinking water by adding directly to the manufactured feed or drinking water should provide

[70]

This dimension is used in the feed does not specifically limit in the technology field, normally used for feed can be used. The above examples, nonrestrictive grains of feed, logistics, food processing, pharmaceutical logistics, birds, fibrous Busan Busan, logistics, maintenance, starch, vegetable, such as plants or grain feed for Busan logistics; Protein, weapons and logistics, maintenance, mineral, maintenance, single-cell protein, such as food and animal feed, middle or zooplankton. They are used alone or mixed with two or more can be used.

[71]

This dimension is used in drinking water does not specifically limit in the technology field, commonly used in drinking water can be used.

[72]

[73]

According to another, this invention dimension, the aforementioned phage Φ C J 27 active ingredients, which include disinfectants or cleansers with. Formulation of the above disinfectants or cleansers specifically does not limit, the technology sector has been manufactured as known formulations can be used.

[74]

The above disinfectants are Admiral firing in order to get rid of the e. coli can be sprayed the animal's activities on the territory, slaughterhouse, our area, cooking, cooking facilities, etc., or can be sprayed shall not be limited.

[75]

The above have been exposed to e. coli intestinal cleanser poison plastic is likely to be exposed to the animal's skin, such as a surface or body wash each part can be used, but shall not be limited.

[76]

[77]

According to another, this invention dimension, the aforementioned phage Φ Φ C J J C 27 or above, phage containing the active ingredient composition 27 can use Zhang poison plastic water e. coli by preventing or curing infectious diseases provides a way to.

[78]

How to cure or prevent a reminder of this invention specifically, have been infected by e. coli infection chapter reading, there is a risk of ectopic human phage Φ above the object, except for C C J J 27 or above, phage Φ as the active ingredient composition, including 27 million the amount of doses available to the geopolitical stage. Enterobacteria phage Φ J C 27 or above, containing a total of 1, suitable for usage of water composition is correct to the extent determined by the physician in a medical judgment can be a person who has knowledge of the relevant technical field you're in commerce is self-evident.

[79]

Enterobacteria phage Φ C above a certain animal for phage Φ J J C 27 of 27 or above, with the active ingredient composition of water, including the amount available, specific medicine want to achieve geopolitical reaction types and to an extent, that object's age, body weight, general health status, gender or expression, of course, the aforementioned phage Φ C J 27 doses of water containing the composition or the time, route and water ratio, the treatment period can be determined by taking into account and, at the same time, or used in conjunction with the drug at the time of composition, including the elements of water and several other drivers and is a well known in the field of Medicine of similar arguments can be varied according to the.

[80]

This may remind you of Bacteriophage Φ Φ C J J C 27 or above, phage containing the active ingredient composition 27 water sprayed on the animal in the form of expression about the forensic formulation nasal dose, or animal feed or drinking water is added directly to the eating can be administered in a manner which, in the form of feed additives or drinking water additives are mixed in feed or drinking water can be administered.

[81]

This may remind you of Bacteriophage Φ Φ C J J C 27 or above, phage containing the active ingredient composition 27 water route and dose approach is not limited to, specifically the organization mentioned above for the purpose of phage Φ C J 27 or the containing composition, this arbitrary route and dose which can be reached on the way you can follow. In other words, the aforementioned phage Φ Φ C J J C 27 or above, phage containing the active ingredient composition 27 water oral or parenteral doses throughout the various paths of the sphere can be, he was administered an example, oral, rectal, nonrestrictive route topical, intravenous, and intramuscular arteries, celiac my, my, my, or transdermal, non-side will be administered through inhalation.

[82]

[83]

This, for example through implementation details rather than this invention. However, these implementation examples are described in this notice to the poetic invention this invention is limited to the scope of these implementation examples.

[84]

[85]

[Implementation example 1]-Admiral firing an infected e. coli Bacteriophage separation

[86]

[87]

Examples of conduct < 1-1 >

[88]

Remove a single phage Bacteriophage screening and

[89]

Chungcheong hongseong County mineral area Yang Jong-farm region samhwa money fecal and environmental samples can be filled from ㎖ to 4,000 rpm for 10 minutes on 50 sample centrifugation and then to 0.45 ㎛ filter filtration equality by preparing the sample fluid with soft baby overlays (soft agar overlay) methods. The aforementioned soft baby how Iran top-PUD overlay (top-agar, 0.7% agar attach on top of a solid using the badge) that grow in host cells using a Bacteriophage is a fungus for observing how says.

[90]

Specifically, konkuk University Veterinary chapters separated from the poison plastic Escherichia coli (UK27) binge culture (OD600 = 2) 150 LB ㎕ and 10 x badge (tryptophan (tryptone) 10 g/ℓ ℓ yeast extract 5 g/;; NaCl 10 g/ℓ) 2 ㎖ the above sample filtrate 18 ㎖ mixed and 30 ° c during 18 hours of culture from the culture fluid above 4,000 rpm, at 10 minutes centrifugation to 0.45 ㎛ filter filtration fluid that equality. Then, LB flat badge above 0.4% (w/v) agar 5 ㎖ and Admiral firing Escherichia coli (UK27) binge culture (OD600 = 2) 150 ㎕ of the mixture and pour in the liquid sample above, over the entrenched, he is an enemy and 30 ° c 10 ㎕ 18 hours and half to form the sterilization for culture medium.

[91]

[92]

One of a kind of fungal half for phage known to exist, because there is a single from the aforementioned fungi for half-formed phage removing. Specifically, the above 400 SM solutions of ㎕ (half of the fungus for NaCl 5.8 g/ℓ; MgSO47H2O 2 g/ℓ; 1M Tris-Cl (pH 7.5) 50 ㎖) and 4 hours at room temperature to the enterobacteria phage solution accomplishes. Subsequently, the aforementioned phage solution 100 ㎕, 0.4% (w/v) agar 5 ㎖ and Admiral firing Escherichia coli (UK27) binge culture (OD600 = 2) 150 LB 150 mm diameter ㎕ and mixed and flat badge has performed a soft sweetheart using overlay method, sterilization for culture until it completely. After the end of the cultivation, the aforementioned LB flat badge and 5 SM of the ㎖ solution at room temperature for 4 hours as far as the number of phage solution.

[93]

Recall the above solution, 1% (v/v) to chloroform, and then for 10 minutes to mix and 4,000 rpm by centrifugation for 10 minutes as the master output as far as you can, and be subject to, such as the aforementioned award 0.45 ㎛ filter filtration can be end-use as far as samples.

[94]

[95]

Example 1-2 conducted < >

[96]

The bulk of the cultured and refined phage

[97]

The above example can be filled in with a 1-1 Bacteriophage conducted Jean poison plastic Escherichia coli using bulk (UK27) cultivation and refining from the phage.

[98]

[99]

Specifically, Admiral firing Escherichia coli (UK27) binge culture to 1.5 x 1010 cfu in the on-the-fly so that the 4,000 rpm for 10 minutes after centrifugation, 4 SM solution at-rich ㎖. Here's a reminder that Bacteriophage 1.5 × 107 pfu by inoculation and MOI (multiplicity of infection) = 0.001, then titrated with a 20-minute politics at room temperature. Subsequently, this 150 LB badge in the 30 ° c inoculate ㎖ 5 hours culture.

[100]

After the exit, the final volume of the cultivation of 1% (v/v) chloroform and 20 minutes stirring RNase DNase I enzyme and A limit has to be a final concentration of 1 ㎍/㎖, respectively, and 30 ° c during 30 minutes in politics. Then, the final concentration of each 1M and 10% (w/v) sodium chloride and polyethylene glycol to 4 ° c for three hours in an additional 12,000 rpm at 4 ° c, as the political representation for twenty minutes the water was suddenly settled by centrifugation.

[101]

Be filled above the sediment suspension in a solution of 5 SM ㎖ and politics at room temperature and then for twenty minutes, 1 ㎖ chloroform the agitation and 4,000 rpm for 20 minutes at 4 ° c by centrifugation to equality as far as you can. 0.45 ㎛ filter, such as the aforementioned award amount and then glycerol density gradient (density: 40%, 5% glycerol) second centrifugation (35,000 rpm, 1 hr, 4 ° c) refining the phage.

[102]

[103]

This is the amount of money the inventor minutes take a sample, from Admiral firing Escherichia coli for having to kill the enemy above unusual phage "Bacteriophage Φ C J 27" naming, and October 25, 2013, South Korea microbial Conservation Center (Korean Culture Center of Microorganisms, 1-Dong, Seodaemun-Ku, Seoul hongje 361-221) deposited no. KCCM11465P was deposited by an arc.

[104]

[105]

Conducted by the < example 2 >

[106]

J C 27 forms of observation Φ

[107]

(4) in enterobacteria phage Φ C example 1 carried out above J 0.01% gelatin solution diluted to 27 and 2.5% glue tar aldehyde (glutaraldehyde) solid solution. This carbon coated MICA plate (carbon-coated mica plate (ca. 2.5 mm mm×2.5)) is a 10-minute adaptation of the enemy, and then washed with distilled water, sterile. Carbon coating (carbon film) into the grid (grid copper) copper 2% URA Neil acetate (uranyl acetate) in 60 seconds-dyeing and drying, transmission electron microscope (JEM-1011, 120 kV, magnification x 200,000), Speculum (Figure 1).

[108]

Figure 1 is a Bacteriophage Φ C transmission electron microscope photos of 27 J as shown, long hair (elongated head) and a tail (contractile tail) contraction, consisting of forms as indicative of mold (morphotype) Mai o irregularities (Myoviridae) belongs to.

[109]

[110]

Conducted by the < example 3 >

[111]

Φ C J, the size of the DNA analysis of the entire genome of 27

[112]

(4) in enterobacteria phage Φ C example 1 carried out above the J genome DNA extraction from 27.

[113]

Specifically, refined phage Φ C in 20 mM J 27 culture EDTA (Ethylenediaminetetraacetic acid), 50 ㎍/㎖ protease (proteinase) K and 0.5% (w/v) SDS (sodium dodecyl sulfate) and 50 ° c is the same as for the 1st time in politics, and volume of phenol (pH 5.0) and the stirring, and then a 10-minute 12,000 rpm at room temperature centrifugation to the equality as far as you can.

[114]

The same volume of fluid, such as the aforementioned award, PC (phenol: chloroform = 1:1) and 12,000 rpm at room temperature mixed and 10 minutes centrifugation to the equality as far as you can. The above equality is the same volume of fluid mixed with chloroform and 12,000 rpm at room temperature 10 minutes centrifugation to the equality as far as you can. The above equality in 3M acetic acid sodium (sodium acetate) 10% of the total volume (v/v) to the mix, and 95% ethanol with twice the volume of the mixture of cold and then, for the 1st time in politics-20℃. 12,000 rpm for 10 minutes at 0 ° c, followed by centrifugation and then master output can be filled by removing sediment, and then 50 ㎕ TE buffering (Tris-EDTA, pH 8.0) by melting. DNA is extracted from the above 10 x dilution in the OD260measures the concentration by measuring the absorbance.

[115]

Then, 1 ㎍ 1% of DNA for PFGE (pulse-field gel electrophoresis) gel loading, AGA Los Bai long (BIORAD) PFGE System 7 program (25 kb or 100 kb size range; switch time lamp (switch time ramp) 0.4 seconds to 1.2 seconds, linear (linear shape); forward voltage (forward voltage) 180 V; reverse voltage (reverse voltage) 120 V) was deployed during the 20 hours at room temperature (2).

[116]

2 C DNA genome of the phage Φ J 27, are enterobacteria phage Φ electrophoresis photo of C J 27 represents the DNA genome of 98 more were able to confirm kb. Also from 2 M is the standard of measurement of the size of the DNA.

[117]

[118]

Conducted by the < example 4 >

[119]

Φ C J 27 protein pattern analysis

[120]

1011 ㎖ pfu/Titus (titer) refined phage Φ C J 5 × SDS sample ㎕ solution 15 27 solution 3 boiled for 5 minutes then mix and ㎕, 12% SDS-PAGE performed (Figure 3).

[121]

Also 3 Bacteriophage Φ J C performed in SDS-PAGE that represents the results to target the 27 electrophoresis photo, about 27.9 kDa, 51.8 kDa and 74.5 kDa were able to confirm the size of the key proteins.

[122]

[123]

Examples of conduct < 5 >

[124]

Genetic characterization of Φ J C 27

[125]

(4) in enterobacteria phage Φ C example 1 carried out above J 27 never learn the characteristics of genes, DNA, gene of Bacteriophage Φ C J 27 analysis equipment FLX titanium sequencer (the sequencer titanium) (Roche). (C) macrogen (Macrogen Inc.) GS and de novo Assembly in software (de novo assembler software) (Roche) uses the combination of genes. Warrior (open reading frame) is interpreted GeneMArk.hmm, Glimmer v3.02 and FGENESB was performed using the software. BLASTP and inter-Pro scan (InterProScan) using the program interprets the frame named the Warriors (annotation).

[126]

The above sequence of Bacteriophage are reported in the phage (Enterobacteria phage HX01, complete genome) sequence and a variety of polarization of the similarities, all 100% match fragments phage was determined to be no. This is a new detached the aforementioned phage Bacteriophage was able to confirm.

[127]

[128]

To table 1 Bacteriophage Φ J 27 C of the sequence and the other enterobacteria phage Detox base shows the comparison of orthology to the column.

[129]

[130]

Table 1 [table 1]
Query Subject 　 Identities
Name Length Start End Description E-Value Match/Total Pct.(%)
Sequence number 1 171637 144696 167103 Enterobacteria phage HX01, complete genome 0 21905/22425 97

[131]

Enterobacteria phage Φ C J manufactured 27 above, DNA, gene analysis equipment, analysis of the sequence as a whole by using the column list in the results column number 1.

[132]

[133]

<실시예6>

[134]

PH C Φ J 27 according to the reliability of the survey

[135]

Enterobacteria phage Φ C J 27 gastric pH as low as in the conditions within the reliability in order to determine whether it can hold a variety of pH range (pH 2.0, 3.0, 4.0, 5.0) reliability in surveys conducted an experiment.

[136]

In order to experiment with various pH solution (sodium acetate buffer solution (pH 4.0 and pH 5.0), sodium citrate buffer solution (pH 2.0 and pH 3.0) produced by each of 0.2 M.

[137]

Above each pH solution 180 ㎕ and 2.4 × 109 ㎖ PFU phage solution of 20 ㎕/Titus mix makes the concentration of each solvent pH 1M, for 2 hours at room temperature in politics. Contrast with the military in the same way as 2.4 × 109 PFU/㎖ enterobacteria phage solution mix to 20 SM solution ㎕ gave ㎕ after 180, for 2 hours at room temperature in politics. Next, dilute and how those steps can use a soft sweetheart overlay each stage drops back by 10 diluent ㎕ 30 ° c for 18 hours, to cultivate the bacteria was measured over whether Titus (4).

[138]

Also 4 Bacteriophage Φ C J 27 of my acid test results. 4, as shown in, in comparison to the control phage Φ C pH pH from 5.0 to 2.0 J 27 active were able to confirm that the stable without losing.

[139]

[140]

Examples of conduct < 7 >

[141]

The temperature stability of Φ C J 27 according to survey

[142]

Enterobacteria phage product formulation of feed additives in the process of formulation of the Bacteriophage as heat may cause heat to determine the stability of the experiment performed.

[143]

Specifically, 2.4 × 108 PFU phage Φ C J 27/㎖ concentration of solution temperature 60 ° c 200 ㎕ to 0 minutes, under conditions, 30 minutes, 60 minutes and 120 minutes of politics. Then, the above processing experiment to cultivate a soft sweetheart overlay way dilute the steps each step down followed by 30 ° c diluent 10 ㎕ 18 hours over whether the sterilization for culture medium Titus measurement (5).

[144]

5 heat resistance of the phage Φ C J 27 shows the experimental results. 5, as shown in, the phage Φ C to 60 ° c during one hour in J 27 I have been exposed to active until about one log reduction in the exposure of 120 minutes, a reduced need to be active for at least 1 log.

[145]

[146]

< Conducted for 8 >

[147]

Remove the main chapter of the wild poison plastic Escherichia coli infection of Φ for C J 27 range survey

[148]

Admiral plastic used to experiment with Escherichia coli in addition to medical school, Seoul National University (UK27), konkuk University School of medicine and quarantine in the House of representatives to remove the State chapter of the wild poison plastic removing e. coli Bacteriophage Φ C J 99 weeks, against 27 for the bacteria to determine whether the active. This separation gives F-serotype F4 F5 type the type (type) 37 weeks, 31 weeks, 7 weeks, F18 F6 type type is 5 weeks 19 weeks and other bacteria.

[149]

Specifically, each sterilization caution binge culture (OD600 = 2) 150 ㎕ to mix soft baby overlay can proceed 109 ㎖ pfu phage Φ of C J Titus/27 drop back by 30 ° c 10 ㎕ solution at 18 hours culture and observe the presence of the fungus, for half-formed.

[150]

To the above results indicated in table 2.

[151]

[152]

Table 2 [table 2]

[153]

[154]

[155]

As indicated in table 2 above, phage Φ J 27 C is a general good causes of diarrhea as a pig money farmers in a significant portion of the F-F6, F5, F4, serotype F18 seems to have infected about the type field are expected to represent a superior efficiency.

[156]

[157]

The list of columns in the documents submitted by attaching

[158]

The scope of a claim

[Claims]

Admiral plasticity of Escherichia coli (Enterotoxigenic Escherichia coli) having to kill specific, new phage Φ C J 27 (KCCM11465P).

[Billing section 2]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P), which includes the active ingredient, Admiral firing due to e. coli prevention of infectious diseases or therapeutic composition.

[Billing section 3]

In paragraph 2 of the aforementioned Admiral the Escherichia coli infectious diseases caused by e. coli plasticity witnesses, infectious diseases for treatment or prevention of the composition.

[Billing section 4]

Under paragraph (1) above, phage Φ C J 27 (KCCM11465P) as the active ingredient, including antibiotics.

[Billing section 5]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P), which includes the active ingredient, feed additives.

[Claim 6]

Paragraph (5) feed additives, animal feed.

[Claim 7]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P), which includes the active ingredient, drinking water additives.

[Billing section 8]

7 protest drinking water additives, drinking water.

[Claims 9]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P), which includes the active ingredient, hand Sanitizer.

[Claims 10]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P), which include cleaners, as the active ingredient.

[Billing section 11]

Under paragraph (1) above, phage Φ J 27 C (KCCM11465P) or paragraph 2 above with the exception of humans, animals, water composition for administering Admiral, including steps for firing due to infectious diseases of Escherichia coli prevention or treatment methods.

[Billing section 12]

In section 11 above, infectious diseases is Escherichia coli Escherichia coli is one of the witnesses, Admiral plasticity in the prevention of infectious diseases or treatment methods.