KR20180103788A - Novel Pseudomonas aeruginosa bacteriophage Pse-AEP-3 and its use for preventing proliferation of Pseudomonas aeruginosa - Google Patents

Abstract

The present invention relates to grape beetle versus bacteriophage Pse-AEP-3 (Accession No. KCTC 13165BP) isolated from nature, having the ability to specifically kill Pseudomonas aeruginosa and having the genome of SEQ ID NO: 1, and And a method for preventing or treating a disease caused by P. aeruginosa using the composition containing the same as an effective ingredient.

Description

Novel Pseudomonas aeruginosa bacteriophage Pse-AEP-3 and its use for preventing proliferation of Pseudomonas aeruginosa}

The present invention relates to a bacteriophage isolated from nature capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa and a method for preventing or treating infection of Pseudomonas aeruginosa using a composition containing the same as an active ingredient, and more particularly, to kill Pseudomonas aeruginosa. Pseudomonas aeruginosa using a composition comprising the bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) isolated from nature and having a genome represented by SEQ ID NO: 1, and the bacteriophage as an active ingredient. It relates to infection prevention and post-infection treatment methods.

Pseudomonas aeruginosa ) is a gram-negative bacillus that is commonly present in the natural environment, but is an opportunistic infectious bacterium that can cause sepsis in people with reduced immunity due to surgery, burns, trauma, and chemotherapy treatment. Pseudomonas aeruginosa is known as a major strain of nosocomial infection and is a causative agent of various diseases such as endocarditis, pneumonia, and meningitis. In particular, in the case of cystic fibrosis patients, Pseudomonas aeruginosa infection is very fatal, and it is known that infection with this bacterium in infants and toddlers can seriously affect lung function.

In general, vaccines and antibiotics are mainly used for the prevention and treatment of infectious diseases of Pseudomonas aeruginosa, but treatment with antibiotics is a situation where the effect continues to decline due to the increase in resistant bacteria. Accordingly, there is an urgent need to develop a drug that can be used to prevent or treat infection of antibiotic-resistant Pseudomonas aeruginosa.

Recently, the use of bacteriophage as a countermeasure against bacterial diseases is receiving great attention. In particular, it is getting more attention because of its excellent antibacterial activity against antibiotic-resistant bacteria. Bacteriophages are very small microorganisms that infect bacteria and are usually shortened to phage (Phage). Bacteriophages have the ability to kill bacteria by proliferating inside the bacterial cells after infection with bacteria, and destroying the cell wall of the host bacteria when progeny bacteriophages come out of the bacteria after proliferation. The bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific bacteriophage can infect only a specific category of bacteria, and due to this, a specific bacteriophage can provide an antibacterial effect only for a specific bacterium, thereby killing only a specific bacterium and not affecting other bacteria. The bacterial specificity of such a bacteriophage provides an antibacterial effect only against the target bacteria and does not affect the environment or flora in animals. Conventional antibiotics, which have been widely used in the treatment of bacteria, have an effect on several types of bacteria at the same time. This caused problems such as environmental pollution or disturbance of the normal bacterial flora of animals. Unlike this, bacteriophages work only against specific bacteria, so the use of bacteriophages does not cause disturbances in the body's normal flora. Therefore, it can be said that the use of bacteriophage is very safe compared to the use of antibiotics, and the possibility of causing side effects by using it is relatively low.

Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer. In addition, in 1917, d'Herelle, a French bacteriologist, discovered that there was an action to dissolve Shigella dysenteriae in the filtrate of a person with a disease. Through this study, he independently discovered bacteriophage and eats the bacteria. It was named bacteriophage in the sense of. Since then, bacteriophages against various pathogenic bacteria such as Shigella, typhoid, and cholera have been continuously discovered.

Due to its special ability to kill bacteria, bacteriophage has been expected to be an effective countermeasure against bacterial infection since its discovery, and there have been many studies related to it. However, after the discovery of penicillin by Fleming, as the spread of antibiotics became common, research on bacteriophage was limited to some Eastern European countries and the former Soviet Union. However, after 2000, due to the increase in antibiotic-resistant bacteria, the limitations of existing antibiotics appeared, and the possibility of development as a substitute for the existing antibiotics emerged. As a result, bacteriophage is again attracting attention as an anti-bacterial agent.

As described above, bacteriophage has very high specificity for bacteria. Due to this specificity, bacteriophages often exert antibacterial effects only against some strains, even if they belong to the same species. In addition, the strength of the antibacterial activity itself of bacteriophage exerted according to the target bacterial strain may be different. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to secure an effective control method for certain kinds of bacteria. In order to develop an effective bacteriophage utilization method in response to Pseudomonas aeruginosa, of course, it is necessary to secure various types of bacteriophages that can provide antibacterial effects against Pseudomonas aeruginosa. It is also necessary to select and utilize bacteriophage, which has a comparative advantage.

Accordingly, the present inventors developed a composition that can be used to prevent or treat infection of Pseudomonas aeruginosa using a bacteriophage isolated from nature capable of killing Pseudomonas aeruginosa, and also use this composition to prevent infection of Pseudomonas aeruginosa or After trying to develop a method of treatment, a composition comprising the bacteriophage as an active ingredient after securing the gene sequence of the genome so that the bacteriophage suitable for this can be separated from nature and the separated bacteriophage can be differentiated from other bacteriophages to be specified. After developing, the present invention was completed by confirming that the composition can be effectively used for preventing or treating Pseudomonas aeruginosa.

Therefore, it is an object of the present invention to have a genome represented by SEQ ID NO: 1 having the ability to kill Pseudomonas aeruginosa ( Podoviridae ) bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) isolated from nature Is to provide.

Another object of the present invention is a composition that can be used to prevent infection of Pseudomonas aeruginosa containing as an active ingredient the isolated bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa and this composition It is to provide a method of preventing infection by Pseudomonas aeruginosa using.

Another object of the present invention is a composition that can be used to treat diseases caused by Pseudomonas aeruginosa containing as an active ingredient the isolated bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa And it is to provide a method for treating a disease caused by Pseudomonas aeruginosa using the composition.

Another object of the present invention is a pharmaceutical composition for the purpose of preventing or treating Pseudomonas aeruginosa infection using compositions containing as an active ingredient the isolated bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa. To provide.

Another object of the present invention is to provide a disinfectant comprising an isolated bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa as an active ingredient. This disinfectant is particularly effective in preventing nosocomial infections.

In addition, another object of the present invention is to provide an antibiotic comprising an isolated bacteriophage Pse-AEP-3 (accession number KCTC 13165BP) capable of killing Pseudomonas aeruginosa by infecting Pseudomonas aeruginosa as an active ingredient.

The present invention is characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill Pseudomonas aeruginosa, Pse-AEP-3 (accession number KCTC 13165BP) isolated from nature, and It provides a method of preventing infection or post-infection of Pseudomonas aeruginosa using a composition containing this as an active ingredient.

Bacteriophage Pse-AEP-3 was separated by the present inventors and deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on November 29, 2016 (accession number KCTC 13165BP).

In addition, the present invention provides a pharmaceutical composition comprising a bacteriophage Pse-AEP-3 that can be used to prevent or treat infection of Pseudomonas aeruginosa as an active ingredient. As an example of the pharmaceutical composition, a disinfectant or an antibiotic may be presented, but it is obvious that the present invention is not limited thereto.

The bacteriophage Pse-AEP-3 contained in the composition of the present invention effectively kills Pseudomonas aeruginosa, so it is effective in preventing (preventing infection) or treating (infection treatment) diseases such as urinary tract infections, wound infections, bacteremia, and endocarditis caused by Pseudomonas aeruginosa. Represents. Therefore, the composition of the present invention can be used for the purpose of preventing and treating diseases caused by Pseudomonas aeruginosa. Diseases caused by Pseudomonas aeruginosa in the present specification collectively refer to urinary tract infections, wound infections, bacteremia, endocarditis, and the like.

Pseudomonas aeruginosa in the present specification may be sensitive to existing antibiotics or may have resistance to existing antibiotics. In other words, it is irrelevant whether or not resistance to existing antibiotics is acquired.

The term'prevention' or'prevention' as used herein means (i) prevention of Pseudomonas aeruginosa infection; And (ii) means to inhibit the development of a disease caused by Pseudomonas aeruginosa infection.

The term'treatment' or'treatment' as used herein refers to (i) inhibition of diseases caused by Pseudomonas aeruginosa; And (ii) all actions of alleviating the pathological condition of a disease caused by Pseudomonas aeruginosa.

"Separated", "separated" or "separated" in the present specification refers to separating a bacteriophage from a natural state by using various experimental techniques and separating a bacteriophage from other bacteriophages to secure a characteristic capable of specifying the bacteriophage of the present invention. It refers to, and in addition to this, it includes proliferating the bacteriophage of the present invention to be industrially utilized by biotechnology.

Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but are not limited thereto. . The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The composition of the present invention can be used as a method of applying or spraying to a diseased area, and may be administered by oral administration or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration , Subcutaneous administration or topical administration may be used.

Suitable application, spray and dosage of the pharmaceutical composition of the present invention include the method of formulation, mode of administration, age, weight, sex, severity of disease symptoms, food, administration time, route of administration, excretion rate, and It varies depending on factors such as response sensitivity, and usually an experienced physician or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

The composition of the present invention includes bacteriophage Pse-AEP-3 as an active ingredient. The bacteriophage Pse-AEP-3 contained at this time is 1×10 ¹ pfu/ml to 1×10 ³⁰ pfu/ml or 1×10 ¹ pfu/g to 1×10 ³⁰ pfu/g, preferably 1× 10 ⁴ pfu/ml to 1×10 ¹⁵ pfu/ml or 1×10 ⁴ pfu/g to 1×10 ¹⁵ pfu/g.

The composition of the present invention is prepared in a unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can also be manufactured by putting it inside a multi-volume container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The composition of the present invention may be implemented with a disinfectant or antibiotic, but is not limited thereto, depending on the method of use. In the present specification, the term “antibiotic” refers to preservatives, fungicides and antibacterial agents.

Bacteriophages capable of providing antimicrobial activity against other bacterial species may be added to the composition of the present invention in order to increase the efficiency for this purpose of use. In addition, other types of bacteriophages having antibacterial activity against Pseudomonas aeruginosa may also be added. Even bacteriophages having antibacterial activity against Pseudomonas aeruginosa are different from each other in terms of the strength of the antibacterial activity or the range of antibacterial activity, so an appropriate combination of them can maximize the effect.

The method of preventing or treating Pseudomonas aeruginosa using a composition containing the bacteriophage Pse-AEP-3 of the present invention as an active ingredient can provide an advantage of having very high specificity for Pseudomonas aeruginosa compared to methods based on conventional antibiotics. This means that it can be used for the purpose of preventing or treating Pseudomonas aeruginosa without affecting other useful flora, and it means that there are very few side effects from its use. In general, when antibiotics are used, common organisms are also damaged, resulting in various side effects. On the other hand, bacteriophages have the same antibacterial activity, even if they have the same antibacterial activity, in terms of the strength of the antibacterial activity and the range of antibacterial properties (in terms of various strains belonging to Pseudomonas aeruginosa), the antimicrobial activity of the bacteriophage is exerted against individual strains. range. Typically, bacteriophage can exert antibacterial activity against some strains belonging to the same bacterial species. That is, even if they belong to the same bacterial species, there may be differences in susceptibility to bacteriophages according to individual bacterial strains], so the present invention can provide a differential antibacterial effect compared to other bacteriophages having antibacterial activity against Pseudomonas aeruginosa. have. This provides a big difference in its effectiveness when used in industrial sites.

1 is an electron micrograph of the bacteriophage Pse-AEP-3.
Figure 2 is an experimental result showing the killing ability of Pseudomonas aeruginosa of the bacteriophage Pse-AEP-3. The transparent part is the lysis plaque formed as a result of lysis of bacteria.

Hereinafter, the present invention will be described in more detail based on examples, but these examples are only examples of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Isolation of bacteriophage that can kill Pseudomonas aeruginosa

Samples secured from the natural environment were used to isolate the bacteriophage that can kill Pseudomonas aeruginosa. On the other hand, Pseudomonas aeruginosa used for separation of bacteriophage are those that were previously isolated by the present inventors and identified as Pseudomonas aeruginosa.

When the bacteriophage separation process is described in detail, TSB ( T ryptic S oy B roth) medium (casein digest, 17 g/L; Soybean digest, 3 g/L; dextrose, 2.5 g) inoculated with Pseudomonas aeruginosa at a 1/1000 ratio /L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) were added together, followed by shaking culture at 37° C. for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. Pseudomonas aeruginosa was inoculated into the recovered supernatant at a ratio of 1/1000, and then cultured again with shaking at 37°C for 3-4 hours. When bacteriophage was included in the sample, this process was repeated a total of 5 times so that the number of bacteriophages (Titer) could be sufficiently increased. After repeating 5 times, the culture solution was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. It was investigated whether there is a bacteriophage capable of killing Pseudomonas aeruginosa through a usual spot assay using the filtrate thus obtained.

The instillation experiment was conducted as follows. Pseudomonas aeruginosa was inoculated in TSB medium at a ratio of 1/1000, and then cultured with shaking at 37°C for one night. 3 ml of the culture solution of Pseudomonas aeruginosa prepared in this way (OD ₆₀₀ is 1.5) was added to TSA (T ryptic S oy A gar) plate medium (casein digest, 15 g/L; Soybean digest, 5 g/L; NaCl, 5 g/L) ; Spreading was carried out on agar, 15 g/L). The smeared plate medium was left on a clean bench for about 30 minutes to allow the smear liquid to dry. After drying, 10 μl of the previously prepared filtrate was dropped onto the plate medium plated with Pseudomonas aeruginosa. It was left to stand for about 30 minutes and dried. After drying, the instilled flat medium was cultured at 37°C for one day, and then it was investigated whether a clear zone was formed at the location where the filtrate was removed. It can be determined that the filtrate in which the transparent ring is generated contains bacteriophage capable of killing Pseudomonas aeruginosa. Through this investigation, it was possible to obtain a filtrate containing bacteriophage having a killing ability against Pseudomonas aeruginosa.

Separation of pure bacteriophage was performed using a filtrate in which the presence of a bacteriophage having a killing ability against Pseudomonas aeruginosa was confirmed. For the separation of pure bacteriophage, a conventional plaque assay was used. To explain this in detail, one lysis plate formed in the lysis plate analysis was recovered using a sterilized tip, and then it was added to the Pseudomonas aeruginosa culture solution and incubated at 37°C for 4-5 hours. After incubation, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. Pseudomonas aeruginosa culture solution was added to the obtained supernatant in a volume of 1/50, and then cultured again at 37°C for 4-5 hours. In order to increase the number of bacteriophages, this process was performed at least 5 times, and then finally centrifuged at 8,000 rpm for 20 minutes to obtain a supernatant. Using the obtained supernatant, lysis was performed again. Since the separation of the pure bacteriophage is not usually achieved with only one of the above steps, the previous step was completely repeated again using the lysing plate formed at this time. This process was repeated at least 5 times to obtain a solution containing pure bacteriophage. Typically, the separation of pure bacteriophage was repeatedly performed until the size and shape of the formed lysis plate were all similar. And finally, it was confirmed whether the pure separation of the bacteriophage through electron microscopy analysis. The above-described process was repeated until pure separation was confirmed by electron microscopy analysis. Electron microscopy analysis was performed according to a conventional method. Briefly explaining this is as follows. A solution containing pure bacteriophage was buried on a copper grid, negative staining and drying were performed with 2% uranyl acetate, and the shape was observed through a transmission electron microscope. An electron microscope photograph of the purely isolated bacteriophage is presented in FIG. 1. When judging by the morphological characteristics, it was confirmed that the newly secured bacteriophage belongs to the bacteriophage Podoviridae.

The solution containing pure bacteriophage confirmed in this way went through the following purification process. Pseudomonas aeruginosa culture solution was added to a solution containing pure bacteriophage in a volume of 1/50 of the total volume of the solution, and then cultured again for 4-5 hours. After incubation, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. This process was repeated a total of 5 times to obtain a solution containing a sufficient number of bacteriophage. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter, and then a conventional polyethylene glycol (PEG) precipitation process was performed. Specifically, PEG and NaCl were added to 100 ml of the filtrate to become 10% PEG 8000/0.5 M NaCl, and then allowed to stand at 4° C. for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. The thus obtained bacteriophage precipitate was suspended in 5 ml of a buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0). This is referred to as bacteriophage suspension or bacteriophage liquid.

Purified pure bacteriophage could be obtained through the above process, and this bacteriophage was named bacteriophage Pse-AEP-3, and then deposited with the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center on November 29, 2016 (accession number KCTC 13165BP). ).

Example 2: Bacteriophage Pse - AEP -3 genome isolation and sequence analysis

The genome of the bacteriophage Pse-AEP-3 was isolated as follows. For dielectric separation, a suspension of bacteriophage obtained in the same manner as in Example 1 was used. First, in order to remove DNA and RNA of Pseudomonas aeruginosa that may be contained in the suspension, 200 U of DNase I and RNase A were added to 10 ml of the bacteriophage suspension, and then left at 37°C for 30 minutes. To remove the activities of DNase I and RNase A after standing for 30 minutes, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added, and then allowed to stand for 10 minutes. And it was allowed to stand at 65°C for an additional 10 minutes, and then 100 μl of proteinase K (20 mg/ml) was added to disintegrate the outer wall of the bacteriophage, and then reacted at 37°C for 20 minutes. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added and then reacted again at 65° C. for 1 hour. After 1 hour reaction, 10 ml of a mixture of phenol: chloroform: isoamylalcohol having a composition ratio of 25:24:1 was added to the reaction solution, and then mixed well. Then, this was centrifuged at 13,000 rpm for 15 minutes to separate the layers, and then the upper layer was taken from the separated layers, and 1.5 volume ratio of isopropyl alcohol was added thereto, followed by centrifugation at 13,000 rpm for 10 minutes to separate the dielectric. Precipitated. After recovering the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried under vacuum, and dissolved in 100 μl of water. By repeating the above process, a large amount of the genome of the bacteriophage Pse-AEP-3 was secured.

Using the thus obtained genome, the genome of the bacteriophage Pse-AEP-3 was performed by using a Pac-bio instrument at the National Instrumentation Center for Environmental Management at Seoul National University. Sequence information was obtained. The finally analyzed bacteriophage Pse-AEP-3 genome has a size of 45,213 bp, and the entire gene sequence is shown in SEQ ID NO: 1.

Based on the obtained genome sequence of the bacteriophage Pse-AEP-3, using BLAST on the Web, we investigated the homology with the previously known bacteriophage genome sequence. As a result of BLAST investigation, the genome sequence of the bacteriophage Pse-AEP-3 was confirmed to have relatively high homology with the sequence of Pseudomonas aeruginosa bacteriophage PaP4 (Genbank Accession No. KC294142.1) (identity: 93%). However, while the bacteriophage Pse-AEP-3 has a circular genome, Pseudomonas aeruginosa bacteriophage PaP4 had a big difference in the genome shape with a linear genome. ), whereas the number of Pseudomonas aeruginosa bacteriophage PaP4 was 70, and had a different number of open reading frames than bacteriophage Pse-AEP-3.

Based on these facts, it could be concluded that the bacteriophage Pse-AEP-3 is a novel bacteriophage different from the previously reported bacteriophages. Along with this fact, from the fact that the strength of the antibacterial activity and the range of antibacterial activity that can be provided are different if the types of bacteriophage are different in general, it was determined that the bacteriophage Pse-AEP-3 can provide antibacterial effects different from other previously reported bacteriophages.

Example 3: Bacteriophage Pse - AEP Against Pseudomonas aeruginosa of -3 Mortality Research

The ability of the isolated bacteriophage Pse-AEP-3 to kill Pseudomonas aeruginosa was investigated. The killing ability was investigated in a manner of examining whether or not a transparent ring was generated through the drop experiment suggested in Example 1. Pseudomonas aeruginosa used for the investigation of killing ability was isolated by the present inventors and identified as Pseudomonas aeruginosa, and 1 strain distributed from ATCC (American Type Culture Collection) ( Pseudomonas aeruginosa ATCC 15692) for a total of 10 weeks. Bacteriophage Pse-AEP-3 had a killing ability for a total of 9 weeks including 1 strain distributed from ATCC out of 10 strains of Pseudomonas aeruginosa. Representative experimental results are presented in FIG. 2. On the other hand, the bacteriophage Pse-AEP-3 is Staphylococcus aureus (Staphylococcus aureus ), Pasteurella multocida (Pasteurella multocida), Clostridium perfringens (Clostridium perfringens ), Lactobacillus planta room ( Lactobacillus) plantarum ), Streptococcus uberis ) and Enterococcus faecalis ) was also investigated for killing ability, and as a result, the bacteriophage Pse-AEP-3 did not have killing ability for these species.

As a result of the above, it was confirmed that the bacteriophage Pse-AEP-3 has excellent killing ability against Pseudomonas aeruginosa and can exhibit antibacterial effects against a number of Pseudomonas aeruginosa. This means that the bacteriophage Pse-AEP-3 can be used as an active ingredient of a composition for the purpose of preventing and treating Pseudomonas aeruginosa infection.

Example 4: Bacteriophage Pse - AEP -3 for Pseudomonas aeruginosa infection prevention Experimental example

Put 100 μl of 1×10 ⁹ pfu/ml bacteriophage Pse-AEP-3 solution in one tube containing 9 ml of TSB medium, and the same amount of TSB medium in the other tube containing 9 ml of TSB medium. Further added. Then, a culture solution of Pseudomonas aeruginosa was added to each tube so that the absorbance at 600 nm was about 0.5. After Pseudomonas aeruginosa was added, the tubes were transferred to an incubator at 37° C. and cultured with shaking to observe the growth state of Pseudomonas aeruginosa. As shown in Table 1, the growth inhibition of Pseudomonas aeruginosa was observed in the tube to which the bacteriophage Pse-AEP-3 solution was added, whereas the growth inhibition of Pseudomonas aeruginosa was not observed in the tube to which the bacteriophage solution was not added.

Inhibition of Pseudomonas aeruginosa division OD ₆₀₀ absorbance value 0 min incubation 60 minutes after incubation 120 minutes after incubation No bacteriophage liquid added 0.5 0.7 1.6 Addition of bacteriophage liquid 0.5 0.3 0.1

From this result, it was confirmed that the bacteriophage Pse-AEP-3 of the present invention has the ability to not only inhibit the growth of Pseudomonas aeruginosa, but also kill Pseudomonas aeruginosa, from which the bacteriophage Pse-AEP-3 is a composition for the purpose of preventing infection of Pseudomonas aeruginosa. It could be concluded that it could be used as an active ingredient of.

Example 5: Bacteriophage Pse - AEP Infectious disease of Pseudomonas aeruginosa using -3 Treatment example One

The treatment effect of the bacteriophage Pse-AEP-3 in animals caused by Pseudomonas aeruginosa was investigated. Forty two-day-old chicks were divided into two groups and reared separately for 14 days. ^{Feed containing Pseudomonas aeruginosa at the level of 1×10 7} cfu/g for 3 days from the 5th day from the start of the experiment was fed in a conventional feed feeding method. Pseudomonas aeruginosa was identified in both groups in feces from the last day of feeding containing Pseudomonas aeruginosa. From the day after 3 days of feeding containing Pseudomonas aeruginosa (the 8th day from the start of the test) to chicks in the experimental group (bacteriophage administration group), 1×10 ⁸ pfu/g of bacteriophage Pse-AEP-3 was included. The feed was fed according to a conventional feed feeding method. On the other hand, the chicks of the control group (the bacteriophage non-administration group) were fed in the same manner as the feed of the same composition that did not contain the bacteriophage Pse-AEP-3. From the ninth day from the start of the test, the number of Pseudomonas aeruginosa in feces was measured for test animals. Pseudomonas aeruginosa selection medium (Pseudomonas Cetrimide agar plate; Oxoid) was used in the measurement of the number of Pseudomonas aeruginosa in this example to prevent interference by other contaminating bacteria. The sample was plated on a selective medium and incubated for 18 to 24 hours at 37°C. A method of identifying Pseudomonas aeruginosa through polymerase chain reaction (PCR) after selecting colonies presumed to be Pseudomonas aeruginosa from the cultured selective medium and separating them purely (colonies identified as Pseudomonas aeruginosa through polymerase chain reaction). If the number of ^{Pseudomonas aeruginosa is greater than 10 2} cfu/ml: 2, the number of colonies identified as Pseudomonas aeruginosa through polymerase chain reaction is 10 ¹ to 10 ² cfu/ml or more: 1, of colonies identified as Pseudomonas aeruginosa through polymerase chain reaction When the number is 10 ⁰ ~ 10 ¹ cfu/ml or more: 0). The results were shown in Table 2.

Pseudomonas aeruginosa count results (average) date D9 D10 D11 D12 D13 D14 Control (no bacteriophage administration) 1.1 1.0 1.2 1.2 1.1 1.3 Experimental group (bacteriophage administration) 0.2 0.2 0.1 0 0 0

From this result, it was confirmed that the bacteriophage Pse-AEP-3 of the present invention is also very effective in the treatment of diseases caused by Pseudomonas aeruginosa.

Example 6: Bacteriophage Pse - AEP Infectious disease of Pseudomonas aeruginosa using -3 Treatment example 2

The therapeutic effect of the bacteriophage Pse-AEP-3 on diseases caused by Pseudomonas aeruginosa was investigated as follows. Forty eight-week-old mice were divided into two groups, 20 mice per group, and the mice of each group were individually reared in a cage of mice for 7 days, and the experiment was conducted for 7 days. On the second day of the start of the experiment, 0.1 ml of Pseudomonas aeruginosa suspension was administered by intraperitoneal injection to all mice. The administered Pseudomonas aeruginosa suspension was prepared as follows. After culturing Pseudomonas aeruginosa at 37° C. for 18 hours using TSB medium, only the cells were recovered. The recovered cells were suspended in physiological saline (pH 7.2) to a concentration of 5×10 ^{9 cfu/ml.} Two hours after Pseudomonas aeruginosa administration, mice of the experimental group (bacteriophage solution administration group) were administered 10 ⁹ pfu of bacteriophage Pse-AEP-3 by intraperitoneal injection. The mice in the control group (non-administration group of bacteriophage solution) were administered 0.1 ml of physiological saline by intraperitoneal injection. Feed and drinking water were fed equally to both the control and experimental groups. From the administration of Pseudomonas aeruginosa to the end of the test, the survival of the mice was examined every day. The results are shown in Table 3.

Survival rate (%) measurement result Days elapsed after administration of bacteria D0 D1 D2 D3 D4 D5 Control
(Bacteriophage liquid not administered) 100 75 35 10 10 10 Experimental group
(Bacteriophage solution intraperitoneal injection) 100 95 95 95 95 95

From this result, it was confirmed that the bacteriophage Pse-AEP-3 of the present invention is also very effective in the treatment of infectious diseases caused by Pseudomonas aeruginosa.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

[Accession number]

Depositary Institution Name: KCTC

Accession number: KCTC 13165BP

Consignment Date: 20161129

SEQUENCE LISTING <110> iNtRON Biotechnology, Co. Ltd. <120> Novel Pseudomonas aeruginosa bacteriophage Pse-AEP-3 and its use for preventing proliferation of Pseudomonas aeruginosa <130> KP090505 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 45213 <212> DNA <213> Pseudomonas aeruginosa bacteriophage Pse-AEP-3 <400> 1 tgatgacccg acgttgtagc caggtctaac ccaacgggcc ctgtcgggcc ccgttgtttc 60 tccgtaggga ctacatcatt agccatccaa ctgactttgc aattgacgaa gaccttgggc 120 tctttatccg aggataccga gaatcctgta cagcactcct agtccgtcac gcaagagcgc 180 cactctgtca acagataacc gaaaggataa tatgcgcaag gtagcaagta gtacaccccc 240 ctggttaagt cctacggcct acgtcgccgc gtctgaaggt ggacccatcg accttaacct 300 tcggttgtga cccagaccat gccgcacgat ttacccgttg gggcttaatc gaatcgagat 360 aaatggatac ctctgaggca cgggtgtaat gaatggctac gatcaataac gtaactgatc 420 tcgctattgc tgctatccag tggtctgacc gtcaggatct tacccaagaa cttctgatgt 480 tgttcattgg gaacactact gaccgtctta accgattgct acgaggaggg agaacgaaca 540 cttcgaaact ctcatggcct tcggtggtgg cattgagatc cctgagcact ttgttgctct 600 aaggtctatc agggggtctc tctctatcgg tggtcgctac tctctcagta catcacccaa 660 gatatcttca cccactatgt taactacaac taccaacctc agggtgtaac tactacactc 720 ggttgtgtaa cttcgttggc gtgcattgcc gttgttcgac ggtcccgttc attgcgaagt 780 cactgtctgt actctctaga gctgagacct ccctaacaaa ccgtcgttga gccagagcta 840 ggatccgcag atgtatccat ggatgtactt gatcggatct gagtatctgt acaccatgga 900 tgaagctcgg tcccagttct ggggtcagaa actcgaacgg gctgttatgg aactccagaa 960 cgaggagaat gccgctgact tcgctagcac tcgactggcg atcaaagaca ttgaacgtta 1020 aggagcttca gatgcctatc tttctaaaaa ccctcgcaat caaccttctg gctaccctct 1080 tcccaaccaa ggtaatcgct aagtctatcg tgactggagc taaggccctg gcagagaaga 1140 ccaactccaa ggttgacgac gagttcgtaa aagtcctgga agacaacctt aaggagaaga 1200 cggaggatgc aacttctaac tgataacctg ggcagcatcg tcagtgccct ggtcgtatcc 1260 tcggtagtga cggtaggttc ctcggttgtc ggtcagaatg ttcatcaggc tctgcttgaa 1320 agaaacatcc aggccacgga gaagctcacc caagcggtga gcgaactcca gatttctatg 1380 gcaatctttg gggagcggta tgtcacccga aaagaaatga aagaagagat aaaggaggcc 1440 aagaatgggt cttgaggtcg caacttatat taaccagttg gtgcctacga accctaccgg 1500 ctctgatctg aaatcctttg gggatgacca cctgcggctc attaagagtg ctattaagaa 1560 taccttcccc aatatttctc aggctgtcac tgtaacggca gcccagctaa acgcagtcgc 1620 tgatacaacc cagtatgtta agcctgggat ggtaatcatg tgggctgggt cattagctca 1680 gatcccagca ggttggaagc tttgcaatgg ggtaggtact acctctaacg ggattcctgt 1740 ccccaacctc attggtgcat tcccttgggg tatcgatggt acttcacagg ctgtaggtac 1800 tagaggtggt agcgctaaca ttgtatggga tgggttcact gaagggactg cccttaccct 1860 agcccagatc ccagcacaca cccacacttg gagatcccga ggggctacta ctctaactgg 1920 atctgcgggt gactcgggtg ctctaacggg tggatctggt aacgcagcca ataccaatct 1980 agagactggt cctgctggtc aaggtcagac tcacaaccat gcagtaaaga tcaacatgcc 2040 gttgggtaat atcccaccgt tctgttctgt gttcttcatc attaagaact gaggtataca 2100 atggccctag agagacaaga ggtcaagaac cctacgggga ttgtgaccga tatcaccccc 2160 gctgacctgc cactggagaa atggtccttc gggaacaacg tccggtttaa gaacggcaag 2220 gctcagaagg ctctgggcca tacgcctatc ttcgatacag cccaggcccc aatcttggat 2280 atgtttcctt tcatccggaa taacattccc tattggctcc tgtgttctga acaaaggttg 2340 tatctagccg acgggactac aatcatcgat gtatccccag gaccttactc tgctagtata 2400 actaacaggt ggtctgtagg gtcgttcaac ggggtaatct ttgctaacga tggggttaac 2460 cctcctcatc atcttccacc gtcagaatca acctttaggg tactccctaa cttcccagcg 2520 aatactactt ttaagaggct taagtctttt aagaacttcc tggttggctt aaacgctaca 2580 agtaactctg tagaaatgcc acagatggtc tggtggagta cttcggctga tgctggtggt 2640 gtacccgctt cttgggaccc aacggaccct actaaggatg ctggtcagaa caccttggct 2700 gataccaatg gtgctatcgt ggatggtgtg aagcttcgtg actccttcat catctacaag 2760 gaagactctg tatactccat gcggtatatc ggtggactgt atatcttcca gttccagcaa 2820 ctgtttaacg acgtgggtat ccttggtcct aactgtgcaa tagagtttga tgggaaccac 2880 tttgttgtag gtcacggtga tgtatatgtc cacaacggtg tgcagaagca gtctgttatt 2940 gatgcccagg tccgtaagtt cttcttctcg gatattaacc ctgacaacta tcaacggaca 3000 ttcgtacttg cagaccacgt gaatactgaa atgtgggtat gctactcttc cactaggtct 3060 gagccaggta agcactgcga cagggctatc atctggaact ggaaggaaaa cacttggagt 3120 atccgagatc ttcccaacgt tctcagtgga gcctacggga tcatcgatcc taaggtgtct 3180 aacctctggg atgacgaccc caacccctgg gataccgata cttcggtctg gggtgaaggc 3240 tcttataacc cagcaaagtc cagtatgatc ttctcgtctt tccaggataa gaagttgttc 3300 ctctttggta ataactctac attctctgga cagaacttcg tcagtaccct tgagagatcc 3360 gatatctatc taggggatga tcgaatgatg aagacagtga gtgccatcat tccacacatt 3420 actggtaacg gtacttgtaa catctgggtg ggtaacgctc aggtacaagg ttctggtatt 3480 cgatggaaag gcccttaccc ataccggatt ggtcaggact ataagattga cactaaacat 3540 gtgggccggt atatagcctt gaagttcgac ttctcttcgg agggggattg gtacttcaac 3600 ggatacacta ttgaaatggc cccgaaggca ggtatgcgat gagtcaaaag tacagccctt 3660 cgattccacc acaggaagag gaggaactgc tgcccttcct taatgaagaa ttcgttaggg 3720 ttggacagac tcttaatgac ctagctgacg gctactgggg ggtctctatg gagcccccta 3780 agaagctcaa gccagggact gtaaagtatt ttgcccccgg agtggtaggt ccagtatccg 3840 gcatctatca ttacgacttg gataaccaat ggagactcgc aggtactaaa cccaaggacc 3900 tgccggggga ttttatccta tttaccccac agaacaacca tcagccgatg ggtacctgtg 3960 cctataggat gaacactgct aaggatgaag tatggataac catgctcatg tctggtggta 4020 actacactaa cggggctact gtcctggatc taccccaggc gtattggcct cctgctgagt 4080 tgtttatccc ggcctattcc agcatcatcc cagcgcagag cactattact tatccacctc 4140 cgtctgaccc taacgcacct ccgttggatc aagtctttga tgttctcaac agggctacta 4200 ttcagactgg ggtggttaac caagcaatgt ttaagatcac agctaatggc cgtgttctaa 4260 tccaaggaat tccccaagga gcagtcttcg ggggtacatt cacattcccc ctggtggtga 4320 ctccatgatt catttgatta cccgtgagaa tatcgatctg cttcctaccg tagtccctgc 4380 tctggcccga gccttcaata ggacggacct cggtaagttt tgggacttcg aacacttggt 4440 tcactccttg gttaactacg aggcctatgt cttctaccaa gaagagagtg gctacgctgg 4500 tgtaattcaa gtgtcccaag cacccctggg taacatcctt cacttcttct ggagtggtaa 4560 gatgcctggg aatgaaaccc cggtagatta ctcggaggta gacgacttcc tcggacagtt 4620 cgcccaacga gttaactgtc ggtttatcca atgcgaaggt cgtcggggct ggaagcctac 4680 cctagagaaa ctcgggtaca ccgaagactc cgtatccttc tatcgtgagg taactccaga 4740 tgaacttcct ccaatttaat gtaaagcgtc tgatggggtt cgatgtagag gatgccctag 4800 attctcgggc ttacaagggt ggtagtaaga agcaaaagac cacctctgtc tctacaccct 4860 atcagcaagg taactataat gaactactct cgggtgccag tgattggcta cataacgggg 4920 gctttgaccc caattacggg ggcgatccga actttgaccc ggtggctgac caaaatactc 4980 tccagcttgg tggcatcgaa gggctgggtg gtctcggggg tgcccttcag tctctcctgg 5040 gtagctcagg tgttagctct ctggctgatt accttggtcc ttacgatccc aacaaaactg 5100 gcttggcgaa cgccattggt gcagccaacg agcagatgca atgggacttc gacactacgg 5160 tacgtccaga cctgcgagca ggggccacta atgctggtca gtatgggagt tcccgagcgg 5220 gtgtagcaga aggtattgct accgctagat tgctccagaa tcaacagaat aacgcctccc 5280 agttggcttt ccaagaccag caggcttaca accagaatag gttgaatacc ctgggtaacc 5340 tgtcggctat tgctaagggc ctcggctctg gtaatgccat gcaggtagat gctggtagta 5400 tgcttcagaa ccaggagcag caggaaatca acggggctct acagaagtgg gcctatgaga 5460 ataacgtcag cctcaacgac ctcctcgcct ataaggagct tatctccggc gatatggggg 5520 gaaccaacgt tactacctcc tcgggtggtg gcggtggtgg tggcctcggg tctgccctgg 5580 gtgctctcgg tggtgcttcc ttgggtgctc tcttcggtgg tccagctggt gcttctgttg 5640 gtatgaacgc aggtggtagg gtagggggcc ttctcttcta atggcttccc tccctcagaa 5700 actgttcgct ataggacaga acataggtgg tgggcaggag cgggttcagt tgagtcgtca 5760 aggctcctac cgccccaccc accttgggac gatgcaatcg ggagagtcta gtggacaatc 5820 aaaccctttt ggcgcaatgg gcggagcagc actcgcggct ctcctcgggc aaggaagtga 5880 gccttcttca gaagcagtac caagcttttc tgtcgagggg gctagaggag caagcgaacg 5940 aggcgcagcg gaagttgcag caggtatggg agcgggagtg ggaatccttc ctagcgcaga 6000 agaactcggt ttcggacagc agccaaagtc tggaatccta agtaaactat ttggaggtta 6060 atcctatggc cagcatggct tatgaagggt ccccaattcg ccctagcatc ctccgggctg 6120 ctcagaatga actggacatg gcgcgaatcg caaggaacaa actgccgctg gctgtagagg 6180 gctcaggaat ccctgatcgt gtccgccggg ctgctcaggc tgccctagag aaccctgacc 6240 gttggtctcg ggctgcccaa gaggtgaccc ctgcggctga agctacgggt cgtggtgccc 6300 tgggccgtat cgcaggtatc ctcggtggac cggtatccgt tggcatccaa gcggctgtaa 6360 ccccaggaga acttggggat gcagagcgta ctcgtgcaga agaaatagct caggctagtc 6420 aggctgtgga gaatatgggg cctgaggttg ctcaagaggc aaaccaatgg gcccagggtg 6480 taggccaacg tgcagcccag aacgccacag ggggccctac gggggctgaa ctcctgtcct 6540 atggggtaac ccctaaccaa ccctctatcg agcctgagat caccccagag gtggcttccg 6600 aggctggtgc tgctgtagca gatgaggagg aggctaatcg ccaggtcatc caacagggtg 6660 cagctgaagg tcttcgtact ggtgctgtaa gtcgtcctga aatggcccag gctgtagttg 6720 aagctgatgc ccagagagag ggtgtagagc ttaaacccca agagcttaag aaccgggtga 6780 acgaagagtt gacccagatg aggactatgg acaatgatga tctgtctcgt tatgtatctt 6840 acgctctcat tggtactggt cttctggctt cggccctaga caagactggt aaagccgggg 6900 atatgttcgc tgcttcctac gagcgtcagc tggatcgtaa cctacaggct gggatcaacc 6960 agcagaagat ggctgccgca gctgctgatc gtcagatcaa ggaaaaggat ctggagcgta 7020 aggtggctaa ggatgctgct gatgtacgtc taggcgaagg gaacctggag gtcaagaagg 7080 gaacccttaa ggaaacctct cgtaagaaca ctggtcttct ggataggtgg actgaggagg 7140 ctgctcgggg ccgagccaac cttgccctaa cccagcgagg tcaggatatg gctaaccaac 7200 gggcccagct acaggctgaa acaacccgtc gtggccagga tatgtctcaa gagaatgccc 7260 agcttagttc tgctgtacgt ctgaagactg ctaagattag cgctcaggca cgtcaggcag 7320 cagctaaggc agcccgaggt gagccggtta ctaccaaaga tgccttggga atcctcagtg 7380 aagtcagtgg atctcaggcc ttgggtggta agaagttggg taagactgcc caacaggcta 7440 tagctcagac ccttcgtaac gaaatgaggg ctaaccctgg ggctaaccct attggtatta 7500 tccagcggga ggctgctaag cttcaaccta cgggtaactg gttctttggt ggtgatctgg 7560 actatcctgc cccaacccga taaaaggtac ttgacaaagt atccaaaatg tagtataata 7620 gacctatagg ctcttaagat tccttcttcc ttttgactta cttactaaga aggttagtat 7680 cctaagatac atcttaagag cctattctct ttccagcaac tagaaggtga cttatggctc 7740 tttctccaga actgaaggcc gctattgatg cggaactggc tgaggtcaag ggcctcgacg 7800 tagccgctga attggcttcc ttgaatgagc cagaggttat cgaagaggca ccccaggaag 7860 tagctccagc tacccctgag cctgccccag acctgagtgc cctagtaact ccggctgacc 7920 ctaattctat tagctctgcg attggtcgcg gggttgatac catgcagtct aacatcggtg 7980 gtaccattgc tactctcggt gagttgactg gcagtgacta cctgaaagac tacggcacac 8040 agatggccga agagaatgct caggaagcgt cacagtacgg aacccccgat gttaggtcct 8100 tcgctgatat ccaagatatc ccatccatcg gttcgttcct caagaataac atcgtagagg 8160 cactcccctc gatggctcct gtcctggctg gtggtgcagc aggggctaag gctggctctg 8220 tatttggagc cccaggtcgt atgggtggtg ccctcattgg ttccttccta agctccatgg 8280 gtatcaacgt gggtgccctg agcaaccaga tgaaggagct agaccccgac caaagcaacc 8340 catggaccgc agtcctaggt ggtgctggtc tgtctgccct ggataccgca ggggctgggg 8400 ttatcgctgg tcccctgttg aagcaccttg ggaaagacgg ggcataccat atgctggtac 8460 agtccggtct acccaagcag actgctattg aagctgttac ccaagctggt aaacacgctg 8520 tagtctctgg tgtggctgaa ggtgttacct ctggtgccca gcaggccctc caggacacta 8580 tcgcgtatga tgccgttggg cagacccaat cccctgagca gttcatggat aacctcctga 8640 cggctgcatt cactggtact gctatgggta ctgccggtgg tgctgtatcc tcggggatgg 8700 acaccctggg tcgtcatcag gactccgctg gatctgctgt ggttgatcct aacgcccctg 8760 cggcccctcc taaggcttct gagtttgagc ctaggggtac ggtcaagaag gcttgggatg 8820 ccctaggaaa cgaagctacg tcgctcctag agccactcgc taaggcctcc cctatcgctc 8880 gtgagttctc tgagaccttc cgtgctgata tgagtggtaa gcgggcctct ggtaagacta 8940 tctttgagga ccaggaacta caggcaggta agtggaactc tgaactagat aacatctttg 9000 agggtaagtc ttccaaagag atcgatagga ttatcgctga tacatccgct ggtgtcaaca 9060 cccccgaggc tacccgtcta cgttctttga tggatgacgt aaggaatgag gctgttaatc 9120 gtggtggcat gtctgttggg accatcccca actatatgcc ctttgggttg tcccctgaga 9180 aggttcaatc tccagagttc ctgaacgaca ttactccgta cttccagaac cgacaagctg 9240 ctgaagatgc tgtagctaac tggctggttg aagtctcgga tgatactcgt ggtaacactg 9300 cccctgaggt taaccgattg gttactcaga accagcagac cggggcttgg gaagtagacc 9360 ctcgctaccg tatccagggt gatccggata cccttcgtgg acggtttgcc cagagtgatg 9420 cagtacctaa gtatggtcag ctggaagaga gtcgggcctt tggctccgtg cctcaagaga 9480 tcctcaacaa gtattctctc aacgatactc ctaagaagcg tctccaggaa atccgagatt 9540 acttcgaagg ggcttcccac cggattgcct ttaccgaacg gtttggtatc aacggagaga 9600 aggctaacgc taagattgct tctgctgtag ctgaggctca acgggctggt aagagagtca 9660 ctaaggaaga agtggatcgg atgtatgacc tagtggatgc ctataatggt atgcacggtc 9720 gtatcaaaga tcctaacctt aagaagctag cggctgttac gtcgggtgcc ctcgtgctct 9780 ccagacttcc cctcgccggc ttctcgaccc taaccgagtt cagcctacca ttcgctaagg 9840 ctggggttat gcctaccctt ggggctgtac tcccaaccat gggtgaggta gtaaggcaag 9900 cagcccgcag gatctacagt ggggttccaa agtctgagac cggtcggttt atgagtgata 9960 tgaaccacac cctggcctct gctacctctc tgatggctga ccgggttggc gctgaggtgt 10020 ttaactctac catccaaaag gcgatccgtg gtcagttcct catcaacggc ttgtccatcc 10080 taacccatgt taaccgagtc tttgcaacag agactgctaa gcgggtctat cagaacaacc 10140 tgatggatct agccgcaggg ctccccttca gttctgccaa tggtgctctg aaggttgctc 10200 agcttcggga aatgggggtt aacatcggta gccagcagga tgccctcaag ttgatctccc 10260 cggctacccc atctgaagtc ctgatggcta acaacgtcaa gaccttggcc atgcgtcggt 10320 ttgtagatca ggtagttctc gaccccacct tcgctgataa gcctatgtgg atgagcaacg 10380 gaaacgttca gatgttctcc ctcttgaagg gttacccagc agcctatggt aacatcatcc 10440 taccgatgtt ccgtagacga atgagcccac acttcgctgg ttcctggacc aacgctggta 10500 tgggtgccgc aggtgtagcc ttcactcttg gtctgatgat gagtctgggg tatctccagg 10560 atgagcttcg tcaactggct aagttcggtg ggtctagccg agaggatacc cgtagtccag 10620 aacaacggat gatggacgta gtgatgcaac agatgccact ccaggcttct atgatctacg 10680 acatgctgac gggttaccgt cgagggacta cccccgcaga ggtactattg ggtccagtag 10740 ctggtgctgc aactgagggt gctatggctg ttggtaaaac tatcgcttcg tttggggatg 10800 acccatccgc aggtgaaatc tggaagttct tgtataagca aactccagca cgtcctttcg 10860 ttgccggtat ggaagcgatg gaagatgctc tggatcttta aggagaagta atatggcaac 10920 ttatgtagaa actgatgccg ctggtcctgg tggccgtgcc tacgtggttg atgtgccaaa 10980 agtcttccga cgtaatgccg atggtgatct cattgagatt acctcggatg ccggtcgggt 11040 aaatactaac atcgttaccg ctacagagtt cctccctgac tattatgcag ttaaggagaa 11100 cgcagccctg gtagtagaga acgttacagc ctccttcccg gagaatccct cagaggccgt 11160 tatcccctct gtcctggtca ataccctcgt gggtgctcct ggagaaacct cgggtggtaa 11220 ccgtgaccct ggccacgtga ttggtctgtt ctctgagacc atgattgagg caccttgcgg 11280 tactgcattc ggtagtgaat tccgagtaga ccctcgacgg gaacacctgg gtgtttacgt 11340 ggctattaag cacgtgattg gcccggatga ccaaaacggt ggtaccatcg gggattacat 11400 tatcgagcag tttgacgaca tgcgtggtcc tgtgaagaac attggttctc ttcaacagaa 11460 ctacctggat ccccgactgg tgactactca cctcggtggt aacgttgtca acacccagca 11520 actcacccag aacattaccc ttactaagca gcagtctggt tggttcttca tgtcccaggg 11580 gactacggat attaccgtta ctcttggtcc ggatgtaacc ccaggttgtc acttccactt 11640 tatccaaggg tctacagcta agatcaagtt cgctgtatct ccggacaagg cttggtatgc 11700 taagggtaat cagacggaga ctgatggtca atttggggaa tgcactgtcc gagtgtatcc 11760 tttcggtggt accgtgggta ccttcaaatc ggcagtttaa taggagttag attatggcag 11820 gttatatcgc tgacaacgac ggcaacagtg tattcgttat tcaatcgaag gctattgctg 11880 ttcgtaatga ggatgggacc ctccgacaag tcattggccc gaaagacttt gagccagtga 11940 agtgggagga tattgagggg aagcctaagc ttctggaggt gggtgctaag gctagtgagg 12000 ctaaaccagg taactggaag cctaaggcta gcgaagtgag tggcctatat gaagccattg 12060 cggcttccat ccaggataag atatctgaga tccccgtggc ttcctatacg gatactcacg 12120 aggcactcgt ggataagttc aatacactac tgaacgctct ccggagctaa agataaggcc 12180 ccttgggata acacccttgg ggcctttttt tggttactcg acggggtggt agataggagt 12240 atcttcggat accctgtcca tgtggtcccg gatgaagcaa cagctatcct gtaggttctc 12300 gaagactaca ggaagggact ctagatatga gctgacacag tggatcccca tcccctgttt 12360 tctctgctcg aaattcctcc aacgaaacca ttgcttatac tgagggaaga agaacttccc 12420 cacttgcttc actcgatact tcataaggaa cctgcccaag ataacacttc gatgaggagt 12480 ctggatattt ctcccagggt caggagtaac atcagtcagc tataccctac atacaccagt 12540 agggcaaaca aagctgctac cgcaatgtag gtccaaggga atccaccctt acccttttga 12600 tctttctgca taacccctcc aatctccgta gaatacagaa gccctgaggt ctactagatc 12660 tagcatccct tcgaggaatt caatctgtct tttagcttct tcagccagtt cttcaaagga 12720 aaaggccttc gcctcagcct tcgcaagagc gctttgcgtc tcttcaagtg ctttctccag 12780 ttcagccttt gtcatagtac accaccttgt cactgcccct gagaacatac tcagaatcaa 12840 tagtccaact catggtaggg catttgattc gaatcatctt cggagtaaaa ccatcaactg 12900 taccaatagc taggtgggag gaggtgttcc tgtggaccag gaagactaca ctgtccccta 12960 cctctagctt acgtcccact cgatctttca tagattaccc caagatatcc ggaaggtgaa 13020 cgctggggat gtagaggatc tccaggactt cgatggaata catccctcgc ctaagcccct 13080 cctcagcacc ccctttccca aacttcatat ccagccagag ttctgcggag atcggcgtct 13140 taaacgccaa cccccgttga ccgaagtcat gctcagactt aatgtagtac aggtctggca 13200 agaacttact cacaggaact cctccacgct catcagcttg aggtcatact ctccatcctt 13260 gtggttccgg aggagggcta cacctcgcca gtggtcgtta ccctgcggtc ccttgtagga 13320 ctcatcgtgg gtatagaagc ttccgactac cagcccgtgg attcgcttct tacctactgc 13380 ctcgatgtgg tacttgaatc cttgctcatg gccttggacg aagctgcgct tgatcttatt 13440 gaggcgatgc tcggcactcc cgccgtatgg ccgaccactg ttgggattgt agaagtagtg 13500 ggcgaaggcg acaccttcga taacacccac gtcgaggaaa tcatggacaa tccaatcgga 13560 cagatcaaaa tgatcgtacc caataaaacc ttggagagca gggttagaat tttcataccg 13620 cttgatacgc tcctcgtggt ttccgatgaa gaagtgcatt tctggtcggt agatacgctt 13680 cttacaaccc gcttggtgtt gctgtaggcg gcgaagagga tcgagcagaa ctcgcatcgc 13740 atcattacca gcttgtatgt cagcgaggac tcgacggcct tcgatcttag cggtaccacg 13800 gtcgtagctt gacagactgg gcatgtccca atggtcccca atatgaacaa ttcgcttggg 13860 gcggttacgt gcgatccact ccccaaggtt cccgaggtga tcgatgttga cctcggatcg 13920 aacttgggtg tccgcaatga cgagagtgtc ttcattatac tttttactca ttagggaagg 13980 actccttgaa agtctctaga gtaaaccagc ggaatccgtt acgttcggcc cattccgcca 14040 tggattgctt tgtgccatct ttacgcttac taagccaatg gcaagctgtg ttaggacgct 14100 cgaagataaa cacaagttca gtatctgggg ggagagcctc cctcacccag atgtacttag 14160 atgcctccga ggcttcctgg aagtacccct taacctctac caacaggtgc ttcgtcgatc 14220 cacaaggttg tgaggtcgat accttgaagt cgggcttgta cttgtgctgt atgacgtagc 14280 tcaccgggtc tggcttgaac tgcatccagg gcatcgtctc gtggacgatc ttctcggttt 14340 cgctgtcgta cttcgtccca atcaatgact tcagtttggg tctgaagaat ctcccctgct 14400 ttgccaatga gccctccttc ttgaagcttc cagataaccg agagtgtccc ttcagtcagg 14460 atagaattag cccgaaccag aagccagatc caatccccca ggcacacccg gaactccgga 14520 tacttcagtt tgatacgttg gatatgagag acaacctttt caacttggtt gtcagttagg 14580 ggctgagagt gatccagaag agggtctaaa actcggttaa gagtaatgca cttctcttct 14640 ttatcaaact cataagccct tgaacggtag atttgagatt tcagtccacg gatacttatg 14700 ggccagcatg aaatctcaca cagtcctaca tcgaactctg ctggatggga gtgagtactc 14760 acgacaaggt tgacgggtag gtcaacacca atggattccc acacatcaaa ccccgcagga 14820 tacccatcag gcttatcccc agtcctacac cggaccttct tgtggcccag cttgttgtag 14880 aatcgagtga cctctacctc tagggtatcg tcatcacagg ggatcaaccg cggtccaaag 14940 cgactacgcc gagtatcagt ggggtcctcc acgtatggcc tacggacata gaggtcgata 15000 tccttgggtg caaccccgtt gtctgcatcc cgaaggaaac caccagcgac cttgtagtca 15060 gtaaagttaa gaccctcaag gatctcaaag aggccttcac agattttctg tgcttcctca 15120 atagaatatt tcttcatcgt catagggaat ctcccaatgt ttcttcagtg agcagtgata 15180 ctcgttaccg gactgctgaa tatcaagcca agagcggtcc cgaatgaggt agaggagacg 15240 aaaattctcc agaaggtact ctttccaatt atctccgtac ttctgttggt aggcgtaagc 15300 gcaagctgcg aagagatctc tctcactaga aagctcccca agcaaatctg ccgctgtctt 15360 tggtcccgtc cctgggaggc ctggtatgtt atccacggag tcaccaacaa ggagttgccc 15420 gtagaggaat gccgtaccgt tgccctcaag cttccatgat ttctgcggct tcttaacacc 15480 atacattctc caagaagcgg agactcttcc aagaccatca acttgaaacg gtcccaactc 15540 cggctgggaa tctccacagg gccaactgta atggtagcat cctgggactt ggcggatatc 15600 cttgtctcta gaacacgctg taaagttaac tccctcttca gtgcctcgaa tcccgagcca 15660 gtcatcagcc tcgataccgt ggaaagtgat tgctccccac ttgtccttaa gtctggctga 15720 cacggtttcc cagtgatgag gcttttcaag accgattcga gtacccttat attgcttgat 15780 tttcgcaagc tccagcctaa aatttccttg gccagtaagg tagatttcac actcatctgc 15840 attcaccctt ttgataattt gttcgacttt atcatcgacg agcttgtgga cttcttcctc 15900 aggccagggc ttctccactt ggatatcgaa gatgtgttcc ttcgacatag ccacgtggcc 15960 tagctcatac ctaagaacgt caccatcgat accagcaatc atcacgttac ctcttctacg 16020 acctgagact ctttgagctt ctcttcccag aagacacgaa gggcaaaggc ttcacccttg 16080 gctcgatact ctgggtgctc taccaccggg ttaaccagtg cctcatagcc cttccagtcg 16140 taatattcac tttcttgacg cttggccttg tccatagctt cctccgttag gctgtagcct 16200 ggaggaagcc ccaggtctac atcgatgccc attacagagt agttactagg aacagtctta 16260 gcccttggat ccaacagaag ttccattgga gtcctcctct tgctgaatga cgagttccag 16320 ggacgctagg gcgttccaca cttcgtgata ccggtggggt agcttacttt gggggtccaa 16380 agcctcaccc ttgtgtcgct caaggaggtg tctcccgaag gcagcatcat atcgctggac 16440 tccgttctcg acagacctcc aacccccgcg actatacttg ctggctccga agttagcaac 16500 ttgtgccact gcatatagag ccctcgggaa cgcttcgaag atgattccaa catccacctt 16560 gccgctatca agtttagcgc ccggctcacc cggcttgatt ccattaggat cgacctctac 16620 tttattatta gatgcagctg tagaactgac cacccttaca ccccacactc tctcgaattc 16680 aattaagcag taggggcagc ccaatgggca tacccccgcc tcgtgatcct ccaagccctt 16740 agataggagc gtcttcgtca tcgaatacct cgtcgaatac atcctcgggc tcttcctcgg 16800 tgaaggcacc accgaacagc ggatcatcct caccgtcacc accggataca atctcgtgga 16860 gtcgtacacc gcgcagtcgc agggccgtca tagtaccctt gcctcgtggg cccttttggg 16920 tctcgatcag gatgccaact tcaccagtag atccacccca cgggttgggg ccatcatacg 16980 gcttgttgtc cgaggtcaga accaccggct tcattacagc agacttccgc tcaccatctt 17040 gcatccaatc cacgtgcttc ttctgcttga ccttgaacag gtactcgccg tcacgctctt 17100 ggtacttgat ggtacccttc agtcgggcat ccttggactc catctgctgt accagatcgt 17160 cggcctcttc tttggtcgga atgcagatgg ttacttggta gacttcaccg tactcggtgt 17220 ccggagttac gatgtagggg aagatcagct tcgctaccgg gaaggtgaat cgttgggata 17280 ctttcttaga catatcttaa acctctcgta gggaagaagg tattatacca caaagggggc 17340 tgaaagtcaa cccccagatg tatcagtgtg tgtcatacca cgagcgacca gcgatggaat 17400 cactggccaa aggacagtta accccaagga gcactccagc atcccggaca cagatttcca 17460 tctgtccacg tagcgcaatg agatcctctg ggtggcattc ccactgacct tcgtcatgga 17520 tatccaggac tttccaagcc cgaaggttca acctccggac ccgttcatct aggagcacca 17580 tggcccactt catgacaatt gcacctgccg cttggaggag ggtgttcaat gccttatgga 17640 tcatcacgtc gccagactca gaacgtcgca tggttagctt ccgtccgtct agcccttcga 17700 gataacctct ctcggcttcc tgcttaaccc tctcaatcaa tgcagcaagg tcagggttag 17760 cctcaaggaa ctgggcccgg agcctagccc catctgccct tgtgccccca ataatagtcc 17820 cgatcttggc atccccagca ccgtagatga aggcgtagat gaacgtctta gcatcgtcac 17880 gggttgggag acctgcggct atctggttgg ccgtgtgtac atcaccctcg accacctctt 17940 tggtgtactc tgggttattg atgtaggatg caagcatccg aagctctagt ccagcaccgt 18000 catagcccac gaagaccatt tgattcgctg ggacgtatac cttgtgcttt cctacaggct 18060 tccacttacc tttcttgagt tcctccacaa tgttagtgaa gggcctaacc ctttcgttct 18120 ctccaatgtc tcggcggagg acagtccatt tccattcacc ggcatcaggt gtcccctgga 18180 acaaccctcg gatttctggt ccaaagggag aacgagcagc ggggatatta actactacac 18240 ggtgcctcat acggcccgta ggtgttgcac aggggttagc actcccctct aggcgtccat 18300 cgggcctcac ctgatcgaca agcccctgca agagggaccg tcggtgggcc caaaccattc 18360 gctgcatcac cagctttccc aggtctccct ggacggtatc catcgactct tccgtcatct 18420 tcggagtcgt caataccttt cgttgccttt ctagctttct cttgacctct ccgatggtag 18480 tcttcccagg gatgattccc tggagcttca ttcggagtcg tccagattta ctctgtcgga 18540 gatcctcaat gtacttattc aaggactcat taaggtcgga tccacgtagc ttcttgccat 18600 cagttccgac agtaatatcc ttgatgttcc attggtcagg aatccatccc tgatccaaca 18660 tccaatcctt gaacttagca gtcttgccaa ggtcgaaggg aatgtactcg attgcagtga 18720 atggtccaga tacctcgaag tgcccaagac gttgccataa ggactggagc ctttgattcg 18780 gcttaccact ctttaggaat ggcttgctga aggccccagc ctttacgatc atcatcggca 18840 tctgggggac ggcctcaagg tcaatcttga ggattcgttc ggtaagcaca tggatcagcc 18900 acttggcccg ctgagtcttg aagtggactc cacgtttact ctggcgactg ataatatcag 18960 ctaccttgtg ctcaacgaac agtgactttc tccaatccac ttagtccccc ttaagacgct 19020 ccgccagatt ctctacgact acctccacgg cctcgtcgga gataccctcc aggtaggaga 19080 ggtagtccac ggttccgctt cggtccagtc gccattgctg ctccaagaac gctacagcaa 19140 cttgcttgag caggaagtca tctacccgct ccatgagaac acttttgatc ttgctcatgt 19200 tatcaccagt cgaaaatgaa gtaggaacat ccacgctcac aggcgaggtc taccaggtac 19260 caggataggt caaccttgcg ctggatatcc ttgatatcac tcagttcttc atcggacaga 19320 ttgagggtat caccccgctt ctccttaagg aattgaaact cccatttctt ctcctcttgg 19380 gagacaacca gggtatcccg aggatccagc tgggcgcccc tgtccacgta gaagtcatcg 19440 tagaggctca tatccttgtc acgaatccgg gcgtttcgac gatccgagta ggaactcagg 19500 ttcaggtgag agcctgtgaa gtagctcacc cccaggaatc cctctggcag gctcaggaac 19560 acccgacgct tctcccgtag ggtaatgtgg ggactgctga ggaacttccg gatgttagtc 19620 ctttgtacct ccatgctctt atgtagggca gaagccatat tacacaggta cttaccacgg 19680 acatgtttgg agtagcttac atggtcactc atttgtatct cctatagggg taaagcaacc 19740 cccggcacct taaggttatt atatcacact ttgctggatt tgtcaagact atttgtggaa 19800 aaccctgtca taccacttgt agaagtattg agcagaggta atcccaatag cacctcccca 19860 cccggacacg aagataaacc atccgatatc caagtgggag gaggctaccg ccttgatgta 19920 agcaaactga gccagcgtga tacaccaaga cactacgaac ccagccttga tcttgtcatc 19980 ccggagcagc ttagagttca atcccaggag gaatacctgg aagaatgcac tccagaagat 20040 gacaaggact tgtagagcta ttgacatgac tcgcatacct cgggatctag gccacaggca 20100 ctacccaagg ggaagtcatc agcatcgatc tcggggccac cccagctacc agagaactcc 20160 ttctccaggg caatctcctt ccgtgcctga tcctccaggc tttccttctt actcgccatc 20220 gttaatctcc tccacagaaa cccatccacg gacagcccca acctcgttgg cccgtacggt 20280 cattggattg gcatcggaat cacccaggaa gaaccgaatg atatccaact cgattcgata 20340 gaagtccgca gtgaacacct tggtgtcctg gaaccccacg ttaacccgat acttcttcat 20400 tcttacctcc caggagttct tcgaggttag ccagtacctt ctgggccttc aggttgtcct 20460 gcatgacctg ggtacgctca gccttaagca ggctaatctc gttgtagatg cgctcagact 20520 cctccgaaga ctcttggatg aaggtcttca ggttcaccag aaccttgtcg aaggagctaa 20580 ggatgttact gatgctaagc tttcggttaa acatagagag tcctcggggg cccgaaggcc 20640 cctccttagt tactttttct tacgaatgcc tttggcctta cgattgccca cacgaccacc 20700 atcagcagcg ttagaagaat gactacggac gcggagatta gaatccccgt taccgccacc 20760 atctttaaca cgtttgatat gatcgacaac ctcatcggct ccgagtttcc ttccaagacg 20820 cttttccacc tttcggcggg ctcggtgccg ctgagcatct ccagacttag accccacgcc 20880 cgtttcgccg cgtcgaatag ccagttccct ttcacgcttg tagttcctca ctcgcttttt 20940 agccaatcta cagtcctcca ttcaacaagc tcttgccaag tcatgccgag tacccggagg 21000 aggtagttga acagtgcgtg actgaccact ccatcctgaa ggcaatactc aaacatttct 21060 tcggagaact tggagaagtc gttgaaatca cccttgtgac acttcaggaa tctaccccag 21120 gcacccaggg aatgaccctg gggtcggtct ggccaagtca acctcgatgc aaccaaggta 21180 tcgtagcttc gatccggagg gatgatgatc ccatgaagtt tctcaaggac ccggttgtcg 21240 tagtttagga tgttatgccc gaccacgaac tttgcatggg cgatgatgcg gtagaacacg 21300 tccatttcct cgggccggaa tccccggact accccagtcg gaatgtctac tgcaacagcg 21360 caccatagcc tcgtgacctc attgaagagt ccgtccccct caaggtcata gatcacgtaa 21420 tcaaattggg gtgtcatcgt cagcaggtac ctcgtcgtaa ctgccctcac gctcaaccaa 21480 gcgacccgtc tcagggttat acttggtgaa cactaggcca gtgttaccgt agttcctgtc 21540 cttgattaca cggatccgac tctcgtgctt ctcttcaccg tcagcctgct tgttccgctc 21600 gaagccgatc ataagctgac accaccgctg catagctcgg gaaccagtga actggctttc 21660 ctttacttca gcgccctcct cgtgggtacg gttacccttg ggtgggttaa ggtgggagaa 21720 gatgaagatc cgcagtccta gctcgtcggc catccctgcg agttctgtac agatacgggc 21780 tatctccgtg ttcatttcag aaggactaag gtggttggtc atggcggtca tgttatccag 21840 aaggatagtc ttcacatcca tggctacagc ccagaagcga atacactcct tgatatgatc 21900 ccaatcgttc tgacccttgt tcttccacat gaagagtttg ccacggagtt tacccgcagc 21960 atctttaaag gcttgctcat cccactcgat atccggtcgg tggaagggca cgttggctac 22020 cttccccgcg atattcttaa gtgtcatggc tacctgctcc tccaagagga aggtccctac 22080 gttctccccg tgctcaatgc aattccaggc agcaatctcg tgggccaaca gggtcttacc 22140 gataccagta ccaccgccta ccccgatgat ttcaccatcc cgctgaccat aggttaggtt 22200 ggttaaacct tcccacgggt aactcttgcc ccacttgggg ggttccagag cctcagcgta 22260 tacctcatcg acagtaacgg aacagtccgg agacttgacc ttgctgccga atcggatcat 22320 ctggtaaacc tcatcccccc ggttacgcat aagcatgtca ttagcgtcct tcagaggaag 22380 ctcgacaaac ttacactcgg ggtacagggc acgtaccttt tctactgact tacggccttc 22440 gtcgtccgca tcgtggcaat agatgacctc gttgaaccgc tcaaggacac cagactcctt 22500 aaggaatgca atgtcccgtc cagcagaacc aacacccgag gaccaactaa tggatgcctg 22560 ccgaagatgc ttccacttct cgggcagctt cgactccatg atctgggcca cagacatagc 22620 cgacagttca tcctcgcaga tgactaaccg caggtgtccc atatccttat gccgaagggt 22680 attatacccg aaggggtcta caccgccctt ggggcttcca cggtagtaga aagctttggg 22740 ttctaggatt cggacgttga acgctcggta ctccccttcg cgagtgcgtg gatagtaatg 22800 ctcgataacg tcctcaccat tttcttgtga tagaccaaca cgtaccccgt accgttcagc 22860 gattgatttg ctgatctttc gagtatcgag ggctttagtt ggatactcaa ggacttcgtt 22920 gacttcatgg atgatatcct cttcagattt ctccctccgc tctggtacct tgaaaccctc 22980 ttcgaggact tcgtagtgac cacaacggtt gcaacttccg aaccgagtac ccttttctgt 23040 atcaacgaac aagatgaggt gatttcctgt tttatcccca cctttagctc ggcaacccgg 23100 acaagccgta tcaccgatga ttttcttcac ttacccctcc agttgactct tgagaaccag 23160 gcttggtaca gcattcacgt tgcccggtag ggttttatag ggagttgtta cgaaggtacc 23220 gtagacttcc tccagttccc gaagacgatc ctcgattttc ttcaatcgct cccacatgaa 23280 gcgaatgcca atggattcgt cacagtaccc accgtagctg gtctcgggac tgtccgaggc 23340 ctccggaatc ttggagacct cgatggagtc ttggaggtgt cgctgggaag ctacaaggcc 23400 caggatctcc ttgaggagca tcttctgggc atgtagctct tggtgtccga aggtgttcgg 23460 ggtgtcgcgg tagacttcta ctgctttgtc tacggtcatt tcgttcatta tactgctcct 23520 tgtcgttttg ccaggtgtgc cttccagtcg ccatcttgga cgatatccat cgccatgtgg 23580 ctatcctgat ggtacaccag ggccttgatg cgttccccat tgattcggaa atccttgagg 23640 gtacgatcat accaacccgg ataaccctct agggcatcca ggttctccag catttcatcc 23700 cccgcttcgc cctcggggag ttgccagatt tcacctacga tgagatcagc acgggaggcg 23760 aaggagagga tggggaagcc gccagcggag tacatcagac cccgctcgca ggatacagca 23820 ttgccgatga actcggcccc ttccagaagg tagtggttgt acaaacctga caggagagta 23880 ccataaacga aaacacgaac agccattaga tagtctcctc gaagattttg ctgaagccct 23940 tgacgtatgc ctccagggcg ctgccttcag cgtccagacc tggggcggtg ttgatctcca 24000 ggacaaaggc ccggccagtg gcgtgctgga agatgatatc tactgcaccg aagtccagcc 24060 cagccacctg gattgcctct gccgctgccg acatagcctg ctcagcacca tcacggtcca 24120 ttgcatcgtt aaccccgtag acccagccgc cagctacgtt acgaacgatg ctttgtcctt 24180 cgttggtcac catttcattg gcaacacggc ggagtttgac ttggagaagt actacttccc 24240 cacggatcat atgggctcga aactcggtac gcttaccgac gattccctgg gtgaacagtt 24300 gtgccctggt gataacatcc gggatgtggg tgtgaccgta gatgtgtacc gggtagtggc 24360 cctgtctacg gagagcatcg atcgcagcaa cttctgcatc attgacagta cacaccaggt 24420 gaatgccgcg accgctatgg ccgttcagat cggtacgtgc atacatgcga ccagagatat 24480 tgagtacacc caaggcatct tcccaacgct cggtccaacg gggaatcagg tgaggggcat 24540 tggcctcgaa gtggcggaag aactccagct tgttagagca gcggtaaaca actgccgggt 24600 cgttgaggaa cacaggggca atatcccgga gtcggtgagc ctctgcacca acggtacccc 24660 agttgattac cgcagtaccc gcacgacctc gataggtaga cccctcggag cgaaggattc 24720 gagcacccag ggcgttacga agagcgacag caccagcaga ggggcgggca cctttataga 24780 tatagaacat ggcaaatctc ctgtttctgt tgatggtagt gaagccagca atagcccctg 24840 gaatcctaag ttagatccca tgtacgcatc ttaggaaccg gcatgggtcg gccaggggct 24900 attactaacg cactacctta ctcgaagtag tcttcgaaga gtcggtcgat agcttccgca 24960 atgactaccg gggccggtac caggaattcc tcgtccgggt tggcatcatt gagttcctgc 25020 tccttgactc gacgaatcga ctggatcttg tccagggtgt cacgggagaa gggatactta 25080 cgggttgcgt cggtctccat ctggatcact acttgacgct tcatcgttac ttgggtcatt 25140 ttgacctcct atttattttc gattacacac tcaccacaga tgaactcgca ctcatccagg 25200 ctaccagtga acttggcacc ttcgtatacc gtcagatgac gattacgctg gtcatatgcg 25260 gttgggatct tcccgcaaga tgcacatccc tgggacacga agtcccgaaa gacatgcacg 25320 ttgttgaagg tgtggccttg caccttcagg gggtaggtga tgttggccgg aggggtgttc 25380 cccgaggtgc cgccgacttg aaccctgggt ttagaatggg atttcgtctc cgatgagatc 25440 gttccaatcc tcaacactgg ggtagtctcc ccaggagtcg gccgatgctt tgggtccagg 25500 cagcttaccg ccagatcctc gacagtaacc ttgagatcac ggtcttggcc gacgaagtag 25560 gcattggaga tactccctcg gaggacggtg tatccgtgga ctttggcgac gttgatgcca 25620 tgagcttcga tgagaaggtt ttcttcacgc tcgattccag tgacagttcc gtactcgctg 25680 ccgtttgcgt aggcctcgat cttgacaaca tcgaaggtga cgagttgccg gtgcttgaag 25740 gtgctacctg ctgcgtcgag gttgttgttg atccggaggg tgttccttgc gtacgcatcg 25800 ccagacgttc catacgacga gcccgcttgg ctgcctttcg ttcccgcttg gctgccttgg 25860 cttcggctgt agtcatcgta gtccgagaca ccgacgtagc gactccacca tgccgctgct 25920 tgcctttcgc tttcgggaat cgggaggtcc agaaacgtga ccggagtggt ctggggttct 25980 gcaccctttc gcactccggc ctccgtgaac gggatagtta ccaggacttc ggccttcagt 26040 tcagcgatgg gagaacgcag gcggatctta gcattgggtc gctccaggaa aaccttgagc 26 100 atcccaggct ccgatgccca tacgatggtc ccggtggtgg taaccgccat gaacagagga 26 160 cgatcttcgt tacggatgaa gttcaggcta cgttccttgg agtcccacca gacaaccgca 26220 gcagcaccag agaacttctc ctcgaacgtc ttcttggcac cgtggttggc aatggtagcg 26280 cacagcatgt tggagtccac gttgtactta tcattgccgt ccagcaggtg cagtccccag 26340 gtatccacgg tgccgttatg taccatggtg atgtggtcta cctggaaggg gtgggcattc 26400 ccatggtccg tcttgtcgcc catcgtggcg taacggttgt gacccatgta gatgcgagcc 26460 cgacccttgt ccaggaactc cttggtttca ggatcagcca ggaagtcgta ggcatctact 26520 gcccgcttgt gaattcctac ctcgttctta accgggtcga tcttggccaa accggtggcg 26580 tgagcaccac ggataatatc cacggccagg agggatttga gaagagcgat ttcgttatca 26640 ctcgcgttag ttgtggcaca gaagcctacc aatccacaca ttgtatttct ccttagtaca 26700 tatctggttc gattgttacg ccggtatggc gctgaaggtg ctgcgggtta tccatgagca 26760 gtacgaaggc atcttgaaga gcctcttggt cctccagaag gtctcgtaca cgcacttggt 26820 gaccaatacg acgcagagaa gttaccgcag taatcgccgt atccagtcga acctgggtac 26880 ctgggaccat tgccaagttg taccgctctg ctgcaccatc gagtagtctg gaggtagtag 26940 atcgagccag cagcagccta cgatccaaaa ccctctgacg ctcacgttcc tgctcccgga 27000 gacgtcgctg acggttctgt tcatcctcca gaggatctac cccggagcga acaaagccca 27060 tggttgccac cgcgagagta gcactggcga tccccatttc acgaccaccc ggagacattt 27120 catagtccgg agataccccc aggaagaaca catcacggag attcacctcg gagagcacct 27180 tgaggaactc ttccaggggg gtttctgcgt gagccatggc gaaggccttg agacacaaca 27240 tacgctgcat caggcctacc atttcctgtc gagtagtcag ggcgtgagca ccacggaact 27300 cgatggaccc gaagttgtgg gttacctgga ggttcaaggc cgaatacttg ggccaggagg 27360 taagacccaa gactttctct cggtcacctt ccggggccag gatacggcga ccgaaggtct 27420 gggccaacca gtcgttgctg aagaacggga tgcagaagtt actatcccga cgatacggct 27480 ggcagtgatc gaagaaaaca tcctcgaatg ccatgtaagt caggaccgtt cgttcgtaca 27540 ccggccactc tacgtcccgc atatccatgt gcaggtgggt ggagcaacgg aaggtaggag 27600 ccggaggggt ttcttccatc gaacttacaa aggattcaac ccgagtgatt gccctctggc 27660 cagaggcggg accggagaag acatactcca taccctcccg cagagagccg tcacccttca 27720 gatcccaccc atccacttct ggccagttgt tgaggccttc cagttcgact tcgaacccga 27780 aggaagcaac cgcaggaagt tcctcgtggt gttccggggc gatgatctgg ttcttacgga 27840 accctatctg tgtgccaata actgacatag caagtcctcc tggttcttga attgttcatc 27900 tgcggacagc cgcccatcca ccatctggcc aaccttcact cccttccagt gtacaacccc 27960 atccttgatg aagaagtccc ggtgggaggg agcagagatc atttcctcgg cctgggtgaa 28020 gattcggatc atcatttcgc cggtgatagc ctgttggcca tgattcagca agatgttccg 28080 gggatctaca cccttgcgct gggtacggtt gttcacgagg aaggatacat aggtatcacg 28 140 aaccccacga ccaaagacgt catagtaccc actggagggc cgcacgcttt ctacctcgct 28200 gtagacaacc cctccgtagt agttactgcc gtcggggcga tacacgttgc caaggagacg 28260 aaacccgtcg aattgacggt cgtcaccaat ggactcggta tcgtccacgt acactacccg 28320 aggctcgccg tccacccgga agaaggcata ggtggaggag tagtacatgt tgaagtctcc 28380 ggacaaccga tggcgattat tcaggtcatt catataccac ccccagcgct ttaagtgcag 28440 ctaccgccgc ggcgccgtcg ttctcgttga tgattcgctg gacttcatcg ccagatacga 28500 tggacttgta ctcctccagg aggtgcttgg aggtgtaagc ccgtactgcc atttcataga 28560 cccactcggt caggtctaca gagaagatcc agaagttcga caggctacgg tactcgcctc 28620 cgtaaggctt atagcgcatt gcgccagcct tgccgtacag cttcttacgc tgggtgtcac 28680 cgtcgaggat gacagaggcc aggcccagga ggtagtcaca catctgcatg acttctcggg 28740 tgttcgcctt ggtgactcgc tcgatgtggc tgaagccgat atggacgtgg ccgccagcag 28800 tgcgcagggt cggaggggca tccttcggca tcacgttctg acgacgagtc cagcagttgt 28860 agtccggctc gcaaccgaag acgtaagcct ggggaccgaa gctgcggagg agttcctcgt 28920 cgtacaggtg agaggacagg ttctcgatga tgttcatgcc gaaggggtgg agcatctggg 28980 cacccaggag tcgaacggaa acgatacgct tcacgaactc ttccttggtg ctggccgggt 29040 cgatgttgta ctccagaaga acgttgtctt cctggacggc accaccggct acagccaagg 29100 gggcttcctt ggttccaccg atgaagccga tgacgctctg ggggttacgg tcgtagccga 29160 cgaaaacttc gggatctgca ccgatcaggg ggattgcgtt gttcattgca tagtctccag 29220 tttttcgagg atttcgggga tgctgtaggg actggtgggt acggtctcag acctttcccg 29280 acctgggtca cctacgtcga tagtgatagg tgccatgatg gagaggatcc gatcattgtg 29340 tacaccttcg agcccgtcgc attcgatcag gtaatcgtcc ttggtatacc cttgccacaa 29400 ctcagcgtcg tgatgcttga gcttgtcctt gaactcttcg cgagtccgcc catggatgct 29460 cttgatatcg aagtaggtga tctccttgta cccacctacg ataccatcca tgacttcgct 29520 gatgtagctg tcatccactt cccatccgtg actctcgcat ccttcccggt attcaccagc 29580 agtcaccggg aacatggcag gatagctgtc gcaatgggca gtgctaccta cctgggcaca 29640 agccgcgagg gtgaggatct ctgcattgca gcccaggatc ttatcacgat tttcctgggt 29700 gatgcgacgc atggaatcac ctacgcacca cagacgatcg cccattcgga gggcacagat 29760 gatggccgat gcgttcttcc tgaggtccac ccgtacatac actgcctgcc ggcgtcctgg 29820 gggagagaag tggtcgggat cattgagatt cttgagatca tcattgtaat cccaccaacg 29880 agtaggccag tcgatagagg ccgagaggat accaccgtcg gtaatcctgt tgaccaaatc 29940 actgttaagg aactccacca gtttaccaga ggtattctcg tgggtagata ccagcaggtg 30000 gccaaccccg gtaacccact cacctctgat accgccgtag gtctggtcgc aataggcccc 30060 gaagcaattt gtatattcgg ccatcatctt gaccgagtag tggtgtacca cagacccttc 30120 ggcgtcatag tacacattat gggctgcata cattcccatt tgatttctcc ttggttgtca 30180 gaagtttttc tcgatgaagc cttcgagggc cagtgagata tcccttatgc cgcttcgcca 30240 gccaatatca cccatgatcc ccagcatgac tactccgtat ttttcacagg taggatgaga 30300 aaggcggatg aaacccccca gctcactatc tacggtcgtg tagggatctt tctttccgaa 30360 gacggttggc tgaggggcta tggtaaccac ctccaccttg tccaactgaa tcttcaggaa 30420 ttcagaccgc cagggtgtac tccttaccca ctcacgtcca cctggggcat cccgattggc 30480 catccaacga ccgaatcgca tagactcttc agcggattgg atgtggatat cccccttctt 30540 gagatcatcc caatcgctac tgaacatgta gttagagagg ggatacccgg tgtagccatt 30600 ggctatcagc ccgtggaagg acattgcgag gtaggacttc cagaagccat acttctcagc 30660 aagcttcaga tggacctcct cctcacgacc tagatagtcg agtccgctgg atatcatctt 30720 gaagaggaac ttcatgatgg ggtaccagga atccagggtg tgtaccttgg ggttgaagta 30780 gataccgcag acaccttcct ttccgtgatg ggtgggggaa atcatttccg tcccgttaga 30840 atacttctcg aagggggaat ccttggcctt gctttcgtaa gaccagaagt gaaccttcgg 30900 gatcttgaag gccttgccgc cgatggtgat ggtcttgagt tcaccttggg tacacttgtt 30960 gagtacatcg atgaacctct tgcttcggcg ggagcagatg aaggtccaac cgtcgagctt 31020 atagccgtgg gttaccggac cttccattgt accgaagcag gaggaagagg tcattacctt 31080 gagggcttta tcctctccat tcttggactt cataccgaag gcgtacttga tacgtgccat 31140 ttgttttctc cttggttgag ttagccagca atacccccag gacaggaata ctaggggtat 31200 tactagcgac ctctaagtta cttagaaggc gccacggcgc tgttgcttgc ctttggtcgg 31260 tgccgacacc ttggtggcct tgaggttgcc gttcagggtc aattcaccgt tggagcagag 31320 acgctcgtag aaaaccttac gcttacccat tgttcacctc cttagcgaac gttgatgaaa 31380 ggcattgctt ggccgccggt gacgtaggta ggcagcttgc cgtcccactt ctcgatggca 31440 ttcagttcta ccacgccggg gttctggcgg agggcttcac cctggatgcg gaggctttcg 31500 gccttacctt gtgccaccag gatagccgct tgcttctcac cctcggctgc cgcgatagcc 31560 ttagctgcct cggcctgggt ctgacgaagt tcgttctcgc gctgcatcgc cgtctgagtg 31620 gccttgatct tggcattgat cgcctcgacc acctgcgccg gcaggccgat cataccgttg 31680 aggtacaggc tttcgacgat gatgccacgg gtggagaagt gctcacggac ggcagcttcg 31740 acttccttca cgaaggcttc cttgcccgga ccatagatgg cctcggcgga cttcttactg 31800 ctggcgttgt tcacggcgtt acggaccacc tgcggcacgt tcacgcttac gatctcgttc 31860 atgcccttgc ggtaggtctg gaacagcagg ggtgcgctac ccggagcagc ccggagggtg 31920 acgccgatgg ggaagtccag cttcaggccg tccttgtcct gggcagtaac caccgccaga 31980 tcgaagttct ggttgaaggt ggggaacttg aacaattcct cgttgggggt caggaacttc 32040 cagccgaccg gggtttcctt cagatcgacg cccttggagt cgcccatcat gttgacgacg 32 100 acgccggtat agccagcggg aaccttggag caacctgcgg tgattgcgag ggcagcaatg 32160 gccaggaggc cgaggatttt cttcataggg attccttact tgtagagaag aggggtgatg 32220 tagaccatgc acaatgcgat gaacatgacg aacatcagta catagaagcc ccaatcatgc 32280 gggggcttac ctgggtggcc cagcaggccg tctatcttat gtatgttgtc gtagaggtcg 32340 gaggtatctt ctccgatgcc ctctaaatcc ttttggattg cctcacggag gcggcctcgc 32400 atctgttccg tcatagtaca tcagacctcc acccgtttac ctgagagctt cttggagtag 32460 gaagccatgt ggctagcaac ccagagggag tacaggatgg aaaggaccag gaggccggca 32520 cccatggcgt ttaccatggt gttagccggg gacagcatct ggggtactac ccagaggatg 32580 cttatccatg cggggatagc caggagcatt acccgagtgt agagcttgag gaccattgtt 32640 cttacctctt tcttaagatg aagaagatag taggttagaa ccttaggtat cctagggata 32700 taaagcaacc ccggacacct taaggttatt ataccatact tccagcagaa tgtcaaggcc 32760 tagatgttac agacctttac agagcttgtc gaggtactcc gagtgctcga cggtaccggg 32820 gtagtcctgc aaaccaccga ggtgacgacg gagaacccca tggttgatga ggatttctgc 32880 cgcagccttc acgttgatga cgtggacccg gaccttggtc gagtggttgt cgctggatgc 32940 ccaggggttg tagtggcttc ccatgcggcg gaggatggtg tcggctacca cctgctcgct 33000 gttcaccgtg aagaccatcg taccgtggcc ctcgttggat tcacgacgga tagcgccgat 33060 cagttccttg gcgaaaccac gggggctgaa cccgttggat gcgaggccgg cgtaatggtc 33120 agcggtaccc gagggtccca tgccgatgta gatttttgcg gtacagcagg agggaaagtc 33180 tctgcggtgc attagtaatc tcctttgatt agtcgtttgg ttgacttgat gattgaaacc 33240 aagggttgat agataacaac cacgaggcac atgtacggaa gggatactgg ccacaaggct 33300 cccaggatgg ttgagatatc cctttccttc tggctcaact cacctgagtg cgagtcgcaa 33360 acagcaagac cgacgatacc agctacgagt agtgctatcg ccatgtagat caggaggaac 33420 acgatgaaca ggagtatgta gatcatggct tcatcactcc gtcaatgcca gcgccacaag 33480 cactgacaca acaaagaaga ccagggggag tccgatgatg aacaggaaca gagaggtcat 33540 ggtttcatta ccctcctctt aacatcacca cagattttac actgtaggtc gtaatagaca 33600 gattcgccta ccacccgacc tttgtgcatg aagtcgaagg gaccagtcct gatgatcttg 33660 taatcgtgga ccccgaagaa gcaccccagc cgaccgaaga ccatcaccaa tccctcgcgc 33720 agttgcaaca acgaccctct ttgggaccga agactggaat gcagtggggg caaggctccc 33780 cggattggat catagcctcc ctggcgtggc gcaggtaagc cgatcggctg gactccatac 33840 ctacggcagc caaggtgaaa ccaagctcac cacggggctc ctgggggcct tcctggaggc 33900 tcatgttgaa cccccgcttt tctacacgac gcttcatgcg gcgctcctgg gccattatct 33960 ggccgatcat gggtttattc ttcatacggc ccccacatac aggaattcac cgacaaccat 34020 ttggccggga atagctcaag gtgagtaccg atttccttgc acttgaggat gttgaagcca 34080 tccttgtgga tgatggtaac gagttcgccc ttcttgaggt aaaccaggtc gccagacccg 34140 gaatcacccg aatggacgta ggcaccatcc gccatctggc tgatgtggaa ttcaaccctt 34200 ggctttgcta cagacacgct gaaagaagta ttggtcatgg cgtcacctta ctgaacccga 34260 tgaacgatgt ggtccaccgg gatgccgagt tcgaagaagg tttcggtgat gaactgcccg 34320 aaattgatga cctttacgtg gcgctcaccg tccttgctta ccaaccagaa aacctggggt 34380 ttagtggagt tcatagacga tacctacacg tttgccgaag atagatacac ggatgtgggt 34440 cacgtaccaa ccagggtacg ccttggggtt aggctggaac ctgcggatgc tgaccaagga 34500 ttccaaccag aggaacagat acagggcggg cgtcatagta cactagaccc tcacggtcat 34560 ttgagcgtcc accgggatga cctcgcagtc gctatggatg ctgcaaatga taccagcctc 34620 caggcaaacg aagtaaacat caccgtcatt ggggaggtag tggttgtggt catcccgaag 34680 gaccatccca tggatgttct caacacggaa aacatctgta gccttgaggg tatgcgccgg 34740 aaccttcttg ggcttttcta ccacattaat cttcatcgga ttacctcacg atgcaaaagg 34800 gatgctgtcc aaggtcgagg acaggacgaa gagaaccacc cacactacca gcaacaaccc 34860 attggaacgt gagcgtatgg cggcacatgc aatatacttc catgccatca ccgcgccctg 34920 ttcttaacca aagatgcccg cttggcacgg agcttgttca actccagggt tttaccaagg 34980 tggctaccat tggtattggt aagctcacga gttaccttgg caatcttgcg gtcgatggcc 35040 tcgatggctt gcatgttgcg gcttacctta cgatagtcgt tgcgtttcac gggatagttt 35 100 cctttgttga tttcccaaga ctccccagcc tgcgaccata ggtccggtgg gggtttcggc 35 160 caggaatcac catggcctca tcagttgggt cagttgatac ccatctcttc cttccagtcg 35 220 cggaagaaga agtatccgat gcctacacaa gacaccacga tagctatcgt catcgcttca 35280 acctcttaat caattcttgc tgttcttgct tccaccgggg atcgggatgg accctcagca 35340 tcctagtgag cttctgcaac cgattaacca tcagtgccct ttcgtgggcg gcttcgtagg 35400 gggacttcat ttggtccacc cgcagaggcc catgcaacgg ccttccacat agacaccaca 35460 ggcgaactta ccatcgacga aagcccaacg gtaaaccacc tgttcaaccc cattgctatt 35520 gtacgaacgg gagttacaac gacctctcac gtaatcgttt gtaacagcta ccagaaagct 35580 accaaaaggg gtccacacgg ttttatcctc ggttagttca tccatacaat acacggacct 35640 accatgtact ctagggagga accaaggggc gcgtcacagt acgcccctgg ccctgtccgt 35700 taggccgcct tcgtaggctc ttcgaacttc agcgccttga tctgtacgat acactcgtcg 35760 tgctcttcca gttggccctt gtcgatggcg cgttggatct tggcacggaa ggcagcgatg 35820 gccgcgtcga gggagaactc gtcggcaacg tcacgttcct tgcggtatcc gaaccacggc 35880 ttttcggtgg cacgctccag gttagtagcc ttgctgccgt cgtacacgaa ggggaactcg 35940 gccgacttgt tcttgtccga ctgagccttg atcttgccgt gatcgatgaa ccacgaggcc 36000 agagcgttgc ggcgggagcc cttgggcatg gccttgaaca gcttgcatgc caacgagact 36060 tcaccatgct cgtcgatgtg cttgagaatg tccaggccgg tagtttgaat cagcttgtcc 36120 agggatgcac cacgctgggc gatggtcttg atgatcccat tgatacgctt gtagtcgctc 36180 atagcttata cctctggttt gatgtgggtg acaggggagt tcttgcactt gctaggcttg 36240 taccaattgc gtagcttagt gtcctcgcgg ttacgccaat cggtattcat cagagccttt 36300 cgcttgcgtg ctccagcctt ttcctgaagg gaaacatact tgctgtccag gatcttgtta 36360 caattggaaa tgatcccgaa cttctgttct gcccgctgct gagctttgcc aaggtgccca 36420 atgttaatac cttgagcacg agccttgcgg ctagccttgc ggcgcagctt cttatcgata 36480 gcagcgttca tcgtttcatc cttttggaat catcagccgg gacacgcatc ccgatacgat 36540 aaaggggtta gtcggcaaac cttggcattg gcgtagccgg cccctttccg cgttagtatc 36600 gctgttatgg tctgtcctct ggctatcctc gggggtcttc gtgtctactc gcccctacct 36660 tcgccagtat ctaccaagct tcgccctctg cgctaagcca ttgatactaa cccaacatac 36720 tgcccatcac gattccacca tgggctctgc taattgttta cgctggagca ggcaaccagt 36780 cggatagaca taaggcggcg tctatcaaag ccagcgctct aaaggagtat gcgctattcc 36840 ctcagctttc gggtttctcc cctgttcagt ccaaggaccg tcgagggtac actttcccca 36900 ggtggccgtc acctgatggg tgcccatgtt agagccttgg gcttggcttg tcaacccttt 36960 gacgggggtt agttcgtcca gttctctggc ataggctctt ttgctgagtg gccatcagtc 37020 ttgcttgtca aggctacgct aatccctaag gagtcatgca cgttatgccg ttgacgtggg 37080 gcgaactata ccgcttcacc gatcccttgt caacccccga tacctgccgg ctttacactt 37140 gggcgacttg ggcgctcttc cctggttggc agcccccgga tagcactgcg gcaaggggga 37200 tggtggccga ggtagaacgt gggggccatt agacccggtt ccgcggggtt tgtcaagccc 37260 ctaggggttt cccccgatgc ctatcggttt ccctcaaggc ggagccatta ggccatattc 37320 tttacggggt tgtctactgg tttttcatac agtctaagga ctatcagtcc cccagcaaac 37380 ccccggatat gaacagtctt tgactaacat agggtatcaa cggaagtcaa taggggatca 37440 aggggttatc aagggatatt cccccggtct ccccctggac tcccctaaga tttttcttta 37500 ggtctatccg atgaacggta tctctggggg ttgacccccg gactagcctt atggtatcct 37560 agggaggggc taggggctat acccctgggg ataggccacc cccactttat ctgggggact 37620 cctgggggga tattccccag atacaccgga ggataaaata gacttttggg gtaacccccg 37680 gttataggcc ctggatcaaa gcacccttcg atacctgttg gtatattact ggataaaggg 37740 gcaataggcc aggacatagc aaacagaggt agtctagggg tatacctatg gaacaacctc 37800 agggagtacc tatagaacct ggatcaaggg aaccctatgc cacttacccc ttaccagcaa 37860 cctacgaagg gtagtcttag ggtatatcta tagaacccta tcataccgtt cgtcgggtaa 37920 atcatgtgat tatcaaagaa atatcaaaaa gtacttgaca aggtatccaa aatgtagtat 37980 aatagaccta taggctctta agattccttc ttccttttga cttacttact aagaaggtta 38040 gttaccttaa ggttatctta agatagtctt aagaaccact tccccattcc agcaaaaccc 38 100 ctaacaaatc cgggtcttgc gactaaaaat aaaaaagatg aaaaagggct tgacaaaata 38 160 gaaaaagtgt gatataatag tattataagg ttgagaaatt agccttaatg gtgctttaga 38220 attcattggg atgtagctca gttggtagag cagggagctg ttaactctca ggtcgtaggt 38280 tcgagtccta ccgtcccagc cattatggcc cattaactca gcggttagag tgctcccctg 38340 tctagggaga agccgtcggt tcgaatccga catgggtcgc caacaaaggt cgttgtagag 38400 gtagatggct actaccatcc ggctgtaacc cggacgccat aggctagagg ttcaattcct 38460 tccttcgacc accattaact cctcgtagct cagttggtta gagtgcctgt cttggacaca 38520 ggaggtcgta ggttcgagtc ctaccggggt gaccatttag agagatcgga gcagtgtatg 38580 tcagacgaaa aggttgtctc gattggggct gctcctcttt ctgctaaaga aaagctagac 38640 ctctattgtg aggctctggc agatggtatg aacaaaaccc aagcatatat agccgctggg 38700 ttctctccta atcatgcgca acgcaacgta gctgcatacc atcgaaagca tgccgaatac 38760 atcaacgcct ttatctctga gcgcatcggg agtcatgtcc ctatggcgct tcgtgtggtt 38820 gtagagatcg ctagcaaccc agaagagaaa ggtgggattc gcctcaaggc ggcccaggat 38880 atcctggatc gtggaggctt cggagctaag cagaaactgg agcttactac taagaatgtg 38940 gaagatatgt ccgcagagga tctaatcagt gaagttcgac gaattctcga cgaagagcca 39000 gagttggcta aagtcatttc tttcccagcc tcttaagtac cttgatctaa tcaagcgaag 39060 agaggggttg gtactaaagt ggtacaaaga ctccctcggg aaagtaactg gtggttatgg 39120 ccacttgcaa ctccctgggg aagacggacc gattacccta gcacgagcag agacttggct 39180 agaaaaggat agtaaggcag cctatgatgc agcccaaggg caagtatctc agctgccttt 39240 ttgcactcca gagctattcg atgccctcgt tagcgtcaat ttccagctgg gtacagcgtg 39300 gacgaagaag ttccccaaga cgtggaatct tctgaaatcc ggaaggtttg atgaggctgc 39360 atgggaggca gaggatagcc tctgggctaa acaaactcca gtgcgcgtaa gggacctcca 39420 acgggccctg tggcgtgcat caacagtagg tgacaaggtg gtctaatgga tacccaagag 39480 cggctacgga atctagtcag ggagttggcg gagcggcaga agtatttccg catgaaccag 39540 tatactcctt atgggtggca agagaagttt atcgcagctt cctcgaactg cgcccagttg 39600 ctggctatga ctggtaaccg ctgtggtaaa acctacaccg gggcctttat catggcctgc 39660 cacctcaccg gtcgctaccc tgagtggtgg actggtagaa agtatgatcg tccagtgaac 39720 tgctgggcgg cggggatctc cacggatacc actcgggata ttcttcagtc cgaactacta 39780 ggtgactgga agaaccctga ggcttttgga acggggatga tccccaagga agatatcgta 39840 gagacgattc gtagggaagg taaacccgga tgtgtgcaag cagtagttgt taagcatgcc 39900 tccgggggcc tctcgtccct tatcttcaag tcctacgaaa tgtcccagga caaattcatg 39960 ggtactgcca tcgacgtcat ctggctcgat gaggaatgcc ccaaggatat ttatacccag 40020 tgtgtaaccc gaacggctac tactgggggt attgtatatc tgacgtttac cccagagcat 40080 ggtctgacgg agatcgtcaa ggacttcctc caggatctta aacctggtca gttccttgtc 40140 catgcaagct gggaagacgc tccacacctc agtccagaag ttaaagagca gctactctcg 40200 gtatactctc cagcagaacg caggatgagg gccgagggtg ttcctatgct cggatctggt 40260 gtagtcttcc ccattctgga agagaagttt gtatgtgagc ctttccagat ccccgatcac 40320 ttccatagga tcatcggtat cgacctcggg tttgaccacc ctaacgctat cgcctgtgtg 40380 gcctgggacc ctgagaaaga caaatactac ctctatgatg agagaagtga gagtggtgag 40440 acccttggga tgcacgctga tgctatctac ctgaagggtg gtcaccagat cccggtagtt 40500 gtcccccacg atgcctttaa gcacgatgga gcaacctctg gtcgtagatt cgtagacctt 40560 cttaaagatg accacaacct caatgtagtg tatgagccct tcagtaaccc accgggtcct 40620 gatggtaaac acggaggtaa ctctgtagag ttcggtgtta actggatgtt gacccgtatg 40680 gagaatggtg atctgaaggt gtttaatacg tgtacgaact tcctaaaaga aatgaaaatg 40740 taccaccgaa aggacggaaa gattatcgac agaaacgacg atatgatatc cgctacccgg 40800 tatgccctgt tgatggcttc ccggcatgca cgtcctggtg ctgtacgaaa cagtggatac 40860 tacaggagtg atactgcaaa gttgacccct gattggtttg ggagtattgt ctaatggcta 40920 agcgtcgtcg caagattaag cctatggatg atgaacaggt acttcgtcat ctagaccaac 40980 ttgttaacga cgcccttgat ttcaactctt cggaactttc caagcagcgt tctgaggccc 41040 tgaagtatta cttcggtgag cccttcggta atgagcgccc tgggaagtcc gcgattgtat 41100 ctagggacgt ccaagagact gtagactgga ttatgccttc tcttatgaag gtattcacgt 41160 caggcggtca agtagttaag tatgaacctc agactgccga ggatgttgaa caggcagagc 41220 aagagactga atatgtgaac tacctcttca tgcgtaagaa cgaggggttc aaggtaatgt 41280 tcgattggtt ccaagacact ctgatgatga agaccggggt tgtaaaggtc tacgtggaag 41340 aggtcctgaa ccccaccttc gaacgattct ctggtctctc tgaggaaatg gtagcggata 41400 tcctggctga tccagacact gagattctag cacagagtgt ggacgaggat ggaacctaca 41460 gtattaaaat tcgcaaggac aagaagaagc gagagattaa agtcacctgt atcaagcctg 41520 agaacttcct ggttgatcgg ttggctacct gcattgatga tgcacgcttc ctctgtcacc 41580 gtgagaagta taccgtaagt gacctgaggc tcttgggtgt tcccgaggat gtactagatg 41640 agcttccata cgatgagtat gaattctctg atagtcagcc agaaaggttg gtacgtgata 41700 acttcgatat gactggccaa ctccagtaca actctgggga tgatgctgaa gccaaccgtg 41760 aggtatgggc ctccgagtgc tacacccttc tggacgtaga tggggatggt atctctgagt 41820 tgcgccgtat cctgtacgtg ggcgactaca tcatcagcaa cgagccttgg gattgccgtc 41880 ccttcgctga cctgaatgcc tatcgaattg cccataagtt ccacgggatg agtgtctacg 41940 ataagatccg agacattcaa gagatccgtt cggtgctcat gcgcaacatc atggacaata 42000 tctaccggac caaccaaggg cgatctgtag tcctggatgg ccaggttaac ctcgaagact 42060 tgctgaccaa cgaggcagcg ggtatcgttc gggttaaggc catgaactct atcatgcctc 42120 tggagacccc tcagctgtct ggggaagtct acggtatgct agataggcta gaggctgata 42180 ggggtaagcg gacaggcatc actgatcgta cccgaggcct agaccaaaac accctacact 42240 ctaaccaagc ggctatgagt gttaaccagt tgatgactgc tgctgagcaa cagattgacc 42300 tgattgcccg gatgtttgca gagactggtg ttaagcgttt gttccaactt ctgcatgacc 42360 atgccatcaa gtaccagaat caggaagagg tcttccagct acgaggcaag tgggttgcta 42420 ttaaccctgc caactggcga gagagatccg acctgacggt taccgttggt attggtaaca 42480 tgaacaaaga ccagcagatg ctccacctta tgcgtatctg ggaaatggcc caggctgtag 42540 tagggggtgg tggtcttggg gtccttgtct ctgagcagaa cctttataat atccttaagg 42600 aagtcacaga gaacgctggg tacaaagacc cggatcgctt ctggactaac ccggattctc 42660 ctgaagctca acgggctaag gccatcagag agcagaagga agcacaacct aagcctgaag 42720 atatcaaagc ccaagctgat gcccaacgtg cccaatcgga cgctatggct aagcaagcgg 42780 aggcccagat gaagcaggta gaggctcaga tccgactggc tgagatcgaa ctgaagaagc 42840 aagaggctgt ccttcagcag agagaaatgg cccttaagga agcggaacta caacttgagc 42900 gtgatcgttt cacttgggaa cgggctcgta acgaagcgga gtatcatctt gaggcaactc 42960 aggctcgggc tgcttatatc ggagacggta aggtacctga gactaagaag ccctcgaagg 43020 cagtgaggaa ataatcttgg gagaacgttt tggctatagt ctagtgctga ctgatgcggc 43080 acaacagatg gtacgtgaga acgttcttcc tgagcttttc cgtattgtcc aagaagagat 43140 cgagggagag tggaagtcca cggcacctaa ggattcagaa tccagggagg ccatctacca 43200 cgaactacat gccctcaacc gcgtacaact tcgtattcag gctatcctgg attcgatcac 43260 tctaaaggag tataagtaat ggaccttgag aatcaaggca tggatgaatt cgaggcagcg 43320 gaagccttcg gagatttgct aggtgatgac ctcctggccc ccgtgatgaa accgtggatg 43380 gtaaagtcgt aggggaagaa gaggaggaag aggtagtaga cgaaggtact ccagaggaca 43440 ccgaagaggt tgagtccgag gaggaagaag agggttctga ggaagatccc gaggttgagt 43500 ccgaggaaga tcctgaggat aaactcttcg agatcccgat tggggatgaa gtctacgagg 43560 ttaacttcga agagcttaag agtggctacc ttcgcaatga agagttcgta actcgacaag 43620 ctgaactaga ggatcagtat gcagctaagt tcgaagagct tgatgctgaa cgctctaatc 43680 tactggccga gcttgagcag tatgctgtta ccgctatcgc tggtgctaat cagtatgaca 43740 atgtaaactg ggagcagctt aaggcccagg atccagagaa gtatcaaacc cttcgactgg 43800 aagccctgga agcccgagac cgagcccagg ctcttatcaa acgtcgtaac gacattaagg 43860 ctatgcaaga gaagcgagcc gagattgtcc actctgccta cgttaagcgt cagacagagc 43920 tagccaagaa acttatcccg gaaatgacta cggatgagtc ttggggtgac aagattgtgt 43980 cctacggcaa atcgattgga tactccgagg atgagatccg agggatatct gatgcccgcc 44040 aactggctgt actcgatgca gcacgtaagt gggctgaatc tcaggtccgt cgcaaggctg 44 100 ccctagagaa gaaggaggag actgaactac ctgctgctgt taagcctgta gctcgtaggg 44160 cggaagcctc agagggttct aagcgggtta aagctgcccg tgctaatctg aggaaagacc 44220 agtcggttga agccgctgct gctctcttct cctctctcga cattttctaa tagaaggaag 44280 tacattatat ggcaactcca actaatgccg tctccactgt agagatcaac ggtaagcgcg 44340 aagaccttat cgatatcatc tacaacatcg caccctatga caccccgttc atgactgcta 44400 tcggcaaggg cgtagctacc gctatcaccc acgagtggca gactgatgaa ctccgccagc 44460 cgggtaagaa cacccgagtt gaaggtgagg atgcaaccat caaggctggt agcttcacca 44520 ccatgttgaa caactactgc caaatctcgg acgagaccct gcaagtcacc ggtaccgcag 44580 ataaggtcaa gaaggctggt cgtaagaacg aactggcgta tcagctggct aagaagtcga 44640 aggaactgaa gctggacatg gaatacgcca tggtcggtgc ccctcaggct aagatccagc 44700 gtaacaccac tactccgggt cagatggcaa atatctttgc ctactacaag accaacggtt 44760 cggtaggtgc taacggtacc ctgccaactg gtgatggttc cgacactggt accgctggtg 44820 acctccgtct tctgaccgaa gacatgctcc tgaatgcctc tgaggctatc tggcgcaacg 44880 gtggtcaggc taactcgatc cagacttcga gctcgatcaa gaaggccatc agcaagaaca 44940 tgaagggtcg tgcaactgag atcaccctgg acgcctcgga caaccgcatt gcgcagaccg 45000 tggacgtcta cgagagtgac ttcggtaagt acaccattcg tgccaaccgc tggttccacg 45060 aaaacaccat gttcatcttt gatccgaaga tgcacgccct ctgctacctg cgtccgttct 45120 tccagcatga actggctaag accggtgaca gcgagaagcg ccagctgctg gttgagtaca 45180 ccctgcgtgt aaacaacgag aagtccggtg ctc 45213

Claims (5)

A grapebery versus bacteriophage Pse-AEP-3 (Accession No. KCTC 13165BP) isolated from nature, having the ability to specifically kill Pseudomonas aeruginosa and having the genome of SEQ ID NO: 1. A composition for preventing or treating diseases caused by P. aeruginosa, comprising the bacteriophage Pse-AEP-3 (Accession No. KCTC 13165BP) of claim 1 as an active ingredient. 3. The composition for preventing or treating diseases caused by Pseudomonas aeruginosa according to claim 2, wherein the composition is used in the form of a pharmaceutical composition containing a disinfectant and an antibiotic. A method for preventing or preventing diseases caused by Pseudomonas aeruginosa including the step of administering a composition containing the bacteriophage Pse-AEP-3 (Accession No. KCTC 13165BP) according to claim 2 or 3 as an active ingredient to an animal other than man. How to cure. The method according to claim 4, wherein the composition is administered to an animal other than a human being in the form of a pharmaceutical composition containing a disinfectant and an antibiotic, to prevent or treat a disease caused by P. aeruginosa.

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