CN108179136A - Coliphage Esc-COP-9 and its enteropathogenic E. Coli Proliferation Ability purposes - Google Patents

Abstract

The present invention relates to a long-tailed virus family phage Esc-COP-9 (entrusted number: KCTC 13131BP) isolated from nature, characterized in that it carries a genome with the ability to specifically kill Escherichia coli and is marked with sequence number 1, And the present invention also relates to a method for preventing and treating pathogenic Escherichia coli infection by using the composition containing the phage as an active ingredient.

Description

Escherichia coliphage Esc-COP-9 and its pathogenic Escherichia coli proliferation inhibition use

technical field

The invention relates to a method for isolating bacteriophage capable of infecting and killing Escherichia coli from natural environment and using a composition containing the phage as an active ingredient to prevent and treat pathogenic Escherichia coli infection. Specifically, it relates to a long-tailed virus family phage Esc-COP-9 (entrusted number: KCTC 13131BP) that is characterized by carrying a genome that has the ability to specifically kill Escherichia coli and is marked with sequence number 1. And a method for preventing infection of pathogenic Escherichia coli and post-infection treatment using the composition containing the phage as an active ingredient.

Background technique

Escherichia coli belongs to the family Enterobacteriaceae, and most of them decompose lactose as Gram-negative bacilli, catalase-positive, oxidase-negative, facultative anaerobic bacteria. Escherichia coli is serologically classified into cell body (O), flagellar (H), and capsular (K) antigens, and these antigens are known to be involved in the pathogenicity of Escherichia coli. Pathogenic Escherichia coli refers to a small number of Escherichia coli that acquire virality, and is generally divided into enterohaemorrhagic Escherichia coli, enterotoxigenic Escherichia coli, enteroinvasive Escherichia coli, enteroinvasive Escherichia coli, There are 5 types of pathogenic Escherichia coli and enteroaggregative Escherichia coli. Pathogenic Escherichia coli can infect domestic animals of various ages and make them sick. The main symptom is diarrhea, and the case fatality rate due to extremely severe dehydration is also very high. The diarrheal disease caused by this pathogenic Escherichia coli is a major disease that spreads among almost all farmers in my country, and the loss brought to the animal husbandry industry is quite large.

Usually, methods for preventing and treating infectious diseases caused by pathogenic Escherichia coli include vaccines and antibiotics. However, treatment with antibiotics continues to be less effective due to the rise of resistant bacteria. At the same time, due to the strengthening of regulations on the use of antibiotics in animals, it is urgent to develop an effective method other than antibiotics.

Recently, the utilization of bacteriophages has attracted great attention as a countermeasure against bacterial diseases. In particular, attention to phages is higher than ever due to a pro-environmental approach. Phages are extremely small microorganisms that infect bacteria, and are often referred to as antibiotics (Phage) for short. After the bacteriophage infects the bacterium, it proliferates in the cell of the bacterium. After proliferating, when the progeny phage dissociates from the bacterium, it kills the bacterium by destroying the cell wall of the host, that is, the bacterium. The bacterial infection mode of phages is highly specific, so the types of phages that can infect specific bacteria are limited to only a part. That is, a specific phage can only infect a specific range of bacteria. Therefore, specific phages can only kill specific bacteria and have no effect on other bacteria. The bacterial specificity of this phage will only have an antibacterial effect on the target bacteria, and will not affect the normal flora in the environment or in animals. Typically, existing antibiotics that are widely used for bacterial disposal affect multiple bacteria at the same time. Therefore, problems such as environmental contamination or interference with the normal bacterial flora of animals are caused. In contrast, since phages are only effective against specific bacteria, the use of phages does not cause problems such as disturbing the normal flora in the body. Therefore, compared with the use of antibiotics, the use of phages is not only safer, but also the possibility of side effects during use is relatively low.

Phages were discovered in 1915 by the British bacteriologist Twort when he was researching the cause of the phenomenon that Staphylococcus (Micrococcus) colonies became transparent after being dissolved by a certain substance. In addition, in 1917, the French bacteriologist d'Herelle discovered that there was a substance that dissolved Shigella dysenteriae in the stool filtrate of dysentery patients, and through research on this, he independently discovered bacteriophage, and from the fact that it can swallow bacteria Starting from a meaning, it was named "bacteriophage". Since then, a variety of phages targeting pathogenic bacteria such as Shigella, Salmonella typhi, and cholera have been discovered one after another.

Because phages have the special ability to kill bacteria, after the discovery of phages, researchers are looking forward to developing an effective solution to bacterial infections, and have carried out a lot of related research. However, after Fleming discovered penicillin, with the widespread use of antibiotics, research on phages was only carried out in some Eastern European countries and the former Soviet Union, maintaining a glimmer of life. However, after 2000, due to the increase of antibiotic-resistant bacteria, the limitations of existing antibiotics were discovered. Phages are receiving renewed attention as antimicrobial agents, with the potential to develop alternatives to existing antibiotics. In particular, with the recent strengthening of regulations on the use of antibiotics at the government level on a global scale, people's attention to bacteriophages has increased, and cases of their use in industries have gradually increased.

As mentioned earlier, phages are very specific for bacteria. It is precisely because of this specificity that even for bacteria belonging to the same species, phages often only exert antibacterial effects on a part of their strains. In addition, depending on the strain of the target strain, the strength of the antibacterial effect of the bacteriophage itself will also vary. Therefore, in order to obtain effective control methods for specific types of bacteria, it is necessary to ensure that there are a variety of useful phages. In order to develop effective phage utilization methods against pathogenic Escherichia coli, it is of course necessary to secure a variety of phages that can provide antibacterial effects against Escherichia coli. Furthermore, among the ensured variety of useful phages, it is also necessary to screen and utilize phages with comparative advantages in terms of antibacterial force strength or antibacterial range.

Contents of the invention

The problem to be solved by the invention

Therefore, the present inventors have devoted themselves to developing a composition useful for preventing or treating pathogenic Escherichia coli infection, which utilizes bacteriophages isolated from natural environments capable of selectively killing Escherichia coli, and using the composition to develop The method for preventing or disposing of pathogenic Escherichia coli infection, through unremitting efforts, finally isolated the bacteriophage meeting the above conditions from the natural environment. At the same time, in order to be able to distinguish the isolated phage from other phages and to specify its properties, after ensuring the gene sequence of its genome (Genome), a composition using the phage as an active ingredient was developed, and it was confirmed that the composition can It is effective for preventing and treating pathogenic Escherichia coli infection, thus completing the present invention.

Therefore, the object of the present invention is to provide a long-tailed virus family (Siphoviridae) phage Esc-COP-9 (trustee number: KCTC 13131BP) isolated from the natural environment, characterized in that it carries a genome marked with SEQ ID NO: 1 , has the ability to specifically kill Escherichia coli.

Another object of the present invention is to provide a composition and a method for preventing pathogenic Escherichia coli infection using the composition. The composition uses the phage Esc-COP- 9 is included as an active ingredient, and the composition can be used to prevent infection by pathogenic Escherichia coli.

Another object of the present invention is to provide a composition and a method for treating pathogenic Escherichia coli infection using the composition. The composition uses the phage Esc-COP- 9 is contained in the active ingredient, and the composition can be used to treat the infection of pathogenic Escherichia coli.

Another object of the present invention is to provide a disinfectant using the composition for the purpose of preventing and treating pathogenic Escherichia coli infection.

Another object of the present invention is to provide a drinking water additive using the composition for the purpose of preventing and treating pathogenic Escherichia coli infection.

Another object of the present invention is to provide a feed additive for improving feeding efficiency by preventing and treating pathogenic Escherichia coli infection using the composition.

problem solving method

The present invention provides a long-tailed virus family phage Esc-COP-9 (entrusted number: KCTC 13131BP) isolated from the natural environment, which is characterized in that it carries the ability to specifically kill Escherichia coli and is labeled with sequence number 1 genome. At the same time, the present invention also provides a method for preventing and treating pathogenic Escherichia coli infection using the composition containing the phage as an active ingredient.

Phage Esc-COP-9 was deposited in Korea Research Institute of Bioscience and Biotechnology (trustee number: KCTC 13131BP) on October 17, 2016 after being isolated by the inventor.

In addition, the present invention provides a disinfectant, drinking water additive and feed additive, which contain bacteriophage Esc-COP-9 that can be used to prevent and treat pathogenic Escherichia coli infection as an active ingredient.

The bacteriophage Esc-COP-9 contained in the composition of the present invention can effectively kill Escherichia coli, and thus has an effect on the prevention (prevention of infection) or treatment (treatment of infection) of diseases induced by pathogenic Escherichia coli. Therefore, the composition of the present invention can be used for the prevention and treatment of diseases induced by pathogenic Escherichia coli.

The meanings of terms such as "prevention" or "prevention" used in this specification are: (i) preventing pathogenic Escherichia coli infection; (ii) inhibiting pathogenic Escherichia coli infection from developing into a disease.

The meanings of terms such as "treatment" or "treatment" used in this specification: (i) suppress the disease induced by pathogenic E. coli; (ii) alleviating the condition of the disease induced by pathogenic E. coli Behavior.

"Isolation", "isolated" or "isolated" in this specification refers to the use of various experimental methods to isolate phage from the natural state; and to ensure that it can be distinguished from other phages to specify the characteristics of the phages . At the same time, it also includes the use of bioengineering technology to multiply the phage so as to be used in the industrial field.

The pharmaceutically acceptable carriers contained in the composition of the present invention are substances commonly used in the production of preparations, including but not limited to: lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, Alginate, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methylcellulose, Methylparaben, Propylparaben, Talc, Stearin Magnesium acid and mineral oil, etc. In addition to the above components, the composition of the present invention may also include lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives and the like.

The composition of the present invention contains phage Esc-COP-9 as an active ingredient. Wherein the phage Esc-COP-9 contains 1×10 ¹ pfu/ml～1×10 ³⁰ pfu/ml or 1×10 ¹ pfu/g～1×10 ³⁰ pfu/g. Preferably, it contains 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 utilizes pharmaceutically acceptable carriers and/or excipients to make preparations according to the method easily implemented by those skilled in the art of the present invention. Manufactured in containers of high capacity. Its dosage form can be in the form of solution, suspension or emulsion in oil or aqueous medium, or in the form of extract, powder, granule, tablet or capsule, and can also contain dispersant or stabilizer.

The composition of the present invention can be implemented as a disinfectant, drinking water additive and feed additive according to different usage methods, but it is not limited thereto.

In order to increase the efficiency in this purpose of utilization, bacteriophages that provide antibacterial activity against other bacterial species can also be added to the composition of the present invention. In addition, other kinds of phages having antibacterial activity against Escherichia coli may also be added. Even phages that have antibacterial activity against Escherichia coli differ from each other in terms of the strength of the antibacterial power or the range of antibacterial activity. Therefore, proper combination of them can maximize their effectiveness.

Invention effect

Compared with the existing methods based on chemical substances such as antibiotics, the method of preventing and treating pathogenic Escherichia coli using the composition comprising the bacteriophage Esc-COP-9 of the present invention as an active ingredient has the advantage of being resistant to Escherichia coli The specificity is very high. This means that it will not affect other useful normal flora, and can also be used to prevent or treat pathogenic Escherichia coli infection, while the side effects produced by it are very small. Usually, if chemicals such as antibiotics are used, they also cause damage to the normal normal flora, which eventually leads to reduced immunity of the animal, thus showing various side effects with the use. In addition, because the present invention isolates the bacteriophage already existing in nature as the active ingredient of the composition, it also has the advantages of being very close to nature and environment-friendly. In addition, even if the strains capable of exerting antibacterial activity among phages are the same, in terms of the exertion of antibacterial effects, in terms of the strength of antibacterial power, or the scope of antibacterial [in the side of various bacterial strains (Strain) belonging to Escherichia coli, phages to The range of antimicrobial activity exerted by individual bacterial strains. Usually, phages can only exert antibacterial activity against a part of bacterial strains belonging to the same species. That is, even if they belong to the same bacterial species, individual bacterial strains have different sensitivities to phages] There are differences at the level. Therefore, the present invention can provide differential antibacterial effects compared with other phages that have antibacterial activity against Escherichia coli. This means that in actual industrial applications, the resulting effects can vary greatly.

Description of drawings

Fig. 1 is an electron micrograph of phage Esc-COP-9.

Figure 2 is the experimental results showing the ability of phage Esc-COP-9 to kill Escherichia coli, and the transparent part is the final lysed plaque formed after the test object bacteria were lysed.

Detailed ways

Hereinafter, the present invention will be described more specifically based on examples. However, the illustrated examples are merely examples of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Isolation of phages that can kill Escherichia coli

When isolating bacteriophages capable of killing Escherichia coli, samples secured from natural environments were used. In addition, Escherichia coli used for phage isolation has been previously isolated by the present inventors and identified as pathogenic Escherichia coli.

Next, the isolation procedure of the phage will be described in detail. Inoculate the TSB (Tryptic Soy Broth) medium (casein digest, 17g/L; soybean digest, 3g/L; glucose, 2.5g/L; NaCl, 5g/L; Dipotassium hydrogen phosphate, 2.5g/L) was added together with the collected sample (sample), and then cultured with shaking at 37°C for 3-4 hours. After culturing, centrifugation was performed at 8,000 rpm for 20 minutes, and the supernatant was collected. Escherichia coli 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. In the case that the phage has been contained in the sample, in order to ensure that the number of phage (Titer) can be increased sufficiently, the above process was repeated 5 times in total. After the above process was repeated 5 times, the culture solution was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered through a 0.45 μm filter. The existence of phage capable of killing Escherichia coli was investigated by a conventional Spot assay using the filtrate thus obtained.

The spot test was carried out as follows. Escherichia coli was inoculated into TSB medium at a ratio of 1/1000, and cultured with shaking at 37° C. overnight. Coli culture fluid 3ml (OD ₆₀₀ being 1.5) thus prepared is smeared (Spreading) to TSA (Tryptic Soy Agar) plate culture medium (casein digest, 15g/L; Soybean digest, 5g/L; NaCl, 5g /L; agar, 15g/L). Place the smeared culture medium on the clean bench for about 30 minutes to dry the smear. After drying, 10 µl of the previously prepared filtrate was spotted onto the culture plate coated with Escherichia coli, and left to dry for about 30 minutes. After drying, the plate culture medium subjected to the above dripping operation was statically cultured at 37° C. for one day. Then, it was checked whether a clear zone (clear zone) was formed at the position where the filtrate was dropped. When a filtrate with a clear ring is formed, it can be judged that it contains bacteriophage capable of killing Escherichia coli. Through this investigation, it was possible to ensure that the filtrate contained phages with killing capacity against E. coli.

Isolation of pure phage was carried out using the filtrate confirmed to contain phage capable of killing Escherichia coli. Pure phages were isolated using conventional plaque assay (Plaque assay) techniques. Specifically, a lysed plaque formed during the lysed plaque analysis was recovered with a sterilized tip, then added to the E. coli culture solution, and incubated together at 37° C. for 4-5 hours. After culturing, centrifuge at 8,000 rpm for 20 minutes to obtain a supernatant. After adding Escherichia coli culture solution to the obtained supernatant according to the volume of 1/50, culture was carried out at 37° C. for 4-5 hours. In order to increase the number of phages, the above process was carried out at least 5 times, and finally centrifuged at 8,000 rpm for 20 minutes to obtain a supernatant. Using the obtained supernatant, the plaque assay was performed again. Usually, since the isolation of pure phage cannot be completed through the above-mentioned one-time process, the entire preceding steps are repeated again using the formed lysed plaque. This process was repeated at least 5 times to ensure a solution containing pure phage. Isolation of pure phage was routinely repeated until plaques formed were all similar in size and shape. The pure isolation of the phage was finally confirmed by electron microscopic analysis. During electron microscopic analysis, the foregoing process was also repeated until pure separation was confirmed. Electron microscopic analysis was carried out by a conventional method, which is briefly described below. The solution containing pure phage was stained on the copper grid, and then negatively stained with 2% uranyl acetate (Uranyl acetate) and dried, and its morphology was observed by transmission electron microscope. The electron micrographs of the isolated pure phages are shown in Figure 1. If judged by their morphological characteristics, it can be confirmed that the obtained novel phages belong to the Siphoviridae phages.

The solution containing pure phage identified in this way was subjected to the following purification process. Add Escherichia coli culture solution to the solution containing pure phage according to the volume accounting for 1/50 of the total volume of the solution, and then incubate for 4-5 hours. After the cultivation, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. This procedure was repeated a total of 5 times in order to obtain a solution containing a sufficient number of phages. After filtering the supernatant obtained through centrifugation with a 0.45 μm filter, a conventional polyethylene glycol (Polyethylene Glycol; PEG) precipitation process was implemented. Specifically, PEG and NaCl were added to 100ml of the filtrate to make it 10% PEG 8000/0.5M NaCl, and then stood at 4°C for 2-3 hours, followed by centrifugation at 8,000rpm for 30 minutes to obtain phage precipitates things. The phage precipitate thus obtained was suspended in 5 ml of buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% gelatin, pH 8.0), and this was called phage suspension or phage solution.

Through the above process, the purified phage was obtained. The phage was named as phage Esc-COP-9. Since then, it has been entrusted to the Korea Institute of Bioscience and Biotechnology (trusted number: KCTC13131BP) on October 17, 2016.

Embodiment 2: Genome isolation and sequence analysis of phage Esc-COP-9

The genome of phage Esc-COP-9 was isolated as follows. The genome was isolated using the phage suspension obtained in the same manner as in Example 1. First, in order to remove DNA and RNA of Escherichia coli that may be contained in the suspension, 200 U of DNase I and RNase A were added to 10 ml of phage suspension, and then left at 37°C for 30 minutes. After standing for 30 minutes, in order to eliminate the activity of DNase I and RNase A, 500 μl of 0.5M ethylenediaminetetraacetic acid (EDTA) was added, and then left still for 10 minutes. Then, it was left still at 65° C. for 10 minutes. Next, in order to disrupt the outer wall of the phage, 100 μl of proteinase K (20 ㎎/ml) was added and reacted at 37° C. for 20 minutes. Then, 500 μl of 10% sodium dodecylsulfate (SDS) was added, and then allowed to react at 65° C. for 1 hour. After reacting for 1 hour, 10 ml of a mixture composed of phenol (Phenol): chloroform (Chloroform): isoamylalcohol (Isoamylalcohol) according to the composition ratio of 25:24:1 was added to the reaction liquid and fully stirred evenly. Then, it was centrifuged at 13,000 rpm for 15 minutes to make layers, and then the upper layer was taken from the separated layer, and 1.5 volume ratio of isopropyl alcohol (Isopropylalcohol) was added thereto, followed by centrifugation at 13,000 rpm for 10 minutes, and the genome precipitation. After recovering the precipitate, 70% ethanol (Ethanol) was added to the precipitate, and then centrifuged at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, vacuum-dried, and then dissolved in 100 µl of water. By repeating the above process, a large number of genomes of phage Esc-COP-9 were obtained.

Using the genome thus obtained at the National Instrumentation Center for Environmental Management of Seoul National University, the next generation sequencing analysis (Next generation sequencing analysis) was performed using the illumina Mi-Seq instrument, and the phage Esc-COP- 9 genome sequence information. Finally, the analyzed phage Esc-COP-9 genome had a size of 51,102 bp, and the entire gene sequence was marked with SEQ ID NO:1.

Based on the obtained phage Esc-COP-9 genome sequence, use BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) on the Internet to compare the similarity between it and the currently known phage genome sequence (Similarity) was investigated. The results of BLAST survey showed that the genome sequence of phage Esc-COP-9 has a relatively high similarity (identity: 93%) to the sequence of Shigella phage pSf-1 (GenBank Accession No. KC710998.1). However, in contrast to phage Esc-COP-9 which has a circular genome, Shigella phage pSf-1 has a linear genome, and the two have a large difference in the morphology of the genome. The number of open reading frames (Open Reading Frame, ORF) on the phage Esc-COP-9 genome is 89, on the contrary, Shigella phage pSf-1 is 94, and it has the same structure as phage Esc-COP -9 The number of different open interpretation frames.

From this fact, it can be concluded that the bacteriophage Esc-COP-9 is a novel phage different from the existing reported phages. At the same time, in general, if the types of phages are different, the intensity and range of antibacterial power they can provide are also different. From this fact, it can be judged that phage Esc-COP-9 can provide antibacterial activity that is different from the existing reported ones. Other phages have different antibacterial effects.

Embodiment 3: the investigation of the ability of phage Esc-COP-9 to kill pathogenic escherichia coli

The ability of the isolated phage Esc-COP-9 to kill pathogenic Escherichia coli was investigated. The killing ability investigation was carried out by investigating whether or not a transparent ring was formed by the spot test proposed in Example 1. The pathogenic Escherichia coli used for the investigation of killing ability was isolated and identified as pathogenic Escherichia coli by the present inventors, and a total of 10 strains were used. The phage Esc-COP-9 has killing ability against a total of 9 strains among the 10 strains of pathogenic Escherichia coli used as experimental subjects, and representative experimental results are shown in FIG. 2 . In addition, the effects of phage Esc-COP-9 on Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Streptococcus mitis, Streptococcus uberis were also investigated. (Streptococcus uberis) and Pseudomonas aeruginosa (Pseudomonas aeruginosa), the results showed that phage Esc-COP-9 did not have the ability to kill these bacteria.

From the above results, it can be known that phage Esc-COP-9 has excellent killing ability against pathogenic Escherichia coli, and can exert antibacterial effect on most pathogenic Escherichia coli. This means that bacteriophage Esc-COP-9 can be used as an active ingredient of a composition for preventing and treating pathogenic Escherichia coli infection.

Embodiment 4: the experimental example of the infection of bacteriophage Esc-COP-9 prevention pathogenic escherichia coli

Add 100 μl of 1×10 ⁸ pfu/ml standard phage Esc-COP-9 solution to one tube containing 9 ml of TSB medium, and add only to the other tube containing 9 ml of TSB medium An equal amount of TSB medium was added. Then, a culture solution of pathogenic Escherichia coli was added to each tube so that the absorbance at 600 nm was about 0.5. After adding pathogenic Escherichia coli, the hose was moved to an incubator at 37°C for shaking culture, and the growth state of pathogenic Escherichia coli was observed. As shown in Table 1, in the hose added with the phage Esc-COP-9 solution, growth inhibition of pathogenic Escherichia coli was observed. In contrast, growth inhibition of pathogenic Escherichia coli was not observed in the tubes to which the phage solution was not added.

【Table 1】

Growth inhibition of pathogenic E. coli

From this result, it can be confirmed that the phage Esc-COP-9 of the present invention can not only hinder the growth of pathogenic Escherichia coli, but also has the ability to kill it. It can be concluded that the phage Esc-COP-9 can be used as an active ingredient in a composition for the purpose of preventing pathogenic Escherichia coli infection.

Embodiment 5: utilize bacteriophage Esc-COP-9 to treat the example of pathogenic escherichia coli infection disease

The therapeutic effect of bacteriophage Esc-COP-9 on pigs that induced disease caused by pathogenic Escherichia coli was investigated. Four weaned piglets 25 days after birth were divided into one group, and divided into two groups in total, and then they were separately raised in the experimental breeding piggery (1.1m×1.0m), and the experiment was carried out for 14 days. The surrounding environment is controlled under the thermal insulation facility to keep the temperature and humidity of the pig house constant, and the ground of the pig house is cleaned every day. On the 7th day from the start of the experiment, all the pigs were orally administered the pathogenic Escherichia coli solution using an oral infusion tube. Oral pathogenic E. coli solutions are prepared as follows. After culturing pathogenic Escherichia coli for 18 hours at 37° C. using TSB medium, only bacterial cells were recovered. Then, it was adjusted to 10 ⁹ CFU/ml with physiological saline (pH 7.2). From the next day after oral administration of pathogenic Escherichia coli, pigs in the experimental group (group injected with phage solution) were orally administered 10 ⁹ PFU of phage Esc-COP-9 twice a day in the same manner as the injection of pathogenic Escherichia coli solution. , while the pigs in the control group (group not injected with phage solution) were not treated. The same feed and drinking water were provided to the control group and the experimental group, and the occurrence of diarrhea was investigated in all test animals every day after the injection of pathogenic Escherichia coli. The investigation of the incidence of diarrhea was carried out by measuring the diarrhea index. The diarrhea index was measured by measuring the conventional fecal consistency (FC) score (normal: 0, soft stool: 1, loose stool: 2, severe diarrhea: 3). The results are shown in Table 2.

【Table 2】

Diarrhea index test results

From this result, it was confirmed that the phage Esc-COP-9 of the present invention is also very effective in treating infectious diseases induced by pathogenic Escherichia coli.

Embodiment 6: the manufacture of feed additive and feed

The phage Esc-COP-9 solution was used to manufacture the feed additive, and it was ensured that every 1 g of the feed additive contained 1×10 ⁹ pfu of the phage Esc-COP-9. The production method of the feed additive is as follows, adding maltodextrin (50%, w/v) to the phage solution, followed by freeze-drying. Finally, it is crushed into a fine powder. The drying process in the manufacturing process can adopt reduced pressure drying, heating drying, and normal temperature drying. For comparative experiments, a feed additive without phage was produced in the following manner, that is, the buffer (buffer; 10 mM Tris-HCl, 10 mM Mg SO ₄ , 0.1% gelatin, pH 8.0) used in the preparation of the phage solution was used instead of the phage solution.

Two types of feed additives produced by the above method were mixed with 1,000 times the pig feed in a weight ratio to prepare two types of feed.

Embodiment 7: the manufacture of drinking water additive and disinfectant

Drinking water additives or disinfectants are only used differently, and their dosage forms are the same. Therefore, both are manufactured in the same way. Use the phage Esc-COP-9 solution to manufacture drinking water additives (or disinfectants), and ensure that every 1ml of drinking water additives (or disinfectants) contains 1×10 ⁹ pfu of phage Esc-COP-9. The manufacturing method of the drinking water additive (or disinfectant) is as follows, according to the method of adding the bacteriophage Esc-COP-9 solution and mixing it thoroughly, ensuring that every 1ml of the buffer used in the manufacture of the phage solution contains 1×10 ⁹ pfu Phage Esc-COP-9. In order to carry out the comparative experiment, the drinking water additive (or disinfectant) without bacteriophage was manufactured in the following manner, that is, the buffer solution itself used in the manufacture of the phage solution was directly used.

The two kinds of drinking water additives (or disinfectants) produced by the above method are diluted with 1,000 times of water according to the volume ratio and then used as final drinking water or disinfectants.

Embodiment 8: confirm the feeding effect of raising pigs

It was investigated whether the feeding effect of raising pigs using the feed, drinking water and disinfectant produced in Examples 6 and 7 was improved. In particular, this survey was carried out from the perspective of case fatality rate. A total of 30 piglets were divided into groups of 10 each into 3 groups in total (group-A fed with feed; group-B supplied with drinking water; group-C treated with disinfection), and the test was carried out for 4 weeks. Each group was subdivided into subgroups consisting of 5 heads, and each subgroup was subdivided into a group using phage Esc-COP-9 (group-①) and a group not using phage (group ②). The piglets of this test are weaned piglets 20 days after birth, and the piglets of each test group are raised in different independent pig houses separated by a certain interval, and each group is distinguished and instructed according to the situation shown in Table 3.

【table 3】

Group distinction and representation in pig feeding experiments

If feed with feed, just use the feed manufactured in embodiment 6 to feed according to the conventional feed feeding mode according to the distinction in Table 3. If feed with drinking water, just use the drinking water manufactured in embodiment 7 to carry out feeding according to the distinction in Table 3 according to conventional drinking water feeding mode. If it is disinfection treatment, it will be disinfected three times a week in turn with the existing disinfection methods. The day of spraying disinfectant of the present invention just need not carry out disinfection with common disinfectant, test result is as shown in table 4.

【Table 4】

It can be confirmed by the above results that feeding with feed and drinking water manufactured according to the present invention and carrying out disinfection treatment according to the present invention can effectively reduce the mortality rate in the process of raising pigs, and it can be concluded that using the combination of the present invention It can effectively improve the performance of pig raising.

In the foregoing description, specific parts of the present invention have been described in detail. Those skilled in the art will understand that the above detailed description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto. Therefore, the actual scope of the present invention should be determined by the terms of the claims and their equivalents.

【Trusted Number】

Trust institution name: KCTC

Trustee number: KCTC 13131BP

Entrusted date: 20161017

<110> Intron Biotechnology Co., Ltd.

<120> Escherichia coli phage Esc-COP-9 and its application of pathogenic Escherichia coli proliferation inhibition

<130> DFP16KR2908

<160> 1

<170> PatentIn version 3.2

<210> 1

<211> 51102

<212>DNA

<213> Bacteriophage Esc-COP-9

<400> 1

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gtgtggcagt aaggccgccg gactcgtgca tatatgagga gggtgatccc gttagtgtag 960

ttagtagagg tagtgtgtgg tgcttaactt ctgagagcga agcgccagaa tatggagata 1020

aagtatttgt aacgcaagat ggagatgcgg catacagtga aggcgacttg ctgattggtt 1080

ggatctttac tggcgaacat gtaaaggttg atcatgagtc atatattgtc ggggtaagca 1140

ttagcgcata acacaaaatt atgttgcacg tttaacaaaa aatgctatca ttggggcgtt 1200

acatactaac agcctttata tatggaggaa gaaaatggaaaaagaaaaat ttgatcagct 1260

agatgcggat attgtttcta gctatctggc tactcgcggc gttaagggtg atgcttctga 1320

tatgggtatc tggacggcgc aagagcttca caagatccgc tctaatgctt acgagaaaga 1380

atatccggca ggttcagcgc ttcgtgtatt ccctgtaact aatgagcttt ctgatactga 1440

taagactttt gaatatcaga tgtttgacaa ggttggttac gcaaaaatta tcgctgatta 1500

caccgacgac ctaccgaccg ttgacgcgct gatgtcaagc gaatttggta aagtgttccg 1560

cctgggtaac gcgttcttga tctccattga cgaaatcaag gctggtcagc gtactggcaa 1620

gagcctttcc actcgcaagg caaacgcagc gcaaaacgct catgatcagg aagttaaccg 1680

cctggtgttt aagggttcaa agccgcataa gattctttcg gtgttcgaac atcctaacct 1740

gacaaagatc gtgtcaactg gctggttaag tgctgatgaa aacactaagt tcccgcaggc 1800

agcatctgat gaactggaaa aggctattga aaccattcag accatcacca atggtcagca 1860

ccgagcaacc aacatcctga ttccgccatc tatgcgcaag gttctttccg ttcgaatgga 1920

aaacacccact gaaagttatc tggaatactt ccagaaacaa aacagcggaa tcaccattga 1980

ctcaatcgca gagcttgagg atattgatgg cacaggaacg aaaggttgcc tggtatatga 2040

gaaagacccg atgaacatgt cgatcgaaat tccagaagca tttaacatgc ttcctgcgca 2100

accaaaagat ctgcatttca aagttccgtg tacttcaaaa tgtactggcc tgacgatta 2160

ccgtccgttt acgctagtgc ttattactgg cttgaaaaaa ccaagttaat cgtataacat 2220

tggggaatcc ttaactggat tccctttttt ttatggagg ttatgaaatg gctaaagaaa 2280

aaactgtaac gctaaaggtt tccggcgtgt gccttatcat tgttgatggt gagcattatc 2340

atccaggcaa agagtttgac gtagaagaat caaaattgaa aacaagcgcg tttgaatatc 2400

taattgcaaa aggcgatctt gaggttaaag acaactccgc attgaacgaa gaaatcaaac 2460

agagcgcagc aagcaaacgt aaaaaagacc cgcgagagggg caagagcaaa gccgaacttg 2520

aagacggcgg catctattaa taaagagggc gcttatagcg ccctttgtca tatctggagg 2580

aaacaaaatg gttgatgaat ttttcactga cgcagaaatc acgcagcaaa tcgtgaaatt 2640

agctccaccg atgaaacaga ttgatccaga tttaatggtg gcatggattg atctagcaaa 2700

ggaatttgtt tgcaagaagc ggtttaaaga atcatatcct aaagcggttg ccctttatac 2760

tttgcacctt atgacgcttg atggagctat gaagcaagaa ggggaaggcg tagagagtta 2820

ctcacggcgc gtggcgtcat tttccctgac tggtgaattt agccagacat ttgatcgcgt 2880

gtccggcgat tcaagcggaa aagagattag acaaactccg tggggcaaaa tgtatgagac 2940

gttaaataga aaaaaaggcg gtggatttgg ccttgtgact ggatttcgga ggcgctgcaa 3000

atgaactata acgagatcgc aagaatggca accgaaggaa ttaacttctt tagtgatagc 3060

aacggtgagt ttaagtgcat aacgcagcgt ggaagcgttg aaataatcgg aggcgaagaa 3120

gtacaaaagc cagagattag tgtaatgatt aaaggtttaa ttcgctcacc aaaagtacgc 3180

gaggttgacg gcgagacaat acgagtaaac gataagctgg gagttttcaa caataatgtt 3240

gaaattaaga acgggtatca tattgatgtt gatggtgaat tatatgttgt agttgaagca 3300

agaccaatca gacaaaccaa tgtcacagtt gcttatcggc ctattttgag aaggatttcc 3360

gtgcatggct aaaaactaca cgataaggga atttcatggc aacattgatg cgtggataaa 3420

cgccgttgat aatggactaa aagattgcgt ggaattgttc gccgaaaaag tacacacaga 3480

tttggttaag cgttctcctg tagatactgg tcgttaccgt gcaaactggc aggtaacagc 3540

aaacaatccg ccattgtatg cgcttaacca gtacgataag cacggcgaca aaacgatcgc 3600

ggaaggcaag cgcgccatat acgcaatatt acgaggtggc ggagcagtaa gagccattat 3660

tttttccaat atgcttattt atgctaacgc gcttgagtac ggtcattcaa aacaagcgcc 3720

agcgggtgtg cttgggatg tagctgttaa gttgagatct tatatggctg aagcaataaa 3780

agagtcgagg gctaaaaatg cactatgaat taatgctatc ggcgcgtaag gcgctggcga 3840

ctgaatacga aagcagattc atgatcgctt atgaaaacgt agagtttacg ccacctggtg 3900

acggttcgcc gtggttgaag tttgactatg ccgaagttga cacagaatac ttgtcattgg 3960

atcgcaaatg cgtttcttat atcggaatga ttcaggttgg tattgtgttt ccaccaggtt 4020

acggaactga caggccgcgc gttctcgcta aagagattgc gcaattcttt tacgatggta 4080

aaatgctgga gcatggttat atatatgagg gtgcaagagt tcacaagccg ctcaagagtg 4140

aaagcggttg gattctccca ataaggtttt atgttcgaat tgaaacaaag gagtaaaaat 4200

tatgcattta ccaaatggat cgcaaatttt cgttgagagc aatcgcggaa gcgaaatcca 4260

agcaacagca gtgtcaaacg ccaaagatcc tgttttcact gttgcatctg gcggcactgc 4320

gtacaagaaa ggtgattatg tgatcatcac tgcgtcttct tggggtaagc tgattgatcg 4380

tgttatgcgt gtaaaggcta acggtgaaga aacaagcgtg actcttgaag gcgtagatac 4440

tactgatcag aacgttttcc cgactggcgg cactgcatct tttgccaaaa ttgatgcatg 4500

gactgagatc ccttgcgttc aagatttgtc gcaggacggc ggcgaacagc aatactacac 4560

ttatcagtgt ttggcagacg atcaggaaca gcagttacct acctataaga gcgccgtttc 4620

tcttacctac acttttgcgc acgaatacga taacgcgatc tatccgctat tgcgttcagc 4680

agatgaatca ggcgaagtaa ccgctcttcg catgtatgta ccaaaagcaa aagagatgcg 4740

ttgctgggct ggtgtgcttt cgtttaacga aatcccgcag accacagtta acgaaatgga 4800

aactgtttcc ctttctgtat cactgaaagg tcgattcacc tttctaccct ctcaagtaga 4860

gtaataacaa ggggcattgc gcccctttttttgttactgt acaatcatga tacacgattc 4920

ataatcaatt cttttaacaa aaagtgctat caaggagaaa gaaatgtcaa aaatgaaatt 4980

gacgattggg ccgcttcctg actttaaatt accagtgaaa tttgcaatgc ctaacggaga 5040

agatcaaaca attatcttca cagttcgcca ccgcaaaaca agtgagattc acgagcgcta 5100

cacgtcagat actccaatga gcgatgttga aatgatcacc ttccttgctt ccggttggaa 5160

tcttgatgat gaatttaacg aagaaaatat taaacagctt cttgattact accccagcgac 5220

agcaatcgga ttaactagcg catacatgaa agcgcttgcg gggcagcgag taaaaaacta 5280

aaaagggcgg tttacctgtt ttatcagaaa ccgccgacag atgcagagct tgaggccgtt 5340

ggccttacaa gggcagacta tgaaggagaa gatccgccag aggttatatt tgatgaaagc 5400

atgatgcaat catgggatat attttgcgca atgcaaacgc aatggagatg ttcaggcggt 5460

ggcgcttacg gatttgatta taacgtcttg cctatgcttt ttgagatta caaggttgag 5520

gatcgcgaga tggcgctaaa cgacttgcga atcatggagc aaaaagcact tgaaatgatg 5580

cattcaaaat aagcgcctac gggcgctttt ttattacctg gaggattaat aatgtcagaa 5640

caatacgcag gcttgacgct tggggttgat gtttctcaac tcaacaatgc tgtaaagtct 5700

ttgcagcaat tcaaaaaggc aaacgacgat gcaaagggaa gcgttgagag ttttgttgat 5760

tcagaggttg ttgcaagaca aagagccaaa caactggctg aggaattggc aaagcagaag 5820

caagaattta aagcaattca gtcggcaata gatccgacag caagcaaaat ggataagttg 5880

cgccaggctg cgacacagct tgatgcgctt tggaaaaaag gaattgtgcc ggatgataca 5940

ttctttgagt taggatcaat tcttgagacg cagcaaaaca aactgatcgc aactaaaaag 6000

gcgctaactg aagaaggccg cgcggcgctt gaggaagcaa aaaacaaggc aagggcagag 6060

gctgaggcaa gaaagtttat tgcggcattg caggcgcaag ctgacgcagc ggaaaaaaca 6120

aaatcagagc ttgtcgaaat gagagccgca caacttggag ttagtgcaga ggctgcgcca 6180

tttattgcaa aaatgaagga gcaggaaaaa caagcctcaa aacttggcgt ttctatgggg 6240

cagtacaagc aagcaatggc gcaattgcca atgcagatca ctgacgttgt tacttctctt 6300

gcttcaggaa tgccagtatg gatgatcgca atccagcagg gcgggcagat caaggactca 6360

ttcggcggtg ttgctaatac ttttaaagcg ctaatgacat ttgtcacgcc gctaagtgtt 6420

ggaatgacgg cgctgactgg tgctcttggg tatgcagcat ataacgctta caaagccaac 6480

gcacaactga aggagatcac aaaaaccgtt caggatgcaa caggtctttc cggtgagttt 6540

gcagagcgga tcgcaactgg aatccaggcg ctatctgaca agacaggaga gagcgcagac 6600

aatatcgcaa aggcttacat aagcactaag gatggagcaa gcgaggctat acaaaaactc 6660

gtcgatgttg gatttaccta tgacgaagca aaggcaaaag taaacgaata caaaaacgca 6720

tcaagttttg taagtcttaa caatgaaatt gctgatcaca aaaacaaagt tcttgagctt 6780

ggcgattcat ggtatgcggt cttaaaagca aaacgtgatt atgcttcacc gtccggcggg 6840

ctgttgggga aagagcttgg ttatgtgaat ccaatgctgc aatttgcgaa gaacacctat 6900

gaggatatag gaaagatagt aaaagatgcg aataaggata tggcggaacg agcagcacgc 6960

atcgacagag aaaatctttc attaaacaga gttcgagcgg cgcaagaggc tttaaacaaa 7020

gccatagagg atcagaagaa cgtagcaagg acagcagacg aagagttgaa aaagcgcgcg 7080

gctgagaacg tggagttcag gcgaaaagag cttgaagagg cgaagaaggc gcagcagcaa 7140

aaggagaaag ttggtggagt tgtaaaagca ccaacagagc agcttgataa ggagctatat 7200

gttctcaagg cgcaacttga aacgctgaaa gagcaccgaa ctgtaaatga tgttatttca 7260

cgccagaggc aatctttgtg gagcattgaa aagcagatcc agatccttga ggaagcgcaa 7320

agtaaacgca agctgacaaa agcggaacag gcgttactaa acgagcaaaa agcagttatt 7380

gctatggcaa aagaaaaggc agagataggc gatcagattg ttttgcaaca gagaaagaac 7440

aagcaatacc aggaagggct taaattcatt cagcaaacat cagatgctat tgacgctatg 7500

aacttgcgac agtctggagc tacagatctg caaatccagc gagagcttga gttgaagaag 7560

ttgcgaactg attatgttgc tggcggcggt agtatcgacg atgaaattta tcagcagatg 7620

gaagcaaagc tgaaggagta ttacgccact gaggatcagc ttagaaataa ctggattgca 7680

ggagcaaaaa acgcatggga ggtttacgga caagatgcaa tggatatgta cggcaacgtg 7740

caagatattg caagcgaggc gctcaacggt ctaaccaacc aaatggctac attccttgca 7800

acaggaaagg caaactttaa gagcttcgca acgtcaatca tccaaatgat tatccagatg 7860

atcacgaaga tggttatctt taatgccata tctggtgcga ttggaggtga cacttggacg 7920

attggaagcc ttcttaaaaa tgttggattt gcaactggtg gatacactgg cgacggcggg 7980

aagtatgagc cagcaggtgttgtccataaa ggcgagtttg tcatgacgaa ggaggcaacg 8040

aagaggatcg gagttggaaa tctttacaaa atgatgcgcg gttatgcaaa tggcggcgtt 8100

gttggcggca cttcataacac tggcggcgga gtttcatctg gagcaacaaa tcttaatatt 8160

ggtgggatta gcgttgatat taacaacgga aacgatccaa aagggttgga gactggcgta 8220

aaaatgattt tcactgatat gattaagcgt tcctgtacgc agggtgggga agtttacgaa 8280

tttgttatgt ctaagcgggg gtgatagtga aacttgagca attcaaatgg tgcacgcaaa 8340

cgcaaggagg cggcggcact atgaccacat caaataacga cagggaaatc tcatttggta 8400

atggttacac gcaggttgcg tcaggagggt ttaacactgt acgcaggggag ttttccattg 8460

tctatgtggg taatgattac cgagatgttg ttgacttcct gaatggtcac aggttgaagc 8520

cgtttttatg gtttatgcct gacggtcaac ctggtctatt cagggtaaaa tctggtagcg 8580

ttgggttaac tccaatatca gcaaccgttc aggaagtgaa agcaacattt actgagcaat 8640

ttacatcaat gcaataattc aagccgcctt tgtgcggctt ttttatgat gatacaatgt 8700

acgaaaggag gtgcaattat ggctaatgaa acaacaggtc gcgcggatct tgaaaattgt 8760

ctgcaaagcc tttaccctgg cgagattatc acgctaattg agattgacgg tacaaagttt 8820

ggcgcaaaca tttaccgcat acacaatgag aatatatcat acactgcgga agaattatta 8880

caggcgcgag aaactggagt tcttcctccg aaagagatta cattccgtgg cgaggtttac 8940

ggcgcgcgcc cgtttggaat atctggaatc aacttcacaa gcaacggaaa ggctgataag 9000

ccacaattga tactatcaaa ccttgatagc caggtgagcg cgatgattcg caactttaac 9060

ggcatgatgc aagctaaggt tacaatatgg atcacgccag cggaattaat ggggaaagat 9120

ggcagcatta aagatggagc ctcaagaaag ctggtttact aattgagcg accaagccat 9180

tacaatagaa tgatggcgaa atttgacctg acatcgcctt atgatatgga tgggataatg 9240

attcctccgc gaataactca aagcgtttgt tattgggcgc agcgtggatg gtatcgaagc 9300

ggaaaaggtt gcggatacaa tggatcgcga atgtttgaca aagacaacaa tcctgtaacc 9360

gatccatctc aagacttttg cgccggaact gtaacagcgt gcaaacttcg ttttggtgca 9420

gataagcagc ttgattttgg cggcgcacca gtagcaagcc tgttaaggag aaatcagtga 9480

tgattagtgc aaagataaaa cttgaaataa tgcagcacgt aaaagatgaa tacccgcgcg 9540

aggcgtgcgg ggttatcact caaaagtcgc gggtacagaa atatcaccgc ataacaaacg 9600

tgcatgatga tcctgaaaat cattttgaga tgggcgcaac tgaatacgtt gaggcgtgtg 9660

aaagcggtga gcttattgct gttgtgcata gccacacagg agacggggca agtacaattc 9720

caagcgccca tgacacatgc atgtgtgatg aaatgggtgt ttcatgggtt atgtgtgtcat 9780

ggcctgaagg tgatatgaga atcattgagc ctgaatctcg tcctctgatt ggtcgcccgt 9840

ggtcgcttgg ggcgtatgat tgctgggggc ttattatggc atggcacaag caacatggcg 9900

ttatcctgaa tgatttcagg aaaccatatg aatggtggaa gcctgagcac ggcgaaaatc 9960

tttaccagga aaattatctg aaagagggtt ttgttgaaac aggagagccg ccaaaacctg 10020

gtgatatggt tatctttcag ctttccgcgc cagtgtggaa tcatgcaggc atttatcttg 10080

gcaacaatca gttgcttcat catgcctttg gtaagttgtc gagggttgat ttgtattctg 10140

gatggtatca ggaacatgca aaaatggttt gtagacataa ggatctgaaa tatgactttg 10200

aaggtaatta aattatctgg atctttaggc cgaagatttg gagtatttca taagctggct 10260

gttgattcat accccagaagc aatacgcgca ctatcttcac aggtggaagg attcaaggac 10320

tacatgcaaa gcgaagtagg atcgcgaatg cgttacgctg tattgttga cggtaagaat 10380

gtaggacagc acgatgaaaa agcgtggcaa tgtgcaaagg aagtgaggat tattccaatt 10440

ccaacaggtt caaagtctgg cgggttgttt caggttgttc ttggcgcggt tattatggcg 10500

actgcatttt tcactggcgg ctcttcactt gcgttaatgg gtgcttttgc atcgtctgct 10560

tttatgatgg gtggagcaat ggtacttggc ggagtaatgc aaatgatctc accgcagcaa 10620

ggaggatcaa gattgtcatc ccaatcagca gaaaacaagc cgtcttatgc tttcggtggc 10680

gctgttaaca caacggcagc aggataccca atacattgc catatggtca aaggaccgtc 10740

ggtggagcta tctggtcggc tgggagttat gcagaagata aggcttaata taaaagagtc 10800

gcgcgttgcg cggctttttt tttgcccgta taattcaaca aatcaaatag cacaaaaggt 10860

gaaaaagcat ggctgaaaat atgataacgg gcagtaaggg tggatcatca aaaccttatg 10920

tgccaaaaga gatggaagat aacctgatct caatcaacaa aatcaaaatc cttcttgccg 10980

tttccgatgg tgagtgcgat ccagatttta cacttcgcga tctgtatctt gatgatgttc 11040

cggtaattgc agacgacgga actgttaact accagggtgt gaaagctgaa tttcgacctg 11100

gaacgcagac gcaagattac atccaagggt ttactgacac atcaagcgaa gtgacgctgg 11160

cgcgtgacat tacgacgaca aatccttatg taatttccgt aaccaacaaa aattatcgg 11220

ctatcagaat caaaatgcta atgccaacag gcattaagca agaggataac ggcgatcttg 11280

tcggcgttaa ggttacttat gctgttgata tggctgttga cggagactct tacaaagaag 11340

tattgctaga caccatcgaa ggtaaaacgc gttccggtta cgacagaagc cgaaggattg 11400

accttccggc atttaatgat cgcgtattgc ttagggttag aagggttacg gcagacagcg 11460

catcatctcg cgttactgat ctgattaagc tacaaagtta cgctgaggtt attgatgcaa 11520

aattccgtta tcctctgact ggtcttgtat acgttgaatt tgacagtgag ttgttcccta 11580

accagatccc taacatttca atcaagaaga aatggaagct gattaacgtt ccgagtaact 11640

acgatcctgt tttgagagag tatcatggtt catgggatgg tactttcaag aaagcttggt 11700

cgaacaatcc ggcgtgggtt ctttatgaca ttatcacaaa ccagcgatac ggattagatc 11760

agcgagaact tggtgtgcag gttgacaaat ggagtcttta cgaagcggcg caatactgcg 11820

atcagaaagt gccggatgga aaaggcggta cagagccgcg ttatctatgc gacgttgtga 11880

ttcaaagcca gattgaggct tatcagctta ttcgtgatat ttgctcaatc ttccgaggca 11940

tgagcttttg gaatggcgag agcttgtcaa tcgtcattga taagccgcgc gatccgtcgt 12000

acatcttcac caatgacaac gttgttgatg gtgattttca gtacacaaca gcaagcgaaa 12060

agagcatgta cacgcagtgc aacgtgacgt ttgacgacga acaaaacatg tatcaacagg 12120

acgtagaggg cgtattcgac acagaggccg cattgcgctt tggatacaat ccgacaagca 12180

taacagcgat cggatgtaca cgcaggagtg aggctaatcg gcgcggtcga tggatactaa 12240

aaaccaactt gcgcagcact acggtaaact ttgctactgg actggaaggc atgatcccat 12300

caataggtga tgtgattgct attgctgaca actttcacag cagcaacctg aagttaaacc 12360

tgtcagggcg cgtgatggaa gtttccgggt tgcaggtatt cgttccgttt aagattgatg 12420

ctagaccagg tgatttcatt atcatcaaca agccggacgg taagccagtt aagcgcacaa 12480

tctcaaaggt aagcggtgac ggaaaaacca ttgagctaaa cattgggttt gggtttgaag 12540

ttaaacctga cacggttttt gcaatcgacc gcactgatat tgcattgcag caatacgttg 12600

taacgagtat cggcaaaggt gatgatgatg atgaatttac atactccatc acggctgttg 12660

aatatgaccc gaacaaatac gacgagattg attacggagt aaacattgac gacagaccaa 12720

cctcaattgt ccagcctgac acaatggcag caccggaaaa tgtgcaaata tcctcatact 12780

cgcgaattgt ccagggtgca agcgttgaaa caatggttgt gtcgtgggat aaagtacctt 12840

acgcatcgct gtatgaaatg caatggcgaa aaggtgatgg caactggctg aatacaccac 12900

agacagcaaa caaagaaatt gaggttgaag gtatttattc aggaaactac caggtaaggg 12960

ttagatctgt ttctgcttcc ggttcaacgt cgccatggtc cagaattgtg acagcttcac 13020

tgactggtaa ggtaggagag ccaggcgcgc cagttaactt aactgcatca gataatgagg 13080

tgtttggcat tcgtgttaag tgggggatgc cagaaggtag cggagacacg gcatacattg 13140

agcttcatca gtcgccagat ggaacggcgg aaaactcaag tctgttaacg ctggtcccat 13200

atccacaata tgaatactgg cacgggacgc ttccggctgg tcatgttgtc tggtatagaa 13260

tccgcagcgt tgacagaatc ggcaacgttt ccggatggac tgattttgtt agaggcatgg 13320

cttcagatga tgtggaggct gttttaggcg atattcttga taagattttt gataccgaag 13380

caggaaagga tctgaaagag aatgccattg atagcgcaaa caaaatcaag gatcaggcac 13440

aaagcatcat tcaaaacgca ttggcgaatg atgcagacgt taggattatg aggaaggaaa 13500

acggaaaacg caaagccgag tttagacaat caatccaatt gatcgcaagt gaaactgagg 13560

cgcgcgttac cgcaatgacg caacttaagg ctgaatttga cgaggaaata actagcgaag 13620

taacgagact cgatcaggca attgcaacag aatcggaaac gcgagcaaca gccattgagg 13680

aattgaaatc acagattggt gatgatattc aggggcaatt aacgcgggtt gaggaagcga 13740

ttgcaaacga aacggaggcg cgcgtttcgg ctgatacagc attaacagcg aagtttggag 13800

atgttgaatc agcgcttaca gaaaaacttg attcatgggc tggcgttaat ggagttggcg 13860

cacagtacgc aatgaaactt ggattgacat acaacgggca gaagtacagc gccggaatgg 13920

tcatgcagtt atcaaacagc gctcaagggt tgatttcaca aatccttttt gatgctggaa 13980

gatttgctat catgacgagt tctaccggag ggtcgtatac attgcctttt gtggtcgaaa 14040

ataaccaagt tttcattaac agcctgttag tgaaagacgg atcaatcaca aacgccatga 14100

tcggtaactt catccaatca aacaattacg tttggaatca aacaggatgg aggcttgata 14160

aaaacggcac gtttgaaaat tatggttcta ctcctggaga gggagccatg aaaatgacaa 14220

acgaaacgat cagcgttagg gatgcaaacg ggcgcttgcg tgttcagatt ggtaggctta 14280

ctggtacgtg gtaaaatcaa agcggggcat ttgcctcgca tttatggagg tttaataatg 14340

gctgaatatg gtgtttcaac gtgggatgct aacgggaagt ataacaacta cggaatcaag 14400

ccagtttccg ttgttggtgt cattagcctt gccgctggtc agacaagcgg atcgtggagc 14460

tttagcattc caagtggttt taaggttggg tatgttgttt cacttgatga aggtgccagg 14520

ggagttggaa gggaaatagt agcatctgga aacacaataa gaattaatcc tacatcgtca 14580

gttggtgcaa atagatattc ttcttcaaag tgcgagttag ttgtttttct tgagagggct 14640

taaaatggct gaatatggag caatgttatc tttatcaaat gggaatccat ttatcacacc 14700

aaaatcaacg ccattttgct tatatggcaa gtacacttat tcatcttcag gtacttctgc 14760

ctatcatagc gcaagcgcaa atataccagt aaaccaatct tatccatgca tggcatttat 14820

aaaaacaaca aatacacaac agccaactgc attaatagct tacaggaatg gtggtaatat 14880

ttatgtaaac ggaggaaatc catatggtca atcatttaca atgactgttt atatatttgc 14940

catattccct caaactctac caaaatatgg tatggctata tgggatgcaa gcggcaagtt 15000

agttttaact aatgaaagca gggtgctaac agatcttgtt acaattggta ctcctggttc 15060

ttcaggtgga acgaatatag atcaaacgtt atctggttct tatgccgttt gcccttcaag 15120

gcttggggcg gttataggta tgggcgcttc tgatatatat acatcatgca ggtataatgg 15180

ttcaagcaca aggattggtg cagcaaggac aacgccagga acaggttcta taacaaataa 15240

cggaaattca ataattgcca tcaagactga tattatacgac caataaaaag gggctttcgc 15300

ccctttattt ttattaccag actgcaatca gcgtttttaa agttgtcaat gcttacccac 15360

tgaaaattaa acgggtagcc agcattaaca agcgtgcgat ttccatctcg ttttactcca 15420

aaaatagcaa ctgaatactg catgccacca ttttcataaa tagccgtgca ttcacgttta 15480

ggcatatttg cgcaaccagt catggcaacg gctgccatca aaatggcgat taactttttc 15540

atcttggtat ctccttcgtt gttgttggtt gcaactatac acgatcttgc ggttggcgtt 15600

tagcaaaaag tgctattctt gttctttttg ttatctggtg ttctcaaatc tgcggagtct 15660

taaatttagc tcaaaaagcg aacaacaata gagcgcaata catacagata aatatataca 15720

tacatataaa tcaataagtt agtagtatat atattcatgt ttgttcttta ttgttatcta 15780

tgttttgtgt gattttgtgtgtctggtgat ttttctgtga ttatgcatgt gtcattggtt 15840

atgtgtgtct atatatctat atccggcgca aattgaggaa caagagaaca aaaagacaac 15900

aaagatattt atcaatcact tacatgtaag ttttgttatc caagatagct agaacgttgc 15960

tagaacagaa aaaacaaaat aactattgac taaacgcatc aaatagctac aatgcagaca 16020

taacaaccaa tgagggtaaa tcatgaataa gtttatggcg tacaccagcg aagaaatgag 16080

taatgagctt tatcacgatc cagaagcgtg gactgctgat tatgttagtg gatcaagcct 16140

tgcggagatt tacagcacat gcccggcggc gtggaagttc aagccgcgtg acgataagag 16200

taaggcgcta gtttttggta cacagtcgca taccaacttt gaaagcaagg agcttttcga 16260

aaaaacctat cgccgtgcgc cagcgccaga agactttaag gatctgataa ccagccaaac 16320

ggcgcttgct gcaaagctga aatcatttgg cctgaaaggt acaaccggaa agacttaccc 16380

cgaacttata aaaatgatgg ttgactgcgg agaagacctt aacgtcatgt ggttaataga 16440

catgatcgca gaaagccagg ccagggccga tggggttcag ttaatcgagg ctaaagatta 16500

cgattcgtgc gtggcaatgc gccaggttct ggaatcaata ccggaacata acgcatgtat 16560

gaatagcaag acggcacaac gtgaattatc gttgttcggt gaaattaacg gcgtaaaagt 16620

caaagtgcga tgcgatcacg ttgacgtaac aaagaacgtc accgcaacgc tgattgatgg 16680

ttacgatgaa aaaggtcagc cgatttgccg agatattatt tatccagagg ccattgtgat 16740

cacagactac aaaactacaa tgagcgcgaa tccggctgag tttatgcgcc tggcttacaa 16800

tcacggctat tatttgaaaa tggcgttgca atgtgatta ttcagaaaag cgtatccaga 16860

agaaaagcga cctatagttg tgcgactgtt agcgcaagag aagaaagagc cttatttgcc 16920

gttggctttc cgcatgaata gcgagcaatt gaaaatcggg cgcattcagt atatgagcgt 16980

aattaaccag tttgccatgt gccagcaaca tgacgtttgg ccttcgtatt caaacggcga 17040

gccggaggtt tgccttgata ctcctgattg ggtgcgacgc cagtttaagc aatatcttat 17100

ttaaacagca caaatagcta aacaaatgaa aaatgcggtg ttataattca acgcataagt 17160

taacaacaag cgtgaggaaa taaacatgga aaacaaacaa gtgtcagagg tggcagttca 17220

tatcaacaat tttgcaactg gcatggctat gttgcttcgt gactttgtag cgccacttga 17280

tccaacggca ggcgaagaag aaatggagta catcagaaaa gtaattgatg cggtcgataa 17340

cgtcgtactt gtcgcaacaa tgagcgaaga caacgaggaa gcaatcaagg ctgtaaaaga 17400

aagttctgat cgcatgatgg aaaatctgat taaattacac accgaagaag aaacaaagca 17460

ttaacagcaa taaggcggcg aaagccgcca tgaattacgg gaatcaatca tgaaactatc 17520

agagcaattc gacaaggttt taccagcact gcataaggcg cgcagtttgt ttgtaaaggt 17580

taagaaggat aagcaaaata cacacttaaa aaaccgttac gctacacttg atgccgttct 17640

tgatgcaatc actccagcat tgaacgataa cgagctaatg ttgatgcagg atatgattga 17700

aagcgaacaa cctaacagaa tcaaggttga gacaacagtt cttcatgttt ctggtcagtg 17760

ggtgaaattt tacgcagagc taccaatcgt taaaaacgat cctcaaggtg tcggatctgc 17820

attcacatac gcgcgtcgtt atgcagccgc cgcagcattt ggcttaagcc aggcagacga 17880

tgacgcacag attgccgtta aatctgcaaa cgactggaag cgtgacattg agaaatgcga 17940

aagcgtaaac gaattgcaag aagtgcttaa atcagcatgg aaggcaagcg atccggcaag 18000

caagcaggta attaaagagc attacgagaa gcgcaaggca gagcttgaga tcggcaaagc 18060

acgaggattt aatccagcgc aaccaaagca aaatcttgca tcgccagaag ttgacacaaa 18120

aaacgacgag caagtaaaat cgcaaagtat cactgatttt gaatgattta caggggcgat 18180

tgcgcccctt ttttacagga gaataaaaca tgcatattat tactggtgag atccgaaaag 18240

agccgcgagt aaagcaaatg cctaacggca gcacacttta tgtggttgaa ctttcagagc 18300

gatacaaaga taaagatgga aactggcaat atatacaaacta cagtttcttt tttaatgcta 18360

aaacagaagg tcttaagggt tggtatgatg aggcgttcca ggttggcaag gttatttctg 18420

tatcttgcga cacgctgcgc attgaaacgc gtgaatataa cggaaagatg tattcaagtt 18480

taatgccagg aggatttgca aatcttatct tcagtcagcg cggggaaagc cagcaacaat 18540

atcagcagcg aacgcaaggg ggatggggcc aaccacaaca acagaatcaa ccgcaacaac 18600

cgcaacagcc aagacaaagc aatcaacctc ctatggactt tgatgacgac attcctttct 18660

gatcaacaag ggggcattac gccccttttt cactttcagc catcccaaga acggcaataa 18720

ccttaacagc aattcgcttt gcaagttcgg tttgatgagc gttaagtttt ccagtggtca 18780

ttattgacaa cattccggtc atacttccaa gcccgttaaa atctgacatt gccgatttca 18840

tgattgatat tggtgagtga ccttcatcag ctattgtatg cgctctctct gacaggtctt 18900

taactacctg atctattcct tcacttttcg tcattttctt ttccttcagg agttgattgc 18960

tgttctggca ttacaggcca cgggctaatt gattcccttt ctgctactgg cttttcaggt 19020

tcaattccta aaaactttcc tacaaactca ccaacaaaat ttgagctatc gcttacataa 19080

acatgcatcc tgtctagatc tgcaacagta acgctatttg gtgcgatcat gatcctctgt 19140

tggtgaaaac cgatatacat cacattagcc ggacgctcaa gagtccccat atttggcgca 19200

attacgccca ttatccaaca ataatgaatg ttgccatcaa gcggctcaca tcctgcgtta 19260

agccagttta ctttctggtg tgggaatgcc attgatgaat cagtagcatc acgccgtaac 19320

gaccattaa cgccgccaat ctcaatgatt tgtttcggcc tcatcccgtt gaattgagtt 19380

acgccagcgc cattttgatt aatgttttgc atatcggttc accttgttac attgatttca 19440

ttttacgcaa tgcgcagata caagcggcgg caagatcggt taattctttc tctatgccat 19500

gatgcgagcc gtctgccttt tcggtcatta gctctttgta ttccatttct acaatcgaca 19560

ttaagccgcc tggcttgtca atgtgcgcgt cccaggtctt aggatggtgc ttagttcttt 19620

caataacact tcccataact gaatcatgtt tgctatctcg ctcataatca tcatcatgat 19680

catcactccc gattgaaact cgcggcaaat gaatgcgcgg tattttatag ccagcataac 19740

ggatcttcat tatctcgccc tcttgatgaa aagcgccccg aaaggcgctt gatttattat 19800

cagccgcaac agccagtagg cggagtctgc ggagtaggta acttgaagtt caccaattgc 19860

tctacctggt tgattttgca attcagtgcg gcggtctggt ttgcctgaga tagagcaaaa 19920

cgcgcttcct gcaactgagc tttcatatcg caaatcaaaa cagcctgaga atcagcaaac 19980

tgttgacgaa tcagatctcg cgttgcgtta ccttgtcgct caatgttcag gttagtctcg 20040

cagcaacatc tctccgctgc aatctgtgcc tgataggaac gctcaagagc attaacatca 20100

gcagcattaa tcgcagcaac cgtctggtta gtgccgctaa caatcgcggt attcagtccg 20160

gcgaaacctt gcaccgatgc aagcaggttt tgcgtattct ggctggtaat gccgttaaag 20220

gttgatgcgg cggagcgctc aacggcaagg tttgtggagt tttggccttg cagagtttgc 20280

agccccaggt tgtttacgcc tgtctggatg ttattaatac catcaagaac agcgttagca 20340

ccgacagcaa cagcagcgcc atcaccacca tagccaccac gaccattaaa gccgttacca 20400

aaccatgacc caatcaggcc accaacagca ccgccaagac cagccgcgcc agcttcgcca 20460

ccaaaaccac caccagtagg gagtaaagtc atatctgaca tagtaaattc ctctttgacg 20520

ttaaaaaaat taaaaaaagt ttgcaacatt tatgttacga caaagagtat gcgcccaatt 20580

accaggaata gatataaaat atttgtaagc gatatgtaag caactggcat aaagacgaaa 20640

aaagcgcagc atgagccgcg cttgagttaa aaatattgtt atttattgag cagtgcagca 20700

acagccgcct ttaattcgtc tatctccttt tgctggtttt cgatcattcc tcccatgtaa 20760

accatagcgc aagccatatc agccatgatc acgttgttat caagagcaag cgtatcatct 20820

gctttgtcaa ccacaatttg acgatctaca tattcttcac cttcatcatt aacgcctata 20880

acatctgcaa aaatgtcccc gccaggtatt agctttacat attcaggatc tatttttagc 20940

agatcctggg ctatcatccc gaatcgctgg atgttgcttc cattgtattt aaacattgtc 21000

ggaagccact gcataacgcg atcatagctt tcttttccat ctgtataaat tatgtcgtgc 21060

tttaaatctc tgtcagagtt agcggccttt tgaaaaatga aatttccact tgatgaccat 21120

gtggttatat cgtttgaggc tggctgaaac tggaagttac agaaaaaact accatcacca 21180

cgaagtctta acgagcaaaa aggccaagcg ttattgcctt gtgggataag acccccatgtt 21240

gcgcgaattg gataaccacc tgttgattga gtaccccatg atagacctgg cgcccatcca 21300

ccatcattgt taggaataat ctcgccccaa acaagagcat taccccaata gttaggatca 21360

tcccagccca tttgctgggt tttgccagtg tattgagcag aatgcgttct gccgtcaggg 21420

taaaacttgt gcatatagct ttgcacgcca tgtttataca gttcaatccc aaacgactca 21480

atttgtgcag caccgcctct gatcaaacca gcgatagcgt aatcgttata ccagttgtaa 21540

cgacttacac cagtccatga gttcattggt ggattcatga ttgaatatag ttgtgacgga 21600

ttgtgttcat tcccgtctac ataaatacca gccccaggag ctaaggcaat acttttcgct 21660

cctgaaaaag caccattttc attaaactga taatactgtg ataacccatt atgaaatacg 21720

cctattgtat gctttcctac gcctacttga gtctcaaaat aagactgaga agacccaata 21780

tcttcgcctt gtgtactctt taattttgaa tggtatac ctgcgtttgc agtagctgca 21840

tttccagttt cattttcaac attaaggctt aggaattttg gtgcatgttt ttcgccaagt 21900

cctatattgt ttctgatgcc atttgcgtca atagctccag taccaccaga agaaatagct 21960

aaaggcacag tttcaaatgc accagatcca gaaatatctc tctgaaaaac aagaagacca 22020

tcatttctga taacaagcct atattctcta tttctagagt acaagatagt atgtacgtaa 22080

tcctgaaaaa atgaatcaat actaagcgtc tggcgagcag attcaatgtc agaaagatct 22140

ttaagattct gatccttatg taatatattt tcaggattta cctgttgcgc ccactgtcga 22200

gcttcatcac gcgcagtttc agcgccagcc tttgcattat ttgcatcaac ggcatgattt 22260

ccagcagcat cacgcgccaa tacagcctga tcgcgagcgc cgaaagcatc ttgtttaatg 22320

gcttccgttt cgattatagc tgaatcttta atctgttgcg tttgcgcctt gattgcgtca 22380

gtgtctgatt taatctggtt tgtttcagca acagcagaat cctttatctg ttgagtgttg 22440

tttttcagtt gttcagtagc agcgcgatca ttggccgttt tctctgcatc acttttaacc 22500

tgattagcca gatcttgcag ctgctttagg tcaaagtttt tgaaaaattc aatagcttcc 22560

tcgatctgag tttctttgct ttgatagtaa cgcagcgttt ccgcaacatc ttgtgctagg 22620

ccgtcaacag tgattgagtc atgcagcaaa ataatgtagt tgcttcgccc aatttcagcg 22680

ccgccagttg tgatggcgcg aatttgtgta tcgctgataa cttcactgat cacggccgct 22740

tgaaatggag catcaatgaa aaagattgtt gcaccagggc gaatgagcgt tagttgctct 22800

cgccattttg tgtcgtatcc agtgatataa ccttgcgcgt ccattgacgc ttgtcctgtt 22860

ctgtaaattg ccatgcgttt tctcccgtgttgcatgttaa tgtcaatgca gataatagca 22920

tttgctaagg aaaaaaaaaa ccccgcaatg cggggtaaaa gtagcagtga catttagaga 22980

agagattcag gtttgtaaac cttcctctct atcccttgcc tgtaattatc atgaatcggc 23040

atcataattt caagtcctct ttcctcggca gcaatgatcg cgtcacggtc tgtactctta 23100

cagacaactc gcattcttct gttgccttta tacctgtaag ctacaatttc aatgttagtt 23160

gcatcaaagc aggcccaaac ctcctgacca gttgcaaggt gaatatgttg cgcatcaatc 23220

cagtcaacgc aaagatagat cgtcttatct gtgcttcctg ttctaacaac cgacccccgc 23280

gtgtagtcct ttgcgagaaa agacttatta ccttcttcat caatgaaaag cacattgcac 23340

atttcatcat caatttcatc tgaatgtacc aggtaacatg ggagcgcgtg aatgtatcga 23400

ccatctttaa cgccgcaatc ataatgacca tcttcaggcg ggtatattcc ttcatatgtg 23460

cttaacggcg tgcgatcaaa ttttagtgtt tttgacataa cgcgctcgca gcattcatgg 23520

cttgcctgtt tcccaaacga ataaccagaa tcacgagata ctcgcttatt tgccttgatt 23580

ctatagtcct gcggaacttt tccaagaaaa cggcccaaaa tattgatcac ttcgctgtaa 23640

ggttctccgg ttagcttcat gatccaacca attccggtat cattaccgca cacgttgcag 23700

atcgcaccgc catcgccttt tttatcgcca agtttatccg tccatctgaa gcgatccttt 23760

ccgccgcagt gggggcaaga ttgatgcttg ccgttaaaaa cttcatttgg aagaccgcaa 23820

atactttgta gcgcctcacg ccacaatcct tgcatatatg ggagaacatc ttccttttga 23880

taaaccataa agttttcgtt gttcatatgc acctcaaaca aaaagcctga cagatgataa 23940

catcatgcca ggcattgttt ttagcaatta gtgctttcag gtttatacag gaatccgcca 24000

ggttcataat ctgacattct taccactcgc aacatttcac gcttatcaca tctctttgtg 24060

agcggttttc cattggaatc aaatcgcaga tctggacggc agaatgaagc gcgatatcct 24120

tgacagccat actttttgta atcgttatgc accttttgag cgccagctgc tgaaatcata 24180

ccgcgaactc gccactgatt tacagtttgc tggctaacgt taagacgttt tgccattgct 24240

gacacgctgc cgtaatactc aataagaata tcaagccgtg cttttagtcc ggcgcgcact 24300

tcatctttta gaacataata acctgtcggt cgcttgcgct tcttcttgtc cttgcctcgt 24360

cttgttccgt tattgccatt aattgtgcgt ttgtcaatct taccagttga tactgcgatt 24420

cgctgttctt tcattattca ttctccaata gcattttttg ttaaacgatg atttaatgat 24480

tgtgtattat acacgccaaa tggcattact caaaggtttt agaatgattc caaacattga 24540

aaagcaaata tcagcacttg gtgaagcggt aattaaatcc attcaggagc gattcactgt 24600

tggagagatt gtgccttacc cttaccagtg cgttgcatac gctgagatcg caaagcgtat 24660

gaaaaattat gagcatccat tctttgttaa agcgtccgta tcggctggca aaacattgat 24720

gtttgcaatg gttgctcacc agtgccggaa aatgggcttg aaaatgatgg tccttgctcg 24780

acaggctgag attgtggatc aggattctga agagataaca aatcttggtg tgccgaactc 24840

catctattgt gcaggactga agacaaaaag cgcatacttt ccgatcgttg ttggttctga 24900

gggaactgta gttaatggac tgtttaaagc gcttggcgac tatgttccgc acgttatcgg 24960

gatcgacgag tgtcaccagg tggattggga agaccttgcc gacgcaatag agaaagacga 25020

gtcattttta caaatgacaa ctaagaaagg cgaaaaagta ccaaatccag attatgacat 25080

aacaaaggga agcaggaaca gaaatacaga gttcctgatt ggtgaggatg gcttgccaat 25140

ggagggaaca ggccgcacac aatacactgt cattatcatg gaaatgatgc gccgctgccg 25200

gaaaacatat ggtcatgaac tgcgcatatt tggcatgact ggatcggagt ttcgcggcgt 25260

agtcccgatc ctggtagaag acaaaaaaca gaaaggattc tggcgtgaac aggttactaa 25320

tattgacacc aactacctga tcaaattcgg ttctgttgtg ccaacaaatt ttggcgacgt 25380

tggcgatcta gggtatgacc tttcagagtt tgaggcatcc agtgaggatg gcgttgcaga 25440

ctttgatgct aaaacgcttc gcaggatgga acaaaaaatt catgaagaag cgaccatgac 25500

aaagcgcatc atggcgaaag ttcacgagat ctgtaaaaac cgcaacggcg tacttgtgac 25560

atgtgcagga gaaaggcatt gcaaagaggc agcggcagca ttgccacctg gaacgactta 25620

caggatcatt actggtaaga ctggcgacaa tcagcgcaag gagtggttga aggaagcata 25680

cgaagggaag gtaaaataca tattccaggt tcaggcgcta actactggcg ttaacgtgcc 25740

gttttgggat acgtctgtta ttctgcgtaa gattggatcg ctaactttgc tgattcagct 25800

tttaggacgc ggaatgcgac ttcttaagaa atggcataag gagcaaggct tcaagaaaga 25860

tgatcacctg gttatggatt tttcaggaac gatggacgag ttaggtgagc tttattttga 25920

tccgatactg gaacaggcgc aacatcagaa gcgattcagg aacggtaaag atccgaagcc 25980

ttgtcctgtt tgcggaactc ttaatagcta ttatgctcgt cgttgtatgc acgtagacga 26040

aaacggcaac agatgcgagt ggttctttaa attcaggaca tgtgatgatc agattgaccc 26100

gcgcactaaa aagattattc agcgcggttg tggaacgaag aatgatattg cggccagggt 26160

atgcagacat tgcgacatgt cattgattga ccctaacgag aagttaagcg gaaagcacta 26220

cacaaaaaac gactggttcc aggtgcaatc tttctcggtt gacatgacga aaaaccagaa 26280

agggataata tttaattatg agctttccga tggcatagac actttcaggg cgagagagat 26340

atttttccct gaatctgaga gccaaatttg ccgcgcaaaa tggcgaagcg ttgctcttaa 26400

gcacatacca gatcgccgtg tcgctggcat ggtggcgagc tatcgtaatg cacgtaaaat 26460

catgcaatat gtgaatcaca tcatgccacc atcgcgtgta actcaccgca agactagcaa 26520

gggtgaagat aatttgtaca agaaggactt taattatggc aatgactgat aaaggcgatt 26580

atctggagtt ttacgaaaga gatccgacgg atacgctaaa ggaggagtca caccagatcg 26640

gggcgtttca atggttaact tatgctcatc ctgaatttct tgcctggcat actaaaaatg 26700

agggcgacaa ggggatcgcc accgcaatga tggatcagca agccggacta gttaagggcg 26760

taagtgattt catcatcctg attggattaa aaggccgcta tccttttgca gcaattgaga 26820

tgaagcgcgt caataagtca ggcaagggga aggcttcacc agtcagcaag gagcaaaaag 26880

ccttcttgcg ccgtgtgcgt gagcttggcg gattcgctgc cgtaacatac ggatataagc 26940

aattcatgat cgcggtagaa tacatgatga aatagcactt tttgttaaaa caggcgcgaa 27000

ggattgcgcc ataataacac cagttcaaca agcaacaggt aattaagaaa tgagtaaaga 27060

aactgaagtg aatttgaac aaatagaacg cgaaacattc attggcaatg ctcttgctac 27120

tggaggccat taccaagccg ttaaaccaaa tcaatattat aaagtcactg gcaaccgcta 27180

taacggcagc aatacgccgg aattgtgcg cgatttgtgg tctacgccgt atgagcttgt 27240

agcatggatg gaaagcgaat acggtgatta tgacatcgac gcggcggcaa gcaaagaaaa 27300

tgctgtatgt gaaaaattct acaagcaagga aacaaattgc ttaaagcgtt ggtggggtag 27360

taacaagcat atatggctga atccgccgta cagcaatata acgcctttcg ttaagaaagc 27420

gattgagcaa atggagcaca ataaccagat cgacattctg ttaccttgcg acacatcgac 27480

aggatggttt tatgaggcac aacagaaagc agcggaaatc atctggatca ctggcgaagt 27540

ttaccaggag gacggaacag aatattcccg cactggacgc ctggcgttca cttcagcgct 27600

tacaggtaaa ccagttcagg gaaacaacaa aggcagcgtt attttcatca tgcgcgaact 27660

taaagaaggt gagcagcaga aaacgcgata cgttaaaatc agcgacattt gcccgtcagt 27720

ggcagagcgt cgcgcacgca aacggagcta aaaccatgca gaaagaaaga agagttttac 27780

ttaatgaaga tggcgaattt cttttgtatc gcgctatggt atgcgatgca cttgataaaa 27840

accctaacgt gaaaaagatg attgattgcg atccgtggga attttcaagc tcattagata 27900

tgagctttga ggagacaaaa aaactcccac ttgaaaaatg gcacgaacaa atcagaaatg 27960

acatgcaaga atttgtcgat tcgtggaatt ttcataacgg aatgatccac caatagcact 28020

ttttgttaaa acgcccggcc ttgtgtcggg cataattatt tcatcaaaac gaacaagagg 28080

aaaacaaaat ggttgtttac gatccacgcg cttttaaaat tgctcaagaa gtttcacgcg 28140

aatctgttgc tggtggttcg gttaacggct atcagtttga ttggtcggca gcaatgaccc 28200

tgcttaaggt tgcatatggt cacgcgccaa ttgaaacagc ggaggaatat tataagcatg 28260

aaggttgaac aaggtaggca ggccgtatgg gatcacgcaa aggagtgcgg gatctctgaa 28320

gacatagcca ggatagcaaa atattttgat attgctgaca taagcattat cagcggcgac 28380

aaaatgacat ttctcaatga acgaccgcgc aagatgcatc gcgtaccagc aataccaaca 28440

aagattgatt tcaaagaggc tatggcgaaa attcgcgagc cgcgcaaata ctacaaatga 28500

ggattattat catgtggcgt ttgttacttt tgcccttacc tgttatgatg gcgatctcta 28560

ttgtatacgt tgtcataatg agataaggag aatttttgat gaagcaaatc aaaatcacag 28620

atgaacaatt tatcaatgag cgcaagcagg gaaagacata taagcagatc gctaatgagt 28680

atggtatgaa cgttcgcagc attgagcgac gcgcggcacg attagcaaag caaggtaaag 28740

ttacaaccat cggatcacct ggttttggtg ttactggaga atcaaagcta attgataaag 28800

atggcaatgt ggttatgaca tggattaaga caagtaagga ccgcgaacag ttagaagcat 28860

taatgcaggc cgctatggat gcatttagcg aagaagttcc tcgacttgat ccacagccag 28920

aatcacaaaa ggattatagc gagacgttat cactgtatcc gatctttgac atgcacttgg 28980

gagcaatggc gcataagcat gaatgcggcg agaattggga tacagcaaca gcagagcgcg 29040

taatgaataa ttttattgat tattccatcc agtgcgcgcc ggatagtgaa aaagctgttt 29100

tgctgattgg cggtgatatg cttcacagcg acggactgga agcggtgaca cctgcaagcg 29160

gtcacgtatt agatcaagat agtcgatacg caaaacttgt ttatgttgcc atccggtcag 29220

tgcgtcgagc aatcacaaag ctgttatcaa aacacaaaaa cgtcgagatt cagattattg 29280

aaggaaacca cgaccaaagc ggcatgatct ggttgcgcgc agcaatggcg gcagcatatg 29340

agaatgagcc acgagtgcat gttgatgtat ctccgcgagt agttcatcac acacaatatg 29400

gcaaaacatt cctggcatac catcacggggc atactatccg caaacctgaa atattgctca 29460

tgatgtgcgc ggcagactgg cgcgaagact tcggcaattc aaaaatgatg tatgctcacg 29520

tagggcattg gcatcatcaa acagtaaccg aaacaagcct gggcattgtt gaagtgcata 29580

gcactatggc ggcaaaagat gcatatgctg cgcgcggtgg atggcgttct cgtcgccgtg 29640

cggctgttat tgtttacgat aaagaatacg gagaagtagg gcgattatg catttatccag 29700

aaatggctgg ttgaactttt aatcattaaa atataaatat aaatcatcaa tttaattggg 29760

cgtggcattt tgcttcgccc tttttttat cctttttgtg cgttcaatgg ttaggataca 29820

atcaattaat tgttgacttt tatctacagg agatctaatc atgaaagatt ttttaaacgc 29880

tgcaacttcc ggcactggcg gcgcgtcaat cactggcgca gtaactggtc aaacaactat 29940

cgcaatagcc agcttagttttgatggctgc atttggcatg tggggcgctt atcttcgttg 30000

gcgtgatagt aaggcgctac gtgatgcgct tgaatgcggt gatattaaga aagctattga 30060

gatcagagggg aaataatgag tataaaacaa agagtgaccg ctgcggcttt tggcgttgct 30120

cttgccctta cttctccgct actggaggaa atagaaggag taaaacataa gccgtacaaa 30180

gatattgctg ggatatggac ggtttgcgcc ggaataactg gacctgacgt aatacaaggc 30240

aaaacgtaca cacaaagaga gtgcgatgca ctactggcaa agcatatcaa gatcgcaaag 30300

gatgaagttg ataagcgtgt aaaggttgac attcctgata caatgcgcgc agcaatgtat 30360

tcattcacat acaatgctgg aactggagct tttagaaatt caaccatgct taagctgatt 30420

aatagcggtc gttacatgga agcgtgcaat cagttatggc gatggacaaa atacaccaat 30480

ccaaaaacag gaaagaaaga aacgtctaaa ggactgcgca accgccgcgc ggtggaattt 30540

aaatattgca ttaaggatct gtaatcatga gaaagttatc agcaatcgcg atcgctgtta 30600

ttttgtctgg atgttcaagc gttacgccgc tgaccggatt aatcggtagt aagccggaaa 30660

tcacagcaca ggcaggagcg gagaacgtaa agcagaacgt tggcgttacc gcaaagcagg 30720

acaccagcac aaaacaggaa actacaataa aggaatctgc ggttgataag gtggacactt 30780

ccagcaagaa ggatttcact acgtcaacca ttcaggcaaa caccataaag gctgataaaa 30840

tccaggttgt gcagggtaat aacggcagat ggtacgaccc cataataata tgcgtggtag 30900

tgttccttgt gctgatttgt ctttactgga gggaaaagaa aaaggaggct taacgcctcc 30960

tttgttttat agtgaatcca tcaaatcttg aaagctgttt ccgctataat cattctcgac 31020

aagaatgtaa gcacctttct tctttatatc ccataacatt tcattgtatt cgtaacgatt 31080

tatttcaaac gtgctgccat cttctcttgt cgcagttcct ttgtcgttga aaacatcaaa 31140

agttacattg atgatttttg tctctttctt tgttactttg atagccattt ttaaaatcct 31200

catattcgtt tcgatgaaac aaatataccc gattcgagat cgggagtttt aacaaaaagt 31260

gctattcaat acgctttaca gtaaccaaca aaacgccgtc ctcatcacac aaattatgct 31320

ctgatatgtt aaacgctcgc atgtcagaat gaattagcgt taacattgcg tgcatgaaat 31380

cgtcatgtga gattgaactt acggcgcaga acttcaatat tttgtctatt agcatttgac 31440

gtaaacaatt cttcatcgct catttctccg atccttaaaa catcccttac ccattcgttt 31500

tgcatacctt ccaggctgta aagttcaaac gagaaggggc gatttgctga tcctttctta 31560

caccagaact ttccaccaac tccatcaaga taaccagtgg tcatcattct tgcgcaaaac 31620

tcttttgaaa cggcggttgc aaacattctc ggcgtaaatc ctgcgaccct tgcgagccgc 31680

tcacattctc tatgctgaaa tataaatttt gctatatgtt gacgtgtaaa ccattcgaat 31740

gactcacacc agcgatacag atccagaaga aacataatta ccccagcaat gttggattca 31800

cgaaaacaac gccattaatc acacaaatat aatttctttc ctcaagcata ggaagcagtt 31860

gcgtttccat gcgcttcatt acgccagcct gaccaacaaa cggcttaact tttcgtgcag 31920

cctcatatat ggcgcgcaca ttcaagatcc ccttgttagc cttaccgtgt cgagtgataa 31980

tctcgattag tttacccatt tcagcatcat caccagcata gccagaagcg ttggcggcgc 32040

taatgtatgt tttgctcaac tcgctaaaca tcaacagtgc ttcctgaacg gtttcaacct 32100

caatctcttt cgacttttga gggtatccgc caggattaaa ccaattgcgg atagtgtgca 32160

aaaccgaagc aatcctgatc acctgcttat ccattttgcc aagagcaccg cgcagcattg 32220

tatgcgaata ctttccgcca tcagcaaggt gcggctccat ctcctgacga gccatgttaa 32280

gaactcgcat tgctgatttg ctgatcttaa gctggatgtt tgattcactc atgatgttat 32340

ggatcagctt gaaatattgg cttttcagtt ccccatcaac aggctcataa gtagaatcac 32400

cgttatcatc aacaaacttt ctacgaccta aaaatgattc ctcacgcaca agcaaataac 32460

gctcacttac accgataccg cgcgatcctg catccatgat ggcattgatt gtttcatcct 32520

gggcaattac cgccatacag ccaagagctt taaaactcat attgttgtca gcgtttgcac 32580

gagctataga aacatgaccg tgatcccatg cttttaatac aagttcgctg ttggttttac 32640

gttcgctatt ggcatatgtc agacctaaaa ggctgttaat acttgttgcc tcgtctgaaa 32700

tcacagcaaa gttaccctgg cgattgttga ttcttgcaag tccttccggc gttgtgtcgg 32760

acacagggaa aacgatgtca cacatctttt ccagtttttc ttccagatct tctttctcct 32820

ggtacagaga ttccatatca gcgccggagc gctcgctttt catctctttt gcaagacctg 32880

ccaactttgc ctgaatcttt tttctttcct tctttcgctg atcattaatt ctttccactt 32940

cagcaaccat aggagcaagt gccaggctgt taacggcaga tttaccagtt gatggaggct 33000

ggcttgtgac aacgtaaaga gcggttggtt gatccgttcc gtgatactca accgtgaagc 33060

gtccaagcat tgcagcagac acgcagccga taaaatgcat atatgccgat gattcaggga 33120

actgaactga tcgagcaatg ttgcgcgcca acttaccaac aacatcaaca tcattgccaa 33180

gagaaattac agggtagcga tcgtttccgt tattaacatc attaacctca ccccaaaagc 33240

tggacgattg acggtatccg tttgcctgaa tggacacgcg caccggagac aatccttgtg 33300

catcagctaa ttcgataatt tgttttggtg ttagtttatt ggtttcaaac gaaaaattca 33360

ttgatgataa ctcctttttt gatgcggcaa taataccgca ccaatgctac ccgcatttaa 33420

caaaaaatgc tatttgatcc gctctatgct aactaagatt ttttctggtt cccttgcgct 33480

atagcaatgc gcggttacgg aatcagcaaa atcaaagtaa agtgatttgt ggtaagaaag 33540

accaatcagc catccaacca tcgttttctt tattggcctg actatatcgc caggcttgaa 33600

ggtttttgtt gccgaatgag tcaccttata gcgatattca gccttgatca ccttatacata 33660

gcctcaaaca aatgacagcc accagcggaa aatccaactt cttcgcggta tagcgtccag 33720

cggtcgccag attcatcaaa aacataacca gcaacagcgc caagcgcgcg gccttgctca 33780

atctggtagc gcttcccttc tttgaaatgc tttttacacg ggatcttgtg atctacaaag 33840

gtgcatttaa tcgtttttgt cttgatatga cgatagtcag actcaacagc gttttgccac 33900

ttgctaccct ttgtaatgct ctttacctga aattgttcca actcgatcac atcatcgcta 33960

ccagcaattt tttcgtatac agctttataa cgacagttca tcaaaacgtt ggttttattg 34020

cagataactt tcatcgttta tttctccttt cgtttcggtg aagtgattat gccaggcttt 34080

tacacctggc ttttaacaaa aagtgctatt ttgcgtttgc ttcaaagacg gcgcgagcaa 34140

aacctcgcgg agtgaggggag cggatcatct ttgtgcgaga tgattttccg ccagttttgg 34200

cccaaccagg attatccttg tcatcctctt taggaaggat tgttgcgcgc ggaggaataa 34260

caaatccgtt tccagtccaa aggcatgttt tcttaaagta ccgatcccgc gccggaataa 34320

tatcagggaa gtcgggatgc ttatcatctt ccggcagata gccagcataa tcgcacggat 34380

ggaacgtata atcaggcttg cgccataacg ttgacagctt gcctacagga ttctctatca 34440

tgtacggaac accgaaataa tcagcgatat gcgccgcaat tttgcaagtg tcggcggctt 34500

catattggaa gttaggattt tccatgcgct ttttagccca atgacgagat ccgctatttg 34560

ctaattgtgt gcaaggcgga aatgccataa tgaaatcagg ctaccccaa ttttcaagtt 34620

ttgcggtata tttgaatagc tcatcaatcc acacattaac gtaaaaaata ttgtcgtgga 34680

ttatgcgcac tgattggtaa tcgccatgat ctgcatcatc gtaattaaag caaatgcatt 34740

tatatccagc atcagcccaa tctttaacgg ccttaccaga tccatcaaac aaagagaata 34800

ttaaaccttt atacatttca tcacctcaaa acggaaagca tctaggacag ttaggatcaa 34860

aattgcaacc acaatcatta acaatcatag ttggatctgc aaatgcagcg ccgcattcat 34920

ggtccatatc catttcaccc aacgcatcat caagcgtcct gattttatat gcgacttcaa 34980

gcgcggcctc ttattcagt ccggcatctt ctgctttttg cagtcgctca aaaaacccgt 35040

cttgactcat ttaataaact ccttaataac agcaataata attacaagcc cagcagtaaa 35100

aacaagcaat ccgacaaggc caatcataac accaaataaa atatcaagca tcttaatcat 35160

ttattcacca ttgataaaac gggaggcggc aacctcccat tgattaatca tacagccttc 35220

cagctttcaa taaacatgct gttttcttcc agcagtttaa tcaattcttc gcgtgtatat 35280

ttgcggattt tactacctgg atgtgagcca acaacaaaca cacctttacc actcgtgatg 35340

gttaactggt tgattccgca agattatcc atcttaatgg cgacgcggtg atgtgcatca 35400

acagtgtgca tgatgttagt gactttagct ttcattatgc gatcctcatt gcacttagtg 35460

tagggcatta tatttacttc aaattctcga tcttgttctt tccaattgag ttgtgaacgc 35520

gatgttcttg cgtctcttta tctccatgaa catcaatata ataaccaggt atggcagtga 35580

aaaaccaatt tccacgccag aaaaaataaa ggccgtgatt gcttcctttc gcgcatttgt 35640

ttgcattttt aggtattgga agtccgtgta ctctccgcaa ttctacattc ttgttaaaat 35700

ttggcattac acaacctcca taaaagtatt gaatccacgc agtttgaaag cattgccatg 35760

tttaacaacg ttccatgtca ggccggagtg atacgcaacc atatcctcaa gatcaatggt 35820

gatgacagtg cctttcttaa agattccttt ctcgtgagtg ttgtcacgct gtacaacttt 35880

aacatcaact tgattttcca tttttaaatc ctcgttcgtt gtcgatgcgc ttaatatacc 35940

aatcagcagc gcatcggttt taacaaaaag tgctattcaa caacagatcc agttaaagag 36000

ataagctgcc ttgtgatctt gtgtctgcga tactggcgcg caacaaccgg aagattttca 36060

ccgctttgat gaccgaagca gatcggcaac tcaccctcaa gagagcggaa gccgccaaat 36120

tcaagatccc gcactggctt tgagcgccac gcttcaataa atccgcgttc gtcacgcgtg 36180

atatagcggt atatgttagg gaggggcaat tgtatcccct caaaatccac agtatcggta 36240

tagctgtaaa acttagaact catagtcaac aaccccaaat tcgttaacaa tttgcgcctt 36300

atagaagccg ccatttttcg ccaggttgta gcaggccgct acagtttcaa attcgcgcgc 36360

ttccggctgg ctgttttcgt gttcccaggt aatcagagta accattttca aatcctcgca 36420

ttcgttgtcg atgtgtgtaa tatatcggat tgcgaggatt gagttttaac aaaaagtgct 36480

atttagtcca ggagttcgat ttcgcgctcg ccagttaacg gatctttaat ccagaatcca 36540

agcgcctcac cgtaaatacc gccatagaat ccaagcgccg acagatcctc acctttaaca 36600

aataccagat ccccgctacg gcttacgtga gatacgttaa cgataaccgg aaaggtgcga 36660

tccttaaatg caggccgcag agcgtaacac ccgttattga gcaaacgtgc tttgcgctta 36720

actggaatag gtgcattgat tcgcgccatc tttattctc cttcattgtt gatcgtgttt 36780

aacggccatc agaacatcac aggactcgtt aatgaattta gccatgcgtt tatcatcacc 36840

aacagggatt agcgcgttaa actcgcgttt gctactgaat accgtcactt tatgaaacga 36900

tgcgaacggc atcgacggga actcgactgt ttgcatcaag atccgcaatc ccatcttttt 36960

ggcccttacc tgggcctcgg caagttcaaa atccacttcg ctaatcattt cttgatctcc 37020

tcagcaataa acttcaggac tttgcgcaaa aacacttcat tattgatggt cgctggcaat 37080

tcctctttca cttcttgcgg cagatagata ttgccgtgcc aggtcaaaca atcgtagttg 37140

atacgataag agcaaagcca ctcgcaatcg ttatctgctt cccaaatgca gagttgcaga 37200

acatcgccaa aattctcaac agtcaggtcc agaccatgac gcttggcgaa gttaaaagtg 37260

gttttgctga tgttcatttg ctttctcctg atgggtatct cgtttcgatg tgtgtaatgt 37320

acccgatcag gatgattcag ttttaacaaa aagtgctatt tctcaactgg cgattattcc 37380

agctattcca ttaatcagga ataacaagaa taactcattc gcgatcacct ggattgtgag 37440

actttgagtt tgatcaattt taatcaagtg attaactttt tgtgtggagg ggagagaa 37500

acgcggtcct cttgcatcct ttccggcaat cctggcgcgc aacatgttca ttatgtttcc 37560

agccgttcac gcaaataagc ctccgcagtt cttacacgtt tcatgatgtc atgcacacaa 37620

aagaaacaaa gtaaaatcta tataaataca cattaaatca ataagttagt tatatatgta 37680

tttatatttg tttcttattg ttcttgttgt gtgatggtta ttggtgtatg ttgcgatttt 37740

tgttttggtg gatattgatt gtttattgtt cgatttggtt ggtgatcatg attgctcatg 37800

gttgcgtatg attcttgatg tattatgta tatatccgcg aaaattcgtg aacagccaca 37860

cagccaaaaa acatggcgag gattcttttg aaatcatgtg cttaagtgtg aattttgttt 37920

ccgcgcaatc tccacacgta tagcaaacaa ttagaacgct tgacatagac acggcgcggt 37980

ggcaacattg caatcaccaa caaacaaggc aaataggagt tacactcatg actatgacaa 38040

gtaaagaatt ttgcgatatg ttgcgagtta atgcagcctc actagctaac ggggatgtta 38100

gcgaactgca aatcaaggag cttgacttac aatgtaatat tgcgccggaa gacttgagga 38160

aaattaacgc ctcatacatc agaacgatta tgaatcgaac gccggaggtg aaagctgttg 38220

gcgcggtgaa ggttgagcgc gttcgaggtg atgcggatat tcctgattgc tatcgcgtaa 38280

ccattaacca gaatccaaag cgccgcttgc tgacagatga agatctgcca aaacttgaaa 38340

gtaagtggcg agcaaagttt attgctcaac tactgaaaac acagccgcga atcagtgatc 38400

tggaaggtga gcgcctggaa ggtgcagcaa tcgcacttga gcgaatgaat gaaatgctca 38460

acgagatggt aaaggtgggg gatgaataat gaataaggca ttaattggag ttttgattgc 38520

aattgccacc ctgaccggat gcgaaaaggc atctgaatca acagttgtcg taggagtgga 38580

gtcagatttc aaagtcggtc gcctgtttac tgttgatgga tgcactgcgt accgattcta 38640

tgacaacggt cgcgcgatct attacaccaa ttgtaatggc gcaactgata gcacataccg 38700

cagcggcaaa aacgtaatgc accaacaagt cacaacagat attaaatagc actttttgtt 38760

aaaactcaaa aacgataacc aggcataata gcctcatcga aacgagattg aggaaaacaa 38820

aatggcacgt cgcatcacca aagaccttaa agtcctgaat aaagaaaacg tagttaaaat 38880

cctggttatc tggggctaca acgaagaaac agcaaagcag aaggtagagg caggttatga 38940

cctggctgta aaagcaatgc caaacgacga cgccaaaggc attgcaaact atgtagcttt 39000

cttttaatta gcaggtgaat aaatgaaaac gaaattagtc cataaatcag aaatcaagat 39060

cggcgacact gtgattcaca acagcgagct tagaacggtt ggcaaagagt caatacgcca 39120

cgatgaattt atggggatct tgttgttcgg tgactcttat cggcttggct ataaaatggt 39180

tgagcttgtg gaagaggtaa aattctaaat agcacttttt gttaaaactc aaaaacgata 39240

accaggcata atacatcaca tagggcggca cggcgtcgcc cagttaaacc aatcaggaga 39300

aagtaccatg ttaaaattgg ctgacatcaa attcccgatc accttcgaat ctcgcggaat 39360

tggccattac acgttcacag ataagaacac ttgtcataag ttttggtcgc atagcgacaa 39420

gccagagtta tcaactatgg aaattgagca cttcattgat gcgcataata gcctttatgt 39480

gaaaacgaag gactatggct acgcaatgcc aggatacact tgtttgcact acaaaccata 39540

taaaggccaa gtgttaccag tgcgctcaac tgagcgatca gagattcata cggtcgagat 39600

gaaaacctgc gactacaaaa agctgtggtg cttaagtgtg gacaatatca agtatccgat 39660

cacgttctac acggttggta attcaacgat tcgcttcaca tcgcgcgaca atggcgttta 39720

tagcgatggt cagaaatcgg agttgaaagt tgatttcttt gttaatggtc acaacaaatt 39780

caatccgcac aatgaaaagg ataaggtgta ctacgtggaa gatattacaa aacacttgtt 39840

tggagaaaca ccattcggcg gcggaaagac gtcaattcaa caaatcattg gcgaggagat 39900

ccacaaggca gccgttaaaa tgaaagcgcc tggaggctgg cgtattgaca agagcggctt 39960

tgccaatcaa gaaatcgtaa cgcagggcga aatcgaagac atgccgctcg gcgcttcaat 40020

catcggcatc agagacaata acggcgactg gaatgatgtt tcattccctg tcaagcggat 40080

cgagttgcac gaagacatga tttgcatcat ggcgcatcat ggtagctata aggacaaacc 40140

attcgccgca gagcttaaca tcaaacgcgg aacgattgtt aagtggaagt ttaaagtgta 40200

aatagcacga attgttaaaa ctcaaatgtg gggccaagta taatggcctc actttcaacg 40260

aaggggataa aacgatgtac attagcaaga agatgaaatg cgtttccgtt aattatggtt 40320

gtgctggaat gttcaagcct ggcgaaatct atacggcgca aaagttgaaa tcatcaaccg 40380

gatcttttta cgtcagcaac agtaaagggc atcgcatgtt cctgaacggc ggcgaaggca 40440

ctaaggttat ggcgcacgct atggtgattg ctgaatttga ggaagtgaaa gatggaaaca 40500

aataagcgcg ttaaacacaa gtttattgat aatcatggcg atgcacttga ggtatacaag 40560

ttcggcgatc gcgtgtttat tgatggcacg tttcaaggtg gaagcatgcc ggattttagg 40620

tcaatactca ccattgacca ggtgcgcaag ctggcggata agttaaatga tctggcagat 40680

gaaatcgaat acaagcaaat tcattttaat tgataggttt gataatgaaa aaatacgaat 40740

ttgaactttg gggcagcaaa taccattttt ccacgagcaa gccgattgtt atcgttgacc 40800

tggacggaac gttatcagac ggatcgcatc gcttgcacct gcttccgaaa gaagatttgc 40860

accttacaga aagctggtct gagttcaata aggcggctgt tggcgattcg ccgattaata 40920

gcacggttgc agtggttaac ggtttgtgga tgtcaggatt tgccatcgtt atcctgaccg 40980

gacgcagcga cgaagtgatg gcggatacct gcaagtggct tagtgaaagc ggcgtcaagt 41040

acgacgcgtt aatcatgcgc cgcaaggaag acaaccgcaa agacacgatc atcaaagaag 41100

aagtattgcg cgctattggc ctggacaaca ttgtgtgcgc ttttgatgat tcacctaacg 41160

tggttaagca cttccgcagc ctgggaatca caacctatca ggtcacggaa tacgataagc 41220

cacataatca tatccaatcg catggcgtgg aggaattgca gaaatgatcc tgaaagtagc 41280

atatgctaac cctcgcggct gcaatatata cggctttaag gttggcgcga tggtcaacgc 41340

caggataaac gaaaaaggcg tattagttgt caagtctcct aaaactggca tggatatgcg 41400

catcaaggag aacggcggaa gattgtttat caggtcaatg gctggtcaaa cgctcgcaac 41460

gttcacaaaa tagcactttt tgcaaaaaca gattgtcggg cttgcggtat agtaagccca 41520

tcgacaacaa acgaggatta aatcatggtt aataaagttg agtttttcaa agcggtagac 41580

aagattgacg gcgcgcaaag tttctttgct atccattcca ttgacggcga agacgttgca 41640

tacaacgtga ttagtcgaga cggtatatca aaacttagtg acctggtggg ttatattgtg 41700

gcgaacggga aatgtgcatg gcccgatttg ccacgagagc cgcgccgccg agacatgcaa 41760

aatccagtcc tgctttacac gctggtaatt gaaggagtca acaaatgatt tggttatacttg 41820

tattttggt ggttttcttt tatatttcag gcttattcat cttccgtgcg ctggtgaaaa 41880

catgcgattg cacggataaa gatcagcctc ttgttctgat gttctggttt gtgtggtttt 41940

gggtggcgct ttaccagatt gtacgggatg aaagcggctt taagtggtga ttcttactat 42000

agcgcattca cgcagtgcgc tatgttgaga taacaacatg agatcatgaa gatgcgaaac 42060

ttcgaaaaaa atcgtatcca aaaagaagcg atacagctac cacgagcacc aagagatgaa 42120

tcaacgctat cgcaataagc gccagagcaa aggtaaacat tccggtaaac atggagaaaa 42180

tcaatatgaa acgagacatt atcatcctta acggaccgcc aggagttggc aaagatacgc 42240

ttgcggcata cttaacgggt catcgttacg cagcggttaa agcgtcgttt aagcaaccga 42300

tgtttgatat tgcattctcc atgcttggcg tttaccgata tgacgagttc attgatctgt 42360

ataacgaccg cgagcagaaa gaaaagcctc aagcaatctt gcaaggtaag tcgccgcgac 42420

aattcatgat ctggattagc gaagaagtca tgaagccagc ttttggcgag caatatttcg 42480

gaaatcgcat ggttgagcaa gtacacgaga tgtacagaga tttggctgtt gtaattagtg 42540

atggcggatt ccctgaagaa atcaagccgc tcgttaaggc aggccatgag gttcacattt 42600

gccgactgca tcgcgaagga ttcacgtttg atggagattc gcgaaactat attgacctga 42660

gcggatacca tcacagagtc aaacattacg acttcacaat gactgatggc gagccagaaa 42720

agactgttga cgagatcatc aaaactgtcc agtggaaaca cattaagatc aaatagcacg 42780

aattgctaaa agcggggcgc aatgcctcgc tataatgcaa acaccaatca acgagaggaa 42840

atcatcatgg tagagttaaa cgacattcgc gttggaacaa aatttcgcgt aacctgggca 42900

gataaatatt gcggagttag caagggccag gttgttactg tggatagtat ttatcgcgga 42960

tgcagcaaag actttcgccg cccacgcatc aagaatggct atatcattac tcgacgtttt 43020

ggctttgagt gttattgcgt agtggcgact caaggagttt tgattgagct tcagcgaatt 43080

agcgatcatc gcggctgtca tgtcaaaaca acgaagatac catctcagcg atcgcgatac 43140

gatgcgcgac ggatgcgccg actcgctcga aatgcgatca agttcaagaa gcatggtggt 43200

aacttttacc gaatgtacaa agggattgcg cgaaatgctg gaaaataaca acaacgcaac 43260

caacccaaag ccgccatttg attttcatga aatcatgatt gtgttattct tgattctctt 43320

agttgtttat tcagtgaggg ccgcaatatg gtttttaatg cactaaaacg aatctttaaa 43380

cctgaatacc ggatcgttgc ttgcacagaa tgcaagattt attatctgca acgccgccgc 43440

gttattagtg gggattggga atatctgacg gatgatgctt tcggcttttg ggctatggag 43500

ttcaatagct acagcgaagc gcgtgagtac gctaaaaagt tgcacaagtt cagaccttac 43560

aagctgcgtt tgctggttgt caaaaaatgc gatgtgtggg atcacgtatg aaaagagtaa 43620

ttataacttt tattgctctt atcgctggtt ctcacttggc ttatgggtat agcggcattg 43680

tcgagtcaat cggcggaatg attatcttcc tggctggcgc atgttacggc tacggcgatg 43740

gatttaaaaa tggcaagcgt tacatcctga ctggcaagaa ataatgttaa ccgcgccgct 43800

tcggtggcgc taaaaggagg aaacaaaatg gctaaaagca atcgtaaaat gttgattaac 43860

gcattcgaaa aggcagcgct tgagcgtggt tggaatgact catttcacac agcaaactgt 43920

atccgccgtt acggattcaa gaattgccgc tcctgggcgc gtcaaatggc ttcatggtat 43980

gatcttgatg cgcattacct tgatatggat tgcgccctgg ttgaaatgat tgagcaggca 44040

gcgttagagg atcgaccatt aacgcaatcc gattttgacg attttgttcg cgatgaaatc 44100

tattacatga gctaatagca ctttttgtta aaacttccgc gagggcattt gttaaagtgt 44160

cctcattgaa gcgaaacaga ggaaaacaaa atgaaaattc gaatcaccag ggttgacatt 44220

gaccgcaatg acggaagcat tacccttgag caatgcggat tcaaagttgg tgacattgta 44280

gaggttgacg gattctttcg cgatggttct tactgcgtgt tagcaatccg caacacggaa 44340

gaaattcgaa tcggtgataa catcggcgta aaccatgacg aatgcgaggt agtagaggaa 44400

tgatcacgat taacctttca gaagagcaag caaagaaact cctgcatgct gtaggttctc 44460

gtgcactttg cggatcaact gatgaaatga tgattgatca tgaggttgca agggagcttt 44520

ttactcagct tgagcaaaaa ttctcaccac gtccgactga cacggcagaa actgcaaaat 44580

ggcttgctga gaatggatac acggctttaa aagttcacaa ctggaagcga aaaacgaagc 44640

gttggagaat gctggatttt aattaaaatt gaatgcctca tacagcgaac aggacgcatt 44700

ttgacgaggc attttttagg taaggtgatt caatggataa ctacataaaa ccgtcgcagt 44760

ggtgcgcaca gaagcaagag gaagcattag agcgcggcga tactgaaaca gcaatgcact 44820

atttcgaaat gtacaacttg tggaagggta gaggtctgta atgtttggaa tgaacgaggc 44880

gcaattcaac gctgcaaaac gccaggctaa gaagtgcggc gaggcaatga aggcggatgt 44940

tgaaaagcgc gggaagtatg ttgatgaagt catgaagggc attattgccg aacattatca 45000

gccagttgcg ccaatgctga caattaccca attcatctgg ttgtgtggtt atcttcgtgg 45060

tagatgggga aacgcatttg accgcgaatg attgacaacg atttcctgat tggttagcat 45120

attcagcgtt tacataagag cggcattagt gccgaacggg tggcgcggct gccacgcaac 45180

aacggcgata tgcaggtttt taccgtaaga cgtatcgcct ttttaatagg tgaatatatg 45240

gaaaagtaca aattaacttt catgtttgac aatatggtga tgaatccctt aaatggttac 45300

gcttgcaata gcttttctgt taccgtgaat gcggatgaaa ttgaggatga tggcaaattt 45360

atcctaatag gtgattacat ttatcctatt cacaatatca attgcatcaa acgcgagatc 45420

gtaaaagatg aagtacaatg taagattgac gatcaggcga atggggcgta attgctcgtc 45480

atgtagacag agttttgaat gtgaggttga ggcgcgcgga gagcttgagg cagcagcaag 45540

agcaaaagag ttgtccggcg ctaatcaaga cactcaccag ttttcaatag atcttgtaag 45600

gaaaatatca tgagttctgt aattattggt ttggttgctt taatcattgg tgttctaatc 45660

ggctttgtcg ctgtatcttt cgtggtggct gttggtgttc gctataagtc aaaaaaaggt 45720

gaatttgctt tagcgttttg ggatgaagag cagaaagttt ggcaggttcg cggtcactat 45780

ttatcaatcg gtggcaggat tcacagcact atgaaaacaa atccagaaaa agtaaaatac 45840

aagtattgac caactacccg ccaaatggcg ggttttttat tgcctgcaat ttgtctataa 45900

tgaggctttg ttataaagga gggcgcatta tggctaaaaa cgttgagcca gcagtaaagg 45960

cttgcaattt taaaaaacta tataataagc aatatggcga tatagcaaaa atgacaaagg 46020

cgcacaagta cacgccggaa caggtgtttg atctggctgt tcgttacttt acgtggggctg 46080

aggaaaacca tatacaggct gcggaaacgg catcctttca gggtgacgta tacgaaagca 46140

agattcacaa gccgcgcgtg ttcaccttaa acggattccg attattcgct ggcctttctg 46200

cctccgtgct tgaaaaatgg cgacgcgagc caggatttag cgatgttatg gactttatcg 46260

acggcgttgt ttacgagcaa aaattccagc ttgctgcaaa caatattgtt aacgccggat 46320

tcattggtaa agagatcggc attgaaaagc cagccaccgt taccattgag aataacacaa 46380

gcgccagcgt tgacgctgta acggcggagg aagtcaaaga ggcggtaatt gacattctgg 46440

agaagatata atgctgattt gggaagactt aacagcggcg caaaaacgcg ccattaagga 46500

aatgagcgaa tattcattcg aaaagatgat ccgcatttgg tttcaacttc tccaggggca 46560

aaggttttta ggaaactggc atttcagcta cctatgcagc gaagttgaaa aaatcatcaa 46620

aggcgagtca cagaatgtaa ttttcaacat aacgccaggc agtggtaaaa cagagatatt 46680

ctcgatccac atgtcacctt atgcgtcatt gaaaagtaag aaggtgagaa atcttaattt 46740

gtcgtttagt gatggacttg tccagcagaa tagcaaccgc attaaggaga ttatggttc 46800

tccagaatgg caggagctat ggcctggaaa gttggcaaag gcgagcgcaa aggatttgat 46860

tgtaacagat ggcggcaagg ttaaattaca agtcaactcg cgttctattg gtggtcaggt 46920

tactggtctg cgtggcggct acatggacga tggtttact ggtatgctgg ttcttgacga 46980

tccagacaag ccggatgaca tgctttctaa ggtgaagcgc gaagccggac acatgcgcct 47040

aaagaacacg gtgcgatcgc gtcgaatgaa ggacaccacg ccaattgtga tggttcaaca 47100

acggctacac gtcaatgatt caacgtggtt catgacaaac ggcggcatgg gcggcatcca 47160

gtttaaggtg gttagtattc cggcgctcgt tactaaggaa tatcgcgaaa cgcttcctga 47220

ttggttaaag cctgagtttg atcgcgatgt tttgtccagt aatccagtga tgattgatgg 47280

tgttgctcat tattcgttct ggcctgctaa ggaaagcgcg gaggaattac ttgcacttcg 47340

aaatgctgat ccgtacacgt tcgccagtca gtatcagcaa cagcctgttg cgcttggtgg 47400

taacgtgttt aaaacagagt ggttccagta ttacggcagc agcgagaaat gcacgctacc 47460

aaaaccggat cgctttgaat atacatttat cacggttgat accgcgcaga aaacaggtga 47520

gcttaacgac tactccgtga tctgttattg ggggatgtat aaagatcgtg tctacttcat 47580

tgatggcgtt cgcggaaaat gggaagcacc ggaccttgag acgaattttg ttgcattcgt 47640

taatcagtgc tggaagcgca ataaagaatg cggaacgttg cgaaggattc atgttgaaga 47700

taaatcatct ggcactggct tgattcagag tgcagcgaaa aaaatcatga tcaagatcaa 47760

tcccgttcag cgcgataaag acaaggttac gcgagcaatg gacgcagcgc cagtaatgcg 47820

cgccggacgc gtagcacttc cagaatccca tcctatgctg gctgagatac tagctgaagt 47880

tgcggcgttt acattcgatg atagccatcc tcatgatgat attgttgata acatcattga 47940

tgcggttaac attgaaatga acatggcaga tgatccggtt ggaagaatga aaaaattggc 48000

aggtttgcgg aacaaatagc acgaatcgac aaaccaatat ataatcaaag gctggaaaat 48060

ccagcctttt ttatggagg aaagataatg agtaaatcac gaatcgttaa ggctgatggc 48120

tataacgaga tctttaaagg cgagaattgc aaatcgcgag tagagcaacc attctacatg 48180

caatcaatgc catacaagac acttgctgac ttttacgaaa aagacggcct ggcaaaaagg 48240

gttattgacg tagtaccaga ggaaatggtt tcccctggct tcacggttga cggcgtggca 48300

gacgaggcag cattccgttc tttgtgggat gagaagcgat tgaatgcaaa aattattgat 48360

gcgctttgct ggtcgcgttt gtttggtgga tctgcaatca ttgctcttgt tcaggatggg 48420

cgagcgctca aatcacctgt aaagcctggt gccatgttgg aggatgtgcg agtttacgat 48480

cgctatcaga ttcgcgttga agcgcgcgaa actaatccac gcaaagttcg ctatggtgag 48540

ccagtgcttt acactgtaac gccaggcgga gacttgccgg aataccaggt acactacacg 48600

cgagtttgca ttatcgacgg tgagcggata cctaacgcaa agcgacgcag caatgacggc 48660

tggggtgagt cagttcttaa caagcggctt gttgaggcta ttgttgatta taactattgt 48720

gaaacattag cgacgcaatt attgcgaaga aaacaacagg cagtatggaa ggccaaaggt 48780

cttgctgact tgtgcgatga tgaagaaggt gttagcgcgg cacgcttgag acttgcgcag 48840

gttgacgatg agggcggcgt aggcaaggcc attggtattg atgctgagga tgaggaatat 48900

gatgttctta actctgacat tagcggcgtt gattcttttc tggaaaagaa aatggatcgc 48960

atagtttccc tttcaggtat tcatgaaatc atcctgaaaa ataaaaacgt tggcggcgtt 49020

agcgcaagcc aaaacacggc gcttgaaaca ttctataaac ttatgagcg aaaacgcgtt 49080

gaggattaca agcctatcct tgaatttcta ttaccgttca tcattagtga gcaggagtgg 49140

agcattgagt tttcaccttt gagcgtgcct agcgataaag atcaagctga gattctgaat 49200

aagaatatcg actcaatcag caaggctatt gatggtcagt tccttgatgt tgaagaagcg 49260

cgcgacacgt tgcgagcaat cgccccaagc gttaaactaa aggacactaa taaaatcaag 49320

ctgccggagc caactgagcc ggagccaggc acacaggggga atgagtaatg aaagtgaaag 49380

gcgttgttaa acaatggcgc tttcctgaag caagcgagcg gcaatttagc cgctcaattc 49440

aggaggcaat cagagatctt gtagttctca tgcgcaagcg aacaaaagcg atgaagtttg 49500

acgcaacaga taatgagatc aacagcgcag aagatgaaat taacagcatg gcaactgacc 49560

ttatagctgg cattgtttcc actcttcctg caatcgcgtt gaccattat aaattcaatg 49620

cgaagcagtt tatcaatgtt gccaagtcaa ctggcggaaa ggataatacg gcggtaattg 49680

ttttgatcgc ggttggcgct aacgctaacg aggattggta tcagacgctc tacggtcaat 49740

ggcacggggct tacagaatca tcgctaagga agttattcac aaacattgtt tctgattggt 49800

caacaaacat tcgcaatgca aactttcgcg gaagcaatga taaacaggtt aatgatcttg 49860

cagaaaagag atttgctgtt tatagctctt ggggtaaaac gagatcagaa aacattatcg 49920

gagcgtggaa tagtcgcctg atgcgccagc gcctttatga tgctaaggta actcattatt 49980

tctggcatgg aatgttagat gacagagagc gcttgcagca tgtgttatgg gaaggtaaaa 50040

gaatagcgct tgatgcaatt catgatttcc ctggtgagcc gtggggttgc cgttgctggg 50100

cgattccaga ttggaatagt aaaggagaat aattaatgaa ggcaaaacaa agatttgata 50160

gcgtccaggt taaggcgcat tttgatgata atggcttttt agtggatcgc ccaattgtgg 50220

cgcgaattgg tttgcaggag taccgcacac cgtatggaat cagacgagag ttcagaccag 50280

cgtccgaggt gttcaaggct gattcccttg ccacattcgc aggtaagccg atcactatcg 50340

gtcacgtcac ggtaacgcca gacaatgcgg atcaggttgt tgttggttca tgcgctggtg 50400

ctggtgttcc aaatggtgtt ggcgttgaag ttccgttaag tattactca aagcgagcaa 50460

ttgagagcgc aaaaaagaaa gacacggcgg aaatttcagt tggttacaca tcaattgata 50520

ttgataaacc aggttggggc aataataaaa ctggcgatta cttgtttgag gaagacatta 50580

aggaagactg gaagcctgat tcccctgatt gggttaaatt tgacgccatt cagacgaata 50640

tccgtgttaa tcacattgca cttgtctttc gtggtcgtgc tggcattgcc aaattaaatc 50700

ttgatagtga acaagatttt ccgtatagtg acgaggttct aaatgacaaa ggagatgagg 50760

aaatgacagt aaaaattaaa cttgatggcg cggtagaatt tgatgttcca aaggccgttg 50820

ctgatcatat tgaggcgtta aaggcagatg caaaagctgc aaccgaaaaa gctgatggcc 50880

ttgaggccga gcgcgacgcc ctgaaaacta aagtcgatgg cattccggcg cagattgcag 50940

aagctgttaa gaaggcaaag actgacgcag aagaacacgc caagctagtt gcagatgcaa 51000

ctgaaattgg catcaagtgt gacggcttgg acgctaaagc gatcaaagtt gcttacgtca 51060

aagaagtcac tggcgctgat atttcagaaa aagccgatgc at 51102

Claims (5)

1. a kind of Styloviridae phage E sc-COP-9 (numbers of being commissioned detached from natural environment：KCTC 13131BP), It is characterized in that：

Carry the ability with specific killing Escherichia coli, and the genome marked with sequence number 1.

2. a kind of, for preventing, enteropathogenic E. Coli from infecting and the composition for the treatment of enteropathogenic E. Coli infection, feature exist In：

The composition is with phage E sc-COP-9 described in claim 1 (numbers of being commissioned：KCTC 13131BP) it is active ingredient It is included.

3. according to claim 2 be used to prevent enteropathogenic E. Coli from infecting and treating enteropathogenic E. Coli infection Composition, it is characterised in that：

The composition is used with manufacturing the purposes of feed addictive, drinking water additive or disinfectant.

4. a kind of prevent that the side infected as caused by enteropathogenic E. Coli is infected or treated as caused by enteropathogenic E. Coli Method, it is characterised in that：

Including：It will be with the phage E sc-COP-9 (numbers of being commissioned described in claim 2 or claim 3：KCTC 13131BP) The step of composition being included for active ingredient is supplied to animal in addition to a person.

5. according to claim 4 prevent from infecting or being treated by enteropathogenic E. Coli as caused by enteropathogenic E. Coli The method of caused infection, it is characterised in that：

The composition is supplied to animal in addition to a person with feed addictive, drinking water additive or disinfectant applications.