CN113249338A - Acid-resistant escherichia coli bacteriophage and embedding process thereof - Google Patents

Abstract

The invention discloses an acid-resistant Escherichia coli bacteriophage with the preservation number of CGMCC No.21991, which is found to have no virulence genes and lysogenic genes after sequencing of the whole gene; the bacteriophage has good high temperature resistance and wide acid-base tolerance range, and can effectively preserve the activity of the bacteriophage when passing through a digestive system; provides a phage source for the industrial production of phage for the prevention and treatment of pathogenic escherichia coli BE-20166 in the poultry breeding environment; the invention also provides an embedding process of the escherichia coli bacteriophage, and the embedded bacteriophage has more stable property.

Description

Acid-resistant escherichia coli bacteriophage and embedding process thereof

Technical Field

The invention relates to the technical field of biology, in particular to an acid-resistant escherichia coli bacteriophage and application thereof in pathogenic escherichia coli infection of poultry animals.

Background

Avian colibacillosis is caused by some pathogenic or conditionally pathogenic escherichia coli, is one of the most serious bacterial diseases harming the poultry industry, and is most prone to generating drug resistance in the process of preventing and treating diseases, so that a series of problems of increase of the dosage of antibiotics, drug residues of poultry products and the like are caused. The disease can occur all the year round, but is frequent in winter and spring and in variable temperature periods, which brings serious economic loss to poultry industry. The avian colibacillosis is mainly prevented and treated clinically through antibiotics, but due to blind addition or excessive use of the antibiotics, drug-resistant strains of the colibacillosis are continuously increased, the drug resistance spectrum is continuously expanded, the treatment effect is further reduced, the breeding benefit is reduced, and meanwhile, the food safety is threatened. Currently, colibacillosis remains one of the major bacterial infections that are harmful to livestock and poultry farming. Therefore, based on the considerations of breeding benefit, food safety and public health, it is urgent to find a new antibacterial preparation which has a definite therapeutic effect on colibacillosis, has no residue, and meets the national culture resistance reduction or resistance-free development direction.

Bacterial resistance is becoming more severe, but the speed of development of new antibiotics is far behind the speed of increasing bacterial resistance, and new alternative antibiotic bactericidal substances are sought to prevent and treat resistant infections to further reduce the use of antibiotics. Many studies have demonstrated that phage therapy is a good alternative to antibiotic therapy.

The bacteriophage has the unique advantages of strong specificity, no cross resistance with antibiotics, no residue, no infection to human and livestock, easy screening and acquisition and the like, and becomes an option with huge potential. Phage preparations are a new antibacterial product, and are receiving more and more attention because they have the advantages of causing dysbacteriosis and reducing the problem of drug residue, unlike antibiotics.

The invention separates a coliphage with good acid-base tolerance, strong cracking performance and wide bacteriophagic spectrum from the sewage of a farm, preliminarily knows the biological characteristics of the coliphage, finds no genes with virulence and lysogeny by whole gene sequencing, proves the safety of the coliphage on the gene level, analyzes the biological characteristics of the coliphage and provides a theoretical basis for the research of the control of the coliphage on the colibacillosis.

Disclosure of Invention

The invention aims to provide an acid-resistant Escherichia coli bacteriophage RDP-EC-20140, and aims to solve the problems that poultry in a farm are not treated timely due to Escherichia coli infection and acute disease, and drug taking is ineffective due to drug resistance of pathogenic bacteria and the like. Another purpose of the invention is to provide application and an embedding process of the Escherichia coli bacteriophage RDP-EC-20140.

In order to achieve the purpose, the invention adopts the following technical scheme:

an acid-resistant Escherichia coli bacteriophage RDP-EC-20140 is observed through an electron microscope, wherein the Escherichia coli bacteriophage RDP-EC-20140 has a polyhedral and three-dimensionally symmetrical head, is wrapped by nucleic acid, has a diameter of 70nm, has a tail with a length of 120nm, has a tail sheath, and is a myxoviridae with uroviridae, wherein the neck is connected with the head and the tail; the gene length is 52804bp, and the phage has no virulence gene and lysogenic gene, is preserved in China general microbiological culture Collection center in 03-25.2021 at the preservation address of: the No. 3 Xilu No. 1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 21991.

Further, the Escherichia coli bacteriophage RDP-EC-20140 is treated at 65 ℃ for 60min, and the titer is still maintained at 10⁸pfu/mL, treated at 80 deg.C for 30min, and the titer is maintained at 10⁵pfu/mL, better tolerance to temperature; the titer is still maintained at 10 by culturing at 37 deg.C for 1h in the pH range of 4-10¹⁰pfu/mL, with highest potency at pH 7, phage retained high activity at pH 12 and pH 2, and the phage was acid-base tolerant over a wide range.

Further, the Escherichia coli bacteriophage RDP-EC-20140 has a synergistic effect with antibiotics.

The invention also aims to provide application of the coliphage RDP-EC-20140 in preparing medicines for preventing and/or treating diseases caused by escherichia coli.

The invention also aims to provide application of the coliphage RDP-EC-20140 in preparing feed for preventing and/or treating diseases caused by escherichia coli.

The invention also provides a bactericidal composition for preventing and treating escherichia coli, which comprises an effective amount of escherichia coli bacteriophage RDP-EC-20140.

Another object of the present invention is to provide an application of Escherichia coli, which is realized by using the Escherichia coli bacteriophage RDP-EC-20140 as a potential therapeutic drug or health product for bacterial infection caused by Escherichia coli.

Another object of the invention is to provide an additive for preventing and/or treating diseases caused by Escherichia coli, which is realized by that the effective component of the additive comprises Escherichia coli bacteriophage RDP-EC-20140.

The invention also provides a cleaning agent or disinfectant, which comprises the coliphage RDP-EC-20140.

The invention also provides an embedding process of the Escherichia coli bacteriophage RDP-EC-20140, which comprises the following steps:

(1) weighing the following raw materials in parts by weight: 35% coliphage RDP-EC-20140 (lyophilized powder), 62% starch, 1.5% microcrystalline cellulose and 1.5% sodium starch; placing the raw materials in a mixer and mixing for 10 minutes at room temperature;

(2) putting the raw materials mixed in the step (1) into a swing granulator for granulation, pouring particles discharged from the granulator into a swing granulator to complete granulation, then putting the granules into a low-temperature fluidized bed for drying at the drying temperature of 40-42 ℃ for 30-40 minutes, and removing oversize and undersize particles through a vibrating screen after drying;

(3) and (3) putting the particles obtained in the step (2) and 30% by weight of palm oil into an embedding machine together to finish embedding.

The invention has the beneficial effects that:

(1) the invention separates and obtains Escherichia coli BE-20166 from the liver of white feather broiler of Shandong one farm, and through drug sensitive detection and PCR detection, the pathogenic bacterium BE-20166 has drug resistance to various antibiotics, contains a plurality of virulence genes, and has strong pathogenicity. The Escherichia coli bacteriophage RDP-EC-20140 is obtained by separating from sewage of a white feather broiler farm, and the bacteriophage has good acid and alkali resistance, certain tolerance to temperature, wider cracking rate and higher cracking rate through relevant experimental verification, thereby providing a bacteriophage source for the industrial production of the bacteriophage for the prevention and treatment of pathogenic Escherichia coli BE-20166 in a poultry breeding environment;

(2) the Escherichia coli bacteriophage RDP-EC-20140 provided by the invention does not have virulence genes and lysogenic genes, and the bacteriophage has better safety according to the gene level; the gene of the phage contains a plurality of lyase genes, and the cleaved product contains lyase expressed by the genes, so that the related escherichia coli can be cleaved again in the using process of the product;

(3) the Escherichia coli bacteriophage RDP-EC-20140 is treated at 65 ℃ for 60min, and the titer is still maintained at 10⁸pfu/mL, treated at 80 deg.C for 30min, and the titer is maintained at 10⁵pfu/mL, with respect to temperatureBetter tolerance; the titer is still maintained at 10 by culturing at 37 deg.C for 1h in the pH range of 4-10¹⁰pfu/mL, with highest potency at pH 7, phage retained high activity at pH 12 and pH 2, with a broad acid-base tolerance range.

(4) The bacteriophage RDP-EC-20140 provided by the invention and the antibiotic have a synergistic effect, so that on one hand, the bacteriophage can improve the sensitivity of the medicine, and the medicine can improve the cracking property of the bacteriophage, and the bacteriophage has very important use value in the process of treating animal diseases.

(5) The coliphage RDP-EC-20140 can be used alone or in a mixture as a biological bactericide, a feed additive or a therapeutic drug, a health product and a medical apparatus for bacterial infection caused by colibacillosis; the Escherichia coli bacteriophage RDP-EC-20140 can be applied to treating or preventing infectious diseases caused by Escherichia coli.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description of the embodiments with reference to the accompanying drawings:

FIG. 1 is an electron micrograph of bacteriophage RDP-EC-20140 according to the present invention;

FIG. 2 is a schematic thermal stability diagram of bacteriophage RDP-EC-20140 of the present invention;

FIG. 3 is a schematic acid-base stability diagram of bacteriophage RDP-EC-20140 of the present invention;

FIG. 4 is a schematic representation of the genetic stability curve of bacteriophage RDP-EC-20140 of the present invention;

FIG. 5 is a schematic diagram of a one-step growth curve of bacteriophage RDP-EC-20140 of the present invention;

FIG. 6 is a lysis curve of bacteriophage RDP-EC-20140 of the present invention against host bacterium BE-20166;

FIG. 7 is a schematic diagram of the synergy test between the bacteriophage RDP-EC-20140 of the present invention and antibiotics;

FIG. 8 is a comparison graph of the storage stability of lyophilized powder and embedded particles of bacteriophage RDP-EC-20140 of the present invention.

Detailed Description

The invention is further illustrated by the following figures and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The Escherichia coli BE-20166 is obtained by separating the liver of white feather broiler chicken from certain farm in Shandong province, and through drug sensitive detection and PCR detection, the pathogenic bacteria has drug resistance to various antibiotics, contains multiple virulence genes and has strong pathogenicity. The coliphage RDP-EC-20140 is obtained by separating from sewage of white feather broiler farms. Relevant experiments verify that the bacteriophage has good acid and alkali resistance, certain tolerance to temperature, wider lysis rate and higher lysis rate.

Example 1 isolation and identification of pathogenic bacteria and phages

1. Isolation and identification of pathogenic E.coli BE-20166

Sampling from a transverse breeding chicken farm of the Jimo field, taking the liver of a sick chicken by aseptic operation, streaking on a selective medium (Mackanka agar medium), culturing at 37 ℃ for 18-24h, forming a red colony which is round, flat, neat in edge and smooth and wet in surface on the medium, picking a typical colony, continuously streaking and purifying for 3 times, picking a single colony, inoculating into 5mL of LB broth, and carrying out shaking culture at 37 ℃ and 200rpm for 8h to obtain a uniform and turbid bacterial suspension. And the serotype is determined to be O78 through 16sRNA molecular identification, and the serotype is further determined to be O78 through serum identification, so that O78 has strong pathogenicity, can be transmitted to human beings through methods such as a food chain and the like, and has strong pathogenic infectivity. It was named BE-20166 according to the laboratory nomenclature and stored in a-80 ℃ freezer.

Further, the virulence genes were determined by PCR, and BE-20166 was found to have genes of virulence factor uptake system irp2(3), iucD (6), virulence factor-temperature sensitive hemagglutinin tsh (5), and virulence factor-colicin V cvAC (1).

2. Isolation and characterization of Escherichia coli phage RDP-EC-20140

(1) Sewage treatment: collecting sewage from transverse culturing farm of Jimo field, centrifuging at 10000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter, and collecting filtrate.

(2) Preparing a mixed bacterial suspension: 0.2mL of the bacterial suspension and 0.3mL of the filtrate were added to 5mL of LB broth, incubated overnight at 37 ℃ with shaking at 200rpm, and then centrifuged at 10000rpm for 5min, and the supernatant was filtered through a 0.22 μm filter and collected for further use.

(3) Phage separation: separating bacteriophage by double-plate method, mixing 0.2mL of mixed bacterial suspension filtrate and 0.2mL of Escherichia coli bacterial suspension, water bathing at 37 deg.C for 10min, spreading double-plate, culturing at 37 deg.C for 8-12h, and observing result. If there are phage, there will be clear plaques on the plate. Selecting a single plaque in 1mL of physiological saline, carrying out water bath at 37 ℃ for 30min, centrifuging at 10000rpm for 5min, filtering the supernatant with a 0.22-micron filter, taking 0.1mL of filtrate and 0.2mL of bacterial suspension, carrying out water bath at 37 ℃ for 10min, paving a double-plate, culturing in a 37-DEG C incubator for 8-12h for purification, purifying for 3-4 times according to the steps until the plaque is uniform in size, and obtaining a strain of bacteriophage which is named as RDP-EC-20140. The plaque diameter formed by RDP-EC-20140 on the double plate is 1mm-2 mm.

3. Preservation of phages

Phage multiplication fluid was mixed with 60% glycerol according to 1: 1, and storing in liquid nitrogen.

Example 2 morphological Observation of phages

Applying a phosphotungstic acid negative dyeing method: phage liquid was dropped on a paraffin plate (titer 10)¹⁰pfu/m L)100 mu L, placing the surface of the copper net with the film on a phage droplet, taking down after 10min, naturally drying in the air for 2-3min, then dropping a 2% phosphotungstic acid (PTA) aqueous solution on the copper net for dyeing, taking down after 10min, drying in the air for 10-15 min, observing by using an electron microscope, and selecting a clear phage image for photographing. As can be seen from the electron micrograph of FIG. 1, the phage has a polyhedral, three-dimensionally symmetric head, which is coated with nucleic acid, has a diameter of 70nm, a tail with a length of 120nm, and a tail sheath, and has a neck portion connected with the head and the tail, which is the myxoma muscle with uroviridaeCarnivora family.

Example 3 measurement of phage thermostability

Subpackaging the phage stock solution into 50 ml sterilized conical flasks, incubating for 30min and 60min at 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 80 deg.C and 90 deg.C, diluting with physiological saline 10 times in gradient, and spreading double plates to determine titer; the results are shown in FIG. 2.

As can be seen from FIG. 2, the titer of the phage RDP-EC-20140 is stable within 35-55 ℃, and is kept at 10¹⁰pfu/mL, treating at 65 deg.C for 60min, and maintaining titer at 10⁸pfu/mL, treated at 80 deg.C for 30min, and the titer is maintained at 10⁵pfu/mL shows that the Escherichia coli bacteriophage RDP-EC-20140 has better high temperature resistance, which has very important reference value in the production, transportation and storage of the bacteriophage.

Example 4 determination of phage pH stability

Adjusting pH of normal saline with dilute hydrochloric acid and dilute NaOH solution to obtain buffer solution with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and diluting phage with prepared buffer solution to 1 x 10¹²pfu/mL, the diluted solution was incubated at 37 ℃ for 1 hour in a water bath, diluted 10-fold with physiological saline and then plated on double plates to determine the titer. The results of the inverted culture at 37 ℃ for 4-6h are shown in FIG. 3.

As can be seen from FIG. 3, the titer was maintained at 10 when the cells were incubated at 37 ℃ for 1 hour in the range of pH4-10¹⁰pfu/mL, with the highest potency at pH 7, and phage still retained higher activity at pH 12 and pH 2, indicating that phage RDP-EC-20140 has better acid and alkali resistance. The bacteriophage DP-EC-20140 has good application value in the process of treating diseases by the bacteriophage, can effectively preserve the activity of the bacteriophage when the bacteriophage passes through a digestive system, and cannot reduce the activity of the bacteriophage due to acid and alkali, so that a sufficient number of bacteriophages can be ensured to pass through a digestive tract to crack corresponding escherichia coli in an intestinal tract.

Example 5 genetic test for stability

The RDP-EC-20140 is continuously passaged for 29 times, the same amount of RDP-EC 20140 and BE-20166 is added into each generation of culture medium, the culture time of each generation is 6h, then double plates are paved, and the titer of the medium is determined.

As can be seen from FIG. 4, the titer stabilized at 10 after the continuous 29 generations of RDP-EC-20140¹¹The pfu/mL is higher than that of the phage, so that the phage has better genetic stability, the titer is not gradually reduced due to excessive passage times in the continuous passage process, and the phage keeps higher activity, which indicates that the RDP-EC-20140 is suitable for industrial production.

Example 6: drawing of phage growth curves

Taking an escherichia coli suspension in a logarithmic growth phase, inoculating escherichia coli BE-20166 and bacteriophage RDP-EC-20140 according to the proportion of the optimal multiplicity of infection, carrying out water bath for 10min at 37 ℃, then centrifuging for 5min at 12000r/min at normal temperature, discarding supernatant, removing free bacteriophage which is not adsorbed on a host, and washing twice by using LB broth culture medium. The pellet was resuspended in LB broth at 37 ℃ and quickly placed on a shaker at 37 ℃ and 200r/min for shaking culture and timing. And (3) sampling at regular time to measure the titer, drawing a growth curve by taking time as an abscissa and a bacteriophage titer logarithm value as an ordinate, obtaining the incubation period and the lysis period of the bacteriophage, and calculating the average lysis amount. Mean lysis is end phage titer/initial host bacteria concentration.

As can be seen from FIG. 5, the titer did not change significantly within 60min after the phage infected the host bacteria, indicating that the incubation period was about 60min, and the titer of the phage increased significantly within 60-100min after infection, and then became stable. Through calculation, the phage lysis amount is about 186 PFU/infected cell, and the phage RDP-EC-20140 is characterized by short latency, strong lytic capacity and high replication.

Example 7 phage vs host lysis curves

The host bacterial suspension cultured for 6h is inoculated into 100mL LB medium at a ratio of 2:100, and cultured at 37 ℃ for 2h (OD about 0.15, about 5.0 x 10) with shaking at 200rpm⁸cfu/mL) the bacterial suspension was divided into 4 sterilized Erlenmeyer flasks, and 0.2mL of phage-proliferating solution was added to 3 of them at the optimum multiplicity of infection, while taking the average value of the control without phage addition.

As can BE seen from FIG. 6, when the Escherichia coli phage RDP-EC-20140 is added into the suspension of the host bacterium BE-20166, because the phage has a stronger lysis effect on the host, the OD600 absorbance gradually decreases after the phage is inoculated, the host bacterium starts to propagate without being lysed with the time increase, and the OD600 absorbance gradually increases until 36 hours later, and the two tend to BE consistent. The bacteriophage can effectively inhibit the growth of host bacteria within 9 hours, and the bacteriophage has very important application value in treating animal diseases.

EXAMPLE 8 phage and antibiotic synergy test

3% of the host BE-20166 was inoculated into LB broth, kanamycin monosulfate was added to a final concentration of 30ppm, and 1 x 10 was added¹⁰The change in OD600 absorbance was measured periodically for pfu/mL phage RDP-EC-20140.

As can be seen from FIG. 7, the OD600 value gradually increases with the time, but the absorbance of the combination of the phage and the antibiotic is smaller than that of the other two groups, which shows that the combination of the antibiotic and the phage can inhibit the growth of host bacteria more effectively, the phage and the antibiotic have synergistic effect, the phage can improve the sensitivity of the drug, and the drug can improve the lytic property of the phage, which has very important use value in the process of treating animal diseases.

Example 9: cracking rate test

Under aseptic conditions, 1mL of sample and 1mL of host bacterium solution (1X 10)⁵CFU/mL), incubating for 15 minutes at 37 ℃, mixing uniformly, and diluting to 10 with physiological saline^-1-10^-3Each gradient was applied in an amount of 100. mu.L, spread on LB agar plates, incubated at 37 ℃ for 24 hours, and each gradient was repeated twice. Simultaneously taking 1mL of physiological saline and 1mL of host bacterium liquid (1X 10)⁵CFU/mL) as a blank, repeat the above steps. Plates with 30-300 colonies were picked for enumeration. The test was repeated 3 times and the average was taken. The phage lysis rate was (1-number of treated colonies/number of control colonies) × 100%.

The calculation shows that the cracking rate of the RDP-EC-20140 reaches 99%, the RDP-EC-20140 has a good cracking effect on a host, and the RDP-EC-20140 is suitable for being used in the culture process.

Example 10 determination of optimal multiplicity of infection

The phage multiplication solution and the host were added to the LB broth at a ratio of the multiplicity of infection of 100, 10, 1, 0.1, 0.01, and the total volume of the culture system was ensured to be the same. After culturing at 37 ℃ for 8h with shaking at 200rpm, the supernatant was centrifuged at 12000r/min at room temperature for 5min, and the titer was determined by plating the supernatant on double plates, and the results are shown in Table 1.

TABLE 1 best multiplicity of infection test

As is clear from Table 1, when the infection count is 01, the growth medium is relatively clear and the titer is the highest after 8 hours of culture, indicating that the optimum infection count of RDP-EC-20140 is 1.

Example 11: the industrial production process of the Escherichia coli bacteriophage RDP-EC-20140 comprises the following specific steps:

1. seed preparation

(1) Preparing a host seed: selecting a single colony in an aseptic environment, inoculating the single colony in 5mL of LB liquid culture medium, culturing at 37 ℃ and 200rpm for 4-6h, inoculating the cultured host bacteria in 5% of the inoculum size in 100mL of LB liquid culture medium, culturing at 37 ℃ and 200rpm for 4-6h, inoculating the host bacteria in 5% of the inoculum size in 1000mL of LB liquid culture medium, culturing at 37 ℃ and 150rpm for 4-6h, and placing the cultured host strain seed in a refrigerator at 4 ℃ for later use.

(2) Preparing phage seeds, namely, after properly diluting the preserved phage seeds, putting 200 mu L and 200 mu L of host bacteria into an upper layer culture medium, pouring a double-layer plate, culturing for 8-12h at 37 ℃, selecting a plate with a cloudy plaque, washing the upper layer culture medium by using 5mL of LB liquid culture medium, then, centrifuging at 12000rpm for 10min, filtering through a 0.22 mu m filter membrane, and collecting clear liquid.

Inoculating the clear liquid and the host into 5mL of LB culture medium respectively in a proportion of 2%, culturing at 37 ℃ and 200rpm for 4-8h (until the proliferation liquid becomes clear or floccules appear), inoculating the cultured phage and the host into 100mL of LB culture medium simultaneously in an inoculation amount of 2%, culturing at 37 ℃ and 200rpm for 4-8h, inoculating the cultured phage and the host into 1000mL of LB culture medium simultaneously in an inoculation amount of 2%, culturing at 37 ℃ and 200rpm for 4-8h, and placing the cultured phage seeds into a refrigerator at 4 ℃ for later use.

2. And (3) phage multiplication:

inoculating the prepared host bacteria into a seed fermentation tank according to the proportion of 3%, culturing for 3-4h, then inoculating the host bacteria into the fermentation tank through a seed transfer pipeline, wherein the inoculation amount is 5%, culturing the host in the seed tank for 3-4h, inoculating the host into the phage according to the inoculation amount of 5% when the OD is 0.7-0.9, culturing for 6-8h, and after the dissolved oxygen is reversely increased, the pH value tends to be stable, and ending the increment.

Note: fermentation tank parameters: the seeding tank is 200 ℃ and 300rpm, the aeration is 1:0.6-0.8vvm at 37 ℃. Fermentation tank: 120-150pm, 37 ℃, 1:0.6-0.8 vvm.

The components of the culture medium: 2.5 percent of yeast extract powder, 0.3 percent of glycerol, 0.8 percent of sodium chloride, 0.2 percent of monopotassium phosphate, 0.2 percent of dipotassium phosphate, 20ppm of calcium chloride and 20ppm of magnesium chloride.

3. Phage post-treatment process

(1) Centrifuging: and (4) centrifuging the proliferated phage by using a tubular centrifuge at 14000rpm at the flow rate of less than or equal to 30L/H, and removing uncleaved hosts, bacterial debris and the like.

(2) Filtering and sterilizing with 500nm organic membrane.

(3) The concentration was carried out using 100kD wound membranes.

(4) And (3) filtering and sterilizing: the concentrated phage were sterilized by three-stage filtration (0.45 μm polypropylene, 0.2 μm double-layer polypropylene, 0.22 μm polyethersulfone).

Example 12 embedding process of coliphage RDP-EC-20140, comprising the following steps:

(1) weighing the following raw materials in parts by weight: 35% coliphage RDP-EC-20140 (lyophilized powder), 62% starch, 1.5% microcrystalline cellulose and 1.5% sodium starch; placing the raw materials in a mixer and mixing for 10 minutes at room temperature;

(2) putting the raw materials mixed in the step (1) into a swing granulator for granulation, pouring particles discharged from the granulator into a swing granulator to complete granulation, then putting the granules into a low-temperature fluidized bed for drying at the drying temperature of 40-42 ℃ for 30-40 minutes, and removing oversize and undersize particles through a vibrating screen after drying;

(3) and (3) putting the particles obtained in the step (2) and 30% by weight of palm oil into an embedding machine together to finish embedding.

Example 13 storage stability testing of phage Embedded particles and lyophilized powder

The experimental group is phage RDP-EC-20140 embedded particles obtained in example 12 of the invention;

comparison group: adding 10% of skimmed milk powder, 3% of trehalose, 3% of pyrrolidone k-30 and 0.5% of Vc as protective agents by adopting a conventional freeze-drying process, and freeze-drying to obtain phage RDP-EC-20140 freeze-dried powder;

the test and control samples were placed at room temperature and their titers were measured periodically.

As can be seen from FIG. 8, the titer of the phage after lyophilization declined relatively rapidly after 80 days, while the phage after particle embedding was relatively stable. Because the embedding process is carried out at a lower temperature, the bacteriophage has better tolerance to the temperature, and the surface of the embedded bacteriophage has a layer of grease, so that the embedded bacteriophage can effectively isolate moisture and oxygen in the air, and the product is more stable.

It should be noted that the specific embodiments are merely representative examples of the present invention, and it is obvious that the technical solution of the present invention is not limited to the above-mentioned examples, and many variations are possible. Those skilled in the art, having the benefit of this disclosure and the benefit of this written description, will appreciate that other embodiments can be devised which do not depart from the specific details disclosed herein.

Sequence listing

<110> Ray union (Qingdao) bioengineering Co., Ltd

<120> acid-resistant escherichia coli bacteriophage and embedding process thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 52804

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

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gcaacatcat tgtgtacgac tacgctgatg tcaacgatgt cgctggtacg gaaagtaaca 120

aagtgctgta caattacttg ttcgtcggta ttaggactga cgccctgaaa ggtcttgagt 180

gtgtgaaaca ttggttgtat ctccttaaca gaatgtaagt ctattatgta tataaaggag 240

gggaaagtaa atagatataa atgtataaaa tacataaaag tctatttact ctggtttaat 300

aacgtgtata taatgagagc ctcacaaaca agaggagaga ctcaatgata aagccaaaac 360

tgttgcggca tattcgcaac ggtattaagc tactcgccga aggcaatgaa tatgcctgca 420

agcaacgcgg tattacaaaa gtcggcctgt ttaataaaga gttaccgact cgctacttta 480

ccaaatcacc attaacactc gtacctgcag cggagtatta taaagacccc gaagcagctc 540

gggctaaaat caaggaaggt cttgaccgac ttgtagagga aggctatttg acagtacgtc 600

aacacactga atcatcttat tactacaagc caacttctaa ggtaaccagc aatgacaatg 660

acgagtcagc cgagtgatat tctcggtctg aaatacttta agccagaagg gattccccaa 720

gtcttcaaag atatgaagcg ctgggctatc tttgagctaa cgtacgaaga cgagactctt 780

gaagttcaat ccaaaccgaa gaaacctgca agagcgccat caggccgttc cttcagcaca 840

actgtgcctg aagagtggat gactttcgac gacgccctga cacgttgtat tcgcaatcgt 900

gaaatgaaga ttaaagctgt gcaggggaat atcacctttg tgccggcttt ggttgttcct 960

cgccagtggt actttgttga cctggacaac catgatgata atccagacat tgaagcaacc 1020

cacaaagcga taattgaggg gactcgtggt gcttatgctg agacgtcaat ctctggcaaa 1080

gggcaacaca ttgcaatccc tttaccatgg actgcagcaa catctaaaga gaaagacgaa 1140

cagctggaca tcaaaatcca aaaggctgag atgttcctgg taatgacagg caatgtattg 1200

agcgagttca atgctaatcc tgtttcagcc cgtgaatggc atgcggcaat tgagaagttc 1260

tttcttgaag caagcgcgcc tgatattgat gttgagtttg agcaagacga agaccgtggc 1320

gaagagtatg acaaagagct gtacgaacag ttagccgaca atactcgctg ggctttagag 1380

cactacctga atgagcatgc accagacggt gttggtgtta gtaatgacgg ttccgaacgt 1440

ttgagccgca tactcaaaga cttgcttcgt gtaacccgta actacgaagt tacacaacgc 1500

atcttcatgg ccagcaaagc tgcagcatac gaaggtcgca gaccaagccg acataacttg 1560

tctgttgata agtatcatca atggttcagt cgtgttggta agactgtgct gaaagaaatg 1620

caacgtgacg gcttattcgt taagaccaag tttgctcttg acatcaacaa agagttagcc 1680

aagaattcag gtgtctccct ggctgagaat gttgagttca aagtcaacga cgatattctg 1740

ccgtctgatg tattcgtcag aacggccccg tccgggttta aacggcttat tgctgagata 1800

caggataaca tatctcctgc taacagggtt aacgactatg ccatcggcac agctcttaac 1860

atcctgagca actgcgctgg tcgcaagtat gtttgtccag taggcggtca tgctaacttc 1920

ctggtaacca acatcattct tgtgggtggt tcatcaatcg gtaaatcgct gtataccgag 1980

ttgttcccac aaatccaggc aagtgtgcca gacacatcgc ctatcagcct taaccgtatc 2040

ccaagggaac agacttttgc aaccaggacg tttgctgaac tgatgtccaa cccgtcatac 2100

cattcagtcc agttgttcta ccctgaattt ggtctggctt taggttctgg cttacgcatg 2160

aatcctaaca acccggacaa ctttcagaaa gctctgatgg acgcttcaac caagcgtaag 2220

gttggcggta ttctgacagg tatcaaacgt gctaatgctg acaatgatgt taagactgta 2280

agcgagccgt gttactctat cctgggtgac tccactcaag agcttatcct tgacaacacc 2340

cggaagcaag acttctcaag tggctttttg ccccgcttcc tgttcatccc taactatgaa 2400

cgagcggagt ttgccaaacc tgagaaggta ggacgtcgcc agcagattcg ccgtactcaa 2460

ttctccaaag agctgattga gaagctggaa gcaatcgctt atgtgaatgc tatcccgcct 2520

aacggtaaac taaggcacga cccgattcct atttacgacg aatcggacga tgatgacttc 2580

ctgtacgagt accagtgcaa tattaatgac atgcgccagt attacaggga taacgaagta 2640

gcatctgcct ttgtgggccg aatgggcgag tatgtattca atatagcagc actgattgga 2700

ttactcgaca actgggatgc tcctgtaatg accagggaca acatcgaatg ggcttacaag 2760

tatgtgcttc gttgtataac cgcctgggtc aataacacaa gcaggatagt cgcaccacca 2820

accaccaacg ccgaagtagt ggacggcttc ctaaaagtct acagggatat tttggccctt 2880

tacaccaaac gaggatggtc tggcattgtc aagcattatc ctgagggtgt cgtgagaagt 2940

atcagggaag aacacctcca gatgaatggt ttaagcatat atgctattcg tcagtctttg 3000

agctggttta aatcgaatta cggttttaat attacagcta ataacaaagt cattgacggt 3060

atgctgcagg atatgattga ccaggactat ttagcagtcc aaatatataa gcccacaaga 3120

ggccgatctg ctaacattta ctgcctgaca gagcgaggat atgacactgc taagaagtta 3180

aaataagtag gtaaatcaag ggcttgggta attgtaaaaa tcctaagccc ttatttttta 3240

agcttatttt acaaactgat taatatttaa actataatta aatatatgga ccctaagagt 3300

atgattttat tactctttta caaatcataa tatatgggct ccctataaaa agattaaggg 3360

ggtaataagg ggatccaagt accggataaa agggggtata tatttatttt attattatta 3420

ttattattat atattattaa ttatattaga agtaataaaa acagctactt agaggcgtct 3480

atatgtatga tccggtttat ctctttatta atatcagaac cggatgaaaa aaatttacgc 3540

tggcgaaaaa aataaaaaag ccggattgat aaataaatct gtagacgttt acactataaa 3600

aaagatgtgt tacaatattt atcaaataca gcaattatca ttgactaaca ggattgaatt 3660

atggtaaata ttcgtgttat tggccagaac tttgaaagaa aaatggctaa aaaattcaac 3720

gaatcttttg gacttgactg ccagcgtaac ctgcagcaat caataaatgg cggggatgac 3780

ttgcttggta taccgttttt cagcgttgaa ctgaagaagc acaagaccaa gtctatcgga 3840

acatggtgga gacagtgtgc ggcatcggca aaggctcaaa acaagatgcc ggtactgata 3900

tacgaactac caagaaaaca gcctaccgtg gtgatgaata aggctgatgt gattgactgt 3960

tttccaagtg gcacgacttt taacgaggca tcgaaccgcc tctttgtggg tctagtagaa 4020

ttaacatggg aacaattttg ccgggtctac aactattacg ttgcgcccgg cactcaatat 4080

cacgtcaatt aatcttgttt cagctcgtca caaatgacac gctgattttt ggcagggata 4140

ataccaagat ttagcagggt gtatggacta tcagcgacac attgttcaac ggtagtccat 4200

tcaggtttgg attcaacaat tttgtaacta caatcatgag tagccacaca tgttgccagg 4260

attaagccaa tcacaagtaa tcaccttcaa ccaggaactc aaacagctgg ttgtaaacgt 4320

tgcaaccttt agcgaaagag ctttcagcag gtttaccgtc gcggacgatc atcatatact 4380

caccgccatg gatgtccacc caatattgca tgccaccttt gaccggatta cgttgtaaga 4440

atttaaacat tatttgctcc tgattccaag tttattcaat aaccaaattg tcatgtcaac 4500

ccaggttaac ggtacagttt tcgggggttg ataattccag cagcttgcag ttctttgtat 4560

ttctgcttgg cgccgctccg cttcgcgtaa taccgcatcg tcagaagtgc cggcagaaaa 4620

aagatcggta acaccagata aatcgcgttg attaacacgt cgagcaatgt catataaatc 4680

atccttagaa aacataagcc aataccataa gaattatcat aaagacccca accagcttcc 4740

tcatcgcccg accttcagca tatgcaggat tcacaacaga atactcaaca acatgtttac 4800

agcggaccat aaggcctcca cttgtgggtt taactcaagc atgaggactt tcatcaaatc 4860

ctcaggattg aattaattac cagcttttaa tcaagccctg tcagcgccag gacttgacta 4920

atatcactct atcattacct gaactactat tatacactct ttattgaagc gagtaaacac 4980

ttttccacca ttagtgcgtt tagccccaga ttttagagct gactggcacc agatatgcca 5040

ctcctcaact gtcaagaatt tttgctcagc tttgggtggc aatccattgg caaggtctac 5100

cgtagtaaat ttgactagtg gcatcacttg acctcaaatt ttgactcgtc gtattccgga 5160

tcagtcacgt cccgggtcgc acaatttttg attgcttccc agtagcgttg ggcatcatca 5220

ccatcgacgt aaaccgtcca gtggccgtaa ccaccttcaa atctgtccca ggcttgttcc 5280

cagactttgc catcttccag cttgtaccaa gttggacgac tgtcaaactc gcggattgga 5340

tggtacccca acaagggatt catcaggtca ataatgtctt cccgactaat ctcctggttc 5400

tcccacaaca ccgtcatgaa ttgttcaaac ttgaaggcgt ttggcagaac tttaagtact 5460

tcaccgtcac aatatacggt atagatacca tcttcgcatt ttgcactgta catgtttctc 5520

tctcctgttg atataatttc aatcatgaac tatcacaact ttggatagtt cattgttgaa 5580

actatactaa ctttgttaca atctgaattg tcagccggaa gtcttccatc aagttatcaa 5640

actctttgga accgccaaca gcgtcagaaa tttgatcttt cacgatttgg gcatttgatt 5700

caagcagttc aatcaggtgc gccaattcgt cttcagtcaa tttcagatct ttcataatgc 5760

atccacccaa gccataactt ttgccagtct tacaacgtct tcatggtccc gctcgccgtc 5820

agaaaattcg ctggcgatga tacccataat tgacgccaga ttcatgttga tgatgtaggc 5880

gtgttggcgc acttcatcaa ttcctgaatc tatcaccagt tccgcaaatt cttgtgaacc 5940

ttcatcccat tcagcccaat actgctcagc ataaaagtcc agttcttttt tcatctcaat 6000

gtcagtcatt ttgattactc ctcagaacct gaaaatttgt catcaatggc ttcaataata 6060

taatcaagag ttgaagtgtg agaactgtca gaaaaagtgc ccatcttctc cagctcaatc 6120

atcatatccc gtaaagccat tgcctggtct ttggtcagtt taattttaag catgtctctc 6180

tcctgttgat tgtgaataat ttcaattgat ggcactactt gagtgccatc tcaaaaatta 6240

ctcttcagtt tcttgtgctt caggttttac ttcagtttca ggtttttcac cagttactga 6300

cttgccctca gtcactttac cagattttac atcattccgg tagtatgaca tatatgactg 6360

gataactctt ttggctttga tgtcatccgc ctccatccca caggctttca tggcatcaac 6420

tagtgccaat ttggaatctt ccttatccat caggttataa tccttaaaca ccagataaca 6480

agtgtccttg gttgatggac cagatgactt ccgggtcccg ccattggaag attttgatgc 6540

cacaactttg acctcttcca cttccagttg gttcagttca tcaaacaatg ccttgaccaa 6600

cacttctttg gagtccttga cagttacaga actttcatca ttaagggact taatcatctt 6660

aatgatctgg ttattggcaa agtactgaag actatccatc tgcactacaa tagtcccaag 6720

actttcagta gtcagaccag attttacaga ctcaacagaa aaggcggaag tcaggtaaac 6780

atagttagtc attgatgtat cctcattgat agaatttttg ataagtgtca atccttgaca 6840

agtaagtcca atcatgagga cttttaccaa gtccccattg ttggaaatta cttcaactta 6900

tccagaattg attggagtaa cttgaccctc acttcctggt ctttgacatc ccttccactc 6960

tccctgtcat attccaacag gtcactttcc ataaataaag tggtgtccaa ttggtcaacc 7020

aataattcca gttcttcttg ggacaattct aaaactttca tcttattact cctgatattt 7080

aataacttgg gattggataa agtcaacaca atcttccaca tccattgact caaagtctgg 7140

tactggacca ttgtttatat tgacacacca aagcccttca ttggtatcat accaaagtgt 7200

gtagttgtcc atcttaatag tcacctcttg tactagtttc cagttgtgac tactgttacc 7260

aatagtcact ggttgaaact attactagtg ttactatcaa caagaaacat gttaccagat 7320

tgttaaagag caaaaggtac cattgacatc taccagatgt acctgttgac tggttaagta 7380

ctacctaccc aaattaaacc atactaatac cctccagacc agttgactgg tcacttggtc 7440

atcacattca ataaatatat aatagtccaa tccctcttga ttgtacacct tatatttgta 7500

agtttatgca agtttttaga aatgataatc acttgcgatt gatagtggtt ctcatataca 7560

attgatatag tttcacatgt aattactcaa ctcaaaatgg tcggcagggg ttataataac 7620

ataggggtgc cacttaaagg agccggcgaa attatttcta aaaatataat ttcaatctga 7680

attatagacg ctttgtacta taacggccga agtagttcac acggtaattg taccaatcat 7740

aatagttacc agagtaatca tttcaattga aataggtgcg gcggggggta atttcgcagg 7800

ggtgctacct gccaggagcc agcggaattt ttttattttt tggtattttt gccttatcag 7860

gaccaatcat catcagagtg atacgacagg agctaacctt ttgcttaaca caataataag 7920

tgaagcccct ctaaaatttt tttatatttt tcggaaattg tgtagacact cactaaaaag 7980

tggtgtactt tctctcctcc acgcgctatc attaatgtga atcatcaaac ggcctcacgg 8040

tctcctactt gtgggtaaca ttatgaaaat tgttcaatta gagccatatc cgcctcaatt 8100

ggctatcgtt tgcagcgaaa aagagcacga agagctccaa aaactgatgg atttgcctga 8160

ttttggcaac tggaatgacc gagaaacgcg tactgcatct acctttacgt tctctcgcgt 8220

taactatccg tacctgctaa ttgtcgtcga actgtaccac ccggacgacc tgaagtacaa 8280

caccatttcc cacgaaggca ttcacgtaat gtcgtcgctg atgaactatg ttggccttaa 8340

atacgaccca gaaaacgatg aatggtacgc atatcagcat gatttcatcg taaatgccat 8400

ttgtaaggcc catgacgagt acctggagcg ccaaaaaaac gccaaaaact aagccaaaaa 8460

tcgaaacagg taaccatcct gcgatactgc cacaagacgt aatgaactcg cttttgactg 8520

acactactgg ggacttttta ggtgattaat ataatctttg caatgggcca aaatggggaa 8580

ttcgggttat ctccggagtt tatgaagcat caggttgact cgtgcaagac catggaagag 8640

gttcgtgcgc ttcctaaatc gggtctgccc tggaaatgtc cgtcagacat gctctttttc 8700

aggactatga ctaattcaat tgcgacagaa ccaggctata atgcatatca gcaactacgt 8760

ggacaattcc caaatgacca gttgcgaaac atgatagcag atgtaacggt aaaaatggtc 8820

ggacatagtg tattgttagc cggtcgtaaa acgtatatga ctatgagtct gcctatgtca 8880

gcccacagaa tactgctgcc ggtatctcgc cagattatgg cttcagaagg ctacaataat 8940

ctgggtgaga tggcagcaga ccttgaatcc agaggtactg acgtctggat tgtaggtggt 9000

gccgagttga tactttcggc ccttgaagag catgcaaaag acttactccg tatcgacaac 9060

atgtttatca gtacaatcaa gcaatcaggt ccaagtgata ttttaatgga tcgtagcaaa 9120

ttaatgatgt acattgatag cgacttcact tataatgatg aatatgttga taacaaacaa 9180

gttctgatac aacgatacag gagtttacac aaatgaacat cataatcgag ggccctgata 9240

atgctggtaa aagtacactt attgagttcc ttaaatctca tactggccgc agtgttattc 9300

ataatactgt tgataaggac cccgaatcgg tcatggctaa acaggccaaa gaaaacgcgt 9360

tggaaggcga ttgcatttat gaccgtagcg ctgttatatc tgagtatatc tattgcctgg 9420

ttcttcaacg tacgcctgtc atcaatatta atgtatcaca tgtagcagat atgtgtgaca 9480

acgccatcgt tatcttttgt atgccgcctg ttgaaaaggt actggcaact acaaaagacg 9540

aaatgcctgg tgttgtggaa aatcttgagg cgttgtacaa tcagtatgac aacctcattg 9600

acgagctggt tatgatgagt aagcagttct tcatttacga ctggactgtt gaagaagacg 9660

gtcctgaagc agttctcgaa tatatcaacg aacgtattaa taaggagtgg aacaaatgag 9720

cccagtatac aatagcacaa tcacactgtc agcaaccgta ggcgacgtca acaagactgt 9780

tactgtgggt tcaactaacc caaacctgtc aggtgaagac ctgctgcgcc tggccaaacg 9840

tgctcttgaa gataacgaag tgactggtac tgaaggcgtc agcatcgaaa ttaacgtcga 9900

aggatgtcca cacagcatca gtattgctac ctcttcattg ccggatgatg aaactcgtga 9960

acagctggaa acaaaaaagt tcaacgacga gctggaatat cgtactggtg agatgttcag 10020

cagtgcgtta aactggtact tgtgcaaggc ctcgggttat actggtgctc ctatgtcaga 10080

ccgcatgtta gctgatatgt cagcacgtga aattgctaaa ggctgggcgc atgtacagat 10140

gctgcaggag catatctgca agctggaaag ctgcaataaa gatgcgtctg agcttattac 10200

tgcctataag cggcagatta acgacctgca gcaactgatg ggtaaacgtg ctgaagagta 10260

tgaggctgag attcgtagcc ttaacggtac agctgttgct aataaccgtg ctttcagcga 10320

agagcgtaaa gcgcttattg cccagcggga cgctgaaaaa cgtcgtgcag acgacctggc 10380

agaccgtctt aactttatcc gtaagcatat ggatgctatg gaaggggtaa tttgatggac 10440

ctgttcgata tggtcgaaca tcctgaagta aacggcttgg cggaagctaa gccttttagc 10500

tgggatgaat tcgtagcagg caaacctgct gtaacccaca acgggcagat tgcatattac 10560

ggaggccata gcgaacatcc gatgtgcact cacctgtaca acgctatcac aaccatatcc 10620

gacgaaaacg ttattaagta cggaaaagaa gtaatgcctt ttcttgtaga tgaacgcggt 10680

tacttcgaag gtaaatggtt cccgtttctg gtagaaatgg cctaaaaacc ggataaatat 10740

aaaggcgtct aagctagttc ttcatctata taagctactt attcgccttt ctactgtaga 10800

ctttctcatc atctctctag acactccatt tttatattct gtttacttac ttcgcgtaac 10860

tcgttataat cccgactatc cggattaacg aggacgatac caatggcaaa accgaaaaaa 10920

acccgtgagg caacagaaga gctgttggcc acgatgcgac gtatccaact ggacccgttg 10980

gaagttatgc aacgagctat tatccttgct gaacgtgaag atgactataa agcaatgatg 11040

gatggtgcgc ttggcgttat gccgtacatg tatcctaagc tgaaagagtc cgttgttaaa 11100

gctgacatcg atcagaactt gtctggcaac ggtgtcaacc tgaatattac aatcggtgac 11160

cagaaagtgg tagacgttac tgaggaagag taataaatga accttgactt tcacctgcat 11220

gacagacaag gtgccgctct gcagtctcag gcaactgaga ttctgtacgg tggcgccgct 11280

ggtggtggta agtctcacct tatgcgtgta atgtcgatta tcatgtgctg catgatacct 11340

gacttgcaga tttacctttt ccgccgaaac tataacgacc ttacggctaa ccacatggtt 11400

ggtcccacaa gttatcctgc cttgctggac ggtatgtgca aagctggcaa gtgcaagatt 11460

aactactcga gcaacacgat taaattctgg aacggtgcga acatcaagct ctgccacctc 11520

cagtacgaaa aagatattta tacataccag ggtgcggaga ttcacgttct gctgatggat 11580

gagcttactc acttcacaga aacgatgtac cgcttcttgc gtaaccgtgt ccgactcggt 11640

gctctcaaag tccctgagaa ctttttccgg ccactgccac tcatcttttg tgggtcaaac 11700

cccggtggtg ttggtcacaa ctgggttaag cgcactttcg taactgctgc tcctccgatg 11760

aaaatcgtta agcaggaaaa acgcgaaggc ggtatggcgc gtcagtacat tccggctaag 11820

atgactgata accctacgct gatggaaaac gaccctgact acggtgaccg tcttgaaggt 11880

cttggtgacc cggctcttgt tgctgcgatg aaagagggtg actgggatat tgttgcaggt 11940

ggtgcgcttg acgacgtatg gtcaaccaag tgtattctgc cgcgtatgcg tattccgaaa 12000

ggctggacta tcgaacgttc tcttgactgg ggttctactc acccgttctc tgttggtatc 12060

tgggctattg caaatggtgc ctcgcttaca gacgacgaag gcaatgagta ctttattccg 12120

cgtggttcaa ttatacgtat agctgaatgg tatgggtgct ctgaaaaaga agctaacagg 12180

ggtattaaat tatctgctgc cgaagtcgct caaggtatta ttcgtctaca ggaaaatttg 12240

caggctggtg gctggattca aagcaacgtt aatcctggtc cggcagatag tcagatttat 12300

gcgtgcaagg aaaaagacgt cgagactatc gcgaagaaaa tggaagacga aggcgtaacc 12360

tgggtcaagg ccgacaagtc gcaaggttcg agaataaatg gcttagagct attccgtcag 12420

cttctgaaaa atgttaagat ggacgaaacg gaaaagccgg gcatttattt catggaaaac 12480

tgtccagcag ccataaacac gttgccggtt ctgccgcgtg acaagaacaa cacggaagat 12540

gtcgacacga actctgagga ccacgtatac gatgattgtc gttacatggt gttgcgtggc 12600

atcaaccgtt atactaccaa gctcaaaata aatatgggac ggtaaaatgt cgataacccc 12660

aagcagcacg ccgaacgtcg gataccagcg tcctgagctg actaagaacg ctggtaagta 12720

ccgaatgatt aaggactgca tcgagggtca ggaagctgtt aaagcacgcg gtgattttta 12780

tctgcctcgt cctgacccta cgaactgcac agaagaaaac ctgatgcggt acaattcgta 12840

cctgcgtcgc gctgtgttct ataacgtact acagcgaact ctttcgggta tgactggcct 12900

ggtattccag cagcagcctg agatgttgat gcctgacgat atggaagtgc tgaaagtaaa 12960

tatcgacggt gcaggcattg gtgctattca gcaaatgcgt aagtgcttca agtctgttct 13020

ggcttttggt cgcgcaggtc tgctgactga cttccctaaa gcgcgtatcg gtgagtttgg 13080

cgagtccatt ggctttaccc gtaaagagct gatggacgga gatgtacagc cgactgtaac 13140

gctgtatatg ccatggcaga ttataaactg gcgggaagag tatcgcaacg gtaaagtgat 13200

gctcactcag gttgtcattt gcgaacagat accgggggtt actgatggct ttcagcaaga 13260

ctacacaccc gtgtggcgtc acctttacct ggatgaagaa ggcaattgca accagtcttt 13320

gtgggtcaat aagggcgaca agaatgcgca gaacgtctcc ggtggtgcgt tgtatgtcac 13380

gcaagaaaat gttgtcttac gcgactccaa cggcttaccg ctgaagttta ttccgttcca 13440

gtttattggt tctgaaacaa acagcgctga gattgaccct ccgccgatgt acgacttagc 13500

tgtgcttaac attgcacact accgtaactc tgctgactac gaagagtcat gcttcgtaac 13560

aggtcaaccc acagtatggg cgtctggtct tactgaggca tgggttgaag atgttctgaa 13620

gggtgaattg cgactcggtt cgtttggtgg tattccgctg ccggctaacg ccgctgtagg 13680

tattatccag accaacccta atacccagcc gtttgaagcg atgaagcaaa aagaagcaca 13740

gatgattgca atcggtgctc gactgattcg tgagaatggt aaagtagaac gacgcgaagt 13800

tgagattaag aatgaagcgg cgtctgaagc ttcgcttatc gtaactgttg cgcagaacgt 13860

tgaagctgct tatcgtaagg caatcgaatg gtgtggcctg ttctacggac accctaaaga 13920

agaaattacg attaacctca gctacaactt catctatagc cagttgactt atcaagaacg 13980

tcagcagctg gttaacgaat ggattgccgg tgctatcacg tttagtgaaa tgcgtagcaa 14040

tcttaaacaa gctggcattg ctaagctcac cgacgaagaa gccaagaagc taattgatga 14100

agagctggct gaacgcgatg cgcgtgaggt agcaaaagaa gcagaaaaga acgctcaagc 14160

agctaagaac agtgatgggc aggaaaataa tccagaaaaa actgaataaa gctatttact 14220

ttcggtttac ctggctatat aatccttcat gtcaaccgcg caacagccgg ccacgagagt 14280

ctgcagggct cagtggattt cggatggctg gctgcgcggt ttgacccaaa cctgttcgga 14340

gcccacttga gtggattgag cccctctcat ataggattgt aaaattatgg cacttaaaac 14400

agttgtaact gaccttaccg aagttgatga aggcttccgt ggcctgtata tcaaagacga 14460

agcatctggc gcattccgtt tggacgttga accagacgaa aaaacgctga aagaattaga 14520

agacctgcgc aaagaaaaac agcgtatgga acagcacacc aaaaagcttc tcgaagaaaa 14580

gaaaaaagaa gctgaacgtg ctcgcctggc tgaagaagaa cgcgcacgtc gtgagcgtga 14640

tgttgaatct cttgagcgct ctatcaaaga gaagcatcag gctgaactgg aacagtatac 14700

taatcgtcag gcacagctgc aagctcagct ggaagcgcaa atggttgata acctggcgct 14760

gagcctggct aaccaactct ctgacacccc ggcacttatt atgccacaca tcaaggcccg 14820

actgcgcgct caggaaattg atggtaagtg gcgtactact gtcgtggatg ttatgggtaa 14880

cccgactgct acaacgccag atgaactagt ggaacatatg cgcgctgata agcagtttgc 14940

acctcttgtg cgtggaacca aggctggcgg tggcggtgcc aacggcggcg gagctaatcc 15000

tggtgggaac ggcggtggcg aacagcacac tttcggaggc ggggacgacc gcatagcacg 15060

tgctcgtgct aaaatccaaa acacacaatg gggcaaataa tttatgtcac tgtatatttt 15120

taacgaacaa gtacgttcta ccgccaccga actggtcgac caggacgtac agaagtttaa 15180

cgaagcttct gctggtaccc ttatcctggg taaccagtac gtaatcggtg attacgttga 15240

gcaggcaatg tggcagctgg ttgacggcat cgctacccgc cgtaatgctt acgctggcac 15300

caacaaagtt gatgcgcaga ctctgggcca gattctggac cgctctgtta aaatcgacgg 15360

ccgtgttggt cctctgaaca ttaccggtac tatgatgcgt cgcatcggtg caggtgacac 15420

tgaaccagca gctgttgttg ctgcacaggc ttctgctgca atgttccagg attacctgaa 15480

taccactgct gcatgtctgg tagctgctat cggcgcaaac gctgatgttg ttctggacgt 15540

aacttcaggc tccggtgctg ctgcaaacat caccatgcag aacctgaaca aagtcaacgc 15600

gaagatgggt gaccgttctc agcagattcg tgcgtacatc atgcactcta ctgcgttcca 15660

ctccctcatc gatcaggcta tcaacaacac ggaacacctg ttccagattg gtgacctgaa 15720

cgtgtacaaa gacttcctgg gtcgtcgtta tgtcgtttct gacatcccgg ctctggttga 15780

tggcgccggt gcttccgcta agtaccgcac tctgggtctg accccgtctg ctgcaatcgt 15840

tcaggtcggc ggcctgtacg acatggttac ctccgagcaa accggtggtg aaaacatcct 15900

gcgtcagatg cagggtgaat acgacttcaa cgtgtcgctg aaaggctaca gctgggtcgg 15960

tcacaaagct ggtcagtctc cgactgatga agacctggct gctcaggctt cctggtctaa 16020

agttgcgact tccaacaaag acaccgctgg tgttatgttg ctggctaagt ctgcaatcta 16080

acccacaaac gggactaaca aaaggcgagc tgacgagctc gcctttttta tttacacgta 16140

taaagttctg gtatataatc ggactaaact tgctggaggc tttctatggc atacacactt 16200

gttgtggaag acggttcaat tgttccgggt gctaactcgt atgttagcgc agcagaagtt 16260

gatgaattta tcactaacga aggtattctc attcagccag gaactgaccc gtactaccaa 16320

gctgccaaag cagctattcg acttgataac cagtttaact ggctaggtcg taaggtcagc 16380

aaaacacaga acatggcttg gccccgtact ggcctcggtg catgcaatgt tgacatcgcg 16440

gagtctgtaa ttccgcgaga agttaagctg gcacagcttt atatgatggc cagcgtattg 16500

agctacgacg gcgaagaagg tgtgcgtagt gaccaggctt tgcgccgtaa gaaagttggt 16560

aacctggaga ttgaatactt cgactccaag tcaggcgtta ccggtgcaac tgacctctgg 16620

acgtcagacg tcaaggtcat gctcaagcac cttgtgggtg catcactgag agcacggaga 16680

gtataatgat tcgtatacgc cttgaacgtc gcgacaaaca aaagttcgag ttcacctacc 16740

gtgcctttaa gtcgtttgag ggtcaaggcg tatcagtagg tgttcatcca gagcacaaca 16800

gacgtgtaaa tcccagggac cgtataacaa acgccgagct agcaatggtg catgagtacg 16860

gtcttaacgg tctgccagaa cgaagctttc ttcgtagcgc tgttggtggc aggggtaaag 16920

gccgacaggc aataaacaaa gctttccgtg ataatgtacc tgctgttctc agaggaagca 16980

tgactgcgca tgagcttaac gacaaaatag gtcgtatgct tgttgacgct gttcacgaca 17040

gaatggacag tgacgtgcca cctcctaaca ccgagctgac agaagaacgc aaacgcgggc 17100

ctggtacact tagagaatct atgcagttgt acgattcgat agggtacaaa gttggcagaa 17160

actggaggct ataatgctca agataacttc gctaatgaag gataaaacct ttgtagcatt 17220

aacagatgtt cttctccgaa atgattcgcg cttaccaaac ggcgacttcg aagacgaata 17280

tacagctgca aagtatctgt gtaacattca acctggtgca gctactaacc agcctaacct 17340

gttgactaaa gatgatggtg agcatgagtt cccacgtatt gcgatatact gtggacaaga 17400

gctgaagctc ggagacttcg ttatctacga agaccagcca taccgtgtat tccgtatcga 17460

cccatggagt cgtcatggcc actactacgt tacagcaatt ctccataaag gacctcaggg 17520

agctcgttca gaagctttta acattacctg atggttcatg cattattggt gaagagtctg 17580

gtacagttga tggtacaccg tatgtttact tgcgccctgg cgacgcatat gacttcggac 17640

ctcctagaac cagacaaggc tctgacggct ttgagtacgt cagcaagcca atgatgacgg 17700

ctgtacgtat tacagctgtc ggtaagaatg ctgctgagct aatgcgtaag ctacacatag 17760

cactgggtag ttcaccggct aagcagttta accgtttacg acattatgca ataaccaaaa 17820

tatcgaagat tgataatgtc ggtggtgcta ttggtgcagg ctatgcgcag aagtcaatga 17880

tgactgttga cattagctat gtgcacaaag ttaaaattga gcaaccgtac atctacaagg 17940

ttgacatcac ttgtatggat gacaacggta acaaagcgac tggtacagta gaggaaccgt 18000

aatggcaact ggagtagacc cggaactgca agcgcagctg gataagctaa caactgctgt 18060

ccagctcatt catgacttcg gtatgtcaga caacccaaac atccctaacc cggaaggcgg 18120

tgtcatacgc acactcgaag gcattaacgc tgcaatcaac gaagctatac caaactttga 18180

aaacgctaac gaagctgcta tccaggcgcg tgctgctcgt gacgcggcta ttgcagccaa 18240

agaagctgca gaaacggcag ctgaacaggc cacagtagct tcgacgtcag caatctatga 18300

gtacggcaac gttgcaagtg cagctacagt gactatacca gctgcatcta aaacaattgt 18360

cagccttact cttaaccagc caaacaccgt aattaatatc ggtggtgtat ctgacccgtc 18420

tactgttgcg cgtcagatta ctattgctat taagcaaggc actggttcta acaaagtgac 18480

tgcttgggat cccagaataa aatgggctaa taacagaaag cccgttctgt cattccttgt 18540

gggttacaca gatgttgtta cgttgcttac ctttgactca ggcgcaactt ggatgggttt 18600

ctttaacggt ggatggatta aataatgcac acgatgttgc ttaaagacgg atcagaccat 18660

cgagctaatg tcgaaagcct tatcgaaggt caccaccatt ttctagagcg taatactggc 18720

agaactgacg atgctacgcg tcagcactat atcttcaacc cagaaggagt agtgtcgaac 18780

aaccgccatt ttattgcaca caccatgatg gagtatcaac ctaacggaga tgctccgtct 18840

gaaagtcagt cgctgctaat actcggcgaa attcacgcgt accttgctac taaggaacag 18900

cggtacctgg acaaagcaat tgagtacttc aatgcttacg tcaagtacta ctacgagggg 18960

gacccaatcc ctgatactcc tcgccgctgg atcgctaact ggctttgtaa tggcaaagag 19020

cctgtgttag ccagcttccc aatcaaccct gactcaccaa cacaaggcgg ctataaaaat 19080

gtgccagtta aatttgttaa cggcaaagca caaatccctc atggttctcc tttctggggc 19140

gagtacctgg atgttgctac ttgggcccac aggggacaca tggcttggcc tgctattaat 19200

ggcggtgtcc gtgttattaa aaataaagtt gactgggacg atatttacaa caattatcgc 19260

gtcactacca tgccagctga cccgtggaat cagttagcct ggattgactg gccgaggtat 19320

ctgggcgaac caagctacac tgtggactgg tcagctgctg aacctcctgt acatctcatc 19380

gagtatattg ttgcatggac caataacaag attggtgttc tgcctggcaa aaacgatgag 19440

ctatggggag gcgagattct tgagactgga ctgcctaact ctcgcagagg tgagattcaa 19500

ctccgtgacc acactgtcaa cggtgtgtat ctgcttaact atgccgttaa gcttcctgtt 19560

gaacacggtg gttatttgct taagcgaaat gaagtatggc acaacagacc ggtaaacgtt 19620

ccattgtacg gcgagcttca gcgcggtaat gctgcggatg ctgagttgtg gttctgtgac 19680

tgctgctata tgctgtggcg aattactaac gagaaaaagt attacaacgc ttggcaagct 19740

gttctgttta cgctggagga gtatattgat attgataaac aagaccagtt cttccgtcaa 19800

gactctcacg cgtcatctcc gtttactgat ggcatctctt atgattggtc ttatcctgaa 19860

ctggcacaag ctatatattc acgtgatgaa aatggttata tagttctacg ccagaacaaa 19920

aaagctcaat catctctgga gcagcaggct gtatggttta agtgtaagcc aacagctaag 19980

atacgtacag aggtcggtgg caaagatgac gctggtcaga acatcaacgt caaggtggaa 20040

atgtacatga accgtgaaaa ggttgacgtc gagcaaaaca tctatcgttt tgtcgcacag 20100

ctacctgctc ttggtgcttc gcctgctaac tatgacattc cgttaagtga ttttgtcgca 20160

ggtactaagc cagatggttc tgagtatatc ttagctgata ttcgttcatc cactgattat 20220

ggcaatgcct ctattgcagt gtcttaccgt actgacatac ttgacaacag acgtggcaac 20280

actgttcgta tgacaatacc ggatagttcg tcaggtgcca ttataggctt ttggctactg 20340

gaatcttccc aggcaccttt aaactcgctg acgtatactt ctacaggtcc gttgtgcatc 20400

aaagtaactg acgcgcaagg ctggagttgg gtacgtgatt tacctgccgc gccaggatgg 20460

gtaactcagc agattcgcac ctgggagttc aggctggcgt cataccagga aaacacaggt 20520

acaccaccta ccagcccaca attcggtgag attaagcagt tcgcgtttgt actgcctgac 20580

ggtgttaata cgtcgacaag tattagctgg tactgtgtga atgatgttcc gcctcgctat 20640

aactggccaa tcggctatac gattaagtat cgtcttactt tctcggctga taacccgtac 20700

acagctaagc taggcgactg tactgtgctg gactatgaga atgataacct tgcttacacg 20760

ccaggtgtta ttccgttctc caacatttat aaccctgatt ctaagcagtt cgatggttgg 20820

catggtatac cttacccagg ctatcagtat ccgtttgttt ttacgcatga cgatactgca 20880

gcaggtacaa tcaggctgaa caacatggtt aactttttgt acgattctca gcaatggtac 20940

tttaagaagt ttggtcaact aggtcctggt gcgtctgctt ttatctggaa ccgttgggat 21000

aactttaaat atggtgtacc tgatacgttt accatgtatc actggggtaa tggtagtgca 21060

tggtcaggtt accagcctcg tgcttatttc tccgcagcac gtggatggta tgagctagta 21120

aaccgtggta agccagttcc gagtaagctt attgcttatg tggataactg gtcaaaatgg 21180

ttgcttgact tcatggaaag gtcaggaggt gtgtcacctt ctaacttccc gcaagaaggt 21240

gttgctctgc cagtcccttc agacttcaca ggtcacatgt gcggactatg gcttgctggt 21300

gcagctatgt cagccctttg tgggtcaaca gtatcaggac tggataagct gatggacatg 21360

cttgtcaatg agctggaaga ccactatgac ataactggta ttcctggaca cgtaatggat 21420

ggtggttggt cgccagcgtt acgacttgaa actggtacag gacctgaaag caacgccatg 21480

tactatggtt tttactctgg cgaaatattg cgagggttgg gcttatacct gctgtacaaa 21540

aacaaagggg ctaaagcaga tatttatgat ttaggcccgt aaccactatt tacttaaccg 21600

aggcgccagc ctataataag ctggcgcacc caataagagg attctccaga tggcaaatcg 21660

tactacagcc aaccgtcctg ttaaagacgc agacctgcgt caagagtcgg ttactcagtt 21720

cccgttatcc aagccaacca agcttgacga gaacggaaaa ttcattccaa ccccagttac 21780

cggtgtaact gctacgccta agactaagtc aattgctgtt ggtgagttta caactctgtc 21840

agcagcagta gcaccggcag acgcagatga cgtcaccgtg ttgtgggaaa gcaatgcgcc 21900

tgacaaagcc actgttgatg gttcaggtcg tgttcagggt atcaaagttg gttcagcaac 21960

tattactgct aaatccaagc ttgacccttc caaaaccgat actgtagcaa ttacggtaac 22020

tgctccgcct gtagccgtca ctggtgttac gttagcacct aagtcgccaa gcgttgtagt 22080

aggctccact gtacagctga ctgcaactgt actgcctgct ggcgctacta acaaagctgt 22140

tacctacgaa tcaaacgatc aggctaaagc taccgttagc gcaactggtc tggtgaccgg 22200

taaagcagct ggtgtcgtca ccatcactgt caaaacagct gatggcagct tcactgacac 22260

cgcgtcagta actgttactg ctgagtaagg agctgattga tgtcgcttcc acttaatgag 22320

gttgtcgacg tccagttcag taagagcaac gggggtaaag gttttacccc caatgttccg 22380

ctggaaggta taatgctttc caactacgag tactcaatcg tagcgggtga tgaccctgtt 22440

cagctagttg tgcgttacat tccaaccaac gctactgagc gcggtatggc atggaaatct 22500

agcgacccgg acgttatcac tgttgatgag ctcggatttg tgaagccact ggcggaaggt 22560

gttgcatacg tgactggtac gtctgttgac ggcggttata cagtacgagc aaagtttaac 22620

attgctcggg cacctatcct tgttgaatct gttacagtgt ctcccacagg cgtgacgctt 22680

gaagaaggca agactcaacg cctgacatac acctatttac ctgctaatgc agacaaacct 22740

gatttctact ggatttcaga tgatgagtct gttgctatcg ttaacgacag tgggcttgtc 22800

actgccattg gagatggcga aacaactgta aacgtacagt acggtgttga cagctctata 22860

ttcgatagct gtactgtaac tgttattccg cctgttgtac acgttacagg agttgagttt 22920

cttactcagt cacctcttaa cgttaatatc ggaagcacag ctgggactag tattcgtata 22980

tcgccagaag atgccactga caaaacagtg acatacgaaa gcagtgcgcc tactgtagca 23040

actgtggata ctgacggaaa tgttactggt attatagctg gtaatgccac tgttactgtt 23100

aagacgaatg acggtgataa gactgacact cttgacatta tagtcagggt gcctgctgta 23160

aatgttaccg gtgtaagtat tgacgaaggc gatacagctt ccatggaagt tggtgacact 23220

cttcagctac atgccactgt gactcctgaa aatgctactg acccatctgt tgtatggagt 23280

gctcctgtat cagaagttgc gtctgttact gaagctggtt tggtaaccgc taacgctgca 23340

ggcttaattg atgttactgt tatcactaac gacgggaact ttactgatag aatcgaggtt 23400

aaagttacag accccagaat cgcagttacg gatgttgcat gggctcctga ctctccgact 23460

aaagtaagtg ccggccatca ggtgcaaact aaaattgtta taacacctga caacgcaact 23520

aacaaaaaag ttagctacaa tagttcaaac acagacgctg ttactgttga cactaacgga 23580

gttatatatg gtcaatctcc tggaacttcg actattacag ttacaacaga cgacggtgga 23640

cttgttgctt cgtttgtagc gacttgcaca gaggtggctg ttattacacc agatggtctt 23700

ggtggtattc cagtcggtgg tactcagcag ttcacttata ctgttaacca gcctgatgct 23760

gaactgacta atattcgtta tgagtctgct aacccagata ttgcaactgt tgattcgtca 23820

ggcgttgtaa ctggtgttgc tagcggcgga tgtaacatct ggatttacgc aactagcaat 23880

ggcgctgaca ttagcgcagc tacttgggca caaatctctg agaaggtaca ggtatatcct 23940

cagtacgccg aatggcttgt gggtaacacc aaacagcttg atgttgagat ttatccgtcg 24000

gactctaaga gcgttaccgt tacttatgaa tcgtcaaatg acactatcat tagtatagat 24060

gcaaatggct tgatgactgc taagtctgaa ggttcagcaa ctattactgc taacgttgtg 24120

atgaacggtg ttgctgcttc tggtacaacg tctccttacg tgtctgagct ttctgttact 24180

actgattcgc tgcctgcact tactgttggc aacacgcagc agctggttgt aacagttaga 24240

cctgaatacg ctaagacaga cccaggctat aacattcagt atacaactac agacgatacc 24300

gtcgctactg taagtaatac cggtgttatt acgggcgttg gtaacggtgg atgtcgtatt 24360

ggtgccacag taactgtgag aaatgctaca gcgtcggaca gctcttatca gtccgtcgat 24420

gcagcataaa aattaattaa ctttgcactg gccatttact tggttaaact tactgagtat 24480

aatggccgga aagtccaccc aagaggtaaa caaaaatgcc gttgaatgta aaccagattg 24540

taaacgttca actgaacaac actccaactg gtatcagcaa aggtgacttc ggtaagctgg 24600

ctcttttaac tcaggaccct ggcactgctt ttcctgacag cgacaacgga cactatgttg 24660

aaatcggcag ctactctgaa gccgagtctt attggggtac tggttcagtt gtacttggcg 24720

cgctgaagat tttctttgca caatctccgc gtcctcgttc agtagttatc gcctcatgga 24780

gcaacacctc tactctggat gcggctatta aagcattcaa cgatgtgtat cctgactggt 24840

atgttctgcg tccgcttccg cctactggcg agctgtctga cgatgacatc agtttgctgg 24900

cgtcaactgt tgctgcttac gataaaaaac gttgcagcta cactaccact aaagcaactc 24960

atattgaaaa cagcgactcc aacccgatta aagttctggc tggtaaaggc tacaacaata 25020

tgtggcttca gtatgataag aacaaccaac actatgcaga catttctgct atggcgcgtg 25080

ctctgtctgt taacttctca ggtaaccgca ctacgattac catgaagttc aaacaagagc 25140

ctggtattac accgaacaac actttaacgc tcactgaagc taacaagtgt aaagcgctgg 25200

gtattaactg gtacacctac tacagcacct ttgctatgct tgcagaaggt actgtcctcg 25260

gtacgtccaa gccgccgcgc ttctgggatg aagtacatgg tctcgactgg ttctgtaatg 25320

ctgtccagac ttatgtcttc aactcaatcg ctacgtcacc cacgaaggtg ccgcagacga 25380

acaaaggtca cgagcgcctt accggtgctg cagctgttgc ttgtcagcag gctgtcgaca 25440

acggcttggt tgcacctggt atctggtacg gtgatgagtt tggtacactg tcttatggtg 25500

accgactcga aacaggtttc tacatctaca tccctgacgt agatgaccag cctctggatg 25560

agcgtgaagc tcgtcaggcc gttgtaatgc aaatcgcgat taagctggct ggtgcaatcc 25620

actccagcga catcctgatt aacttcactc gctaaggagc agatcaatga tcatcgattc 25680

ttctgaatgc gtgttgagcc tgaatggtgt tcagctgtcc gactgggcag acggtgccga 25740

tgtcattgac gtgcaatggg tggacccagc ttccacctat cttgcaggtg cagatggcaa 25800

tggcattcag attatgaacc cgtccaaagc cattaagcta actatcaagg ttaagcaaaa 25860

tggtccggat gccaaatggc ttaacgacca gtacaacctg cagcgcaaca acattcgtgg 25920

attcgtcccg tttgtgctgc aagccaaaga cctgttaaac caggatttgg catggggtaa 25980

aaaaggttac tttaccgatg gttccccgtg gactcgcggt aacggccata acccgactac 26040

cttcgctatt gtgttccacc aaggtggtat gcagttgact gaaggctttg accagtaaca 26100

atagaggaaa gcacaaatga ctgagcccaa atcatttgac atcgacgacg taacgtacaa 26160

catgacactc gcaaacgcgc tgcaagcctg gacagtcctg aaaaaagcag gtaagctgtt 26220

caaaggcatt ggcaacgcgg acattgtggg taaagacggc aaagtggacg gtaagaaagc 26280

aatgctggtc ctgttggacg ctgttctgga gaacgcaggt tctgacgaga tgacagctat 26340

tgaggaacta atcttccgca atactgttgt tgtagttgat ggcaaaccgc gtaagctgtc 26400

tgactgtaaa gaccaacact tcaacgcaca tcgtagccat atcttcggtg tgctcaagga 26460

gggattggta tatcagttcg cggatttttt caaagggaat ggcctgtcag gtctagcggg 26520

tctagccacg cccttgacga aataaaacgc aagcaacagt cagggacgca agaaactgac 26580

tggttcgttt ggcttcctat tctcaagggt catgtgaccc ttgcagaatt aaagtcgtct 26640

tacagcttgt gtgatttgtg cgacctgcat attgctattc tggactatgc cgagttagag 26700

gaaatccatg gcgaccataa tcgatagctt tctggtaacg cttgagagta aagccgatag 26760

ttctggcttt gacctcttcg accagctgtc ccgccgtttt gtgattaccc tcggcgatgt 26820

gatcaatctt gctgagcgtg ctgctggtgc gctcggtaag atgtttgcgc ctgcgttaga 26880

ggctgacgta ctcgctgcca agattaaagg cttcggctac gacgtcgagt cgacgtggaa 26940

aacgctgatg gacatgtctc tgcaaatggg tacaccattc caggaggcac tagacggctt 27000

tactaaactt aaatcgtatg gcgttgaccc gttaaacggt tcgctacaaa tgctcaaaac 27060

aatcactgct gcaacaggcg gtgatttggc acgtaccatc gttgcgtatg gccaggcgtc 27120

tgcgatgggt tcattgcagg gccaggaaaa gaaccagttc attaacgccg gtgttgacat 27180

ctggggcgca ttgtcccgct acactggtaa gaagctcggc gagcttcagg atatgatgcg 27240

gcagaagcaa atcactgccg acatactgac tagcgcgctc aaacaagaag ccgaacgttt 27300

aaaaccaatt gcccacgaag tgtcacagac actcaaagcg cagttaacta acctgagcac 27360

aatttggtat gctgctcgtt ctaagattgc ccagaccaga gtatgggatg aggtaactaa 27420

aggcgcgata agtatggtta agtcactcac agcagtgctt aacgatacca cacttactgc 27480

ggcgtttaaa aagttcgacg aagttgtatt caatacaatt gaaaacgtaa aacgacttat 27540

ctacgttatc ggggataact tcccgttaat ggttgctata acctcatacg cactgctgtc 27600

acttattccc gtgctagcgg cacttattcc ctggctagct aaggtcggca acgcctttgt 27660

gggtttagct accaaaatgc ttatgaatcc actcacgtgg tatgcactgg ctatcggcac 27720

agttgttgct gtactcactg acctcgatga cgctctggca ggacgtgaga cagttgttaa 27780

gtggtcaaaa gacgttgtag ccgcatgggg aattgtcaag cagtatatcg tagttattgc 27840

agactttatt ctgtcaatat ttgacggtcg cttatggaac aaactcggta actggtggca 27900

aggtttcctt gttgaagtta agcaaggcat agagcagata aagctttggt tgtctgaagc 27960

actttaccag ctgcctggtg gtaagttcct gatggcgttg tttggtaagc tagattctac 28020

actgggcttt gctggcaaca aacttgtggc aggtgctgaa gctactgcta atgctgtatc 28080

gcctacaact gagcagggcg gaagcttctg gagtagggca ctcaactggt ctatgcgtaa 28140

cttcaaccct atcacagggg agatgacagc tggtggtcag gctatgcagt cagccataaa 28200

tactgctaag caaaaccaac agcagaacgc aactgttatt caccaggata atagcgttaa 28260

agttgatgct actattaccg gtgtacagaa cccacaagaa gtaggtcgtg aggtcaatga 28320

ccaggtcaaa caggcgcaga aagagaatgc tactgtaagg gctaacaaca aaacggcggt 28380

aacacaatga gtgacatctt tgctggaatt actgatgctg ccaataactt gtaccaggat 28440

gcccgtgaca gactcatggg cgacggtaca accaccttac gcttccgcaa ctgcggaggc 28500

attaagatgg atgctgtcac taatgaagag catcagtctg accttgacat cgctgacaat 28560

gaacttgagt ccggggctaa ggcttcggac catgctgcag tacagccaaa agttattact 28620

gtaactggtg ttgtagttgg ctatatcgac gagtcgattc aagagtcaac agttaacgac 28680

gtaaccggct tacggtctat ggatttcttg gatgatattc agcttccaca ggtagtgtct 28740

actgtcctcg aaggaacaag ggactttgct gttaacaagc tagtctcatt catagactgg 28800

ggcggtttag acgctaccat ttcacgctct ttagttccat ggcttcctga ctttagtata 28860

gctgaacaac ttaagtcttc ggacaacatg aggattgaac aggtataccg tacactgctg 28920

gatttacaga aaaacgttat ttactgtgac gttgatactg gtatcttcaa gtatacgaat 28980

atgcttttga agtctgttcg tgttgtgcag gagaaagatg gttcagctac gtttaccctt 29040

acgttccgtg aagtcattga agtaccgatt gtagttacaa acgctgtagc cgctaagtcg 29100

acaggtcgcc ctgctaataa cggcaccaag aaaagcggac gtgcacaagg tcagggtgat 29160

gccaccagta agaaggacat aaacacaact acaccgatta gcgagaaacg tgtaaacgat 29220

aaccgcggtt ttggtgtaac actcggtgac atgtttggct ttaacttacg aggcatattc 29280

tgatgatact taccacaact gctttccttg acattatagg taatgatggt tctggtattt 29340

atgacttgga acctacctcc gaaggtaagc aatcaatatc aatttctatc gctggatata 29400

cgtataacat tgagttgtac tataacacgg taggtcagct ctggcaggct acagtgactg 29460

acgtatcaag taatgctgta ctttgttcca gttatgcttt acaatatggg cgtattgctt 29520

ttggtaactg ggacacgcgt atagcgttgc tggtaagtga caaatcctcc agccgcatcg 29580

gtcctatgac tacagatgac atgggaggac gttgcagtgt ctacatcgtc gacaaaacag 29640

cctacccgtc agtttggccg cgcgttaaag cttgaggtca gcaacggtaa agatgaaggc 29700

ataatcatct caaatcttcg tgttaccttt gacattcaaa agggtaacac gaagcatcca 29760

aataaagcga caatcacagt gtttaacttg actgaatcca accgctataa gttggcgtca 29820

ggtacctggg attacgtccg tctctctgtg ggttacggtc ttgacaacat agtttatcgt 29880

ttgatattcg aaggcgatat tactcgagct aacgaccaac gaaacggtct tgacatacag 29940

actgtcctcg aatgtggcga tgcccgtaag tcttacaacg atggctttgt taatacgaca 30000

ctagctgctg gttctaccta cgacagtgtt gttgatgttt gcctggccag tatgccaaaa 30060

gttgaagaag ggtacgtctg ccttgacagc aataaaccag ctttcctgcg agcacgtact 30120

ctgttcggac aaacacacaa catactcgac caggttgctg ctgccaacaa ggctacctgg 30180

tctatacaag acaataaact caatatcatt cctaacgact ctgttactga ctacgacgtt 30240

gttgtgctgt cagaatcgtc tggtatgatt ggtgctcctc gcggcacaga caaaggtctt 30300

gaggttactt gcaacctgaa tccttctctg gccatcggtg gtcttgtaca agtaaattct 30360

atgtttacac agtacaacgg acagtataaa atcgacacta taagatttcg gggtgacaat 30420

aaaccgaaag gaccgtggcg tgctgacttg acggttctcg gtggacagtt cgcgaagaaa 30480

gttcgtcgac tcaagaagcc acgcaaactg tcaaagattg agtacgacta tgttactgaa 30540

cccggtgttg accatacagt aggcggaggt agctgatgtc agctattgac tataacacag 30600

caacacttga tgaggccatt gaggctgcac tgtttgctgc aatgattggt aagcgccact 30660

gtttacctgg ccgcatagta tcgtttaaca gcaagctaca gactgttgtt gttgaaccta 30720

tggttagtgc cgaaaacgaa gatggttcaa tgagacagct accaccagca gctgatgttc 30780

ctttgttcca gctaggcggt ggtgattttg ttataacgct tgaaccaaag aaaggcgacc 30840

cgtgcttact tcttgttgcc gacagatgta ttgacgtatg gtatgagctg gctgaaaacg 30900

ctataccagg tgacttccga cagaacgacc tgtctgactg cttctgtctt gtgggcttca 30960

gacctaagcc tctgaagata acaaactgga tggaaggtgt aactatccgt aaggttgacg 31020

gcagtcacta tatcaatatt aataatgctg gcaaggtatc gataaaggct acagcagtgg 31080

acttgcttga tacaccaaca cttaatgcac caaacacagt ggccaagttt aaagaagttg 31140

acattggcgg cgttaagatg tccaaacaca gacaccaaga aaacggtgat ggtggtggta 31200

ttactaatgg cccgcaaaac taggtaaccc acaatgcgaa ctagacgact cgattcaaac 31260

tgggactgga attttgggcg tggccgactt gactacgccg atacgtctga gtccattgaa 31320

caacgcatcg tgcagatact ccgctgttat actatggact gggtacatga cctggactat 31380

ggcattgact ggttcggact aatggagaag cctgttcgat gggctgacct ggaggctgca 31440

atcaagacag caatacttgc ggagtatgga gttagttcta tcgagagctt taccctaagc 31500

ttcgaccctg actcacgtca cgctgtttat tcggttgagg ttaatgatat ttaccacaac 31560

acaacctcta ttaacagaag tattaattta accggagaaa actgatgggc tctgtaacta 31620

gcacaggcta tgttcccgtc aagctcgacg actggatcag cagattcgaa acgctcttcc 31680

gtcgtgtgta cggcgcggat attgatttgt cagctaagtc tgctgacgga caaatggttg 31740

gtattctggc gcagatgttt gcagaccagg acgagcaagt tgcaattctg ttttctatgc 31800

ttgaccctaa ccaggcatct ggtgtatacg tagaccagtt ccttgcctac gtagcgcttc 31860

agcgtaaagg cgagatgtca acgctgttga ccagggttaa gattgaaggt gaccgtaaca 31920

ctattatacg tactccttac tatgtaactg acagctcgaa taacgtgtac cagcttaact 31980

ccactgttgc gctggacgat aacggtgtag tcgaaacaag cttcagctct gttaagactg 32040

gccgctttgt cgtacttgca aacgatgagc ttaagccaca gaccatcgtt tctggtgtgc 32100

gtaaagttac caacatgcaa gtgtcgtctg gcggtgcgtc cactgagctg gactctgagg 32160

caatagaccg tgcgtataac tcttacggca aacctggcac gaacagcata gacggtattg 32220

ttgctgagat tcgtcagatg actgatgtta tcgcgtgtaa cggttacgag aacgaagaag 32280

atactcggga cccgttaact ggcttacctc ctcacagtat gcagattgtt gtggatggtg 32340

gttcgcctgc tgctattgct gactgtatct tccgccgtaa gactaatggc tgtaagcaga 32400

ttggcactgt gtcttatcag attacggata agtcaggtac acagcgcaca atgaaatgga 32460

gtcagcctac ttatgttgag gcttatgctt ctctcgttgt agcccgtaag gaactgttta 32520

ccgacgtttc cgaggagctt attaagaatg ctattctgtc caaggtatac aatatcggtg 32580

aagaggtatc aacctacgaa ctgggtcagc tgattcaaga agcgaatggc gtaagcccta 32640

acttctacat caagactttc tttgtgggta aatcagctaa tccgtcgaca gatacgattc 32700

tgcgtatcac tgctcttgag cgtgcaatat tcgctgcaga ccgtatcact attgcagtgg 32760

tgaatgctga tggctgaata tcataaacta cttattcaac agtacgctga taagccaaaa 32820

gctgttgcaa ctattaaggg cttcgatgat gcaattcaag gtatctggga cggtgcccaa 32880

caagttgttg atagccttaa tatcgataaa gcttttggta aaggtcttga taatgttgca 32940

gcccgcgtcg gcgcatctcg tgttctcaag aaaggcgtag accgtaagtt cttcggcttt 33000

actaacaaag taccagacgt gcttggcttc ggtggttcgt ctggtaataa aggtggtccg 33060

ttctatcgct acggcgcatc agtatgggac ccggtaattc ttgacgacat tgacctgcgt 33120

acttacatca agatgaaaat ctggaagaac atgcaagacg gtactatgcc gtatttaatc 33180

gacttcctga ttgaattctt cgggatgaat aacttcgtta taactgacaa cgaagatatg 33240

acaatgactc tggagatata cagtactctg acaccactta agcaagtgct gattcagaac 33300

tacgatatac ttccgcgccc cacaggggtg aagattgaac ttaacacgag cgttgacgtc 33360

acggatagtc tgtaccgtta cgctactgta aacttaccag cacaggtgaa tgtatatggc 33420

tgatgtacaa acaagacccg ataacgacat cttcgccagc tccgcgggta ctggtgaact 33480

gctgccgttc ggtgaccttg ctcgtggctg gggtgcgacc cttggctact cagaaggtat 33540

tcctccgatg gagtggttta actttattgg taaccgtact gacaaaggta tccactacat 33600

ccttcagcag ggtgttgata tatgggtaga aactgagacc tacccggttg gtgcactggt 33660

taaagttaac gccgacaata tgctgtaccg ttgtctggtg cagaacatta atatgccacc 33720

gtctgctaac cctacacagt ggcgccctgt actcgatctg actgcggcaa ctgctgcagc 33780

acgacaggtt ggtaaagcct ctggtaacgt aatccaggtc ggtgcatttg gcttaggtgg 33840

caacgctgag gtagttacta atgctaacag cattgtaaac gacggtatct accagcttcc 33900

agcgtcaggt actggcggac ctactaacgg cgttgagact gagattctgc atatcaactg 33960

ggatgagaaa aacgcttcac agtggggacg caccaagggc aagcgcgtaa tagccatgcg 34020

tgagaaggtt aataatacct ggggcggatg gtcatactct tacgggcctg ataataaacc 34080

tacggcagct gagctgggta ttaaccagga cttcatccct atggatggtt ctgactcaat 34140

ccgcggtaac ttaggtactg ttggctggtt gcatattgca cgtactactt ccgacttgct 34200

acgtgctgct aactccgccg gcgtaggtac tgttgagtat tctggtaaca tcgcaggtgg 34260

tactgctgtt caggagttgc tgacttcagt ttcggttgcg gctaacagta atgttatcac 34320

tgcggctcgc tggggttctg tgcctatgtc gcatatccag cacattctta cggcaggtgg 34380

tgctacacag attaacatgg cggtatgtcg ccctggcggt attgccgacc gccgtacagt 34440

agctttgcag gtggatggcc ttaacagccg agtaactacc acaaatagct ttattttatg 34500

tgagaatggt gttagagtat attcacctaa caacccgcag cctatcgata cttcagccgg 34560

taacgttcgg ggtattcgac tcggcgctcg tattgctgct ggtgtatccg agtcaacgtt 34620

ctaccctggg catgtaatga ctggctggcg cttcggcaat aagaaagagc tgcgcggtgg 34680

tacatactgg tcagcgccgt tacagtatta tatcaacggc acttggatta acgtaagcaa 34740

cgtatcgtaa aggttactat gactaagcat aaagtgttcc gtaattttac catgtaccat 34800

cctgaggtaa cagaagagaa cgagaacatt gtaaatgctg gtgcaatctt cctcaaggat 34860

gaggaaggtc gcgactggta taatctcgca aaagagttcg ctatcaaata cgaaggcgca 34920

acgttcttgc aggtagataa gaacggagtt gtaattgcgg cgcataccga cccgactatg 34980

ttccatcctg tgggttgcga tatggttgta acgtacgacc cgcccttcaa catcttcgag 35040

acagttggcg aatggctgtt tgacggtaaa gagtttacgc ttaacgcacc aatgatgaag 35100

gagatcgctc gtgagcgtaa agaacaagct ctgtccatcg cttcgaccaa ggtcagtgtt 35160

ttacgtgaca cagttgatag tggcaatgct accgatgaag aaaaagagct atttgctaaa 35220

tggcgtgatt acaggattgc tgtcaatcgt cttgacatta accttggttc cgctatggtt 35280

gttccaccta tgccggagta aaccatgcaa ctgtcatttt tcaactttac agaatacaac 35340

ccacaaagga cggcggagac acttccgctg attaagcaag gcgttgtatt catcaaggat 35400

aacaatggta atgactggcg caagctggct aaagagctgg ctaagaataa agagctggtt 35460

cattacattg ttgtggacaa gtacaatgct gttgccgcgt atgctaacga tcctacgcta 35520

ctgtttcctg ttaattgcga tttgcatatt gttgagtctg tccctgttgc taagtttgcg 35580

gattgctatt acgaatggtc gtacgttaat ggcaagtttg ttaagcactc tgaactgcag 35640

aaggtgattg cagaagaacg cctgcaggaa gaacaggctt gggttgaagg acaactcgac 35700

actgaagaag atgaggaacg tattatcgct ctccataaat tccgtgccga tttgttgctg 35760

cttgttatgt cagatgcgcc gcatatcgac tggcctgtac gaccgtaact aattgctacc 35820

agcaagcttg tgcagttgct ggtagtgttt cttttacgct tgagtcgtgc tataatccac 35880

tctgggtata caataaaccg gagatatggg atgtttgact acaacgacaa gtcttccctt 35940

attggccagt ttgtggcttg ggtaacaata acagtaagta ctttatcact aagtgattgg 36000

ggtactctta tcggtatact cagttccttg gtgatgctga ttgttgttac gtataaccaa 36060

acacggcgaa ccaaaattat gtcggattat tacaatcgta agaagccgat agaggatgaa 36120

gtcgcatcga aagaagaggt gttgcgtgag tctgaaatca gcagcgacta aaggaggttg 36180

tagcgtgctc gctatcatcg ggcttgtatt gtcgatgaca agtacgcatg tacgtactaa 36240

taaagaaggt cttgaaataa tcggtaatgc agaaggttgt atgcgcaacc cgtatgtatg 36300

ccctactggt tatttaacag ttggcattgg tagccgcatc tacagcgatg agcccgctgt 36360

acggcgcgaa ggcctgacag accaggagat tgctgaccgc tgggtcaaga atattcaaga 36420

ggccgaaaaa tgtgttaatg actggttcca cggtaaagat atgaacacga accagttcag 36480

cgccatgacc tctatggtat ttaaccatgg ttgcactaga ctccgtaaga acaaggatgg 36540

ttcacctacc aaaatatata ctgctgctcg taaacaaaac tggactgaaa tgtgtaacag 36600

cataactgac tgggatatgg gtggtaagta caggggcttg acaatacgta agcaaaagga 36660

aaaagcgcta tgtctaaaac cggtgcaata attgcagtta ttctcggcct cgccattgtg 36720

ttcctgtctt tgtgggtacg tgagctgaaa ggtgatatta ccgaccgcga cggtataatc 36780

aaacagctta acaagactaa tggcgagttg tctgctgcta atcaaacgct gattaattct 36840

gtaaaggatg aacgtgaggc ctcagccaaa cgcgccagta agatagctga acttgaacaa 36900

caactgcgtg ataagaaggg taaatatgac gaagcaacaa aagatgattc gtgcgctaat 36960

accgttgctc cttctgccgt tcttgaactc atgcgctgat aagcctgaag ttgtttatgt 37020

tgacagggta gtcaaggttt atccgcctgg tgcatacctg gttaaatgcc ataagcctga 37080

attaacaggt aatacctgga aggcaatagg tcagttagct attgaacgtg gaacagctct 37140

tgatgattgt gcagacaaga tggacagcat aattaactgg agtaacaaag gggccgagtg 37200

atggcccctt tatttgatta agcctgcagg aagcctggga acatatcagc gcgtaaagca 37260

gcgcccttca gtaaccagat ttggtcttta gcctgtgcaa gcgcattgtc ctgagctact 37320

tgtttgccga tgtctgcatt gaactctgca gggtcaacac aaccagaagt acctacacct 37380

aactggaagc catccagtgt tgccacacat acagttacag ttgtattcgg tacctgccag 37440

aaatgatact ccagtttatc aacaagagca ttaacttctt taaaatctac tttcattatc 37500

gctttccttt cttctcgtta tactgcgcgc aggatgcaca caatactgta ccgggtaaga 37560

gctgacgtcg cttttcttca atatcatccc cacactggta gcacgtcagc gggcttacgc 37620

ctgtgtatct gacacggttc gacagcgctg cgtcccgggc aacttgttca gcttcagaag 37680

ccatatcagt aatgtccgat gtgcgcatag tctcaatcat tattttccct cttcttcaca 37740

atacagggtt tctaattcat cgcatagctg ttgcgtccag taatcatcct cgccacgtat 37800

ctggaagcac agcacaggaa tatcctgtcg taaaatatcg ctcttgtaaa tctttttggc 37860

aacagcttct ggttcttcct ggaatatctt cataaaccag tccaggcctg acgtgcagcc 37920

tgaggcgtct agtatctcct tcgcccttaa cttaagcgtt tcatctacct catactctaa 37980

acctatctta gacatattat agacctttaa ttagtgtgcc tctgcccagt tactttctat 38040

cttagcatca gtaagaacag gtacacgaag cggtagtaca gtttccatga cccgtacgaa 38100

atcatagaat ccatcttctg cttgcttacc agacggtaga gagaagttaa gctcatcgtg 38160

taccgtcagg cgaggtacgc caatcacgtc aaagtaaccg tcttcatacc ctttcagcat 38220

tgcagacttc atctgatctg ctgacgaccc ttgcagcact cggttgattg ctttgtgggt 38280

gtctgccagt ttaatgttcg ggccatactc ctgacgtgct tcttcaaatg gtaaaggcat 38340

cttaccgaat ccaccacgtt ttgggaccca tttgttgaac cacgtcttcc ggcccataaa 38400

ggtttctaca tacccctggc tatagcataa atcttcataa tgcttcatgg tagcaccaat 38460

aaatggtgca ccctgatggt atgccttcag aacaggttcc gcttcttcag gcgtcattgc 38520

taacttacgg gcaagagcat taagcatcat gccgtacaac aaaccaaagt tgatggtctt 38580

agccggttta cgttctataa cgtgacctga ggcttccttg ataatatcga ccataacatt 38640

gtggtaatct gcttccgggt tagccgcgaa aattgctcgt aactcatcag cgcccgggcc 38700

gactgcaaaa tgcgccatga atcgatattc aatttgggcg tagtctgctg atccccatcg 38760

gtgtccgaca tccggtacga aacaagcacg aaggagtggt cccagtattt tgtgtcgaga 38820

cggtatattc tgcagattag ggtcagatga gctaaaccga ccactaacgg ttccgccgtc 38880

atcaccgcgc atttgatgga attgaccgtg aaggaagcca tttgtattca tccccaatat 38940

atagccacga ataaacgtgg actggatttt ctcataagac ttaatctcag acaccagttc 39000

cgcaaacgga tggtgcaagc ctgctaagaa gtctgccgta aacgacggat tgcccgccgc 39060

tgttctgaag tactccagac caagatggtc aaatgcaaga gctaagtccg gacctgagtt 39120

aacgttaatg ttaaagccaa tctggtcttt aatctgctgg tgcattttcc ttatttcagc 39180

gctaagcgta tcgtcagcct ctttggcctt gtcaaggtca acacgcacac ctgcaaaacg 39240

catcttaagc attaatggaa tgagcttgca ctccatatag aatacatctg ttaaacacca 39300

gcgacttaac aaaggccact gggctttgat aatgtgtatt ggtaagtcgg cgtcagattc 39360

agcatagtaa cccacaagac gaggaggcgt tttgtagatg tactttctgg cttcacctgt 39420

tggtttacag ccataagcat ctgaaatcca ttggtacatc tcggctgagt ctttaccttc 39480

gcctacatac tttcgtccaa gtccttcaag tgcaacacgc gcgcctgggt caagtaatgc 39540

ttccgcgaat tgtacatcca tgcacaatcc tttaacctcc acgccttctg ctgcaagcca 39600

gcccaagtca tatgtgatgt tagcaccgac tttaacttgg ttaggtcgcg agagttcgag 39660

ccgaagccaa ccaagtactt cctcagctgg aaggttatcc tcttcgccga gctcgtgtct 39720

gattgggtag taagctttga agccgttgtc cgtagcgata ctaactccaa cgatatgacc 39780

ctccctcctg gcccaaccac taccatttgt atccatccct gggtcgtatg tttcacagtc 39840

aagaccgata atctttgcag aagacaagtc cgggtaatgc gcgcgcggcc gccatccagt 39900

atccctgacg tatggtatag gacggttgat aggtttaaca ttctttgcac cttcgaagct 39960

atcgaataaa aagctcatca gtcccctcta acatacgcaa ttacaactgt acgagttgag 40020

ttttccattt tgagcgcctg actacccagt cctattcttt ccatgccttt agccagttta 40080

ctcatctgac taataacaca gttgaacggt ttactaaact tagtcgactg gaacttaatg 40140

tcgacggaca tctttggaat aatcttatct ggtaagtctg cctttgcagt tgactcacct 40200

gttatctcca ggatggcttg accttcgtcg tacccagtaa gctggtccat gacttccagg 40260

aactcaggag tgatggcccg catatccatt tccatattga agaacttcga tacgtctggc 40320

catttggctg caagtaccgg ggcatccatc tgcacaccat ccgcaaagcc gaagaacatc 40380

tggccacggg cgtatcctac acttgtgggt tccatcttca acttaaccag agcctcaagc 40440

attggtttgg ctatggcaca ttcaaattca aaatctacgt tcagcttctc gcgtatcata 40500

tacttaccgt tagtggcgta agcataacca tcgcgtaaaa gtaaggatgt tgcccatacc 40560

tgcggtgctt cgttaggtac ccatttggct aaggcagtta ataccgggag taattcgcca 40620

tgcgctgaat accagttgtt aactgtagca agtggcggta ttggttcatt aagcgcttgc 40680

attcttgttt taagtacacc tgctgtgact gtgaggtttc cagcggctgt aatgttaaaa 40740

gtaatctgtt cagttcttgc cgcattaacg gccttacgaa atgattcagc atccacttgc 40800

aggtctggga atcctttaca tggcgctcgt agtaatacgt tcttgtagta cgaatggatt 40860

gttccgtctg caatccatat tttgcccata agaccactat cagtatactt gatgggacga 40920

gcttgttgaa cggcagtctg tatttgtttg gtgtcatagt taaacttgct catagtataa 40980

cggggccgaa gccccgctcc atcttatacg gccatttgga gtttgatgtt tggcattggt 41040

ttgtacttct caaccattgg cgtaacatcg gcaggagtga agtcaaacac tgacacgtca 41100

taaattgcat taatgtcagg acgaccttca aagttagtgt ccagtgtagt atcagaattg 41160

cttacgatac gttgcaggaa aatctttgcc gcatcgatat gattctgata gatgtgataa 41220

tcacccagcg taacagtcat atcacctacc ccagtaccca actcagcagc aatggtaatc 41280

tgcaacatct gatgcaacag aatatcgtga ggcagaccaa ggataacatc cgcgctacgc 41340

atgtggaaca tcagctctaa ttcattacgg ttgttaatga acagctgaaa gccatggtaa 41400

caaggcggaa gggccatggc gtccatctct tgtgggttcc atgccgtaat gtatgcccgg 41460

cggtcagtag gcttaacctt cagcgtttca ataacgtctt taagctggtt agtaagaaca 41520

ccattcttat agaatggctt aatccattgc acgccgtaca caggacctaa gtcacgattg 41580

tctggtgtac cccaacgttt gtttgcatcc tgcagattac cttcccacca gttgcagcca 41640

aagcctttga gtgtgtcgac gtttgaaatg ccgttgataa agcaataaag ctcagcagca 41700

acagccttga tgttaacagg cttaaacacc ggcaccatca gtttgttgtc gttgaaagcc 41760

agcttaaatg ctgtaccaaa acaacggcgg gtaccaacac ctgtacggtc tacaacatcc 41820

tcacctttac gcaggataaa agaaatgatg tcgctgtaaa gagaatcgac agtgctgtat 41880

gttttcatta aaacagttcc ttttgagccg ggttaacgtg tgcgtcgatg atggcctgga 41940

tgtcaggtgc tttccatcct tcaggcttaa caatatcctg accagactta tgcttgctgt 42000

tgcttgcatc aggcttagcg cgttctttcg ccatgttagc agcctgtacc gcatcccatg 42060

ctgcgtcaag tggccagttg ttgtttgcac accaggtaat caggtggata attaactggg 42120

ccagacggtc gcaatgagtg tcttcagtat ccttaacata tacaataagg ccgtcagcgt 42180

cgaagcggtc cataaatccg catggcgtag ttaacagaat cggatactca aattcgttgg 42240

tgaaccatgc agtctggtca tacaagtata acgtacctgc ggcaatgtaa attaaatcta 42300

caataccatc taagaagcca gctttgtcgc cgtccttctt agcttgttta atttcttcca 42360

tctcctcaat aagacggtcg taacgaagct tgaactcagg tttaccgcct tgactaacag 42420

gcttgaggtc aaacttctca tgaaaagcgg taacgcctgc agcaatttta ctgaatcgca 42480

tttcgtaact ccttacaaac agtttcgata aacttacgct taatttctgt agaccagata 42540

atatgacgag cctgttctaa agtgtcccaa cgttctttac ccacaggaaa gggaacaatg 42600

aaaaggccgg tacgggatat gcgcttgata aacgggaatt tacagcgcat ggttgtttca 42660

aacttgataa agtttgtttg cacttcgcgt gtaaacttga ccttatcaaa ttcatgttca 42720

agagtttgag caatgttgca tacttcctgg aaagaagatg caggcaagta atcttcgcgg 42780

ccgacggtgt tcacgtagtc gatttggccc tctagccgaa gtatccaaat cctgtaataa 42840

gcttcagcaa cacgttgtag attcattgta tctctcctct aaaagataac ttattatacc 42900

acatcactct ctagtggtaa agcacttttt acgcttctgg taaaaataaa aaggaccagc 42960

gttaactggt ccttttggca gggtggcgta aggagataca aggcatcgta actatgcaac 43020

caggcagcgc atatgagatt ggccaaaaga tttaccattt taatccccgc gacgagacga 43080

aaaggaggtg tcatcgagta gccaatcaca tatgcgttac ttgctgtgtg ctctcaccgc 43140

ggaaagtata gagcgttgca cttattacac cgatggagtg gcaaaagcac acagcgagta 43200

caaccttagt tgtggtgtct tagcacctga gttattgtgg ataacctgca cccgacgggt 43260

gtcaaaaaga tacgccaaaa caccacaact aaaattgtga ccgccgtcta caccaggcgc 43320

ctggggtcaa ggtagcgagc caataccctt ttcagcgtcg gaggaatcga acctcacaca 43380

tcacggcttt taccaaatct cggtcagcga gcagaattga actgcattca ctggacaccc 43440

tcactgtttc aacacaccag gcttaattga ttgttgataa gaagagagat gtaaggatgt 43500

ccattgaatg cacctccgta gaggtgcgtt cagatcagcg ctattattca gcaacttctt 43560

cgttgtcgcc ttcgatcgcg ccagccagtt tagcagcttc agctttagca gcttctgctt 43620

tctggcgagc tttttcagct ttctcttcag ctttgcgaat ctgctcagca gctttcgctt 43680

cggctttttc agctttgatg cgagcacgct cttcagcttt acgctgtttt tcagcggctt 43740

tagcagcatc gcgttctgct ttagcggtat caacacgctt agccagaacc ttaccagtgg 43800

ctttacgcca gtaggtcagc tgggaagaaa cagtaccacg ggacatttcg gtagtggcaa 43860

caacttcgtc cataacgtca gagctggact tgccttcttc atgggcttta tcagccaggt 43920

cccatacata acgtgcacca gtaccagctg ccggatacag tacgccgtta acttcttctt 43980

taccagttac aaaagtcagt ttctcgctca taattttttc ctcgtctaca gggcttaatg 44040

tttgttcgtt acgtgagtta atatacgtca tttaaaagag tgtgtaaaca ccttttttaa 44100

acttttttca ttagtgtcta gaaaggcaca tcatagtcaa aaagtttaca tccggaagac 44160

aggactgccg gcggtggtgt tttgcctccg gataacaggc aacctcctgc ctgaaaacta 44220

cggcagtttg cacagctcat atcctgcaag tatataataa gctgtcggcg aacatgccgg 44280

ggtaaagctt tagcctgggt tgctacccgt tcaaatagtt ctctgtcatc gagcgccata 44340

accgtttccc cagtcgtagt tcatgatttg tgggtagggt cgtttaatcc ataccttgat 44400

gcgcttagga atagccagct cacccttacg gcgaataaac tcatcgcatg tttcaggcgg 44460

ttctgttcct gcagcattac gccaccaggc atctgccatt tgcttagcat tgccatgggc 44520

ctctatattt acccaggttg tatacacttc caggtttgag taatacacaa cctttatact 44580

gtcaggtcgt ccctgcttct ggaataaaga atagtttacc agcgtaacat ctttatccac 44640

aacacgaagc tgaccgttag tcattacctc ttcttctgat gaagtgaagg caatggcagg 44700

tccgttaact gtaaactcaa attcatgtcc gcacatcgga tatacaccat tctcgtctgg 44760

cgtacttcct ttagccggat gcttacacgt acgagcagca tagccgctta cctggtgaca 44820

gatagggcac tctttaccgc caggtgtttt ctctgaccgt ttctttttct tcttaccaag 44880

acgtggcgga actgccgggt cgtcaatagg tccaaggtca atggtgttag atgtgaagtc 44940

cataacaaga cagtcatgct tgcctggcgc tgtacgtaat cctcgaccaa gtatctgcac 45000

ccacaaagca acagacgtgg acgggcgcaa gatgccgagc atatcaatct caggatagtc 45060

aaatcctgta gtcaatacgt ttacgtttac acagactcgt gctcgtccgc ttgtaaactc 45120

ttccagtgct gcttcgcgtt ctttctttgt aagaccgcca tgcacaacta cagtctgcca 45180

cccgcgcaga ttaaattcgt ctgctatgtt gtgggcgtgc tcaacacttg tggcaaatac 45240

aagaacgtgg ttcctgtctt taccgtagtg catcatctca cgaatagccg cgactgttat 45300

actatgctgg ttggctacct tctcaagctc tgacgggata tagtcaccca tcctttcgcc 45360

aacattactg gtgtcaatct tggttaacac tttcttgttg accaggcgcg acaggaaacc 45420

ttcatttacc agcctggtga atgcgtcagg tgttgtaagg tcatagcaga tgtcggtaaa 45480

tataccacag tccagcaagt gaccacctgc caggcgatac ggagttgctg tgagtcctgt 45540

tacctttacc ttaggattaa gctctttgaa gtgagcaata accttacggt atgttgtttc 45600

agacttctca ggaacaagat gggcttcgtc aatgaatata agattgaact tacctgcttc 45660

ttccaggttc ttaataattg actgtaccga tgctacaacg atacggcctg aaaagtcttt 45720

gtgtcctgca cctgaagagt agattgatac tggtgcttgt ggccagtgac gaacaatcgc 45780

tttggcgtct tgttcaacca gctccttgac gtgagtcagt ataagaatac gctgacgggg 45840

atagctggta agaatatcct tcatcagcat accaagcaca ggtgatttac ctgaacctgt 45900

cggcatgaca ataagtggat taccactata agaattgaag tacgaccacc aggctgtgac 45960

cgcttcttgt tgataccagc gagcttcaaa agccatttat catctcctca ttaactttat 46020

atattatacc acttccttgt ggtaaagtat acttttattt taaggaaagt ttaacgggct 46080

tctcatcctg cgggaagaca tcagcggggc gataatcatc acaagctgct aattgctctt 46140

cgattgtttc gagctctttc tggtgcttgt tacaatacca cactttgccc tgaagcggtt 46200

tactatagtg gcaggttctg cagttcctgt ctggcattgc tcctttgtgg cacaagtggt 46260

ggtactcgca cattcctggc ttgcactccc accagatagg cgattcagaa atcttcggca 46320

tatccaaacc agattccgca tatacaacag cctccgcttt actcagttgt atgtcgcttt 46380

ccagtaagtc aatctcgacc acctcaaaat acatctcgtc tgtgttcttg ttgattgcga 46440

tgtacacagc ttgattcagc ttaagcttcc gcatgtatac ctgcatctgg gcatagtgta 46500

ccggcttact tgctttaaca gacttgagct tgactaactc tttaaacgac ttatcgccat 46560

gcgtcttgaa ttcagcaagt gcccatacgc ctttgtattt aattacacag tcacaagacc 46620

cagagaagat gccattgaat tcgtgtatgc gccactgctt accagtcttg ggattgacgt 46680

ccagcacttc gtcacatacg gaacgaagcc actttacaat tctgctttct tcaaggttac 46740

cacgttcaaa cagacgaagt tgcttagctg taaacttctc cttgaacgac cagtgcagaa 46800

cgtgccaaag atagcgatag cagttgtgtc ctatctgcga tgctccaaga tgcccgcgtc 46860

gttcttctct ttcgtgggct tcgactatct tgatgtcaat acgtttctgc aggtcgtcaa 46920

acggacttgg cattgttaaa ctcctcaacg ctgaattcat cgtacacaat attatgagcc 46980

cacaagtgag cgacggcatt tgccatactg tctttccatt tggattgtga agctgtatga 47040

gccaatgcaa ctacacgtac caggttaagg ttggctatca gcatgtctgc acaatgttcg 47100

cacggcgggt aggttatgta gatggtaacc gggcctaatg aaggagtgtc taactcgctc 47160

cttatgttag ctaaggtgtt tatctcgcta tgaatagtta gagcgttctt aaggctatta 47220

ggcactattc ttatagagtc tagctgcgta ggtagaccgt tatatccagt ggaaatgata 47280

cgcttgttct ggtccacaag aacgctaccg acttgacgtt tcgggtcctt agaccagctt 47340

gcaacttcac aagctatgcg cataaaccgc ttatcccact tgtccatctt cttcttttgc 47400

atggtagtct ccgtatacga aaataggggc cgaagcccct attgaatatc tactcggcag 47460

ggattattct tcccaaggag ctgctgcacc gtcgtcagca ccaggtgccg ttgcagtagc 47520

actcgccttc gttgaagtac ccgcacctgc acctactttc ttcgtcgtcg atgcagttgc 47580

ttgagaatga ccacctactg cagtagttgc ttcagttgac gctttgtatt cgcgaatatc 47640

gttttgtggg tcgtactcac cggtcttgtc gatacgaaca ccaactttac cagctacaat 47700

acggttttcc agtaagtcgg tagaagtcag ttcaacaata cccagcgcac gacacaacgc 47760

agacagtttg cgatgaccga tttcaacagc ttgtgcatta gggttggagt aggtaatcat 47820

gtcaaataca acacggttag cgaactcacc ttcctgcact ttcagacgca gggacagata 47880

agaaccagtg cctgctttag tcggcttcag ctcaacctct tcaatcatga agttgtacca 47940

gcctttcggg atagggtcaa aagactgttg cggttcgacg gtagagctgt tgaagtttaa 48000

ttcagccatt gtttggtatc ctgtttaagt ttagtttaat ttcgtttaac gctttaccgg 48060

cagcgttcgg ccgtgatgct tattatacgc cactttttat catggcgtaa acacttttat 48120

tcgctggttc ctttctcagc ttgagcggct ttcttcgcca tccaggcttg gtgcttctct 48180

ttcgattcgt ccgaaccaat tttagtgagc tgcacgttcg ggtcgtcgtt ctgcatctgt 48240

tctgcagctc gacctgtcag aagcagataa ctgtctgact ccggatgata gtaccacagc 48300

tggtaaggat gatagccttg cagcttgtca taaatccagc ccaggtcagc aggttcacgt 48360

gcatccagtt cacgacctgg tgcgttacgg cacttagctt cccattggta gtcgggctgg 48420

gtctgaatgt agcgctcgct gttaccattg gcatcaggtg cagtcatacg taaagcaagc 48480

accgcatcca taagatacgg cagttgttta ggtaactgct ggccaggcat aagtggtgag 48540

tacaacattg caccactaac ctcgtccttg ttcttctcaa gcttgcacag gtatgcaacg 48600

ctgaaaggca ggtcacggaa ctgacgcatg atatacatca tcttgtcgtt ggtattacca 48660

taagcctgtc gtttgtcgtt gacctgcgac ttctcatagt ccagacaaac ttcagccacg 48720

tctgataacg agtcgaaaat cagatgctca tacttctgcg catccttact ttcagtaagc 48780

catttataca gcgccagaat gtcgttgaac gtattgattg gcgtagagtc gatgtcagca 48840

tctgcaatcg acatcaaacc gccttcagcg tcaatgacaa gaaccttatc gtgaggcaag 48900

gtcttagcca ggctggtttt accaacacct gatgcgccgt aaatagcgac cttaggtcgc 48960

ttatttccta cggcttgttt agtagataca atctgaacca ttaatgatag tcctcgtaaa 49020

gtgattcttc atcgcagtac atacgataaa gggcttcttc ggcttcttcc agggtgtcaa 49080

actcctcgcc acagacggag tattttgtgg gttcatcgaa tgactcgacg atgtgccagt 49140

catgatgaat accgtatgtc cacaagtcgc agtcatcttt tgcaacaagc ttacgtgtgg 49200

aacgaaattg tgctagagta ttgatgctca tattctctct cctcatgtgt atagaggtat 49260

tatacaccac ttacatgggg taagtacact actattttac actaacagct tgacacgata 49320

gccgtgacaa accgaggttg gctatgacgc catcaagcaa tactcctgca tgctcctcat 49380

tgtatgcttc tacccacaaa gggatggtga cacccttcag atggtcgctg tacacgttga 49440

acctatatac ctgtttgtgt gctttgtcaa cacgtacaac agtaaggtta ccatcctcgt 49500

aaggaggaag ctcaacgcct ttcttgctcc agaagttacg gaattcagca ctattaacaa 49560

tgccgtgtac aacacgctca gtaagttctg ttttgaggtc agcacgtgcc tttgtactga 49620

taaagaaacg acaagagaag aaagcattga cgtttatcac atcaaccgca tacacaacgt 49680

tatgctcttc agtagttggt gctttataaa caatgtattt agttttgaac aggtccatag 49740

tactctccag ggaggaagac tggcgggctt gcacccgcct aagttattac agcagttctg 49800

ctttcagcgc agcgatacca tcgagcaatg ctttgatatc cgggttagat gccgccaggt 49860

cagcaacaac tgactgacga cgacgttcca gctctttctc aacttccagg tcgaggcgtg 49920

ctgacgcttc acgcagtaag tcttcaacct gttgacgacg ctgatacttg ttaaagtcag 49980

gcacgtcaac cagccatttg agagttgcag tgttgtaacc agaagtattg gctgcactta 50040

cagacacaac atcaacgagc ttggtgccat tcagctccgg atagttaaca ttgcgaacgt 50100

caacaaccgc tttgtcacct acacttactt tgccgatgta cttgtaagtg tacacacgac 50160

cattcggttc aaacttaaca ccaacaaact tcagtttgtc gtctgcaata acctcttcct 50220

caactttgaa gttgattgca gtgttcttga caggcgcagc cgctttgagc ttagcaacca 50280

tgtactgttc tgaatgagac ggacggcaca gacggaagac gtcttcattg taggtcagcc 50340

agttaccggt ctcatccacg catgcaatct gattagtgcc gtccagtgaa cgaacaccag 50400

ggcgattctt ctcaacacga tacagcttac cttctttcag gttagcgtaa gaacggcggg 50460

cgatacccca gttgtggttt tcagattcgt acttagcgaa acgatcaacg ctgaagttct 50520

gcttcagacc acggtcgtcg attacccata agttatcgta agccccgccg aaagagccga 50580

ggttggcata cactttgccc tgcgtcaaca gggaagtgtt tccctcgtca acacagataa 50640

caaaacccag agcttcgtgt gctttaccgc tgcggtcaaa tactgtagtc atttcttgtt 50700

tccttgtata ccagtttgat tgtatgccag agatattccc tgcgcatgtg ctttattgct 50760

taaccgttaa agacattata tcattagtga gtggtgacgt acacatttat tttaaggcag 50820

aagtgcaaat agcatcacac aaacaataat gagcacggtc gtaatcttca aacaagaaca 50880

cttcgccatt ggcgactact tcattactgt tgacctgcag gttaatacct gaatgcagaa 50940

gcacatccat agcctggtcc ttgttcttgt acgggtccca aataagctgg aagcctgacc 51000

cacaaaggag acatcccttg agctccggga gagttgactc cttcacagtc acattacaaa 51060

tcttagccag tttagcacga gaatctagag tactcattta cgtttacctt tacgtttgtg 51120

tagcggagta tatgttttct ttggcacgcc aaacatggcc gcgtatgtac cgacttcttt 51180

ctttgttggc ttatccacaa tctcgtcaat cacgattaag tcttggtcaa tattgcgaaa 51240

gacttcagcc gctcttttct caacaggagt aaagtcaata accagctctt tgcctatcag 51300

gctttctttg agcttgtcaa atagccattt gtcacgcgga ccaggattgc ccataaacat 51360

aacacgcatc tgcttattgt ccatggatga tataccccgt tatttgtgtt gccaggtaca 51420

aaccgtatac cacacaccca acgagtatgg tatcaagaat cagatgagct atcttcacgt 51480

ttgacctccg gaaggaatga gttatcaacc ttaatcagta cgttacggct gttaaccact 51540

tccaggctgg tgccatcctt tttgtgggtt acacgaataa acgcaacacc ctcagcctgt 51600

gcctgctcaa tagctttatt cagcacaaca ttatcctgta ccatatgacg tacgcgagga 51660

aggatattgc aaagaggttc catctcgtcc attcctgcag tacgcatgac ggacttgaga 51720

atgccaagat gatgcttgta acggtcttca agattatact tcttgctgtt atctgatgcc 51780

atgtaagtct ccaaaagtta gggggcttgt gctaagcccc gtttaattac tcggcttctt 51840

ccgtagctgc cggggtgaat gttacagtcg gaataccagg cgaggtcttc acaacagggt 51900

cgacgatagc cagttgttct ggtgtcaggt tacggtatgc agtaactgac atttcatact 51960

tggttttaaa cagcttggca acaacagtct tcggtaactt cttcattacg tcaggcacaa 52020

gcttctggtc tagtgagcgg ttaaccttct gctcaacatt gagtttacca ggtacgccat 52080

gcatgctgaa ctcaaccgtt tgtgtacctt ctttaccacg attgataatc atgtcacgtt 52140

caagacgtgc tttaatggtc cggcgcaacg tctcctggtc agctttcgct tgtgccatcc 52200

agcggttcag attcgccagc ttgtcgtaca gctgttccgt gtccatcgat tccagttctt 52260

gtgggttcaa tgccatcagt tgtttcctca ggttgcatgt gcatagttgc acctattagt 52320

agacaataat tattgaacat tgtacgtagt gcctggcccg caacaatatc cgttatgtct 52380

attataccac ttttagtggt gacgcgtaaa gctttttctt tctttgtttt ctccacaaat 52440

gcaatgctga tgatttcttc cgcatgaatt acgtggatgg tacctgcaaa ctcaccagtc 52500

ttagctggtg cctggtacac taacgacata gacggttggt tactcactta attgctccta 52560

tttatataag tccatccagt tacgatactc gtcataaaac cggtcaaact cgtttttggt 52620

gagtgcaata gcaacaccat ttaccagatg catggttcgt gttttgatgt tgacatacat 52680

gacgttgctg tacgggaaca agctaccgtc atagtcagtg aagtaagtcg gtgtaacatg 52740

aagcacatcg cgcaatgcaa ccagcgcttc aacagcttct gcatggcctt cgcggcggaa 52800

cgtc 52804

Claims (10)

1. An acid-resistant Escherichia coli bacteriophage is characterized in that the bacteriophage is RDP-EC-20140, and the preservation number is CGMCC No. 21991; the Escherichia coli bacteriophage RDP-EC-20140 has a polyhedral stereosymmetric head part, a nucleic acid coating part, a tail part with a diameter of 70nm and a length of 120nm, and a tail sheath, wherein the neck part is connected with the head part and the tail part, and the Escherichia coli bacteriophage RDP-EC-20140 is a Anoviridae of Anoviridae.

2. The coliphage of claim 1, wherein the coliphage RDP-EC-20140 is treated at 65 ℃ for 60min to maintain the titer at 10⁸pfu/mL, treated at 80 deg.C for 30min, and the titer is maintained at 10⁵pfu/mL, better tolerance to temperature; the titer is still maintained at 10 by culturing at 37 deg.C for 1h in the pH range of 4-10¹⁰pfu/mL, with highest potency at pH 7, phage retained high activity at pH 12 and pH 2, and the phage was acid-base tolerant over a wide range.

3. The bacteriophage of claim 1, wherein the bacteriophage RDP-EC-20140 is synergistic with an antibiotic.

4. Use of the coliphage RDP-EC-20140 as claimed in any one of claims 1 to 3 in the preparation of a medicament for the prevention and or treatment of diseases caused by escherichia coli.

5. Use of the coliphage RDP-EC-20140 as claimed in any one of claims 1 to 3 in the preparation of a feed for the prevention and or treatment of diseases caused by escherichia coli.

6. A fungicidal composition for the control of escherichia coli comprising an effective amount of the escherichia coli bacteriophage RDP-EC-20140 of claim 1.

7. Use of a bacteriophage according to any one of claims 1 to 3, wherein the bacteriophage RDP-EC-20140 is used to provide a potential therapeutic or nutraceutical for bacterial infection caused by e.

8. An additive for preventing and or treating diseases caused by Escherichia coli, wherein the effective component of the additive comprises the Escherichia coli bacteriophage RDP-EC-20140 as defined in claim 1.

9. A cleaning or disinfecting agent comprising the coliphage RDP-EC-20140 of claim 1.

10. The embedding process of the coliphage RDP-EC-20140 of claim 1, comprising the following steps:

(1) weighing the following raw materials in parts by weight: 35% coliphage RDP-EC-20140 (lyophilized powder), 62% starch, 1.5% microcrystalline cellulose and 1.5% sodium starch; placing the raw materials in a mixer and mixing for 10 minutes at room temperature;

(2) putting the raw materials mixed in the step (1) into a swing granulator for granulation, pouring particles discharged from the granulator into a swing granulator to complete granulation, then putting the granules into a low-temperature fluidized bed for drying at the drying temperature of 40-42 ℃ for 30-40 minutes, and removing oversize and undersize particles through a vibrating screen after drying;

(3) and (3) putting the particles obtained in the step (2) and 30% by weight of palm oil into an embedding machine together to finish embedding.

Images