KR20230164829A - Composition for inhibiting proliferation of Salmonella enteritidis comprising bacteriophage - Google Patents

Abstract

The present invention relates to a composition confirmed to inhibit the growth of Salmonella Enteritidis in food using bacteriophages strained from repopulating full culture-based bacteriophages, using the bacteriophage ΦSEP1 of the present invention (accession number KCTC18931P). , bacteriophage phiJR14 (accession number KCTC18927P) and bacteriophage phiJR16 (accession number KCTC18928P) have excellent antibacterial activity against Salmonella Enteritidis contained in food, so they can be usefully used as food additives and feed additives.

Description

Composition for inhibiting proliferation of Salmonella enteritidis comprising bacteriophage}

The present invention relates to a composition confirmed to inhibit the growth of Salmonella Enteritidis in food using bacteriophages screened from repopulating full culture-based bacteriophages.

Salmonella ( Salmonella spp.) is not only an indicator of hygiene in poultry farms, but also pathogenic to chickens. Among them, Salmonella Enteritidis and Salmonella Typhimurium are classified as pathogens important to public health because they cause food poisoning not only in chickens but also in humans. According to the FAO/WHO report, 77.1% of poultry-borne food poisoning was reported to be Salmonella , and in fact, Salmonella occurred in Europe in 2006. As a result of analyzing about 16,000 cases of food poisoning, Salmonella Enteritidis accounted for a high proportion of 62.5% and Salmonella Typhimurium accounted for 12.9%.

Salmonella in poultry Various antibiotics are being used for control, and antibiotic resistance is also showing an increasing trend. In fact, of the 83 Salmonella (9 serotypes) isolated from chickens and ducks on domestic farms in 2015, all 83 had resistance to at least two or more antibiotics, and in particular, 66.3% and 57.8% had nalidixic acid, respectively. ) and streptomycin.

Among various antibiotic alternatives, the specificity, proliferativeness, and abundance of bacteriophages (or phages) are characteristics that distinguish them from other antimicrobial agents (ex. antibiotics, etc.). With the recent rise in the problem of misuse and abuse of antibiotics, phages are used as harmful bacteria. Efforts to use it to control people are in the spotlight again. Due to the safety tests of bacteriophages and their effectiveness in controlling pathogens, the FDA granted GRAS status to anti-listeria phages, and efforts are underway to use phages in the treatment of antibiotic-resistant bacteria. In addition, unlike pathogens that are resistant to antibiotics, it has been confirmed that pathogens that are resistant to bacteriophages often have reduced pathogenicity, which means that the severity associated with resistance is relatively lower than that of antibiotics.

The countless different wild type strains that exist in nature may have different characteristics from laboratory strains, and this remains a limitation of various microbial inhibitors. Furthermore, the problem of regrowth by antimicrobial agent-resistant variants and mutants is a common problem for all microbial inhibitors. In the field of controlling harmful bacteria using bacteriophages, a method of applying bacteriophages with various different characteristics (particularly distinct host ranges) as a cocktail is used to solve these problems.

The useful bacteriophage screening method using the 'random screening method' and the 'mutant-base screening method' failed to isolate bacteriophages that can effectively inhibit the growth of Salmonella.

(001) Republic of Korea registered patent KR10-2396828

Accordingly, the present inventors completed the present invention by combining bacteriophages screened from repopulating full culture-based bacteriophages and confirming that they inhibit the growth of Salmonella Enteritidis in food.

Therefore, the present invention seeks to provide a composition for killing Salmonella Enteritidis containing bacteriophage ΦSEP1 (accession number KCTC18931P) represented by bacteriophage SEQ ID NO: 1.

In addition, the present invention seeks to provide a composition for killing Salmonella Enteritidis containing bacteriophage phiJR14 (accession number KCTC18927P).

In addition, the present invention seeks to provide a composition for killing Salmonella Enteritidis containing bacteriophage phiJR16 (accession number KCTC18928P).

In addition, the present invention seeks to provide a composition for killing Salmonella Enteritidis comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).

The present invention provides a composition for killing Salmonella Enteritidis containing bacteriophage ΦSEP1 (accession number KCTC18931P) represented by bacteriophage SEQ ID NO: 1.

In addition, the present invention provides a composition for killing Salmonella Enteritidis containing bacteriophage phiJR14 (accession number KCTC18927P).

In addition, the present invention provides a composition for killing Salmonella Enteritidis containing bacteriophage phiJR16 (accession number KCTC18928P).

The present invention provides a composition for killing Salmonella Enteritidis, comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).

Provided are food additives and foods for killing Salmonella Enteritidis, including a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).

Provided are Salmonella Enteritidis feed additives and feeds containing a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).

Bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P) of the present invention have excellent antibacterial activity against Salmonella enteritidis contained in food, and are therefore used as food additives and feed additives. It can be usefully used.

Figure 1 is a graph showing the results of a Salmonella Enteritidis proliferation inhibition test in which bacteriophages (phiJR14, phiJR16) isolated from the bacteriophage pool were applied to the cocktail:
SE348 inhibition experiments were conducted in liquid medium with combinations of ΦSEP1+phiJR14, ΦSEP1+phiJR16, and ΦSEP1+phiJR14+phiJR16. The initial bacterial concentration was fixed at 1x10 ⁴ CFU/ml, and the phage concentration was fixed at 1x10 ⁶ PFU/ml.
Figure 2 is a photograph taken with a transmission electron microscope of ΦSEP1.
Figure 3 is a photograph showing the plaque morphology of ΦSEP1, phiJR14, and phiJR16:
From the left, plaque forms of ΦSEP1, phiJR14, and phiJR16 (host strain cultured overnight in TSB, diluted phage, and 0.4% agar medium were poured onto a TSA plate and allowed to solidify) Check the results after incubating for 6 hours at 37℃).
Figure 4 shows the circular genome of ΦSEP1, where the outer ring represents the ORF of the forward strand and the inner ring represents the ORF of the backward strand, and the functional genes are indicated.
Figure 5 shows the whole genome DNA tree with ΦSEP1, a LASTN search was performed with ΦSEP1 DNA sequence in NCBI, homologous sequences were selected for “distance tree of results” analysis, Neighbor Joining tree method was selected, and the results were “ It was downloaded in “Newick format” and FigTree (version 1.4.4.) was used to generate the entire genome tree.
Figure 6 shows the results of phylogenetic analysis of ΦSEP1, which shows the major envelope protein (A), terminase large subunit (B), and DNA polymerase (C) of ICTV-class Salmonella bacteriophage, a member of Siphoviridae.
Figure 7 is a graph showing the results of experiments at (A) 4°C and (B) 25°C confirming the inhibition of Salmonella Enteritidis growth in egg yolk.
Figure 8 is a graph showing the results of experiments at (A) 4°C and (B) 25°C confirming the inhibition of Salmonella Enteritidis growth in egg white.
Figure 9 is a graph showing the results of experiments confirming the inhibition of Salmonella Enteritidis growth in milk, with (A) at 4°C and (B) at 25°C.

In the present invention, we analyze the characteristics of bacteriophages (ΦSEP1, phiJR14 and phiJR16) that kill Salmonella Enteritidis isolated through the 'repopulation full culture-based bacteriophage screening method', and isolate them from foods (milk and egg yolk). and egg white), it was confirmed that it can effectively inhibit the growth of Salmonella Enteritidis, and a cocktail of ΦSEP1, phiJR14, and phiJR16 was previously reported, even though it was applied at a relatively 'low concentration'. The present invention was completed by confirming that it had an inhibitory ability similar to or better than that of some bacteriophages.

The bacteriophage ΦSEP1 of the present invention has host sensitivity to Salmonella Enteritidis and can kill them.

Bacteriophage ΦSEP1 of the present invention consists of the nucleic acid sequence represented by SEQ ID NO: 1.

The bacteriophage phiJR14 of the present invention has host sensitivity to Salmonella Enteritidis and can kill them.

The bacteriophage phiJR16 of the present invention has host sensitivity to Salmonella Enteritidis and can kill them.

The present invention provides bacteriophage ΦSEP1 (accession number KCTC18931P), which has a specific killing ability against Salmonella bacteria. In addition, the present invention provides a composition for killing Salmonella Enteritidis comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).

Salmonella is a gram-negative bacillus that mostly proliferates in the intestines but does not produce toxins. It is mainly infected through ingestion of contaminated food and water, or from feces to the oral cavity. It is mainly caused by food ingredients such as raw meat, poultry, eggs, vegetables, and dairy products that are not properly cooked. Representative diseases that can occur due to Salmonella infection include gastroenteritis, sepsis, enteric fever, and subclinical infections.

The bacteriophages ΦSEP1, phiJR14 and/or phiJR16 of the present invention have host sensitivity to Salmonella Enteritidis and are capable of killing them.

In addition, the present invention provides a composition for killing Salmonella Enteritidis containing the bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16). The composition for killing Salmonella Enteritidis of the present invention can be formulated by a conventional Salmonella preparation manufacturing method, and there are no particular limitations on the method and means of formulation, but when administered to the human body, within the pharmaceutically acceptable range, When administering to livestock or fish, it can be formulated within the range permitted by veterinary medicine and the administration route can be determined accordingly.

The composition for killing Salmonella Enteritidis of the present invention may, in addition to the bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16) of the present invention as an active ingredient, include a carrier, other auxiliaries, etc., as needed. The carrier may include physiological saline solution, distilled water, etc. , feed, media for bacteria, etc., and supplements can be used by adding buffer solutions for purposes such as pH correction.

The composition for killing Salmonella Enteritidis of the present invention can be manufactured in the form of liquid, semi-solid, powder, etc., but is not necessarily limited to the above three preparations and can be formulated in various forms.

The composition for killing Salmonella Enteritidis of the present invention can be administered in a conventional manner through routes such as orally, intravenously, intraarterially, intraperitoneally, intramuscularly, transdermally, intranasally, inhalation, or rectally. The dosage of the composition for killing Salmonella Enteritidis of the present invention refers to the amount required to achieve the effect of killing Salmonella bacteria in the administration target. Therefore, the amount depends on various factors including the type of disease, severity of disease, coordination of dosage form, age, weight, general health status, gender and diet of the administering subject, route of administration, time and period of administration, and concurrently used drugs. It can be adjusted.

The bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16) of the present invention or the composition for killing Salmonella Enteritidis containing it can kill Salmonella regardless of resistance to existing antibiotics, thereby treating diseases caused by Salmonella infection. It can be used for antibiotic-resistant Salmonella bacteria, and when used against antibiotic-resistant Salmonella bacteria, it can kill Salmonella-resistant bacteria that could not be removed with existing antibiotics, so it can be useful as a replacement for existing antibiotics. In particular, it can be provided as an alternative to the use of antibiotics in the food industry, livestock industry, and fishery, which can solve the problem of antibiotic residues in food, livestock products, and marine products.

Additionally, the present invention provides a food additive or food containing the bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16). The food additive or food is not particularly limited in its formulation as long as it contains a bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16), but it is more preferable that the target food is a food that is susceptible to contamination by Salmonella.

In addition, the present invention provides a feed additive or feed containing the bacteriophage bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16). The feed additive or feed is not particularly limited in its formulation as long as it contains a bacteriophage cocktail (ΦSEP1, phiJR14 and/or phiJR16), but it is more preferable if it is a feed additive or feed targeting livestock susceptible to Salmonella infection. Examples of feed additives include binders, emulsifiers, and preservatives added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, buffers, colorants, extractants, and oligosaccharides added to feed to increase utility. etc. may be included, and in addition, feed mixtures, etc. may be additionally included.

Hereinafter, the configuration of the present invention will be described in detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.

<Example 1> Experimental method

<1-1> Bacteria and bacteriophage strains and their propagation methods

Bacteria and bacteriophage strain information and propagation methods used in this experiment are summarized in Table 1 and Table 2. In the case of bacteria, unless otherwise specified, they were cultured at 37°C and 160 rpm using TSB (or TSB agar medium [TSA]) medium.

<1-2> Regrowth pool medium preparation and subculture

A bacterial inhibition experiment was performed on Salmonella Enteritidis wild strain SE348 and Salmonella Typhimurium wild strain ST288 and ST393 on liquid medium using each isolated phage, and the re-growing bacteria that occurred after 24 hours of growth were treated with PBS buffer 2- After washing three times (phage removal) and resuspension by adding an equal amount of TSB medium, glycerol stock (15% glycerol) was prepared, which was named Regrowth Pool medium.

For subculture of the regrowth pool medium, 100 μl of the regrowth pool glycerol stock was inoculated into 5ml fresh TSB medium and cultured overnight, then an equal amount of 30% glycerol was mixed and stored and maintained as 15% glycerol stock.

<1-3> Isolation and subculture of single plaque from bacteriophage pool

The bacteriophage pool dilution confirmed to inhibit the growth of Salmonella for 24 hours and the regrowth pool in the form of 300 μl glycerol stock cultured overnight were mixed with 0.4% agar medium and placed on a previously prepared solid medium plate ( plate) (1.5% agar) and incubated at 37°C for 6 hours. The single plaques produced were plate eluted from the repopulating bacteria, turned into stocks, and stored. Single plaque isolation was performed more than three times on repopulating bacteria to complete single plaque isolation.

After inoculating 300 μl of the stock phage, 4 ml of 0.4% agar medium, and 100 μl of the full culture glycerol stock re-proliferated in TSB, 300 μl of the overnight culture was mixed and placed on a solid medium plate ( 1.5% agar) and incubated at 37°C for 6 hours. Plate elution and filter sterilization were performed and maintained.

<1-4> Dotting assay to confirm host range

300 μl of the indicator culture grown overnight was added to 4 ml of 0.4% agar medium, poured into a previously prepared TSA (1.5% agar) plate, and left at room temperature for about 30 minutes to harden the 0.4% agar medium. 10 μl of bacteriophage sample diluted to a concentration of ^{10 6} plaque forming unit (pfu)/ml was dotted on 0.4% agar medium and cultured at 37°C for 24 hours to confirm lysis or plaque formation.

<1-5> Large-scale preparation of bacteriophage

Bulk-volume preparation in liquid culture, polyethylene glycol (PEG) precipitation, and CsCl gradient ultracentrifugation of ΦSEP1 were performed as previously described, and high-titer phage stocks were prepared.

<1-6> Transmission electron microscope analysis

Bacteriophage stocks purified with dialyzed CsCl were observed under a transmission electron microscope. 10 μl of bacteriophage ΦSEP1 purified with dialyzed CsCl was dropped onto carbon-coated copper grids and incubated for 10 min. Excess solution was removed with filter paper, and grids were negatively stained with 10 μl of 2% uranyl acetate (Sigma, St. Louis, MO, USA). Samples were visualized by Bio-TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV and magnification of 200,000x.

<1-7> DNA isolation of bacteriophage, whole genome sequencing and base sequence analysis

Genomic DNA of ΦSEP1 was extracted using PEG-precipitated bacteriophage stock (filter-sterilized) after treatment with DNase and SDS and ExgeneTM Cell SV DNA purification kit (GeneAll Biotechnology Co., LTD., Songpa-gu, LTD.).

Whole genome sequencing was performed at LAS Inc., Gyeonggi-do, using the MiSeq sequencing system (Illumina, San Diego, CA, USA).

Open reading frames (ORFs) were predicted using RAST, Phanotate and ORF finder. If two or more of the three programs matched, the predicted ORF was selected.

ORFs were compared with a non-redundant database using the BLASTP algorithm, and functional annotations were organized. tRNA encoding genes were identified using ARAGORN v1.2.41. The circular genome map of ΦSEP1 was created using CGView.

<1-8> Identification and classification of bacteriophages

Comparative analysis of the entire genome sequence of ΦSEP1 was performed using Gegenees 3.1.0 [Agren et al. 2012] using other Salmonella -infecting Siphoviridae bacteriophages classified using the International Committee on Taxonomy of Viruses (ITV) (Virus Taxonomy: International Committee on Taxonomy of Viruses, 2020 Release) ( Salmonella virus SP31 [NCBI Accession no. NC_021775.1]; Salmonella virus AG11 [NC_041991.1], Salmonella virus Ent1 [NC_019539.1], Salmonella virus f18SE [NC_028698.1], Salmonella virus Jersey [NC_021777.1], Salmonella virus L13 [NC_021317.1], Salmonella virus LSPA1 [NC_026017 .1], Salmonella virus SE2 [NC_016763.1], Salmonella virus SETP3 [NC_009232.2], Salmonella virus SETP7 [NC_022754.1], Salmonella virus SETP13 [NC_022752.1], Salmonella virus SP101 [NC_042065.1], Salmonella virus SS3e [NC_006940.2], Salmonella virus wksl3 [NC_041992.1], Salmonella virus SE1Kor [NC_042025.1], Salmonella virus 35 [NC_048632.1], Salmonella virus 37 [NC_029045.1], Salmonella virus 118970sal1 [NC_031930. 1], Salmonella virus BP12C [NC_031228.1], Salmonella virus BSPM4 [NC_048655.1], Salmonella virus Chi [NC_025442.1], Salmonella virus FSLSP030 [NC_021779.1], Salmonella virus FSLSP088 [NC_021780.1], Salmonella virus iEPS5 [NC_021783.1], Salmonella virus KFSSE1 [NC_048683.1], Salmonella virus Siskin [NC_048727.1], Salmonella virus SPN19 [NC_019417.1], Salmonella virus ST101 [NC_048648.1], Salmonella virus YSD1 [NC_048666.1] ], Salmonella virus Lumpael [NC_048113.1], Salmonella virus 9NA [NC_025443.1], Salmonella virus SP069 [NC_042062.1], Salmonella virus Sasha [NC_047786.1]) were used.

To classify ΦSEP1 at the genus level, we performed a BLASTN search with the ΦSEP1 DNA sequence in NCBI and selected homologous sequences for “Distance tree of results” analysis. The Neighbor Joining tree method was selected and the results were downloaded in "Newick Format". This used FigTree (version 1.4.4.) to generate the accompanying whole genome tree.

To determine ΦSEP1 at the species level, the DNA identity (%) of ΦSEP1 with other ICTV-classified Salmonella-infecting bacteriophages (Family, Siphoviridae) was calculated using the BLASTN algorithm and the following equation.

To confirm the previous results once again, the phylogenetic position of ΦSEP1 was determined using the above ICTV-classfied phages and the amino acid sequences of the capsid protein, terminase large subunit, and DNA polymerase of ΦSEP1.

<1-9> Salmonella in liquid medium Proliferation inhibition test

50 μl of Salmonella cultured overnight was added to 5 mL of fresh TSB medium and cultured at 37°C with shaking (160 rpm). When the OD ₆₀₀ is about 0.3, dilute the bacteria concentration with PBS buffer to 5x10 ⁵ CFU/ml and add 100μl to 5ml TA medium to make the initial bacteria concentration 1x10 ⁴ CFU/ml, then use SM buffer. Then, 100 μl of bacteriophage diluted to a concentration of 5x10 ⁷ PFU/ml was added (final bacteriophage concentration, 1x10 ⁶ PFU/ml). Bacterial growth was monitored by measuring OD ₆₀₀ while culturing at 37°C and 160 rpm.

<1-10> Analysis of Salmonella Enteritidis growth inhibition using bacteriophages in various foods

① Growth inhibition test of Salmonella Enteritidis using bacteriophage in egg yolk and egg white

Fresh eggs were purchased at a supermarket in Jeonju. First, the surface of the egg was washed with 70% ethanol, then the sac and egg white were separated using a sterile pipette, and contamination was removed with ultraviolet light for 30 minutes.

Salmonella grown overnight was added to 5 mL of fresh TSB medium (1% inoculum) and cultured at 37°C with shaking (160 rpm). When the optical density (OD=600) at 600 nm reached 0.3, the bacterial concentration was diluted to 1x10 ⁶ CFU/ml using PBS buffer, and 100 μl was added to 10 ml planting material, making the initial bacterial concentration 1x10 ⁴ CFU. 100 μl of phage(s) diluted in SM buffer was added to a final concentration of 1 x 10 ⁶ PFU/ml. Inhibition of growth of Salmonella Enteritidis was confirmed through CFU counting at designated times while culturing at various temperatures.

② Growth inhibition test of Salmonella Enteritidis using bacteriophage in milk

Sterilized milk was purchased from a local supermarket in Jeonju. The sterilized milk was sterilized again with ultraviolet light for 30 minutes and plated on TSA to confirm that there was no bacterial contamination.

Salmonella grown overnight was added to 5 mL of fresh TSB medium (1% inoculum) and cultured at 37°C with shaking (160 rpm). When the optical density (OD=600) at 600 nm reached 0.3, the bacterial concentration was diluted to 1x10 ⁶ CFU/ml using PBS buffer, and 100 μl was added to 10 ml planting material to make the initial bacterial concentration 1x10 ⁴ CFU. 100 μl of phage(s) diluted in SM buffer was added to a final concentration of 1x10 ⁶ PFU/ml. Inhibition of growth of Salmonella Enteritidis was confirmed through CFU counting at designated times while culturing at various temperatures.

<Example 2> Experimental results

<2-1> Isolation of single plaques from bacteriophage pools in various samples using repopulation pool medium

After plating, single plaque isolation was performed from the effective bacteriophage pool using regrowth pool medium stored in the form of glycerol stock. A total of 16 plaques were isolated (phiJR1-phiJR16), and among the combinations with ΦSEP1 previously used to prepare phage pools, a combination that could inhibit Salmonella Enteritidis SE348 in liquid medium for more than 24 hours was selected. The bacteriophage cocktail selected in this way consists of ΦSEP1, phiJR14, and phiJR16.

Phage name Host Strain
(=Propagation strain) Sources Description References ΦSEP1 SE348 Jeonbuk National University The host strain culture cultured overnight (37℃, 160rpm) in TSB is cultured with various samples, then centrifuged and sterilized by filtration, and then plated with the host strain and stocked through single plaque isolation and plate elution. Gwangpyo Kim, Yubin Choi 2021 phiJR14 Regrowth pool
culture Bacteriophage pool Regrowth pool culture cultured overnight (37℃, 160rpm) in TSB is plated with the Bacteriophage pool. After 6 hours of incubation, when clear lysis is confirmed, the plate is eluted, filtered, sterilized, and stored at 4℃. Gwangpyo Kim, Yubin Choi 2021 phiJR16 Regrowth pool
culture Bacteriophage pool Gwangpyo Kim, Yubin Choi 2021

<2-2> Host range analysis of phiJR14 and phiJR16

As a result of host range testing of phiJR14 and phiJR16 using a dotting assay, 16 Salmonella Enteritidis, 10 Salmonella Typhimurium, and 20 Salmonella Gallinarum wild strains were inhibited, as were SEM1 and SEM2 isolated through mutant-based screening and regrowth pool media.

Species Phage
Bacteria ΦSEP1 phiJR14 phiJR16

Sal .
Enteritidis wild type strains SE344 + + + SE345 + + + SE346 + + + SE348 + + + SE349 + + + SE351 + + + SE352 + + + SE360 + + + SE373 + + + SE376 + + + SE377 + + + SE378 + + + SE382 + + + SE383 + + + SE384 + + + SE385 + + +





Sal .
Typhimurium wild type strains ST288 - + + ST289 - - - ST290 - - - ST292 - - - ST297 - - - ST298 - - - ST299 - - - ST301 - - - ST304 - - - ST305 - - - ST314 - - - ST335 - - - ST357 - - - ST362 - - - ST364 - - - ST367 - - - ST368 - - - ST370 - - - ST371 - - - ST392 - + + ST393 - + + ST395 - + + ST398 - + + ST399 - + + ST404 - + + ST405 - + + ST406 - + + ST407 - + + ST416 - + +



Sal .
Gallinarum wild type strains SG462 + + + SG463 + + + SG464 + + + SG465 + + + SG466 + + + SG467 + + + SG468 + + + SG469 + + + SG470 + + + SG471 + + + SG479 + + + SG480 + + + SG481 + + + SG482 + + + SG483 + + + SG484 + + + SG485 + + + SG486 + + + SG487 + + + SG488 + + +
Salmonella
spp. SEM1 - + + SEM2 - + + Regrowth
pool
culture + + +

<2-3> Test to confirm the ability of phiJR14 and phiJR16 to inhibit the growth of Salmonella Enteritidis

When Salmonella Enteritidis SE348 was suppressed by simultaneous administration of ΦSEP1, phiJR14, and phiJR16, 24-hour suppression was possible in all generations. This is the result of measuring the OD ₆₀₀ value after 24 hours after conducting an experiment on liquid medium with various combinations of ΦSEP1, phiJR14, and phiJR16 (ΦSEP1+phiJR14, ΦSEP1+phiJR16, ΦSEP1+phiJR14+phiJR16). In the case of ΦSEP1+phiJR14, the OD ₆₀₀ value was about 0.3, ΦSEP1+phiJR16 was about 0.2, and ΦSEP1+phiJR14+phiJR16 was less than 0.05, showing complete inhibition even after 24 hours.

Furthermore, it was confirmed whether the ability to inhibit the growth of Salmonella Enteritidis was maintained during subculture of phiJR14 and phiJR16. For subculture, first, inoculate 100 μl of glycerol-stocked regrowth pool culture into TSB and culture overnight at 37°C and 160 rpm. Next, 300 μl of culture medium and 300 μl of phiJR14 and phiJR16 were added to 0.4% agar medium, respectively, poured onto a TSA plate, and cultured at 37°C for 6 hours, followed by plate elution and filter sterilization, and stored at 4°C.

Subculture was performed for more than 7 generations, and when Salmonella Enteritidis SE348 was suppressed by simultaneous injection of ΦSEP1, phiJR14, and phiJR16, 24-hour suppression was possible in all generations (Figure 1).

<2-4> TEM analysis

Salmonella enteritidis HJL348-infecting phage ΦSEP1 was found to be a member of the Siphoviridae family with an isometric head (diameter 59 ± 5 nm long, n = 20) and a non-contractile tail (length, 132 ± 10 nm long, n = 20) ( Figure 2).

<2-5> Analysis of single plaque morphology of ΦSEP1, phiJR14 and phiJR16

In order to determine the single plaque form of isolated single ΦSEP1, phiJR14, and phiJR16, the host strain cultured overnight in TSB, diluted phage, and 0.4% agar medium were poured onto a TSA plate, allowed to solidify, and cultured at 37°C for 6 hours to check the form. (Figure 3).

<2-6> ΦSEP1 complete genome sequencing and functional annotation

The genome of ΦSEP1 is 42,730 bp in length, has 62 ORFs (17 on the forward strand and 45 on the reverse strand), and no tRNA is predicted (Figure 4, Table 1). Among these, 33 ORFs (53.2%) were functionally annotated using the BLASTP algorithm, which were identified as structural proteins (ORF23, ORF24, ORF29, ORF30, ORF31, ORF36, ORF37, ORF38, ORF39, ORF40, ORF41, ORF42, ORF43, ORF44, ORF45, ORF46, ORF48, ORF49), DNA replication, transcription and repair (ORF10, ORF11, ORF12, ORF16, ORF17, ORF18, ORF21, ORF22, ORF35), DNA packaging (ORF51, ORF52, ORF53), host lysis ( ORF1, ORF2, ORF47) and others. Genes for integrase and antibiotic resistance have not been identified, which shows that it can be useful in food, etc.

<2-7> ΦSEP1 complete genome sequencing and functional annotation

TEM analysis revealed that ΦSEP1 was a member of the Siphoviridae family. Therefore, we compared the genome sequence of ΦSEP1 using ICTV-classfied Salmonella phages among members of the Siphoviridae family (Table 2).

When compared with other ICTV-classified Salmonella phages of the Siphoviridae family using Gegenees (v. 3.1.0.), ΦSEP1 showed high similarity to Salmonella virus SS3e and Salmonella virus wksl3, members of the Guernseyvirinae subfamily (Table 5 ).

Next, a BlastN search using the whole genome sequence and homologous sequences of the ΦSEP1 DNA sequences selected for “Distance tree of results” analysis showed that ΦSEP1 is related to members of Jerseyvirus (Fig. 5).

Finally, BlastN analysis comparing the entire genomes of ΦSEP1 and Salmonella viruses (Table 2) shows that ΦSEP1 exhibits 95.65% nucleotide sequence homology with Salmonella phage SE2 (Table 6). In addition, phylogenetic analysis results using major capsid protein, terminase large subunit, and DNA polymerase showed that ΦSEP1 is closely related to Salmonella phage SE2, a member of the Jerseyvirus genus phage (Figure 6).

Considering these results, the phylogenetic identification of ΦSEP1 was concluded to belong to Family Siphoviridae, Subfamily Guernseyvirinae, Genus Jerseyvirus, and Species SE2.

<2-8> Inhibition of growth of Salmonella Enteritidis in food using ΦSEP1, phiJR14 and phiJR16

① Inhibition of Salmonella Enteritidis growth in egg yolk (refrigeration temperature, 4℃)

10 ⁴ CFU/ml of Salmonella Enteritidis and 1x10 ⁶ and 1x10 ⁷ PFU/ml phages were treated and stored at 4°C, and the CFU of Salmonella Enteritidis was monitored for up to 6 days.

As a result, when bacteriophage was not treated, the CFU of Salmonella Enteritidis did not change for 6 days. On the other hand, in the case of 1x10 ⁶ PFU/ml treatment, after 24 hours of incubation, the CFU of Salmonella Enteritidis decreased by about 0.5 logCFU when treated with ΦSEP1 alone, and by 1 logCFU when treated with cocktail (ΦSEP1+phiJR14+phiJR16). A decrease in severity was confirmed (Figure 7A).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, the CFU of Salmonella Enteritidis decreased by 1.5 logCFU after 24 hours of culture, and decreased to 2.5 logCFU after 48 hours of culture, and then some growth was confirmed again. On the other hand, in the case of cocktail treatment, the CFU decreased by 2.5 logCFU after the first 24 hours and maintained the same level of CFU, but began to decrease again from the 5th day and decreased by 3.5 logCFU on the 6th day (Figure 7A).

② Inhibition of Salmonella Enteritidis growth in egg yolk (room temperature, 25℃)

In the absence of bacteriophage at 25°C, the CFU of Salmonella Enteritidis increased to about 2, 4, 5, and 6 logCFU after 1, 2, 3, and 4 days of culture, respectively.

In the case of treatment with ΦSEP1 alone at a concentration of 1x10 ⁶ PFU/ml, inhibition of the growth of Salmonella Enteritidis could not be confirmed for 6 days. On the other hand, in the case of cocktail (Φ SEP1 + phiJR14 + phiJR16) treatment, Salmonella Enteritidis concentration was confirmed to be approximately 0.5 logCFU lower during the culture period (Figure 7B).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, proliferation inhibition was very similar to the cocktail 1x10 ⁶ PFU/ml treatment, whereas in the case of cocktail 1x10 ⁷ PFU/ml treatment, 2-3 logCFU compared to the control during the 6-day culture period. A lower concentration of Salmonella Enteritidis was confirmed (Figure 7B).

These results confirmed that the growth of Salmonella Enteritidis in egg yolk was very different depending on the culture temperature, and that the same cocktail treatment was more effective in inhibiting growth or killing than ΦSEP1 treatment alone under all conditions.

③ Inhibits growth of Salmonella Enteritidis in egg white (refrigeration temperature, 4℃)

Egg white was treated with 10 ⁴ CFU/ml of Salmonella Enteritidis and 1x10 ⁶ and 1x10 ⁷ PFU/ml phages and stored at 4°C, and the CFU of Salmonella Enteritidis was monitored for up to 6 days.

As a result, when bacteriophage was not treated, the CFU of Salmonella Enteritidis did not change for 6 days. On the other hand, in the case of 1x10 ⁶ PFU/ml treatment, after 24 hours of incubation, the CFU of Salmonella Enteritidis decreased by about 0.5 logCFU when treated with ΦSEP1 alone, and by 1 logCFU when treated with cocktail (ΦSEP1+phiJR14+phiJR16). A slight decrease was confirmed, and the decrease in total CFU continued until 48 hours and was then maintained at a constant level (Figure 8A).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, the CFU of Salmonella Enteritidis was reduced by 1.5 logCFU after 24 hours of culture, decreased to nondetectable after 48 hours of culture, and maintained until 6 days. In contrast, in the case of cocktail treatment, it decreased by 2.5 logCFU after the first 24 hours, decreased to a nondetectable level after 48 hours of incubation, and was maintained until 6 days (Figure 8A).

④ Inhibition of Salmonella Enteritidis growth in egg white (room temperature, 25℃)

When bacteriophage was not added at 25°C, the CFU of Salmonella Enteritidis approached 6 logCFU after the first day of culture and showed a slight increasing trend thereafter (Figure 8B).

In the case of treatment with ΦSEP1 alone at a concentration of 1x10 ⁶ PFU/ml, the level was maintained at 4 logCFU/ml until the 2nd day and increased to 5 logCFU/ml from the 3rd day and maintained at the same level thereafter. On the other hand, in the case of the cocktail treatment, a decrease of 0.5 logCFU was confirmed after day 1, and slight growth occurred thereafter, but the level of Salmonella Enteritidis was consistently lower than that of the single treatment. The count was maintained (Figure 8B).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, a decrease in total CFU of more than 2 logCFU was confirmed after 24 hours of culture, but it steadily increased thereafter, reaching about 3.3 logCFU/ml on the 6th day. On the other hand, in the case of cocktail treatment with the same concentration, nondetectable results were confirmed in 24-hour culture, but afterward, the cells continued to proliferate, reaching a concentration of 2.5 logCFU/ml on the 6th day (Figure 8B).

These results show that Salmonella Enteritidis grows more actively in egg white than in egg yolk, and that at the same concentration, cocktail treatment can inhibit the growth of Salmonella Enteritidis more effectively than single phage treatment. Furthermore, phiJR14 and This result confirms the usefulness of phiJR16.

⑤ Inhibits growth of Salmonella Enteritidis in milk (refrigeration temperature, 4℃)

Sterilized milk was treated with 10 ⁴ CFU/ml of Salmonella Enteritidis and 1x10 ⁶ and 1x10 ⁷ PFU/ml phages and stored at 4°C, and the CFU of Salmonella Enteritidis was monitored for up to 6 days.

As a result, when bacteriophage was not treated, the CFU of Salmonella Enteritidis did not change for 6 days. On the other hand, in the case of 1x10 ⁶ PFU/ml treatment, after 24 hours of incubation, the CFU of Salmonella decreased by about 2.3 logCFU when treated with ΦSEP1 alone, and by about 3 logCFU when treated with cocktail (ΦSEP1+phiJR14+phiJR16). has been confirmed. Afterwards, the total CFU continued to decrease, and in the case of ΦSEP1 treatment alone, it reached a nondetectable level from the 4th day, and in the case of the cocktail treatment, it reached a nondetectable level from the 2nd day and was maintained until the 6th day (Figure 9A).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, the CFU of Salmonella Enteritidis decreased by 3.5 logCFU after 24 hours of culture, decreased to nondetectable after 48 hours of culture, and remained until 6 days. In contrast, in the case of cocktail treatment, it was lowered to a nondetectable level after the first 24 hours and maintained until 6 days (Figure 9A).

⑥ Inhibits growth of Salmonella Enteritidis in milk (room temperature, 25℃)

When bacteriophage was not added at 25°C, the CFU of Salmonella Enteritidis approached 8 logCFU/ml after the first day of culture, and then increased slightly to over 9.5 logCFU/ml on the fourth day (Figure 9B).

In the case of treatment with ΦSEP1 alone at a concentration of 1x10 ⁶ PFU/ml, 4 logCFU/ml was maintained until day 1, then increased to 5-6 and 7-8 logCFU/ml on day 2 and 3, respectively, and maintained at the same level thereafter. . On the other hand, in the case of cocktail treatment, the overall growth pattern was similar to that of ΦSEP1 treatment alone, but Salmonella Enteritidis growth was confirmed to be up to 0.5 logCFU lower (Figure 9B).

In the case of ΦSEP1 1x10 ⁷ PFU/ml treatment, a decrease in total CFU of about 0.7-0.8 logCFU was confirmed after 24 hours of incubation, and thereafter, the level was maintained at the same level, with regrowth occurring from the 5th day and reaching 7 logCFU/ml on the 6th day. reached. On the other hand, cocktail treatment at the same concentration showed a similar overall pattern to ΦSEP1 treatment alone, but maintained a lower CFU/ml of more than 0.5-1 logCFU (Figure 9B).

Therefore, the results of this experiment confirmed the superiority of the cocktail mixing ΦSEP1, phiJR14, and phiJR16 because the effect of killing Salmonella Enteritidis was excellent even at low bacteriophage concentrations.

Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)

Accession number: KCTC18927P

Trust date: 20211013

Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)

Accession number: KCTC18928P

Trust date: 20211026

Name of depository organization: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)

Accession number: KCTC18931P

Trust date: 20211026

<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Composition for inhibiting proliferation of Salmonella enteritidis comprising bacteriophage <130> DHP22-154 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 42730 <212> DNA <213 > Artificial Sequence <220> <223> SEP1 whole genome sequence <400> 1 aatcccggta agggtcagtt accgggataa aggagctaga gggatgaata agcaggagca 60 ggtttagtat cttaggaatt ctcctatgtg tcaacctact ttgcttcgcg gccgatggct 120 tccgcctgtt g ccacggcat accgtcgaat agcgcaacac gacccgcggc acgccgacgc 180 aatccaagga gtgatttgcc gttctgataa tggaactgag atagcttatt ccgcagtgtg 240 gcaacgtcgc ccttacgcag ggcctgccct gttccggtag aagccgcaat cacaccggcg 300 ccagcgttat acaccaggtc gcacatcgca tcgaactgtg actgattgag cgacggatgc 360 gctacagcgt ctaccgcagc tacggcctta gccatatcct tgtgcagcag cagaagaccc 420 tgtccctcgg taatcttctg gccttctttc acatctgcgc cgtagtggcc atagccgata 480 gtaaggtact tctcactctt cgttgccttg tacgcggttc cgcggaaacc ttcgaacgcg 540 gcggtgaatt tgatgccgtt gttactgatg tttcggtttg acatacatcc tccggtattt 600 gtagtagcga gcgcctttca cgcaa atgtg acgcaccgat ttaagcgcga atatagcgac 660 gagagtcgta attatgaacg gtggaatggc accggtaaag agatgggcga gacctccggc 720 ggcggtaacc accgacgcca tgtacagcag gcggccgaaa agaccgtctt caacccagtc 780 ggcgtagata ttaattaacg aggtggcgat gatggcaacc aggcaaatga tgccaattga 840 tgagttgagc atgttatctc ccccacggta acttaaagtt gctgagggat gagt cagcag 900 cttcgaataa cttaagccag aacataccta ccccgaaagg aaccaggtac tgaccctcag 960 actgtgataa ttcatagtaa cttataatga tgggggatgc gtacactgca caacaggcgg 1020 aagcggtgag atggcacaat cgcaaccatg gcgaaatagc ctcacgcttc ttaatct gag 1080 atacggcacc cccggcgagg cccgccacac taagccaaag gtattatatcg tccacaataa 1140 aaaaatctcc gaaagttggt ttatccttcg gagattagca tttatacgac aattttagta 1200 acagataatt cggatttatc ctagtcgatg tgttatcacg cttccatcgg taccagggtc 1260 aaacctcttc gcaacctcta ccgcttccgt tgccgtcgcg cccatgaaca t ggctgtaag 1320 cgcatacgga gccccggagc ctacggcagc agcgttacct gcaattggta taaccgagct 1380 taacggtgcg tcccacgccc tacagaattc taccgcgaaa atacggttat cctctttaac 1440 aaacaccagc gccgaaaagt cgaagtcgta gtgcttaggc gtcagta tct catcatcggc 1500 aaccaacaac atacccaccc cggcgtcccc ggatacccct ataaacaaagt ggtcgttctc 1560 gtaaatcttg gtatccgtgt ggtatttgta attgccggtg acacaggtat cacaggccat 1620 ggttttgccg tcgaaagcta tcgtagtcat ataaattacc tgaacgtata aagtctgatg 1680 gatggcttgt gcggggatatc tgagtgcccg cgtttacgcc acagtttgta cagt ttaacc 1740 gccatatgcg cccgcgcctg gctactgaac tcgccaaggt ttaccacttt cttcccgtcg 1800 taagagtgcg cacgccacag acctttgcct acacaatact taggctggtg caccccaggg 1860 atgcgcggat acacgtccgg catctcatta actggctttg cg cgcggttt aggtttacgc 1920 ttaacctttt ctttaacgcg agtaaccttc tttgttaccg ccgatgaagg aggtgccagg 1980 ttgatgccgg cgtgtttcag tatatcggtt gcgagactca taatcacttc tcctgtttac 2040 cgagtcgctt aggcgaacag atagcgcgta cttctgaatc atttggcttg tcgccctgaa 2100 acaggaagtg cgcattctcg gcggcgcgtg ccgctgaccg gcacgcctct aatgtgtaaa 2160 acgtttccga tgccgcgagt tgcatctggc ctgcggacag taccgata aataagatac 2220 tggtcatttc tttaacccgc atattctgca catcatcgac gcacttgtat cgtaaaagta 2280 ccaatcgtgc tcgcacccct gagcatcttc tttacggaaaa ccagattgct ccggcccgtg 2340 ttcacctttg cgcgcaagtt tgtacgcctc cacgtcgccc gtagctttta ccggcgccat 2400 acgcaactcc ttaacttgcc ctgtcaggtg tgatatgtgc tcagatttct taatgttgtc 2460 ttcctgtaac tcagccacca gcaccgacac atcacgtaac tgattcttaa gtgactgcac 2520 ctcctgcatc aggtcttcac actggttgct gtttaccccgc tgccgtagct ggtcaatcag 2580 gtactctaat acc tcgtctt tgttatattcac ccccacactc ctctatctcg tttcgataaa 2640 gtaatagtac cctattatgt tttgttacgt caatactatt ttacgattaa tttatcccga 2700 cgcaccgtaa gataacgggg tttcttcgca tgagcacgcc accactcaac acgggcgtag 2760 atgccctgaa gcgatgttac tttgccgtgt ctgatttctc cgttccgccc ggtccaggtt 2820 acaaggtcat ttggtttcat tcgttagtct ctgttatcga ttcgacgaag tccctaagtt 2880 tcaggtaaca gtatgtccac tccttcgacg attcttcatg cgcgtcgtgg tcacccccgg 2940 cggtatagga catagccgcc gaagcatcat caacggcgtg gacaagtacc atcagatagg 3000 ttgcctgttc tttagtaata attgt tgcgt ctcctgcaac aattgcgttg attgttgcat 3060 ttcgtgaaat agaaaaatat taacaaaaat ggtacgctgc tactcagcag cgtaagtcac 3120 tcagtaaaat cctaaactac tgttacaagc ttcctgcggt tccgataaca gcgcccaggt 3180 gc ggccccac caccacgata ccccaggaac tatccaccca tcatcatcgg cgtccggatg 3240 cggccccggg gaccactaaa accctagcca taaacggcaa cctcctttaa gacccgagaa 3300 tcaccggcgc ggactcttta gaatcttttg ccgtggggtt atttacaggg aggtccctgc 3360 cccattagag tgcctttatt ctcgtccctc gtcaggtaag cgcttgccgc aaggcaagtg 3420 cgttaatccc tgagtagtag actaagaatc tttc ggcatt tcagttacgt tgccgttgtc 3480 cagtgttgtg cctttattct tattacttgg taactaaagg gcaggcctta agcctgccca 3540 ctaatcgtta catttttacg gttgcgaatc tacggcacat caaggaattc agatacttaa 3600 gagatatgct gccgatgaga atatagctc g cccagggcag ttcagatact aaggaaggga 3660 agttactgag ttttaagttt gcgggtttca gtaagcctta acgcgtatcc gcttggggcg 3720 gacccgctaa gctctcacaa ggtgaaacag aaacttaagt aacttgaagt tacaagagac 3780 aacttacacg aaaaaaatga aaatgtcaat aggtgtgttt atagttgatt tcccattatg 3840 ccgtgggtat acttgcttta ctaactgaga tggaggattt gagaatgaca caga atgaag 3900 tagctgagct tattggggtt acccgccgca cgctgaataa ctggttaaga gacggcaagt 3960 tcccggattg ttgtgtccgg attatgggtc gccggatgcc gggtacgttt gaccgggaga 4020 aagtggaagc gtggattagg gagaatgtaa agtgaccgat ttccaga aaa gggtattcag 4080 aactgtagtc gcgcttacgc gtaaaaaaagg gtcgtgtagt gttcttgact tgcgccgcgc 4140 ttattttcag tactatagtt cctctctcat cgagaggtcg ctggaagttt tggttagaga 4200 cggagcagtt aaaagtaagg ccggaaaata ttcggcagta gctgaagtta aaggctccca 4260 ggcgacccca gaagaccttg aataaaagaa aagccccaac gcgcgaacgt cagggcc tta 4320 aacactacaa aggaaaaagca catgtcaaca ggcaatttag catactcatg gtcgcgtcgc 4380 aatcgccgtg aggaacgcgc tgacattaac gtttacacca cgacctccgg cacctgggat 4440 gagtttgtcg aacttatgca accgcttaaa cgttcgcgcc ggaaccc caa gacagacccc 4500 ggctacatta ccgccgcttg taccgccacg gtaagctcta ccggtaaaga agccgccgaa 4560 ggtatgttct atcgctgcaa tgcgtctgtt acgtcttcat ccctggccta tgccgacgtg 4620 gacagcgcga cgccggaaga gttcgcaacc gactgtgaga tggtgcgcga gtcacgttac 4680 gcaatgatgc tctacaccac ggcatcccac accgaagaag cgccgcgcta cc gcgtcgtt 4740 atgcctgtac gcactccggt aaccggtggc gacatcatcc gcatccggta cggcctgttg 4800 gcacacttcc ttaaaggtcg tgacgtggat agcgccgggt tcaccctgtc gcagcctatg 4860 taccgccctc cggtaggaag ccaggtcatc gtg tctgaaa gtagccgcat gattacggcg 4920 agcaagctta tggaggaagt tcctgaaatt aacgttaccg gtgcttctga ttataaagtt 4980 ccggagggtg aacaatccga attaaccgac ctgtttgaag agttcgcttt tgaattcggc 5040 ggacgtatga ccgaccgcgg cctgcaaatg cccgccacgc cggagcacgc cgcccaatac 5100 accaccggcg aacctaagca ggacgacttc ctgttctgtt ggccgcgcga cggcttcga g 5160 cgacccaacg ttaccctgta ccacgatacc gacctggtag ctacgggcgg gatgaagcct 5220 ggcggacggg atatgtgggc ttacgcttgt gcggccaccg gcttaccttt tgaccgtgtg 5280 gaggtggcgc ttgggtgggc ggagggggtc acttgcg acg aagaagacct ggacgacgaa 5340 gaaccaccag caccacaagc ggacttcatc gtcgagggct atatgccgtc taattgcatt 5400 tgggatatcg tcggtgaatc cgggacgtat aaatcgttct acacactcgg catgatgtat 5460 atcagcgccg ccgggtatcg gttcgcgggg gcagacaccc gtaaagccca tcatttctat 5520 attgatggtg agggtgggga gttcacgcac acccgtatcg ctgcgttggc cgctaaatac 5580 ggcgatgaag gtatgcgata tatgcatgtg cttgacgctg gtgaattcgc ggatacgaag 5640 aaactggtgc gtaaaatgcg ccagatagcg ggcagtgaac ctgtcgggat ggtcgctttc 5700 gacactctta accagacctt cgggaactgg atagacaagt tcaatgagaa ctcagccgga 5760 caggacggta tgggccgtgt cgtggctatg ctcaaagaag tgcgcgacgg tactaaaaggc 5820 gctgtaggcg ttgtccacca caccccgaaa ggtggaagta aggcgcgtgg aagtggtgcg 5880 ctgtatgccg gtgtcgatgt ggaactaacg cttgtccgcg ctaccgagaa acaaataaac 5940 gttgcgcaca ccaagaacaa aaacggtatg cagcagaaaa caataggcat ggtgcttgaa 6000 ccagtacagt tt cgcgaggc cccgccgccg aaagagttcc aggcggtaga attcgctggg 6060 ggtgagggat acggggagat agtcaacctc gaccttccgg agccgcataa agctctcgta 6120 cttatgccgt ggggcttcca gccgttcgaa acggacgagg aaaaagagcg aaacgaagga 6180 ttggatggta aaggtaaaga ttccgtaaag gatacggtta aacggtctaa agacgcttcg 6240 gcccgtgagt cggttatgtc cgctctcgag gatttacagc aagccgatga tacgggacga 6300 gggttcacac aacggcaaat agtcgcgaga gctggagacc acagtatcac aaacctggtg 6360 cttgagaaga tgctacgcga gggtgaattg attcttggtt gcgacgaaaa cggtgaagtt 6420 gtaacgaata cttacaggtt gccgatgggt atagac gacc gtaagcgacc gaagaaccgg 6480 tatgaaccta acgacaacat aaagacaaca gaaggggatt tagagtaaaa agaaaggggc 6540 catcaggccc ctgtatcacc aaagcttaac catcgagggt caacaccgag agcatcggca 6600 atgtggaaga ttttaaaaca gtctacctta cacacccgg c cggttgccag gtgggtaatc 6660 aggctttggg atacgccagc tttgaaagca agctcagttt gacccatgcc gatttcttta 6720 cggcggcgct ctatgcgagc gcctagctca gattgctgca tatgtttact ccttagttag 6780 atatgcagta atagtacagt aataaaatac acgacgcaat ttacgaaaaa gttcttgcat 6840 aaataataga gtacgattat agtctttatc accaactaaa caggagaaga aataatgtta 6900 gaccaattct taaaattact tgaacgtttc gtagtcgccc atgaactgat tgctgcgaat 6960 agtgtgaaac agacgtctaa caaatccgta agcgaaatga ctgttgacgt ctccgttgcc 7020 ggtgtcgaga cagttaaaaa agaactggct aaagcactgg aagaagtc ga gaaagcaaaa 7080 gaaattccgg tcgaaggtga ggacatcgtc gacactaaac cggcagaaga ggagaaacca 7140 aaacgtaagc cgcgtaaggc aaaagtagag gaaccagtac aagaagagaa ggaagaagta 7200 gattaccagt ctcttcgtga ccagattaag gctatcgacg acgcaattaa tgaaggtccg 7260 agcgatgccg cgtgtgacga ttcagatgaa ctgctggaag agttcaccgg ta agaaaatg 7320 aagaattgcct cgattaaaga cgaagacctg gccgagtacc tggaacgcct gacggcaatc 7380 aagaacaagt atttcgaaga agaataaata taccgcggcc ttcgggccgc tttaactgag 7440 ggtcggaatt atggctattt tagctaagaa aagtggtctt atgattgcag gg cgagttat 7500 agaagaaaca gataactttg tatgcttcca cgctacggat aacaaacgtc ctacgtatgt 7560 acaaaagtcg gataaaacac aaaaagtttt cgatggtgat ttttctatgg atgacgtttt 7620 cgcgtggatg gattctgtga gggtaaagaa atgatttacc aactctaccg cgccgtagac 7680 aggcggggata acacagaagc actatggctg ttgcgtgcat cgtccggtgc gcacctgatg 7740 gaagagatgg cgtacttagg caaagtgcca cggcctaaag atataggccg tcacgtgtcg 7800 cagattaagc gcacgacttt cgcaaaacct gacttttatg tcttcgagtc gatgtatgga 7860 tgggctatgc attgcgacca taagacgcga catttaattg accagtggga gaacagggta 7920 tgattttaaa ggaacgcggt ggcaataacg atgtgcacgc cctgctgtcg ccgtctggcg 7980 ctaaaaagtg gcttagctgt gccgcatcac tggcatgcga aaaagacatc cccaacacgt 8040 ccggtaaatc cgccattaca ggaaccgcat gtcacactat agccgaggtc catctcaacg 8100 cctatatccg cggcactgca ctgccactag agcgagaagt cggtgcttac gtgctggatg 8160 agggtaaagg cc agattaag gcgctaatca gcccgatgaa aggcgcggta ctgattacgg 8220 cggacatgat tgagcaggtg cgcaagtaca ctgactactg caaaccaatt atcgacgtag 8280 cgacttacgc caagctcgag atgcgggtaa atcttactga ggtattgcat ccgggttacg 8340 aaggcg ttga gacattcgga acggctgacc ttgttgccgt tcaggaactg gctaactccg 8400 acgagcacat gctcatttatc ggcgacctta aaaccggacg gcatcgtgtt gaagcgaaag 8460 aaaacaagca gcttatgctt tacgctctcg gtgtttatcg ccgactcaag agacgttata 8520 acatcaccgt ggttcgtctg gtcatcttcc agccgtatgc tggcggggcg tccgagtggg 8580 acatct cggt tgaaggtctg gaactgttcg ctaagttcgc acagaaacgt gcgctgttag 8640 cgcttgatgc ctatttccgc ggtaagaaga acctgaaatc gtcggacttc aaaccgtcgg 8700 tcgatggttg tcagtggtgt cgtttctcag aacagtgcgc cgcgcgtaca aaaaccgtta 8760 actctgtact ggcggaagaa ctggaggacg actttgcgct ggaacttaca ccggagcaac 8820 tcgtagctga gtatgagaag ttgccactgc tgcgccagca catcgacaag gtcgagaaag 8880 cgatggctgc cgcgttgcat tccggtaaga aagtgccggg gtacaagctg gttgaaggcc 8940 gcccgggtaa tcgtgcgtgg aaagataccg atgcgctgct ggaaacgcta tcacattttg 9000 agttgggcgc tgaa atgctt cataaagaag tactaatgac cccgaccgag gccgagaagc 9060 agcacaaagg ttctgagcta tgggcggcac tggagaaaca cgtaacccgt aagccaggag 9120 cgccgtgtgt gacaacggtc gaagacaaac ggccagaatg gaaaaaatgtt actgaagacg 9180 atttaga ggc ttgacatctt aatcgtactc tattatatta ctaatcactg accgggcagc 9240 tccccggata aactctaaaa cgcgagaaaa ctaaaatggg aattaaactt aatcttcgta 9300 aagtacaaac cgcatggctg aacgtattcg aacgcgctaa agaccgtgaa aataatgacg 9360 gttccgttac taaaggtacg tataacggta cttttatcct gactccggaa cacccacaaa 9420 ttgaagagct tcgcgatacg gtattcgcag tagtgtc gga agcattggga gaggccgccg 9480 ctgagaaatg gatgaagcag aactacggcg aaggtaagca tatggataaa tgcgccgtgc 9540 gcgacatcgc cgaacgtgat aacccgtttg aagacttccc ggaaggtttt tacttccagg 9600 ccaagaacaa acaacagcca tta atcctga cttcggttaa gggcgaaaag caggtagaac 9660 cagacttcaa cattgatggt gagcagattg aaggtgagca ggtatatagc ggttgtgtgg 9720 ctaatatctc aatcgaaatt tggttttccg agcaatataa agttttaggc gcaaaactaa 9780 acggcatcaa atttgcaggt gaaggtaaag cgtttggcgg ttcttcggtt tctgccagcg 9840 ttgatgacct ggaagatgat gaagaagatg aaaccccgcg ccgcgaacgt cgccgtaacc 9900 gttaatatat tctcatttca tttaaggcgg cttcggtcgc cttttctata agggtcaaat 9960 gaatgaactt gctttatctt gatactgaaa cattctccga agctgatttg aaaaaagtcg 10020 gctcctatgc ctacgctgaa catc ccacta ccgaaattgt tatttgcacc tacgctttcg 10080 atgaaggccc cgtgcaggta tgggatgcca ccgacgggag tgatatgccg cgtgatttgc 10140 gccgggcgat gctaaagctg caaaaaccag acagcaacct caaactggta ggccaaaact 10200 tccttatgtt cgaccgacca gttattaagc attgctgggg attcgaactc ctgatagaaa 10260 acatcatcga cactatgata gttgcattcc gccatgccct tcctgggtca ctagccgcgt 10 320 tgtgcgaggt tttaaacatt gacgccagca tggctaagga taaacgtggt aaggcgctta 10380 tacagcgatt tagtaagcct acgcctaaga actataagat tcgacgctac actgccgcta 10440 cccacccaaa agagtgggcg gaatttattg catacgcaaa aagcgacatt acatccat gc 10500 gtgaagtgta taagaaaatg cctaagtggg ggaattctga gttcgaagac agcgtgctgc 10560 gattagacca gattattaat gaccgtggat ttaaggtaga cgttgcgctg gcggaagccg 10620 cgattgaggc ggtcacgcgc cataaggaag agttgcaaga agaagcacaa cgcaaatacg 10680 gcggctcact aacaggcaaa gacttcctgc ctattttaca ggagctagcg ccagcgca cc 10740 gcatacacaa cgcacagaag tcaacactaa acgatttgct ggcggatgag gatttaccgg 10800 acgacgcccg cactattatc gagatgcgtc tcggggctgc ttctaccgca tcgacgaaat 10860 acgcgccgtt gctgttaggc cgttcttcag atgaccgcc g ccgtggttgc ctgcaatacg 10920 gaggagcgaa gcgaacattg cggtgggcgg ggaaaggttt tcagccgcaa aacctggcgc 10980 gcgggtatta tcacgacgat gaactggata ggggtattgc cgcgttactt aaaggccgtg 11040 cacaccgccg ttttgatgtg gccaagctaa cagcgtctac cgtccgaagc tgcattatcc 11100 cggaagccgg gcgtaagttt gttgttgcgg attactctaa cgtcgaaggc cgtgggctt g 11160 catggctggc gggcgaagaa accgcgcttg atacgttccg cgccgggctg gatatttact 11220 gcgtaaccgc aggtaagatg tttggcatgg accccgacga tattaagaaa gaacgtaaag 11280 acttacgcca gataggtaag gcttgccttc accgtcatac ccaagt tttg accgatggtg 11340 gatttaagga tattatggcg gttacatcag catataaagt atggaatggc gaaaaaatggg 11400 tgaatacaaa aggcgcgcac cttatggggt ggaaaccggt aataaatgtg gacggggttc 11460 taatgaccga agaccacaaa atcttgacgc attcctggaa ggcggcaaag caactcgttt 11520 caaacaaata tatgatgggc ctcgccctgg ggagaggtgg ggacgcctgg ttatcctacg 11580 tgagctacca aaac gacaag gccaaagaca actactcgtc caatgtgatt gcggagagat 11640 gccggggcgg gtctattatg acaatgtccg agcgggtaaa accactcaat gcaacatctg 11700 cgcccttgaa gcaacaaaaaa aatatcgtaa aaaatatttc tgctacgaag acgcaatgcc 11760 ggat gatgcg cacagaacgc gattgctcaa ccggttgtcc gcggcaatcg tgcggacaac 11820 aagccccgga aacaagagtt acaaaaacta tggggctagg ggtatcacgg tatttgacca 11880 gtggagggcc gataaaaggt cgtttcttag atatgttcaa actttggaag gctgggatga 11940 cccaaacctc gaaatggacc gaatcgacac agacggtaac tacgagcctg gcaacattcg 12000 gtttgtcagc cgttct gaaa actgccgtaa cagacgtcga atacctgagc ttcaacgaaa 12060 gtatgatgca gccgttgccc gcattgctga gcttgaacgg gaaattaacc tactgcgagc 12120 cagtttacga cctgattgat gtggaagacg gcaaccgttt tcttatagcc tcagactcgg 12180 gctttcttgt ggcgcataac tgcgaactgg gcctcggcta cgagggaggt gtcggagcgt 12240 tcgttacgtt tgctaaaaac ctgggtcttg accttattga gatggcaaaa acaatggacg 12300 ggactttccc cgaccacatc tgggctgcta ctgcacgtgg gtatgagtgg gcgcgtatcc 12360 aggaagccaa gagaccaccg catcccggtg aaaaggatga caggccgtcg tatatacttg 12420 acaagaaagt atggcgcaca tgtgacgcga t caagcgtat gtggcgtgag tcacaccctg 12480 aaacagtagc gttctggcgc gaccttaaag acggaatttt agccgctgtt cgtaatcctg 12540 gtcgtgaatt ttgggctggg gcacacttgc gccggaatgg tgaaagggct atccgcatat 12600 ggcgta ccgt agaatttgat tcgtcgggca ggaaggttcc tggctggtgg ttgtgtatgg 12660 agttgccgtc aggccgtatc ctatcgtatc cgggaatcgg cgtcagcgtg acaaaggaaa 12720 ccgacgaaga cgggcgggta aacaccaatg taagaattaa gtaccagggc gagaaccagt 12780 taacacggca gtggactacc ctgtacacac acggcggaaa ggcttgtgag aacatcgttc 12840 aggcgttttg tcgtgactta ttggcctacg caatgctttaa t gttgaggct ggcgggtacc 12900 ctatagttct ttctgttcac gatgaactgg tatgcgagac tccggataca tcagattaca 12960 cggtagctga actggaaaaaa ctaatgtgtg cattgccaga atgggctgat ggttttcctc 13020 ttgtagcgga aggtgcggag ttaaaac ggt atgctaagta aactgattat ttattatttc 13080 agggaagaag actgtcgaat ctgtccgcgc tgtggggtta agcacacgaa acgtgagggg 13140 tgcatagatg agtacgcctg agggccgcgt ccagaaatat gcaaaagagc gattcgaggc 13200 cctggggggc ctcgtacgca aattgtcgta cgaaaataga gtgggcgctc ctgacctgct 13260 ggtgattctc cccggtggca tcatctggtt cgtcgaggtg aaga aagacg aaaacacgaa 13320 gccagacccg caccagttgc gcgagcacga gaggatgcgt aaacgcggcg caaatgtttt 13380 tgttgttggg tcgaagaaac aggttgataa attaatagaa cactattata tttagttaac 13440 accaaacaga aataaggaat tgagaaatga aatatgaata tgaccgcaaa ccagcacgtg 13500 acatcgtacc aggcgatatg attttcaacg ttaaaacccg ccagccggtt gccgttgata 13560 ctgtgttcgt cgagtcaaac ggtaaactgg ttatcgaaga tgtaactggc aatgttacag 13620 cgttcgggcg gaaggaactg gtactggtgg cgaaatgagt aaattcacta gaaggcctta 13680 tcaaaagctc atgacctcgt tcatgctgcg acacccgcgc tgtaatatct gggcaagtat 13740 gggcagcggc aagtgcctga aacgcg ggac tgaagttatt atgtttgacg gtacgaccaa 13800 gaaagtagaa gacgtcatcg tcggtgacgt gcttatggga ccagattcaa cgcctagaaa 13860 tgtactgtct ctcggtagag gccgggagat gatgtacgag gttaaaccgc gaaaaggcga 13920 gtcgtatacg gtaaacgaaa gccatatatt atcactgcga actactacag gtatcgcaaa 13980 aggttcatgg ccagataaca ccgttttcga cataagcgtc cgcgactggc ttaagttgcc 14040 gaaatatgta accgggccta atgggtatct taagggttgg cgggtacctg tcgattttcc 14100 aaggaaagag caggacgagg ctttattgcc cccgtacctt atggggctat ggctgggcga 14160 cggtacttcg tcgtctgggg ccataacatc aggagagaac gaaaagga aa taagggcgta 14220 tcttgagtcg tacgcagcac gtaatgggat gcaaatacga aaagaaggac ttacatggag 14280 tatatctcac ggcaacacag gtcataagaa gcacgggttt acgcacgcgc tgaaatcggc 14340 gggtgtcctg aacaataagc atataccgca taattacaaa tgcggcgacc ggagacagcg 14400 tttagagttg ctggcagggc ttctggatag tgatggttac tgcgatttgt ctaaagcagg 144 60 ttttgactgg atttcggtaa gcgagcgact cgctgatgat ttttgctatc tatgccgttc 14520 tcttggtttc gccgcttaca agaagaaaac tcgtaagcgt tgcgcaaaca ctgatgtttg 14580 gggggattac ttccgcgtat cggtatcagg cgatt tttct gaggtgcctt tcgtgcgtgg 14640 tcgccaccag aatttaccga agcgtaatat taataaaaac gtacttaacg tcgggataga 14700 atctataacc ccggtcggtg tagatgatta ttacggcttc acatcgacg gtgaccaccg 14760 gtttttatta ggtgacttca ccgtaacgca caacacaagc tcgacaatgt gggtgcttag 14820 ccgcctgttc cgtaatgggc aacttaatga cgacgaccga gtgttaattc tggccccttt 14880 acgtg ttgcg tcaggtacgt ggccagcaga acaagagaaa tggaacttcc cgtgtctgag 14940 tgtagtagat gcaactggtt ctgagaagcg acgcatcgcg gcgctggagt cagacgctaa 15000 cgtggtttgc acaaattacg aagttataga atggcttatt gactactacg gcaaagac ga 15060 ctggcctttt accgttatcg ttgccgatga gagcacgaaa ctgaaatctt tccgcagccg 15120 ttcaggcggt agcaagcggg caaaggcgct tagtaaggtg gcgttcggta aagttaagcg 15180 tttcattaac ctgaccggta caccatcacc aaacggcctc aaagacttgt ggggtcagaa 15240 ctggttcatc gacgcgggtg aacgccttgg gtcttcatac acggccttta ccgatagatg 15300 gtttaactcg gtacagaaag acaaatctgc gatggcgcgg gggtaccatg ctcgcccagg 15360 cgcggataac gagattcacc agaagatgaa ggatatcagc cttaccattg atgccgccga 15420 gtggttcggt tgtgaagcac cggttattgt accggttgag attgacctgc cgaagaaagc 15480 gc gtcaagcc tacatcgata tggaggagga gttattcgcg gaactggaga gcggagaagt 15540 tgaagcggct aacgccgccg ctaaaacggc taagtgcttg cagattgctt ccggtgccgt 15600 gtatgtgtcg gggccggatg gtgaagcaac gaaagactgg gagaaagtgc acgacgcgaa 15660 actcgatgcg ttagagtcca ttgtcgagga gttgcagggt gcgccgctgc tggtggccta 15720 tcagttcaag cacgaactt g agcgcattct taggcgattc ccccaggcgc aggcgtttgc 15780 gaaaggtgct aagggtaata agcagatgga atcttggaac cgcggggaaa tcgagatttt 15840 gtgcgtgcac cctgcatcgg cgggccatgg tttgaattta caggacggcg ggcatcatct 159 00 ggcgtttat tcgcaaggct ggaacctgga gcactatttg caggttgtcg agcgtatagg 15960 tcctgtacgc cagaaacagg ctggccacga gcgtccagtg ttcctgtatc acatagtcgc 16020 taaagacacg ctggatgagg tcgttgccgc gcgtacgggc gagaaaaaat ctgtccagga 16080 agagttgctt aattatatga agagacgagg taagagatga ataatgagtt tgatatcgac 16140 gcttgcaacg aattgataaa agacgccatt aattcccgcg agcaacttct agctatgcaa 16200 ttaaagcgag aaataaaacg tatcagggaa ctcgaagaag aggttttacg gctacggcag 16260 caaagagacg ctgctaacgc gcaacttgac tggctactag aacaacagga agttgacaaa 16320 gagaggggcg ctaaatgaaa tactcaatt g gtgcgaaatg ctttttaata ggctacggat 16380 tggctgcggt cgctttactt atcgctgctg tggttctgtt atcataaatt aaggcccctt 16440 acggggcctt tactatttat gccaaagtta atcttgtata gcttccatcg gcccgcttaa 16500 ctaacgcctt aaggctatcg ccttccagat acagtgatat agacccgtta tccctgacag 16560 cgttgtctgg tagggcggac atcgctaccg gtatctcaac agcacgataa t cgcccctat 16620 ttatttttaa tgacacggca tcgggcaagc tgcccgaaat agctgtgact tctgtccacg 16680 ctgaaccaga acctgggccg ccgttaatgt gggagtacac ggacccacta tcaagggttt 16740 ttgacagttt atgaatacga aaccgaagcg ccgcagaacc attattgaaa ctgtttatgc 16800 gagagttacc aagaccttcc tccatcaaat tggctacgtt aatccttgag gggtccacga 16860 agcccgttat tccgcttaca gtggagttag gtgcgtctat cgtcatgccc tgttcggtag 16920 ttgaacggat gccgaccaaa cgaaggccgt ttacacgaca ggcccctgag atatatatct 16980 ggttcgcggg gaagttttta gtattggtgt cgattatgc gatattagtg aatactgt tt 17040 cgtgggcaag aatataagcc ccagaaccag cgcaatcctc gacggttatg ttagagacat 17100 acagaccttg cccgtccata ccgaaaccta cccccagcga acctctaaca aatagattgt 17160 ctatcaaatg gtttaatggg agcatatgta ccgggtactg agaaatgggg aaatcccctg 17220 ggcggtcatc ttcggggttc atgtcagtat ctgcgcctaa atcgaagccg tcccatacag 17280 ggtatagcac tgctgaatcc cggaattgca ggttgtagtt acgagaggtc gtagaaccta 17340 ccgtaccttg ccacgtctta acaccacttt cccccgcacg atacgaagta aacccgatga 17400 ccccgccatc gcgcgcgaaa ccgccattgt ttcgtaagaa ttgagcgcta cttaccgaac 17 460 cgtaacttgt gcgcccgcca ataacatagt tacccttgcc ccaatcgccg ctcaggtttt 17520 caaaggtaat tacgccatct tttccgccgc tcgggttgtc agcgtctacc atcttacaga 17580 aatggcatcc gcggaacagg aaacacgcca taagaccact cgccc ggtga acctcgacgc 17640 ctgtacattc ccgaatttcc agggtagaag atatgctttg ccctttagct tccggaggga 17700 gaagggattc tataccagga aacttggcgt aatcgtttac cgtcggctgg tatccatctg 17760 ttttagactg tttaagtgtg gccacgattg ccgcggggtc ggttatccac tgattatcgt 17820 cggtccacgg tttaatcacc cacggcgttg tagcactctc cataaagggg gctattacaa 17880 tggaaccttt accta attgc gtaaatacca ggtttccgtc gccgataaac ttcgctttac 17940 agtcgatggt cagggtcttc ccgccgaagt taacggtctc attgttgctg aagttgtaat 18000 cgcggtcgat aagaacacta tcaacggcgg cgtctgccaa ttgttgtaac gtagtaa agt 18060 cagacagctt aacggagtac ttaaatttct tatccgcttc cgcacggaag gtgatgtccc 18120 caaaagctac ccaagcagaa tcagaaatgc caccggttgt tagaggggta gacgccgcgg 18180 ggataacttt cggcagggag ccacgccatg cgtaatagtt accgtccccg ccatcttctt 18240 tcgaccaaag aaccgcttta ttggcgtcgt taattcccag ggttccgccg gttgtgaagt 18300 taaaagacgc cggagagaaa ccggcgtcac gcaagaccgc gggcagcgtc ttctgcgtct 18360 gcccggttac ctggttagta gcgtagtcga tatctgcgcc acccgctaca ccaccctgtc 18420 tgccggtgat cacctcggct tcgaaaatct ggtgtttttt agctacctgt aaatcg taaa 18480 gtgacaatac atcaccgcaa ccactagaca tatgactatc ctcttagtta aaaccgttat 18540 cgaatccgtt agagaacgca cgaccgaaag gcggtacgtt gtcgaactta tagaaatcct 18600 tgtcatagtt aaacccggtt attttgactg ttctgtcatc gccggggtct accgtagaaa 18660 caagaatcat ctgagcatta tgccttgctt cgttgccgaa tgaaaattca gttttcaggg 18720 cgctattacc cgtgtaaatg gcttcctgc g gtacagaagt cataatcacc tgcctgtcgt 18780 gcgctccagg gactacatta acactttgca cgccgccgtc acgtaacttg agaatcaaag 18840 aatggtcatc tcccgaggtg aacttaactg gttgtgacag ttctacggtg agcccgttta 18900 ccgcgg ttac gtagccgtcg taggtcgcca tacgcgaacc cttaaccaca ctaacggcac 18960 gattcggaag ggcaaaaata ccctcttcag tggcggtaaa cgacaccgaa attttcttta 19020 aaatgttctt ctggtggcga cggtttgccg cccagaaagc ctgcttatag ttgcggattc 19080 cttttgagtc gtaagtctcc gttttaaggc ccccggtttc gggtatggtt atagtctctt 19140 tgacgttcgt cttagggtct atgtac gaga atttaaggct gtcaaaaacc tgagaatcgt 19200 taaatgttct ggtccacttt tcagaagtac cggccttgct tcggtgggtg aacaccatct 19260 cggggcccat gcgagggcgc tcaaaatcga gaaggatatc cgccccacgc cgatatgggg 19320 tgcagaatat ggcgtctgct atagtactaa ctatgtcctg catggtggtt ttatagtcat 19380 caaacgtgta gcaaaattcc cccgcctgtt tgtcaccgaa atacgcttcg acctcgttct 19440 gcacggcaag taacttatcc atgttgcgta ccgtcaggtt taaaccaccc acgtccgggt 19500 cacgtgccag gcggatgagg gattgtacag cctgtgtatt aggtgtcatc gtatcttcga 19560 aaacaccgtt accaaggtat ttgtgtacca tttcggtggc aat catacgt aactgtggtt 19620 gcttaacttc cgcagcacgc ggtgtctgtt tacgcatcga gtgtacggta gttctgttgc 19680 cgtagtgcgg agtgttatcg cgtgtttgcc cgtacaagtt aacgtacgtt acttcatcaa 19740 ccacagac cc ctcgaaatta aagtcgaggt ctgttaccct tctggcgcgc acccgcaccc 19800 gtgacacaac cggcaggtcg gcgtaaatag tgacgccgtt ataatctgga gtacgcccgg 19860 acacggttcc ttgtgcggtg tatattggcc cgtaaggaac gctattttca tcgagtagct 19920 ggtactgtat ctcagcggta acggatgcca gtgtttttga cttgccgttg tctttataca 19980 taccattggc agccgcgata ttggcgacta tgcgctcaac ctcggtgcgg tttatagaca 20040 cccaatcggt taatgttttc tcgtacgtgt tatcaggttg cagcgacgct tccccgtcac 20100 cgcggaaaaa cgaaccgggt cttatttctt gccacctacc aatgagacca ccgggtacta 20160 taaaggtaac ggtcccttcg gacac gctga ctacctgata tcctccaccg gagaggtcaa 20220 cgtctgtatt tgtccacgcg gacaccagcg aaaacgtgtc attaggcacc agtacccccg 20280 aaaaatccga accacctgtt gggtcatata tcgtgccaat gtttccagac aaagacggat 20340 acccggacat gtaagagaaa gaaatgccca taccgttcgg tgctttaaga accacgccgt 20400 caacttcgtt agattctacg gtaatgtata ggccttgctc tatggggtcc ccaaccatga 20460 cctg cggtgt agatgtatta tttggtgacg tatacggcgc gtacaccgca accgacgtgc 20520 ctgttatatc tgatacacgg gtatccccgt ccgttatgcc atccgggtgt atgtcgaggt 20580 aaccacgccc ggcgtcgtaa tagccgtact ctacgatttt accgg cagcg ttaaacacct 20640 tgtaagtaga cataaggtta ttggggatag tttgcaccgt cccacagatg tcgtagctgc 20700 gctcgtatgg gcgggccttg ttgttacggt cggtgatact gttgttcggg gaatccgcct 20760 gtgagttcgc gaggttagat gatgcccctt tcacggacgg cgacaagagc ttcgccaacg 20820 gtttaagaat aacgctaaaa atcttcatga cgcctttaat ggcgccaccc ccggcggatt 20880 ccacaatgt g gaaagtcgcg ttttctttca gtgcttcgaa gtcttctgtt acgtcgttat 20940 cttctccgat ttcgtcaatg aaaacacgca ccggcacccc gtccggaaca tggttcacga 21000 cgaagttcat tggattatcg cgataacgtt tgacgtcaaa tgt gccgtct tcgttacggg 21060 tatagtggat tactagcgcc aaaattcaat ctccgaataa gtatctttaa ggtcttccag 21120 actatccagt ctaacctgtc tggacgccag ctcacagtgg ctaaccatcc cttcgtaata 21180 taccccagcg tgccacacta ttcgccctct atgtttcaca cccagtaaaa ccgcgtcaaa 21240 attctgaggg ataaacgcgc gggttagtcc tttcgggtta gcgtggccgc catcaaaggc 21300 ggcgtctatt gccgttg ggc ttgttacatc gaacattgga gtcgataacc ccgcgtcagc 21360 ccggacgttg cggacgtggt gccaacaatt acggcggcgg aagtcatacg gcaggccagt 21420 gtaatcatta atattcacga tgccagtatc ccgcggagta aaggtatttc ttctggtgtc 21 480 atcaggattc cggtagcccg ctggttaagc atcggcgtac cagtttccgc cgtaaatacg 21540 cctttttctt gcgtgagtgt ttgtaactcg taaaccaccg ggccgtcgca tggataggat 21600 aggtcggtac tcacatagcg acggaagatg aatttaggta actccttatt atcatagggg 21660 atgcggtcca tttcctcatc cagtatgtta agcacatccg gcagcgaaaa ggatgcggtc 21720 tggtccatat cgttattgtt ggccgcgttc t tggcctcca ttggcgtacc ttcgaatgtc 21780 acgacctccc cggtctcaag agtagctgtt aaatcgtcag ccccgcgcac aagtaaccac 21840 cgtttcgaga gaagtggatg gtatatctca agagttataa agtccatctc accgtcggga 21900 ttag acgcta gtttacgtcg gtatgctgct tctactgatt cctgactcat tgcatagggt 21960 cccagattcg ggggaatgta gtctgataca ctccgtatgt tttaagaaat tcgcccaggc 22020 aatcaccata gcacccgtac aaatcgggta ggttctggtt caggcaagca ttgttggttt 22080 cctggaatgg tgatttctcg gcggtggccg tgaaggtgat tacccaattc tttccgtcat 22140 ctgtggactc gttccacgtg gatgttaacg ttacc tggta atcctcaata ccctgaccca 22200 agtcgtgctt catccagaaa ctggacgcac caccgtctac cttttccatg aaactgagga 22260 atgcctgccg ccccaacggc gatacgacca gggtaacgtt aatcggaaac acgtcaaaat 22320 aggtgtcgcg tccctgg cga accccgccac cggccaggtc tacccgccac acgttgttac 22380 cgcgggtcat cgagtagcct ttcgatacta taggcctcaa agaggccggg aaatggtaat 22440 cgctcatatc aatatcctgg ttggccgcgt gtagcgcgac gtgacttaga aatggcgctg 22500 ttactatctt gtaacgctga acttacggtt tcactgatta taatacgtaa acgaccttcg 22560 tcatcgcgtt ctgttgcagc cgaatcaatt cttccagtgg tgtt gttcac gatagtaaca 22620 ttatctccac cggatttagc gccattctcg cccataatct gtcgcatctg ctccgcagtg 22680 cggacacgtg acgcgctagc tggcatgatt acttccggtt taccgcgttc agcaatagta 22740 gacatctgcc ctgcggcgag gttacc gccc tgctcgcgcg cggagcgaat cttacccacg 22800 ttggcgagac ctgcggctac ggcagcagca gcagcaactg gcgctaaaaa tggccctact 22860 actgggatgg ccgccgttga tttatacgct tctactgctg acgtgtacgt agcgattgtc 22920 gcctgagcga ttgcgaatgc cttgtacgcg gtggacgctt cacccagtgc agacccgatg 22980 ttgctcgcca tattgccgaa cgcggcgcct gttgctt tag cccggtctaa tgcgtaggta 23040 tcattcattg cattcagggt ctgctggtag gtttcttccg cgattaaacc ctgttcatgg 23100 aaatcattaa gtttttgcag cttaacttca tactggcggt ctaactccgc aaattctcct 23160 tcagcaaggg cctgcatctg agc gacatac gcatccgcgg agaactgttt ttccaggcgc 23220 tctttctcac gtttatccaa ctcggtctgg cgggccgtat ccgcctcaag cataatctgc 23280 gttttagcgg tttcgtactg ttggtcgcta aggaggcgat tgccgtaaaa ctcctgtagc 23340 tttgtaagtt gctcttcttg gttagcttgt atcttctgga atacgtcgtc gttaaggtgg 23400 attgactgat aatagaaatc ctcagcggct ttcttctggc gttcgaatgc gtcgat ggat 23460 ttcttggcgg ccttgtctgc ggaatcacct gtagaaccgc tgtccttacc tgatacctta 23520 aacttaccca gaccttcggc cttagccgct tctttatcaa tgtcgtaagc agcgcgcttt 23580 aacgcgatga ggtcttcctg ggcttctacc tg ttccccgga aacttttcct cgtcaggtcg 23640 gtaacacccc tttgggtatc catagcaagc gctaactgga agttaaggtt cttggcctcg 23700 accaccgact caccgggggt cagcgccgcc tttaccagac gaccggcgtc ttgtgcgccc 23760 ttaaccatat cttcaaaaaa ccgctccatc gccgtgaatt cctgggcgat aaagtccagg 23820 gcgtccgccg cggattcggc tatcgtagaa gcggtttctt ctccgctccc ggtgatgctt 23880 tcgctggtat agttccaagc ggagtttaaa gcgtttaggg cgtctccaac catcccgaag 23940 gcggcgtcga atgaggcctc tatagcacct ccgacttcgg acgccatatc accccacatc 24000 ttcatctcag cgacaaactc acctgacgct acccgagaat ttatctcctg tatcaggtca 24060 tcaacatagc gtaaaggttc cgccagtgac cctacatcta tactagtggc aagggtcatc 24120 ttcaactgag accatgagtc ttctgcgcta gcaatagcgc cattgagcgt gttcgcctgg 24180 tctgccatag cgcccgcgaa gttcacgtta cctatgttaa gaagatactg ctcgatatcg 24240 gcggcatttt tcttaactac agtggttgtt ccctggaagg tgaattcgat atccttttgg 24300 ttctgcttgg c cttgatacc gaattctttc agacgttcga attcaaatgt actcgcatcc 24360 gccaccgctt caatcatctg gctaaggtct ttacccatcg ccgacgcggt attgccgtaa 24420 gagcggagcg cttcttccga tggagttagg cctagagcta ccagcttacg gaatccttcg 24480 accgcttgct caagtccgta aggggtgtcg cgggcgaagt cctgtaagat actcagggcc 24540 tgctttgcgc cttgcgtgct tcctgttagt gttttcaggc tggcggacat cttgtcgagt 24600 tgccgttgtg actcgactag ttcctgcgcc gccttatagg ctgtagcggc tacaacggcg 24660 gccaggccgg taacagcggc gcccgcaact tttgcggatt tagagaggtt atcaagacgg 24720 tctgatgtgg ccttcgcgcc ttgtt cggtt acctttacta ctaagctagc tacatcagcc 24780 atcgtttcta ccctcaaaaa tggcgtctaa gcccatgata atttcagatt caaacatccc 24840 tatattctga cctgaaatag cgctatacgc cactaaatct gaccaactaa gttgctctct 24900 tgggtatagc tttacgccgt c gtcatcaac gcggcgggtg aatttaacat cacgatactt 24960 ctcaaaagtg gtgagtagtt caggggggca ttcaggcccg gcgtcctgcg ttgtttcttc 25020 cgcgtcttct attaccccca tagcgataag cgcggctttg tgcccgtcgg ctatactgtc 25080 aaacttgcgc ctttcgtggc gggttatgaa gttccaacgg gcgaactgaa acaacgcgtc 25140 tacttttcct gcaattggcg caactgctca t ggtggaata cgactacatg ttctgccagg 25200 cgcttatatt gggcgagaag agtctcaaga ttttccttag tgaactcgtc atcaagactc 25260 cagccattca ccaattcgag ggccaattgc cggtttaggt ctcccgccgc gtcttccatt 25320 tttaaattgt attctgagaa gtctttttgc tcttcgcatt tatcccgaag cggttttaat 25380 ttacctaccg ccgctcggta cgccagggtg aaggcccgca tagctttaac cgcaacatca 25440 gcctcaggag agacaacgtt caaccatcc cctgaatctg taccatcttt taaaggaatc 25500 ggcatacgcg cgcctttctc ggcttctgct tcgtagtaaa aatcgctaag tttcatagta 25560 ataccctttg gttagtcggt tactggttgg tagttgcgcc aggcgggaac cacccgcttt 25620 tccggtgcga ccgtaggcgc caaataagaa taacaaaata gcttgcataa ccgttaatag 25680 tggcctatta tccttacgtc aactaaacaa tatgaggtta agaacatggg cgtttctatc 25740 ggtgttatcg cgtttatcta tttcatcccg ttcc tggtgg ccttactgcg taatcataaa 25800 gctaaattag gaatctttgt atgcaatctt ctacttgggt ggattcttct gccgtgggta 25860 ggcgctctca tctgggcctg taactctaac gttaatggta aataatctgg cttaattaaa 25920 attacattat tagaatcaga tttgtcttaa acaatcagga aataagcatg gtcactcgca 25980 aaataacaga tgaacagtta cagcaagaac taaatgctgg ccttggcccc acagaaatag 26040 ctaaaaagta caatatg tcc cgccgtaatg ttcagcttcg ttctgcgcgc ctggcgaaga 26100 aaggcattgg ccacgggcgc gacgtaagcc accttgtacc tgatgggtat aagattaagg 26160 gtacgtcgtc actggtagat gaattcggta acacgaaact tcaatgggtg aaaaccgata 2 6220 ccgacgcgga acgccaggtc gagttgatga aagccgttgt agacgagatg aaaaaagatt 26280 tacctaagta taagcctgtc actttgccca aaaatggcac agatgacgac ttacttaatc 26340 tttacacaat aacggaccat cacattggga tgctggcgtg ccaagaagaa ggtggagatg 26400 attacgacac cacaatcgcg gaatctctcg cagttgactg gttcaacagt gcggtttgta 26460 tga gtcctcc gagtggtgaa tgtatcatta acttcttagg agaccaaatg cactacgacg 26520 gcatgaaggc aatgacccct atgagcggtc atattctttc agcggatagt cgcctgttta 26580 aaatgatacg cacggccatt aaggttatca agctggctgt ctcgctgtgc ttacga aaac 26640 acaagaaggt taaacttctt atctgtgaag gtaaccacga ccttagttct tctttgtggc 26700 tccggggagat gttctacgag gtgtatacaa atgagcctcg cgtcgaggtt aaccgggagg 26760 tgtcgccgta ttatgcgtat aagttcgggg attgtatgat ttcagcacat cacgggcatt 26820 gttctaactt tactaaagta gagcagtcta taataggtaa gtatcgggag atgtacgggc 26880 agtg taagtt cacatatgtc catacgggtc acctacacca ccgggcggtt aaagagacta 26940 atcttctaat tgttgagcaa caccagacat tagcggctaa agatgagtat tccagtaaag 27000 gcggttacta ttcaggtaga agcgcaaatg tgattacata ccataaacgt tacggggagg 27060 tgtcccgcat aagtatacct gttgaaatgc tgcgagacat aaaccccaaa tcaacatact 27120 aaaatgtagg tgaaaggatg gattggagtg aggtttttag ctacaaaaac ggctataa at 27180 aggacataaa gatgactaac aaatacaatc gcacaatgac aaatactgac ggagatagca 27240 ttacctgtga tgtgtacgac gttctgaggg catttgatat ccgcgacccg gcactacagc 27300 atgcgttgaa gaaactgttg tgtatgggct tgcgggg gca caaggacaca ggaaccgact 27360 tagcagaagc aattgaaagt ctggagaagt tacggaaata ccgtagtaat attgatgagt 27420 gagaaaaagg cccctttcgg ggcttttttt tatcgtcacg ataaagaatc tgatttgaac 27480 gataagaatg ttcgaagtta aatatatcta caccaacaag aggggatgaa accatgaacg 27540 aattaatgaa tgttagtgaa gcacaaacca tgtccagccg tgagattgcg gaattgacgg 276 00 ggaaagaaca cgacaacgta cgacgcgata tattaaaaat ggcacaggaa ctttccctca 27660 attttgagga aaaggttttg ccttcaaacg gtggcagacc aagtaaggta tttctcttag 27720 acaaagaaaa tacatttaatc ctgatttcag ggtacagcat caaaatgcgc g ctgccatca 27780 tccgccgttg gcaggaactg gagtcacaag cgagcaaacc atccttaccg gtgccccaga 27840 caatggggga ggctctaagg ttggctgcgg acttgtggga ggagaaagaa cgccttgcgc 27900 ttgagaacaa ggaaatggca ccaaaagctg atgtctacga ccgcatcatc gacagaaata 27960 acttgtacaa cgcaacgcag gtggcccaga agtttggtca atctgccgtg tggatgaata 28020 aac aacttga acaatttggc gtatacaatc gctccgtaaa acgcggtcgt gtcttccagc 28080 aatggttcat cgataagggt tacggcatta tgcgtgaaac agaaactggg cactcgcagg 28140 ctatgttcta cgcagaaggt gaaatgtgga ttatctgtaa gttaacggca gaaggcttga 28200 tttaaaacta aggccccgaa aggggccttt ttttttctta ggtgtactta atgcgctgga 28260 taacaattga agactggaac tggttgccgg ttgcctggcc ctcaagagac agggtgacag 28320 attcgggacc accgatttcc ggagttgctg atgtcaacga agcgcgtttt aaggtgaaac 28380 tcatggcacc gtctgggccg tccagaacag aagatacctc catttgcgtt tcgttcagga 28440 att tattcaa taacgtcatg tcgtacagct tacccgccaa cgagaaggtg ttagccgcgc 28500 ggccacgttc gacaaatgcc acgctgttat ttcctaactc gaactgtgcg gacgcgctgt 28560 tgtcgttagt aatggtgaac gtgtcgataa gtttaagcgg ag cggcgccg tcataggcgg 28620 atacgtcaac cgatgcaaac ggacgggcgc taaaattggt cgtgaaatct gaaccactcg 28680 gcggtacttg taggatttcc tgattcaagc caatgaacgg gaaagagccg gtcaccatcg 28740 cgttaactgc ctgttcgata gtgaacccgg taaactcgac tccgcgggtt atgatgtatg 28800 aatctgggtt cccgcattta cctttcaacc atgtcaagat tgaataggtt ttacacaagt 28860 taccagtttc aagtttat ct gcgatacgca aattggcttg aatatctgat tcggcggtaa 28920 gggtatgctg gatgcccgtc ccggttacga ctgtagcggt tactgcggta acgaggaaag 28980 ctttgtcgtt attcccggat aaaccatcga attgcaccag gtcacctact tcaacgccgt 29 040 ctgtcacaaa gctaccggta gcgcgtgtaa aagttttcgc tgctgggtct acggtgatac 29100 caatgccggg tagagttgac ccggccaccc atgaactagt catcgcaccg gccaacagct 29160 cgtcctgact ggccgcgctc agttcaatag cgtattcacc tgttacctga cggttgccgg 29220 tgcggatgga cgatgtttca cggctgccgt cgagctcatt ggaaatgagg gcgtcgcgcg 29280 ttacggccgg aatgcctcca gtgttgcgga gcggctgcca taccgggttc gctggcgtca 29340 cccccggcgt cgtctcaaga acgtaaaatt gcgcggtcat cgcgcctttg tatggttgta 29400 acgccattat ctaatcctcg ctgtaaaggc tatgaaatta attgagagag gcctcttcgc 29460 ccatccattc tcaacaataa gaggaccgag gcttactgat tgtacctcag cacagatttc 29520 gttacgacta aaacagttac cggcggcgaa ggcggcgttt agcttatcag ctaatcggtt 29580 aataggagcg ctacccaagg ccgacccgac gttaatgtct acctgataaa cacccgcccg 29640 ttgttcagtc cagaacaagt cagcttgttc agtatctgac aacagcatat aactcgcaag 29700 atatggggta tctgtagacg taggtgcgtc tatgttct cc agtgcgacct ttatcccgtt 29760 gttcgtgcca aatgctacca gtgccgtgtc gaatgctttt gttaaatcct cgaaatatgt 29820 tgccattatt tcaccttaga ggcttcttca tttaataatt gctggaaacg cgatacatta 29880 actctaacga agccttgt gg cgcttgctgc gaccaaccat actccaggcg ctgcgcatac 29940 ggtaaattgt tagttagagt aaacgtgtgc cagtcagcgg catttagcac gaaattagcg 30000 gcattactgg tcgccgtggt gccagcttta tcggtagcgt ccgtagttcc gtcggcggga 30060 gtaccgccgg acgccatcca gttcatacga aagcgacctg tatctacagg actcgctttg 30120 ataatcgcag aaaacaactt gatagatacc tgacgaatta ccttttcagg att cttctta 30180 gccttttcca cgaacttgga cacatcaaga gcaaaactca ttttctcacc tgaataaagt 30240 acgctacaac gtcatcgtta accatcttct tctcgatggc gaccacagac cactgttcgc 30300 cattgaattg tactttatca tccatcttag gaattacgct gtaatcgg cc ttaaccacca 30360 tatcccctgc ctgaatggtg gtcccgttta ccagccccgc gttaacaggt accggaactg 30420 atttaagagg taccgtagtg tccgcccccc aaacgtactc acctaaaaca gggtcccatg 30480 ttttttgacc tttctgaatc aagattacag tactgccgta ttttgatagt aggcgagttc 30540 ctacaccttg catccgttta ctaaaggcgg tgcccattac gcagcctcca gtctcgagat 30600 aaccaataag gctgacggcg ctacacccca ggaagtaact gttgccgctt gcgggtacag 30660 gccgccgaag ttagagccgg cggcatcgcg cataatctct acggtaaatg tctgcccggc 30720 tgcggcattc acaacaacac gagattcaat ggggattgtg gattccgcac t cgccagttt 30780 agtcgcggct ggtgatccga attgcgcacc tccgagaaga acacgcgaca gaaggatgga 30840 tgttccgctc gctccggtgc gcccagcctg caatttaacc cggatagcgt agttgaacgt 30900 aactaccccg gatgcattta tcattacagg gtctgcgctt gttttctgcg cacccccgaa 30960 ggatacggtt aacgctgtac cagtggtagt tggcgcctgg atatctgtag acgccgcgcg 31 020 gaggacctcc acttccttag ccccgtacag catagaatcc gccatctgag tggtaacttc 31080 gcgcaacttt tccggtgtaa ttagcccgga ctggttatca gggaagtttg ccccaattaa 31140 ggcgaatatt tccgatttag tcttagccat gtttagcccc tgaaaacatt aaaggagtag 312 00 gcattattgc taccgcataa gagaggtcgt agcgcatcat cggctgccgt gatgcttact 31260 gtgccgcctg agtagccatt cttaaagtag gacaccgtta ctgcaccctc aacccgttcg 31320 gtctgaacct ctcgcccgtc tgtagaccct cgaacgtcgg taccagcgcc atactcgacc 31380 gcggccatta cctgagcttg aataactaac gatggaatta cattggatgg ctgagggaac 31440 ccgtgtaaag taacac cggt tcgagggaat gccagtgcct ggtttgcgga tacgcgacga 31500 ccgcacatct gagactcaaa aaggccaaca tataccgcgc cattacggag agaggcctcc 31560 gctgcgatgt catcttctgg cagctcaaga ccgtacttag aggccatgga cctcgcgtct 31620 t ccaggctga cgtaagagtc agcattcggg atgccttgac cggtttccac gataagcggc 31680 ataaattatt cctctacgct tttgcggcga cgacgctgct taggttctcc gccgccgttg 31740 taatgcggtt cttcggccac tggctgattt tcgattaatg cttccgggtt cgcttcttcg 31800 cgcatcggga ctaactgccc gtcaacctcg accacacctt tgtattgttc ccgtactacg 31860 taattatctg tcatg tctaa tccttaaagc ggcccggagg ccgcgtgtat ttaacttacg 31920 gtaactaccg tactatctga aatgatatta ccgtaaccgt cgtgcgctac gactttgtaa 31980 gtaccggaat ccgccgctgc ggagttggcc ttagtgtaag tcaaagcggt tgcacccgc g 32040 atagcgttat tatccttata ccattgaacg gttttagggt ccatgccgtc ggcgagttcc 32100 acggtcagtg tgatagcctg cccggcggtt accgcggtag tatcattgag accggtagag 32160 aaacgcaacg gcattacgtt ttccatatca atctcaacct gcccgtcctg cggactatca 32220 tcagatacac caacaatacg acgtttaatt acatcaacca tttttatcct cctcaggata 32280 cagtaccagc gtttttaagg gcggttaaca gattagtaac ggca gtgcgc aatgaggtta 32340 catcggtgcg caacttattg tagttagaca ccaacgcgtc aaactcttct ttcgtaggag 32400 cggctgctgc ggctgcacca ctaccggcac taatcgctgg cggtgcggct actgtagcgg 32460 actttttaac ccctccgatt gtcgttgtgg tagcggcggg aatatcgaat tccgcgttaa 32520 ccaggctgcg gggtaaccct ttacctgtct ttgacattat attgcctccg tataaatgaa 32580 aagagggacc gaagtccctc aagagtatac cctacgttta agcaccgacg ccagttacca 32640 ggaacgcaat cggtacgtgc ttacgggcta ctacacgatt ccagttggtt gcgttagcca 32700 ggtcctgcca gctggcggag cgggcgatag tctcagta cc gttgccggtg attacggcgc 32760 tggtgaagct gtaaccaaac ggatgcagca accacgtttt acgcgtccat agggtttcaa 32820 cgccgccgcc gttagcgcga gatgcttcac gctcgtattc caacggcatc tcaggattgc 32880 cttcgccata g ccgatagcg ccctggccga agatgacgga gatgaacttg cgctgcgcac 32940 cctggcctac tacggtcatg ctgtcatcga cgataacacg gtagccctgg taggtggcga 33000 acatggtgtt gttctcagcg tcacggatga agtcgataag ctgagcttta cgcgcctgcg 33060 catatacgaa gctgtgcatc gcgatagcac cgagaacctc accaccatta cccatcaatg 33120 cgtcacccat agtctgggta gcgtcaatga atgcacctgc gtcgaagcc t gaagttgcag 33180 aaacgtcaac aaccatgtcg ttctgctcgt gatacgcatc agtagcggat acgttgtcgt 33240 tgtacaggcc gagtgcagtt gcaataaggc gacgctgcgc ctgacgctgc cagaagttat 33300 ccagacggga tgcgacgg ac tgcaacggat tctggctggt aagttctaca gtcaggtctg 33360 cctggccgaa accttcgttc agatatgcaa cgcgggccat catttcaccg gtctgaatag 33420 cgcgcggggt cgcgatgtcc tggtatacgt cattcgagta gttaggctcg atagaagtat 33480 cgatagcttt ccagaacggc aggttagcga tgttagacgg tccgcgggca atctcggcag 33540 catacggagt tggggtcaaa attccggagt tgaaaaacgc ggttttctct accgggtctt 3 3600 ccgtcatata ggacgccagt acagggatat tgccagttac gatgttacca atagtggtga 33660 ttgccattat ttatttcctc agggctttaa gttgccgttc aaattcggca gggttagatt 33720 tatagagagc caaccgctca ctttcactca tgtctttaaa cgctggtgc g gccccgccgc 33780 ctttatccc ggaagccccg ccaccggaag ctgcatttgc tttaatcaaa tgcgaaaaag 33840 ctttatgttc gcgcaggtat ttgcggaact gctcagggtc ggtagtaaca acattgccat 33900 ccgcacccat gaatttagtc actacgtctt cgccttcgaa ctcggtctta acgaacggtg 33960 ccaggatttc taccgcttcc ggggtgatga aatcaccggc gaaagaaccc aaaacagcct 34020 ta cgttcact tccaagaatt cgcgcggcta aagaagcgta gcgttcttct ttctctttca 34080 gtacgggttc atactgacta cgaatcgtct tttcaaattc gtccatttta ccagcagcct 34140 tcagggcctc ctggtgggcg cgctgccgtt cttcttcggc t tctttagcc cggcgagcag 34200 cttccttttt ctcgctaagg agcgcttcct gatttgcctt gagcccggcc acttctttct 34260 caatcaaggc ctgcacttct tcggcggtgt acatttttgg tgcatcacct gcacccgatt 34320 tatcttctac tccagcttct tcctggagtg gatagtgtaa aaaacgattc atagtcaata 34380 tgtcccctgg actttaggac accgggccac ccggtgtttc tgtaacaaga ataaattatt 34440 ccacttacta aggcaactat t ccagaatat tccttatgta ctcctgcaac cggaacactt 34500 tcagacgtaa ctgccgtgtg cactccgcgt tacgtacgtc gatggccagg tcttcatcag 34560 cgtcactgct cgggggaata agcttgcacg gcggttgcat tagtgtcata tccggggatg 34620 gaattagcat tctcagtggc gcgtcgtttg attgacaccc gttcagaagg aaactcgcag 34680 acagtacggc caggacggat aacatatttg atgacttcat gggtgatagc ctccgaattt 34740 tctttgcctt ccgattcggc ggaggcggcc ttagtatcat tctgctgctg ccgttgtgtt 34800 ttcttcgtca gctcagcttg tgccttcttt tgttgctgtg aaacgagatt ggcgcggcct 34860 tcta cccacc cgcttctgta gttgtactgg ccgtataacc acaaggagag gataccggct 34920 gccgtggcta ccgcggttac tttccaattc atagtatccc ctttctttat acgctaagcc 34980 cgacgaagtc gggcgttaat ctcgtatatc tgtaaacatc atacggcgaa tct tagctaa 35040 atactgaaag caatatagct cggcccttcc gaatgcgttc gcggttacgc tagggcgttg 35100 tgacgcccgc ttcacgcgac ccgcaccgcg gtacgctgaa agcgtagtta gcgtagcatt 35160 taaaataata gaaatctact ttttgaataa agtattcctg aatttacatt ggtaaggaat 35220 aactggaata atcgctggta aaattaaggc cccgtcaagg ggcctttcgt tacttaggtt 35280 ctgagacctt agccgctttg ctaaccaccg acacacc gtc ggcatctact gccttgacga 35340 aatattctcc ggggtcttta acggttagtt ccccttcaac gtaaggaacg tttattacct 35400 ctttaccatc tttaaaccat tgcaaatcgt agggcgcttt accgcctttt acaactacgg 35460 ttaattttgc agaaccgtct t tcagctcta catctgccgg ttgcagatcg aagtacacat 35520 ctcccgcacc atccagatac ggtacttcgt acaggatgcc gtctgagatt gtggtcagtc 35580 cggtcttatc cgcaaacggc atttcatcta ccgactcgcc gagaacagat tcgtctttaa 35640 tataaacaac gccttcaccc gcaccagaca cacgtgcgta ctgcactact cgacgtgacg 35700 gcacatcttt aactttgaaa aagcccatct ttattcctct ttca gataat cggcaacacg 35760 tttatcgagc tccgccatct ctttaagagt taacgggcgc ccgaatccgt ctaccgatat 35820 tacacgaaat tcttccggtg ttatcccact attacgaaaa atcttacccc ttacggggcc 35880 aagtgcttcg tcctggaacc acgccggtt g ttgtttaagg aattcgtagt agctcgtgtc 35940 cgcgcttacc tgctgacctc catctgcacc cctggccgcc cgttttgcac ccttatccag 36000 gaagtcgaat tctggactta ttaccggtgc tgtgctcgac cgacagttag gatgcgctgg 36060 cggtagcgga cctttaccta tttcgtattg catagaatcc cgcgaccgac aaacggctga 36120 ggttctggag tcaagggtag atacccactc gtatttcgtg atgatgtcgc c gttctgagc 36180 gtaaacctgt tgccgtgctt cgttggatac gtgcgccagc gctgtccgga taactgtagc 36240 agcgttacgc tcggagatat cggccagccc accgggcccg acaacgttct taactatctg 36300 tctggtggtc ataccctgta cgaaccc aga cttaaccccc atgaccaggc gatttacctc 36360 ggtttgccgc cagccactca ttagcgaaac aaaatcaacc ggtttttcgc ttagttcaag 36420 cggcgcgaaa gtcgcggcgg cccatacttg ttcgggggcc ggtgttacga aattagcgtt 36480 aacgttggtc gacagggttt ttacgttcca gttaatttca tagtctgaaa gctcgcgggc 36540 atcggccagt agtttctcat accattcaga tgtaactccg tttagcgatt tctcaag ttc 36600 ccgcagcatg acggtgagtc tcgctgccgt cctgctgtcg tccccgaaca gcaacacctg 36660 tcgcttaacc tcatcgcgca tccgcttgat gaatggcgcc aggtctttca cttcgccgga 36720 tgcggtacgt tgtaaccata tctga tggct gataatcgat tctaaaagac tcatataccc 36780 ctcctgtctc tacgcaacaa tcatatcagc cagtagggta tagacataat catgcacatt 36840 tatagcttaa aagatcattg acaaatggta tatctatata taccatttaa gcgtacattt 36900 aaacaggagt gatatacatg aaattaaaaa cattagagtt cccgtatggc gaaatcactt 36960 tcgaccgtga caactttatt aacgtaaacg agttggtcga ccagatgaac cgttggcgct 37020 tagacaatgg tatgggcttt aagcacctac ggcagctaat gacgacagat gggtttaaag 37080 atttcaaggc tgagtgtgaa aagcagcttg gcattaagga tgccgtcagg attacacgag 37140 ggagaaacgg ttcaacattc acctgtctac atttggcagt gtatatcgcc gagcagtaca 37200 gcacctattt ccattcctc gttatagacc gatttattac acaacggcag gtagagctgc 37260 gtaacatcgg cgcggttagc ttcgtagagc ttaatgccgc cgttagccgg atgctcgagc 37320 gcacagaagg gcgaataggc catagtgggc atttcataca ggtagcgaaa gcaatcaagg 37380 agtctataga tattcgtgaa gtcacaggtt tcgatacttg ggactctcag gacgcaaaaa 37440 ctaatcaatt gcggagc gag attcaaaagt ctatggtgac cttgttggat atggaagcag 37500 tcaactcgtg ggatgagctt aaagaggcaa tacctcgtgt tgtacgtaaa tgcgccgccg 37560 gtgtatgcta aaactacggc cccgttatgg ggcctttttg tttactactc ctgtt gttgc 37620 gccgcctgcg gtatttcccc ctgaacctca gtggcgaccg gtaatggctg gtcagccaca 37680 gcgtccttaa tatcagcgtc tgtccagtcg gtgacaccgg ccttacgcaa cgcggcatag 37740 taggccgtgg cgggcagcag cccggcgtta atgtccgcca tccatgcagc ccggtcttgc 37800 gcggtcatag gttgcaggaa gaaatccatg ttaaggcgga actcgacttc cgtatcttca 37860 ggtttaccga gcattat cgc cacccaacgt aaagcatctg tatacgcctg gcttacgtta 37920 cgtgcgattg ttgccatgac ggacgtatcc gcgccgcgtt ggatgcgtgc cgattctgcg 37980 gtaatttgct gagtcggggt gataagctgc gcaccaatct ggatagcctg ctgctcttta 38040 tccaacatat tctggcgggc caggttattc tcgccagcct gaataagctg ggcgcttccg 38100 ccgtacccca ggttatgccc gcaccgacta ccaaatttga taccgttagg gttcgcttcc 38160 ttgaatgatt gaggtgttaa gttctcgcct gggtagataa acagggtcgg ctgcccaaca 38220 acgaagctgg actcctcgtt atcagcgcta ttgcggtaat gacctatgtt aagctctgcc 38280 agcggcaaca aaggcgcgtc g tcgatggtg gcgtcgttat tggtcgcccc gataaaggta 38340 aacggaatta cccctcgtaa tgattcccct aagtccgggt aaatctccac gacgtcttcc 38400 tgcgcaccac cttccgcatc gaaacgaaaa agacgttgac ggtagttgcc gtcgctatcg 3 8460 atgtccagaa cgcgatactg ttcgccgtat ttcgtttcga actcgtttcc cggttcgttg 38520 tactcccatg tctcacgcag cacgaccata gttacgcggt ttacggaacc tacgcgcgtg 38580 agtcgccagt taacgatatt ctcggtagtg tagaaagcga ttgtagggtt gagcagaccc 38640 gcgttctgtt cggccgctgt agctgcgccg gtttccgggg cgtcaacaag aagaccacca 38700 cgacccactg agtcaatctc catgagcgtg tcctgc gcgt gctgtattag gcctacacca 38760 gacccatctg cgtttttaag aagatactcc agctccttcg ggatattgat ttccggttct 38820 ttgcgcatga cgctgccaac catccctgac agcgtgcgcc gggtgaagtt atagacgata 38880 ccgcccgctt catattct gc ctgacgcgct tcaccgtaag ctttgtccgg ttcgttcagt 38940 cctacgttgc gtagatagct aaccaattca cctgcaagcg cgtggcgcac cttctgccat 39000 ttaggcgcgt agtgcagcca ttcacggtgc ttggttttta cgccagaacc ctggccgttt 39060 gctgttaaca tttaacaatc ctcttaaagt gcgaaagtaa ccgggatatg ggatacaggt 39120 ttaaccaacg gcatttcgta ggcgataggg tatcctgtcg cgtcgttctg gtggtcgttg 39180 ccgcttgtct tatccggtat accgttttta tcgtaagcct gttgctccag gcaacgtgct 39240 gtaaccgggc aagcctgctc gttgaccatt aatctaccag actccagcgc ttattcacc 39300 gatgctacac gg tctttaac tgcagggtta actgatttcg cacgtatctc gaaaccagcg 39360 ttctgtaatt gggcaatatc cgacgtactg gcatcagtcg atttacggtt cttgccgctg 39420 gcgtccgggt acatgacgat gtggtggccg tgtcgtttcc accgctcggt aatctccctg 39480 accacatccg gggtgtcgaa catatccacc agctcggcta ccgcgtgcca gacatactca 39540 cgctgaacgt atacggtgct agccatatgc ccgacgttga agtcctgacc g atatacaaa 39600 gtttcaccag gttgtatagt ctcccggctg ctgttcttgc gtcggtcgta agcataatac 39660 acgctgccgg atgtcaggtt gacaaattca ccatcgatgt aggcatcaat taactggcct 39720 ggatacgtat cccgcaacga ctgcacataa tct tccggta gaaagggatt ggatgttgtc 39780 gaggcctgaa tcatttcata gcctggtttc ttttttacag cccatctgtc gtgcacaaaa 39840 cggaagcctt ctggcgtcgt aaataccgaa actgtattag cgggcttcgg ggtaattgga 39900 cgatatgtac gcggcaactg acggttacgg gcaataactt tattccaggc gtgctcggcg 39960 tggtctttat ttaacgtgtc caactcgtcg atttttgcgc ggaacgattc gtagccaaca 40020 at tcgtgctg gattatccaa tgtgcgaagg acaaaatccc cgaattgccc ggatgaagta 40080 taaatgatgt tgtcagattt attgtacttg tagcgaatac cccaatcgga tagcttctct 40140 tccatacgag gggcgaggat aagacgtaca aggtcatacg tcggttcgta catggca ata 40200 agggaatcgc taccgccctc catgctgtcg agtagggctg agttacacat gacctccgat 40260 ttacctgtgc cgaagcccgc aacgaaggcc gggaatttac aatgcagatt aaggaacgcg 40320 ccttgcggtt ctgttgctgt gatatcaacg ttcaccgggc accaccttaa tagttacttc 40380 gctaattggc tcatcgcgcg tttcttccac aacagtttta ttaagcccaa gttttgccgc 40440 ggca aacgta gcggatatac cggcggcacc ggtctcagtg aaatacgcct cttccagggc 40500 ttgtgccgtc tcatatgctt cggcgaatgc aggaacctct cggagccata gcttgataat 40560 cgggatggtt acgccgatgt gtaatgcgaa gcgggccagc gacggcggtt tgtcctggat 40620 aagcgggcgt tcgtcgcctt tagacgtggg tacgagttcc catgacgcgc ggtcgaagaa 40680 cctgattaac tcgtcgcaat agtccggatc ccatag ttcg gcggaatgtc tggatgactg 40740 atacaggctt tgtttaccac gcgggcgttt acggcgacgg tttgcactaa ccgcttcttc 40800 gtgcgcggcg gccactactt cggcgtcagg ctgttttaat ttgagtttca tgcaatcctc 40860 t cttaaatgc gtatgaccat catagcgcat cagacgcccc ctgagcgtct attaaggatt 40920 gtacatggga atgaaggcct gtgtacagaa aagcccgcag aagcgggctt gtgtcagtta 40980 tataggccgt gtcgtagtgc tcttctacca tcgccgccgt aatggtcgtt agctgcggcg 41040 ataacccagg cgttcgggat gattagcaat ctcatttgaa aaaccacagc aggatgccgg 41100 ccgctcctga tacacccaag cctaaaaaca taggccacca cagatatcgg ggaagactgt 4 1160 taaacacagt cggttgtttt attagcgcac tgagaaaggc aaaccaaaac atcaggtcgc 41220 cagtggcatc ggtagccttc aaaatctcaa tcattctcca ctctcctctt tataaaccca 41280 ccatttacct acggcttccc atcgccaccc gcgactatgt atggcttcga acctacgacc 41340 aaatactgtt tccccgtacc acatgacaaa tagcttcacc gcggcaccag caccagcttt 41400 atgagcattc aaacggcgat acgctagtga catcctcgta agacttaccg cctcaacaaa 41460 tggccaaaga aacaaaatcc acatcaggta catagcaccg gcaagcagta atgttgtgaa 41520 tcctataccc atgaggatga gaaaactatc catcacccct ccagcttcat acggtcgttg 41580 cgcgataatc cacgtttcgt gtagatgacc ggtgacgttt tcgtatgggt catcagtcgg 41640 ttgttataga tgacatgttg ctcgcatttt gtgtcatggc ggtacttgga caatgtcatc 41700 tcattaagtc ccgtctcccg gcacgtatca gccagcgtgc cgtg ggtctc tattagtttt 41760 gggatactag taatcattcc tgttcgctct ccttctcaag tcgttccagc agcatcttgt 41820 atgcttctaa ctggaattct tcgttcattg acaggcccag cttacttaca gactccaaat 41880 acgtgatgcg ttggtgcaac gtttctttgc atactcgttt catcgtttaa tcgccccgca 41940 cttaacacac tgtccgcaca tatctttagg attgttaaca aaatcgtgct cgcacttatt 42000 caggtcataa cggttgataa gctcccggcg atactcatta aggtagtcaa gcgcagcctg 42060 gtgtgcctcg atttgcgcgt ctactgcttc aatcttaaac ctgccgttgg cggcttctac 42120 catcatcttt tcatattcgt tgttagtcat ttcttcttac cccacattcg attgaga tac 42180 ttgttcttgt ccggtcctgg gaaactgtta cgtttaatca gttcctcgcg tgtcgggaag 42240 ggcgtgttac ttaccttacc gcccacctta aacgttaccg ttacttgccc gttgtcgctc 42300 attcctgcac cccccatccg agtacatcta cgccgtcata gtaatgtgag gtgcgctcca 42360 gctcggcggc ggcttcggcc ttttcttcgc tgagataaac atcaagaacg taagtgtccc 42420 cgtcgtga tg ccatctggat acgatatata ctttcatttt tgttctcctg cataagctgc 42480 tttcagtgtc tgcatcgcgg cgaaccagaa cgcatccgct ggaatccgtg tacttaggtt 42540 attaacggca atgcgtgcca tcagttgtgc gtctttgaat gcttcggtg t ccagtagcgg 42600 tttgcaatgg taaaaaactt tatcgctttg taagttcatc ttctctactc cgttctcgtt 42660 gtcgatgaac taaatataat agggttaatg ttactagtca actattattt ctgaatacag 42720ataaaaaaaaa 42730

Claims (8)

A composition for killing Salmonella Enteritidis containing bacteriophage ΦSEP1 (accession number KCTC18931P) consisting of a nucleic acid sequence represented by SEQ ID NO: 1. A composition for killing Salmonella Enteritidis containing bacteriophage phiJR14 (accession number KCTC18927P). A composition for killing Salmonella Enteritidis containing bacteriophage phiJR16 (accession number KCTC18928P). A composition for killing Salmonella Enteritidis comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P). A food additive for killing Salmonella Enteritidis, comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P). A food for killing Salmonella Enteritidis containing a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P). A feed additive for killing Salmonella Enteritidis, comprising a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P). Feed for killing Salmonella Enteritidis containing a mixture of bacteriophage ΦSEP1 (accession number KCTC18931P), bacteriophage phiJR14 (accession number KCTC18927P), and bacteriophage phiJR16 (accession number KCTC18928P).