KR102224896B1 - Novel Polypeptide and Antibiotics against Enterococcus spp. Bacteria Comprising the Polypeptide - Google Patents

Abstract

The present invention relates to a novel polypeptide and uses thereof as an antibiotic. More specifically, the present invention relates to a polypeptide derived from a bacteriophage, and an antibiotic against bacteria of Enterococcus spp. comprising the polypeptide. A novel endolysin and/or bacteriophage expressing the same provided in the present specification can be usefully used as the antibiotic against Enterococcus spp. bacteria, such as Enterococcus faecium.

Description

Novel Polypeptide and Antibiotics against Enterococcus spp. Bacteria Comprising the Polypeptide}

Provided herein are novel polypeptides and their use as antibiotics. More specifically, a polypeptide derived from a bacteriophage and an antibiotic against bacteria of the genus Enterococcus comprising the polypeptide are provided.

In general, Enterococcus (Enterococcus) genus of bacteria is a Gram-positive bacteria, merchandise from the intestinal tract of warm-blooded animal or human, and is discharged through the feces. In general, the pathogenicity is relatively low, but it is known as a causative agent of subacute endocarditis or urinary tract infection, periodontal infection, intestinal tract infection, wound infection, sepsis, and nervous system infection.

In addition, bacteria of the genus Enterococcus can cause infectious food poisoning, so they are also used as contamination indicators for food or public water. In particular, bacteria of the genus Enterococcus, which are resistant to vancomycin, are attracting attention as the causative bacteria in various intestinal, skin, and respiratory infections or as the cause of complex infections.

Enterococcus faecium (Enterococcus faecium) is a representative example of the bacteria of the genus Enterococcus, forms a colony in the human intestine and causes diseases including enteritis. In particular, it can cause sepsis in patients with reduced immunity. Varieties of Enterococcus faecium that are resistant to various antibiotics are found at high frequency, and in particular, a variant resistant to vancomycin (Vancomycin-resistant Enterococcus, VRE), which is called the last antibiotic, frequently appears, so it is reported as a case that cannot be treated. .

On the other hand, 　 bacteriophage refers to a bacteria-specific virus that inhibits and inhibits the growth of infected bacteria by infecting specific bacteria. Bacteriophages are bacteria by infecting their host bacteria and proliferating inside the bacterial cells. After proliferation, when the progeny 　 bacteriophages come out of the bacteria, endolysine, the protein of the bacteriophage, is used to destroy the cell wall of the host bacteria. It has the ability to kill people. The bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific 　 bacteriophage can infect only a specific category of bacteria, and because of this, a specific 　 bacterio phage has an antibiotic effect against a specific bacterium and does not affect other bacteria.

Therefore, the use of 　 bacteriophage as a countermeasure for bacterial diseases is receiving great attention. In particular, after 2000, due to the increase in antibiotic-resistant bacteria, limitations of existing antibiotics appeared, and the possibility of development as a substitute for existing antibiotics emerged, and bacteriophage is again attracting attention as an anti-bacterial agent.

One example provides a novel polypeptide. The polypeptide may include the amino acid sequence of SEQ ID NO: 2. The polypeptide may be derived from bacteriophage. The polypeptide may be one having endolysine activity derived from bacteriophage.

Another example provides a polynucleotide encoding the polypeptide. The polynucleotide may include the nucleic acid sequence of SEQ ID NO: 3.

Another example provides a recombinant vector comprising the polynucleotide. The recombinant vector can be used as an expression vector.

Another example provides a recombinant cell comprising the polynucleotide or a recombinant vector comprising the same.

Another example provides a bacteriophage comprising the polynucleotide. The genome of the bacteriophage may include the nucleic acid sequence of SEQ ID NO: 1.

Another example is,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

A polynucleotide encoding the polypeptide,

A recombinant vector comprising the polynucleotide,

Recombinant cells comprising the polynucleotide or a recombinant vector comprising the same, and

Bacteriophage containing the polynucleotide

It provides an antibiotic comprising at least one selected from the group consisting of.

The antibiotic may have an antibiotic effect against bacteria of the genus Enterococcus, for example, Enterococcus faecium.

Another example provides a pharmaceutical composition for the prevention and/or treatment of infections of Enterococcus bacteria or diseases caused by Enterococcus bacteria including the antibiotic as an active ingredient. Another example is the step of administering a pharmaceutically effective amount of the antibiotic to a subject in need of prevention and/or treatment of infection of Enterococcus bacteria or diseases caused by Enterococcus bacteria. It provides a method of preventing or treating diseases caused by infections or bacteria of the genus Enterococcus.

Another example provides a feed additive comprising the antibiotic as an active ingredient.

Another example provides a disinfectant comprising the antibiotic as an active ingredient.

Another example provides a disinfection method comprising applying the antibiotic to a subject in need of disinfection.

Another example provides a detergent comprising the antibiotic as an active ingredient.

Another example provides a washing method comprising the step of applying the antibiotic to a subject in need of washing.

When the bacteriophage, which can be used as a natural antibiotic by proliferating the bacteria as a host, penetrates the bacteria and finishes proliferating from the inside, the completed phage particles are released to the outside of the bacteria. The enzyme that makes the pathway is endolysine. All bacteriophages have these endolysine genes in their genome, and use the endolysine protein expressed during proliferation. Bacteriophages that proliferate bacteria into a host and endolysine derived from bacteriophages having properties of degrading cell walls can be used as natural antibiotics.

In the present specification, a novel bacteriophage is isolated, and the antibiotic effect of the bacteriophage and the endolysine derived therefrom is verified, thereby providing an antibiotic and/or a use related thereto of the bacteriophage and endolysine derived therefrom.

Hereinafter, the present invention will be described in more detail:

One example provides a novel polypeptide. The polypeptide may include the amino acid sequence of SEQ ID NO: 2. The polypeptide may be derived from bacteriophage. The polypeptide may be one having endolysine activity derived from bacteriophage. The polypeptide may have a molecular weight of about 38 kDa (38 kDa±2).

Another example provides a polynucleotide encoding the polypeptide. The polynucleotide may include the nucleic acid sequence of SEQ ID NO: 3.

Endolysine refers to a peptidoglycan hydrolase encoded by bacteriophage. Endolisine is synthesized during late gene expression in the lysis circuit of bacteriophage proliferation and mediates the release of progeny virions from infected cells through degradation of bacterial peptidoglycans. In terms of enzymatic activity, endolysine is typically glucosamidase, muramidase (a type of lysozyme), transglycosylase, amidase, or endopeptidase. It may have an activity of one of (endopeptidase).

In the present specification, a polynucleotide (which may be used interchangeably with “gene”) or a polypeptide (which may be used interchangeably with “protein”) is “including a specific nucleic acid sequence or amino acid sequence” or “a specific nucleic acid sequence or amino acid sequence. "Consists" may mean that the polynucleotide or polypeptide consists of or essentially includes the specific nucleic acid sequence or amino acid sequence, and the original function and/or the desired function of the polynucleotide or polypeptide To be construed as including "substantially equivalent sequence" to which a mutation (deletion, substitution, modification, and/or addition) has been added to the specific nucleic acid sequence or amino acid sequence within the range (or does not exclude the mutation). I can.

In one example, the polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence" or "consisting of a specific nucleic acid sequence or amino acid sequence" means that the polynucleotide or polypeptide is (i) the specific nucleic acid sequence or amino acid sequence. Consists of or essentially includes a sequence, or (ii) 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more of the specific nucleic acid sequence or amino acid sequence , 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more of an amino acid sequence having a homology consisting of, or essentially comprising the same, and means maintaining the original function and / or a desired function. I can. In the present specification, the original function is an endolysine enzyme function (e.g., peptidoglycan hydrolytic activity) (in the case of an amino acid sequence), or a function of encoding a protein having an endolysine enzyme function (in the case of a nucleic acid sequence) The desired function may mean an antibiotic activity against bacteria of the genus Enterococcus, for example, Enterococcus faecium.

In the present specification, unless otherwise stated, as a'comprising the amino acid sequence of SEQ ID NO: 2'or'consisting of the amino acid sequence of SEQ ID NO: 2', a polypeptide having endolysine activity,

(1) a polypeptide consisting of or essentially comprising the amino acid sequence of SEQ ID NO: 2, and/or

(2) 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the amino acid sequence of SEQ ID NO: 2, Consisting of or essentially comprising an amino acid sequence having at least 99.5%, or at least 99.9% homology, endolysine enzyme function (e.g., peptidoglycan hydrolytic activity) and/or bacteria of the genus Enterococcus, such as Enterococcus Polypeptides with (retaining) antibiotic activity against faecium

It may mean that.

In addition, in the present specification, unless otherwise stated, a polynucleotide encoding a polypeptide'comprising the amino acid sequence of SEQ ID NO: 2'or'consisting of the amino acid sequence of SEQ ID NO: 2',

(a) a polynucleotide encoding a polypeptide consisting of or essentially comprising the amino acid sequence of SEQ ID NO: 2, and/or

(b) 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the amino acid sequence of SEQ ID NO: 2, Consisting of or essentially comprising an amino acid sequence having at least 99.5%, or at least 99.9% homology, endolysine enzyme function (e.g., peptidoglycan hydrolytic activity) and/or bacteria of the genus Enterococcus, such as Enterococcus passi Polynucleotides encoding (retaining) a polypeptide having antimicrobial activity against um, and/or

(c)'Containing the nucleic acid sequence of SEQ ID NO: 3'or'Consisting of the nucleic acid sequence of SEQ ID NO: 3'polynucleotide

Can mean,

'Containing the nucleic acid sequence of SEQ ID NO: 3'or'consisting of the nucleic acid sequence of SEQ ID NO: 3'polynucleotides

(d) a polynucleotide consisting of or essentially comprising the nucleic acid sequence of SEQ ID NO: 3, and/or

(e) 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the nucleic acid sequence of SEQ ID NO: 3, Consisting of or essentially comprising a nucleic acid sequence having at least 99.5%, or at least 99.9% homology, endolysine enzyme function (e.g., peptidoglycan hydrolytic activity) and/or bacteria of the genus Enterococcus, such as Enterococcus Polynucleotides encoding (retaining) polypeptides with antimicrobial activity against faecium

It may mean that.

In the present specification, the term "identity or similarity" refers to a degree of correspondence with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). For homology to nucleic acid sequences, for example, the algorithm BLAST (Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) according to the literature or FASTA by Pearson (Methods Enzymol) by Pearson (Methods Enzymol). ., 183, 63, 1990). Based on this algorithm BLAST, a program called BLASTN or BLASTX has been developed (see: http://www.ncbi.nlm.nih.gov).

Another example provides a recombinant vector comprising the polynucleotide. The recombinant vector can be used as an expression vector.

Another example provides a recombinant cell comprising the polynucleotide or a recombinant vector comprising the same. The recombinant cell may be obtained by introducing (transforming or transfecting) the polynucleotide or the expression vector into a host cell.

Another example provides a method for producing endolysine, comprising the step of expressing the polypeptide in an appropriate host cell. The step of expressing the polypeptide in an appropriate host cell may be performed by culturing a recombinant cell containing the polynucleotide or a recombinant vector containing the polynucleotide. The method for producing endolysine may further include, after the step of expressing, separating and/or purifying the expressed endolysine.

Introduction of the polynucleotide or vector into a host cell may be performed by appropriately selecting a known transformation method by a person skilled in the art. In the present specification, the term "transformation" means introducing a vector containing a polynucleotide encoding the polypeptide into a host cell so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides may include all of them, whether inserted into the chromosome of the host cell or located extrachromosomally, provided that it can be expressed in the host cell. As long as the polynucleotide is introduced into a host cell and can be expressed, the introduced form is not limited. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include expression control elements such as a promoter, a transcription termination signal, a ribosome binding site, and/or a translation termination signal, which are usually operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. In the above, the term "operably linked" may mean that an expression control element (eg, a promoter) and a polynucleotide are functionally linked to perform transcriptional control (eg, transcription initiation) of the polynucleotide. . Operable linkage can be accomplished using gene recombination techniques known in the art.

The method of transforming the polynucleotide into a host cell may be performed by any method of introducing a nucleic acid into a cell (microorganism), and may be performed by appropriately selecting a transformation technique known in the art according to the host cell. The known transformation method includes electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol-mediated uptake. ), DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method, and the like may be exemplified, but are not limited thereto.

In the present specification, the term "vector" refers to a DNA construct containing a nucleotide sequence of a polynucleotide operably linked to a suitable regulatory sequence so that the desired protein can be expressed in a suitable host. The control sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and/or a sequence regulating the termination of transcription and/or translation. have. The vector can be transformed into a suitable host cell and then expressed independently of the host cell's genome (genome), or can be integrated into the host cell's genome.

The vector usable in the present specification is not particularly limited as long as it is replicable in a host cell, and may be selected from all commonly used vectors. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and as a plasmid vector, pBR system, pUC System, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, and the like may be exemplified, but are not limited thereto.

The vector may further include a selection marker for confirming whether to be inserted into the chromosome. The selection marker is for selecting cells transformed with a vector, that is, to confirm whether the polynucleotide is inserted, and selectable phenotypes such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins. It may be selected and used among genes conferring. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, and thus transformed cells can be selected.

The polypeptide provided herein may not be of natural origin, and may be recombinantly or chemically synthesized. When the polypeptide is produced recombinantly, a conventional signal peptide, a cleavage site, a tag, etc. may be combined for purification. Thus, in one non-limiting example, the polypeptide provided herein is a signal peptide, a cleavage site, a tag (e.g., His tag, GST (glutathione-S-transferase) tag) that can be commonly used in the process of recombinant production of a protein. , MBP (maltose binding protein) tag, etc.), or a purified form from which they are removed, or any amino acid residue (eg, glycine, etc.) is N It may be in a form added (bonded) to the C-terminus (eg, N-terminus) and/or.

Another example is,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

A polynucleotide encoding the polypeptide,

A recombinant vector comprising the polynucleotide,

Recombinant cells comprising the polynucleotide or a recombinant vector comprising the same, and

Bacteriophage containing the polynucleotide

It provides an antibiotic comprising at least one selected from the group consisting of.

The antibiotic may have an antibiotic effect against bacteria of the genus Enterococcus, for example, Enterococcus faecium.

In the present specification, the term "antibiotic" encompasses all forms of agents having a growth inhibitory ability and/or killing ability against bacteria (ie, Enterococcus genus bacteria, such as Enterococcus faecium), unless otherwise specified , Antibacterial agents, preservatives, disinfectants, etc. can be used interchangeably.

Another example provides a pharmaceutical composition for the prevention and/or treatment of infections of Enterococcus bacteria or diseases caused by Enterococcus bacteria including the antibiotic as an active ingredient. Another example is the step of administering a pharmaceutically effective amount of the antibiotic to a subject in need of prevention and/or treatment of infection of Enterococcus bacteria or diseases caused by Enterococcus bacteria. It provides a method of preventing or treating diseases caused by infections or bacteria of the genus Enterococcus. The prevention or treatment method may further include the step of identifying a subject in need of prevention or treatment of an infection of a bacteria of Enterococcus genus or a disease caused by a bacteria of Enterococcus genus before the administering step. . The bacteria of the genus Enterococcus may be Enterococcus faecium.

The disease caused by the bacteria of the genus Enterococcus may be selected from among all diseases caused by infection of the bacteria of the genus Enterococcus, for example, enteritis, sepsis, nervous system infection, urinary tract infection, bladder infection, prostate infection, epididymal infection. , Wound infection, bacteremia, endocarditis, meningitis, bloodstream infection, intestinal tract infection, periodontitis, gingivitis, abscess, etc., but is not limited thereto.

As used herein, a pharmaceutically effective amount refers to the content or dosage of an active ingredient capable of obtaining a desired effect. The content or dosage of the active ingredient in the pharmaceutical composition depends on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration interval, route of administration, excretion rate, and response sensitivity. It can be prescribed in various ways. For example, when the active ingredient is a polypeptide, one dose is 0.001 to 1000 mg/kg, 0.01 to 100 mg/kg, 0.01 to 50 mg/kg, 0.01 to 20 mg/kg, 0.01 to 10 mg/kg, 0.01 To 5 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 1 to 100 mg/kg, 1 to 50 mg/ kg, 1 to 20 mg/kg, 1 to 10 mg/kg, or 1 to 5 mg/kg, but is not limited thereto. In another example, when the active ingredient is a bacteriophage, a single dose is 1*10 ³ to 1*10 ¹⁰ PFU/mL, 1*10 ⁴ to 1*10 ¹⁰ PFU/mL, 1*10 ⁵ to 1*10 ^{It may be 10} PFU/mL, or 1*10 ⁶ to 1*10 ¹⁰ PFU/mL, but is not limited thereto.

In another example, the content of the active ingredient in the pharmaceutical composition is, based on the total weight of the pharmaceutical composition, 0.01% to 99.9% by weight, 0.01% to 90% by weight, 0.01% to 80% by weight, 0.01% to 70% by weight %, 0.01% to 60%, 0.01% to 50%, 0.01% to 40%, 0.01% to 30%, 1% to 99.9%, 1% to 90%, 1% to 80% by weight, 1% to 70% by weight, 1% to 60% by weight, 1% to 50% by weight, 1% to 40% by weight, 1% to 30% by weight, 5% by weight % To 99.9%, 5% to 90%, 5% to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 5% to 40% by weight, 5% to 30% by weight, 10% to 99.9% by weight, 10% to 90% by weight, 10% to 80% by weight, 10% to 70% by weight, 10% to 60% by weight %, 10% to 50% by weight, 10% to 40% by weight, or 10% to 30% by weight, but is not limited thereto.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier is commonly used in the formulation of a drug containing protein, nucleic acid, or cells, and may mean a carrier that does not stimulate an organism and does not inhibit the biological activity and/or properties of the active ingredient. have. In one example, the carrier is lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, It may be one or more selected from the group consisting of syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. The pharmaceutical composition may further include at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and the like, which are commonly used in the manufacture of pharmaceutical compositions.

The subject of administration of the pharmaceutical composition is humans, primates such as monkeys, rodents such as mice, rats, dogs, cats, pigs, cattle, horses, sheep, goats, livestock such as chickens, ducks, geese, pheasants, quails, turkeys It may be one or more selected from mammals, including poultry, such as, or cells, tissues, or cultures thereof derived therefrom.

The pharmaceutical composition may be administered by oral administration or parenteral administration, or may be administered by contacting cells, tissues, or body fluids. Specifically, parenteral administration may be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since the protein or peptide is digested, the oral composition should be coated with an active agent or formulated to protect it from degradation in the stomach. When administered intranasally, the pharmaceutical composition may be diluted and administered through a nasal spray that is absorbed into the nasal cavity through a nebulizer or a nebulizing system, and examples of the nasal spray or respiratory formulation for the nasal spray include an aerosol agent. .

In addition, the pharmaceutical composition may be formulated in the form of a solution, injection, suspension, syrup, emulsion, extract, powder, powder, granule, tablet, capsule, aerosol, etc. in an oil or aqueous medium. For this, a dispersing agent or a stabilizer may be additionally included.

Another example provides a feed additive comprising the antibiotic as an active ingredient.

Another example provides a feed comprising the feed additive composition.

The feed may be prepared by separately preparing the antibiotic in the form of a feed additive and mixing it with the feed, or by adding it directly when preparing the feed.

The 　 antibiotic in the feed may be in a liquid or dry state, for example, may be in the form of a dried powder. The antibiotic is 0.005 to 10% by weight, 0.05 to 10% by weight, 0.1 to 10% by weight, 0.005 to 5% by weight, 0.05 to 5% by weight, 0.1 to 5% by weight, 0.005 to 2% by weight, and 0.05 of the total feed weight. It may be included in an amount of to 2% by weight, or 0.1 to 2% by weight, but is not limited thereto. In addition, the feed may further include conventional additives that can increase the preservability of the feed in addition to the antibiotic 　.

In the present specification, the feed to which the antibiotic may be added is a commercially available feed, or grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starch, meals, grain by-products, and proteins. It may be selected from the group consisting of oils, inorganic logistics, oils, minerals, single-cell proteins, zooplanktons, food leftovers, etc., but is not limited thereto.

Another example provides a food additive or a drinking water additive comprising the antibiotic as an active ingredient. By mixing and supplying the antibiotic with drinking water, it is possible to reduce the number of bacteria of the genus Enterococcus, such as Enterococcus faecium.

Another example provides a disinfectant comprising the antibiotic as an active ingredient. Another example provides a disinfection method comprising applying the antibiotic to a subject in need of disinfection. The disinfectant is a generic term for agents to prevent pathogen infection, and is a general living disinfectant, disinfectant for food and cooking places and facilities, buildings such as poultry farms and barns, livestock, drinking water, litter, egg seat, transport vehicle, and tableware. It can be used for disinfection of articles, etc.

Another example provides a detergent comprising the antibiotic as an active ingredient. Another example provides a washing method comprising the step of applying the antibiotic to a subject in need of washing. The antibiotics Enterococcus spp, such as Enterococcus passive help because of the antibiotic activities against (Enterococcus faecium), Enterococcus spp, such as Enterococcus passive help objects which can be exposed or exposed to (Enterococcus faecium) It can be applied for cleaning (washing) the skin surface or parts of the body.

The novel endolysin provided herein and/or the bacteriophage expressing the same exhibits excellent growth inhibitory and killing ability against Enterococcus bacteria, such as Enterococcus faecium, and enterococcus bacteria, such as Enterococcus faecium. It can be usefully used as an antibiotic for

1 is a cleavage map of an expression vector expressing an endolysin (LNT201) gene of a bacteriophage PBEF80 that infects Enterococcus faecium.
Figure 2 shows the purification process of endolysine (LNT201) derived from the bacteriophage phage PBEF80.
3 is a photograph showing the halo zone of the Enterococcus faecium culture medium formed by endolysine (LNT201) derived from the bacteriophage phage PBEF80.
4 is a graph showing the antibiotic effect of endolysine (LNT201) derived from bacteriophage phage PBEF80 on Enterococcus faecium in vitro.
5 is a graph showing the antibiotic effect of endolysine (LNT201) derived from bacteriophage phage PBEF80 on Enterococcus faecium in vivo.

Hereinafter, the present invention will be described in more detail by way of examples, but these are merely exemplary and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the embodiments described below can be modified within the scope of the essential gist of the invention.

Example 1: Isolation of bacteriophage capable of killing Enterococcus faecium bacteria

1.1. Culture conditions of the strain

Enterococcus faecium (CCARM 5502) was used as a host, and cultured with shaking at 37°C in BHI (Brain Heart Infusion) medium.

1.2. Isolation of bacteriophage

In order to screen bacteriophages infected with Enterococcus faecium, samples were collected at Mohyeon Sewage Treatment Plant in Mohyeon-myeon, Yongin-si, Gyeonggi-do, Korea. The collected sample and Enterococcus faecium were cultured with shaking at 37° C. for 3 hours, and then centrifuged at 500 rpm for 20 minutes to recover the supernatant. Subsequently, the supernatant was filtered through a 0.45 μm filter, and a double afar layer plaque assay was performed.

Briefly explaining the assay, the culture solution of the host bacteria Enterococcus faecium and bacteriophage was mixed with 0.1 M.O.I. in 5 ml of the top agar, poured onto an agar plate, and incubated at 37° C. for 24 hours to obtain plaques. Purified pure bacteriophage could be secured through repeated execution of the above process, and this bacteriophage was named bacteriophage PBEF80.

Example 2: Genome isolation and analysis of bacteriophage PBEF80

Sequence analysis was performed on the genome of the bacteriophage PBEF80 obtained in Example 1. After incubating Enterococcus faecium in 200 ml of BHI medium to OD ₆₀₀ =0.5, the bacteriophage filtered ^{therein was infected with 10 9} pfu/ml or 0.1 MOI to lysis. Thereafter, sodium chloride was added thereto to a final concentration of 1 M, and then left at 4° C. for 1 hour. Then, after centrifugation at 11,000 xg for 10 minutes, PEG (Polyethylene glycol 8000) was added to the precipitate at 10% (w/v), and left at 4° C. for 1 hour. Then, after centrifugation at 11,000 xg for 10 minutes, the supernatant was removed, and the precipitate was suspended in SM buffer [100 mM NaCl, 10 mM MgSO ₄ (heptahydrate), 50 mM Tris-HCl, pH 7.5]. Here, chloroform was added in a ratio of 1:1, vortexed, and centrifuged at 3,000 xg for 15 minutes to obtain a supernatant.

Into a polycarbonate test tube, 3 ml of 40% (w/v) glycerol was added, followed by 4 ml of 5% (w/v) glycerol, so as not to be mixed. Here, the prepared supernatant was added and centrifuged for 1 hour at 11,000 x g at 4°C. Then, after removing the supernatant, the precipitate was resuspended in SM buffer to obtain bacteriophage genomic DNA. Bacteriophage genomic DNA was isolated using a phage DNA isolation kit (Norgen biotek corp.) according to the manufacturer's manual. Using the genome sample separated as described above, the nucleotide sequence of the genome was analyzed (LAS, Illumina MiSeq platform).

The finally analyzed bacteriophage PBEF80 genome has a total nucleic acid sequence length of 44,131 bp and 41% GC content. The full-length nucleic acid sequence of the bacteriophage PBEF80 genome is shown in SEQ ID NO: 1. Based on the genomic nucleic acid sequence information, the homology with the previously known bacteriophage genome sequence was investigated using BLAST on the Web. As a result of BLAST investigation, the genome sequence of the bacteriophage PBEF80 was confirmed to have low sequence homology with the Lactococcus bacteriophage phiL47 (GenBank accession No.: KF926093.1) (query coverage: 1%, identity 82.52%). Based on these facts, it was confirmed that the bacteriophage PBEF80 is a new bacteriophage that has not been previously known.

Example 3: Cloning and purification of LNT201 endolysine

Through an ORF search for the analyzed genome sequence of the bacteriophage PBEF80 (SEQ ID NO: 1), the ORF of 1,044 bp (SEQ ID NO: 3) at positions 1,664-2,708 is estimated to be an endolysine gene, and endolysine derived from the bacteriophage PBEF80 and Genes encoding this were designated as LNT201 endolysine and LNT201 genes, respectively.

PCR (polymerase chain reaction) was performed on the genome of the bacteriophage PBEF80 using Primer (F: 5'-ATGCGG ATCCATGAGTAAACATTTAGCAGTATG-3' (SEQ ID NO: 4), R: 5'-CAGGCTCGAGTCTTTTCGGAATT GTCAAAACG-3' (SEQ ID NO: 5)). Then, the LNT201 gene (a 1,044 bp long nucleic acid sequence at the 1,664-2,708 site in SEQ ID NO: 1) was obtained. The amino acid sequence of LNT201 endolysine encoded by the LNT201 gene is shown in SEQ ID NO: 2 (347 aa). The PCR was performed under the following conditions: step 1: 94° C., 5 minutes; step 2: 94° C., 30 seconds; step 3: 52° C., 45 seconds; step 4: 72°C, 1 minute; step 5: repeat steps 2-4 30 times; step 6: 72℃, 10 minutes. The obtained PCR product was cloned to the BamHI/XhoI site of a pET-21a vector (Novagen) with an N-terminal 6X His-tag to prepare an expression vector (pET-LNT201 plasmid) for LNT201 endolysine expression. The expression vector pET-LNT201 is schematically shown in FIG. 1.

_{After transforming the prepared pET-LNT201 plasmid into E. coli BL21-pLysS strain (Novagen), OD 600} = in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) Incubated to 0.5. Thereafter, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added, followed by shaking culture at 37° C. for 4 hours. After cell harvesting, resuspended in lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole), 1 mM PMSF and 1 mg/ml lysozyme were added and allowed to stand on ice for 30 minutes. The cells were lysed by sonication, and centrifuged at 13,000 rpm for 40 minutes to obtain a supernatant. This was passed through a column packed with Ni-NTA agarose resin (Qiagen). Afterwards, wash with wash buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 30 mM imidazole), elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 300 mM imidazole), and elution with LNT201 protein (6xHis tag). Included) was a tablet.

The purity of LNT201 protein was confirmed by 15% SDS-PAGE, and the concentration of LNT201 protein was measured by Bradford assay. The results of confirming each reaction product obtained during the purification process through SDS-PAGE are shown in FIG. 2. The molecular weight of the purified LNT201 protein confirmed through SDS-PAGE was about 38 kDa.

Example 4: Investigation of target bacteria spectrum of bacteriophage PBEF80 and endolysine LNT201 derived therefrom

In this Example, in order to investigate the target bacterial spectrum of the bacteriophage PBEF80-derived endolysine LNT201 (Example 3) selected in Examples 1 and 2, Enterococcus faecium KCTC2022, CCARM5117, CCARM5502, CCARM5506, CCARM5512, CCARM5202, CCARM5504, CCARM5514, CCARM5563, F133, F288, and F382; Staphylococcus aureus KCTC1916, CCARM3689, CCARM3A047, and CCARM3A017; Bacillus cereus KCTC1661; Acinetobacter Baumani ATCC19606; E. coli ATCC8739, E. coli O157 ATCC700927; Enterobacter cloase CCARM0252; Klebsiella pneumoniae KCTC2261; And Listeria monocytogenes ATCC19114 were tested for antimicrobial activity. In addition, the antibacterial activity of the bacteriophage PBEF80 against KCTC2022 and CCARM 5502 was also tested.

The target bacterial spectrum of the bacteriophage PBEF80 and endolysine LNT201 was confirmed by a spot test. Spot test was carried out in 4 ml of top agar (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, 0.7% agar) _{and 100 µl of each bacteria cultured to OD 600} = 0.5 were added to the LB plate. After the top agar was hardened, 10 µl of bacteriophage PBEF80 (1 x 10 ⁸ PFU/ml) or 10 µl (2 mg/ml) of LNT201 were spotted and incubated (cultured) at 37° C. for 18 hours.

Figure 3 shows the culture in this way. In addition, the formation of halo zones formed as a result of the culture was measured, and the results are shown in Table 1 below:

Host LNT201 PBEF80 E. faecium KCTC2022 ○ ○ CCARM 5117 ○ NT CCARM 5502 ○ ○ CCARM 5506 ○ NT CCARM 5512 ○ NT F133 ○ NT F288 ○ NT F382 ○ NT CCARM 5202 ○ NT CCARM5504 ○ NT CCARM5514 ○ NT CCARM5563 ○ NT B. cereus KCTC 1661 - NT A. baumannii ATCC 19606 - NT E. coli ATCC 8739 - NT E. coli O157 ATCC 700927 - NT E. cloacae CCARM 0252 - NT K. pneumoniae KCTC 2261 - NT L. monocytogenes ATCC 19114 - NT S. aureus KCTC 1916 - NT CCARM 3689 - NT CCARM 3A047 - NT CCARM 3A017 - NT

(Above, in Table 1,

○: Halo zone creation,

-: No halo zone,

NT: Not Tested)

As shown in Fig. 3 and Table 2, it was confirmed that the bacteriophage PBEF80 was infected with Enterococcus faecium KCTC2022 and Enterococcus faecium CCARM5502, resulting in a halo zone. In addition, it was confirmed that endolysine LNT201 forms a halo zone in all of the 12 Enterococcus faecium strains tested. The results show that both bacteriophage PBEF80 and endolysine LNT201 derived therefrom have antibacterial activity against microorganisms in the genus Enterococcus, and in particular, enterolysin LNT201 has a broader target bacterial spectrum.

Example 5: Investigation of killing ability of endolysine LNT201 against Enterococcus faecium

As in Example 4, when performing a spot test on the host bacteria Enterococcus faecium CCARM 5502 with the bacteriophage PBEF80 and its derived endolysine LNT201, it was confirmed that the host bacteria were killed to form a halo zone (Fig. 3 Left-most photo). In addition, it was confirmed that LNT201 clearly formed halo zones in Enterococcus faecium KCTC 2022 and F133 strains (three photos on the right of FIG. 3).

In this example, the bacteriophage PBEF80 and endolysine LNT201 in Example 4 were both tested for bacterial killing ability of endolysine LNT201 on Enterococcus faecium (CCARM5502), which was confirmed to have antimicrobial activity.

In vitro test

To this end, 0.13, 0.53, or 2.11 μM concentration of endolysine LNT201 and Enterococcus faecium (CCARM5502) 1 X 10 ⁵ CFU were added to a reaction buffer (20 mM Tris-Cl, pH 7.5) to a final volume of 200 μl. And left to stand at 37° C. for 1 hour. After 30 minutes, 1 hour, 2 hours, and 20 hours, the number of colonies of Enterococcus faecium was confirmed, and the results are shown in FIG. 4. As shown in Figure 4, it was confirmed that endolysine LNT201 has a killing ability against Enterococcus faecium at all the tested treatment doses and times, and this Enterococcus faecium killing ability was found to be dependent on treatment time and dose. In addition, as a result of performing a minimum inhibitory concentration (MIC) test of LNT201 using the broth microdilution method, the minimum inhibitory concentration for Enterococcus faecium of endolysine LNT201 was found to be 3.1 ㎍/ml (80.98 nM).

In vivo test

The ability of endolysine LNT201 to kill Enterococcus faecium was also confirmed in vivo. Galleria mellonella was used as an animal model for the in vivo efficacy evaluation. The Galleria mellonella model is a healthy group (non-infected group), Enterococcus faecium-infected group (drug-free group), LNT201-administered group administered with LNT201 to the Enterococcus faecium infection model, and Ciprofloxacin (Sigma-Aldrich) in the Enterococcus faecium infection model. ) Were divided into ciprofloxacin-administered groups, and 10 Galleria mellonella were used for each group. Infected group and infection model were infected with Enterococcus faecium CCARM 5502 with LD ₈₀ concentration of 2x10 ⁷ CFU/larva, LNT201 group administered endolysine LNT201 at a dose of 15 mg/kg 30 minutes after infection, ciprofloxacin group administered ciprofloxacin. Was administered at a dose of 8.3 mg/kg. As a result of observation for 72 hours, the survival rate of Galleria mellonella is shown in FIG. 5. As shown in FIG. 5, it was confirmed that the survival rate increased by 2 times in the LNT201-administered group compared to the infected group (non-drug-administered group).

From the above description, those skilled in the art to which the present application belongs will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

<110> LyseNTech <120> Novel Polypeptide and Antibiotics against Enterococcus spp. Bacteria Comprising the Polypeptide <130> DPP20200615KR <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 44131 <212> DNA <213> Artificial Sequence <220> <223> PBEF80 genome <400> 1 ttaaccggtg gatatttaaa ccttactttg aaagatgata gtggtactta taacggaaca 60 ctcgatgcga aagcattaac gaatatgccg tgggtcaatc tagtaatgga tagcgcattc 120 gaaacagcgg aatcaaatcc accgcaatat aaaatcattt acaatttgga cggtagcaaa 180 accatcaatt tccgtgggca agtaaaactc aaatcgggaa caatgagtcc aggaagagca 240 taccaaccat ttaaaacggc atccggagcg cattcgttgc cggcagaggt acggcctaga 300 tcaacaacat tcgcatatgc gccagttggt gtggctagtg gaacaacttt cggcggtcag 360 tgggctatca catcatccgg atatttctca ttaggggtag cgcaatcaaa caccacctat 420 gccgttttgt cgccgataac ctacgttata gattgatcat tttcgattat gctatccgac 480 aacaaagaac aacggttatg agattatggc ataatgaaaa ggaaaggagg tggaagccta 540 tgcattacac gaacaattca gtcgatgaaa gagggaaacc ggtataataa caatccgggt 600 tttccccaat ccggattgtt tcatgtggaa caatcggagg tggaaaaagt gaaacgagat 660 aatagactaa gctataaagt tggttttgtt tccttttttt attggctatt tgtatatatc 720 tatccaacgg atcgcctcat ttccccggat gggattccgt tgaatgggat tggtttgata 780 attgataaat ttaacgatgt gtttgtttct ctttttgtca tgatggttgc atccctttac 840 ttggtggcca tctatgcatc gaaagagaaa ctaagaatcg ttatggccgg cattttgttt 900 ggcgtatggg ttacgtcaac gagtgcgatg gtttggtggg ctttcatgcg tggcctagtt 960 tcgccatcat tagcaatcta cgccggatat tgcgtttcaa ttttcattga tattcggacg 1020 ggggttgata ataaatggat tggcaaaaaa tagttttggc cataatcgca atgatttccg 1080 gatcatatgg ggtcattaca ggtttcatca actacaaaaa agatgtgaac aagggaagaa 1140 accaacgtga tcaaatagcg tgggaaacga tggaaaaaca atacaaaaac cttgaaaaaa 1200 gttatgatga tctatcgaaa aaagtttcac agctagaaaa aaagttggat gaattccggg 1260 aggaaaacac agcgaaagac aaaatcattt tagaaaaaga tcgaatcata atgaaaaaag 1320 atggcgaaat cgaagacttg aaagaagagg tttccgatct taaaaagaca aataaagcgt 1380 tgagagttaa aatggcagag atgggaggtg tcaaaacgtg aaagatcaga tcatgcaaat 1440 gtttaatttg tcggacgttg cttataaccg tttgaaatgg acagtaagca ttgttttacc 1500 ggcaatcatc acttttttcg gaattctagg tcaaacaatg ggctggaatt tcactgataa 1560 agtgatcacg attggaacgg cttttgttgc gatgttaggg atcattttag gtatttcaac 1620 aaacgaatac aacaaaaacg aagaaaacaa ggaggaaaac aaaaatgagt aaacatttag 1680 cagtatgcgg acatggacag ggcgatcctg gagcaatggg aaacggatat ggcgaagcgg 1740 aattcacacg cacaatctta aagccaaaaa tggaagcatg ggcgaagaaa ttgaaacata 1800 attcaattga atggtatgac acaagcaaag acatgtacca aaacagcaat gcaggcggtg 1860 gagcgtattc agtcagcaca agttatgcaa gtgtgacaga attccatttg gatgcagtag 1920 cgagtggaag cgcaacaggt ggccacgtga ttatctcatc acaattcagc ccagatgcga 1980 acgatctagc tatcgcaaaa gttttggaga aatatgtagg tttgtgggga tcatcaaaac 2040 caacaggaac atataaacga aatgacttgc tgaatttgaa tgtattcgca aaacgtggca 2100 tttcttatcg tttagttgaa ttgggattca tttcaagcaa aaaagacgtg gataacattg 2160 tgaaaaacat cgatgccatc gcaaaaggta tggtcgaagc gattacaggg gaaaagatcg 2220 aaggtggttc gtcaaacaat aacagcaaca acaatgcgaa accatcaaca ccaaaaacac 2280 acgatcaaat catcgcagaa agtaaaccaa aaaccgttgg aaactacgtt ggtaaattgg 2340 aagtattcaa cgaattaaaa tggggcattt tccggattgc tggttggtta gtgccgatca 2400 acggtgcgcc ttatttgaat catggatatg tattttggaa agaagcaggc acaggaaaag 2460 aaattggacg ttgcccatca aaaggcatca tgagaaatga tgctaataaa gcatatggat 2520 tgcctaacgg cttgaaattc ggccttgatg gcacgctaga cattaagaaa tttgcaggta 2580 aaaaagtata tccatgttta cgcagaacaa acgacaaagc aggaaacagc attgcaggca 2640 aagcaaatgg caatggttgg atcgatatcg agtttccgga atacgttttg acaattccga 2700 aaagatagtt tattctttgt tttgttgtaa taattggcat gttacttcct aagtgaaaag 2760 ttcggtcaga aatggccgtt ctttttttat ttatttttta tgtaataaat tgttgcaaat 2820 tgtaacatat catggtatac ttaattcata aagttaaagg aggaaaaaca aatgaaaaaa 2880 gtagaggtca acggatattt caatttatac aaatgcaaag atgggatttg ggatggtgct 2940 tttgtttggt caaacactga ttattcatct ttatacacgt ttttagttga tgtggatagc 3000 ttggaaaaat tgggttacga ctacgaagga tatagacttg tagaggtcaa cggcatccga 3060 tgcattccaa tgacattttc aaatgcagaa gtcgaaagag tttgttaata aaaaaatgcc 3120 gggtgtaaaa tcccggcttt ctaggaggta aaaaatgaag cctagtgtat ctttggaaaa 3180 atacaaagaa attatttcgg gatgtgatca tcaagaatta cacatcgaag caaaaggatt 3240 tttgaatggc ggaataaatg aacacaacga agcatatgcg atcgctttat atgggcggtt 3300 gtgtatgtta ggcagttgga tttcagcatt attaggggag gaaatagaga aatggaaaga 3360 aagcaagtga acacagcaag gattcgtgta caaatgaaaa tgagagggga aacagatgaa 3420 acgatggcaa agcgtttaaa cgtttcacgt gtaacataca acaaattgat caataacaaa 3480 tacgtttccg gcaacgccga tgcttatatc gaaaccatcg aaaaaattct tggcatggat 3540 caaaatatca gataaaaaag gagaatgaaa gatgatagat tttggaaaag tgaatgaaaa 3600 tattttgcgc acacaggatg gaaaagaaac aaaagaatac acttacagcg caactgatgg 3660 cgaacgtggg atgtcagatg aagaagtttt gatgtttcag ctacacaaag aaaatgatga 3720 actgaaaaaa atgatcgagg aatcgaatca agtgatcgat caagcaggca tgataaaatc 3780 gcttgcaaag atggcgcaaa agatcgcaaa agtgatcgag caaacaaaaa cgattgaaaa 3840 atccggttgg aacagtttcc acaaatacga gtatgcaaca gaatcagatg tgaaaaaagg 3900 cgttcgaaac gtaatggccg aaaacaaact gattttgatg aacaacgtta aaaaatacga 3960 agaaaacatc gttcagacga aaaacggcgg aaaagaatca caagtgaaat tgcacatcga 4020 atatactttg atcgacacag aaacaggatt ttcaaaaact ttcacaaaga ttggtgtggg 4080 tcaagatgca ggcgataaag ctttctacaa agccgaaaca ggtgctttga aatacgcttt 4140 gacaacgttg ttcttgcttc ctagtggcga tggcgatccg gaaatcaaca gccaggaaaa 4200 cgatggcaca gatgcaaaaa gcaaaccgca agaaaaagac ttgctgaaaa tgaaaacaac 4260 agccgaattc attttgaagg caacgaaatt agaacaacaa aaaacaatag tcggattttt 4320 gcaagacgta gccaacaaaa acaatatttt ggatgataaa ggcaatgcaa aaaatccgga 4380 tgatttaacg gtggcggatg tggccaaaat gaacaattat ctttctaagt ggaaattgaa 4440 agtcattgat gatctgaaaa cacagcaaaa acaaaacgaa caaatggata gagagagaat 4500 gcaacagcaa acagaacgac aacatcaaca agcaaaacaa caaaaagatg caaaaagcat 4560 tttcgggggt agataataat ggcaaacgaa attcaaaaac aagataacaa cattgcagac 4620 gttttgaaaa tggagatcac gccggccgtg atctctttcc caggggaaaa agaattgcgt 4680 gcttatttag atgcagaaat gaaaaaatat aaaagcctgg taatcactga cgagaatatc 4740 agcgatgcca aaaaagcaaa aacatatttg aataaaatga aaaaagcgtt aaatgatgcc 4800 agaattcaaa aagaacgtga agtgatgcaa ccttttaacg aattcaaagc atcaatcgaa 4860 acattgatca aacagcttga tgaaaccatc gatccaattg atcaaaaaat caaagaagcg 4920 gatatattgg caagagagca gaagaaagag aaaatcaaaa aagagatcat ggaaataata 4980 gatggccacg gtatcgcaga agaagaagca ggatttttcg agatcgaacc gaagtggcta 5040 aacaaaacgg caaagatgaa agatattaga gaatcactcg tcatgcaagc aagatcatat 5100 ttgcaacaaa agaaggaata cgaagacggc gtgaaactta tccacaacct atccgaaacg 5160 cttgacgtga cagaagacaa ttacatcgaa ctttttaaag acggcatgag catcgatgat 5220 gttttagctt tcatgaaaag agagaaagaa aatcgtgacc aattgcgaga aaaagaagag 5280 aagaaacgcc aggaagaaga gctaagaaaa tcccaggaag aacaaacaaa acaagaagaa 5340 caacccgatc cagatttcga ttatttggcc ggcattgatc cagaagaata ccagggagaa 5400 aaatcagatg tgcaattcga tcttgcaggg ctaaaaggtt ttgattttga aacaggagaa 5460 gtctttgaga atgatttgcc ggaaatagaa agtgacgttc aaacggtcac tttgaaacta 5520 agtggaacat atgaaaattt agagcgtttg aataacgtca tgcaagattt gggaatttca 5580 ttcgaattga tcgatcttgc gaatgattat gtacgcatca gataaaccat tcaaatttgg 5640 aacattttcc ggccgatttg gcgttttaaa cgtccggccg gctaattatt catgaaaata 5700 gtaaaacggc ttaaaaacga aaagaggtgg cattttgagc atcgggaaaa taatcggtaa 5760 aaacgaaaaa tatttgatga tcaaaatgga tgaagagata aacgaaaact acatgaagtt 5820 gcttgcagat ggcggaagca attggatcga tgtccggttg atcgataacc ggccacggtc 5880 agtggtgcag aatgcgttat ctcatgcgct tattcgagat atcgcaagaa gtcagctgga 5940 tgatccacgt tatatcgaag aagtgttgaa atacgaattt tatgaacgta ccggcatcga 6000 cttctttcat tccgtggcaa ccgtagacga agccaggaaa tggatttcgt ttttgattga 6060 tctcatgctt gagtttcgta ttccattcaa aaaacgatat gcctatcttt ttgaggattc 6120 cacctggttt tatcaagcat gcaaacatcg tgtttgtgcc gtttgtggaa aagaacatgc 6180 agacatccat cacataacgg ccgtaggtaa cagaaaaaga aaactagtcg atcatcgatt 6240 gtttccgatg gttgcgcttt gcagggagca tcatcaaaaa gcgcatgaat taggtcaaat 6300 attgtttatt aagcaattca aaattatccc ggtttattta tcgggcgttg atctcgttaa 6360 gatcggcatc atgtcaaatg cgcaaattaa aagattggat gaggtggttt gatggaaaag 6420 agaatgaaag cattagtgtt gatcgatttt gccaggtaca aaaaggattt atacaagacg 6480 aaaaagaaac ttgaatcatt tgcagatttt ttgtttagtg tacacgatga atatatgcaa 6540 ttttcggcag ttaattatct aggggaaacg atagcaagag aatatttccc agaagaattg 6600 aaagacgatt tgcatgccat cgtaaaaaat gcaatcgaag ggatcaacaa agagatcgat 6660 caagcggaaa tgattattca aaattatcga gcagacgaag caagcgaagt tttgaatgct 6720 tacaacaaag ccagaatcag aagtgaaaaa ataaaaagaa aaggcggttt ggtgtaatga 6780 agcaaaaaat atcgaatgaa cgaaaatacg tactgatttc aaatagaaat aaaggcgata 6840 aagataaatt gttgttttgg ggtcaattga catgggatga tgaacggcga agcttttcgg 6900 gatatactca cgatttcact aagtgcgaaa gatacacgtt ggaagaaatt caaaaagcta 6960 atttcccgaa agaatatttg gaaaaattca atctcaaaga ttgggtggat tttaaaggcg 7020 aaacatttta ttgtaccgca ttagaattga tcgaaatcat gggagcgcct aagactgttt 7080 ttgtttatta atagcaaagg ccatctttct tgaaaaagaa atttggcttt ttttaatatt 7140 ttacttgtgc aatcagttgt acaatgatac aatgatcttg tgaacaacta attgcacaaa 7200 aggaggggat gaacatggca aacaaagtgg atcgtgtcga gttggaaaaa atgcgactag 7260 acagcggaaa agagtggaaa gaggttgccg aagaagttgg catgacaaag tattatcttt 7320 caagattgat caatggcgga acaggtcaag aagccgttga taaatggtta ccaaaaatca 7380 aaaacgcttt gaagcgataa aatcgtttat aagacgtttt gaagcctttt ggggtaatta 7440 tccatgaaac ggattaaaac gccggaaaca aaaaaagaag tgggagaggg aacgaaatag 7500 catggcaaag atgaaaaagg taaaaaaaga tcattttgtt gtgattgata gcaatctgat 7560 tcgtgatgat cggctttcgt ggaaagcacg tggcattttc ggttatttgt ggtcaatgtc 7620 cgatgattgg gaattctacg aaaaagaagt gatgaagcat tcagcggacg gcctggcatc 7680 tttacaaagc ggattgaagg aattagaacg atatggctat ctaaagcgta tcaaggttcg 7740 tgacaaaggg aaattcaaag gatatgaatg gttattgacg gatacgccag aaatcatcaa 7800 aaatgaagaa aatggcgata tgccaacgaa tgaaccgaaa tcagaaaatc cgatctcgga 7860 aaaaccgttc tcggaaaaac cgttctcgga aaatccgata ctaagtaata taaataataa 7920 gcattatcaa atacaagaaa aatcaacaga agaaaaacct atgagcgaat caaatgattc 7980 actcgcagaa gcgttcgaaa agctttggaa attgtatccg aacaaaaaag gcaaaaagcc 8040 ggctttcaca gcatacaagc gagcaatcaa aaaaggaacg acaaacaagc agattcaaga 8100 tggcattgtc agatacattg cagaaatcaa aaaaagacgt acgccgatcg acaaagtggc 8160 acatggatca acgtttttca atcaagaaag atggttggat gattatgatc aacaagcaac 8220 agtttcagac ggtggaaaca aatattcgaa tttgggatgg tgatacctgg tggaaaacat 8280 gagagattca atgcaaatgc ttatgagcaa gttcatgatc aaaacggatg aaatgtgcga 8340 aatccacgga attgcgaaag tgattgccag aaatcaaaac gacatgaaac ctttttgtgt 8400 tgaatgtcgc aggttggaaa tcgaacagca aaacaaagaa agagcagata aggcattgca 8460 agcagatatc aaacgaaaaa cgtatgatgc actgaaatat gattcgattt gctcggatga 8520 tgatgtttgg aaagcatctt ttgacacgtt taggacggat gggaaagatg cagatgcttt 8580 gaaggtagca aaagacaaag ccagacacat tgccggagag tacctggcga aacgaattga 8640 agttgacgaa aaagggaaaa agaagatcgt tagaaacgaa tttaacacga ttttaacagg 8700 cccggccgga tcgggaaaaa gccatttggc catgtcgatc ctcaaagccg tcaacgaaca 8760 ttccgatccc tggcaaagtt gcattttcgt ttcaacagtc gatttgctga tgcttatccg 8820 ggatagtttt ggcaagccag attcgaaatt ttcagaagca aacatgatca atttgttaac 8880 aaattgcgat ttgctcgtat tggatgatct aggaacagaa tcaagtttgg taagaagtgc 8940 gaatgaatca tcggattttg tgcaaaaagt tttgtttagc atcttatcga ggaaaaggac 9000 aatcatcacg acaaatcttt catcgggtca attgatggat atgtacaatc ctaaaatcat 9060 ttctcgaatc aataagggag taggatttga cggaaagcac gtgatcaaga tgccggatga 9120 tgtcgaagac aaacgcattt tattctaatg tttcacatgt aacaaaaaat cggagggaaa 9180 ttttatgaaa ttagttatca acagcgaaaa tttgaaagta tcgatcgaag caaaaggaaa 9240 tttggatgaa cgtcatgtga tttcagcgta tcagttagca acaggagatt tcaaagcgtt 9300 gtttgtggaa gaagaaacaa atacacaaac gatcaattta gatggtgcag aagtcgcaaa 9360 aggattgctt tcacaggttg aaagtccttt tcaaaaaggc gtaagagaat ttaaggaagg 9420 caaaaagcct tttgatccga aagaagctga atgggtacaa acagaataca tttgtccggc 9480 gtgtgggatg gaagggagaa gaaacacgat aaaaacaaac aaatatgtca aatgctatga 9540 atgcaatacg aaaatcaaac tagaaagagc cgtgccagaa aacgaagaaa aagaaacgga 9600 caaagcagga aattattaca tcgcaaatga tttctttgaa attcaataaa taatcggatg 9660 aaaggtggaa aaaacatgtg tgaagtatgt aaaggggaaa aagtggtagt tgttaacgtt 9720 ggcggaggaa ttacatcatt tcaaccttgc ttgaaatgca acgaacctcc ttacaatgaa 9780 cgacacaaag aaacatgggc agatttggag cgcattttga gcatatgcga tcctgatact 9840 aaaatcgatt aaatatatag gcgttgctta aaacgcctta aaacgaaaaa tagaggctaa 9900 ttcggagggg taggaaatga caaaaaaaga gtttcgtcgt ttaagatcga tttatcttgc 9960 aatgtatgat caaacaaaaa atacattttg gttcgataaa gtcaaagaac taacaagaaa 10020 gtatgaagaa gaaaatgact atcggcggtt tgatggttat ataaaaatcc actatccgga 10080 cggatcggaa tatttgtatc gcgatttgga gcttgcaggc gatcaattta aattttccgg 10140 atcgaaaatg tttgacatgg caagagcagg taagaagtta agcgatggga cgtttttcac 10200 gttggaagaa atacctaaga aaaatcgttt ggtaacattt gaatatccgg acggttgcat 10260 caaagaattt tcatctatcg taagcgctag caagtcgatc ggattttcaa ttgatacaat 10320 ttctaggtat aagaattcag ataagttgtt gttaggggaa atcaagatca caacgttttt 10380 gcatgaagaa ttataaaaaa atttatcttt ttattgtaca atcagttgca caatgatata 10440 atacatttgt aaggacaact agttgcataa taaaaaaggg agaggtggaa aatgatgtgg 10500 gcaattggat ttttcgtttt tgcattcatc acgttggctt tgctatataa cgaatggcaa 10560 aatgagaaag aagcaaaatt ggaagctttg aaaaaaatca atgatttgga aaaacaagtg 10620 cgagttttgg aagaagtcgc aagcatcacg aaagaagacg gagggatgga gttttgaaaa 10680 aatggtcgga gtttgaaagg gaaatagaac aaacaaatgc atattaccaa agaaaaggcc 10740 gtggagcggt ggcgaaattg ccgaatggtg tgcaaacagt caaacaagca ggcacgatca 10800 aaaatttccg ggtaaaaacg ccgtgtgatt tcatcggaca cgttgacggc gttccggtgg 10860 ctttcgatgt taaaacgaca tcaagaaaga ccgcatttcc aattttcacg agaggaaaga 10920 acaacaaaga aacatattcg cttaagaagc atcaagcaat gtttttatca cagttcgaca 10980 acggcggaag aggttatgtg ctaatctatt caattccgaa agatgtatcg tggcttgtgc 11040 cgatcgaaga atatttgatg ctttgccagg caatgaaaaa cagcggtaag aaaagcatta 11100 gattcagcca ttttatcgga tatgaaatcg aaagaaaagg catgttttgg gattatgccg 11160 aaaaaattat cggaggttaa aaggatgtat attatagggt tcataatcaa aatgattctt 11220 gcaatcatcg tgattgcttt cgttggtttt ggtgctttgt ggatattgtt tccagaaaat 11280 ccgcttgttg tttgggtcgt tgcaattgct ttgccggtta tcatttggca agggtacaaa 11340 gaattggagg aaaatcgatg atcgatgcaa acgaagtgga aaagattatc ggcgttcggc 11400 ttgatgattg gcaaaaagaa atgatcgaca attctaattc gatcaatttc aatgcaaatc 11460 agaaaaggtc ggggcgtgga cttgtgatga tcgttaaatt tttattgcag gttggccatc 11520 catattcctt acctaaaaaa tcaatgttat tagataaaaa taaaatttat gcaatcaatt 11580 atttcgaaat gatggtttat tgctatgaaa aactaaaagc ggaaggcatt ccggttcgca 11640 cgcttaaaaa tacggaagaa tatgaaagag agggaaacaa gtgatctgta taggcgcaaa 11700 acaaccagga gggaacgcac aacctattcc gatcagtgcc aggcctattg atatggtcga 11760 cttgctgaaa cggctaaaac gagcttacac gcaatttcag caagtatcag aatatatcta 11820 cgttaacgga ctgacattga tcgatgtaga actcgaaacg gaacttgatc ggccaatatt 11880 catttgcagg aacttaaatg gcaaactatt gtatctgttt cacgatggtt gtatcaaaag 11940 tgttcggaat aaaaacggta acatggaaat cgaagaaaat catttgtggg attggaggta 12000 aagaattgat atttgttagc tatttgattc ttgtttcgtt ggtcagcggt ttggggattt 12060 ttacgatcag cacgtttttt cgccaggcgt ttaatcacga tcaagaaatc gataatttta 12120 atgcaatatt ttggtttggc atcgcaatcg ttttggccgt tgtcatattg gcttgcctgg 12180 tattcatcgg attggagttg aaacagatat gaaaaagcat gtgttggaaa tcgtagtcaa 12240 aaagaaaaat gtgatcttga ggagaacagt caaacatgca ggatcagaaa aaatcgcaag 12300 agatgtgttt cacgtggaac atgagcgact aagaaaaatg caagtcgaac aagacgtgaa 12360 agaaatccaa aaagcgcaac ggcgtggctt ttatgatgtt tatctaagat atatcaagat 12420 gaaccaaaaa acgaacggtt tgcaggtgtc ggaaagcttt gtggaagaac atttcacttt 12480 tgccaacgat caaaaattcg ggaggatcga tgatgttagc aaagcaaaat gatccgcatc 12540 attatgcttt acttgataaa atcaagccaa ccgaagaaat ggaaaaattt agaaacgaat 12600 attatacgaa agaggtattt gggaaatgga aataattaca agagaagaat tcacagtcaa 12660 aatcaaaaaa atgttagaaa tgcaagatgc acttgatcaa gcaatactca aagaaaaggc 12720 gctaggccta acgaaaaacg attggttctt tttaactaaa acggcactta ttgtcgaagt 12780 gtcagaagcg acaaacgttg cagaatattt caagcattgg aaagatcatc gaggaaacaa 12840 agcagatgca ggaaagacac atgaagaaac gttgtgcgaa gaattttcag atgcatttca 12900 ttttctgtta tcgcttatta atcaaagcgg atcattcaaa tcagccggat catatttaga 12960 caaacaaatt gcaggatggt ttaacgatga tgcaattaat tcaaaaacag gatatttaga 13020 tcagtttgaa acaatataca atttcaattt tgatggatca tcgacagcga tgtttttcag 13080 tattttaaat agcgaattat caagcctatc tttgatatca atgccatccg cttatatacg 13 140 tatgcttttc gattttctag cttatgcaaa gatttgtgat gtaacattca acgatttata 13200 cgaagcttat atgatcaaaa ataagatcaa ttatgaacga cttgaatcgg ggtattgatc 13260 gatgcaacca gtatatattt tgtgggctgt atttatcgca atttttattg gtgtaattat 13320 tttcaaagat ggcgaaggag attagaaatg ggaaaacgtg gcaaacgaat caaaaagaaa 13380 aacagaaaac ggaaagaatc acgtgaaatc aacgaacaaa aaacaaagaa agtgagtgat 13440 caaaatggca ttagtggagt acacagcaac aaaagtcgtt tgtgatattt gcggagcgga 13500 tgcaaatgga tcgtggtatc gaagggaaga agtgaacgga ttttcaaata gattatttgt 13560 aagccctatt gatctatgtg atacacattc gaaattgtat gatcaaaacg aagaattcaa 13620 aaaatacgtt gttttagaaa ataaaaaaga tgtttatcca caggaaaaga aagatgaatt 13680 gattcaagca atgaaacaaa aatgggaggg catggaggaa tggcaaaaag atcaattgaa 13740 gaacaaccat tgatcgcaga aatcaaagca aactacacag ggtttgtgaa taactctttt 13800 atcggaaaca taagcacaag cggacatccc aggattttgg atggtttatt ggtcaacttg 13860 attgcagaca ttatcaaccg caaagaaacg gacgaagagc gccacaatct ttgctatcag 13920 attagtcagg ttatttcaga tcacttgaag caggaggaag atcgatgaaa tatcgggttg 13980 gggataggtt cgtggcaaga gtgaaagggg agttccttat tcgtgaggtg gtcgcagttg 14040 attgcgatca tccacacgga tatcattata aaatgatggc cagcgattca aaaccgtttt 14100 ggcaaacaga aaagcaattg gataaatata ttcacgttgg aaatgttttt cggatagaac 14160 gttcttacga aatatacaaa gcaacacaag aaaaaaaggc gtgaagaggg gttgtggtgt 14220 ggaaatgaat caatttagcg caggcgatcg gatcatttgc aaatattcgg atgttactga 14280 ttggatttac aaagtcgaga aattcgaaaa gaaggatcgc aatggcgtgt atcatttact 14340 ttcgccatcc ggtgtgaatt ttgaatgtca gcaagcgaac gtggaagatg tttgcgaatt 14400 gtatgagaaa ggacgttaac aaatgacaga aatcgaatgg gcgttgttta tcgtagcatt 14460 atttttcggc aatatctcat ttcgtgatag acaaggcgtg ccacgatttg aatatagcgg 14520 attgattgca tatctaatca aaaaggcttt tgaagctata aaaacaatga aagaggatga 14580 tcgatgatgg aaaaatggca atatatgtgc gctttaaaag cagatcaaga agcatggaaa 14640 gcaacagagt gtttcaatac aagaatcgaa gcaattaatg ccggcattca aggtttggca 14700 aaaaaagacg aaggcatttt cggagaaatc ccagaaatag gaacgacaaa attcgcagtt 14760 ggcagaataa acgaatacaa aatcaatttt gaaaacgtgg cagaagagat catggagatc 14820 ataagcgaag atgcctatga ttatctaggg gtatcggcaa cggactatct cgaagacgtt 14880 gaggacgatc aaaagaaaag atttgcaagt gtgatcgaaa aatttttcga ggaagaaaaa 14940 ttgatgccga aatgtttcaa catcgttgac attgaagaat acgacatcac ggacttattc 15000 ggatcatacg tgaaaaacaa tacatttccg gattttacca agaaagaatg gagaagcggt 15060 tttgatgcag acatggctta aagaataccg aatgaaacaa gcaaacgagt tgatccgaat 15120 gatcgcaaat cgtggccggc gtttctttga ttatacgaca gttaagagaa tggataacga 15180 agcacttttg gggagtgtgt cgcatttcat tatcaaaaac aaccgtgtct atttcaaaga 15240 tggttataca cagaaagaaa tatttgctta taaccatctt tacatgaaag aatttacgcc 15300 aggcggaaca ctcgaagcgc ttgtgttaga tttgtcggag ttcattcgaa caggcgaacc 15360 aacaaacgga aacaacggtt ataacggcat attttgccat catgaaaatt ggggctattc 15420 gatcgaagaa atggaagaga tcagagcgta tgcaaaggaa atcggatttg ctaaggagga 15480 atgaaatgga agggttgaat ttgaaaacgt tcgttgtggt aaacggcaat gaatacatgg 15540 tcagcaccgt ttatgtttat gttttgggat gggaaacaat ggttttcaga acaatcggcg 15600 atcaaacggt tgtggattgg caagaaatag ataaatatac caggcgttac ttttccgagg 15660 aaaaagcaag agaaggccat cgggaaacga tcatcgcttt gaggagggat gcttgatggc 15720 tgacatcgtt ttccggatag cgtgtggcat ggcaattgta ttgctggcta tatacgcaaa 15780 aaaagaattt aaagaaacgt caagaagaaa tgaacagttg cgcagaacaa tcgcaggaag 15840 aaggagcaaa tagaatgaac caacgccaga agaaaaaaca actgaaaaag atcgttgatc 15900 taatgaatca aatcgaaatc gatgattatg acggccaggg aaatgaattg ttttatgttt 15960 tggccgaagc aaacgaaaaa catcaagcaa tccttgaaaa aatatgcagt ataacaaaga 16020 cagacaagtt actcgttcac atgatgtacg aatacgaagg aatgatggat gattttgaag 16080 attgggtcgt acaattcgat ctaacggatg tatggatgct tatcaagcaa tggccgaaga 16140 aatatgacat ggcttataat tcgaaaaaag gatttgtgat cgtgagggat cagatgtgac 16200 aaacaaagag atcaaagaag cattgcaaca aaagggaatg ccgatgttaa gaactatgaa 16260 aggaaatcta gttttatatc taccgctagg atcaacagat cgatataaac caatcgcaga 16320 aacctggcac aggaaatgga tcatccttga tgatattgag gttttgggaa acaaggcgtt 16380 tgatgtaata aagctattaa ataaatggga tcataaaaac taattgagga tgtttcacat 16440 ggaacattag tgtataatga acgaagatgc cacggcatcg atcatttggt ttcttctttc 16500 atgattcaag tccgattttt gttggagaag tcggcaacga tgtggattga ttggactact 16560 cacaattttc catttcttat cctatcgaat tcggccgttg gtggtcggga tgaaggtttg 16620 cttaacagca aacctttttt tgttggtttc cgtgtataat agacgtatca aatgcaagaa 16680 aggagaaagg acatgaaaaa caatgatgaa atgatcggat caaaagaaat aaagatcgtt 16740 ccgatcgatc tagtgatccc ttacgaagaa aatccacgct tgaatgataa ggcggtggag 16800 tacgtgaaga attcaatcaa acaattcaaa tttgcacagc cgatcgcagt cgataaagac 16860 ggcgtgatca ttgcagggca tacgagatat agagcatcga aagaattagg cctggaaagc 16920 gtgccggtgg tcgtattgga ctatttgacc gatgaagaag taaaggctta tcgattggca 16980 gataataaag tggccgaatt tgctgattgg gattttgaaa agctgaattt ggaactagac 17040 agcttactag atacagaaat cagcatggaa gattttggct ttgcattcaa taccgaatta 17100 gagttcgaag acgaagaaga gcaggaagca tcggaaatga tacagatcaa atttgcatta 17160 gcaccagagc aagccgagat catggaaggc gtgttccagt tattagaacc gaactcgaag 17220 acatacggca acgaagatca attgggaaac aagttctttt cattagcgga gcagtgggct 17280 aagatgaagc aggaagcgag gggatcagat tgacgaagaa gaagccggcc gatcaaaaga 17340 agcgtgcagg ccgtaaaagc aaatatgaaa cacacgtcaa gccttacttt gagcgcatcg 17400 agctgatgaa gaagaacgga tatcatgacg aagacatcgc aaaagcattg gacgtcaaca 17460 agtcgacatt ttcatccttt aaaagcaaat atcccgaatt aagagaatta ttggaaaagg 17520 caaacgttga tcttgaattg aaagtgatcg attcactagc gcaacgagcc gtagggatga 17580 gatacaccga aaagaagaag atcatgcgca agaaagaagg ccaggtggta tttcaagaga 17640 tcgtggaaac agagaaattc ttaccaccag acgtgggagc aatagcgtta tacatgaagc 17700 gtaacaacat ttacaacaca gcagaagaag aattgacgaa agcccggatc agaaaggcca 17760 acgccgaaac ggatatgcta accggagaaa gccagggaga agatgcaaac aattgggcgc 17820 aaggcgtgct aagccttaca gaaaggcaaa ggatggccaa cggagagcgc aggcgcatct 17880 ttgatgatga ggaaacggac aaaatggaag agtagcgtta tttttatttt gtaccgccac 17940 cggagcgaca tcaaacaaaa gtattgatat atcaacaatc acgaaaaaac ggcaatttta 18000 acagactgtt atttttgcca gaaaggaaag aaatatgact gaaaaagata aaaccgtcga 18060 agcagataag atcgattggg cagagattgt cagtgtatac tacaatgatc cagtcgcatt 18120 cgcagaagac tttttgaatt tcgtgccaga tgattggcaa gcaaaggtta tgtatgatat 18180 ttgtgaccat ccattcgttg ccgtcaagtc cggtcaaggt gtggggaaaa caggggtcga 18240 atcagcagtg atcttgtggt acttatgcac aagaccgaat gcaaggatcg tggcaacagc 18300 accaacgaaa cagcagttgc aagatgtctt gtgggcggag gtttcgaagt ggcttgtaaa 18360 cacgaagctc gaacgattga tcacgtggac gaaaacaaag atatacatga acggtcatgg 18420 ggaacgatgg tttgcagtag ctaagacatc taaccggcca gaaaatatgc aaggctatca 18480 cgaagaaaac atgatcttta ttgtagacga agcatccggg atcgatgatg aagtaatgga 18540 agccgtattg ggtacgttgt ccggtaaaga caataagctt ttgatgatgg gaaatcccac 18600 gaaagtttcc gggatattct acgatgcatt tcataaggat cgcagagatt acaaaacaca 18660 cacagtcgat tccaggaaca gcccaagagc atccaaagaa aacatccggc gtttgatcaa 18720 tcgatacggc gaagaaagtg acgtggtggc agtccgtgtc aaaggcgatt tcccaagtgg 18780 agagcctaac gcattcattt cactgaccga agtagaaaag gccgtatcaa gagatttgga 18840 aggctacgag aaacgcaggt tcgatgctta caagttttat acaaacggcg tgatccaaaa 18900 tatcatcgat attccaaaag atgcagaaat acggatcggg gtcgatattg ccagacatgg 18960 ggatgatgaa acagttctcg caccacgtgt cggcctttac tcgctaccat tgcaaacatt 19020 cggcaagcaa gacttaatga ccaccacagg gcggatcatg ctaatggcga aggaactaca 19080 tgagatatac aaccgcaaga tcgcaatcac attggatgat acaggggtcg gcggtggtgt 19140 cacagaccga ctgaaagaga ttgtgacaga agaaaatttg gattggatgc aggtcgatcc 19200 ggtcaatttt gctgaaaagg gtaacgatga ttatatgggc gttatctctt tgatgtatgg 19260 gaactttaaa gattacatcg attccatcaa gttgccagat gataacgaca tgatcggaca 19320 acttacaatc cgccggttcg atgtcgaaag caaagggcgc atcaaaatcg aatccaaaaa 19380 ggaaatgaag cgccgtggcc atccgtcacc ggatagagcc gaagccgttg taatgtcgta 19440 ttataaaccg ccattcgatg ctactcaatt caagataacg ccacaaatga tcaaaattaa 19500 taagaaatag ggggataaca caatgaattt ttttaacaag agcatccggg aaaagaaaga 19560 acgtgagaaa gccagcaagg aaaagcgaag tcgtgagtta gtcatgcaac agttggatgc 19620 agtaggtaac caggccaggg aaatgatttc ggtcatggcc gttaagaaag ccgaaaataa 19680 tccaaaagga cggcagacag aaccaaaata tgagcagttc aatctaatta agaacatgcc 19740 gatcaaacgt gaatattatc gatcaaaaca agcaatcgaa ttgctttatt atattttgaa 19800 tgtcacgcca gatggatcgc aggcaaagaa caattatttg cgactagcta acaaggggca 19860 ttatgtcgaa gtgttgggat ttgatggtaa accgcacgca gaaggtacag cctacatcaa 19920 cgaagaactt gcaccacgca tcggcaagat atatggcaag ggcgctgatg gcctggtaga 19980 cgtgttgaat ctatcggcat atgtttccgg agcggaagca atggaagtcg aattgacgga 20040 aaaccttgat gatatcgtag actttcacat catttcgcca gaaaccttgc aatacatcat 20100 caaggaagaa gatggaacgt tgcaattggc gcaggtttta acagacggaa caacgaaaga 20160 attgaatgat caagtccgat atatcccaat tgatccgatg attgatgatc cttacggagt 20220 aagcccaatc attccggcat tacaagcttt gatctttcaa aaagaattca tcgatgatct 20280 gaaagcagta gcacatcatc aaggccacgc aagaatggat gtttccatcg tagccgatag 20340 cattatttca tcagcaccag acagcatcaa gaatgatcca gaacgattga caacatttgt 20400 gaatcagcag atcgatcaga ttaagtcaga ttttgaggaa ttagagccaa caagcgactt 20460 tttccacaca gacggcgttc aggtggatat ggcaggcgga acacaaggga gatcgatgga 20520 tttcacaccg ctaatgaccg ttgtcgatcg acaagttatg caggcattga aacaattgcc 20580 aatcttaatg ggtcgaaacg aatcgacaac agaaacacat ggatcgatcc aatggcaaat 20640 ctttgttgat actatcgaag catggcagaa cgtcacaaaa cggatgcttg aatggggata 20700 caacctggca ttgcaggtca aaggcatccc agcaaaagca acagtgacgt tcgagaaggt 20760 acgtgcggaa gatcgcaagg cagaagccga agcatacgaa atcgagatca gaaacaatca 20820 ggctttatac caacaaggca tcatcgatca agaaacgttt gctatgaata ccacaggaga 20880 agacccggca gaagaaacac cacgatccca gggaacaggc acaagcggtt cgggaacgaa 20940 cttttcatcg ctattggcta agtacagcac accggccaat gaagaaggca cagaagaggg 21000 cgaagaaaac gccggtcgta gtcgttcggc atttccctat taagcccgga acgaaccggg 21060 cggaaggatc agacgaattc attgcagaga tcggacagcc gttttcggat gaggtagcca 21120 agcgatcgat aaagatgcaa gatgagatca cgaagctgtt aagcaaacaa cgtggctatt 21180 tcgaaaagat gatcaaagaa tcaacagctt tgccggattt gcttgtggat aagtatgtcg 21240 gagagaaaga agacagcgaa aagcggatcg atttagaaga attcggcttt gagtttgaca 21300 cagagcagga gaaagaagcg ttccttttat ggttcaaagc gaatttttac accgatgaaa 21360 tggataaatt gatgcaagag gaatggacgg aaatccttat gcgttggatc acggacatcg 21420 tgacagtggt cggcgaaatt aatcttgcag ttatcggcat cgatacggat ttcggttgga 21480 tcgatgatgc attagtcgaa caagtcgaat acatggccac cacatcgagc agggaaatag 21540 tgggaacaaa ttatcgatca gttatgaaaa acatcctggg aacggtcaaa aagggcaatt 21600 ggtcgattcc agaagccgtg aagaacctca aaaaagagtt tgccttttca aataagcgtg 21660 caaaagtgat cgcacgaacg gagatattgc gagcgcagtc aacaggccaa tttggatcgg 21720 acattgatgc atatgatcgg ggattgatca tcggcaagga atggcacagc acacacgatc 21780 atcgcacaag agattcgcac gtgtcagcag atggccagat tcgggagttc ctagagccgt 21840 tcgaagtagt cagcaatggc acagtatctt atatgtttta tccaagagat acatcgctat 21900 cagcaccggc aaacgaggtc attcagtgca gatgtttcta tactcgcatc atggcaggcc 21960 aggaacaaaa aatgatcgat gccggatata gcgttgacga aaaagaaagc aactgatagt 22020 attaaatttg tcagaataac cgaacacaat tatgcaaaac taaaaaattc actatttttt 22080 atcggtttaa ctgattcttt ctttttctga aatgcatgtg aggtcgtaag gggaaacgat 22140 cagcggatga gggttgcgaa agcaatcctt ttcgtttata cttatctcat acctactttt 22200 tcatttgttt ttttccatac agggaactct ttcattcgag aagcggtgct gatgttttca 22260 gtaccgtttt tttatgttaa aatgaacata ccatgcggaa acatggcgtg tttaacaaaa 22320 accattcttt cgacaagggg cgatctcatc ttttgatggg gttgcctttt tattttgcac 22380 agaaacgctt gtttgctgat ataatcgaat tgttacatgt gaaacataag gagggaaaag 22440 aaatgatgct aggaagagag cgatgcaaaa caatcccgga tcagaacaaa tccggtaaac 22500 gtccgccagt ttgcaaagat atgtttggct ttccaatacc ggcagatcgc agaccaccac 22560 aaccaccaga accagtgaaa acacaaataa agatcgattt tgaaggccag atgtttgatt 22620 gcgatgtaat tgacggagtg gccaagatac cagatgaaat cgtgcagaaa atgaagaaaa 22680 cgaacctaaa acacgaaaaa acaacagaaa taacaatcaa aaccggatat gacaagcaaa 22740 acgaagatat ttctgctaaa attctagtga agacgaaagg aggtggagaa taaatgaatt 22800 tatacgaaag acaaatggca gttcaaacag catccggaaa cgtggagcaa tacatggatc 22860 gcattaatca aatgtcacat gtgcaattaa gtgcagaaga tgtgttcgtg tatcaaggta 22920 ttatctcatc ggatgcgatg gatagttact acacaaaaat ggatgcagaa acttccttgc 22980 gaaactatgc caacgatctg aaagccggca cgccattaat gacgtatcac gaaacatcac 23040 agtcgccgat tggccgatcc tttgattcat caattgatgt caaagaggat gggaaaacag 23100 ttgttcgggg attgttttac atcgttcgtc atacgaaaat caatggagaa agcacagatg 23160 atcttatccg ccagatcgaa acaggaacat tgacagaaat gtcagtgggg tttggtggga 23220 taaatttatg gtataagtct agttacgatg gcaaagatat ctttgaatca cgctactatc 23280 caggagataa agacgaaaac ggcaatttag tgtattatta cattatggat gcgacgttgc 23340 gagaggtttc tcttgtttac aagggagctt gtccggatgc cgtagtcgaa cgaatcagaa 23400 acgatttgcc gaatatggag gatgccgaaa gacaaatcca gaaatacgaa acacgtttca 23460 acgttcggat cgatatgcca aaacagcgaa gcaaggaggg aaaacggatg aacattgaag 23520 aaatcacgag agcagtagaa aaaggcgatt tgcaacgtgg cgaattgctg aaagcattaa 23580 aagtcgatgc aatcacagac ggtcaacgct cgatccttaa agagttgggc gaagatgcaa 23640 gcatcgaaac tgttcgatca ctgaaagaga aagcagagat cggaaaacga gcattcgaag 23700 aaaaattgga tgagttagtg aaagcacgtg ttgcgattgg tcaagagttc gatcaagaaa 23760 aatacaaaga acaaatgcga tcaatggatg tcgatttcat cgaagatcaa acacgattat 23820 tcgaagacat caagaaagca gagttcaaac caggccgaca aacacaaaaa caaaatgacg 23880 aagaaggcaa agccgttttc gtaagtgaat catacaaggg ggagaaataa aatggcaaaa 23940 agaacgcaag ttttaacacc aacaaacggc atcaacctaa ccgtatatcc taaagccggc 24000 gaagtgatca aacctggcga tgctttagta ttagacacaa cggatgcgct aggttatggc 24060 gttaagttag caggagcaaa cgaagcgcct ttattcgttg ctatgattgg taacgagatc 24120 ggcgaagcaa aaacaaaacg caacgcaatt tccgcaaaag tagtcggcaa atacaacact 24180 gttttatcat tgccaagcga tgatagtgca aacatcggcg attatgtatt gccgaatgga 24240 agtggcgtat ataccaaaga aacagttttg gaagaaggaa cgccaaaagg attcctagtt 24300 gtctataaag acagcaaaaa agtggaggtg ctattctaat gaaaatcttt aaacaaccga 24360 aaatcgaagt gaaaaaccgt ttcggccaat tggtcacatt ggaagaagcg aatttcatcg 24420 aaacagttcg tgaacatgca aaaaatgaac gcaacatcag cgaaaatctt atcaacgaag 24480 attcatccat gatggttcgt tcattcctgg aagataacgg cttgacaatg ggagatttcg 24540 acaatttctt atcacgtgat gtaaaagatg cacgtgttcg tgatttcatt tataatacag 24600 gacttgaacc agtattcacg accatcgtag aatcatatgt gcgtggtcaa tatgaatcag 24660 ttgacgaatt gggcgatctt gtgatgtcac gtgtgccaat ggatcaaatg actcaagctt 24720 attattcagc aatcagcgaa gacggcgaag catatgaaat ggttatcgtg gcagaaggtg 24780 cagatatccc agttactaaa ctagtaacag atacaaaaca catcattgtc gcagagaaaa 24840 aaggccgtgg gctttcattg actgatgaag cagtgaaatc acaacgtatc gatcaattac 24900 aagcattctt ccgcaaattg gggattcgct tgcaaaaatc cgaagttaag caagcaatca 24960 gcgtgctatt aaacggatat ttcggcgata ttgatgcgcc acaaacaatc ggtgtgaaaa 25020 gcacaacatc ttatgatcta actgatttat ggttcgcaca tcgtgtcatg acggatgatt 25080 atggtttcgt tcctaacgaa tacatcatga accgtaagac agcaacagaa tatctagggt 25 140 taaaagatgg ccagggcaac ttcttctttt tacaaaacat tttgaatgca tcattgccag 25200 aaggtatcca aaacgcaaca atccgcatca atgaagaaat cacagatgat aaaatcttgc 25260 tcgtggataa acgcaacgcc ttgcaagaat acgtgttcaa agacttatca agtgaaacag 25320 aacgttcagc aagcaaacaa gcatcaacag tctacacatc taagatcaat caattcgttc 25380 catttgaaaa gaacgcacgc atgatcctag acttgaaaca agcacgcagt taagccggct 25440 attatagccg gtttccatta attttttagg tggtgtaaaa gatggccaaa aagagtgaag 25500 aaacaaagaa gaaaaccaaa gaagaagtga agcaggaaga gaaagcgccg gaagttgaaa 25560 caaaagaaac accggaagaa aagccagaac gctatcgggc gaaagaaggt tatccagttt 25620 tctattacaa atatggcgtg atcgatcacg aatggcgtga acttgatgga gcaatgccat 25680 tgctgatgaa gagaagaatt gaagatggta tgatcgaagt agaatcagag gtgcaatagt 25740 tatgtttata accatcgatg atttgaaagc aaaaggccgt tacaaagaac aacgttcgaa 25800 gatggctgat aatgatctaa acgatttact tgatcggatc gatatttata tcgaatcaag 25860 aaccggatgc agtaagttgg aagaaacgga cgatccgaag atcaagaaac gtttggccat 25920 cgcatcgatt ggcgttgctg attatcttta ttttgtggat aacgtcatga agatcgagca 25980 aaacatgatg ggcgttaaaa gcgaaaacat cggagattat tcttattcgc tatctgatgg 26040 gggtatcgat tacagcaaga tcggcaatcc ggaaatcgat agcattttag atggtttgag 26100 ttgtaagagc gaatcagtgt tcgcatttgg catttcacat ccgaaaaaac acagatgggg 26 160 aaagcggtgg taaaatgttc ggcgatttta tgattcacac agcagacatc acggaacaaa 26220 tcaaagatga gaacgcaggc acagacgaat tcaatagacc aatcacaacc gaaaagacca 26280 cgaggggaat tgaatgccgt tttgtcacat ccattaagcc ggttttagat ggagataagt 26340 ccggattgct agaaacaagc tatattctag tagatgccga cagcgttccg gaaatcactt 26400 tcaatcaaag cacaactgtt tcgaacatcc ggctaaaatc ggatgattct atcgtttcag 26460 atggcacatt cactgtttcc accgttggca tacaaaatgc catgatgggc atccaccacc 26520 taaaaatcta tttggaaagt ggtggttaaa tggccaaaaa atcgatggtt gcatttggct 26580 ttgatgaaga agccaggaaa tggatgaata acctagaaaa gaacgtgcag aaggcttcaa 26640 atgatgcagt cgaagcgatg ggcatggttt gggcggataa tgcgaagctt atgaccagga 26700 acgatcaaca catcgacacc ggggcatacg tgaacagcat tgcatatgcc accagttttc 26760 ccggaaaaga tggtggattc gcagtcggtg cagtgatcaa cgaaaagaag gcattgcaca 26820 tgatggaaca gatcactcta ggtagtgggg tttcatatgc ggtttatctt gaaaaaagat 26880 ataatatatt tgcacgttcg ttggatgcta gtatggcaga aatgaaacgt gtcggcacag 26940 aagtattcaa aaggtcattg ggggtgtgat aagtgcagta caaagatata tcactaagca 27000 tcgcacgtgc ggtcgatcgg gaaatgcctg gcaaattcaa atcgattcgc aaaatgattg 27060 ctgatgatgg ggaaaaaccg cagttagtgg ccaagatggc aggaacacaa acgatcatgc 27120 tatctgttta cactgacaag gcacaacctg gatcggattc attggcttac agcctatgtg 27180 tggatgcagt gaacgtgctt atcgatccca caacggcaat tgatgaaatg attcaaagca 27240 tcgatttgca aagcacgccg gatttggtaa acgatgcaga tcgtaacgcc tggcaggcgt 27300 ggtgctattt ggatgtaact catttatata caggaggtta ggaaaatggc aaaggaagaa 27360 tcaaaagcgc caaaaacaga agcgaaatct aacacattca aagcgaaagt gaaaatgcct 27420 ggttatagca aacgagcaat cgtgacagct aaaatcggca ataaagaaat tgctttgatg 27480 gaaaacgaag ttatcgaagt aacacaacaa gagttcgatc aattaaacct tgcaacgtca 27540 agcgcagtcg aaatcgtaaa agttaaataa gagaggatga agaaaaatgc cagaaacagg 27600 atcaaacgtg atgaaatgga acaaagcgga aattttaggt gggccaggtc gtttagtaat 27660 caacgatgat atttctctac gtcctacaaa aatttctgat gttatggatt taacagcgcc 27720 attcgctttg aaagaaggat ggagagattt aggagcaaca caagaaggga tcaatatttc 27780 acgtggtttc gatacagaag attttgagat cgatcaatct gtacagccta ttgatcgcac 27840 gatttcaagt ttcacaacat cactaagcac aacattagca caagattcca tcaataatcg 27900 tcaattggcc atgatcggtg gaacaatcaa agaaaatgca gccacattag gcgagccggt 27960 tacattgaca gcaccaacgc ttatcggtgc gaaagtgatc aaagtcggag cagtaacagg 28020 cattcaaaaa gatggatttg tgatcgttgg ggatgaaaca cgccaggtcg caaacgttga 28080 tgcaggaaca aaaacagtga cattgaaaaa atcacttgaa aaagcacacg aagcagatac 28 140 attagaacag aaatcagtaa caccagttac cgaattaggt acaaaagaaa tcggctacgg 28200 tgcgccaagc gaaatcgatc actttcaaac agcgatcatt tctaaaatgg acgatggaac 28260 attgcgcttg actgtattct acgatacaac attatcaggc gatgaagtag aaacaactta 28320 cggaaaagat aagaaagtct tgccaatcgg cttgacggcg ttcccagttg atgatctacc 28380 agaagacgaa aatgtgttca aagaattcca acaaactgta taagtttttg ctataatggt 28440 ggtggcgata ttattcgcca tcactgtttc acatgaaaca ccaatcagga gggaaatcaa 28500 atgacagaat tagaaaacca attgaaaagc acagatgtaa caacaaaacc aactcatatc 28560 gagttatcag atggaaaagt tatggcagtt ccgaaattag acgttgggaa aacgttgggg 28620 cttgcacgtg ttatcgcagg cgatggattc gcattatggc gcaaggcaca aggcctattc 28680 acagaagttg aaaagaaaga tgattttggc aatccaatgt tagatgatga aggtcaaccg 28740 atcgttgaaa tcatcgaacc ggaaaatgaa caaattttcg cattcatcgt ggaaaacgtg 28800 ggcgaagatt tattgccgaa agtattaggc attttgttcg gtatcaaagc agaagaagtc 28860 gaaaatatgg aattatacga tgccattgaa ttgttgacag ctttagtcga tgcgaacgac 28920 ttaaagaaaa cttttttagc agtgaagaaa ctgacggaaa aactagtgtc acgtcaggcg 28980 aacgaaccga tagcaacgcc ggcaaattaa catggaaaga acaaattgat tttttaaaag 29040 ggattttcga agcaaacgtg ttgaccatca tccgacaagt ggacgaagtg gcagaccatt 29100 tcaattatac ggatgaatat atcatggatc attcgttgtc ctggttaaaa agaaaataca 29160 gctttgcaat gaacaaacaa aaagatcgat tggttcaatc gcaacgatca acattcagcg 29220 ccattcaagc ggttgtttcg tcattcatgg gatcaaaagt gaacatcgat gaaatactaa 29280 taccacaacg agaaccatcg gaagcagaga aacatctaac ggaattgctt gaaagtctta 29340 aagaaggcaa tacgaaggtt tccggcgtta acgaagaagg gttcgtggtg gatgaatggt 29400 ggaaaacaac ataaattttt ggaaggctat gaatttttcg ttcatagtct ttttttatta 29460 agaaagggga gaaaatcaga tggcagacgg aaaaagaatc ggtggtgcgt ttttagattt 29520 tcaagtggac tattcacagc taaaaacgtt tctaagcacg atgaacaacc tatcagacca 29580 gacgaacaaa caaatatccg gaatcgggtc agtgatcgaa tcaacgatcc agaaaacgaa 29640 gaacttgacg aaggaaacgg caaacggtgc gaatcagcaa aagaatgcca tgagtaacat 29700 catgacacaa tatgcaaacg catcgaaaaa ggtcgaagaa atgaccaaca gcatttcgaa 29760 aacaaaggca acatcatcaa acgccacaaa atcgattcta agcgattcta agagcatagc 29820 aggagaatca tccaaaacag cttttaaaat gtcgcaatcg acatcgcagg cgttcgattc 29880 gatgaaaaaa gacagccgtt ctttgaatca aacacttgtg gccggaatga aagccaacgt 29940 gcaggagttt gggcaaagtt tcaaaaatct aggctcgaca ttcagcggaa tcgcatcatc 30000 gatcattaac acgttcaaaa agattccatc cgggatcgca tcgattccga aaacgatcgc 30060 aacacagatg aaaaatctag ccggatcgat tacatcagga ttcgcacgtg cgaaagattc 30120 ggcaatctca cagataaaga aaataccgga tgcagtacga tcggcaggaa attcaatcaa 30180 gacaggattt gcaacggcgt tcaatacagt tgtttccaca gctacaaacg tagtcaataa 30240 gacgaacaga gcgatccaga cattgccaca aacaatctca cgtgcaggca gtgccatcaa 30300 gaacggattt tcgaatggga tcaaagcaat tccacagatt gcaagcaacg tggctaagtc 30360 agcaggaaac gccttttcat ccatcggaac aaccgtgaag aatgtcgcat cggcatccgg 30420 aagcgcaatg aaatcattcc tatccagtgg gatgcaaaac gtcctttcga aagcacgttc 30480 cgttttccct ggtgtcaaga aacagattca agacggcgtg gacgaaccgg ccaaaacagc 30540 atcgctttca ttaggtaaaa tcgcaggcgc tatcggcgtg atgaaagtcg catccaaagc 30600 cttttcgatg attggatcat cgatggaagg tgcgatcaat cggatcgata cactagcaaa 30660 tgccgatcgt gtgttcgaaa acatgggatt caaggcagaa gaaacacaaa agatgatgca 30720 ggcgctaaac aaatcaataa ccgggcttcc tacggcgctg aatgatgccg tttccggcgt 30780 tcagttgtta gcatcatcaa caggggatat aggcaaatca caaaagattt tcgaagcgtt 30840 gaataatggg attctagggt tcggcggatc ggctgacatg gtaaacaatg ccatcgttca 30900 gttatcacaa tcattctcaa atggcaaagt cgatgccgaa acatggaatt caatgatcaa 30960 tagtggatta gggccagctt tgaatgctat tgcgaaacaa atgggcaaga cggccggaga 31020 gttaaaaaca gggctatcag aaggcactat atccgtcaaa gatttccaag atcgtttgat 31080 tgaattaaac gaaaaaggtg gcggagggct gaaatccttg caacaaatcg cacaggatgc 31140 cacaaaaggg atcaagacat ccattgccaa ctttaaaacg tcagtgacac gtggtgtcgc 31200 agagatcatc aaggcgtttg acaagtttat cacagcagta acaggcaaag gcgttgctga 31260 ttggatcggc acgtttgcat ccggattcga agcaggattg aagaaagttg catccggatt 31320 cgaagcagtc acgccattag ctaagaagct ttatgatacg ttgaaaccat ttgcgccagt 31380 gatcggcgga gtggtggccg gtttgcttgc aatgggcggt atcaactcag caatctttgc 31440 cattagcaaa ctatcgggag tattcgcaat gtttacgaag gcattgtggg caaatccaat 31500 cgggttagtt gtcggcctta tcgtaacatt aggcgttgta ttctataaca cttataagaa 31560 atcagaaaca ttccgcaaga gtgttgataa cttgctgaaa ccgctgaaaa aagtcggaga 31620 aatggcacag ttagcaggca aaatgatcca attgtattta ttcggagaat atacaaagga 31680 taaaggggaa caatttgcta aaacacgcaa agcgttggaa gaaatgttac caaaacaagt 31740 ggtagatgca tttgtaaaac aaattcaacg tgttagcgat gcaaaagaaa tattgacgaa 31800 tatcatcaaa gccatatcgt tatcattcca agatttaaaa ccggaagaat atggcgaaaa 31860 aatgaaatct ttgcatgatt atctcggcca attcattcca caagaacaat tgaatgcaat 31920 ttgggtcaga atcgctaatt ttaacaatgc gataaaccag ttcaaagata tatttagtat 31980 tttctatgat gctttaaaag gtaattttaa agatttcaac caattgtatg aggaatatgg 32040 agatattttc gatattagtc aactgttcga tatagtttat atttctcaaa tattgaatca 32100 agtgggcgat acgtttagat cagcgtttac gcagattaaa caagccattt tgacaggtgt 32160 cacagagggc gattttacgc aactctatgc attgatccaa caaatagcgc caacgattat 32220 cgggatgcta ttgggcggta ttccacagat tgccatcgca ggcggacggc tattgctttc 32280 gattgctgat ggtatgggat tgaatattcc acagttgatc acgaagctaa caactgaaat 32340 cttgccaaac atcgttaaca cattggtaac cggggcaatt acatttgtga atgtcggcat 32400 ccaatggatc aacaacatta ttgccggtat taaccaatca ttgccaacat tattgccaat 32460 cgcatcgggt atcatttcaa acctggtaac gtttatcatg caggcattgc cagtgatcgt 32520 gcaaacaggt tggacaatcc tttcaaactt gatggatggg atcatgcagg cgttgcctaa 32580 cttgatcaac attgccgttc aattggtggt atggcttgcg cagaccatcg cacaaaactt 32640 gccgataatt gtcaacaagg gaatagatat cgttcttaac ttgttgcaag ggattctcag 32700 caagttccct caatggtggg aaatggcagt caagctgatc attcaatttg gcacgaaact 32760 agctgaaaac tttccatcga ttgtgaaaaa aggttgggaa ttgctgacaa aacttgtgca 32820 agggattggc gaaagaattc cggcattgct tgcaatggtc gttcgattta tgggcgttat 32880 gctcggaaag atattggagt atttgccaga cttgatcatt ctaggtgcga aattgatcgt 32940 tgctttgatc aaaggcttat gggagttcgg caaaggattg ggcgaattca tcagcggatt 33000 ttgggattca ctgaaaaaag gttttgctca aattgattgg ggcgaaatcg gtcgaaacgt 33060 aatcgatggg attgtcaaag ggtttacaaa aggattcaag aaggcaaaag atgccgtgac 33 120 cgatcttgca ggaaacatcg gcggatggtt caaagaaaaa atgggtatca agtcgccgtc 33180 acgagttatg gccaagattt ctcaatgggt gccggcaggg gtagcggtcg gagtggaaaa 33240 aggtgcgccg ttagttgata aagcgatgga tgcattaaca gccacaatga cgaaaactgt 33300 acaaagcgcc gatccaaccg caaaaaatga aatcaaagtc gaatcgaaac caacatcgat 33360 gccagtagat acgaatcaga tcacaaacga tggttcgttg aatgcatcgg cctttaatac 33420 cggattgcaa acgatcgttc cgcaagttac gaacacgatc ggcacaatgg cgaaggcagt 33480 tgatacagcc ttgacgaatc aagtgccaga aatgctttca agtggtcgtg aatggataac 33540 aaccatgaac aatggaattg ttcagacagt gccacagctt acaacgagcg tttcgagcat 33600 ggtcaatcaa acaaacagca ggatcgatgc gaacgatgcc acaatgacca gaaacggaac 33660 agaatgggga agaaacatcc tatccgggtt caattcgatt tatccacagt ttatcagtcg 33 720 ggttggacaa ttcacaaatg atactgttaa ccgtttgaga tcgatcaata acactgtttt 33780 atccatcggc agacaataca tgcagttgct tcttaacgga atcaatcaat tgtatggcac 33840 agttgtttca cgtgtaacag cgttgggaaa tcagatggtt tcatccttaa attcaaaacg 33900 tggcggattc tacaatgccg gaacgtattt gatgaatgga ctaatatccg gaataaatgc 33960 cgtcaaaggt tcacttaatt cgacaatgaa cagcgttgca aatgcgatgg tcggaggaat 34020 cggaaaaggt gtgaacggcg ttgccagtgg tgtaaactac atcatgggtc aagtcgaatc 34080 gaaaaaacgg atcaacaatt ggacaatacc gaaatatgcc agtggtacag atggacatcc 34140 gcaagatggg tttgcgatgg tcaatgatca accgggatcg aattatcatg agatcgttca 34200 acgtccgaac ggcgaaacgt ttatccctaa agcaagaaat gccgtgttat ggttagaaaa 34260 aggctcgaag gttgtcaaag catccatgac gaagcaaatc cttaaggcta gaaaaaacgc 34320 cattccacgg tataaaaacg gtgtcgggga tatcgatgtg gcagatttca tcgatgatga 34380 agatgcattg aaacgatttt tccaaagcaa agtggattat tcgaaaatca agtcgccgtg 34440 gaacggtatg acaagaacag ccgttgacat gatcggccat gcgtcaaatg acatggtgca 34500 aacggcgttg agtgatttct ttagtcacgg caatttcgat ggatcattgt ttgcaggtgg 34560 cggagcgttc aacaacgttt acacgtatct tgtagacgtg gcacaaaaac tcatgacgaa 34620 gttccctggt ttagttccca catccggtta ccgtccgggc gatgcttatt atcacggaaa 34680 acaccaggca atcgatttgg cttatcctgg agtttcaaac gatccaagat ataccacgat 34740 cgcaaattac gcttttgaga aattcaagaa tcaaattgct tacgtcatta caaatggccg 34800 agtacgtgac cgcatggggc tatcaggcac aggcgcatcc ggcaattggg taacatggcc 34860 agcacacgat cactttgacc atatccatct aaacggagca tttggcggtg gccaattgca 34920 aagcggtggt ggcggtggcg ttgaaagatg gcgtggccta gctatcaaag cattaagaat 34980 ggaaggacaa tattcgccgg ccaaccttaa tgcactattg aatcaaatgc ggacagaatc 35040 aaatggcgat ccgaatgcca tcaacttatg ggatccaaac gctatgaagg gtacgccatc 35 100 aaaagggttg atgcaggtta tcgatccaac gttccgagct tttgcacgtt cgccatacaa 35 160 taaaaacatc tttgatccta tttcgaacat cctagcatca atcagataca cgttgaaaac 35 220 gtacggaagt ttagttgccg ggtggcgtgg tgtaggctac gaaaacggcg gaatggtcac 35280 aaaagatggg ctttatagaa tgggcgaagg aaacaaagct gaaatggttc taccattgac 35340 caaaccacaa agagcgatgg aattaattat gcaagccatc caatatatgg gcggtggtgg 35 400 attgatcaaa tcgctttcaa atgtcgcaat tccggtcatg gataatctga caggattcgc 35460 aataaacagc gcaggcattt ccggtggtgc ggatgatcca accaacaggg ccattttaag 35520 tgctatcatg gaacaaaatg aaatattgcg ccagattcta gcgaagaaca ccgatgtcat 35580 tttgaatggc cgtgttgttt cacaagaatt gttgccagaa ttggataaac agcgtgcaag 35640 aaataaagtg ttagaaagac gaaaagaagg gaagaaatag aatggtagat caaaaagtag 35700 atcaaaaaga gttccgagga acaaagatga tcgttgatgg gaaaaggctt tcgagccttt 35760 tccagatcaa caagatcagt cggccatcga caccagtcga acacgacaag caagaaacac 35820 aattcggata tagatacatc cggacaaaag aaaaggacaa caccttgaaa attgatataa 35880 ccatccgatc gaacgaaatc aatgactttc gtccggttgc tgatttaagg gatgaaattg 35940 ttcgggagtt atacaaagac ggtttgagaa aattcgaatt tacagatcag ccagatcgtt 36000 attgggaagg taaattggat ggatcgttag aattggaata tatcaacaag cgaaatgcaa 36060 aaactacgat cgaattatgg attccggccg gacatgctat ttcctcatta aatgaaaaag 36120 aattcacgaa tgacgtggaa agcgatcctg gtgtaatcaa tgcagtaaac ggcggaacaa 36180 gacgtgttta tccattggca gaagtttctt tcacgaaaga atgcggatat atcgcattta 36240 tgaacgagag tggcaatcta ttgcaattcg gaaacccgga cgaagtagac ggaactgtca 36300 aagaaaagaa tgaacagtta tttgatgatc acatgttaca agaaagacct ggttgggtaa 36360 gaaaccaggg gatcacaccg ccagtcactc atcgacgaga tcaagttgga acagttagct 36420 atataaacga ttctgaaaaa tatccggata cacctaacga aggatatttc aaaacatata 36480 actatggaga tacatcgttg ccaagttggc acggattggc tatgacaaaa atcattcctt 36540 tgccatccga tggagaatat ccggagaatt ggcacagttc tttcagattc gactttaata 36600 atgacggaga gccgaacaaa aacaagccga gaaagattgg tcatttttcc gttacttttt 36660 ccgatgaaaa cgacaaaatt atttgttcat ttggcatcga ggacaacaat ccatcagcag 36720 aaaaagggga tatgtttttc tatattgggg ataaacgtgt ttttgaagat cgaaaaacga 36780 caaactatta cattaaccaa cgttatcccg gcaagtggat cacagttgaa aaaatcggca 36840 atgtgatcac attaagagcg caagcgtttg gaaaaacaaa aacattcact attccaagcg 36900 atgcaggcat ggggaaactc agaaaagtaa cggtttatat agcacaatac agagataatc 36960 cggtcatgcg taacattgtt tttcgtgctt tggaggttac aaaatacaat acaaaatatt 37020 ggtcagattt taaaaatatt tttcaaccag gagattttct ttcgatcgat ttcggaacac 37080 gatcaatcac gaggaatggc caaccactcg acaaattggg cgcatatggc aacgaatggt 37140 ttcctttagt gcctggaaat aatgaagtgg tatttagtta ttctgattgg gttatcgaac 37200 cgccacaagt gaaaatgaaa ttcaaggagt tgtggtatta atgacagaat tttattttgc 37260 agatagacga atggaaattc aagcagttgg atcaactgat gaaacagatg cgattttaat 37320 tttggaagat gatttacaag aaacactgaa aacagcatcc ggcatctata cgattacaat 37380 ttccacgtta tcggaaaagt acgatccggc agacatacaa gaacacacga aaattttgaa 37440 ttatatcctt gtaaaacgtc cagattcgga taaatgtgat tattatacga tcatggacgt 37500 tgaggaagat tatgaaaatt atacaagatc gctgacagtt gaaaatgccg gacttgatct 37560 tttgaatgaa gttttgccac cgtggggatt gccggcggat caacagaata ttgtttggta 37620 tttagaaaag gccatatctg attccggatt tgtaattggc atcaatgaat acggaaaaag 37680 caaccgaaga aaattgaagt gggatggatc aagcacagca ctagaaagag cgcagtcgat 37740 tgccacgcaa ttcgataatt gtgaattgag tttttctttc gaggtaaaag gcttgcaggt 37800 aaccaaaaaa tatttggata tctacaagca tcgaggatcg caggaaagaa agcaattgtt 37860 tttcggtcgt gagattaaaa acatcaaaaa gactgaaagt gcctatgatc tcgcaacggc 37920 tttgcaagcg acaggcggaa cgccagaagg caaagaaacg ccaatcaatc tatatagtta 37980 tgtaggatca agcgataatg gccgatacta caccgaaaaa aactcatact acgtaaaaga 38040 tcgggaagcc ttgaaaattt acagccgata cattgcgccg aatgaaacag gaaatgacgt 38 100 tggccatatc gttgcgcaat acagctacga aacaacaagc cagtcagagc tattcaatcg 38 160 aaccgttgcc gagcttaaaa aaagaaacca tccaacagaa ggatgggaaa ttgaattgta 38220 ttatatgccg gatggcttat cagttggcga tgaggtggcc atattgcatc cggagcgtga 38280 tttgtatttg gaagccaggg tggaagaaat aacctattcg ataaccggat tgccaaccgt 38340 taaatttgga gatttcttgc aggttgattc cgggatcgat gaagacttgc gaaaaatggc 38400 cgatgccatc gctaacattg aaaaaacgca aatctattat agaattgtat ttgtatcaac 38460 taatggattc aatttcatga atggcgaagg atattcggat gttacggcac ttgtattcaa 38520 aggaaatgaa gacgtaacag aagcattcga accagaacga ttcgtgtggc aaaagtacga 38580 tgatcaaggc gtacacgata ccgaatggga agcacaacat aacggaacag gcaacacgat 38640 tcgcatttat ggaaatgatc cggcaaatta tgacgttaac atcacgaagg aggaatgaca 38700 aatggccaat ttggtttcgg gtagtttcaa atcaacaaat gtaaacgatg gagcagatgc 38760 aaggttgctg atgcttgaaa catcgggaaa tgtgatcgga tttgatgcag aaggaaaagc 38820 agtgccaggg caaaaattag atattgtcgc atacattcaa aatatagagg gttcgccggt 38880 attcacggcg atcccttaca ttggagaaac ggcacaaacg ccgatcacat taaaaggatc 38940 gggaaatgct aggtatatca caggggaaga atggaataaa gattggaaca gattaattgt 39000 ttcggtatcg ttggcaggtt tcacagattc caccacgatt gcaaaggtgc aagatggttc 39060 gggtaaagat ggccaaaacg ctattgtcgg ccttttaacg aacgaagcca tcacgttatc 39120 cgcagatgaa aacggaaata tcccggactt ttccacagct accggagaat ttcaaattta 39180 cgatggattg acgaaagtca cagaaggcgt ttcttttgcg atttccggcc aagtaggcgc 39240 atccggcaca attaaccaga acggcgttta caacgtcaca gcgatgacgg cagacacagc 39300 aacgatcgaa ttcaaagcaa cgtacaaagg aacggagatt gttaagaaat taagcctggc 39360 aaaatctcgc aaagggcaac caggtaaaaa cgccaatttg ctttatttat ccgtttcgaa 39420 caatgtgatt gtatttgatg caaacgatgt cgcatttcct aaccaacgca tttcaatcgt 39480 ggctaagttg caaaatattt ccggcacagc cacattcaaa gcgatccctt acatcaacgc 39540 aacagcacaa ccaggaattg cattatcggg ttcgggcaat gatcgataca tttccgggga 39600 aaattgggat aagcgatgga cgagcctatc agttacggca acgatcggag aattaagcga 39660 tcaaacaacc atttataaga tcagcgaagt aaaaggagcg gacggaaaaa acacgatcat 39720 cggcatctta acgaacgaat cgatcacgat ttcagcggat acaaatggga acgttccgga 39780 cttcacaaag gcaacaggaa cattccagat ttacgatggc atcgcattgg ttgatccgaa 39840 aaacgtcact tatgcagttc agacacagac aggcgcaacg gctacgatcg gcacaaacgg 39900 cgtttataaa ataaccgcaa tgacagcaga cacagcaacg gtcgttttca aggctaccta 39960 tggcggagtt gagagaacaa aacaattgtc cattgcaaaa tcaaaagccg gccaaaaagg 40020 cgaagcaggc aacggcatca agtcaacgaa aacacaatat caaagatcag cagacggaca 40080 aacaccacca acaggcacat ggcttgatga aatcccggta gttccgccag gagaattctt 40140 gtggacgagg gtcacacaaa cattcgatga taatacaaca tcagtcagtt atagtgtttc 40200 aaaaatggga gaagatggtg tggggctaga atcaacagaa atcttttatg taggttcaac 40 260 aaacggacaa acaccgccag aaagcggatg gcaaacgact gttcctaacg tgccacaagg 40320 tcaatacatg tggacaaaga ttgtgtggac ttattcggat gattccataa aaaccgctta 40380 ttcagtgggt aaaattggtg ctaatggagc aaacggaaac gatggcttgc ctggaaaaga 40440 tggcgtaggg atcgctgaca cgttgattga atatgcggtc aacacaagtg gcacagttag 40500 acctacaagc ggttggtcaa caaacacacc aagtgttcca caaggtcaat atttgtggac 40560 gagaatcaca tggacttata cagacaatac taatgaaaca ggctattcag tagctagaat 40620 gggtcaagat ggtgctaaag gtaacgatgg tatagctggt aaagatggag taggtttgaa 40680 atcaactagt atcacatatg ctggttcaac aagtggtact acaccaccaa ccacaggttg 40740 gaacactcaa gtaccaaccg ttcccgctgg tcaatattta tggactaaaa ctgtttggac 40800 ttatacagac aattcaaacg agactggtta ctcagtagct agaatgggtc aagatggtgc 40860 taagggtaac gatggtatag ctggaaaaga tggcgtaggg atcgctgaca cgttgattga 40920 atatgcggtc aacacaagtg gtacagttag acctaccagt ggatggtcca caaacatacc 40980 aagtgttccg caaggtcagt atctatggac tagaaccaca tggacgtaca ccgacaacac 41040 aacagaacaa ggttatacag tagctagaat gggtcaggat ggtgctgacg gagaaacgca 41100 atatacgcat attgcttatg caaatagcac tgacggcgta gatggtttca cgactgttta 41160 tccgaatttg aatttgttag atggtactaa agattttagt ggtacttgga aaaattcaag 41220 tgattgggtg actgagggaa catataaagg cttaactgtt aaaaaacgaa cctggcaatg 41280 gaagggtatt tataaaacat ttactgtacc taaagatggg atttacacat tttcagccta 41340 tataaaaagt tcaggaaatt ccgcaaatgt gtatagatat ggtgcagtaa attctacatc 41400 atctcaagct ccaatacagc aggcaatagg aaataacttt gactggacta gagataacgt 41460 aactttaaat ttaaaagcta atgatgtggt gtggattaga tacgaaataa caggcactgg 41520 ggcggattca attttatgga ctgcaggtca taaatatgaa ttaagctcaa cctctactcc 41580 atatatgccc tcagatagcg aagtcataac tgctgactgg cctatgtaca tcggtcagta 41640 cacagacttt acgcaagctg acagtactaa tccatccgac tatacttgga gtctgatacg 41700 agggaatgat ggggctgatg gtaaagatgg tgtgagtgtt aaaactatca caacgacata 41760 tcaggcaggc acaagcggaa cgacaccgcc aacaggtgta tggctaccga caccgccgac 41820 agttgctgaa aatcaattct tgtggacgaa gatcgaaatt tcattggatg atggtacgaa 41880 aagcacaggt tattccgttg gtaaaatggg agcgcaagga cagccgggag aaaaaggcga 41940 tccaggtgcg ccaggaacgc caggaaacgg catcaaatca tcatctgtta cttatcaagt 42000 ttcaacaagt ggaacaacta cgccaacagg aacatgggta tcgacaccgc cagcagtgcc 42060 acaaggtcaa tatttgtgga caagaacggt gctgacgttc gacaacggac aaagcgtgac 42120 cgcctatgca gtaagtaaag ccggaataga tggcgaaaat ggattgaata acgataacat 42180 ggttttcaac aatcgttttg agaaattcaa tgaatggcca caaaattcga tgggttggct 42240 aaaaactcaa ttcacgcagg caacacaacc agaaacagac aagccaacaa acaatatttt 42300 gaaaattggt ggcgatgcat catcggcatc acgatataca ttttcgagaa atatcgcagt 42360 tgttcctggt actacgatcg aattatcgtt tgatgcgaaa ctatcgagat tattcacgaa 42420 taaaaacatc gctatcattc ggatgtttga tgccgaacat gcgaattcat ccagccaacc 42480 ggacacagtg ggcggaaaca gctatatcaa tctatttaca gatggaacag caaatgccgg 42540 cgcaggcgtg acagaatatg acggaaaaaa tacaccgatc acaaccgcaa attcctggta 42600 tcgatacaag gttcgatttt tagttccgga taacgtgaca aatgtcaaaa tgtttttcac 42660 cacgaccgat ccgaatgcat catcattcct tttcattcgt gatccgcaag gcgtgatcta 42720 caaagattta aacgccaaca cacgaaactt tgcgcttgat tcacgttttc caagcgccaa 42780 aacaatcgga ggaactacga cacgaagaaa gaacattcag ctttcaaggc cgacaaaagc 42840 aggcgaaaaa tatacgattg cattagatta ttcattgatt tccggaacgc taactagtca 42900 aattcaagtt gttttggcag acaagaatga agcaggcaaa ggaaatgttt tgcttaatac 42960 gtccggcaac aaaaacagat tagtcggaga gatcacagca accggcgaag ccgatcattt 43020 gcaaattttc cacggtgtga atggggcaac aacagaagcc acaaattacg agtttgaaaa 43080 gatcatgttt aatttgggcg ataaaacatt cgactatgaa caagcgccgg aagatggttt 43140 cagcacgact aacgtttggc cgaatagttc atggaacatc ggaaaaggca cgtgggttta 43200 tacagatgat cagtttgaaa ttttagcacc agaaacagat aaaccagaat catcgattct 43260 acatggtaaa cctataaacg taagttatca acaaacctac caaatgccac atccaagatt 43320 tgtgaatgca ggggatatcg tacacgtttc atttgattac aaagaaaaaa accgaacgaa 43380 agatactttg cttgctataa tacgaatttt tccggaaaaa gatacagcta acagcgcagc 43440 aaatgcattg gctgaaaaac ctataaaacc atctgttttg ggttggagta tggcaaacaa 43500 taccgattgg gtaaacgtgg actacgtttt ccgatcaacg gtttccgggt ggctcgaaat 43560 cttgccatac gatgatgatg taaccggcaa tcacgagagc ttttatcgtg aaatattcgt 43620 aaccaatgca gggaagttgc atggcgaatg gagtccggcg caaattgact ttgcaacgca 43680 aatcgatggc attccaaaag tgttcgtaca ggcaaacatg ccaatcgatc caagtatcaa 43740 aaatggcgat caatggtggg tacagtccgg aaatagagtg acatcgatca aggtatatgg 43800 aaatggcgca tggcacgatc aagcgatcga tcaagccgtt ttgaatatcg tgcaattgaa 43860 tgccgtcaat atcaacggat cagtcgtgaa tggttcgcag tttatcaata cgtttgacat 43920 taaagacagc gatcattcaa aaatgaaggg tacgactaaa atccaagatg gattcattat 43980 gcaagatgtg gaaaatatcg gtttggatgc ttcaggagca gaaacaaaaa aatttcttga 44040 aagccatatg cgtttgtctt acggtagttt tttatcacaa agcacgttgt ttgatgatca 44100 agggaagcaa acgagcaact catcatctaa a 44131 <210> 2 <211> 347 <212> PRT <213> Artificial Sequence <220> <223> LNT201 <400> 2 Met Ser Lys His Leu Ala Val Cys Gly His Gly Gln Gly Asp Pro Gly 1 5 10 15 Ala Met Gly Asn Gly Tyr Gly Glu Ala Glu Phe Thr Arg Thr Ile Leu 20 25 30 Lys Pro Lys Met Glu Ala Trp Ala Lys Lys Leu Lys His Asn Ser Ile 35 40 45 Glu Trp Tyr Asp Thr Ser Lys Asp Met Tyr Gln Asn Ser Asn Ala Gly 50 55 60 Gly Gly Ala Tyr Ser Val Ser Thr Ser Tyr Ala Ser Val Thr Glu Phe 65 70 75 80 His Leu Asp Ala Val Ala Ser Gly Ser Ala Thr Gly Gly His Val Ile 85 90 95 Ile Ser Ser Gln Phe Ser Pro Asp Ala Asn Asp Leu Ala Ile Ala Lys 100 105 110 Val Leu Glu Lys Tyr Val Gly Leu Trp Gly Ser Ser Lys Pro Thr Gly 115 120 125 Thr Tyr Lys Arg Asn Asp Leu Leu Asn Leu Asn Val Phe Ala Lys Arg 130 135 140 Gly Ile Ser Tyr Arg Leu Val Glu Leu Gly Phe Ile Ser Ser Lys Lys 145 150 155 160 Asp Val Asp Asn Ile Val Lys Asn Ile Asp Ala Ile Ala Lys Gly Met 165 170 175 Val Glu Ala Ile Thr Gly Glu Lys Ile Glu Gly Gly Ser Ser Asn Asn 180 185 190 Asn Ser Asn Asn Asn Ala Lys Pro Ser Thr Pro Lys Thr His Asp Gln 195 200 205 Ile Ile Ala Glu Ser Lys Pro Lys Thr Val Gly Asn Tyr Val Gly Lys 210 215 220 Leu Glu Val Phe Asn Glu Leu Lys Trp Gly Ile Phe Arg Ile Ala Gly 225 230 235 240 Trp Leu Val Pro Ile Asn Gly Ala Pro Tyr Leu Asn His Gly Tyr Val 245 250 255 Phe Trp Lys Glu Ala Gly Thr Gly Lys Glu Ile Gly Arg Cys Pro Ser 260 265 270 Lys Gly Ile Met Arg Asn Asp Ala Asn Lys Ala Tyr Gly Leu Pro Asn 275 280 285 Gly Leu Lys Phe Gly Leu Asp Gly Thr Leu Asp Ile Lys Lys Phe Ala 290 295 300 Gly Lys Lys Val Tyr Pro Cys Leu Arg Arg Thr Asn Asp Lys Ala Gly 305 310 315 320 Asn Ser Ile Ala Gly Lys Ala Asn Gly Asn Gly Trp Ile Asp Ile Glu 325 330 335 Phe Pro Glu Tyr Val Leu Thr Ile Pro Lys Arg 340 345 <210> 3 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> LNT201 coding gene <400> 3 atgagtaaac atttagcagt atgcggacat ggacagggcg atcctggagc aatgggaaac 60 ggatatggcg aagcggaatt cacacgcaca atcttaaagc caaaaatgga agcatgggcg 120 aagaaattga aacataattc aattgaatgg tatgacacaa gcaaagacat gtaccaaaac 180 agcaatgcag gcggtggagc gtattcagtc agcacaagtt atgcaagtgt gacagaattc 240 catttggatg cagtagcgag tggaagcgca acaggtggcc acgtgattat ctcatcacaa 300 ttcagcccag atgcgaacga tctagctatc gcaaaagttt tggagaaata tgtaggtttg 360 tggggatcat caaaaccaac aggaacatat aaacgaaatg acttgctgaa tttgaatgta 420 ttcgcaaaac gtggcatttc ttatcgttta gttgaattgg gattcatttc aagcaaaaaa 480 gacgtggata acattgtgaa aaacatcgat gccatcgcaa aaggtatggt cgaagcgatt 540 acaggggaaa agatcgaagg tggttcgtca aacaataaca gcaacaacaa tgcgaaacca 600 tcaacaccaa aaacacacga tcaaatcatc gcagaaagta aaccaaaaac cgttggaaac 660 tacgttggta aattggaagt attcaacgaa ttaaaatggg gcattttccg gattgctggt 720 tggttagtgc cgatcaacgg tgcgccttat ttgaatcatg gatatgtatt ttggaaagaa 780 gcaggcacag gaaaagaaat tggacgttgc ccatcaaaag gcatcatgag aaatgatgct 840 aataaagcat atggattgcc taacggcttg aaattcggcc ttgatggcac gctagacatt 900 aagaaatttg caggtaaaaa agtatatcca tgtttacgca gaacaaacga caaagcagga 960 aacagcattg caggcaaagc aaatggcaat ggttggatcg atatcgagtt tccggaatac 1020 gttttgacaa ttccgaaaag atag 1044 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 4 atgcggatcc atgagtaaac atttagcagt atg 33 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 5 caggctcgag tcttttcgga attgtcaaaa cg 32

Claims (21)

A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The polypeptide according to claim 1, which has endolysine activity. A polynucleotide encoding the polypeptide of claim 1. The polynucleotide according to claim 3, consisting of the nucleic acid sequence of SEQ ID NO: 3. A recombinant vector comprising the polynucleotide of claim 3. A recombinant cell comprising the polynucleotide of claim 3 or a recombinant vector comprising the same. A bacteriophage comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The bacteriophage according to claim 7, wherein the polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 is made of the nucleic acid sequence of SEQ ID NO: 3. The bacteriophage according to claim 7, wherein the genome of the bacteriophage consists of the nucleic acid sequence of SEQ ID NO: 1. The bacteriophage according to any one of claims 7 to 9, wherein the bacteriophage has an antibiotic activity against Enterococcus faecium. The polypeptide of claim 1,
A polynucleotide encoding the polypeptide,
A recombinant vector comprising the polynucleotide,
Recombinant cells comprising the polynucleotide or a recombinant vector comprising the same, and
Bacteriophage containing the polynucleotide
Including at least one selected from the group consisting of,
Having antibiotic activity against Enterococcus faecium,
Antibiotic. The antibiotic of claim 11, wherein the polynucleotide consists of the nucleic acid sequence of SEQ ID NO: 3. The antibiotic of claim 11, wherein the genome of the bacteriophage consists of the nucleic acid sequence of SEQ ID NO: 1. delete delete A pharmaceutical composition for the prevention or treatment of diseases caused by infection of Enterococcus faecium or Enterococcus faecium , comprising the antibiotic of claim 11. delete The method of claim 16, wherein the disease is enteritis, sepsis, nervous system infection, urinary tract infection, bladder infection, prostate infection, epididymis infection, wound infection, bacteremia, endocarditis, meningitis, bloodstream infection, intestinal tract infection, periodontitis, gingivitis, or abscess. Phosphorus, pharmaceutical composition. A feed additive comprising the antibiotic of claim 11. A disinfectant comprising the antibiotic of claim 11. A detergent comprising the antibiotic of claim 11.

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