KR102233993B1 - Immunologically cloaking Bacteriophage - Google Patents

Abstract

The present invention relates to a bacteriophage containing a self-peptide in the capsid and its utilization, and the bacteriophage of the present invention can evade the immune system and thus circulate for a long time in vivo to continuously and effectively remove bacteria. And can cure infections caused by bacteria.

Description

Immunologically cloaking bacteriophage {Immunologically cloaking bacteriophage}

The present invention relates to a bacteriophage capable of avoiding elimination by immune action, and in particular, to a bacteriophage containing a self-paptide in the capsid and its use.

Several antibiotics are used to treat various infections caused by bacteria. Antibiotics are substances produced by specific microorganisms to inhibit the growth of other microorganisms, and appropriate antibiotics are used for each type of infectious disease. Proper prescription of antibiotics is effective in the treatment of bacterial infections, but problems such as resistance to antibiotics, vomiting, and discomfort may occur due to the abuse of antibiotics.

In recent years, as an alternative to solve the problem caused by the existing antibiotics, such as Korean Patent Laid-Open Patent No. 10-2012-0067096, an antibiotic that uses viruses or bacteria that do not directly harm humans or animals. Research is active. However, since viruses and bacteria are generally recognized as foreign substances in vivo, they can be rapidly removed from the body by the immune action of animals and plants, including humans. For example, phagocytosis caused by phagocytes is a non-specific immune function against microorganisms, so when external microorganisms enter the living body, they can ingest and decompose microorganisms regardless of the type of microorganism.

In order to delay the immune clearance of the virus bacteriophage, a study using polyethylene glycol has been conducted. However, since polyethylene glycol has hydrophilic and inert properties, it can form a three-dimensional barrier that can reduce the interaction between proteins on the surface of the bacteriophage. The three-dimensional barrier formed by polyethylene glycol has disadvantages of limiting cellular uptake, inhibiting endosomal escape, and promoting a secondary immune response. In addition, when the first bacteriophage labeled with polyethylene glycol proliferates, the effect of polyethylene glycol disappears because polyethylene glycol is not labeled in the proliferated bacteriophage. Therefore, it is difficult to expect an effect such as removal of bacteria by the proliferated bacteriophage, and there is also a disadvantage that continuous chemical treatment is required.

Therefore, in the development of antibiotics using viruses or bacteria in addition to the previously used antibiotics, there is a need for a method of avoiding the immune action existing in the living body.

Korean Patent Application Publication No. 10-2012-0067096.

An object of the present invention is to provide a bacteriophage containing a self-peptide in the capsid, and an antibacterial composition using the same, a health functional food or pharmaceutical composition for the treatment or prevention of infection caused by bacteria And it is to provide a composition for controlling plant diseases.

The present invention provides a bacteriophage comprising a self-peptide represented by the following formula (1) in the capsid.

[A ₁ A ₂ A ₃ … A _n ]-Equation (1)

(n is the length (number) of the sequence, 6≤n≤21, _{each of A 1} to A _n is an amino acid, and _{6 amino acids of A 1} to A _n bind to SIRP-α (signal regulatory protein-α) It is a possible amino acid, the remaining n-6 amino acids are arbitrary amino acids, and n is a natural number.)

The bacteriophage according to an embodiment of the present invention includes a self-peptide expressed in the capsid constituting the head of the bacteriophage.

Bacteriophage according to an embodiment of the present invention includes lytic phage.

In the bacteriophage according to an embodiment of the present invention, the self-peptide includes a self-peptide derived from CD47.

The bacteriophage according to an embodiment of the present invention includes a bacteriophage of E, E, L, T, E, E in the sequence of amino acids capable of binding to the SIRP-α (signal regulatory protein-α).

In the bacteriophage according to an embodiment of the present invention, the self-peptide includes a self-peptide linked to the C-terminus of the capsid.

In the bacteriophage according to an embodiment of the present invention, the self-peptide includes a self-peptide of SEQ ID NO: 1 (GNYTC E VT ELT R E G E TIIELK) or SEQ ID NO: 2 ( E VT ELT R E G E ).

The type of bacteriophage according to an embodiment of the present invention is selected from T7, T3 β-phage, Phage C1, H-19B, ΦFC3208, ΦN315, Φ13, ΦMu50A, ΦETA, PVL, PV83, T12, CS112, 315.2, and M30 Includes one or more of which are.

The present invention provides a pharmaceutical composition for the treatment or prevention of bacterial infections comprising a self-peptide-expressed bacteriophage in the capsid.

The pharmaceutical composition according to an embodiment of the present invention includes a pharmaceutical composition for oral administration or injection.

The present invention provides an antimicrobial composition comprising a self-peptide-expressed bacteriophage in the capsid.

The present invention provides a functional food for preventing bacterial infections comprising a bacteriophage in which self-peptide is expressed in the capsid.

The present invention provides a composition for controlling plant diseases comprising a self-peptide-expressed bacteriophage in the capsid.

Bacteriophage according to an embodiment of the present invention is an infection-causing bacteria or antibacterial target bacteria E. coli , Shigella sonei bacteria (Sigella sonnei ) and Salmonella typhimurium , Corynebacterium diphtheria , Clostridium Botulinum ( Clostridium botulinum ) , Staphylococcus aureus , Streptococcus pyogenes And It includes one or more bacteria selected from cholera bacteria ( Vibrio cholera ).

Bacterial infections to be treated or prevented according to an embodiment of the present invention include one or more infections selected from enteritis, bacterial dysentery, diphtheria, acute fever, scarlet fever, cholera, and food poisoning.

The bacteriophage containing the self-peptide in the capsid of the present invention is not recognized as an immune substance in vivo, and thus can avoid an immune function in the body. In particular, since it is possible to avoid the mechanism of removing non-specific microorganisms by macrophages, etc., it is possible to circulate along the blood for a long time, so that the treatment or prevention effect of bacterial infections and the antibacterial effect are very excellent.

Figure 1 is a) mouse macrophages J774A.1 and b) human macrophages THP-1 and T7 bacteriophage (WT-T7: wild-type bacteriophage, Self-T7: self-peptide-expressed bacteriophage in the capsid) Veterinary This is the result of a fluorescence diagram showing phagocytosis according to the ratio.
2 is a confocal microscope image showing phagocytosis according to the ratio of the number of mouse macrophages J774A.1 and T7 bacteriophage (WT-T7; wild-type bacteriophage, Self-T7; bacteriophage expressing self-peptide in the capsid) (Scale bar: 10 μm).
3 is a result of measuring the fluorescence intensity of blood collected from balb/c mice injected with PBS, cy5 labeled-WT-T7 and cy5 labeled-Sefl-T7, respectively.
4 is a result of a plaque-forming assay for determining the number of surviving phages in blood collected from balb/c mice and NOD/SCID mice injected with WT-T7 and Sefl-T7, respectively.
5 is a result of measuring fluorescence images of the whole body of balb/c mice and NOD/SCID mice injected with PBS, cy5 labeled-WT-T7 and cy5 labeled-Sefl-T7, respectively.
6 is a liver (liver), spleen (spleen), heart (heart), kidney (kidney) and lung (lung) of balb / c mice injected with PBS, cy5 label-WT-T7 and cy5 label-Sefl-T7, respectively. ) Is the fluorescence image measurement result.
7 is a result of detection of a fluorescence signal measured at the same point of the great saphenous vein (GSV) in balb/c mice injected with cy5 labeled-WT-T7 and cy5 labeled-Sefl-T7, respectively.
FIG. 8 shows live imaging results measured at the same point of the great saphenous vein (GSV) in balb/c mice injected with cy5 labeled-WT-T7 and cy5 labeled-Sefl-T7, respectively.
9 is a result of live imaging of phage accumulation in the liver of balb/c mice injected with cy5 labeled-WT-T7 and cy5 labeled-Sefl-T7, respectively.
FIG. 10 shows a concentration-dependent symptom change as a result of intraperitoneally injecting different concentrations of E. coli BL21 into balb/c mice, and then measuring the symptoms of disease (Symptoms of Disease) in mice from injection to 100 hours ([ Normal; 0], [reduced activity; 1], [coma, wrinkled fur and hunchback; 2], [coma, wrinkled fur, hunchback and eyes closed with exudates around the eyes; 3], [moribund state ; 4], [death; 5]).
FIG. 11 shows a case where balb/c mice were not infected with E. coli , and balb/c mice were infected by intraperitoneal injection of E. coli BL21 at a concentration of 2 × 10 ⁸ cfu to induce infection (enteritis) in PBS, WT- These are the results of intraperitoneal injections of T7 and Self-T7, respectively, and measurement of the symptoms of disease (Symptoms of Disease) in rats up to 100 hours ([Normal; 0], [reduction of activity; 1], [coma, wrinkled fur and hunchback; 2], [coma, wrinkled fur, hunchback and eyes closed with exudates around the eyes; 3], [moribund; 4], [death; 5]).
FIG. 12 shows a case where balb/c mice were not infected with E. coli and a balb/c mouse that caused infection (enteritis) by intraperitoneal injection with 2 × 10 ⁸ cfu concentration of E. coli BL21 was PBS, WT- This is the result of intraperitoneal injection of T7 and Self-T7, respectively, and culture of tissue fluid collected from the intestine of mice at 100 hours. As a result of culturing the mouse tissue fluid injected with PBS and WT-T7, E. coli colony formation was confirmed, and the culture result of the mouse tissue fluid injected with Self-T7 showed that E. coli completely disappeared.
FIG. 13 shows a case where balb/c mice were not infected with E. coli and balb/c mice that caused infection (enteritis) by intraperitoneal injection with 2 × 10 ⁸ cfu concentration of E. coli BL21 were PBS, WT- These are the results of intravenous injections of T7, PEG-modified T7 (PEG-T7) and Self-T7, respectively, and then measuring the symptoms of disease in mice up to 100 hours ([Normal; 0], [reduced activity; 1], [coma, wrinkled fur and hunchback; 2], [coma, wrinkled fur, hunchback and eyes closed with exudates around the eyes; 3], [moribund; 4], [death; 5] ).
FIG. 14 shows a case where balb/c mice were not infected with E. coli and balb/c mice that caused infection (enteritis) by intraperitoneal injection with 2 × 10 ⁸ cfu concentration of E. coli BL21 were PBS, WT- T7, PEG-T7 and Self-T7 were injected intravenously, respectively, and tissue fluid collected from the intestine of mice was cultured at 100 hours. As a result of culturing mouse tissue fluid injected with PBS, PEG-T7, and WT-T7, E. coli colony formation was confirmed, and as a result of culturing the mouse tissue fluid injected with Self-T7, E. coli completely disappeared.
FIG. 15 shows ^{a balb/c mouse that caused infection (enteritis) by intraperitoneal injection of 2 × 10 8} cfu concentration of E. coli BL21, respectively, followed by intravenous injection of WT-T7, PEG-T7 and Self-T7 for 100 hours. This is the result of measuring the number of E. coli present in the intestine after extracting the intestine of the mouse. When WT-T7 was injected ^{, more than 2 × 10 8} cfu/ml of E. coli was detected, and when PEG-T7 was injected, E. coli was removed, but it was not possible to completely remove E. coli like Self-T7.
Figure 16 shows the T7 bacteriophage 10B capsid sequence into which the sequence encoding the self-peptide is inserted.
Figure 17 shows the sequencing results of the T7 bacteriophage contained in the self-peptide T7 bacteriophage 10B capsid.
18 shows the results of measuring the survival of WT-T7, PEG-T7 and Self-T7 phages in various simulated body fluids at 37°C.
19 shows the results of measuring the optical density of the bacterial suspension inoculated with WT-T7, PEG-T7 and Self-T7 (n=3).

Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in the present specification should be interpreted as generally understood by those of ordinary skill in the relevant field. The drawings and embodiments of the present specification are for a person skilled in the art to easily understand and implement the present invention, and contents that may obscure the subject matter of the invention in the drawings and examples may be omitted, and the present invention is limited to the drawings and examples. It does not become.

The present invention relates to a bacteriophage containing a self-peptide in the capsid and its application.

Bacteriophage is a virus that proliferates using bacteria as a host, and can be used to remove bacteria that cause various infections in humans and non-human animals. However, because bacteriophage itself is recognized as an external substance in vivo, it can be eliminated by various immune systems of organisms such as the reticuloendothelial system (RES). In particular, since removal of virus by phagocytosis of macrophages non-specifically removes microorganisms, bacteriophage injected into the living body can be removed in a short period of time.

The present invention provides a bacteriophage capable of avoiding the immune system in vivo by expressing a self-peptide in the capsid of the bacteriophage, and provides a pharmaceutical composition, a health functional food, an antibacterial composition, and a composition for controlling plant diseases using such bacteriophage. do.

In the present invention, "self-peptide" refers to a peptide that was originally present or inherent in a living body, or a peptide that can block a clearance signal by an immune function in a living body. For example, the glycoprotein of CD47 (Cluster of Differentiation 47) is a'self' marker expressed on the cell membrane of all organisms, and a specific peptide derived from CD47 is SIRP-α (signal regulatory protein) of phagocytes. -α) can interact (bind) to form a complex and send a signal to'do-not-eat-me' that can avoid phagocytosis.

In the present invention, the self-peptide is not limited as long as it is a peptide capable of sending an immune suppression signal to phagocytic cells so as to avoid phagocytosis by phagocytic cells capable of non-specifically removing microorganisms in the immune system in vivo.

According to the present invention, if a self-peptide that is expressed in the capsid of a bacteriophage and can be exposed to the outside is used, the object and effect of the present invention can be achieved, so there is no need to limit the self-peptide to a specific peptide.

According to an embodiment of the present invention, the self-peptide is preferably a CD47-derived peptide, but is not limited thereto. The self-peptide can be isolated from a specific protein, produced from a recombinant microorganism, or obtained through artificial synthesis.

According to another embodiment of the present invention, the length of the self-peptide is not less than 6mer, 6 to 21mer, preferably 6 to 15mer, more preferably 6 to 10mer, but is not limited thereto (hereinafter, the sequence is specifically mentioned. In the absence of this, the sequence recording sequence generally used in the field of the present invention is followed, such as the sequence of 5'-end to 3'-end or N-terminus to C-terminus). Specifically, in the case of a CD47-derived self-peptide, a self-peptide in which E, E, L, T, E, E, which are amino acids necessary for interacting (binding) with SIRPα, are sequentially inserted on the sequence of the self-peptide. Can include. When E, E, L, T, E, E are included in the sequence of the CD47-derived self-peptide, it is self-peptide by any amino acid sequence other than E, E, L, T, E, E. The three-dimensional structure of can change. In addition, the degree of interaction with a protein that recognizes a self-peptide, such as SIRPα of phagocytes, may vary according to a change in the three-dimensional structure. However, peptides containing E, E, L, T, E, E in sequence can function as self-peptides, thereby imparting immune evasion properties to the phage. Such self-peptide can be represented by the following formula (1).

[A ₁ A ₂ A ₃ … A _n ]-Equation (1)

(n is the length (number) of the sequence, _{each of A 1} to A _n is an amino acid, and _{6 amino acids of A 1} to A _n are E, E, L, T, E, E in order, and the remaining n-6 Dog amino acids are any amino acid, and n is a natural number.)

In the above formula, n may be 6≦n≦21, preferably 6≦n≦15, more preferably 6≦n≦10. Alternatively, when a longer sequence is required, it may be 10≦n≦21.

For a specific example of the above formula, when n=6, A ₁ A ₂ A ₃ A ₄ A ₅ A ₆ is EELTEE, and when n=7, A ₁ A ₂ A ₃ A ₄ A ₅ A ₆ A ₇ is XEELTEE , EXELTEE, EEXLTEE, EELXTEE, EELTXEE or EELTEXE, EELTEEX (X represents any amino acid), and when n=8, XXEELTEE, XEXELTEE, XEEXLTEE, XEELXTEE, XEELTXEE, XEELTEXE, XEELTEEX, EXELEE, EXELTEE, EXELTX , EXELTEXE, EXELTEEX, EEXXLTEE, EEXLXTEE, EEXLTXEE, EEXLTEXE, EEXLTEEX, EELXXTEE, EELXTXEE, EELXTEXE, EELXTEEX, EELTXXEE, EELTXEXE or EELTXEEX. In a more specific example according to another embodiment of the present invention , a self-peptide consisting of CD47-derived GNYTC E VT ELT R E G E TIIELK (SEQ ID NO: 1) or E VT ELT R E G E (SEQ ID NO: 2) I can.

In the present invention, the self-peptide may be expressed in the capsid of bacteriophage and exposed to the outside. The self-peptide expressed in the capsid and exposed to the outside can inhibit an in vivo signal required for immune action or can perform an interaction that can avoid immune action. According to an embodiment of the present invention, the self-peptide expressed in the capsid of bacteriophage interacts with various signal regulatory proteins of phagocytic cells, thereby avoiding the removal by phagocytosis of phagocytic cells. More specifically, CD47-derived GNYTCEVTELTREGETIIELK is linked to and expressed at the C-terminal of the phage capsid, and is exposed to the outside from the capsid to interact with SIRP-α, a signaling regulatory protein of macrophages (self-peptide receptor interaction) so that macrophages do not remove bacteriophage through phagocytosis.

In the present invention, the self-peptide is included in the capsid (coat protein) of the bacteriophage and may be exposed to the outside of the capsid. The capsid containing the self-peptide is preferably a capsid forming a head among bacteriophage capsids, but is not limited thereto.

According to an embodiment of the present invention, in the case of T7 bacteriophage, through recombinant of the T7 bacteriophage sequence and the self-peptide, the self-peptide may be linked to the C-terminus of 10B of the coat protein and expressed. . The self-peptide is not linked to and expressed only in the major capsid, which occupies many of the proteins constituting the capsid, and a T7 bacteriophage can be constructed to be expressed in the minor capsid. The difference in the type of capsid that connects the self-peptide may be a difference in the degree of immune evasion of a bacteriophage constructed to have immune evasion, but the desired effect in the present invention does not change.

According to another embodiment of the present invention, when the bacteriophage is constructed so that the self-peptide is expressed in 10B, the capsid of the T7 bacteriophage, the self-peptide is very uniformly expressed in 415 10B forming the head of the T7 bacteriophage to the outside. It can be exposed. This uniformity of expression can be very efficient in transduction of signals for immune evasion and interactions with receptors on the surface of phagocytic cells.

In the self-peptide of the present invention, the sequence of the bacteriophage and the method of recombination may vary depending on the type of bacteriophage.

According to an embodiment of the present invention, the nucleic acid sequence of the self-peptide to be recombined with the sequence of the bacteriophage is designed, and the sequence encoding the self-peptide in the nucleic acid of the bacteriophage can be recombined using a vector and a restriction enzyme containing the same. .

More specifically, in order to express GNYTCEVTELTREGETIIELK derived from CD47 at the C-terminus of 10B, which is a T7 bacteriophage capsid, a sequence encoding GNYTCEVTELTREGETIIELK can be recombinantly inserted into the sequence encoding the 10B capsid of T7 bacteriophage. A sequence encoding a GNYTCEVTELTREGETIIELK ggt aac tac aca tgc gag gtc aca gag ctc aca cga gaa ggc gag acc ata att gaa ctc When aag (SEQ ID NO: 3) or a sequence complementary thereto is inserted into a sequence encoding 10B capsid atg ctc ggg gat ccg aat tca agc ttg (SEQ ID NO: 4) or a sequence complementary thereto, the sequence encoding 10B capsid of T7 Atg ctc ggg gat ccg aat tca ggt aac tac aca tgc gag gtc aca gag ctc aca cga gaa ggc gag acc ata att gaa ctc The aag agc ttg (SEQ ID NO: 5) sequence or a sequence complementary thereto may be included to express the self-peptide in the 10B capsid.

In the present invention, the bacteriophage is not particularly limited unless it is a bacteriophage harmful to humans or non-human animals. Preferably, it is a lytic bacteriophage. The lytic bacteriophage proliferates by lytic lysis of the bacteriophage host, so it can kill the bacteria that cause infection. And, as long as the host bacterium is present, the bacteriophage capable of expressing the self-peptide can continue to proliferate.

As the type of bacteriophage, at least one selected from T7, T3 β-phage, Phage C1, H-19B, ΦFC3208, ΦN315, Φ13, ΦMu50A, ΦETA, PVL, PV83, T12, CS112, 315.2, and M30 is preferred, but limited thereto. It does not become.

According to an embodiment of the present invention, since a lytic bacteriophage containing a self-peptide is used for the capsid, it is possible to continuously self-proliferate by using harmful bacteria as a host in vivo and in vitro, and the safety is excellent.

And, the later-generation bacteriophage generated by self-proliferation may also contain a self-peptide in the capsid in the same manner as the bacteriophage of the present invention, so that the object and effect of the present invention can be retained as it is.

This feature is an important feature that differentiates it from self-peptide-modified nanoparticles. Since self-peptide-modified nanoparticles cannot proliferate on their own, the number of self-peptide-modified nanoparticles is bound to decrease with time.

In addition, depending on the degree of infection, the self-peptide-modified nanoparticles must be continuously injected, and abnormal symptoms in vivo may occur due to the injection of an excessive amount of nanoparticles.

In addition, there is no need for a carrier for delivering the bacteriophage to the target location, and the bacteriophage has been proven to be safe enough to be used as a therapeutic agent.

Furthermore, the bacteriophage of the present invention does not require induction of complex chemical reactions required for the synthesis of nanoparticles and peptides and formation of covalent bonds thereof, as in the preparation of nanoparticles modified with self-peptide.

The present invention can provide a pharmaceutical composition for the treatment or prevention of infections caused by bacteria by utilizing a bacteriophage containing a self-peptide in the capsid.

The term, prevention, of the present invention refers to any action that suppresses or delays the onset of a disease by administration of the composition, and the term, treatment of the present invention, improves the symptoms of the disease or suppresses or alleviates the disease by administration of the composition. And any act that is advantageously altered.

The pharmaceutical composition according to the present invention is not limited, but may further include a pharmaceutically acceptable carrier. In the present invention, the term, 　 pharmaceutically acceptable carrier means a carrier or diluent that does not inhibit the biological activity and properties of a bacteriophage administered without irritating an organism. For example, as an acceptable pharmaceutical carrier in a composition formulated as a liquid solution, as sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol , Ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition according to the present invention may be used as a method of applying or spraying to a diseased area, and may be administered through nasal spray, oral administration, injection or parenteral administration, and in the case of parenteral administration, intravenous administration , Intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration may be used, but this is only an example, and the administration method is not limited thereto.

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

Formulations for oral administration comprising the pharmaceutical composition according to the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrup or It can be formulated as an elixir. For formulation into formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate , Lubricating oil such as calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned substances.

Formulations for parenteral administration comprising the pharmaceutical composition according to the present invention as an active ingredient include injectable forms such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or an aerosol that enables inhalation through the respiratory tract. It can be formulated for spraying, etc. In order to formulate a formulation for injection, the composition of the present invention may be prepared as a solution or suspension by mixing in water together with a stabilizer or buffer, and it may be formulated for unit administration in ampoules or vials. When formulated for spraying such as an aerosol, a propellant or the like may be blended together with an additive so that the water-dispersed concentrate or wet powder is dispersed.

The pharmaceutical composition including the bacteriophage according to the present invention may be selected to be suitable for the type of bacterial infection to be treated or prevented.

In one aspect of the present invention, the type of bacterial infection is not particularly limited, but may be one or more selected from enteritis, bacterial dysentery, diphtheria, acute fever, scarlet fever, cholera and food poisoning, etc., and are limited thereto. It is not. Depending on the type of bacteria causing bacterial infection to be treated or prevented, the bacteriophage using the bacteria as a host may be different.

The type of the bacteriophage is not limited, but for example, select from T7, T3 β-phage, Phage C1, H-19B, ΦFC3208, ΦN315, Φ13, ΦMu50A, ΦETA, PVL, PV83, T12, CS112, 315.2, and M30 At least one of which is preferred, but is not limited thereto.

According to an embodiment of the present invention, it can be confirmed that administration of bacteriophage in different ways such as intraperitoneal administration as well as vascular administration through injection has an excellent immune evasion effect, and has an effect capable of preventing or treating infectious diseases. there was.

According to another embodiment of the present invention, the dosage of the bacteriophage is not limited, but a suitable dosage may be determined by measuring the number of phagocytes in the blood. Preferably, 10 to 100 times the number of phagocytic cells, but is not limited thereto.

The present invention can provide an anti-bacterial composition by utilizing a bacteriophage containing a self-peptide in the capsid.

The antimicrobial composition according to the present invention may be usefully used for humans, animals other than humans, household goods, cosmetics, clothing, and packaging materials.

The term "antibacterial composition" of the present invention may mean a substance that is provided to humans or animals other than humans in the form of a drug to kill microorganisms or to inhibit the development, and at this time, it refers to preservatives, fungicides, antibiotics, etc. Can be used. The animal may include, but is not limited to, mammals including humans, and exhibits specific activity against specific bacteria, so it does not kill beneficial bacteria, can kill only specific pathogens, and does not induce drug resistance or resistance. It can be used as a novel formulation with a longer life cycling compared to the antibiotics of The antimicrobial composition according to the present invention may include a bacteriophage containing a self-peptide alone or an additive appropriately selected according to the purpose of use. There is no particular limitation on the optional use and content of the additive, and it can be appropriately adjusted according to the use method and formulation commonly used in the art. Accordingly, the additive may be a material included in a conventional antibacterial composition, for example, an excipient or a diluent, and examples thereof include saline, buffer, dextrose, isoparaffin, water, glycerol, and ethanol, and are limited thereto. It is not.

In addition, the term “antibacterial composition” of the present invention may refer to a material included in various articles such as household goods, cosmetics, clothing, and packaging. As a non-limiting and specific example, it can be used for articles such as fabric softeners, detergents, detergents, disinfectants, quasi-drugs, fragrances, cosmetics, and packaging films. When used as a cleaning agent, it can be prepared with a spray agent, a cleaning agent, a shampoo, a hair conditioner, a hair conditioner, a soap, etc. In this case, the antibacterial composition may be included in an amount of 0.01 to 1% by weight, but is not limited thereto. When used as a quasi-drug, it can be prepared with a hair growth agent, a hair dye, a hair removal agent, a deodorant, an anti-body odor, and a toothpaste, and the antibacterial composition may be included in an amount of 0.01 to 2% by weight, but is not limited thereto. When used as a fragrance, a volatile material may be further included, and the antimicrobial composition may be used in an amount of 0.0001 to 1% by weight. When used as a fabric softener, the antimicrobial composition may be used in an amount of 0.0001 to 5% by weight. When used as a cosmetic, the antimicrobial composition may be included in an amount of 0.0001 to 1% by weight.

The antibacterial composition comprising the bacteriophage according to the present invention may exhibit antibacterial activity against various microorganisms, and the antibacterial target is not limited, but human pathogenic bacteria, phytopathogenic bacteria, fungi, etc. may be exemplified, and a specific example of E. coli ( E.coli ), Shigella sonei bacteria (Shigella sonnei ), Salmonella typhimurium , Corynebacterium diphtheria , Clostridium Botulinum ( Clostridium botulinum ) , Staphylococcus aureus , Streptococcus pyogenes And It may be one or more selected from the group consisting of cholera bacteria ( Vibrio cholera ), and the like, but is not limited thereto.

The present invention can provide a health functional food for preventing infection caused by bacteria by utilizing a bacteriophage containing a self-peptide in the capsid.

Health functional foods containing the bacteriophage according to the present invention as an active ingredient are not particularly limited, but for example, there are various 　 foods, beverages, gums, teas, vitamin complexes, health supplements 　 foods, etc., powders, granules, tablets, It can be used in the form of a capsule or beverage.

The active ingredient may generally be included in an amount of 0.001 to 15% by weight, specifically 0.01 to 1% by weight of the total 　 food 　 weight, and the health beverage composition is included in a ratio of 0.001 to 1 g, specifically 0.01 to 0.1 g based on 100 ml. However, this is only an example and is not limited to the numerical range.

Health functional food according to the present invention 　 in addition to containing the bacteriophage containing the self-peptide as an essential ingredient 　 food acceptable 　 food supplementary additives, such as natural carbohydrates and various flavors may contain as additional ingredients. . Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Taumatin, rebaudioside A, glysirhizin, saccharin, aspartame, and the like may be used as the flavoring agent.

In addition to the above, the health functional foods according to the present invention include various nutrients, vitamins, minerals, synthetic flavors and natural flavoring agents, flavoring agents, coloring agents and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties. It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

In addition, the health functional food according to the present invention may contain pulp for the manufacture of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The method for preparing the health functional food is not particularly limited, and generally, a widely known method for preparing food may be introduced and used, and may be modified and used as necessary.

The present invention provides a composition for controlling bacteria causing plant diseases, and the composition may use a bacteriophage containing a self-peptide in the capsid according to the present invention.

The composition for controlling plant diseases according to the present invention means a drug used for the protection and growth of plants, and chemical pesticides and biological pesticides used to control diseases, pests, weeds, etc. that occur during plant cultivation or storage It is a generic term for growth regulators used to enhance or inhibit physiological functions, and adjuvants that enhance drug efficacy.

Specifically, the composition for controlling plant diseases may be a bestial injection composition, a biologic composition, a fertilizer composition, a nutritional composition, a plant fortification composition, a plant protection composition, a soil conditioner composition, or a yield enhancer composition. However, it is not limited thereto.

Formulations of the composition for controlling plant diseases include wettable powder, water dispersible granule, dusting powder, suspension concentrate, emulsifiable concentrate, tablet, and liquid ( liquid) or an oil suspension, but is not limited thereto.

The composition for controlling plant diseases may include one or more dispersants together with an effective amount of water, and the dispersants may be included in the range of about 2 to about 60% (w/w). These dispersants can be used without limitation, those known in the art such as emulsifiers and suspending agents, and specific examples of dispersants include polyvinylpyrrolidone, polyvinyl alcohol, lignosulfonate, phenyl naphthalene sulfonate, ethoxylated Alkyl phenol, ethoxylated fatty acid, alkoxylated linear alcohol, polyaromatic sulfonate, sodium alkyl aryl sulfonate, glyceryl ester, maleic anhydride copolymer, phosphate ester, condensation product of aryl sulfonic acid and formaldehyde, alkyl Condensation product of aryl sulfonic acid and formaldehyde, addition reaction product of ethylene oxide and fatty acid ester, salt of addition reaction product of ethylene oxide and fatty acid ester, sulfonate of condensed naphthalene, ethylene oxide and fatty acid ester Addition reaction product of, salt of the addition reaction product of ethylene oxide and fatty acid ester, lignin derivative, naphthalene formaldehyde condensate, sodium salt of isodecylsulfosuccinic acid half ester, polycarboxylate, sodium alkylbenzenesulfo Nate, sodium salt of sulfonated naphthalene, ammonium salt of sulfonated naphthalene, salt of polyacrylic acid, salt of phenol sulfonic acid, or salt of naphthalene sulfonic acid, but are not limited thereto.

In addition, the composition for controlling plant diseases may further include a wetting agent, a binder, a filler, or an organic additive.

The humectant may be included in the composition for controlling plant diseases in the range of about 0.1 to 5% by weight, specifically 0.5 to 2% by weight, and specific examples include alkyl naphthalene sulfonate, sodium alkyl naphthalene sulfonate, sulfonated alkyl carboxylate Sodium salt of, polyoxyalkylated ethyl phenol, polyoxyethoylated fatty alcohol, polyoxyethoxylated fatty amine, lignin derivative, alkane sulfonate, alkylbenzene sulfonate, salt of polycarboxylic acid, ester of sulfosuccinic acid Salt of, alkylnaphthalenesulfonate, alkylbenzenesulfonate, alkylpolyglycol ether sulfonate, alkyl ether phosphate, alkyl ether sulfate or alkyl sulfosuccinic acid monoester, but is not limited thereto.

The binder may be included in the composition for controlling plant diseases in a range of about 0.1 to 7% by weight, specifically 0.5 to 3% by weight, and specific examples include polyvinyl alcohol, lignin derivatives, polyvinyl pyrrolidone, polyalkylpi Rolidone, carboxymethylcellulose, xanthan gum, polyethoxylated fatty acid, polyethoxylated fatty alcohol, ethylene oxide copolymer, propylene oxide copolymer, polyethylene glycol or polyethylene oxide, but is limited thereto. no.

The filler may be included in the range of about 0.1 to 10% in the composition for controlling plant diseases, and specific examples include bentonite, sub-bentonite, attapulgite, kaolinite, montmorillonite, Bauxite, hydrated alumina, calcined alumina, diatomaceous earth, chalk, fuller's earth, dolomite, kieselguhr, loess, propylite prophyllite), talc, vermiculite, limestone, natural and synthetic silicates, silica, or china clay, but is not limited thereto.

The organic additive may be included in the range of about 0.01 to 50% in the composition for controlling plant diseases, and specific examples include macronutrient, micronutrient compost fertilizer, natural element, natural Natural organism, trichoderma, humic acid extract, Bacillus thuringiensis , virus, natural fungus, plant extract, pyrethrum, biological control product, natural It may be an oil, natural extract, mineral or urea group, but is not limited thereto. The micronutrient may be zinc, iron, manganese, copper, boron, cobalt, vanadium, selenium, silicon, or nickel, but is not limited thereto. The macronutrient may be nitrogen, phosphorus, potassium, calcium, or magnesium, but is not limited thereto.

According to an embodiment of the present invention, as a result of liquefying the dry powder of the self-peptide-expressed phage stock and spraying it on the plant where the plant disease occurred, it was possible to confirm the inhibition of the progression of the plant disease. It is believed to be a killing effect.

In the present invention, the causative agent causing the plant disease is not limited in its kind as long as it is a fungus that can be controlled by the composition for controlling plant diseases of the present invention, for example, Cladosporium cucumerinum (Cladosporium cucumerinum ), Fusarium solani , Colletotrichum cocodes coccodes ), Pellicularia Sasaki (Pellicularia) sasaki ), Alternaria mali and Botryotinia perceliana (Botryotinia fuckeliana ) may be one or more selected from, but is not limited thereto.

The types of plant diseases that can be controlled by treating the composition for controlling plant diseases including the bacteriophage of the present invention are not particularly limited, but examples thereof include blast disease, powdery mildew, red pepper anthrax, grape anthrax, apple spot pattern fallen leaf disease, leaf sheath pattern. It may be one or more selected from bottle, black star disease, gray mold disease, red mold disease, sunspot disease, and downy mildew disease, but is not limited thereto.

In the present invention, the antimicrobial or plant disease control includes the steps of treating an antimicrobial composition comprising the self-peptide-expressing bacteriophage of the present invention as an active ingredient, a composition for controlling plant diseases, respectively, to a microorganism or plant (leaf or root). However, it is not limited thereto.

Hereinafter, examples for carrying out the present invention will be described. The examples correspond to one example for carrying out the present invention, and the present invention should not be construed as being limited by the examples.

[Example 1] Construction of T7 phage representing self-peptide and preparation of bacteriophage stocks

The T7Select415-1 cloning vector (Novagen, Madison, Wis.) was used to express the self-peptide on the T7 phage capsid protein 10B molecule. To represent the self-peptide on the T7 phage capsid, two DNA sequences were designed. The DNA sequence of the self-peptide was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT AAC TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC ATA ATT GAA CTC AAG-3' (SEQ ID NO: 6), reverse direction is 5'AGCT CTT GAG TTC AAT TAT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC GCA TGT GTA GTT ACC TG-3' (SEQ ID NO: 7).

In order to represent the self-peptide on the T7 phage, two primers were annealed at 94° C. for 4 minutes to form a double-stranded DNA insert, and ligated to the C-terminus of the 10B capsid protein using EcoRI and HindIII restriction enzymes. The phage containing the cloning vector was constructed through in vitro packaging, and the complexity of the phage was measured using a plaque-forming assay recommended by the manufacturer.

After PCR amplification, the sequence of the recombinant T7 phage was identified. 5'-ATG CTC GGG GAT CCG AATT CA GGT AAC TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC ATA ATT GAA CTC AAG AGC TT G CGG CCG CAC TCG AGT AAC TAG-3' (SEQ ID NO: 8) or a sequence complementary thereto was confirmed. It was used as a WT-T7 control that did not express the peptide sequence on the capsid. After preparing both the T7 bacteriophage representing the self-peptide and the wild type, phage amplification and purification were performed. The cultured culture of E. coli BL21 was inoculated into 500 ml of M9LB medium, and cultured in a shaking incubator at 37° C. to a final OD of ₆₀₀ = 0.7 to 0.8. T7 phage constructed in BL21 was infected in M9LB medium, and cultured until complete lysis of the cells was observed. Thereafter, 12.5 g of NaCl was added and centrifuged at 12,000×g at 4° C. for 10 minutes to separate bacterial debris from T7 phage. Next, 10% polyethylene glycol (MW 8kDa) was added to the supernatant, and stored at 4° C. for 10 hours to efficiently precipitate phage. The above precipitate was centrifuged at 12,000×g at 4° C. for 10 minutes, and the precipitated phage was resuspended in 10% PEG (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The suspension was centrifuged at 14,000×g at 4° C. for 10 minutes, and the pellet was resuspended in 1 M NaCl (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Then, the solution was centrifuged at 14,000×g at 4° C. for 30 minutes, and an aqueous phase containing T7 phage was carefully collected. Phage was ultracentrifuged for 60 minutes at 35,000 rpm at 20°C on a four-step gradient of cesium chloride in TE buffer (10 mM Tris, pH7.5, 100 mM NaCl) (Optima XE 90 ultracentrifuge, Beckman Coulter, Pasadena, CA). , USA). On the 2:1 layer (CsCl:TE), the sharp and cloudy bands of the T7 phage were separated.

In the finally obtained T7 phage expressing the self-peptide, 415 copies of GNYTCEVTELTREGETIIELK self-peptide, a sequence of 21-mers derived from CD47 that bind to SIRPα of macrophages, were expressed in 10B capsid, a major capsid. (Figs. 16 and 17).

[Example 2] Phagocytosis test using fluorescence-activated cell sorting (FACS) in vitro

1. Mouse macrophage J774A .One

^{J774A.1 (1.0×10 6} cells per mL DMEM) was dispensed for each well in a 6-well culture plate, and cultured for 24 hours at 37°C in _{a CO 2 incubator.} Cy5-labeled wild-type (hereinafter'WT-T7' means the same bacteriophage) and self-peptide-expressed T7 bacteriophage (hereinafter'Self-T7' means the same bacteriophage) molar ratios 1:10 and 1:100 Each was prepared at a ratio and incubated at 37°C. After incubation for 30 minutes and 1 hour, excess phage was washed 4 times with 1 ml of PBS. The peptide was fixed by washing for 15 minutes after addition of 1 ml of 4% formaldehyde, and cells were separated from the plate by adding 0.5 ml of trypsin EDTA (Gibco, Waltham, MA).

Cells were washed with 1 wt% BSA in PBS. Thereafter, a flow cytometer (Beckman Coulter MoFlo XDP, Pasadena, CA, USA) capable of detecting ~500,000 cells with an FL-4 detector (670/40) was used.

The results of phagocytosis analysis by J774A.1, a mouse macrophage cell, in vitro are as follows. The fluorescence intensity was lower due to the interaction between SIRPα and Self-T7 on the surface of macrophages, indicating that macrophages ingested less Self-T7. When the number of J774A.1 and T7 phage was 1:10 (J774A.1:T7 phage), phagocytosis was reduced by 70%, whereas when the number of J774A.1 and T7 phage was 1:100, phagocytosis was reduced by about 50% (Fig. 1a). And, the confocal microscopy image showed less cell uptake of Self-T7 (Fig. 2).

2. Human macrophage THP -One.

Using human macrophage THP-1 having 85% amino acid sequence identities and 100% amino acid sequence positives with the mouse self-peptide, in a method similar to the experiment for the mouse macrophages Flow cytometry was performed.

^{THP-1 (1.0×10 6} cells/mL RPMI1640) was dispensed for each well in a 6-well culture plate, and cultured for 24 hours at 37°C in a _{CO 2 incubator.} Differentiation of THP-1 cells was performed in 100 ng/mL of PMA (phorbol myristate acetate) for 2 days, and it was confirmed through attachment of these cells to tissue culture plastics.

Cy5-labeled wild-type (hereinafter'THP1:WT-T7') and self-peptide-expressed T7 bacteriophage (hereinafter'THP-1:Self-T7') were prepared in a molar ratio of 1:10 and 1:1000, respectively, and 37 Incubated at °C. After incubation for 30 minutes and 1 hour, excess phage was removed by washing 4 times with 1 ml of PBS. The peptide was fixed by washing for 15 minutes after addition of 1 ml of 4% formaldehyde, and cells were separated from the plate by adding 0.5 ml of trypsin EDTA (Gibco, Waltham, MA).

Cells were washed with 1 wt% BSA in PBS. Thereafter, a flow cytometer (Beckman Coulter MoFlo XDP, Pasadena, CA, USA) capable of detecting ~500,000 cells with an FL-4 detector (670/40) was used.

As a result of analyzing phagocytosis by human macrophages THP-1 in vitro , it was confirmed that human macrophages consumed less THP-1:Self-T7 than THP-1:WT-T7. In addition, compared to the case where the ratio of the number of THP-1 and T7 phage was 1:10, it was confirmed that the phagocytosis (macrophage) was significantly reduced when the self-peptide was treated at 1:1000 (Fig. 1b). .

[Example 3] Quantitative comparison of phage and wild-type phage circulation expressing self-peptide in whole blood

6-week-old male balb/c and NOD/SCID mice were received and prepared from OrientBio (Sungnam, Republic of Korea). Cy5-labeled wild-type and self-peptide-expressed T7 bacteriophage 2×10 ⁹ pfu were intravenously injected. Blood was collected from each mouse into tubes containing 10% heparin at 0.25, 0.5, 1, 4, 12 and 24 hours after injection. The fluorescence intensity of each tube was measured using the Xenogen IVIS image system (Caliper Life Sciences, Waltham, MA, USA) (FIG. 3). In addition, the collected blood was serially diluted with Luria-Bertani (LB) and examined by a plaque-forming assay to determine the number of phages surviving in the blood (FIG. 4).

As a result of the test, Self-T7 was present in the blood for a longer time, and there was a significant difference from 15 minutes after injection.The number of Self-T7 phage plaques was more than 4 times higher than that of WT-T7, and this difference persisted over time. Became. At 1 hour after injection, the number of Self-T7 plaques was 18.5 times higher than that of WT-T7. The overall number of phages was higher in NOD mice. In particular, Self-T7 was present at high concentration until 30 minutes and decreased after 60 minutes, but showed about 12 times higher concentration in babl/c, and about 4 times higher than WT-T7. These results are due to the interaction of CD47 and SIRPα, which are different for each mouse strain, and it was confirmed that Self-T7 can be maintained for a long time without being removed by immune action in blood circulation.

[Example 4] Measurement of biodistribution using Cy5-labeled phage

6-week-old male balb/c and NOD mice were received and prepared from OrientBio (Sungnam, Republic of Korea). Cy5-labeled wild-type and self-peptide-expressed T7 bacteriophage 2 × 10 ¹⁰ pfu was injected intravenously. Fluorescence was measured for each mouse 2, 4, 12 and 24 hours after injection using the Xenogen IVIS image system (FIG. 5). After 30 hours, the mice were euthanized and several organs (liver, spleen, heart, kidney and lung) were excised. The fluorescence intensity of each organ was measured with the Xenogen IVIS image system (FIG. 6).

As a result of the measurement, Self-T7 accumulated more slowly than WT-T7 in the organ. These results indicate that Self-T7 does not accumulate in the organs and can circulate in the blood for a longer time.

[Example 5] Phage in vivo Biometric image measurement in circulation

6-week-old male balb/c and NOD mice were received and prepared from OrientBio (Sungnam, Republic of Korea). The body temperature was maintained at 37° C. using a heat pad during the experiment. Live imaging of phage circulation was performed after surgical skin incision to expose the great saphenous vein (GSV) in each mouse thigh. Anesthesia levels were monitored during the experiment by tow pinching, and the Zoletil-xylazine mixture was continuously injected. After phage injection, FITC-dex (dextran-fluorescein isothiocyanate) was injected into the tail vein of each mouse to label the vascular lumen. After intravenous injection of Cy5-labeled wild-type or self-peptide-expressed T7 bacteriophage 2 × 10 ¹⁰ pfu, two-color fluorescence signals were simultaneously detected with 2 bandpass filters (FF01-525/45, FF01-697/58, Semrock). (Fig. 7). In the case of live images of phage accumulation in the liver, the processing procedure of the mouse was the same as the previous procedure. FITC-dex and Alexa Fluor 555-conjugated CD31 antibodies (CD31-A555) were respectively injected to label blood vessels and Kupffer cells for 30 minutes before phage injection. A single median abdominal incision was performed up to ~5 mm at the base of the sternum. The cover glass was carefully put on the liver. ^{After intravenous injection of 2×10 10} pfu of Cy5-labeled wild-type or self-peptide-expressed T7 bacteriophage, 3-color fluorescence signal was generated with 3PMT through 3 bandpass filters (FF01-525/45, FF01-697/58, Semrock). It was detected at the same time (Fig. 9). Images were taken with an IVMVL custom confocal microscope in the GSNT department of KAIST.

As a result, the fluorescence intensity of both WT-T7 and Self-T7 increased immediately after injection, and the fluorescence of WT-T7 rapidly decreased within 1 minute, whereas the signal intensity of Self-T7 was more than twice that of WT-T7 lasted ( Fig. 7). The fluorescence intensity of WT-T7 was significantly lower than that of Self-T7 as a result of confirming the great saphenous vein (GSV) image at the same point through the fluorescence microscope image (FIG. 8). These results showed that the amount of WT-T7 circulating in the blood decreased rapidly over time due to phagocytosis.

In addition, WT-T7 was accumulated in the tissues, but Self-T7 continued to circulate along the blood. 10 minutes after injection, the red color of WT-T7 was observed in the liver, not in the blood vessels, and in the case of Self-T7, the red color of the phage appeared in the blood vessels than in the Kupffer cells of the liver (FIG. 9). Through these results, expressed in self-T7 - peptide was found to inhibit the phagocytosis in the in- vivo.

[Example 6] in vivo Treatment with phage in

For each infection experiment, E. coli BL21 was grown from single colonies in 150 ml of LB medium in a shaking incubator at 37° C. until the optical density (OD ₆₀₀ ) reached 1 (10 ^{9 cfu).} Thereafter, E. coli BL21 was harvested by centrifugation for 10 minutes at 15,000×g in 100 ml of a bacterial culture solution. The harvested E. coli BL21 bacteria were suspended in PBS at 4° C. ^{, and serially diluted with 2 × 10 6} , 2 × 10 ⁷ , 2 × 10 ⁸ , 2 × 10 ⁹ , 1 × 10 ¹⁰ cfu (per 100 μL). . Next, a 3-week-old balb/c male mouse (KOATECH (Pyeongtaek, Republic of Korea)) was prepared, and in order to measure the lethal dose, a diluted solution was injected into the mouse by intraperitoneal injection, and then observed for 100 hours (FIG. 10). Specifically, 3 groups of mice were infected with 2×10 ⁸ cfu (per 100 μl) of E. coli BL21 by intraperitoneal injection. Thirty minutes after infection, 2 of the 3 groups were ^{injected with 4×10 9} pfu of wild-type and 4×10 ⁹ pfu of self-peptide-expressed T7 microwave. The remaining groups were injected with PBS as a control. After injection, the mice were [normal; 0], [reduced activity; 1], [coma, wrinkled fur and hunchback; 2], [coma, wrinkled fur, hunchback and eyes closed with exudates around the eyes; 3], [Moribund state; 4], [death; 5] were classified and scored.

The results of confirming the symptoms of the mice are as follows. The PBS-treated control group did not show any symptoms, but the other group mice injected with E. coli died within 48 hours. Mice injected with WT-T7 died within 80 hours, and a slight treatment effect by T7 could be confirmed. In mice injected with Self-T7, symptoms appeared up to 20 hours after injection of E. coli , but symptoms began to disappear after 50 to 60 hours, and symptoms completely disappeared and returned to a normal state after 100 hours after injection of Self-T7. It came back (Fig. 11). These results showed that Self-T7 can survive for a long time in mice and that E. coli lytic activity can be maintained to alleviate symptoms caused by bacterial infection. In addition, in the mice treated with WT-T7, a viscous yellow liquid was discharged, resulting in yellowing and swelling of the intestine, and as a result of culturing the tissue fluid collected from each mouse, E. coli colonies appeared only in the group of mice treated with WT-T7. It could be confirmed (Fig. 12).

^{In addition, 4 × 10 9} pfu (per 100 µl) of T7 bacteriophage surface-modified with PEG was intravenously injected to confirm the effect of treating infections caused by E. coli. As a result, the treatment effect was superior to that of intravenous injection of WT-T7, but E. coli Unlike this completely killed Self-T7, it was confirmed that some of the E. coli survived even after 100 hours (FIGS. 13, 14, 15).

[Example 7] Oral administration treatment using phage

As in the above example, the effect of oral and intraperitoneal administration to avoid immunological elimination was measured, instead of the generally used injection administration method.

As a simulated experimental group, fasted gastric fluid, fed gastri fluid, and intestinal fluid were prepared and used as follows.

1.fasted gastric fluid

0.7% (v/v) 0.2% (w/v) sodium chloride in hydrochloric acid, 0.2 mg/mL pepsin, pH 1.5

2. fed gastri fluid

0.7% (v/v) 0.2% (w/v) sodium chloride in hydrochloric acid, 1.0 mg/mL pepsin, pH 5

3. Intestinal fluid

0.062% (v/v) 0.68% (w/v) KH ₂ PO _{4 in} NaOH, 1% (w/v) Pan Creatine, pH 6.8

For each experimental group prepared as described above, 1 x 10 ⁶ pfu of WT-T7, PEG-T7, and Self-T7 phage were added to each simulated fluid and incubated at 37° C. for 1 hour in a shaking incubator at 100 rpm. . After incubation, the solution of fasted gastric juice was neutralized with 1M NaOH, serially diluted with LB medium, and then E. coli at 37°C by a plaque-forming assay. After inoculating BL21 and incubating for 3 hours, the number of viable phages in the solution was determined.

The ratio of surviving phages for each experimental group was measured and shown in FIG. 18.

From the above results, survival of phage was almost impossible in fasted gastric juice with very low pH, but in gastric juice in which the supply of pepsin was increased through food supply, the pH changed to the extent that phage survival was possible, so that more than 80% of the phage survived. It was confirmed that plaque can be formed. This has a slightly lower survival rate of plaques compared to the case of direct administration to the intestinal fluid through the abdominal cavity, but there is a possibility that it can be used orally with food as an alternative to avoid immunological removal of phages caused by injection into blood vessels. It was confirmed that it was high.

[Example 8] Comparison of phagocytosis (macrophage) of Self-T7 and PEGylated phage (PEG-T7)

From the results of Example 1 above, in the T7 phage (Self-T7) expressing the self-peptide of the present invention, a 21-mer derived from CD47 that binds to SIRPα of macrophages to 10B capsid, which is a major capsid, It was confirmed that 415 copies of the sequence GNYTCEVTELTREGETIIELK self-peptide were expressed.

In order to compare the phagocytosis effect of the phage of the present invention, a PEGylated phage (PEG-T7) using a conventional method as a control was prepared as follows, similar to the PEG treatment process of Example 1.

The cultured culture of E. coli BL21 was inoculated into 500 ml of M9LB medium, and cultured in a shaking incubator at 37° C. to a final OD of ₆₀₀ = 0.7 to 0.8. T7 phage constructed in BL21 was infected in M9LB medium, and cultured until complete lysis of the cells was observed. Thereafter, 12.5 g of NaCl was added and centrifuged at 12,000×g at 4° C. for 10 minutes to separate bacterial debris from T7 phage. Next, 10% polyethylene glycol (MW 8kDa) was added to the supernatant, and stored at 4° C. for 10 hours to efficiently precipitate phage. The above precipitate was centrifuged at 12,000×g at 4° C. for 10 minutes, and the precipitated phage was resuspended in 10% PEG (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The suspension was centrifuged at 14,000×g at 4° C. for 10 minutes, and the pellet was resuspended in 1 M NaCl (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Then, the solution was centrifuged at 14,000×g at 4° C. for 30 minutes, and an aqueous phase containing T7 phage was carefully collected. Phage was ultracentrifuged for 60 minutes at 35,000 rpm at 20°C on a four-step gradient of cesium chloride in TE buffer (10 mM Tris, pH7.5, 100 mM NaCl) (Optima XE 90 ultracentrifuge, Beckman Coulter, Pasadena, CA, USA) and purified.

As a result, it was confirmed that about 48% of exposed lysine was conjugated with polyethylene glycol (PEG) in the T7 phage. PEG-T7 was surrounded by a layer of hydrophilic PEG, reducing the adhesion of opsonins present in the serum.

The self-peptide-expressing T7 phage of the present invention prepared as described above (Self-T7) and control PEG-T7, WT-T7 and PBS were treated in E. coli culture dishes, respectively, and then optically over time. The density (OD ₆₀₀ ) was measured (FIG. 19 ). The number of self-peptides expressed in Self-T7 was only 1/5 of the number of PEG conjugated to T7, but Self-T7 showed remarkably superior macrophage inhibitory effect compared to the PEG-T7.

In the recognition of the host by T7 phage, the tip domain including A518, D520, and V544 of the T7 tail fiber plays an important role in determining the range of the host, and the pyramidal domain is also a lipopolysaccharide on the phage surface. It was found that it can be used as a domain for interaction with (lipopolysaccharide).

From the above results, the self-peptide bound to the Self-T7 phage surface does not cause modification of the phage tail and does not affect bacterial infection efficiency, whereas PEG-T7 has a remarkable bacterial infection rate due to the screening effect on the phage tail. It was confirmed that there was a delay. The presence of primary amine sites in the tip domain and pyramidal domain of the phage tail can be confirmed through PDB ID code 4A0T, etc., and from this, it can be inferred that the binding of non-specific PEG interferes with the bacterial surface recognition of the T7 phage. there was.

[Example 9] Plant disease control experiment using phage composition

The bacteriophage prepared in Example 1 was taken out from the stock in which the bacteriophage was stored, 10 mg of bacteriophage was mixed in 1000 mL of water, and stirred well at 25° C. for 30 minutes to dissolve to prepare a liquid composition for controlling plant diseases.

Subsequently , in order to measure the activity of the composition against Cladosporium cucumerinum , which causes black star disease in cucumbers, 150 mL of the liquid composition prepared above was applied to cucumber leaves and roots, respectively. After treatment by spraying 10 times each, it was observed whether the black star pattern disappeared from the cucumber.

As a result, after 24 hours, it was confirmed that the diffusion rate of the black star pattern gradually began to slow down in cucumbers, and after 10 days, the black star pattern was no longer spreading. From this, it was confirmed that the bacteriophage contained in the composition exhibited lytic activity against the pathogen, Cladosporium cucumerinum, to inhibit the spread of plant diseases.

[Example 10] Expression test according to the length of self-peptide

The self-peptide DNA designed as follows was synthesized and expressed in T7 phage capsid protein 10B. Expression method was performed in the same manner as in Example 1, and in vitro and in vivo experiments were performed in the same manner as in Examples 2 to 6.

The self-peptide DNA sequence of 6 mer was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GAG GAG CTC ACA GAA GAG -3', reverse direction is 5'AGCT CTC TTC TGT GAG CTC CTC It's TG-3'.

7 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GAG ACA GAG CTC ACA GAA GAG -3', reverse direction is 5'AGCT CTC GCC TTC TGT GAG CTC CTC It's TG-3'.

The 8 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT GAG GTC GAG CTC ACA GAA GAG -3', reverse direction is 5'AGCT CTC TTC TGT GAG CTC GAC CTC It's ACC TG-3'.

9 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT GAG ACA GAG CTC ACA CGA GAA GAG -3', reverse direction is 5'AGCT CTC TTC TCG TGT GAG CTC TGT CTC It's ACC TG-3'.

The 10 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT GAG GTC GAG CTC ACA CGA GAA GAG AAG-3', reverse direction is 5'AGCT CTT CTC TTC TCG TGT GAG CTC GAC CTC It's ACC TG-3'.

The 11 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT GAG ACA GAG CTC ACA CGA GAA GGC GAG AAG-3', reverse direction is 5'AGCT CTT CTC GCC TTC TCG TGT GAG CTC TGT CTC It's ACC TG-3'.

The 12 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TGC GAG GTC GAG CTC ACA CGA GAA GGC GAG AAG-3', reverse direction is 5'AGCT CTT CTC GCC TTC TCG TGT GAG CTC GAC CTC It is GCA ACC TG-3'.

The 13 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG AAG-3', reverse direction is 5'AGCT CTT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC GCA ACC TG-3' to be.

The 14 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC AAG-3', reverse direction is 5'AGCT CTT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA ACC TG-3'.

The 15 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC AAG-3', reverse direction is 5'AGCT CTT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA TGT ACC TG-3'.

The 16 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC AAG-3', reverse direction is 5'AGCT CTT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA TGT GTA ACC TG-3'.

The 17 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC CTC AAG-3', reverse direction is 5'AGCT CTT GAG GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA TGT GTA ACC TG-3'.

The 18 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC ATA CTC AAG-3', reverse direction is 5'AGCT CTT GAG TAT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA TGT GTA ACC TG-3'.

The 19 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT AAC TAC TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC ATA ATT CTC AAG-3', reverse direction is 5'AGCT CTT GAG AAT TAT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA GTA GTT ACC TG-3'.

The 20 mer self-peptide DNA sequence was synthesized as follows.

Forward direction is 5'-[phosphate] AATT CA GGT AAC TAC ACA TGC GAG GTC ACA GAG CTC ACA CGA GAA GGC GAG ACC ATA ATT GAA AAG-3', reverse direction is 5'AGCT CTT TTC AAT TAT GGT CTC GCC TTC TCG TGT GAG CTC TGT GAC CTC It is GCA TGT GTA GTT ACC TG-3'.

As a result of in vitro and in vivo phagocytosis and E. coli killing experiments by expressing the above-synthesized self-peptide in T7 bacteriophage, there was a difference between the experimental groups in the effect according to the length of the self-peptide, but the immune evasion and E. The present invention was completed by confirming that it represents all of.

<110> Korea Advanced Institute of Science and Technology <120> Immunologically cloaking Bacteriophage <130> P-15950 <160> 38 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Self-peptide from Cluster of Differentiation 47 <400> 1 Gly Asn Tyr Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr 1 5 10 15 Ile Ile Glu Leu Lys 20 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Self-peptide from Cluster of Differentiation 47 <400> 2 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu 1 5 10 <210> 3 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> coding DNA of Self-peptide from Cluster of Differentiation 47 <400> 3 ggtaactaca catgcgaggt cacagagctc acacgagaag gcgagaccat aattgaactc 60 aag 63 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> T7 bacteriophage 10B capsid <400> 4 atgctcgggg atccgaattc aagcttg 27 <210> 5 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> self-peptide inserted T7 bacteriophage 10B capsid <400> 5 atgctcgggg atccgaattc aggtaactac acatgcgagg tcacagagct cacacgagaa 60 ggcgagacca taattgaact caagagcttg 90 <210> 6 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> embodiment 1 forward primer <400> 6 aattcaggta actacacatg cgaggtcaca gagctcacac gagaaggcga gaccataatt 60 gaactcaag 69 <210> 7 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> embodiment 1 reverse primer <400> 7 agctcttgag ttcaattatg gtctcgcctt ctcgtgtgag ctctgtgacc tcgcatgtgt 60 agttacctg 69 <210> 8 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> self-peptide inserted T7 10B capsid <400> 8 atgctcgggg atccgaattc aggtaactac acatgcgagg tcacagagct cacacgagaa 60 ggcgagacca taattgaact caagagcttg cggccgcact cgagtaacta g 111 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 6mer self-peptide forward <400> 9 aattcagagg agctcacaga agag 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 6mer self-peptide reverse <400> 10 agctctcttc tgtgagctcc tctg 24 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 7mer self-peptide forward <400> 11 aattcagaga cagagctcac agaagag 27 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 7mer self-peptide reverse <400> 12 agctctcgcc ttctgtgagc tcctctg 27 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 8mer self-peptide forward <400> 13 aattcaggtg aggtcgagct cacagaagag 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 8mer self-peptide reverse <400> 14 agctctcttc tgtgagctcg acctcacctg 30 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 9mer self-peptide forward <400> 15 aattcaggtg agacagagct cacacgagaa gag 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 9mer self-peptide reverse <400> 16 agctctcttc tcgtgtgagc tctgtctcac ctg 33 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 10mer self-peptide forward <400> 17 aattcaggtg aggtcgagct cacacgagaa gagaag 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 10mer self-peptide reverse <400> 18 agctcttctc ttctcgtgtg agctcgacct cacctg 36 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> 11mer self-peptide forward <400> 19 aattcaggtg agacagagct cacacgagaa ggcgagaag 39 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> 11mer self-peptide reverse <400> 20 agctcttctc gccttctcgt gtgagctctg tctcacctg 39 <210> 21 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 12mer self-peptide forward <400> 21 aattcaggtt gcgaggtcga gctcacacga gaaggcgaga ag 42 <210> 22 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 12mer self-peptide reverse <400> 22 agctcttctc gccttctcgt gtgagctcga cctcgcaacc tg 42 <210> 23 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 13mer self-peptide forward <400> 23 aattcaggtt gcgaggtcac agagctcaca cgagaaggcg agaag 45 <210> 24 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 13mer self-peptide reverse <400> 24 agctcttctc gccttctcgt gtgagctctg tgacctcgca acctg 45 <210> 25 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 14mer self-peptide forward <400> 25 aattcaggtt gcgaggtcac agagctcaca cgagaaggcg agaccaag 48 <210> 26 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 14mer self-peptide reverse <400> 26 agctcttggt ctcgccttct cgtgtgagct ctgtgacctc gcaacctg 48 <210> 27 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 15mer self-peptide forward <400> 27 aattcaggta catgcgaggt cacagagctc acacgagaag gcgagaccaa g 51 <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 15mer self-peptide reverse <400> 28 agctcttggt ctcgccttct cgtgtgagct ctgtgacctc gcatgtacct g 51 <210> 29 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> 16mer self-peptide forward <400> 29 aattcaggtt acacatgcga ggtcacagag ctcacacgag aaggcgagac caag 54 <210> 30 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> 16mer self-peptide reverse <400> 30 agctcttggt ctcgccttct cgtgtgagct ctgtgacctc gcatgtgtaa cctg 54 <210> 31 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> 17mer self-peptide forward <400> 31 aattcaggtt acacatgcga ggtcacagag ctcacacgag aaggcgagac cctcaag 57 <210> 32 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> 17mer self-peptide reverse <400> 32 agctcttgag ggtctcgcct tctcgtgtga gctctgtgac ctcgcatgtg taacctg 57 <210> 33 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> 18mer self-peptide forward <400> 33 aattcaggtt acacatgcga ggtcacagag ctcacacgag aaggcgagac catactcaag 60 60 <210> 34 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> 18mer self-peptide reverse <400> 34 agctcttgag tatggtctcg ccttctcgtg tgagctctgt gacctcgcat gtgtaacctg 60 60 <210> 35 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> 19mer self-peptide forward <400> 35 aattcaggta actactgcga ggtcacagag ctcacacgag aaggcgagac cataattctc 60 aag 63 <210> 36 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> 19mer self-peptide reverse <400> 36 agctcttgag aattatggtc tcgccttctc gtgtgagctc tgtgacctcg cagtagttac 60 ctg 63 <210> 37 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> 20mer self-peptide forward <400> 37 aattcaggta actacacatg cgaggtcaca gagctcacac gagaaggcga gaccataatt 60 gaaaag 66 <210> 38 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> 20mer self-peptide reverse <400> 38 agctcttttc aattatggtc tcgccttctc gtgtgagctc tgtgacctcg catgtgtagt 60 tacctg 66

Claims (15)

delete delete delete delete delete delete delete A pharmaceutical composition for the treatment or prevention of infections caused by bacteria, including a bacteriophage containing a self-peptide represented by SEQ ID NO: 1 (GNYTCEVTELTREGETIIELK) in the capsid . The method of claim 8,
The pharmaceutical composition is a pharmaceutical composition for oral administration or injection.
The method of claim 8,
The bacteria are Escherichia coli ( E. coli ), Shigella sonei bacteria (Sigella sonnei ) and Salmonella typhimurium , Corynebacterium diphtheria , Clostridium Botulinum ( Clostridium botulinum ) , Staphylococcus aureus , Streptococcus pyogenes And A pharmaceutical composition that is a bacterium selected from the group consisting of cholera bacteria ( Vibrio cholera ).
The method of claim 8,
The infectious disease is one or more infectious diseases selected from the group consisting of enteritis, bacterial dysentery, diphtheria, acute fever, scarlet fever, cholera and food poisoning. The capsid (capsid) contains a bacteriophage containing a self-peptide represented by SEQ ID NO: 1 (GNYTCEVTELTREGETIIELK), and the bacteriophage is lytic, anti-bacterial (anti-bacterial) composition. The method of claim 12,
The bacteria that are the target of the antibacterial are E. coli , Shigella sonnei and Salmonella typhimurium , Corynebacterium diphtheria , Clostridium Botulinum ( Clostridium botulinum ) , Staphylococcus aureus , Streptococcus pyogenes And Cholera bacteria ( Vibrio cholera ) is a bacterium selected from the group consisting of antibacterial composition. The capsid (capsid) contains a bacteriophage containing a self-peptide represented by SEQ ID NO: 1 (GNYTCEVTELTREGETIIELK), and the bacteriophage is a lytic, health functional food for preventing infection caused by bacteria. A composition for controlling plant diseases caused by bacteria, including a bacteriophage containing a self-peptide represented by SEQ ID NO: 1 (GNYTCEVTELTREGETIIELK) in the capsid, and the bacteriophage is lytic.

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