KR101539336B1 - Anti-bacterial composition comprising ADK - Google Patents

Abstract

The present invention relates to an antibacterial composition comprising an adenylate kinase (ADK) protein derived from Mycobacterium tuberculosis as an active ingredient and a composition for preventing or treating infectious diseases. The ADK protein derived from Mycobacterium tuberculosis in accordance with the present invention can be usefully used in producing an antibacterial composition for gram-negative bacteria or preventing or treating infectious diseases caused by gram-negative bacteria by selectively having excellent antibacterial activity against gram-negative bacteria.

Description

An anti-bacterial composition comprising an ADK protein as an active ingredient,

The present invention relates to an antibiotic composition comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis as an active ingredient, and a composition for preventing or treating an infectious disease.

The term " antibacterial agent " refers generally to an antimicrobial agent, and particularly refers to a substance that has an antibacterial effect against bacteria, in particular, a substance that exhibits excellent antibacterial activity by inhibiting a system of bacteria synthesizing cell walls and proteins, it means. Antimicrobial agents are mainly extracted from fungi and are widely used today to treat diseases caused by bacterial infection.

Starting with the discovery of penicillin antibiotics by Fleming in the 20th century, numerous antimicrobials and antibiotics have been developed to escape the diseases caused by bacterial infections. These antimicrobial agents occupy an essential position in our lives, and they are widely used not only as medicines but also as food and cosmetic preservatives. However, in the case of an antimicrobial agent using a chemical synthetic substance, the number of bacteria resistant to the antimicrobial agent is gradually increasing, and the use thereof is gradually limited.

Antimicrobial Resistant Bacteria refers to bacteria that are resistant to specific antimicrobial agents and do not respond to the antibiotics. For example, penicillin-resistant Staphylococcus aureus, which has no effect on penicillin as described above, is applicable. In addition, Methicillin-Resistant Staphylococcus aureus (MRSA) has been reported for the first time in academic circles in 1961 and has since become a major pathogenic infectious disease worldwide. In 1988, Vancomycin-resistant enterococcus (VRE) was first detected in Europe and in late 1990, vancomycin intermediate-resistant S. aureus (VISA), a vancomycin resistant strain reported in Japan, USA, France and Korea Respectively. In addition, vancomycin-resistant staphylococcus aureus (VRSA), highly resistant to vancomycin, the last known treatment for Staphylococcus aureus, the most common causal agent of human infection, was introduced in the Centers for Disease Control By the first report, the possibility of so-called super bacteria is increasing.

Accordingly, the present inventor has made efforts to develop a new antimicrobial agent, and as a result, it has been confirmed that the ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis selectively exhibits an excellent antibacterial activity against Gram- Thereby completing the invention.

It is an object of the present invention to provide an antimicrobial composition for Gram-negative bacteria comprising ADK protein as an active ingredient.

It is still another object of the present invention to provide a composition for preventing or treating an infectious disease comprising an ADK protein as an active ingredient.

In order to achieve the above object, the present invention provides an antimicrobial composition, quasi-drug, food additive, or feed additive for Gram-negative bacteria comprising ADK protein as an active ingredient.

The present invention also provides a pharmaceutical composition or food composition for preventing or treating an infectious disease comprising an ADK protein as an active ingredient.

The ADK protein derived from Mycobacterium tuberculosis according to the present invention has an excellent antimicrobial activity selectively against Gram-negative bacteria, and is useful as an antimicrobial composition for Gram-negative bacteria or an infectious disease caused by Gram- Can be usefully used for prevention or treatment.

FIG. 1 shows the result of SDS-PAGE of the recombinant ADK protein.
Figure 2 shows the effect of recombinant ADK protein on the growth of E. coli .
Figure 3 shows the effect of recombinant ADK protein on the growth of P. aeruginosa .
Figure 4 shows the effect of recombinant ADK protein on the growth of S. aureus .
Figure 5 shows the results of SEM analysis of the effect of recombinant ADK protein on E. coli cell morphology.
Fig. 6 shows the results of TEM analysis of the effect of recombinant ADK protein on the surface of E. coli cells and the degree of destruction of cell bodies.
FIG. 7 shows the result of confirming that ADK protein specifically migrates only to Gram-negative bacteria in an animal model.
8 is a graph showing the results of confirming the bactericidal effect of ADK protein on E. coli in an animal model.
FIG. 9 is a graph showing the results of examining the bactericidal effect of ADK protein on S. aureus in an animal model. FIG.
Fig. 10 is a graph showing the results of confirming the sterilizing effect of ADK protein in each tissue of an animal model. Fig.

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

The present invention provides an antimicrobial composition for Gram-negative bacteria comprising an ADK protein as an active ingredient.

In the present invention, "antibacterial" or "antibacterial activity" means a property of resisting microorganisms such as bacteria or fungi, and more specifically, antibiotics means a property of inhibiting growth or proliferation of bacteria .

In the present invention, the "antimicrobial composition" is a composition having an activity of inhibiting the growth of microorganisms such as bacteria and fungi, and may include all forms used in various fields requiring an antibacterial effect. For example, , Quasi-drugs, food additives or feed additives, and the like. Specifically, in medicines, for antimicrobial, sterilizing and disinfecting purposes in agriculture, antimicrobial, sterilizing, and disinfecting in agriculture, for dandruff prevention, for athlete's foot, underarm It may be used for products directly related to microorganisms such as anti-ingestion, anti-acne, etc., or may be used for preservative, antimicrobial or sterilizing purposes, for example, for cleaning detergents or dishwashing detergents.

The ADK protein of the present invention is derived from Mycobacterium tuberculosis . Preferably, the ADK protein is composed of the amino acid sequence shown in SEQ ID NO: 1 and can be encoded with the nucleotide sequence shown in SEQ ID NO: And includes functional equivalents of such proteins. Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Refers to a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein having substantially the same physiological activity as the protein encoded by the nucleotide sequence shown in SEQ ID NO: 2.

The ADK protein of the present invention includes not only the protein having the native amino acid sequence but also the amino acid sequence variant thereof within the scope of the present invention. A variant of an ADK protein refers to a protein having an ADK native amino acid sequence and one or more amino acid residues having different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. The ADK protein or variant thereof can be prepared by natural extraction or synthesis (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by recombinant methods based on DNA sequences (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

The ADK protein of the present invention can be encoded by the nucleotide sequence of SEQ ID NO: 2, and variants capable of functionally equivalent to the nucleotide are included within the scope of the present invention.

The gram negative bacteria according to the present invention means bacteria which are stained with red when stained with a Gram stain method, and generally have a strong coloring resistance and strong surfactant resistance. Gram-negative bacteria of the present invention includes all types of gram-negative bacteria have an endotoxin (endotoxin), for example, Escherichia O (Escherichia) genus Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) genus, Salmonella (Salmonella ), a keurep when Ella (Klebsiella) genus Neisseria (Neisseria) genus, Enterobacter (Enterobacter) genus Shigella (Shigella), a morak Cellar (Moraxella) genus, Helicobacter pylori (Helicobacter), a Pomona in Ste note's (Stenotrophomonas) genus, genus Vibrio (Bdellovibrio) to beudel, Legionella, and the like (Legionella) in strain, but is not limited thereto. Specifically, Gram-negative bacteria include Escherichia coli , Pseudomonas aeruginosa , Pseudomonas fluorescens , Pseudomonas putida , Pseudomonas chlororaphis , Pseudomonas pertusino , Pseudomanas pertucinogena , Pseudomonas stutzeri , Pseudomonas syringae , Acinetobacter baumannii , Acinetobacter lwoffii , Acinetobacter calcoaceticus , But are not limited to, Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Salmonella enterica , Salmonella bongori , Salmonella enteritidis , Salmonella typhimurium , , Salmonella gallinarum ( Salmonella gallinarum ), Salmonella pool room (Salmonella pullorum), Salmonella emban Dhaka (Salmonella mbandaka), syulseu (Salmonella choleraesuls) salmonella, cholera, Salmonella Thompson (Salmonella thompson), Salmonella inpam teeth (Salmonella infamtis), Salmonella Derby (Salmonella derby), keurep when Ella Klebsiella pneumonia , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , Neisseria gonorrhoeae , age (for example, Klebsiella pneumonia , Klebsiella pneumoniae , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , Neisseria meningitidis , Enterobacter aerogenes , Enterobacter cloacae , Shigella boydii , Shigella dysenteriae , Siegel meningitidis , Enterobacter aerogenes , Enterobacter cloacae , La flex Neri (Shigella flexneri), Shigella hand Nei (Shigella sonnei), morak Cellar Kata Harleys (Moraxella catarrhalis), morak La Raku appears (Moraxella lacunata), morak Cellar Vorbis (Moraxella bovis), Helicobacter pylori (Helicobacter pylori), H. Hale Mani (Helicobacter heilmannii), H. Felice (Helicobacter felis), H. non-steel rail (Helicobacter mustelae), Helicobacter page Nelly Helicobacter spp ., Helicobacter fenelliae , Helicobacter rappini , Helicobacter hepaticus , Helicobacter bilis , Helicobacter pullorum , Stenotrophomonas maltophilia , Stenotrophomonas nitritireducens , Bdellovibrio bacteriovorus , Legionella pneumophila , Legionalla anisa , Legionalla birminghamensis ( Legionalla birminghamensis ), Stenotrophomonas spp . ), Legionella The Mani (Legionalla bozemanii), Legionella Cincinnati N-Sys (Legionalla cincinnatiensis), Legionella two fur (Legionalla dumoffii), Legionella pilreyi (Legionalla feeleii), Legionella pick Manipur (Legionalla gormanii), Legionella and Kerry lover (Legionalla hackeliae), Legionella Israel Legionalla israelensis , Legionalla jordanis , Legionalla lansingensis , Legionalla longbeachae , Legionalla maceachernii , Legionalla micdadei , Legionella oak lead Zensys (Legionalla oakridgensis), Legionella, and Intel Corporation alkylene upon the like (Legionalla sainthelensi), Legionella Took sonen sheath (Legionalla tucsonensis), Legionella ward's Zloty (Legionalla wadsworthii), but is not limited thereto.

In another aspect, the present invention provides a composition for preventing or treating an infectious disease comprising an ADK protein as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Since the ADK protein of the present invention selectively exhibits excellent antimicrobial activity against Gram-negative bacteria, the composition containing the ADK protein can be effectively used for the prevention or treatment of infectious diseases.

In the present invention, "infectious disease" means a disease caused by the propagation or invasion of a pathogen causing diseases such as viruses, bacteria, fungi and parasites to animals or humans. For the purpose of the present invention, Means an infectious disease. For example, the infectious diseases of the present invention include respiratory diseases, gastrointestinal diseases, inflammatory diseases and the like caused by Gram-negative bacteria, and specifically include pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract infection, meningitis, But are not limited to, myocarditis, myocarditis, arthritis, gonorrhea, gonorrhea, bacterial dysentery, enteritis, conjunctivitis, gastritis, otitis media, cystitis, lymphatic tuberculosis, sepsis and the like.

In the present invention, "prevention" means any action that inhibits or delays the infectious disease by administration of the composition, and "treatment" means that administration of the composition improves symptoms caused by the infectious disease, Means any act that

The pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent conventionally used. Examples of the carrier, excipient or diluent that can be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be administered orally or parenterally in a conventional manner. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In the present invention, "administering" means providing a desired pharmaceutical composition of the present invention to an individual by any suitable method.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the pharmaceutical composition of the present invention may be administered at a daily dose of 0.001 to 1000 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. The composition of the present invention can preferably be administered in the form of an injection.

The pharmaceutical composition according to the present invention may further include one or more known substances having antimicrobial activity besides the ADK protein.

In the present invention, the food composition may preferably be in the form of a health functional food.

In the present invention, the term " health functional food " refers to a food group imparted with added value to function or express the function of the food by physical, biochemical, biotechnological techniques or the like, Means a food which has been designed and manufactured so that the body's control function regarding disease prevention and recovery is sufficiently expressed to the living body.

The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

When the food composition of the present invention is used as a food additive, the composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Cloning of recombinant ADK (Rv0733)

The ADK (Rv0733) region was amplified by PCR using the genomic DNA of Mycobacterium tuberculosis H37Rv (ATCC 27294) as a template (primers: 5'- CATATG AGAGTTTTGTTGCTGGGACCG-3 'and 5' - AAGCTT CTTTCCCAGAGCCCGCAACGC-3 '). The isolated PCR product was digested with NdeI and HindIII enzymes and inserted into the pET22b vector, an expression vector. E. coli BL21 transformed with the pET22b vector into which the ADK gene was inserted was transferred to LB medium (containing 100 μg / ml ampicillin) and cultured at 37 ° C for 12 hours. Thereafter, 1 mM isopropyl-D-thiogalactopyranoside (IPTG) was added and cultured for another 6 hours, and the cells were then lysed with lysis buffer (1M DTT, lysozyme, PMSF). The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA, Invitrogen, Carlsbad, CA, USA) agarose according to the manufacturer's method. The final purified recombinant ADK protein was confirmed by SDS-PAGE analysis and was confirmed by sequencing (SEQ ID NO: 1) and quantified using the Bradford assay. The result of confirming the recombinant ADK protein by SDS-PAGE is shown in Fig.

Experimental Example 1. Antibacterial activity of ADK protein

To examine the antimicrobial activity of the recombinant ADK protein obtained in Example 1, the ADK protein was cultured in the presence of Gram-negative bacteria Escherichia coli, E. coli , Pseudomonas aeruginosa and P. aeruginosa , ( Staphylococcus aureus, S. aureus ) on the growth of S. aureus .

More specifically, 1 × 10 ⁵ CFU of the strain was added to 10 ml of LB medium, and the ampicillin and ADK proteins were added according to the following five conditions (Group 1: LB broth media with ampicillin 100 μg / ml , Group 2: LB broth media with Adk 20 μg / ml, Group 3: LB broth media with Adk 50 μg / ml, Group 4: LB broth media with 100 μg / ml, Group 5: Non-antibiotics LB broth media) . After incubation in a 37 ° C shaking incubator, 100 μl of each group was taken at 3-hour intervals, diluted in LB medium in appropriate volume, and plated on LB agar (Biobasic, Amherst, NY) plate. After incubation overnight at 37 ° C, the number of colonies on the LB agar plate was counted and the CFU value for each hour was calculated. The results are shown in FIG. 2 to FIG.

As shown in FIG. 2 to FIG. 4, the ADK protein inhibited the growth of Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa but did not affect the growth of Staphylococcus aureus, a Gram-positive bacterium. On the other hand, the known antibiotic, ampicillin, inhibited the growth of Gram-negative bacteria and Gram-positive bacteria. As a result, it was confirmed that the ADK protein of the present invention showed an antimicrobial effect specifically for Gram-negative bacteria.

Experimental Example 2. Analysis of MIC and MBC

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the recombinant ADK protein were measured in order to determine the optimum concentration of the recombinant ADK protein to inhibit growth of various Gram-negative bacteria and Gram-positive bacteria.

More specifically, colonies of each strain ( Escherichia coli DH5α, Escherichia coli K1, Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa, Salmonella enteritidis (ATCC 13076), Salmonella typhimurium, Klebsiella pneumoniae (ATCC 13883), Staphylococcus aureus USA: One strain was isolated and cultured for 6 hours in 10 ml of MH agar medium (OXOID, Hampshire, UK), and each strain was adjusted to a spectrophotometer OD 625 of 0.1 (MaFarland 0.5) MH medium. The diluted strains were diluted 1/20 in saline (0.09% NaCl). Antibiotics (ampicillin, kanamycin, gentamycin) and ADK protein were added to 96-well culture plates containing 100 μl MH medium, and 9 μl of the diluted strains were added to each well and cultured overnight.

MICs were calculated using REMA (Resazurin microtiter assay). 30 μl of 0.01% resazurin (ACROS organics, NJ) solution was added to each well of the 96-well culture plate and incubated at 37 ° C for 1 hour. When the medium of each well is indigo, it means that the strain does not grow, and when it is pink, it indicates that the strain is growing well. These color changes were measured on a spectrophotometer OD 505 and 50% and 80% inhibitory concentrations were calculated assuming 100% results in wells not treated with antibiotics and ADK proteins.

In order to measure MBC, 10 占 퐇 of each well of the 96-well culture plate was plated on LB agar plates and then cultured at 37 占 폚 overnight to calculate concentrations at which colonies did not grow.

The results of the above tests are shown in Table 1.

As shown in Table 1 above, the ADK protein inhibited the growth of all kinds of Gram-negative bacteria and showed a bactericidal effect against them. However, it was confirmed that ADK protein had no particular influence on growth and survival of Staphylococcus aureus, Respectively.

Experimental Example 3: SEM analysis

To confirm the effect of recombinant ADK protein on cell morphology, Escherichia coli was treated with recombinant ADK protein over time and analyzed by scanning electron microscope (SEM).

More specifically, 10 ml of LB medium was treated with 100 μg / ml ampicillin (used as a positive control) or 20 μg / ml ADK protein, and E. coli K1 was incubated for a period of time at 37 ° C. with shaking Respectively. The cultured Escherichia coli cells were washed three times with 1 × PBS and fixed with 1 ml of a solution of 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO). The sample was observed using LEO 1455 VP SEM (Carl Zeiss, Oberkochen, Germany). The results are shown in Fig.

As shown in FIG. 5, the control group had a bright and smooth cell surface, whereas the ADK protein-treated group showed that the surface of E. coli was peeled off and cell death was observed.

Experimental Example 4: TEM analysis

To confirm the effect of the recombinant ADK protein on the cell surface and the degree of destruction of cell organs, Escherichia coli was treated with recombinant ADK protein and after 12 hours, transmission electron microscopy (TEM) analysis using gold nanoparticles according to a known method Respectively. The results are shown in Fig.

As shown in FIG. 6, in the case of the control (Control), the surface of the cells was kept constant, and the nanoparticles were introduced into the cells and showed no destruction. However, in the group treated with the gold nanoparticles coated with the ADK protein, Cell wall was lost and cell death was observed inside the cell (white appearance).

Experimental Example 5. Selective Antibacterial Activity of Gram Negative Bacteria of ADK Protein Using Animal Model

To confirm whether recombinant ADK protein selectively binds to Gram-negative bacteria in animal models, the following experiment was conducted.

First, E. coli K1 or P. aeruginosa, a gram-negative organism, was subcutaneously injected into the left side of CAnN.Cg-Foxn1 nu / CrljOri mice at a concentration of 10 ⁷ CFU / mice, and S. aureus , a gram- On the right side. 200 μg / mouse of IRDye800-conjugated ADK was intravenously injected into the tail of the mouse. After 3 hours of injection, the movement and position of ADK was observed using a near-infrared (NIR) fluorescence imaging system. The results are shown in Fig.

As shown in Fig. 7, it was confirmed that the ADK protein specifically migrates only toward Gram-negative bacteria.

In order to confirm that the recombinant ADK protein selectively bound to Gram-negative bacteria in the animal model has a bactericidal effect against Gram-negative bacteria, E. coli K1 stained with IRDye800 or S. aureus (10 ⁷ CFU / mice) and FITC-conjugated ADK (200 μg / mice) were intraperitoneally injected into CAnN.Cg-Foxn1 nu / CrljOri mice and observed for sterilization by time of near-infrared (NIR) fluorescence imaging system. After 24 hours of injection, each organ was removed to confirm the presence of the strain. The results are shown in Fig. 8 to Fig.

As shown in FIG. 8, in the IRdye800-conjugated E. coli alone group, a certain amount of E. coli survived in the abdominal cavity of mice until 24 hours after the injection, but the group treated with FITC-conjugated ADK was E. coli . The survival rate of E. coli was remarkably decreased and it was confirmed that it was completely removed after 24 hours of injection. In addition, the ADK protein was also released to the outside after 24 hours.

On the other hand, as shown in FIG. 9, in the case of IRdye800-conjugated S. aureus injected group, the survival rate of S. aureus was not influenced even when FITC-conjugated ADK was treated.

In addition, as shown in Fig. 10, it was confirmed that E. coli , a gram-negative bacterium, was completely removed by the ADK protein in both mouse lung, spleen, liver and kidney tissues.

From the above results, it was confirmed that the ADK protein derived from Mycobacterium tuberculosis selectively has an excellent antibacterial activity against Gram-negative bacteria.

Hereinafter, formulation examples of the pharmaceutical composition and the food composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1. Preparation of a pharmaceutical composition

1-1. Manufacture of Powder

ADK protein 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

ADK protein 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of capsules

ADK protein 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation Example 2. Preparation of Food Composition

2-1. Manufacture of health food

ADK protein 100 mg

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

<110> Konkuk University Industrial Cooperation Corp <120> Anti-bacterial composition comprising ADK <130> KK1-6 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 182 <212> PRT <213> Mycobacterium tuberculosis <400> 1 Met Arg Val Leu Leu Leu Gly Pro Gly Ala Gly Lys Gly Thr Gln   1 5 10 15 Ala Val Lys Leu Ala Glu Lys Leu Gly Ile Pro Gln Ile Ser Thr Gly              20 25 30 Glu Leu Phe Arg Arg Asn Ile Glu Glu Gly Thr Lys Leu Gly Val Glu          35 40 45 Ala Lys Arg Tyr Leu Asp Ala Gly Asp Leu Val Pro Ser Asp Leu Thr      50 55 60 Asn Glu Leu Val Asp Asp Arg Leu Asn Asn Pro Asp Ala Ala Asn Gly  65 70 75 80 Phe Ile Leu Asp Gly Tyr Pro Arg Ser Val Glu Gln Ala Lys Ala Leu                  85 90 95 His Glu Met Leu Glu Arg Arg Gly Thr Asp Ile Asp Ala Val Leu Glu             100 105 110 Phe Arg Val Ser Glu Glu Val Leu Leu Glu Arg Leu Lys Gly Arg Gly         115 120 125 Arg Ala Asp Asp Thr Asp Asp Val Ile Leu Asn Arg Met Lys Val Tyr     130 135 140 Arg Asp Glu Thr Ala Pro Leu Leu Glu Tyr Tyr Arg Asp Gln Leu Lys 145 150 155 160 Thr Val Asp Ala Val Gly Thr Met Asp Glu Val Phe Ala Arg Ala Leu                 165 170 175 Arg Ala Leu Gly Lys ***             180 <210> 2 <211> 546 <212> DNA <213> Mycobacterium tuberculosis <400> 2 gtgagagttt tgttgctggg accgcccggg gcgggcaagg ggacgcaggc ggtgaagctg 60 gccgagaagc tcgggatccc gcagatctcc accggcgaac tcttccggcg caacatcgaa 120 gagggcacca agctcggcgt ggaagccaaa cgctacttgg atgccggtga cttggtgccg 180 tccgacttga ccaatgaact cgtcgacgac cggctgaaca atccggacgc ggccaacgga 240 ttcatcttgg atggctatcc acgctcggtc gagcaggcca aggcgcttca cgagatgctc 300 gaacgccggg ggaccgacat cgacgcggtg ctggagtttc gtgtgtccga ggaggtgttg 360 ttggagcgac tcaaggggcg tggccgcgcc gacgacaccg acgacgtcat cctcaaccgg 420 atgaaggtct accgcgacga gaccgcgccg ctgctggagt actaccgcga ccaattgaag 480 accgtcgacg ccgtcggcac catggacgag gtgttcgccc gtgcgttgcg ggctctggga 540 aagtag 546

Claims (14)

1. An antimicrobial composition for Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
delete delete 2. The antimicrobial composition for Gram-negative bacteria according to claim 1, wherein the ADK protein is encoded by the nucleotide sequence shown in SEQ ID NO: 2.
The method of claim 1 or claim 4, wherein the gram-negative bacteria is Escherichia O (Escherichia) genus Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) genus, Salmonella (Salmonella), A keurep when in Ella (Klebsiella), Neisseria (Neisseria) genus, Enterobacter (Enterobacter) genus Shigella (Shigella), a morak Cellar V. (Bdellovibrio) to (Moraxella) genus, Helicobacter pylori (Helicobacter), a Pomona in Ste note's (Stenotrophomonas), a beudel Wherein the antimicrobial composition is at least one selected from the group consisting of genus Escherichia , genus Escherichia , and genus Legionella .
6. The method according to claim 5, wherein the gram-negative bacteria are selected from the group consisting of Escherichia coli , Pseudomonas aeruginosa , Pseudomonas fluorescens , Pseudomonas putida , Pseudomonas chlororaphis , Pseudomonas stutzeri , Pseudomonas syringae , Acinetobacter baumannii , Acinetobacter lwoffii , Acinetobacter lwoffii , Acinetobacter spp ., Pseudomonas spp ., Pseudomonas spp ., Pseudomonas spp . But are not limited to, Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Salmonella enterica , Salmonella bongori , Salmonella enteritidis , Salmonella typhimurium, Salmonella typhimurium , Salmonella gall inarum), Salmonella pool room (Salmonella pullorum), Salmonella emban Dhaka (Salmonella mbandaka), Salmonella cholera syulseu (Salmonella choleraesuls), Salmonella Thompson (Salmonella thompson), Salmonella inpam teeth (Salmonella infamtis), Salmonella Derby (Salmonella derby), Klebsiella pneumonia , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , Neisseria gonorrhoeae , Klebsiella pneumonia , Klebsiella pneumonia , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , ), Neisseria meningitidis , Enterobacter aerogenes , Enterobacter cloacae , Shigella boydii , Shigella dysenteriae, and the like. ), Shigella flexneri , Shigella sonnei , Moraxella catarrhalis , Morak Cellar Raku appears (Moraxella lacunata), morak Cellar Vorbis (Moraxella bovis), Helicobacter pylori (Helicobacter pylori), H. Hale Mani (Helicobacter heilmannii), H. Felice (Helicobacter felis), H. non-steel rail (Helicobacter mustelae), Helicobacter page Helicobacter fenelliae , Helicobacter rappini , Helicobacter hepaticus , Helicobacter bilis , Helicobacter pullorum , Stenotrophomonas maltophilia, ), watch as Stephen notes Pomona's age Retreat leader sense (Stenotrophomonas nitritireducens), Vibrio bacteriophage as beudel's (Bdellovibrio bacteriovorus), Legionella pneumophila (Legionella pneumophila), Legionella not four (Legionalla anisa), Legionella vir Ming hamen system (Legionalla birminghamensis ), Reggio Nella Bose Mani (Legionalla bozemanii), Legionella Cincinnati N-Sys (Legionalla cincinnatiensis), Legionella two fur (Legionalla dumoffii), Legionella pilreyi (Legionalla feeleii), Legionella pick Manipur (Legionalla gormanii), Legionella and Kerry lover (Legionalla hackeliae), Legionella Legionalla israelensis , Legionalla jordanis , Legionalla lansingensis , Legionalla longbeachae , Legionalla maceachernii , Legionalla micdadei , and characterized in that at least one member selected from the group consisting of Legionella oak lead Zensys (Legionalla oakridgensis), Legionella four Intel alkylene when (Legionalla sainthelensi), Legionella Took sonen sheath (Legionalla tucsonensis) and Legionella ward's Zloty (Legionalla wadsworthii) Antimicrobial compositions for, gram-negative bacteria.
An antimicrobial quasi-drug for Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
An antimicrobial food additive for Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
An antimicrobial feed additive for Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
A pharmaceutical for the prophylactic or therapeutic treatment of an infectious disease caused by Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient Composition.
delete The method of claim 10 wherein the gram negative bacteria is Escherichia O (Escherichia) genus Pseudomonas (Pseudomonas) genus Acinetobacter (Acinetobacter) genus, Salmonella (Salmonella), A keurep when Ella (Klebsiella) genus Neisseria (Neisseria ) genus, Enterobacter (Enterobacter) genus Shigella (Shigella), a morak Cellar (Moraxella) genus, Helicobacter pylori (Helicobacter), a Pomona's (Stenotrophomonas) genus Vibrio (Bdellovibrio) in and Legionella in beudel a stereo notes ( Legionella ). &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
13. The method of claim 12, wherein the Gram-negative bacteria are selected from the group consisting of Escherichia coli , Pseudomonas aeruginosa , Pseudomonas fluorescens , Pseudomonas putida , Pseudomonas chlororaphis , Pseudomonas stutzeri , Pseudomonas syringae , Acinetobacter baumannii , Acinetobacter lwoffii , Acinetobacter lwoffii , Acinetobacter spp ., Pseudomonas spp ., Pseudomonas spp ., Pseudomonas spp . But are not limited to, Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Salmonella enterica , Salmonella bongori , Salmonella enteritidis , Salmonella typhimurium, (Salmonella typhimurium), Salmonella Galina room (Salmonella gal linarum), Salmonella pool room (Salmonella pullorum), Salmonella emban Dhaka (Salmonella mbandaka), Salmonella cholera syulseu (Salmonella choleraesuls), Salmonella Thompson (Salmonella thompson), Salmonella inpam teeth (Salmonella infamtis), Salmonella Derby (Salmonella derby), Klebsiella pneumonia , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , Neisseria gonorrhoeae , Klebsiella pneumonia , Klebsiella pneumonia , Klebsiella granulomatis , Klebsiella oxytoca , Klebsiella terrigena , ), Neisseria meningitidis , Enterobacter aerogenes , Enterobacter cloacae , Shigella boydii , Shigella dysenteriae, and the like. ), Shigella flexneri , Shigella sonnei , Moraxella catarrhalis , , Morak Cellar Raku appears (Moraxella lacunata), morak Cellar Vorbis (Moraxella bovis), Helicobacter pylori (Helicobacter pylori), H. Hale Mani (Helicobacter heilmannii), H. Felice (Helicobacter felis), H. non-steel rail (Helicobacter mustelae), H. Tenerife Nelly in (Helicobacter fenelliae), H. La Feeney (Helicobacter rappini), H. H. party Syracuse (Helicobacter hepaticus), H. Billy's (Helicobacter bilis), H. pool room (Helicobacter pullorum), Pomona in Ste note's malto pilriah (Stenotrophomonas maltophilia , Stenotrophomonas nitritireducens , Bdellovibrio bacteriovorus , Legionella pneumophila , Legionalla anisa , Legionella briminghamensis, Legionalla birminghamensis), Reg Nella Bose Mani (Legionalla bozemanii), Legionella Cincinnati N-Sys (Legionalla cincinnatiensis), Legionella two fur (Legionalla dumoffii), Legionella pilreyi (Legionalla feeleii), Legionella pick Manipur (Legionalla gormanii), Legionella and Kerry lover (Legionalla hackeliae), Legionella Legionalla israelensis , Legionalla jordanis , Legionalla lansingensis , Legionalla longbeachae , Legionalla maceachernii , Legionalla micdadei , and characterized in that at least one member selected from the group consisting of Legionella oak lead Zensys (Legionalla oakridgensis), Legionella four Intel alkylene when (Legionalla sainthelensi), Legionella Took sonen sheath (Legionalla tucsonensis) and Legionella ward's Zloty (Legionalla wadsworthii) Is a pharmaceutical composition for preventing or treating an infectious disease.
A food composition for preventing or ameliorating an infectious disease caused by Gram-negative bacteria comprising an ADK (Adenylate Kinase) protein derived from Mycobacterium tuberculosis comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient .

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