KR101627455B1 - Chimeric peptide having antimicrobial activity and antimicrobial composition comprising the same - Google Patents

Abstract

The present invention relates to a chimera peptide having an antimicrobial activity and to an antimicrobial composition containing the same. An Hn-Mc chimera peptide having an amino acid sequence represented by SEQ ID NO: 1 according to the present invention exhibits a high antimicrobial effect without losing an activity even under the presence of a physiological salt and is not toxic to human cells. Also, strong antimicrobial power is exhibited against antibiotic resistant bacteria, and very excellent in vivo availability is ensured. Accordingly, an antimicrobial pharmaceutical composition, a cosmetic composition, or a food additive composition including an Hn-Mc chimera antimicrobial peptide according to the present invention may be usefully used as an infection treating agent, a food preservative, a fodder additive, or a cosmetic additive.

Description

TECHNICAL FIELD The present invention relates to a chimeric peptide having antimicrobial activity and an antimicrobial composition containing the antimicrobial activity and antimicrobial composition,

The present invention relates to a chimeric peptide having antimicrobial activity and an antimicrobial composition containing the chimeric peptide. More specifically, the present invention relates to a chimeric peptide having antimicrobial activity and an antimicrobial composition containing the amino terminal region of HPA3NT3 peptide, which is a derivative of HP (2-20) secreted by Helicobacter pylori The present invention relates to a novel chimeric peptide for antibiotics synthesized by bonding carboxyl terminal portions of melittin present in bees poison and a pharmaceutical composition for antibacterial use, a cosmetic additive composition and a food additive composition containing the peptide.

Resistance to bacterial diseases appeared shortly after the first use of antibiotics in the 1940s. Especially, all types of antibiotics resistant to all types of antibiotics (MRSA: methicillin resistant Staphylococcus aureus, VRE: vancomycin resistant Enterococcus, imipenem resistant Pseudomonas aeruginosa, penicillin resistant Streptococcus pneumoniae) There is a growing concern that the The occurrence of these resistant strains and the abuse of antibiotics in hospitals, which are the cultivation sites, caused the generation of these resistant strains more frequently, and these "Super-bacteria" were no longer available for treatment. The theme of the WHO World Health Day 2011 is "antibiotic resistance", obesity, diabetes, HIV and other diseases that are threatening the future of humankind, but more threatening is antibiotic resistance. According to WHO, the rates of antibiotic resistance in China are 96.4%, 84.9% in Taiwan, 80.7% in Japan, 78.9% in Japan, 77.7% in Korea, 46.1% in France, 43.6% in Spain and 35.4% in the United States. to be. In addition, recently, the frequency of antibiotic-resistant pneumococcal infections has doubled in the United States, the number of antibiotics in OECD countries has been ranked first, and antibiotics prescription rate to 55% in colds has been a major indicator of abuse of antibiotics in Korea .

In the end of 2010, in Korea as antibiotic resistant alumina erythromycin-resistant Staphylococcus aureus (VRSA), meta ampicillin-resistant yellow pogo phase aureus (MRSA), carboxylic Buffett partyand resistant intestinal bacteria (Carbapenem-resistant Enterococcus, CRE) , vancomycin-resistant enterococci (VRE), the multidrug resistant Pseudomonas aeruginosa (Multi-drug resistant Pseudomonas aeruginosa, MDR PA), multidrug-resistant Acinetobacter bacteria (Multi-drug resistant Acinetobacter, MDR AA) 6 kinds was designated as super bacteria statutory infectious diseases, with the country also super bacteria with it Suggesting that the war had already begun.

Multinational pharmaceutical companies say that they can treat with new antibiotics. However, since antibiotic resistance appears in the intensive care unit of a hospital with weakened immunity, antibiotics cause septic shock due to the endotoxin secreted during the bacterial killing process It may be raised to death. In this reality, scientists around the world are developing new antimicrobial and anti-inflammatory drugs as an alternative to existing chemical antibiotics.

The human body and living organisms are exposed to various microorganisms both inside and outside and are constantly attacked. However, they have an innate immune system that can protect themselves and can sustain life by autoimmunity. One of these innate immune systems is an antimicrobial peptide, which has different classes and amino acid sequences but is secreted or inherited by almost all life forms, from viruses, bacteria, protozoa, fungi, plants and mammals. Antimicrobial peptides are small peptides composed of less than 60 amino acids and act as primary defense agents when organisms are infected by pathogens. The antimicrobial peptide is mainly cationic and has amphipathic α-helical structure and β-fold structure by hydrophobic amino acid residues. Due to such structural characteristics, the lipid component of the negatively charged pathogenic bacterial cell membrane Affinity with membrane lipids and changes the dislocation of microbial cells through the so-called 'barrel-stave', 'carpet' or 'toroidal-pore' mechanism, or breaks the cell membrane by piercing the cell membrane itself. Some of them enter the cell membrane and then attack the target in the cell to paralyze cell function, thereby exhibiting antibacterial activity. Antibacterial peptides have very good antimicrobial activity in vitro and do not show cytotoxicity, but in vivo experiments are often insufficient. This is because for many reasons, the physiological and anatomical conditions in vivo are largely different from the experimental conditions in vitro. First, if there is salt, which is a physiological condition, the activity of the antimicrobial peptide having a low positive charge is remarkably inhibited. Second, because peptides have small molecular weight and size, they are almost absorbed in the kidney and excreted in vitro. Third, it is easily lost by protein and peptide protease (protease & peptidase) existing in all tissues or cells, body fluids and blood in living body and lose activity.

Accordingly, the present inventors have continued research on antimicrobial peptides having reduced cytotoxicity and high utilization efficiency in vivo. While using 17 common amino acid residues, it is possible to produce economically large quantities, (Hn-Mc), which does not lose its activity even in the presence of a physiologically active salt, and has been shown to be effective against not only four kinds of test microorganisms, but also a drug-resistant P. aeruginosa 5, Drug-resistant S. aureus), and thus the present invention has been completed.

Therefore, a technical problem to be solved by the present invention is to provide a peptide which exhibits no cytotoxicity and exhibits excellent antimicrobial activity as a new mechanism of antibiotic for removing bacteria showing resistance to antibiotics.

Another technical problem to be solved by the present invention is to provide an antimicrobial composition comprising the peptide as an active ingredient.

In order to solve the above-mentioned technical problems, the present invention provides an antimicrobial chimeric peptide having an amino acid sequence shown in SEQ ID NO: 1.

Preferably, the antimicrobial chimeric peptide comprises an amino terminal region of HPA3NT3 peptide (amino terminus HPA3NT3 (1-7) of SEQ ID NO: 2) and melitin carboxyl terminal region melitin (17-26) (SEQ ID NO: 3) As the chimeric peptide, the HPA3NT3 peptide is a derivative of HP (2-20) secreted by Helicobacter pylori, and the HPA3NT3 peptide is a carboxyl terminal site of melitin present in bee venom.

In the present invention, the chimeric peptide having an antimicrobial activity is referred to as "Hn-Mc peptide ", and its amino acid sequence is shown in SEQ ID NO: 1.

According to one embodiment of the present invention, peptides having homology of at least 95% with the amino acid sequence of SEQ ID NO: 1 and exhibiting the same effect may be included in the present invention. The peptide disclosed in the present invention may comprise a peptide comprising SEQ ID NO: 1 or a fragment thereof and one or two amino acid altered peptides.

As used herein, the terms "homology" and "sequence identity" are used interchangeably and refer to the degree of overlap of sequences between two amino acids (or similar nucleic acids).

Specifically, the present invention includes an antimicrobial peptide in which each amino acid in the synthetic peptide having the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid having a similar structure, or the amino group at the amino terminus or the carboxyl group at the carboxyl terminus is replaced with another functional group . For example, a peptide obtained by substituting phenylalanine or tryptophan with another hydrophobic amino acid in the synthesized peptide has no cytotoxicity like the peptide having the amino acid sequence of SEQ ID NO: 1 and can maintain excellent antimicrobial activity. Among the amino acid sequences of SEQ ID NO: 1, hydrophobic amino acids which may be substituted for phenylalanine or tryptophan include alanine, isoleucine, methionine, phenylalanine, tryptophan, proline, valine and lucysin.

According to one embodiment of the present invention, the amino acid sequence having the above homology of 95% or more is characterized by having the following sequence.

(i) an amino acid sequence in which the phenylalanine in the amino acid sequence of SEQ ID NO: 1 is substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, methionine, tryptophan, proline, valine and leucine;

(ii) an amino acid sequence in which the tryptophan in the amino acid sequence of SEQ ID NO: 1 is substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine; or

(iii) wherein the phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced by a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, methionine, tryptophan, proline, valine and leucine, and at the same time tryptophan is replaced with alanine, isoleucine, methionine, Amino acid sequence substituted with a hydrophobic amino acid selected from the group consisting of phenylalanine, proline, valine, and leucine.

As used herein, the term "amino acid" includes D-isomers and modified amino acids as well as the 22 standard amino acids that are naturally incorporated into the peptide. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. In another aspect of the present invention, the peptide may include post-translationally modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (such as, for example, beta-depleted deamidation , Deamidation) and structural changes (e.g., formation of a disulfide bridge). It also includes amino acid changes such as changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that take place during binding with crosslinkers to form peptide conjugates.

Alternatively, the peptides disclosed herein may be artificial variants, including amino acid sequences in which one or more amino acids are substituted, deleted and / or inserted as compared to the peptides of SEQ ID NO: 1. Amino acid changes in artificial variants as well as wild-type polypeptides include conservative amino acid substitutions that do not significantly affect folding and / or activity of the protein. Examples of conservative substitutions include, but are not limited to, basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine) Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring interactions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa.

The antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention is characterized by having an antimicrobial activity against at least one bacterium selected from the group consisting of gram-positive bacteria, gram-negative bacteria, and antibiotic-resistant bacteria. The gram-positive bacteria may be any one or more of Gram-positive bacteria including Bacillus megaterium and Staphylococcus aureus. The gram-negative bacteria may be Escherichia coli E. coli , and Pseudomonas aeruginosa . < Desc / Clms Page number 7 >

The antibiotic-resistant microorganism has antimicrobial activity against at least one bacterium selected from drug-resistant Staphylococcus aureus and drug-resistant Pseudomonas aeruginosa . .

The Hn-Mc chimeric antimicrobial peptide having SEQ ID NO: 1 according to the present invention exhibits a high antimicrobial effect without losing activity even in the presence of physiological salt, has no toxicity to human cells, exhibits a strong antibacterial activity against live bacteria in antibiotics, The utilization efficiency is very good.

The present invention provides a pharmaceutical composition, a cosmetic additive composition or a food additive composition comprising the antibacterial chimeric peptide as an active ingredient, wherein the composition is useful as an infectious agent, a cosmetic additive, a food preservative or a feed additive .

The pharmaceutical composition may further contain at least one active ingredient having an antibacterial effect together with the antibacterial chimeric peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention.

The pharmaceutical composition of the present invention may be prepared by incorporating one or more pharmaceutically acceptable solvents in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable solvent may be a mixture of saline, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. In addition, other conventional additives such as antioxidants, buffers, And a dispersant, a diluent, and a surfactant may be formulated into injectable formulations, tablets, capsules, or granules, such as aqueous solutions, suspensions, emulsions, etc., by adding excipients, if necessary. In addition, the polymer can be made into a hydrogel or a film using a biomolecule.

The pharmaceutical composition of the present invention can be administered orally, intraperitoneally, intravenously and subcutaneously according to a desired method. The dosage may be appropriately selected according to the body weight, age, sex, health condition, diet, administration method, excretion rate, The range varies accordingly. The daily dose of the antimicrobial peptide may be 1 to 10 mg / kg, preferably about 3 to 6 mg / kg, depending on the clinical result. Preferably, the antimicrobial peptide is administered once or several times a day depending on the degree of infection .

According to one embodiment of the present invention, the cosmetic additive composition comprising the antibacterial chimeric peptide having the amino acid sequence of SEQ ID NO: 1 may be prepared in the form of a general emulsion formulation and a solubilized formulation. Cosmetics of emulsified form include nutrition lotion, cream, essence, etc., and cosmetics of solubilized form have softening longevity. In addition, the cosmetic additive composition of the present invention can be prepared in the form of an adjuvant which can be applied topically or systemically, which is conventionally used in the field of dermatology by containing an acceptable medium or a base in addition to the above-mentioned antibacterial peptide. In addition, as an excipient used in a cosmetic additive composition, it may further contain arbutin, kojic acid, rucinol, vitamin C (vitamin C) and vitamin C derivatives, And may further contain retinol and derivatives thereof, indole acetic acid and derivatives thereof, adenosine, tocoperol and derivatives thereof, and carnitine. In addition, the excipients include conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances, which are commonly used ingredients in cosmetic additives. Examples of suitable formulations of cosmetics include solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing oil phase in water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) , In the form of a non-ionic follicular dispersion, in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. Also, it can be formed into a form such as encapsulation and grafting by using chitosan, dextran, and hyaluronic acid, which are biocompatible polymers, as a carrier.

The cosmetic additive composition of the present invention preferably contains the antibacterial chimeric peptide of the present invention in an amount of 0.01 to 0.5% by weight based on the total weight of the cosmetic additive composition.

According to one embodiment of the present invention, the food additive composition may be prepared by incorporating one or more known food preservatives together with an antimicrobial chimeric peptide having the amino acid sequence of SEQ ID NO: 1. Examples of the food preservative include sodium benzoate, potassium benzoate, calcium benzoate, dehydroacetic acid, potassium sorbate, calcium sorbate, methyl p-hydroxybenzoate, and p-hydroxybenzoic acid. Sodium propionate, calcium propionate, and the like, but are not limited thereto.

There is no particular limitation on the type of food using the food preservative. Examples of the foods to which the above substances can be added include dairy products including dairy products such as drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

Preferably, the food preservative composition comprises 0.01 to 0.5 wt% of the antimicrobial chimeric peptide, based on the total weight of the food preservative composition.

Thus, the Hn-Mc chimeric antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 according to the present invention shows high antimicrobial effect without losing its activity even in the presence of physiological salt, has no toxicity to human cells, Not only does it exhibit antibacterial activity, it also has excellent bioavailability. Accordingly, the antimicrobial pharmaceutical composition, cosmetic composition and food additive composition comprising the Hn-Mc chimeric antimicrobial peptide of the present invention can be usefully used as an infectious agent, a food preservative, a feed additive, or a cosmetic additive.

FIG. 1 is a diagram illustrating a design of a chimeric antimicrobial peptide Hn-Mc, in which the amino terminal of the HPA3NT3 peptide of the present invention is conjugated with the carboxyl terminal of melittin.
FIG. 2 is a graph comparing the degree of hemolysis (%) of mouse Hn-Mc chimeric peptide of the present invention with HPA3NT3 peptide and melittin peptide.
3 is a graph showing cytotoxicity of the Hn-Mc chimeric peptide of the present invention to mouse skin fibroblast (NIH3T3) in comparison with HPA3NT3 peptide and melittin peptide.
FIG. 4 is a graph showing the antimicrobial activity of the Hn-Mc chimeric peptide of the present invention by observing the degree of destruction per hour by treating the artificial giant liposome with a peptide.
FIG. 5 is a transmission electron microscope image of the Hn-Mc chimeric peptide of the present invention observed 1 hour after treatment with Escherichia coli at a minimal inhibitory concentration.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples and Examples. The following examples and embodiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1 Preparation of Peptide Derivatives

HPA3NT3 (1-7), which has amphiphilic and cationic amino terminal regions of HPA3NT3, a derivative of Helicobacter pylori secretion peptide, HP (2-20), and melitin (17-26), which is amphiphilic and cationic And an amphipathic conjugated peptide having an amphiphilic alpha-helix structure was synthesized by increasing the cation and hydrophilicity.

The amino terminal portion (HPA3NT3 (1-7)) is shown in SEQ ID NO: 2, and the amino terminal portion of melitin (melitin (7-23)) is shown in SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 2 was synthesized in the order of joining SEQ ID NO: 3, thereby preparing Hn-Mc synthetic peptide having the amino acid sequence of SEQ ID NO: 1 (see FIG.

The peptides were synthesized by FMOC solid phase method using a mouseowave automated synthesizer (Liberty, CEM, USA). Each of the peptides synthesized was purified by reverse phase HPLC (Prominence) using a C18 analysis RP column (Shiseido capcell pak) LC-20AB, Shimadzu, Japan) and identified and confirmed using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

Peptides Hn-Mc (SEQ ID NO: 1), HPA3NT3 (SEQ ID NO: 2) and Melittin (SEQ ID NO: 3) as shown in Table 1 below were synthesized.

Peptides order Molecular Weight Hydrophobicity Net charge Hn-Mc FKRLKKLISWIKRKRQQ
(SEQ ID NO: 1) 2062.61 -1.82 +8 HPA3NT3 FKRLKKLFKKIWNWK
(SEQ ID NO: 2) 2847.50 -0.6 +7 Melittin GIGAVLKVLTTGLPALISWIKRKRQQQ
(SEQ ID NO: 3) 2068.67 -0.01 +5

≪ Test Example 1 > Antimicrobial activity measurement of antibacterial chimeric peptide

In order to confirm whether the chimeric peptide synthesized in the present invention can be used as an antimicrobial agent, the antimicrobial activity of the chimeric peptide having the amino acid sequence of SEQ ID NO: 1 prepared in Example 1 was examined using Gram-negative bacteria, Gram- Respectively.

In the case of bacteria, 10 ml of TSB (tripticase soy broth) was cultured at 37 ° C., and then transferred to a new medium for 6 hours. The microorganisms in the logarithmic growth phase were suspended in 10 mM sodium phosphate buffer (pH 7.4) Or a solution containing 10% by weight of TSB (Trycase soy broth) medium in physiologically active saline (PBS, NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 2 mM, pH 7.4) 1X10 ⁶ CFU / ml. To each well of the polypropylene 96-well plate was mixed 90 μl of the microbial dilution and 10 μl of the peptide (10 μl) diluted twice in duplicate and then cultured at 37 ° C for 12-24 hours.

The absorbance of each well was then measured at 600 nm using a microplate reader to determine the lowest peptide concentration that completely inhibited the growth of the microorganism as the minimal inhibitory concentration (MIC) . The MIC values were determined as the mean value of three independent experiments, three at a time.

First, Bacillus megaterium and Staphylococcus aureus as antibiotic sensitive Gram-positive bacteria of the peptide were cultured as Gram-negative bacteria in Escherichia coli and Pseudomonas aeruginosa ), The antimicrobial conjugation peptide Hn-Mc of the present invention having the amino acid sequence of SEQ ID NO: 1, the HPA3NT3 peptide having the amino acid sequence of SEQ ID NO: 2 as the control group and the melittin peptide having the amino acid sequence of SEQ ID NO: MIC values of each strain were measured using the method described in Table 2.

LIS: Low Ionic Strength buffer, 10 mM Sodium Phosphate Buffer, HIS: High Ionic Strength, PBS, NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 2 mM)

As shown in Table 2, the antimicrobial activity of Hn-Mc peptide was similar to that of HPA3NT3 and melitin, but it was confirmed that the Hn-Mc peptide was maintained at physiological activity conditions with high ionic strength.

≪ Test Example 2 > Antimicrobial activity measurement of antimicrobial chimeric peptide

In order to verify the practical use of the chimeric peptide of the present invention as an antibiotic-resistant bacterium for the treatment of an antibiotic resistant bacterium , drug-resistant Pseudomonas aeruginosa , which is an antibiotic resistant bacterium directly isolated from a patient who was given a vaccination at Busan Paik Hospital, The antimicrobial activity against Drug-resistant Staphylococcus aureus was the same as that described in Example 2, except that physiologically active saline (PBS, NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 2 mM, pH 7.4).

The antimicrobial resistant bacteria used and the measured results are shown in Table 3 below as the minimum inhibitory concentration (MIC) required for inhibiting the growth of microorganisms. For comparison, Ciprofloxacin and Gentamicin, which are commercially available antibiotics, were used as a positive control.

As shown in Table 3, the antimicrobial chimeric peptide Hn-Mc of the present invention is a drug-resistant Pseudomonas aeruginosa , which is an antibiotic resistant bacterium , and Drug-resistant Pseudomonas aeruginosa , Staphylococcus aureus ) were found to have strong antibacterial activity against all five species.

≪ Test Example 3 > Measurement of red blood cell hemolytic activity and cytotoxicity of antimicrobial chimeric peptide

To determine whether the chimeric peptide of the present invention exhibited cytotoxicity, the erythrocyte-disrupting ability of the antimicrobial conjugated peptide was examined as follows.

Human red blood cells were diluted with a phosphate buffer solution (PBS, pH 7.0) to a concentration of 8%, to which the antimicrobial chimeric peptide Hn-Mc prepared in Example 1 at a concentration of 1/2, the HPA3NT3 peptide as a control and melittin Each of the peptides was serially diluted and reacted at 37 ° C. for 1 hour. Then, the mixture was centrifuged at 1,000 g, and the amount of hemoglobin contained in the supernatant was measured by measuring the absorbance at a wavelength of 414 nm. To investigate the degree of cell destruction, 0.1% Triton X-100 was added to human red blood cells, and the absorbance of the supernatant was measured. The cell-destructive activity of 0.1% Triton X-100 was determined as 100% Was calculated according to the following equation (1).

In this formula,

Absorbance A is the absorbance of the group treated with the peptide solution at a wavelength of 414 nm,

Absorbance B is the absorbance of the group treated with PBS only at 414 nm wavelength,

Absorbance C represents the absorbance of the treated group of 0.1% Triton X-100 at a wavelength of 414 nm.

FIG. 2 is a graph comparing the results of measuring the erythrocyte destructive ability of the antimicrobial chimeric peptide Hn-Mc, HPA3NT3 peptide and melittin peptide of the present invention. As shown here, Hn-Mc showed hemolysis at 250 μM at 1.1% compared to 69% at 250 μM and 100% at 3.9 μM for the control HPA3NT3. From this, it was confirmed that the Hn-Mc antimicrobial chimeric peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention is a stable peptide which does not cause red blood cell hemolysis.

≪ Test Example 4 > Cytotoxicity measurement of antimicrobial chimeric peptide

To examine whether the chimeric peptide of the present invention was toxic to normal cells, a mouse fibroblast cell line (NIH3T3) was treated with a peptide and MTT assay was performed. NIH3T3 cells (10,000 cells / well) were cultured in a 96-well plate for 16 hours in the presence of 5% CO 2 using DMEM (Dulbecco's modified eagle medium) containing 10% FBS. These cells were treated with a peptide derivative After further incubation, cell viability was evaluated by MTT assay and absorbance was measured at 570 nm. At this time, the cell viability [Viability (%)] was determined by the following formula (2).

In this formula,

Absorbance A is the absorbance of the group treated with the peptide solution at a wavelength of 570 nm,

Absorbance B is the absorbance of the group not treated with the peptide,

Absorbance C represents the absorbance of 0.1% Triton X-100.

3 is a graph showing cytotoxicity of the Hn-Mc chimeric peptide of the present invention to mouse skin fibroblast (NIH3T3) in comparison with HPA3NT3 peptide and melittin peptide. As shown here, HPA3NT3 shows a survival rate of 15.1% NIH3T3 cells at a concentration of 125 μM and melitin shows a survival rate of 1.8% at a concentration of 7.8 μM, whereas the Hn-Mc chimeric peptide of the present invention has a survival rate of 125 μM Showed 82.1% survival rate and showed almost no cytotoxicity.

From the above results, it can be seen that the Hn-Mc chimeric peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention exhibits excellent antimicrobial effect and can be effectively used as a safe antimicrobial agent because it has no cytotoxicity.

≪ Test Example 5 >

In order to investigate the mechanism of action of the antimicrobial activity of the peptide of the present invention, the structural phenomenon occurring over time was measured by treating peptides with a large-sized liposome artificially made of the Hn-Mc antimicrobial chimeric peptide of the present invention prepared in Example 1 .

A giant unilamellar vesicle (GUV) was prepared by electrical stimulation with several phospholipids diluted with phosphatidylethanolamine / phosphatidylglycerol / phosphatidylethanolamine-rhodamine (PE / PG / PE-Rhodamin, 30/1) was diluted. The diluted phospholipid aqueous solution was diluted with 100 μL of diluted lipid on indium tin oxide (ITO) coated glass (25 × 35 × 1.1 mm Sigma-Aldrich, St. Louis, Mo.) . Thereafter, the lipid and diluted organic solvent were removed for at least 2 hours under vacuum evacuation. A chamber was made by inserting and fixing silicone louvers (electroformation chamber, 25x25x1 mm) in the middle of the bonding of indigo tin oxide (ITO) and indium tin oxide (ITO) 5 mM HEPES buffer with sucrose was injected into the chamber. A voltage of 1.7 V and a frequency of 10 Hz were injected into the indium tin oxide (ITO) glass using a current generator (Agilent 33220A, Agilent Technology, US) The frequency was converted and reacted for 10 minutes.

The liposomal aqueous solution was removed from the electroforming chamber and diluted with 5 mM HEPES buffer to which 0.1 M glucose was added to the liposome aqueous solution. This prevents the large single membrane liposomes (GUVs) from popping by the osmotic pressure phenomenon and facilitates the precipitation in the slide glass due to the density difference between the external glucose (180.16 MW) and the internal sucrose (342.30 MW) Contrast is given. Fluorescence microscopy The phenomenon was photographed using a digital CCD camera (DP70, Olympus) with a fluorescence phase contrast microscope (Ix71, Olympus, Tokyo, Japan).

FIG. 4 is a graph showing the antimicrobial activity of the Hn-Mc chimeric peptide of the present invention by observing the degree of destruction per hour by treating the artificial giant liposome with a peptide. As can be seen from the fluorescence microscopy, the shape of the GUV began to change at the same time as the treatment with Hn-Mc peptide. The size of the GUV was reduced by one side of the pericardial membrane in 3 seconds, and it was destroyed with small fragments over time.

In order to examine the effect of the Hn-Mc chimeric peptide of the present invention on actual bacteria, Escherichia coli was treated with Hn-Mc peptide at a concentration of MIC and stained with 1% Uranyl acetate And observed with a transmission electron microscope. As the control group, a group not treated with the peptide was used.

FIG. 5 is a transmission electron microscope image of the Hn-Mc chimeric peptide of the present invention observed 1 hour after treatment with Escherichia coli at a minimum inhibitory concentration. As shown here, the control without the peptide retains the cell shape of the bacteria, but the bacteria treated with the Hn-Mc peptide were found to be destroyed and the contents inside the cells were leaked out of the cells.

These results demonstrate that the cell membrane of the functional small bacterium of the Hn-Mc chimeric peptide of the present invention can be destroyed to kill the cells.

≪ Formulation Example 1 > Preparation of pharmaceutical composition

1-1. Manufacture of liquid agent

Hn-Mc Antibacterial chimeric peptide 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to a conventional liquid preparation method, the lemon flavor was added in an appropriate amount, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 mL, And sterilized to prepare a liquid preparation.

1-2. Manufacture of Powder

Hn-Mc Antibacterial chimeric peptide 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-3. Injection preparation

Hn-Mc Antibacterial chimeric peptide 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 mL) per 1 ampoule in accordance with the usual injection preparation method.

1-4. Manufacture of tablets

Hn-Mc Antibacterial chimeric peptide 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-5. Preparation of capsules

Hn-Mc Antibacterial chimeric peptide 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

≪ Formulation Example 2 > Preparation of cosmetic additives

2-1. Manufacture of softening longevity (skin lotion)

Hn-Mc Antibacterial chimeric peptide 0.25 wt%

Beta-1,3-glucan 1.0 wt%

Butylene glycol 2.0 wt%

Propylene glycol 2.0 wt%

Carboxyvinyl polymer 0.1 wt%

0.2% by weight of phage-12 nonylphenyl ether

Polysorbate 80 0.4 wt%

Ethanol 10.0 wt%

0.1% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

2-2. Manufacture of nutrition lotion (milk lotion)

Hn-Mc Antibacterial chimeric peptide 0.5%

Beta-1,3-glucan 1.0 wt%

4.0 wt%

Polysorbate 60 1.5 wt%

1.5% by weight of sorbitan sesquioleate

0.5% by weight liquid paraffin

Caprylic / capric triglyceride 5.0 wt%

Glycerin 3.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

Carboxyvinyl polymer 0.1 wt%

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

2-3. Manufacture of nutrition cream

Hn-Mc Antibacterial chimeric peptide 0.5%

Beta-1,3-glucan 5.0 wt%

Wax 10.0 wt%

Polysorbate 60 1.5 wt%

≪ tb > < tb > < tb >

0.5% by weight of sorbitan sesquioleate

Liquid paraffin 10.0 wt%

Squalane 5.0 wt%

Caprylic / capric triglyceride 5.0 wt%

Glycerin 5.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

≪ Formulation Example 3 > Preparation of food preservative

Hn-Mc Antibacterial chimeric peptide 0.5%

0.1% by weight of dehydroacetic acid

0.1% by weight potassium sorbate

0.2% by weight calcium sorbate

0.5% by weight sodium benzoate

0.1% by weight potassium benzoate

Calcium benzoate 0.5 wt%

0.1% by weight of methyl paraoxybenzoate

0.1% by weight of p-hydroxybenzoic acid propylate

0.1% by weight sodium propionate

0.1% by weight calcium propionate

They are prepared separately according to a conventional preservative manufacturing method.

Thus, the Hn-Mc chimeric antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 according to the present invention exhibits a high antimicrobial effect without losing activity even in the presence of physiological salt, has no toxicity to human cells, Not only does it exhibit antibacterial activity, it also has excellent bioavailability. Accordingly, the antimicrobial pharmaceutical composition, cosmetic composition and food additive composition comprising the Hn-Mc chimeric antimicrobial peptide of the present invention can be usefully used as an infectious agent, a food preservative, a feed additive, or a cosmetic additive.

<110> Industry-academic cooperation foundation of sunchon national university <120> Chimeric peptide having antimicrobial activity and antimicrobial          composition comprising the same <130> PA-15-0023 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Hn-Mc peptide <400> 1 Phe Lys Arg Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln   1 5 10 15 Gln     <210> 2 <211> 15 <212> PRT <213> HPA3NT3 peptide <400> 2 Phe Lys Arg Leu Lys Lys Leu Phe Lys Lys Ile Trp Asn Trp Lys   1 5 10 15 <210> 3 <211> 26 <212> PRT <213> Melittin peptide <400> 3 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu   1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln              20 25

Claims (5)

A chimeric peptide having an antimicrobial activity comprising the amino acid sequence shown in SEQ ID NO: 1. The method according to claim 1,
Wherein the chimeric peptide is selected from the group consisting of Bacillus megaterium , Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa , drug resistant Staphylococcus aureus Wherein the chimeric peptide has antibacterial activity against at least one bacterium selected from the group consisting of Drug-resistant Staphylococcus aureus and Drug-resistant Pseudomonas aeruginosa . A pharmaceutical composition for antimicrobial use, which comprises the chimeric peptide according to claim 1 or 2 as an active ingredient. An antimicrobial cosmetic additive composition comprising the chimeric peptide according to claim 1 or 2 as an active ingredient. A food additive composition for antimicrobial use, which comprises the chimeric peptide according to claim 1 or 2 as an active ingredient.

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