KR100879211B1 - Novel antimicrobial peptide and its application derived from echiuroid worm, Urechis unicinctus - Google Patents

Abstract

The present invention relates to an antimicrobial peptide synthesized from Urechis unicinctus and its use, and more particularly, to an antimicrobial peptide having an amino acid sequence set forth in SEQ ID NOs: 1 and 2.

Antimicrobial peptides according to the present invention can be usefully used as a safe antibiotic for the human body because it has no cytotoxicity and shows excellent antibacterial and antifungal activity.

Dog fire, antibacterial, antifungal, antibiotic

Description

Novel antimicrobial peptide and its application derived from echiuroid worm, Urechis unicinctus}

The present invention relates to a peptide isolated from Urechis unicinctus and its use, and more particularly, to a peptide having antibacterial and antifungal activity and a pharmaceutical composition containing the same as an active ingredient.

Infection of pathogenic microorganisms is one of the most common and fatal causes of human disease. Unfortunately, the abuse of antibiotics has resulted in antibiotic resistance of pathogenic microorganisms.

Indeed, the rate at which pathogenic microorganisms are resistant to new antibiotics occurs much faster than the rate at which analogues of new antibiotics are developed. For example, which may pose a threat to life, Enterococcus faecalis kusu nose (Enterococcus faecalis ), Mycobacterium tuberculosis ( Mycobacterium) tuberculosis) and Pseudomonas her rugi pathogenic microorganisms such as labor (Pseudomonas aeruginosa) species have been brought up resistance to all known antibiotics until now the (Stuart B. Levy, Scientific American , 46-53, 1998).

Tolerance to antibiotics is a distinct phenomenon from antibiotic resistance, first discovered in Pneumococcus sp. In the 1970s and providing an important clue to the mechanism of action of penicillin (Tomasz). et al ., Nature , 227, 138-140, 1970).

Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result. The resistance is due to the fact that when antibiotics inhibit cell wall synthase, the activity of bacterial autolytic enzymes, such as autolysin, does not occur, which is due to the fact that penicillin does not endogenous hydrolytic enzymes. Activation kills bacteria and bacteria also inhibit their activity, resulting in survival during antibiotic treatment.

It is important, very clinical, if it becomes impossible to eradicate resistant microorganisms is because the effectiveness of antibiotics in clinical infections dropped (Handwerger and Tomasz, Rev. Infec . Dis. Pathogenic microorganisms having a resistance to antibiotics, 7 , 368-385, 1985).

Resistance is also considered a prerequisite to resistance to antibiotics, because of the surviving strains despite antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. Since virtually all resistant pathogenic microorganisms are known to have resistance (Liu and Tomasz, J. Infect. Dis ., 152 , 365-372, 1985), new antibiotics that can kill these antibiotic-resistant pathogenic microorganisms The development of antibiotics is urgent.

As discussed above, new antibiotics are needed to combat pathogenic microorganisms that are resistant to antibiotics, and new antibiotics that act independently of autolysine activity are required. In addition, there is a need for such new antibiotics to provide pharmaceutical compositions for effectively treating infections of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in the process of maintaining homestasis are biologically active substances derived from various organisms.

Many studies have been conducted on a number of physiologically active substances, and among them, antimicrobial peptides isolated from various organisms are known to work in various ways from bacteria, fungi and viruses. There are also antibacterial peptides are known to play an important role in the host defense and innate immune system (Boman, HG, Cell, 65 , 205, 1991;... Boman, HG, Annu Rev Microbiol, 13, 61, 1995).

The peptide used in this study was Urechis , a marine organism. unicinctus ), which is known to have a sequence similar to that of vertebrate neurotransmitter peptides, but studies on antimicrobial and antifungal activity have been insignificant.

The present inventors have completed the present invention by revealing the antimicrobial and antifungal activity of the peptide isolated from the dog open and its mechanism of operation and revealing that the peptide is an antimicrobial peptide that can be used as an antibiotic.

It is an object of the present invention to provide an antimicrobial peptide and its use which are free of cytotoxicity and which exhibit excellent antibacterial and antifungal activity which can be used as antibiotics.

In order to achieve the above object, the present invention is a dog opening ( Urechis unicinctus ) provides an antimicrobial peptide.

The present invention also provides a use of the above antimicrobial peptides for antibacterial and antifungal applications.

In addition, the present invention provides a pharmaceutical composition for antibacterial and antifungal comprising the antimicrobial peptide as an active ingredient.

The antimicrobial peptides according to the present invention are peptides derived from Gaebul and have excellent antibacterial and antifungal properties, and thus are preferably applied as antibacterial agents or antifungal agents.

The antimicrobial peptide preferably has an amino acid sequence represented by SEQ ID NO: 1 or 2.

As a result of investigating the antimicrobial activity of the antimicrobial peptides of SEQ ID NOs: 1 and 2 against the pathogenic microorganisms through the preferred embodiment 2 of the present invention, it showed an antimicrobial effect almost equivalent to that of melittin, a powerful antimicrobial peptide.

Specifically, the antimicrobial peptide according to the present invention has an antimicrobial activity with a minimum growth inhibition concentration (MIC) of 1.25-5 μg of pathogenic bacteria and an excellent antifungal activity with a minimum growth inhibition concentration (MIC) of 1.25-5 μg of pathogenic fungi. Has

Such pathogenic bacteria include Staphylococcus aureus and Enterococcus. faecum), streptococcus free Tansu (Streptococcus mutans), E. coli 0-157 (Escherichia coli 0-157), Pseudomonas aeruginosa ), Vibrio There are vulnificus), a pathogenic fungus is Candida albicans (Candida albicans ), Trichosporon beigelii ), and Marasegia pearl ( Malassezia) furfur ) and the like, showing excellent antimicrobial activity against all of them (see Table 2).

This antimicrobial peptide acts on the fungal cell membrane (see Example 3 and FIG. 1), and it was confirmed that pores could be formed on the fungal cell membrane, such as melittin, which is a strong antimicrobial peptide (see Example 4).

In addition, as a result of investigating erythrocyte destructive ability using the antimicrobial peptide of the present invention, the peptides described in SEQ ID NOs: 1 and 2 show no cytotoxicity, whereas melittin, a peptide isolated from bee venom used as a positive control, is cytotoxic. It was confirmed that this appeared very high (see Example 5).

In addition, the antimicrobial peptide was confirmed to be a safe substance having a minimum lethal dose of 10 mg / kg or more without showing a toxicity change to 5 mg / kg even in parenteral administration acute toxicity experiments in rats (see Example 6).

Therefore, according to the present invention, the antimicrobial peptides consisting of the amino acid sequences of SEQ ID NOs: 1 and 2 have high antimicrobial and antifungal activity, and thus it can be seen that they are effective antimicrobial peptides.

Moreover, since the antimicrobial peptides consisting of the amino acid sequences of SEQ ID NOs: 1 and 2 have no cytotoxicity and show almost harmless results even upon oral administration, they can be used as antibacterial and antifungal agents because they are peptides that can be used in humans. In addition, it can be usefully used as an additive in various food preservatives, cosmetic preservatives and pharmaceutical preservatives.

Antimicrobial peptides of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. The antimicrobial peptides of the present invention may be administered in a variety of parenteral formulations, which are formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero Latin and the like can be used.

In addition, the antimicrobial peptides of the present invention may be used in admixture with various carriers that are acceptable as medicaments, such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran to increase stability or absorption. Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the antimicrobial peptide of the present invention is 1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and may be administered once to three times a day.

The antibiotic containing the antimicrobial peptide of the present invention as an active ingredient may be administered to a patient in a single dose by bolus form or by infusion for a relatively short period of time, and may be administered in multiple doses. dose) may be administered by a fractionated treatment protocol with long term administration.

Since the effective dose of the antimicrobial peptide of the present invention is determined in consideration of various factors such as the age and health condition of the patient as well as the route of administration and the number of treatments of the drug, it is common knowledge in the art Anyone with A can determine the appropriate effective dose.

From the above results, the antimicrobial peptides described in SEQ ID NOs: 1 and 2 of the present invention exhibited excellent antimicrobial activity in both Gram-positive bacteria, Gram-negative bacteria and fungi and did not exhibit cytotoxicity, and thus can be usefully used as antibiotics safe for humans. .

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

Example  1: antimicrobial activity of SEQ ID NOs: 1 and 2 Peptide  Synthetic and Separation Purification

In this embodiment the first marine life urechis unicinctus (Urechis unicinctus ) was synthesized antimicrobial peptide. At this time, the antimicrobial peptide is as shown in Table 1 below.

Peptide Amino acid sequence SEQ ID NO: Urechistachykinin 1 Leu-Arg-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH ₂ SEQ ID NO: Urechistachykinin 2 Ala-Ala-Gly-Met-Gly-Phe-Phe-Gly-Ala-Arg-NH ₂

First, in order to synthesize an antimicrobial peptide, the present inventors have used a liquid phase solidification method of Merrifield using a Fmoc amino group protective container (Merrifield, RB., J. Am . Chem . Soc ., 85 , 2149, 1963). ) Antimicrobial peptides set forth in SEQ ID NOs: 1 and 2 were prepared.

The antimicrobial peptide synthesis method was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of the N _α -amino group of amino acids.

Specifically, the peptide having the carboxyl terminus of -NH ₂ form Rink Amide MBHA-Resin as a starting material, and the carboxyl terminus of the -OH type peptide used Fmoc-amino acid-Wang Resin as a starting material. Elongation of peptide chains by coupling of Fmoc-amino acids was performed by N -hydroxybenzotriazole (HOBt) -dicyclo-hexylcarbodibodiide (DCC) method.

After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM), followed by nitrogen. Dried with gas.

A solution of trifluoroacetic acid (TFA) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added thereto and reacted for 3 hours to remove the protecting group from the resin The peptide was isolated and then the peptide was precipitated with diethyl ether.

The crude peptide thus obtained was purified by reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15μ, 19.0 mm ×) with an acetonitrile gradient containing 0.05% TFA. 30 cm, Waters).

Each synthetic peptide was hydrolyzed at 110 ° C. for 24 hours with 6 N-HCl, and the residue thus obtained was concentrated under reduced pressure, dissolved in 0.02 N-HCl, and the amino acid composition was measured by an amino acid analyzer (Hitachi 8500 A).

In addition, the molecular weight was calculated based on the sequence of the synthesized peptide, and accurate molecular weight was measured using a matrix-assisted laser desorption ionization mass spectrometer. As a result, it was confirmed that the antimicrobial peptide having the correct amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight coincide.

Example  2: antibacterial of SEQ ID NOS: 1 and 2 Peptide  Investigation of antimicrobial activity against pathogenic microorganisms

 In order to investigate the antimicrobial activity of the antimicrobial peptide prepared in the above Example was performed as follows. At this time, melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptides of SEQ ID NOs: 1 and 2 were stored at -20 ° C and dissolved in sterile tertiary distilled water.

[Antibacterial Activity Measurement]

In order to determine the antimicrobial activity of the antimicrobial peptides of SEQ ID NOs: 1 and 2, first, the pathogenic bacterium Staphylococcus aureus ), Enterococcus fascium faecum ), Streptococcus mutans), E. coli 0-157 (Escherichia coli 0-157), Pseudomonas aeruginosa , Vibrio vulnificus ) was diluted with a Mueller Hinton antimicrobial activity measurement medium (Beef extract powder 0.2%, Acid digest of casein 1.75%, Soluble starch 0.15%) to 1 × 10 ⁶ cells / 1 mL of bacteria and placed on a 96-well plate. After 100 μl aliquots, the solutions of the antimicrobial peptides of SEQ ID NOS: 1 and 2 were treated at concentrations diluted in stages.

Subsequently, the minimum growth inhibition concentration (MIC) was measured by measuring absorbance under a wavelength of 620 nm using an ELISA reader while shaking culture for 6 hours in a 37 ° C. incubator. The results are shown in Table 2 below. .

[Antifungal Activity Measurement]

Measurement of antifungal activity of the antimicrobial peptide of the SEQ ID NOS: 1 and 2, the pathogenic fungus Candida albicans (Candida albicans ), Trichosporon beigelii , Malassezia furfur ) was incubated in YPD complete medium (2% glucose, 1% peptone, 0.5% yeast extract, pH 5,5). In addition, the solution was diluted to 2 × 10 ³ cells / 1 mL of bacteria with YPD liquid medium, and 100 μl of the solution was mixed into 96-well plates, followed by the stepwise diluting of the solution of the antimicrobial peptides of SEQ ID NOs: 1 and 2. did.

After shaking for 24 hours in an incubator at 28 ℃, MTT [3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide] solution [5 mg of MTT / mL of PBS (pH 7.4)] to each well and incubated for 4 hours in a 37 ℃ incubator. Subsequently, 20 μl of 20% SDS containing 0.02 N HCl was added to dissolve Formazan produced by MTT, followed by reaction at 37 ° C. for 16 hours.

Next, the minimum growth inhibition concentration (MIC) was measured by measuring the absorbance of each well under a wavelength of 570 nm with an ELISA reader. The results obtained are shown in Table 2 below.

Minimum Growth Inhibitory Concentration (MIC) for Pathogenic Microorganisms of Antimicrobial Peptides of SEQ ID NO: 1 MIC ^a (μg / mL) for pathogenic microorganisms Urechistachykinin 1SEQ ID NO: 1 Urechistachykinin 2 SEQ ID NO: 2 Melittin Gram positive bacteria Staphylococcus aureus 5 2.5 1.25 Streptococcus mutans 2.5 1.25 0.3125 Anterococcus fascium 2.5 2.5 0.625 Gram negative germs Escherichia coli 0-157 1.25 1.25 0.3125 Pseudomonas Arujinosa 2.5 1.25 0.3125 Vibrio Blinpicus 5 2.5 1.25 Fungus Candida Albicans 5 2.5 2.5 Tricosporon Beiglai 2.5 1.25 1.25 Marasegia Pearl 5 5 2.5 [Note] a: Concentrations of the antimicrobial peptides of SEQ ID NOs: 1 and 2, which are not able to grow pathogenic microorganisms

[Antibacterial activity]

Referring to Table 2, it can be seen that the antimicrobial activity of the antimicrobial peptides of SEQ ID NO: 1 and 2 has antimicrobial activity in a wide range of pathogenic bacteria, including gram positive and negative bacteria. In addition, the minimum growth inhibition concentration (MIC) was mostly 1.25 to 5 ㎍, which showed a similar or slightly weaker antimicrobial activity to melittin, a powerful antimicrobial peptide.

[Antifungal activity]

Referring to Table 2, the antifungal activity of the antimicrobial peptides of SEQ ID NOS: 1 and 2 showed a minimum growth inhibition concentration (MIC) of 1.25-5 μg against pathogenic fungi. This means that the antimicrobial peptides of SEQ ID NOs: 1 and 2 exhibit potent antifungal activity with similar or slightly weaker antifungal activity than melittin, a potent antifungal agent used as a control.

Example  3: antibacterial of SEQ ID NOs: 1 and 2 Peptide  Identification of antimicrobial active sites in pathogenic microorganisms

Identification of the antimicrobial activity site in the pathogenic microorganism of the antimicrobial peptides of SEQ ID NO: 1 and 2 was carried out as follows.

Fluorescent material was attached to the peptide using gel filtration chromatography. FITC (Fluorescein isothiocyanate) is a fluorescent substance that has no effect on cells including antifungal activity and is commonly used because it emits green fluorescence when irradiated with confocal fluorescence microscopy. At this time, in order to determine whether the FITC and peptides were combined, trisine-gel SDS-polyacrylamide gel electrophoresis was used.

Confocal laser scanning microscopy (CLSM) was performed to determine the intracellular distribution of peptides using the FITC-labeled peptides. The fungus C. albicans was incubated with the peptide of SEQ ID NO: 1 labeled with FITC for 5 minutes at 28 ° C. After incubation, unreacted FITC was removed by washing the cells with phosphate-sodium chloride buffer, pH 7.4 (PBS) solution.

Subsequent visualization of FITC-labeled peptides was performed using a Leica TCS 4D connected to a Leica DAS upright microscope (Leica Lasertech GimbH, Heidelberg, Germany), and the results obtained are shown in FIG. 1.

Figure 1 is a fluorescent substance in order to determine if the antimicrobial peptide indicating the antimicrobial activity will have an effect on any part of the cells; Candida an antimicrobial peptide labeled (FITC Fluorescein isothiocyanate) albicans (Candida albicans ) cells were processed and then the cells were taken by confocal fluorescence microscopy. At this time, (a) of Figure 1 is a photograph of the antimicrobial peptide of SEQ ID NO: 1, (b) is a photograph of the antimicrobial peptide of SEQ ID NO: 2.

Referring to Figure 1, it can be seen that the antimicrobial peptides of SEQ ID NO: 1 and 2 in accordance with the present invention all act on the fungal cell membrane.

Example  4: antibacterial of SEQ ID NO: 1 Peptide  Examine the effect of pathogenic microorganisms on cell membranes

1,6-diphenyl-1,3,5-hexatriene (DPH) analysis was performed to confirm specific effects on the cell membranes of the antimicrobial peptides of SEQ ID NOs: 1 and 2.

After culturing C. albicans in YPD medium, the cells were diluted with YPD medium so that the cell number was 2 × 10 5 cells, and then treated with the antimicrobial peptides of SEQ ID NOs: 1 and 2. Cells were then fixed by incubation in a 28 ° C. incubator for 2 hours and then treated with 0.37% (v / v) formaldehyde for 30 minutes. The cells were then frozen and thawed with liquid nitrogen twice.

In general, the cells were stained by incubation at 28 ° C. for 45 minutes with a 0.6 mM solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) known to adhere between the lipid bilayers of the cell membrane. After incubation, cells were washed four times with phosphate-sodium chloride buffer, pH 7.4 (PBS) solution, 0.5 ml PBS solution was added, cells were destroyed by an ultrasonic machine for 2 minutes, centrifuged and the supernatant was 350 nm. The amount of DPH was measured using a fluorescence photometer under the wavelength of excitation and 425 nm emission. The results obtained are shown in FIG. 2.

Figure 2 is a Candida albicans ( Candida) antimicrobial peptides to determine how the antimicrobial peptides specifically affect the cell membrane of the microorganism albicans ) and then stained with a fluorescent material (DPH; 1,6-diphenyl-1,3,5-hexatriene), which is commonly known to adhere between lipid bilayers of cell membranes, and then the amount of fluorescent material is measured using a fluorescence photometer. It is a graph measured. Where ● is an antimicrobial peptide having an amino acid sequence of SEQ ID NO: 1 (Urechistachykinin 1), ○ is an antimicrobial peptide having an amino acid sequence of SEQ ID NO: 2 (Urechistachykinin 2), and ▼ represent melittin.

2, in the case of the antimicrobial peptides comprising the amino acid sequences of SEQ ID NOs: 1 and 2 according to the present invention, the amount of DPH decreases as the throughput increases, and in the case of melittin used as a positive control, As the amount increased, the amount of DPH was significantly reduced.

Melitin is known to form a hole in the cell membrane, and through the results of Figure 2 it was confirmed that the antimicrobial peptide according to the present invention is likely to form a hole in the cell membrane of the fungus, such as melittin, a positive control.

Example  5: antibacterial of SEQ ID NOS: 1 and 2 Peptide  Cytotoxicity Measurements on Human Red Blood Cells

The cytotoxicity of human erythrocytes was measured as follows.

Human erythrocytes were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer to prepare 8.0% erythrocyte cell solutions. Then, 100 μl of the 8.0% erythrocyte cell solution was dispensed into 96-well plates, followed by the stepwise dilutions of the antimicrobial peptides of SEQ ID NOS: 1 and 2, respectively.

Then, physiological saline (Saline 0.85% NaCl) was added to the wells so that the total amount was 200 μl, and the cells were incubated for 1 hour in a 37 ° C. incubator. After the incubation, the supernatant was transferred to the side wells by centrifugation for 10 minutes at a rotational speed of 1000 rpm in a centrifuge.

Destructive capacity for erythrocytes was analyzed by measuring absorbance under a wavelength of 414 nm with an ELISA reader. And when treated with 0.1% Triton X-100 as a control, the value was calculated as 100% destructive capacity, and only the red blood cells were counted as 0% destructive capacity. At this time, the breaking ability of the antimicrobial peptide was calculated by the following Equation 1, and the obtained results are shown in Table 3 below.

Determination of Cytotoxicity of Antimicrobial Peptides of SEQ ID NOs: 1 and 2 on Human Red Blood Cells (Human erythrocytes) % Human erythrocyte destructive capacity ^a (㎍ / ml) 20 10 5 2.5 1.25 0.625 Urechistachykinin 1, SEQ ID NO: 1 0 0 0 0 0 0 Urechistachykinin 2, SEQ ID NO: 2 0 0 0 0 0 0 Melittin 100 100 100 83 52 7 A: Cytotoxicity in humans is determined by the destructive capacity of the antimicrobial peptide of SEQ ID NO: 1 on human erythrocytes.

Referring to Table 3, it was confirmed that the antimicrobial peptides of SEQ ID NO: 1 and 2 does not exhibit cytotoxicity even at high concentrations. In contrast, melittin, a powerful antimicrobial peptide, was found to be highly cytotoxic even at low concentrations. These results indicate that the antimicrobial peptides of SEQ ID NOs: 1 and 2 found in the present invention can be used for humans without damaging the human body.

Example  6: On the rat  About Parenteral administration  Acute Toxicity Experiment

Acute toxicity experiments were performed using 6-week-old SPF SD rats.

Each of the antimicrobial peptides set forth in SEQ ID NOS: 1 and 2 of the present invention was suspended in 0.5% methylcellulose solution and administered once parenterally to 2 animals per experimental group at a dose of 1 g / kg / 15 ml. After administration, the mortality, clinical symptoms, and weight changes of the animals were observed, and hematological and blood biochemical tests were performed. The necropsy was performed to visually observe the abdominal and thoracic organ abnormalities.

As a result, all animals treated with the antimicrobial peptide of the present invention had no significant clinical symptoms or dead animals, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, autopsy findings, and the like. In addition, the antimicrobial peptide of the present invention did not show a toxicity change in rats up to 5 mg / kg, and the parenteral administration minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 10 mg / kg or more.

Figure 1 is a fluorescent substance in order to determine whether the antimicrobial activity is shown antimicrobial peptides have an effect on any part of the cell; an antimicrobial peptide labeled (FITC Fluorescein isothiocyanate) Candida albicans (Candida albicans ) cells were processed and then the cells were taken by confocal fluorescence microscopy.

(A) is a photo of the antimicrobial peptide (Urechistachykinin 1) of SEQ ID NO: 1, (B) is a photo of the antimicrobial peptide (Urechistachykinin 2) of SEQ ID NO: 2.

Figure 2 is to identify antimicrobial peptides specifically how the impact with the cell membrane of microorganisms, the antimicrobial peptide of Candida albicans (Candida albicans ), and then stained with a fluorescent substance (DPH; 1,6-diphenyl-1,3,5-hexatriene) known to adhere to the lipid bilayer of the cell membrane, and the amount of the fluorescent substance was measured using a fluorescence photometer. One graph:

●: antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 (Urechistachykinin 1)

○: antimicrobial peptide having an ananoic acid sequence of SEQ ID NO: 2 (Urechistachykinin 2)

▼: melittin (Melitin)

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Novel antimicrobial peptide and its application derived from          echiuroid worm, Urechis unicinctus <130> DP20070172 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Urechistachykinin 1 <400> 1 Leu Arg Gln Ser Gln Phe Val Gly Ser Arg   1 5 10 <210> 2 <211> 10 <212> PRT <213> Urechistachykinin 2 <400> 2 Ala Ala Gly Met Gly Phe Phe Gly Ala Arg   1 5 10  

Claims (6)

An antimicrobial peptide having the amino acid sequence set forth in SEQ ID NO: 1. delete Antimicrobial and antifungal pharmaceutical composition comprising the peptide of SEQ ID NO: 1 as an active ingredient. The method of claim 3, The peptide is Staphylococcus aureus ), Enterococcus fascium faecum), streptococcus free Tansu (Streptococcus mutans), E. coli 0-157 (Escherichia coli 0-157), Pseudomonas aeruginosa , Vibrio vulnificus ), Candida albicans ), Trichosporon beigelii ), and Marasegia pearl ( Malassezia) furfur ) antimicrobial and antifungal pharmaceutical composition having antimicrobial activity against pathogenic microorganisms. delete delete

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