KR101667535B1 - An anti-microbial peptide Scolopendrasin-6 isolated from the Scolopendra subspinipes mutilans and its synthetic peptide - Google Patents

Abstract

The present invention relates to an antimicrobial peptide scolopendratin-6 and a composition thereof isolated from a walnut.
The present invention provides an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 isolated from Scolopendra subspinipes .

Description

An antimicrobial peptide, scolopendrazin-6, isolated from the king's wing and its composition {An anti-microbial peptide Scolopendrasin-6 isolated from the Scolopendra subspinipes mutilans and its synthetic peptide}

The present invention relates to an antimicrobial peptide scolopendratin-6 and a composition thereof isolated from a wangzine. More particularly, the present invention relates to an antimicrobial peptide scolopendratin-6 And compositions thereof.

 Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, unfortunately the antibiotic resistance of pathogenic microorganisms has been caused by the abuse of antibiotics.

In fact, the rate at which pathogenic microorganisms are resistant to new antibiotics is much faster than the rate at which analogs of new antibiotics are developed.

For example, pathogenic microbial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which may pose a life threat, I have developed resistance to all antibiotics.

Tolerance to antibiotics is distinct from resistance to antibiotics, which was first discovered in the 1970s by Pneumococcus sp. And provided important clues to the mechanism of action of penicillin.

Resistant species stop growing in the presence of the usual concentration of antibiotics but do not eventually die.

This resistance occurs when the antibiotic inhibits the cell wall synthetase because the autolytic enzyme activity of the bacteria such as autolysin does not occur. This fact suggests that penicillin is an endogenous hydrolytic enzyme Activation kills bacteria and bacteria also inhibit their activity, resulting in the survival of antibiotics.

It is clinically very important that pathogenic microorganisms have resistance to antibiotics, because the inability to eradicate the resistant microorganisms is ineffective in antibiotic treatment in clinical infections.

In addition, tolerance is considered to be a prerequisite for resistance to antibiotics, which is due to the survival of strains despite antibiotic therapy.

These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics.

Since virtually all pathogenic microorganisms showing resistance are also known to be resistant, development of new antibiotics capable of killing pathogenic microorganisms resistant to these antibiotic resistance is urgent.

As described above, it is necessary to develop a new antibiotic to prevent damages caused by pathogenic microorganisms showing resistance to antibiotics, and to develop new antibiotics that act independently of autolysin activity.

In addition, there is a need to provide pharmaceutical compositions for effectively treating such new antibiotics with an infection of pathogenic microorganisms.

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

Many researches have been carried out on a number of biologically active substances, among which antimicrobial peptides isolated from various organisms are known to act in various ways, ranging from bacteria, fungi and viruses. Antimicrobial peptides are also known to play an important role in host defense and the innate immune system.

Korean Patent Registration No. 10-0491423 (antimicrobial peptide isolated from silkworm) and Korean Patent No. 10-0964136 (antimicrobial and antifungal peptide genes isolated from Woldukwang larvae, and synthetic compounds having antibacterial and antifungal activity) Peptides) have been known, but no studies have been made on novel peptides isolated from Wang Ji Nee and their new uses.

It is an object of the present invention to provide an antimicrobial peptide scolopendrazin-6 and a composition thereof, which are isolated from Wang Ji-nee which can be effectively used as a preventive and therapeutic agent for fungal diseases of the human body as well as exhibiting antibacterial and antifungal activity.

In order to accomplish the above object, the present invention provides an antimicrobial peptide comprising an amino acid sequence separated from Scolopendra subspinipes and represented by SEQ ID NO: 1.

The peptide is characterized by having antibacterial and antifungal activity.

The antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa may be determined by measuring the concentration of the antimicrobial activity of the antibiotic-resistant Pseudomonas aeruginosa , which is expressed by at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157) .

The antifungal activity is characterized in that it appears to Candida albicans .

The peptide is characterized by being non-toxic to human red blood cells.

The present invention is to provide a pharmaceutical composition for antifungal and antifungal preparation containing the above-mentioned antimicrobial peptide as an active ingredient, or an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative.

According to the present invention, there is provided an antimicrobial peptide scolopendratin-6 and a composition thereof isolated from Wangzine.

In addition, the antimicrobial peptide exhibits strong antibacterial and antifungal activity, and is safe for human body since it has no cytotoxicity, so that it can be provided as antibiotics, food preservatives, cosmetic preservatives, medicines and the like.

Fig. 1 is a graph showing the inhibitory activity of the antimicrobial peptide scallopendrathin-6 of the present invention isolated from Wang jingne against Gram-negative bacteria, positive bacteria and antibiotic-resistant bacteria.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified. Hereinafter, the present invention will be described in detail.

The invention wangjine (Scolopendra subspinipes ) to provide an antimicrobial peptide represented by SEQ ID NO: 1.

In detail, in the present invention, in order to identify an antimicrobial peptide gene from Scolopendra subspinipes , Wangzine is first rapidly frozen with liquid nitrogen to isolate total RNA, and then, using RNA seq analysis method, the genes related to Wangzine transcripts Based on the sequence of the translated amino acids from each transcript, the genes that are presumed to be new antimicrobial peptides among the unicamines are selected using the properties of the antimicrobial peptides that have been discovered.

The amino acid sequence of the new antimicrobial peptides are named with a pen driver Shin -6 (Scolopendrasin-6) it to the Driscoll was composed of 17 amino acids with PRQVQVWFQNRRAKIKK-NH _2.

In order to measure the antimicrobial activity of the antimicrobial peptide Scolopendrasin-6, the present inventors used Staphylococcus aureus as a pathogenic bacterium, Escherichia coli 0-157 -157), and the RDA assay (radial diffusion assay) was performed on the antibiotic-resistant Pseudomonas aeruginosa (Multi Drug Resistance Psudomonas aeruginosa ) and the pathogenic fungus Candida albicans . As a result, melitin Or more potent antibacterial or antifungal activity (see FIG. 1).

In addition, the present inventors measured cytotoxicity against human erythrocyte cells to determine whether the antimicrobial peptide scolopendrazin-6 exhibited cytotoxicity. As a result, the inventive antimicrobial peptide scolopendrathin- Melitin, which was used as a positive control, did not show cytotoxicity against red blood cells, but showed high cytotoxicity even at low concentrations (see Table 2).

From the above results, it can be seen that the antimicrobial peptide of the present invention, scolopendrazin-6 and a composition containing it as an active ingredient, exhibits excellent antibacterial and antifungal effects as well as being free from cytotoxicity, thus being useful as a safe antimicrobial agent and antifungal agent Can be used.

Accordingly, the present invention provides an antimicrobial peptide scolopendrazin-6 and a pharmaceutical composition containing the same as an active ingredient. The pharmaceutical composition can be administered parenterally at the time of clinical administration and can be administered in the form of a general pharmaceutical preparation Can be used.

That is, the antimicrobial peptide scolopendrazin-6 of the present invention may be administered in various forms of parenteral administration. In the case of formulation, diluents such as a filler, an extender, a binder, a wetting agent, a disintegrant, Or an excipient. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In addition, the antimicrobial peptide scolopendrazin-6 of the present invention can be used in combination with various carriers permitted as medicaments such as physiological saline or an organic solvent, and can be used in combination with glucose, sucrose, Carbohydrates such as dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the antimicrobial peptide scolopendrascin-6 of the present invention is 0.01 to 200 占 퐂 / ml, preferably 50 to 200 占 퐂 / ml, and may be administered once to three times a day.

In a pharmaceutical composition of the present invention, the total effective amount of the antimicrobial peptide scolopendrathin-6 of the present invention and the peptide having 95% or more homology thereto is in the form of a bolus or by infusion for a relatively short period of time May be administered to a patient in a single dose and may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > antimicrobial < / RTI > peptide of the invention as a pharmaceutical composition.

In addition, the present invention provides antibiotics, food preservatives, cosmetic preservatives or pharmaceutical preservatives containing an antimicrobial peptide scolopendratin-6 as an active ingredient.

In this case, food preservative, cosmetic preservative and pharmaceutical preservative are additives used to prevent deterioration, decay, discoloration and chemical change of foods or medicines, including bactericides and antioxidants, and proliferation of microorganisms such as bacteria, fungi and yeast As well as functional antibiotics such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods and medicines. Ideal conditions for these food preservatives, cosmetics, and medicine preservatives should be non-toxic and have a very small effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

≪ Example 1 > Selection of a peptide gene derived from Wangjing

In order to identify the antimicrobial peptide gene from Wang Ji, the Wang JiNe was rapidly frozen with liquid nitrogen to isolate the total RNA. The gene information of the WangJin transcripts was confirmed using the RNA seq method, Based on the amino acid sequence, peptides showing similar properties to those of conventional antimicrobial peptides were selected from the unicin, and as a result, genes presumed to be novel antimicrobial peptides were selected (Table 1).

The amino acid sequence of the selected antimicrobial peptide was composed of 17 amino acids with PRQVQVWFQNRRAKIKK-NH _2, was designated as "pen driver Shin -6 (Scolopendrasin-6) as a squall.

Scolopendrazine-6
Peptide sequence
(SEQ ID NO: 1) PRQVQVWFQNRRAKIKK-NH ₂

Example 2 Synthesis and Separation of Scolopendrazine-6 Peptide

The antimicrobial peptide scallopendra-6 derived from Wangzine isolated in Example 1 was synthesized, isolated and purified.

First, in order to synthesize the antimicrobial peptide scolopendratin-6, the present inventors prepared a peptide using Merrifield's liquid phase method using a Fmoc amino group protecting container.

Synthesis of the antibacterial peptides is the Fmoc (9-fluorenylmethoxycarbonyl) amino acids of the N _α were synthesized and used as a protecting group (protecting group) of the amino group (amino group) to the conventional method (solid phase method).

Concretely, the peptide having carboxyl terminal in the form of -NH ₂ used Rink Amide MBHA-Resin as the starting material, and the peptide having carboxyl terminal in the form of -OH used Fmoc-amino acid-Wang Resin as a starting material.

Elongation of the peptide chain by coupling of Fmoc-amino acid was performed by DCC ( N -hydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide) method.

After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM) It was dried with gas.

A solution of TFA (trifluoroacetic acid) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added and the reaction was carried out for 3 hours. The peptides were separated and the peptide was precipitated with diethyl ether.

The crude peptide thus obtained was subjected to a reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15 μ, 19.0 mM ㅧ) using an acetonitrile gradient containing 0.1% TFA 30, Waters).

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

The molecular weight was calculated based on the sequence of the synthesized peptides, and the exact molecular weight was measured using a MALDI mass spectrometer (MALDI).

As a result, it was confirmed that the antimicrobial peptide having the correct amino acid sequence was synthesized because the calculated molecular weight and the calculated molecular weight were identical.

<Experimental Example 1> Antimicrobial activity against pathogenic microorganisms of the antimicrobial peptide scolopendrazin-6

A radial diffusion assay (RDA) was performed to measure the antimicrobial activity against the antimicrobial peptide scolopendratin-6. The antimicrobial peptide was stored at -20 ° C and dissolved in 0.01% acetic acid in consideration of the stability of the peptide.

In addition, Melittin, an antibacterial peptide isolated from bees, was used as a positive control.

[Antimicrobial activity measurement]

In order to measure the antimicrobial activity of the antimicrobial peptide of the present invention, pathogenic bacteria such as Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157), Pseudomonas aeruginosa (Multi-Drug Resistance Psudomonas aeruginosa ) was cultured in 3% (w / v) trypticase soy broth at 37 ° C and 200 rpm for 18 hours, and then 4 ㅧ 10 ⁶ CFU (colony forming units) / ml 2 &lt; / RTI &gt; and 30 min.

Thereafter, a sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type I (low electroendosmosis) agarose and 0.03% TSB (4 × 10 ⁶ colony forming units / ml) were added to the underlay gel and poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was punched out to obtain a concentration of 1 mg / mL Lt; / RTI &gt; of the antimicrobial peptide was pored into each well.

10 mL of an overlay gel (6% TSB, 1% agarose) was poured and incubated at 37 ° C again to confirm formation of a clear zone (CLEAR ZONE) after incubation at 37 ° C for 3 hours to allow the antimicrobial peptide to diffuse The obtained results are shown in Fig.

[Measurement of antifungal activity]

Measurement of antifungal activity of the antimicrobial peptides of the present invention, after grown overnight with pathogenic fungi of Candida albicans (Candida albicans) a YPD medium (1% yeast extract, 2% peptone, 2% dextrose) 30 ℃, 200 rpm conditions in , Which was inoculated on a fresh medium and grown for 2 hours to become logarithmic.

Thereafter, a sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type (low electroendosmosis) agarose and 0.03% TSB (4 × 10 ⁶ colony forming units / mL) were added to each well and poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was punched out to obtain a concentration of 1 mg / mL The antimicrobial peptide was put into the hole by 5..

10 mL of an overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C again to confirm formation of a clear zone. The obtained results were shown in FIG. 1 Respectively.

[Results of antibacterial activity]

As shown in FIG. 1, the antimicrobial activity of the antimicrobial peptide scallopendrazin-6 of the present invention showed inhibitory activity against Gram-negative bacteria, Gram-positive bacteria and resistant bacteria, there was.

In addition, when compared with melittin, an antimicrobial peptide used as a positive control, it was confirmed that the inhibitory activity was high in Gram-negative bacteria and resistant bacteria, which means that there is a possibility of being a therapeutic agent for existing antibiotic resistant strains.

[Results of antifungal activity]

As shown in FIG. 1, the antifungal peptide scolopendrascin-6 activity of the present invention showed that CLEAR ZONE was formed in the same manner as Gram-negative bacteria and positive bacteria, and activity was increased in a concentration-dependent manner .

In addition, this indicates that the antimicrobial peptide of the present invention exhibits strong antifungal activity, indicating that the antifungal activity is similar to that of melittin, which is a potent antifungal agent used as a positive control.

<Experimental Example 2> Cytotoxicity of the antimicrobial peptide scolopendrazin-6 on human red blood cells

To determine whether the antimicrobial peptide scolopendrathin-6 of the present invention exhibited cytotoxicity, cytotoxicity was performed on human red blood cells.

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% red blood cell solution, and the red blood cell solution was added to 96- 100 [mu] l of each solution was dispensed onto the plate, and then a solution in which the antimicrobial peptide was diluted stepwise was mixed.

Next, physiological saline (Saline 0.85% NaCl) was added to all the wells so that the total amount became 200 μl, and the cells were cultured in a 37 ° C. incubator for 1 hour and centrifuged at 1000 rpm for 10 minutes in a centrifuge And the supernatant was transferred to the side wells.

The supernatant was used to analyze the destructive activity against red blood cells by measuring the absorbance at 414 nm using an ELISA reader.

At this time, the destructive ability of the peptide was calculated by the following formula (1), and the value obtained when 0.1% Triton X-100 was treated was calculated as 100% destructive ability, The results are shown in Table 2 below.

As a positive control, melittin, an antimicrobial peptide isolated from bees, was used.

[Equation 1]

% Destructive ability = (absorbance of solution treated with peptide: 414 nm - absorbance of phosphoric acid-sodium chloride buffer: 414 nm) / (absorbance of solution treated with Triton X-100: 414 nm - absorbance of phosphoric acid-sodium chloride buffer:

Measurement of cytotoxicity of the antimicrobial peptide scolopendrathin-6 against human erythrocytes
% Human erythropoietin ^a (占 퐂 / ml) 0 6.25 12.5 25 50 100 Scolopendrazine-6 0 2.1 0 0 0 0 Melitin 0 31.2 95.7 98.1 98.7 99.2 [Note] a: Cytotoxicity to human is measured by the ability of the peptide to destroy the human red blood cells

As shown in Table 2, it was confirmed that the antimicrobial peptide scallopendrathin-6 of the present invention did not show cytotoxicity at all concentrations.

In contrast, melittin, which was used as a positive control, showed high cytotoxicity even at low concentrations.

As a result, the novel antimicrobial peptide scallopendrazin-6, which is found in the present invention, exhibits excellent antimicrobial and antifungal activity and is not cytotoxic. Therefore, it can be used as a safe antibiotic, food preservative, cosmetic preservative, it means.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> National Academy of Agricultural Science <120> An anti-microbial peptide Scolopendrasin-6 isolated from the          Scolopendra subspinipes mutilans and its synthetic peptide <130> P2014-0067 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Scolopendrasin-6 <400> 1 Pro Arg Gln Val Gln Val Trp Phe Gln Asn Arg Arg Ala Lys Ile Lys   1 5 10 15 Lys    

Claims (10)

1, which is separated from Scolopendra subspinipes and is the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
Antimicrobial activity against at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157) and the antibiotic-resistant negative microorganism Pseudomonas aeruginosa (Multi Drug Resistance Psudomonas aeruginosa ) Peptides.
SEQ ID NO: 1:
Pro Arg Gln Val Gln Val Trp Phe Gln Asn Arg Arg Ala Lys Ile Lys Lys
delete delete delete The method according to claim 1,
Wherein said peptide is non-toxic to human red blood cells.
delete delete A food preservative containing the antimicrobial peptide of claim 1 or 5 as an active ingredient.
A preservative for cosmetics containing the antimicrobial peptide of claim 1 or 5 as an active ingredient.
A pharmaceutical preservative containing the antimicrobial peptide of claim 1 or 5 as an active ingredient.

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