KR101686428B1

KR101686428B1 - An anti-microbial peptide, Periplanetasin-2 isolated from Periplaneta americana and its synthetic composition

Info

Publication number: KR101686428B1
Application number: KR1020150081053A
Authority: KR
Inventors: 황재삼; 윤은영; 남성희; 안미영; 김미애; 김성현; 이준하; 김인우
Original assignee: 대한민국
Priority date: 2015-06-09
Filing date: 2015-06-09
Publication date: 2016-12-15

Abstract

The present invention relates to novel periplanetasin-2 peptides isolated from cockroaches and compositions thereof.
The peptide chemically synthesized by the present invention (Periplanetasin-1) showed a strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi. The antimicrobial peptide according to the present invention can be used for the development of antibiotics, food preservatives, cosmetic preservatives, and medicines containing antifungal agents because of their low cytotoxicity and strong antimicrobial activity.

Description

An antimicrobial peptide periplanetasin-2 derived from a cockroach and its composition {An anti-microbial peptide, Periplanetasin-2 isolated from Periplaneta americana and its synthetic composition}

The present invention relates to novel peptides derived from cockroaches and compositions thereof, and more particularly to novel periplanetasin-2 peptides and compositions thereof that exhibit antibacterial and antifungal activity with minimal cytotoxicity from cockroaches.

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 Priority No. 10-0625875 (a novel peptide isolated from Aspergillus nidulans and a pharmaceutical composition containing it as an active ingredient) and Korean Patent No. 10-0964136 An antimicrobial and antifungal peptide gene, and a synthetic peptide having antimicrobial and antifungal activity). However, there is no research on novel peptides isolated from cockroaches and novel uses thereof.

It is an object of the present invention to provide an amino acid sequence of an antimicrobial peptide periplanetasin-2 isolated from a cockroach which can be effectively used for the prevention and treatment of Gram-negative bacteria, positive bacteria and human fungal diseases and compositions thereof.

In order to achieve the above object, the present invention consists of the YPCKLNLKLGKVPFH-NH2 amino acid sequence (SEQ ID NO: 1). It is intended to provide a periplanetasin-2 peptide deduced from a gene isolated from a cockroach (Periplaneta americana).

The peptide is characterized by having antibacterial and antifungal activity.

The antimicrobial activity is characterized by exhibiting at least one of Staphylococcus aureus and E. coli 0-157 ( Escherichia coli 0-157).

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

The peptide is characterized in that the toxicity to the red blood cells is less than that of melittin.

The present invention provides a pharmaceutical composition for antibiotic and antifungal preparation containing the above peptide as an active ingredient, or antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative.

The present invention provides antibacterial peptide periplaneta 2 and composition thereof derived from a cockroach, and thus can be provided to antibiotics, food preservatives, cosmetic preservatives, pharmaceuticals and the like because of its excellent cytotoxicity and excellent antibacterial and antifungal activity .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the antibacterial activity and antifungal activity of the novel periplanetasin-2 peptide of the present invention derived from a cockroach.

Hereinafter, the present invention will be described in detail.

The present invention provides a periplanetasin-2 peptide (SEQ ID NO: 1) deduced from a gene isolated from a cockroach (Periplaneta americana).

In order to identify a peptide gene derived from a cockroach (Periplaneta americana), the present invention first isolates whole RNA by rapid freezing of Wangzine with liquid nitrogen, and then uses the RNA seq analysis method to confirm the gene information on the cockroach transcripts Based on the sequence of the translated amino acid from each transcript, the unicin, which is presumed to be a peptide exhibiting antibacterial and antifungal activity, is selected by using the properties of the previously discovered antimicrobial peptide.

The amino acid sequence of this peptide is composed of 15 amino acids with YPCKLNLKLGKVPFH-NH2, which is named Periplanetasin-2.

In addition, the present invention provides a pharmaceutical composition for antibiotic and antifungal preparation containing the active ingredient as the active ingredient, or antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative.

Specifically, the present inventors used Staphylococcus aureus , Escherichia coli 0-157, and Staphylococcus aureus as antibacterial target bacteria to measure antibacterial and antifungal activity of the periplanetin-2 peptide, 157). As a result of the RDA assay (radial diffusion assay) for Candida albicans as an antifungal activity target, it was found that it exhibited a strong antibacterial or antifungal activity similar to melitin having strong antibacterial or antifungal activity (Fig. 1).

In addition, the present inventors investigated the ability of the novel periplonethin-2 peptide of the present invention to exhibit cytotoxicity, and as a result, the novel periplonethin-2 peptide of the present invention has a low cytotoxicity Whereas melitin, a bee venom used as a positive control, was highly cytotoxic (Table 2).

From the above results, the pharmaceutical composition containing the novel periplanetin-2 peptide of the present invention as an active ingredient exhibits excellent antifungal and antimicrobial effects as well as a small cytotoxicity, and thus is useful as a safe antimicrobial agent and antifungal agent Can be used.

Such a pharmaceutical composition can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.

That is, the novel peptide of the present invention can be administered in various forms of parenteral administration. In the case of formulation, the peptide is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . 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 novel peptide of the present invention can be used in combination with various carriers permitted as pharmaceutical agents, such as physiological saline or an organic solvent, and can be used in combination with carbohydrates such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The total effective amount of the novel peptide of the present invention and the peptide having more than 95% homology thereto in the pharmaceutical composition of the present invention may be a single dose by a bolus form or by infusion for a relatively short period of time, , 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 > as < / RTI > the pharmaceutical composition of the novel peptide.

In addition, the present invention provides an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative containing a novel peptide 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.

As shown in FIGS. 1 and 2, the novel periplonetasin-2 peptide of the present invention exhibits strong antibacterial and antifungal activity and is insignificant in toxicity, so that it can be used effectively as a preservative for foods, cosmetics and pharmaceuticals.

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: Screening of cockroach-derived antimicrobial peptide gene

In order to identify the peptide gene showing antimicrobial or antifungal activity from cockroaches, the cockroach was rapidly frozen with liquid nitrogen to isolate the total RNA. The gene information on the cockroach transcripts was confirmed using the RNA seq method, Based on the sequence of the amino acid translated from the dead bodies, the uni - jean was filtrated based on the existing antimicrobial peptide properties. As a result, genes presumed as new antimicrobial peptides were selected. The selected amino acid sequence was composed of 15 amino acids with YPCKLNLKLGKVPFH-NH2 and named Periplanetasin-2. (Table 1).

Peptides Amino acid sequence Periplanetasin-2 YPCKLNLKLGKVPFH-NH2

Example 2 Perryplatanesin-2 synthesis and isolation Purification

In Example 1, the periplanetasin-2 peptide derived from the cockroach was synthesized, separated and purified.

First, in order to synthesize a periplanetasin-2 peptide, the present inventors used Merrifield's liquid phase solid phase method (Merrifield, RB, J. Am. Chem. Soc., 85, 2149 , &Lt; / RTI > 1963).

Method of the peptide synthesis 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 purified by HPLC on a reverse phase HPLC column (Delta Pak, C ₁₈ 300 Å, 15 μ, 19.0 mm × 30 cm) using an acetonitrile gradient containing 0.1% TFA, Waters).

After the synthetic peptide was hydrolyzed with 6 N 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.

Example 3: Antimicrobial activity of periplanetasin-2 antimicrobial peptide against pathogenic microorganisms

A radial diffusion assay (RDA) was performed to investigate the antimicrobial activity against the periplanetasin-2 peptide. Melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20 ° C and dissolved in sterilized distilled water.

[Antimicrobial activity measurement]

Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157), 3% (w / v) TSB (trypticase soy broth) was used to measure the antimicrobial activity of the antimicrobial peptide ) At 37 ° C and 200 rpm for 18 hours, and then cultured for 2 hours and 30 minutes at a concentration of ⁴ × 10 ⁶ CFU (colony forming units) / ml under the same conditions.

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) was added to the underlay gel and poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was pored and ⁵ μl of peptide per concentration .

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 (CLEAR ZONE) The results are shown in Fig.

[Measurement of antifungal activity]

Candida albicans , a pathogenic fungus, was grown overnight at 30 ° C and 200 rpm in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) And incubated for 2 hours to become logarithmic phase.

Then the petri eda Citrate phosphate buffer (9mM sodium phosphate, 1mM sodium citrate, pH7.4) and 1% (w / v) type (low electroendosmosis) agarose, a sterile underlay gel composed of 0.03% TSB (4X10 ⁶ colony forming units / mL) was added, and the mixture was poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was pored and 5 μl of the peptide was added per concentration.

After incubation at 37 ° C for 3 hours, 10 mL of overlay gel (6% TSB, 1% agarose) was added and cultured again at 37 ° C to confirm the formation of a clear zone. Respectively.

[Results of antibacterial activity]

As shown in FIG. 1, the periplanetasin-2 peptide showed activity against the pathogenic bacteria and positive bacteria, which was found to increase in a concentration-dependent manner. Compared with melittin, a potent antimicrobial peptide, all of the pathogenic strains were similar to melittin, indicating that it is possible to treat the existing strains.

[Results of antifungal activity]

As shown in FIG. 1, it was confirmed that a clear zone (CLEAR ZONE) was formed as a result of measuring the antifungal activity of the periplanetasin-2 peptide, and it was confirmed that the activity was increased in a concentration-dependent manner as in the case of a negative strain. It also means that the antifungal peptide exhibits strong antifungal activity due to the strong antifungal activity similar to that of melittin, which is a powerful antifungal agent used as a control.

Example 4: Cytotoxicity measurement of the periplonetasin-2 peptide on the red blood cells of the rat

The measurement of cytotoxicity against rat red blood cells was carried out as follows.

Rat erythrocytes were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer to prepare a 5.0% red blood cell solution.

Thereafter, 100 μl of the 5.0% red blood cell solution was dispensed into 96-well plates, and then a stepwise diluted solution of the antimicrobial peptide was mixed.

Then, phosphoric acid-sodium chloride buffer, pH 7.4 (PBS) was added to all the tubes so that the total amount became 200 쨉 l, and the cells were incubated in a 37 째 C incubator for 30 minutes.

After the culture was completed, the supernatant was transferred to a 96-well plate by centrifugation at 1000 rpm for 10 minutes in a centrifuge.

The destructive capacity of the red blood cells was measured by measuring the absorbance at a wavelength of 550 nm using an ELISA reader.

100% destructive capacity was calculated for 0.1% Triton X-100 as a control, and 0% destructive capacity for only red cell.

At this time, the destructive ability of the peptide was calculated by the following equation (1), and the obtained results are shown in Table 2 below.

[Equation 1]

% Destructive capacity = (absorbance of the peptide-treated solution 550 nm-absorbance of the phosphate-sodium chloride buffer 550 nm) / (absorbance of the solution treated with Triton X-100 550 nm-absorbance of the phosphate-sodium chloride buffer 550 nm) * 100

Measurement of cytotoxicity of antimicrobial peptides of periplanetasin-2 on rat erythrocytes
% Red erythrocyte destruction ability ^a (ug / mL) 0 25 50 100 200 Periplanetasin-2 0 3.37 2.22 2.13 2.41 Melitin 0 106.9 104.9 106.41 104.8 [Note] a: Cytotoxicity is determined by the ability of the antimicrobial peptide to destroy red blood cells

As shown in Table 2 above, it was confirmed that the periplanetasin-2 peptide was destroyed in a concentration-dependent manner, but the degree was not so significant. In contrast, melittin, a potent antimicrobial peptide, was highly cytotoxic at low concentrations.

These results indicate that the antimicrobial peptides found in the present invention can be used as antibiotics, food preservatives, cosmetic preservatives, and pharmaceuticals since they exhibit excellent cytotoxicity and excellent antimicrobial and antifungal activity.

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.

Claims

The periplanetasin-2 peptide consisting of the following SEQ ID NO: 1 and deduced from the gene isolated from the cockroach (Periplaneta americana).
SEQ ID NO: 1: YPCKLNLKLGKVPFH-NH2

The method according to claim 1,
Wherein the peptide has antibacterial and antifungal activity.

3. The method of claim 2,
Wherein the antibacterial activity is exhibited for at least one of Staphylococcus aureus and E. coli 0-157 ( Escherichia coli 0-157).

3. The method of claim 2,
Wherein said antifungal activity is exhibited against Candida albicans .

The method according to claim 1,
Wherein said peptide is less toxic to melatonin than to red blood cells.

A pharmaceutical composition for an antibacterial and antifungal preparation containing the peptide of any one of claims 1 to 5 as an active ingredient.

An antibiotic comprising the peptide of any one of claims 1 to 5 as an active ingredient.

A food preservative containing as an active ingredient a peptide according to any one of claims 1 to 5.

A cosmetic preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.

A pharmaceutical preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.