KR20160119943A - Peptide has antimicrobial, antifungal, and antitumor activity and uses thereof - Google Patents

Abstract

The present invention relates to peptide having antimicrobial, antifungal, and anticancer activities, and relates to uses thereof. According to the present invention, the peptide has excellent antimicrobial and antifungal activities with respect to gram positive bacteria, gram negative bacteria, and an antibiotic-resistant strain, thereby being efficiently used for the uses such as a pharmaceutical composition, a cosmetic composition, agricultural pesticides, a preservative for preserving a feed using the same, and the like. The peptide maintains excellent anticancer activities with respect to a cell strain of ovarian cancer, thereby being efficiently used as an anticancer agent with respect to the anticancer agent.

Description

Peptides having antimicrobial, antifungal and anticancer activities and their uses {Peptide has antimicrobial, antifungal, and antitumor activity and uses thereof}

The present invention relates to peptides which exhibit novel antibacterial, antifungal and anticancer activities, and antibacterial, antifungal and anticancer compositions containing the same as an active ingredient.

Some of the substances that play an important role in maintaining the homeostasis of organisms are physiologically active substances derived from various organisms. Numerous researches on physiologically active substances have been conducted so far. Among them, researches on antimicrobial and anti-cancer peptides are very important fields in life sciences and medicine.

All living organisms are known to produce antimicrobial substances for survival. Antimicrobial peptides isolated from various organisms are known to act in a variety of ways, ranging from bacteria, fungi and viruses to play an important role in host defense and innate immune systems. (Boman, HG, Cell, 65, 205, 1991; Boman, HG, Annu. Rev. Microbiol., 13, 61, 1995). Biologists produce their own antimicrobial peptides (Bevins et al., Ann. Rev. Biochem., 59, 395-414, 1990). The first is the cysteine-rich β-sheet peptide molecule, the second is the α-helical amphiphilic peptide molecule, the third is the proline-rich peptide (Mayasaki et al., Int. J. Antimicrob. Agents, 9, 269-280, 1998). These antimicrobial peptides have a variety of structures depending on the amino acid sequence. The most common of these structures is a structure that forms an amphipathic alpha helical form with no cysteine residues such as an antimicrobial peptide cecropin found in insects . Many studies have been carried out on the antimicrobial activity of peptides isolated from living organisms, and many attempts have been made to develop antimicrobial agents using peptides isolated from living organisms.

In addition to research on the development of antimicrobial agents, studies are underway to develop anticancer drugs using peptides separated from living organisms. Most of the anticancer drugs currently in use are developed by chemotherapy, and their pharmacological actions vary according to the type of cancer. Various toxic side effects are manifested, and the serious problem of toxicity to cancer cells and cancer cell resistance Studies on the development of effective anticancer drugs have been actively conducted. Compared with conventional chemotherapy, the peptides isolated from living organisms have a small molecular weight and thus are less likely to cause an immune response, are easy to penetrate into tissues, and selectively act on cancer cells.

On the other hand, insect-derived useful genes have been put to practical use in industry and attracted much attention in domestic and overseas related fields, and expectations about the current status and possibility of insect-using industry are also rising. In addition, in order to recognize and utilize insects rich in biodiversity as useful biomass in terms of industrial and economic aspects such as securing of genetic resources and development of biomaterials, insect resource secu- rity competition is becoming more and more fierce in national strategic level, centering on advanced countries.

For example, the European Union is conducting a venomics study to identify the components of venom secreted by animals, including arthropods, from 2011 to 2015 under the theme 'Venomes for Health'. In addition, as the claim of national sovereignty of genetic resources is strengthened due to the effect of the Nagoya Protocol, studies on the utilization methods of native plants in each country are proceeding, and in Korea, Research on the identification of unique genetic resources through identification and functional testing of these genes is being steadily strengthened.

The venom of insects, including arthropods, has received much attention in recent years as a rich report of physiologically active substances, especially neurotoxicants. Numerous toxins isolated from the venom of scorpions, spiders and bees have become a means of studying neuronal functions such as the action of ion channels and the identification of receptor functions and are used in research for the treatment of neuropathological disorders (Biochem. Biophys. Res. Commun. 250: 612-616, 1998).

In particular, physiologically active peptides isolated from the venom of arthropods have been attracting attention as novel antibiotics capable of controlling antibiotic resistant bacteria and new anticancer activity factors.

Under these circumstances, the present inventors have made intensive efforts to find new peptides having antimicrobial, antifungal and anticancer activities. As a result, the novel peptides isolated from wasp liquor have antibacterial, antifungal and anticancer activities and thus are useful as antimicrobial agents, antifungal agents and anticancer agents And the present invention has been completed.

Accordingly, a main object of the present invention is to provide a peptide having antibacterial, antifungal and anticancer activities.

It is another object of the present invention to provide antibacterial, antifungal, and anticancer compositions using the antibacterial, antifungal and anticancer peptides.

In order to achieve the above object, the present invention provides a peptide having the amino acid sequence of SEQ ID NO: 1.

The present inventors have investigated the expression genome and the proteome present in the capillary beetle for several years in order to develop a new antimicrobial agent or anticancer agent. As a result, The function of the peptide having the sequence was verified (Figs. 1 to 4).

In the present invention, the term peptide refers to a polymer composed of amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, it means a peptide having a high antimicrobial activity and antifungal activity against harmful strains and showing a strong anticancer activity. The peptide structure of the present invention is characterized by having a helix structure. The peptide of the present invention is a peptide represented by SEQ ID NO: 1 having an amino acid sequence of 'IKWKAILDAVKKVL'.

In the present invention, the peptide is characterized by exhibiting antibacterial, antifungal and anticancer activities.

The peptides have a particular gram-positive antibacterial activity against gram-negative bacteria, and antibiotic-resistant strains, and more particularly Stability Philo nose kusu aureus (Staphylococcus aureus , Escherichia coli ), Candida albicans ( Candida albicans and Botrytis < RTI ID = 0.0 > Cinerea ), but the present invention is not limited thereto (Figs. 5 to 10).

In addition, the peptide has an anticancer activity, and specifically includes a carcinoma, a lymphoma, a blastoma, a sarcoma, a liposarcoma, a neuroendocrine tumor, a mesothelioma, Schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell carcinoma (HCC), squamous cell carcinoma cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, Cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma, , Cervical cancer, ovarian cancer, Liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, It has anti-cancer activity against anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract and head and neck cancer. Specifically, it has anticancer activity against ovarian cancer, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the peptide according to the present invention was treated with a human ovarian cancer cell line (SK-OV-3) to inhibit the proliferation of ovarian cancer cells (FIG. 11).

On the other hand, the peptides of the present invention can be linked to the internalization sequence or protein transduction domain to effectively enter the cell.

Recent reports have shown that a variety of cellular internalization sequences, such as the TAT transactivation domain of HIV virus, transportan, which can transport antennapedia, across the plasma membrane, are known. In addition, polyarginine is known to be an attractive tool for transporting peptides into cells by transporting peptides and proteins across the plasma membrane much more efficiently.

Thus, the peptides of the present invention may comprise a cellular internalization-transporting substance or sequence. The cellular internalization sequence may be any internalization sequence known or newly found in the art, or a conservative variant thereof. Non-limiting examples of cell internalizing transport materials and sequences include, but are not limited to, polyarginine (such as R9), Antennapedia sequence, TAT, HIVTat, Penetratin, Antp-3A (Antp mutant), Buforin II, MAP (model amphipathic peptide), KFGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis- guanidinium- Aniline-threonine-cholesterol).

In addition, the peptides of the invention may also include additional amino acid sequences designed for specific purposes to increase the stability of the targeting sequence, capping motif, tag, labeled residue, half-life or peptide. In addition, the peptides of the present invention can be linked to coupling partners such as effectors, drugs, prodrugs, toxins, peptides, and delivery molecules.

In addition, the peptide of the present invention can be prepared in the form of a pharmaceutically acceptable salt. Specifically, a salt can be formed by adding an acid, and examples thereof include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid and the like, organic carboxylic acids such as acetic acid and halo such as trifluoroacetic acid (Such as acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid) and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (such as hyaluronic acid, chondroitin sulfate, Arginic acid), and organic sulfonic acids (e.g., methane, sulfonic acid, and p-toluenesulfonic acid) including sulfonic acid esters such as chondroitin sulfate.

The peptides of the present invention can be obtained by a variety of methods well known in the art. Specifically, it can be produced by chemical synthesis such as in vitro synthesis, cell-free protein synthesis, or genetic engineering using a protein expression system. However, as described through the production of the following peptides, Is preferably used.

The peptide of the present invention can be produced by a genetic engineering method using the amino acid sequence of SEQ ID NO: 1 derived from the mastoparan protein obtained through genome information or the nucleotide sequence encoding the same.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a peptide of interest in a suitable host cell, including a necessary regulatory element operatively linked to the expression of the gene insert. Expression vectors of the present invention include those elements normally present in a suitable expression vector, including expression regulatory elements such as promoters, operators, and initiation codons. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide and must be operative in the individual when the gene product is administered and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

It may also contain a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

The expression vector may be any conventional expression vector. For example, plasmid DNA, phage DNA, etc. may be used. Specific examples of plasmid DNA include commercial plasmids such as pUC18, pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, YCp50). Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, gt10, gt11 and ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used. Since the expression amount of the protein and the expression of the expression of the expression vector differ depending on the host cell, the host cell most suitable for the purpose may be selected and used.

Also, in the present invention, the term "transformation" refers to genetically stable genetics such that the nucleotide fragment migrates into the genome of the host organism to express the desired peptide.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

The host is not particularly limited as long as it is capable of expressing the peptide of the present invention. Specific examples of hosts that can be used in the present invention is Escherichia coli (E. coli) and Escherichia (Escherichia) bacteria belonging to the genus Bacillus subtilis (Bacillus subtilis), such as Bacillus (Bacillus) bacteria belonging to the genus Pseudomonas is footage (Pseudomonas such as Pseudomonas spp., such as S. putida , Saccharomyces sp . S. cerevisiae ), skiing investigation, Schizosaccharomyces pombe ) and insect cells.

According to another aspect of the present invention, there is provided a pharmaceutical composition for an antibacterial and antifungal agent, which comprises a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The peptides of the present invention exhibit excellent antibacterial and antifungal activity through directly affecting harmful microorganisms, and thus can be usefully used as pharmaceutical compositions for antibacterial and antifungal agents.

Specifically, the pharmaceutical composition has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant strains, and more specifically, Staphylococcus aureus, Escherichia coli, Candida albicans, But are not limited to, Candida albicans and Botrytis Cinerea.

The novel peptide of the present invention 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, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, do. 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. Examples of suppository bases include witepsol, macrogol, tween-61, cacao butter, Liu lingzhi, glycerogelatin and the like.

In addition, the novel peptide of the present invention can be used in combination with various carriers that are accepted as pharmaceuticals, such as physiological saline or an organic solvent, and can be used in combination with a carbohydrate such as glucose, sucrose or dextran Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals.

The effective dose of the novel peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

The total effective amount of the novel peptide of the present invention in the pharmaceutical composition of the present invention may be administered to a patient in a single dose by bolus administration 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 > of < / RTI > novel peptide as a pharmaceutical composition.

According to still another aspect of the present invention, there is provided a cosmetic composition for an antibacterial and antifungal agent comprising a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The peptides of the present invention exhibit excellent antibacterial and antifungal activity through directly affecting harmful microorganisms, and thus can be usefully used as cosmetic compositions for antibacterial and antifungal agents.

The cosmetic composition of the present invention contains, as an active ingredient, the ingredients commonly used in cosmetic compositions in addition to the above-mentioned novel peptide, and includes conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, .

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a flexible lotion (skin), a nutritional lotion (milk lotion), a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder .

When the formulation of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tragacantha, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a chlorofluorohydrocarbon, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tragacantha, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

According to still another aspect of the present invention, there is provided an agricultural chemical containing, as an active ingredient, a peptide having the amino acid sequence of SEQ ID NO: 1.

The peptides of the present invention exhibit excellent antibacterial and antifungal activity through directly influencing harmful microorganisms, and thus can be usefully used as an agricultural chemical composition.

According to still another aspect of the present invention, there is provided a composition for preserving a feed comprising, as an active ingredient, a peptide having the amino acid sequence of SEQ ID NO: 1.

Preservatives for foods and feeds, preservatives for cosmetics and preservatives for medicines are additives used to prevent deterioration, decay, discoloration and chemical change of foods and medicines. These include bactericides and antioxidants. They are used for the proliferation of microorganisms such as bacteria, fungi and yeast , And functional antibacterial agents such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods, feeds, cosmetics, medicines and the like. Ideal conditions for preserving foods, food preservatives, cosmetics and medicine preservatives should not be toxic and should be effective in trace amounts. Pesticides to eradicate the pests and diseases of crops also inhibit the growth of harmful microorganisms and are harmless to the human body, so that a person can safely consume agricultural products sprayed with pesticides. The peptides of the present invention exhibit excellent antibacterial and antifungal activity through directly influencing harmful microorganisms, and thus they can be widely used as preservatives, cosmetic preservatives and pharmaceutical preservatives for foods and feeds.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In the pharmaceutical composition for preventing or treating cancer according to the present invention, the cancer is selected from the group consisting of a carcinoma, a lymphoma, a blastoma, a sarcoma, a liposarcoma, a neuroendocrine tumor, The present invention relates to a method of treating a cancer selected from the group consisting of mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma, of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, Glioblastoma, cervical cancer, ovarian cancer (ovar) ian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, , Anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract, and head and neck cancer. Preferably, ovarian cancer can be mentioned.

The term "prevention" in the present invention means all actions that inhibit or delay the onset of cancer by administration of the peptide or composition according to the present invention.

The term "treatment" in the present invention means any action that improves or alters the cancer by the administration of the peptide or composition according to the present invention.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the patient's sex, age, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, the pharmaceutical composition of the present invention can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. Specifically, the pharmaceutical composition of the present invention is preferably administered orally or intravenously.

Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives are included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In another embodiment, the present invention provides a method of treating cancer in an animal other than a human, comprising administering the peptide or composition to a subject in need thereof.

The method of treatment according to the present invention does not mean that a method of treating an animal other than a human, but such a treatment method is ineffective in humans. In addition, in the case of humans, considering the fact that the symptom can be improved by administration of the therapeutic composition according to the present invention, it can be sufficiently used in human therapy.

The term "animal excluding human beings" in the present invention means animals, such as horses, sheep, pigs, goats, camels, nutrition, dogs, etc., except for humans having cancer whose symptoms may be improved by administration of the therapeutic composition according to the present invention . By administering the therapeutic composition according to the present invention to an animal other than a human, ovarian cancer can be effectively prevented and treated.

The term "administering" in the present invention means introducing a predetermined substance into an animal by any appropriate method, and the administration route of the therapeutic composition according to the present invention may be administered orally, May be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient into the target cell.

The preferable dosage of the therapeutic composition according to the present invention varies depending on the condition and body weight of the patient or the animal to be treated, the degree of disease, the drug form, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 1 to 10 mg / kg, preferably 1 to 5 mg / kg per day. The administration may be carried out once a day or divided into several times.

As described above, the peptide of the present invention has excellent antibacterial and antifungal activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant strains and is useful for pharmaceutical compositions, cosmetic compositions, agricultural chemicals, preservatives for preserving feeds and the like And has an excellent anticancer activity against ovarian cancer cell lines and can be useful as an anticancer agent against ovarian cancer.

FIG. 1 is a graph showing a result of a quality check using Bioanalyzer for horsetail whole RNA.
FIG. 2 is a graph showing mass spectral results of pre-mastoparan A of wasp poisonous liquid.
Figure 3 is an analysis of the amino acid sequence and evolutionary association of mastoparan.
FIG. 4 is a cross- lewissii and Vespa simillima Figure 3 shows the 3D structure of the Mastoparan protein of xanthoptera .
Figure 5 shows that the peptide of the present invention is < RTI ID = 0.0 > The results of the top agar and broth inhibition assays demonstrate the antimicrobial properties of E. coli.
FIG. 6 shows the results of Top agar and Broth inhibition assays showing that the peptides of the present invention have antimicrobial activity against S. aureus.
FIG. 7 shows the results of Top agar and Broth inhibition assay experiments showing that the peptides of the present invention have antibacterial activity against C. albicans.
FIG. 8 is a picture of the B. cinerea potato dextrose plate cultured for 24 hours inoculated with the peptide of the present invention and the control drug.
Figure 9 is a graphical representation of B. cinerea (Left) 48 hours after inoculation and a 24-hour potato dextrose plate (right) after treatment of the peptides and control drugs of the present invention.
Figure 10 B. cinerea (Left) after 72 hours of inoculation and a potato dextrose plate (right) after 48 hours of treatment with the peptides and control drugs of the present invention.
11 is a graph showing the anticancer activity against Ovarian tumor cells of the active peptides of the present invention.

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

Example  1. Active Of peptide  Isolation and identification

1-1. Extracting Bee's Mandate and Poisonous Glands

Some wasps were collected from Gwangju Taehwa Mountain in Gyeonggi Province, Suru Mountain in Gunpo City in Gyeonggi Province, Chiaksan in Gangwon Province, and Songcheon-dong in Andong City in Gyeongsangbuk Province. After the transfer to the bit female wasps collected in Gwangju Taehwasan to the laboratory in a living state in the anesthetized with CO ₂ immediately doknang was separated and self-righteousness. In order to extract whole mRNA, the autologous mucus was isolated from the gastrointestinal tract and the whole mRNA was extracted using Trizol reagent (MRC, Cincinnati, OH).

The female wasps were immediately frozen and stored at 80 ℃ until the venom was collected. The wasps were stored only in the rapidly frozen storage. More than 100 ml of poison was extracted from more than 100 wasps. 20ml and 30ml were used for proteome analysis and metabolism analysis, respectively.

1-2. Expression of a horsetail dog On the transcript  About Transcriptome  analysis

The total RNAs expressed in the horsetail lines were isolated. A total of five live wasps were harvested and rapidly frozen for isolation, and the frozen horsetail was transferred to RNAzol (Sigma-Aldrich, USA) solution to homogenize the tissues.

The total RNA of the wasp extract was subjected to quality check using Bioanalyzer (Bioanalyzer 2100, Agilent, BSK). As a result, it was confirmed that the extracted whole wasp of the wasp was a high-quality RNA capable of performing analysis for a transcriptome (Table 1 and FIG. 1).

[Table 1] QC results for whole hornet wasps using Bioanalyzer

Double-stranded cDNA (ds cDNA) was synthesized using reverse transcriptase (New England Biolab, USA) and random hexamer (New England Biolab, USA) as a template. The synthesized cDNA was prepared using a TruSeq Paired-End Cluster kit (Illumina) and DNA sequencing was performed using Illumina hiSeq 2000.

After reading a total of 6.16 Gbyte sequence expressed in the wasp of the wasp, the adapter sequence and the unanalyzed sequence were removed to obtain useful gene information of 5.7 Gbytes. The obtained gene information was assembled through de novo assembly using SOAPdenovo-Trans and Trans-ABySS program. As a result, a total of 72,510 transcripts were obtained and the transcripts were annotated using the BlaspP program. In addition, FPKM values of the secretory genes were calculated using the Cufflinks program to estimate the expression level of transcripts.

[Table 2] Frequency of Expression Transcripts in Hippopotamus

1-3. Some horsemen present in the poisonous liquid Peptides  analysis

Separation of the peptides was performed using 5 ml of wasp poison. The first low-MW fraction was separated into two fractions using a size exclusion chromatography column (Superdex peptide-10 / 300GL column) under 50 mmol Tris-HCl buffer (pH 7.4) buffer (pH 7.4) for 5 ~ 8 fractions.

First, MS and MS / MS data were obtained by MS ^E analysis using nano LC-ESI-tandem MS and peptide profiling was performed using UniProtKB's hymenoptera-related protein database or 6-frame translation protein database to identify the first peptide Respectively.

For the peptides not identified in the first peptide identification, the molecular weight of the venom peptide was measured using the positive mode of the nanoAQUILTY (C18 RP column) ESI-Tandem (Q-TOF) mass spectrometer and analyzed by MS / MS De-novo peptide sequences were obtained using fragments of a ⁺ , b ⁺ and y ⁺ -type and immonium ions.

Molecular mass information of the peptide was obtained using LC-MALDI-TOF / TOF MS, MS / MS data were obtained for the peptide candidate MS by comparison with the negative control, and de novo peptide sequencing was performed. The sequences and types of active peptides were identified (Table 3 and Figure 2).

[Table 3] Types and amino acid sequences of active peptides present in the wasp poison

1-4. Mastoparan Comparison of two-dimensional amino acid sequence analysis Three-dimensional structure  comparison analysis

Mastoparan of some wasps result of the amino acid sequence comparison of the Vespula Mastoparan Vastus vulgaris was most highly related to Mastoparan-V1 (Fig. 3). Vespa basalis , Vespa orientalis .

Many peptide substances (hormones, neurotransmitters, venom peptides, etc.) in vivo, including mastoparans from V. analis , are produced in the form of prepropeptides and then processed in vivo And is known to have the form of a mature peptide. The free peptides of horseshoe marstoparan are composed of signal peptide, prosequence, and mature peptide. Mastoparan present a three-dimensional structure it has been found to Vespula lewissii and Vespa simillima The xanthoptera Mastoparan is expected to be similar to the Mastoparan structure of Vespula lewissii (Figure 3 and Figure 4).

Example  2. Active Of peptide  Antibacterial and antifungal activity analysis

2-1. The subject microorganism

To test the antibiotic activity against the microorganisms of the poisonous peptides EpVP1, EpVP2a and EpVP2b, Escherichia coli, a gram negative bacterium coli ATCC 11775 ), gram-positive bacteria such as Staphylococcus aureus ATCC 12600 ), Candida albicans ( Candida albicans ATCC 10231 ), which is a yeast mold, was distributed at the Korean Microorganism Conservation Center (KCCM). Mycelium moldy strawberry gray mold ( Botrytis cinerea ) were sold in the Forest Research Institute of the South Forestry Research Institute and used in the laboratory.

2-2. Antibacterial and antifungal activity test

The active peptides of the present invention were tested for antibiotic activity against bacteria and fungi.

1. E. coli  Antimicrobial life span

One colony of E. coli cultured on an LB (LURIA-BERTANI) agar plate was suspended in 2 ml of LB broth and cultured in a shaking incubator at 37 ° C and 250 rpm for 16 hours And the cultured E. coli LB broth was diluted with fresh LB broth at a ratio of 1:10 and subcultured. Cultures were incubated until an OD 600 value of 0.5 was reached.

First, the antimicrobial activity against E. coli was examined by top agar method.

10 ml of LB broth containing 0.7 g of agar was sterilized and cooled to about 40 캜. Then, 0.1 ml of the E. coli culture solution having an OD 600 value of 0.5 was slowly inoculated onto the LB plate, . After a lapse of about 1 hour, a filter paper having a diameter of 5 mm was placed on an LB plate, 20 ml of DMSO was used as a control (C), and 20 ml of each of 10 mg / ml Ampicilin (A) and Kanamycin (K) was inoculated as a control. Then, the filter paper was inoculated with 20 ml of the active peptide of the present invention at a concentration of 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM and 1.0 mM, respectively, and stored at room temperature for 1 hour to allow the LB plate to dry. Then, the LB plate was incubated at 37 DEG C for about 16 hours. As a result, it was observed that when the concentration of the active peptide of the present invention was 0.5 mM or more, the proliferation inhibitory loop of E. coli was produced (see FIG. 5).

In addition, the antimicrobial activity against E. coli was examined by the Broth inhibition assay method. 45 ml of LB broth (OD 600 value of 0.5) and 5 ml of 1% DMSO were added to a 96-well plate of a subcultured E. coli strain diluted at a ratio of 1:10, and used as a blank. 40 ml of LB broth and 5 ml of 1% DMSO were mixed with 5 ml of E. coli culture (OD 0.5) and used as a positive control. Then, 5 ml of the active peptide of the present invention was mixed with 40 ml of LB broth at a concentration of 10 mM, 5 mM, 1 mM and 0.5 mM instead of 5 ml of 1% DMSO, and the mixture was added to a 96-well plate. The change in OD value was measured by incubating each well in a 96-well plate at 37 ° C for 3 hours. As a result, when the concentration of the active peptide of the present invention was 0.5 mM or more, the growth of E. coli did not appear after 3 hours, and the active peptide of about 0.5 mM inhibited the growth of E. coli (See FIG. 5).

2. S. aureus  Antimicrobial life span

S. aureus cell stock was inoculated on a Nutrient agar plate and cultured at 37 ° C for more than 16 hours. Single colonies selected from the cultured plates were suspended in 2 ml of Brain heart infusion broth and cultured in a shaking incubator at 37 ° C and 250 rpm for 16 hours. Brain heart infusion broth of cultured S. aureus was cultured in 1 : 10 in fresh Brain heart infusion broth. Cultures were incubated until an OD 600 value of 0.5 was reached.

To examine the antimicrobial activity against S. aureus by top agar method, 10 ml of Brain heart infusion broth containing 0.7 g of agar was sterilized, cooled to about 40 ° C, and the OD 600 value was adjusted to 0.5 0.1 ml of the cultured S. aureus was slowly inoculated on a Nutrient agar plate and top agar was made. After 1 hour, the filter paper with a diameter of 5 mm was placed on a Nutrient agar plate, 20 ml of DMSO was used as a control (C), and 20 mg of each of 10 mg / ml Ampicilin (A) and Kanamycin (K) was inoculated as a control. Then, 20 ml of the active peptide of the present invention was inoculated into the filter paper at a concentration of 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM and 1.0 mM, respectively, and stored at room temperature for 1 hour to allow the nutrient agar plate to dry. Then, the Nutrient agar plate was incubated at 37 ° C for about 16 hours. As a result, it was observed that when the concentration of the active peptide of the present invention was 0.5 mM or more, a proliferation inhibitory loop of S. aureus was produced (see FIG. 6).

In addition, the antimicrobial activity against S. aureus was examined by the Broth inhibition assay method. 45 ml of Brain heart infusion broth (OD 600 value of 0.5) and 5 ml of 1% DMSO were added to a 96-well plate of a 1:10 dilution of sub-cultured S. aureus and used as a blank. Brain heart infusion broth (40 ml) and 1% DMSO (5 ml) were mixed with 5 ml of S. aureus culture (OD 0.5) and used as a positive control. Then, 5 ml of the active peptide of the present invention was mixed with 40 ml of Brain heart infusion broth at a concentration of 10 mM, 5 mM, 1 mM and 0.5 mM instead of 5 ml of 1% DMSO, and the mixture was added to a 96-well plate. The change in OD value was measured by incubating each well in a 96-well plate at 37 ° C for 3 hours. As a result, when the concentration of the active peptide of the present invention was 0.5 mM or more, the growth of S. aureus did not appear after 3 hours, and the active peptide of about 0.5 mM inhibited growth of S. aureus with the same result as the top agar method (See FIG. 6).

3. C. albicans  Antimicrobial life span

C. albicans cell stock was inoculated on a Potato dextrose agar plate and cultured at 37 ° C. for 16 hours or more. Single colonies selected from the cultured plates were suspended in 2 ml of Potato dextrose broth, cultured in a shaking incubator at 37 ° C and 250 rpm for 16 hours, and the cultured C. albicans Potato dextrose broth was diluted 1:10 And then subcultured with fresh Potato dextrose broth. Cultures were incubated until an OD 600 value of 0.5 was reached.

To examine the antimicrobial activity against C. albicans in a top agar method, 10 ml of potato dextrose broth containing 0.7 g of agar was sterilized and cooled to about 40 ° C., and an OD 600 value reached 0.5 0.1 ml of the cultured C. albicans was slowly inoculated on a Potato dextrose agar plate and top agar was made. After 1 hour, the filter paper with a diameter of 5 mm was placed on a Potato dextrose agar plate, 20 ml of DMSO was used as a control (C), and 20 mg of each of 10 mg / ml Ampicilin (A) and Kanamycin (K) was inoculated as a control. Then, 20 ml of the active peptide of the present invention was inoculated into the filter paper at concentrations of 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM and 1.0 mM, respectively, and stored at room temperature for 1 hour so that the potato dextrose agar plate was dried. Then, a Potato dextrose agar plate was incubated at 37 ° C for about 16 hours. As a result, it was observed that when the concentration of the active peptide of the present invention was 0.5 mM or more, the proliferation inhibitory ring of C. albicans was formed (see FIG. 7).

Antibacterial activity against C. albicans was also examined by Broth inhibition assay. 45 ml (0.5% OD 600) of Potato dextrose broth of subcultured C. albicans and 5 ml of 1% DMSO were diluted to a ratio of 1: 10 and used as a blank. 40 ml of Potato dextrose broth and 5 ml of 1% DMSO were mixed with 5 ml of C. albicans culture (OD 0.5) and used as a positive control. Then, 5 ml of the active peptide of the present invention was mixed with 40 ml of Potato dextrose broth at a concentration of 10 mM, 5 mM, 1 mM and 0.5 mM instead of 5 ml of 1% DMSO, and the mixture was added to a 96-well plate. The change in OD value was measured by incubating each well in a 96-well plate at 37 ° C for 3 hours. As a result, when the concentration of the active peptide of the present invention was 0.5 mM or more, the growth of C. albicans did not appear after 3 hours, and about 0.5 mM of the active peptide inhibited the growth of C. albicans (See FIG. 7).

4. B. cinerea  Antifungal life span

B. cinerea was inoculated on a Potato dextrose plate and cultured at 20 ° C for 6 days. The potato dextrose plate was cut into 3 mm * 3 mm size, and the cultured B. cinerea was inoculated in the center of a new potato dextrose plate. After inoculation, the cells were cultured at 20 DEG C for 24 hours, and a filter paper having a diameter of 5 mm was placed thereon. 20 ml of 1% DMSO was applied to the control (C), and 20 mg of each of 10 mg / ml Ampicilin (A) and Kanamycin (K) was inoculated as a control. Then, 20 ml of the active peptide of the present invention was inoculated into the filter paper at concentrations of 0.1 mM, 0.25 mM, 0.5 mM and 1.0 mM, respectively, and the plate was dried and cultured at 20 ° C. Immediately after inoculation, after 24 hours, and after 48 hours, the untreated and treated groups were compared (see Figs. 8 to 10).

As a result, it was found that B. cinerea radially propagated mycelium in the inoculation area 24 hours after inoculation, and when the culture was continued, the mycelium was spread throughout the plate. Fungi are eukaryotic organisms that are not commonly used antibiotics. In particular, B. cinerea is a gompham that rotates the fruit of infectious plants and is easily found in refrigerators as well as farm households.

The active peptides of the present invention prevented the spread of the mycelium of B. cinerea . In the photographs taken after 24 hours from the inoculation of the active peptide of the present invention, the mycelium penetrates the filter paper in the case of the antibiotics inoculated group or the control group, but the filter paper area in which 0.5 mM or more active peptide was inoculated is normal B . cinerea It was confirmed that the progress of the mycelium was inhibited (FIG. 9).

This inhibition is more evident in the results after 48 hours. The filter paper area inoculated with more than 0.5 mM of active peptide showed normal B. cinerea The progress of the mycelium is inhibited, and the active peptide of the present invention forms a protective membrane to inhibit the progress of the mycelium (FIG. 10).

Example  3. Active Of peptide  Analysis of antitumor activity

In order to confirm that the active peptides of the present invention isolated in Example 1 had anticancer activity, the SK-OV-3 strain, which is one of human ovarian cancer cell lines, was treated with an active peptide to investigate the anticancer effect.

The ovarian cancer cell line was prepared by seeding (1 × 10 ⁴ ) on the day before the experiment and culturing for one day. The prepared ovarian cancer cells were treated with the active peptides of the present invention at concentrations of 0.25 mM, 10 mM, 25 mM and 50 mM, respectively. The anticancer effect of the active protein of the present invention was observed by comparing the treated group with the untreated control group (C) treated with 0.001% DMSO and the DMSO treated control group without any treatment.

After 24 hours and 72 hours, the culture medium was removed, and 100 ml of culture medium and 10 ml of MTT (5 mg / ml) were added to a well plate. ₂ < / RTI > conditions for 4 hours. After the incubation, the supernatant was removed, 100 ul of DMSO was added, and the optical density was measured at 540 nm.

As a result, it was confirmed that the proliferation of ovarian cancer cells was statistically significantly reduced in a medium containing 25 mM or more of the peptide cultured for 24 hours or 72 hours (FIG. 11).

<110> NATIONAL INSTITUTE OF BIOLOGICAL RESOURCES <120> Peptide has antimicrobial, antifungal, and antitumor activity and          uses thereof <130> PN-20150012 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Vespa analis parallela Andre <400> 1 Ile Lys Trp Lys Ala Ile Leu Asp Ala Val Lys Lys Val Leu   1 5 10

Claims (12)

A peptide having the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein said peptide exhibits antibacterial, antifungal and anticancer activities. 3. The method of claim 2,
The antimicrobial and antifungal activity is an activity against Staphylococcus aureus, Escherichia coli, Candida albicans and Botrytis cinerea. Characterized as a peptide. 3. The method of claim 2,
Wherein said cancer is an ovarian cancer. 5. The method according to any one of claims 1 to 4,
Wherein the peptide additionally comprises a cellular internalization sequence. A pharmaceutical composition for an antibacterial and antifungal agent containing a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient. 1. A cosmetic composition for antibacterial and antifungal use containing a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient. An agrochemical containing as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1. A preservative for preserving a feed containing an peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient. A pharmaceutical composition for preventing or treating cancer, which comprises a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 11. The method of claim 10,
Wherein said cancer is an ovarian cancer. 1. A method for treating ovarian cancer in an animal other than human, comprising administering to a subject in need thereof a peptide having the amino acid sequence of SEQ ID NO: 1.

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