JP2017077236A - ANTIMICROBIAL PEPTIDE DERIVED FROM ABALONE β-GLUCAN-BINDING PROTEIN, NUCLEIC ACID ENCODING SAME, AND USE THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel peptide having antimicrobial and/or anticancer activity, a nucleic acid encoding the novel peptide, and a recombinant expression vector having a nucleic acid encoding a novel peptide inserted therein, and to provide a pharmaceutical composition, a cosmetic composition, and/or a food supplement aid as an antibacterial and/or anticancer composition containing a novel peptide as an active ingredient.SOLUTION: The present invention provides a peptide having anticancer properties as antimicrobial properties and new physiological activity, a nucleic acid encoding the same, and the application of this peptide or nucleic acid. According to the present invention, a vector in which a synthesized peptide or a nucleic acid encoding the peptide is inserted is used as an active ingredient, and is then utilized in an antimicrobial and/or anticancer composition, such as a pharmaceutical composition, a cosmetic composition and/or a food additive.SELECTED DRAWING: Figure 3A

Description

  The present invention relates to a novel peptide, and more particularly, to a novel peptide having antibacterial activity and / or anticancer activity, a nucleic acid encoding the peptide, and uses such as industrial applications thereof.

  The immune system as a defense system against external pathogens varies from organism to organism. All immune systems can be made up of systems that distinguish between self and non-self and recognize, dispose, and communicate external molecules. The immune system against external pathogens in living organisms remembers the innate immune system that immediately senses and reacts to the pathogen when it first enters, and memorizes it when the same pathogen invades. The adaptive immune system is an acquired immune system that constructs a defense system against pathogens.

  Unlike vertebrates that have developed an adaptive immune system, invertebrates lack antibodies and lack or lack an adaptive immune system. Instead, invertebrates have a very efficient innate immune system as a defense system against invading pathogens. In particular, marine invertebrates inhabit and proliferate in an environment rich in such microorganisms, which is more efficient than inland invertebrates. The immune system has developed.

  The first stage of the innate immune response in living organisms, including marine invertebrates, is to recognize pathogenic bacteria that are about to enter the body. The cell wall of a microorganism that is one of the typical pathogens is a pathogen composed of glycoproteins such as lipid polysaccharides (LPS), β-1,3-glucan (β-1,3-glucan), and peptidoglycan (peptidoglycan). Although they have related molecular patterns (pathogen-associated molecular patterns, PAMPs), they do not exist in the host or are hidden in various ways. Such pathogen-associated molecular patterns (PAMPs) are recognized by specific proteins that make up the invertebrate innate immune system, such as pattern recognition receptors (PRRs) or pattern recognition proteins (PRPs). be able to.

  PRPs and PAMPs form a complex and induce a series of activated immune responses. Each PRPs recognizes and binds to appropriate PAMPs that are not present in multicellular organisms but only on the surface of pathogenic microorganisms, phagocytosis, nodule formation, encapsulation, pre-encapsulation, It induces a series of immune responses such as proteinase cascade activation and antimicrobial peptide synthesis. For example, peptidoglycan recognition proteins (PGRPs), C-type lectins, lipid polysaccharide binding proteins (LPS-binding proteins), β-1,3-glucan binding proteins, β-glucan binding βG protein, Various forms of PRPs derived from invertebrates such as lipid polysaccharides and glucan binding proteins (LGBP) have been reported, such as lipopolysaccharides and glucan binding proteins (LGBP).

  PAMPs present in the microbial cell wall directly activate the function of invertebrate defense cells, but the immune response that amplifies a series of stimuli caused by invertebrate PRPs is secondary to antibody secondary in vertebrates. It is similar to typical activity. In particular, the prophenoloxidase (pro-PO) activation system shows an important defense mechanism in a wide variety of invertebrates. This immune system recognizes bacterial antigens (sugar sites, saccharide moiiety) such as LPS, peptidoglycan and β-1,3-glucan that are present as major components of fungal cells such as yeast and fungus Based on what you do.

  Some LGBPs have been cloned and analyzed in aquatic organisms. For example, signal crayfish (Pacifastacus leniusculus LGBP), prawns (Marsupenaeus japonicas LGBP), Kouraiebi (Fenneropenaeus chinensis LGBP), Azumanishiki (Chlamys farreri LGBP), Kuroawabi (Haliotis discus BGRP), such as pearl oyster (Pinctada fucata LGBP) were reported. In particular, the prickly crabs (Pacifastacus leniusculus LGBP) are known to play an important role in the activity of prophenol oxidase (proPO).

In general, LGBP includes two polysaccharide recognition sites, a polysaccharide binding motif and a beta glucan recognition motif. Invertebrates recognize bacterial antigens (sugar sites) such as LPS, peptidoglycan and β-1,3-glucan that are present as major components of all yeast and fungal cells.

Antibacterial peptides designed based on the binding or recognition domain of LPS are known. Currently, antibacterial activity has been reported with the corresponding synthetic LPS-binding domain peptides of anti-lipopolysaccharide factor (ALF) obtained from various crustaceans. For example, lactoferrin, a non-hemi iron-linked glycoprotein, has an antimicrobial activity in its LPS-binding domain. The recombined N-terminal domain of Gram negative binding protein 3, GNBP3, binds to β-1,3-glucan and exhibits antibacterial activity.

  However, new types of antimicrobial peptides need to be developed to study these new physiological activities.

  An object of the present invention is to provide a novel peptide having antibacterial and / or anticancer activity, a nucleic acid encoding the novel peptide, and a recombinant expression vector into which a nucleic acid encoding the novel peptide has been inserted.

  Another object of the present invention is to provide a pharmaceutical composition, a cosmetic composition and / or a food additive as an antibacterial and / or anticancer composition containing a novel peptide as an active ingredient.

  The present invention relates to peptides having anti-cancer properties as antibacterial properties and new physiological activities, nucleic acids encoding them and applications.

  According to one aspect of the present invention, the present invention provides a peptide having a sequence represented by the following formula (1).

[Formula 1]
(N-terminal) -WLWKAIWKLLL- (C-terminal) (1)

  In the formula (1), X is a basic amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H), or serine (S), threonine (T), asparagus. A polar amino acid selected from the group consisting of gin (N) and glutamine (Q).

  For example, the peptide of the formula (1) is composed of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

  In an exemplary embodiment, the C-terminus of the peptide represented by the formula (1) is amidated.

  The peptide may have at least one physiological activity selected from antibacterial activity and anticancer activity.

  According to another aspect of the present invention, the present invention provides a nucleic acid encoding the aforementioned peptide.

  For example, the nucleic acid can include a nucleotide composed of the base sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

  According to still another aspect of the present invention, the present invention provides a recombination expression vector into which a nucleic acid encoding the aforementioned peptide is inserted.

  According to still another aspect of the present invention, the present invention provides an anticancer pharmaceutical composition comprising the above-mentioned peptide as an active ingredient.

  For example, the peptide is used as a pharmaceutical composition for treating at least one cancer selected from the group consisting of uterine cancer, cervical cancer and lung cancer.

  According to still another aspect of the present invention, the present invention provides an antibacterial pharmaceutical composition comprising the above-mentioned peptide as an active ingredient.

  For example, the peptide exhibits antibacterial activity against gram positive bacteria, gram negative bacteria and yeast.

  According to still another aspect of the present invention, the present invention provides an antibacterial cosmetic composition comprising the aforementioned peptide as an active ingredient.

  According to still another aspect of the present invention, the present invention provides an antibacterial food additive comprising the above-mentioned peptide as an active ingredient.

  The peptide variants obtained by the present invention show efficient antibacterial activity against various pathogens. Furthermore, the peptide synthesized by the present invention exhibits anticancer activity against various cancer cell lines as another physiological activity in addition to antibacterial activity.

  Therefore, the peptide synthesized according to the present invention can be applied as an active ingredient of an antibacterial, antibiotic composition and / or anticancer composition. For example, the peptide synthesized according to the present invention can be used not only as an active ingredient of an anticancer and / or antibacterial pharmaceutical composition, but also as an active ingredient of an antibacterial cosmetic composition or an antibacterial food additive as a cosmetic preservative. It is expected that it can be used as

FIG. 3 is a diagram illustrating a full-length amino acid sequence and a cDNA sequence of a lipid polysaccharide derived from abalone and β-1,3-glucan binding protein (HDH-LGBP) according to an exemplary embodiment of the present invention. The signal peptide sequence and the poly- (A) signal site are underlined, and the integrin-binding motif and N-glycation site are highlighted with a gray square. The polysaccharide binding motif is represented by red italics and underline. According to an exemplary embodiment of the present invention, lipid polysaccharides derived from abalone and peptides constituting β-1,3-glucan binding protein (HDH-LGBP) and natural peptides were substituted and synthesized. FIG. 2 is a Schiffer-Edmundson helical wheel diagram for peptide variants. An arrow represents an amino acid residue substituted with a natural peptide (HDH-LGBP), and a hydrophobic amino acid residue and a hydrophilic amino acid residue constituting the peptide are surrounded by a square. 3 is a graph showing the antibacterial activity of HDH-LGBP-A1, which is a peptide variant synthesized according to an exemplary embodiment of the present invention. Microbial growth is expressed as a percentage of the maximum absorbance (OD) observed in the absence of peptide. Data were obtained from three independent experiments and are expressed as mean ± standard deviation (SD) values. 4 is a graph showing the antibacterial activity of HDH-LGBP-A2, which is a peptide variant synthesized according to an exemplary embodiment of the present invention. Microbial growth is expressed as a percentage of the maximum absorbance (OD) observed in the absence of peptide. Data were obtained from three independent experiments and are expressed as mean ± standard deviation (SD) values. 2 is a photograph showing antibacterial activity after heat treatment of a peptide variant synthesized according to an exemplary embodiment of the present invention. In the figure, N indicates the result of the radiation diffusion analysis of the non-heat-treated peptide, and H indicates the result of the radiation diffusion analysis of the peptide heat-treated at 100 ° C. for 10 minutes. 3 is a photograph showing antibacterial activity according to salt concentration of a peptide synthesized according to an exemplary embodiment of the present invention. Cell morphology after treatment of HDH-LGBP-A1, which is a peptide variant synthesized according to an exemplary embodiment of the present invention, at different concentrations with respect to the HeLa cell line and the A549 cell line, was obtained using a phase contrast microscope. It is a photograph taken by. In the figure, the upper side is the analysis result for the HeLa cell line, and the lower side is the analysis result for the A549 cell line. Using a phase contrast microscope, cell morphology after treatment of HDH-LGBP-A2, which is a peptide variant synthesized according to an exemplary embodiment of the present invention, with HeLa and A549 cell lines at different concentrations. It is a photograph taken by. In the figure, the upper side is the analysis result for the HeLa cell line, and the lower side is the analysis result for the A549 cell line. After treatment of the peptide variant HDH-LGBP-A1 synthesized according to the exemplary embodiment of the present invention with HeLa cell line, A549 cell line and normal cell line at 37 ° C. for 24 hours, each cell line It is the graph which measured the viability of. After treatment of the peptide variant HDH-LGBP-A2 synthesized according to an exemplary embodiment of the present invention with HeLa cell line, A549 cell line and normal cell line at 37 ° C. for 24 hours, each cell line It is the graph which measured the viability of. It is a graph which shows the FACS analysis result which measured the structure maintenance of the cell membrane and the exposure degree of phosphatidylserine in the HeLa cell processed with acetic acid for 24 hours and dye | stained with Annexin-V-FITC / PI. Cell membrane structure in HeLa cells treated with HDH-LGBP-A1 (concentration 1 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A1 (concentration 5 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A1 (concentration 10 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A1 (concentration 20 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. It is a graph which shows the FACS analysis result which measured the structure maintenance of the cell membrane and the exposure degree of phosphatidylserine in the HeLa cell processed with acetic acid for 24 hours and dye | stained with Annexin-V-FITC / PI. Cell membrane structure in HeLa cells treated with HDH-LGBP-A2 (concentration 1 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A2 (concentration 5 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A2 (concentration 10 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. Cell membrane structure in HeLa cells treated with HDH-LGBP-A2 (concentration 20 μg / ml), a peptide variant synthesized according to an exemplary embodiment of the present invention, and stained with Annexin-V-FITC / PI It is a graph which shows the FACS analysis result which measured the exposure degree of maintenance and phosphatidylserine. 6 is a photograph showing the results of a gel retardation assay for DNA of peptide variants synthesized according to an exemplary embodiment of the present invention. Using an agarose gel, the degree of delay of DNA (50 ng) cleaved with λ-Hind III, a commercial molecular weight marker, was measured, and the binding of the peptide to the DNA was measured. 3 is a photograph showing a result of inhibition analysis of a DNA polymerase of a peptide variant synthesized according to an exemplary embodiment of the present invention. The effect of peptides on DNA polymerase is E. coli 16S rDNA was tested using amplified PCR.

<Definition>
As used herein, the term “amino acid” is used in the broadest sense and is intended to include naturally occurring L-amino acids or residues. Commonly used 1- and 3-letter abbreviations for naturally occurring amino acids are used herein (ref. [Lehninger, Biochemistry, 2d ed., Pp. 71-92, (Worth Publishers: New York, 1975]). Amino acids are not only D-amino acids but also chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not normally contaminated with proteins such as norleucine and amino acids known in the art, For example, phenylalanine or proline analogs or mimetics that allow for the restriction of the conformation of the same peptide compound as natural phenylalanine (Phe) or proline (Pro) within the definition of amino acids. Such analogs And mimetics are referred to herein as “functional equivalents” of amino acids Other examples of amino acids are described in the literature [Roberts and Velaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol.5, p.341 (Academic Press, Inc .: NY 1983)].

For example, synthetic peptides synthesized by standard solid phase synthesis techniques are not limited to amino acids encoded by genes, thereby allowing a wider variety of substitutions for the amino acids provided. Amino acids not encoded by the genetic code are referred to herein as “amino acid analogs” and are described, for example, in WO 90/01940. For example, amino acid analogs include 2-aminoadipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-Aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-Aminobutyric acid (Aib) for Gly; Cyclohexylalanine (Cha) for Val, Leu and Ile; Homoarginine (Har) for Arg and Lys 2,3-diaminopropionic acid (Dap) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylasparagine (EtAsn) for Asn and Gln; (Hyl); allohydroxylysine (AHyl) for Lys; 3- (and 4-) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; alloisoleucine (AIle) for Ile, Leu and Val; 4 for Arg -N-methylglycine for Gly, Pro and Ala (MeGly, sarcosine); N-methylisoleucine for Ile (MeIle); norvaline (Nva) for Met and other aliphatic amino acids; Met and other aliphatic amino acids Norleucine (Nle) for ornithine (Orn) for Lys, Arg and His; citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; and for Phe N- methylphenylalanine (MePhe), trimethyl phenylalanine, halo - (F-, Cl-, Br or I-) contains a phenylalanine or trifluoryl phenylalanine.

As used herein, the term “peptide” includes all proteins, protein fragments, and peptides separated from naturally occurring or synthesized by recombination techniques or chemically. In certain embodiments, compound variants are provided, eg, peptide variants having one or more amino acid substitutions. As used herein, “peptide variants” refers to substitutions, deletions, additions and / or insertions of one or more amino acids in the peptide amino acid sequence (substitutions). insertions) and exhibiting substantially the same biological function as a peptide composed of amino acids of the main body. The peptide variant must have an identity of 70% or more, preferably 90% or more, more preferably 95% or more with the original peptide. Such substitutions can include amino acid substitutions known as “conservative”. Variants can also include nonconservative changes. In one exemplary embodiment, the variant polypeptide sequence differs from the original sequence by substitution, deletion, addition or insertion of 5 or fewer amino acids. Variants can also be altered by deletion or addition of amino acids that have minimal impact on the peptide's immunogenicity, secondary structure and hydropathic nature.

“Conservative” substitution means that there is no significant change in properties such as the secondary structure and hydrotherapeutic properties of a polypeptide when one amino acid is replaced with another. With respect to such conservative substitutions, amino acid mutations are relative similarity of amino acid side chain substituents, such as polarity, charge, solubility, hydrophobity, and hydrophilicity. ) And / or on the basis of similarities such as ambipathic nature.

For example, amino acids have common side chain properties, i) hydrophobic (leucine, methionine, alanine, valine, isoleucine), ii) neutral hydrophilicity (cysteine, serine, threonine, asparagine, glutamine), iii) acidic ( Aspartic acid, glutamic acid), iv) basic (histidine, lysine, arginine), v) residues that affect the chain direction (glycine, proline), vi) aromatics (tryptophan, tyrosine, phenylalanine) it can. Conservative substitutions involve replacing one member of each of these classes with another member of the same class.

Arginine, lysine, and histidine are all positively charged residues, alanine, glycine, and serine have similar sizes, and phenylalanine, tryptophan, and tyrosine, as analyzed for the size, shape, and type of amino acid side chain substituents Can be seen to have similar shapes. Thus, based on this point, arginine, lysine and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing mutations, the hydropathic index of amino acids can be taken into account. Each amino acid is given a hydrophobicity index by hydrophobicity and charge. Isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine, respectively. (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9) ), Tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5) , Asparagine (−3.5), lysine (−3.9) and arginine (−4.5). The hydrophobic amino acid index is very important in conferring the mutual biological function of proteins. It is a known fact that in order to retain similar biological activity, it must be substituted with an amino acid having a similar hydrophobicity index. When a mutation is introduced with reference to the hydrophobicity index, amino acids having a difference in hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5 are substituted.

On the other hand, it is also known that substitution between amino acids with similar hydrophilicity values results in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are given to each amino acid residue. Arginine (+3.0), lysine (+3.0), aspartic acid (+ 3.0 ± 1), glutamic acid (+ 3.0 ± 1), serine (+0.3), asparagine (+0.2), glutamine (+0 .2), glycine (0), threonine (−0.4), proline (−0.5 ± 1), alanine (−0.5), histidine (−0.5), cysteine (−1.0) , Methionine (-1.3), valine (-1.5), lysine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), tryptophan (-3.4). When a mutation is introduced with reference to a new aqueous index, the amino acids are substituted with amino acids having a new aqueous index difference within ± 2, preferably within ± 1, and more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the overall activity of the molecule is known in the art (H. Neuroth, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly performed exchanges are the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Exchange among Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

In the present invention, “polynucleotide” or “nucleic acid” is used interchangeably and refers to a polymer of nucleotides of any length, and encompasses DNA (eg, cDNA) and RNA molecules. “Nucleotides”, which are the building blocks of nucleic acid molecules, can be incorporated into polymers by deoxyribonucleotides, ribonucleotides, modified nucleotides or nucleotides, and / or their analogs, DNA or RNA polymerase, or synthesized by reaction. It can be any temperament that can be mixed in. Polynucleotides can include modified nucleotides, sugars or analogs with modified nucleotides, such as methylated nucleotides and analogs thereof (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

Some mutations in nucleotides do not allow mutations in proteins. Such nucleic acids include nucleic acid molecules that include functionally equivalent codons or codons that encode the same amino acid, or codons that code for biologically equivalent amino acids. In addition, mutations in nucleotides can change the protein itself. Even in the case of a mutation that causes a change in the amino acid of a protein, a protein that exhibits substantially the same activity as the protein of the present invention can be obtained.

The peptide or nucleic acid molecule of the present invention is an amino acid sequence or nucleotide sequence described in the sequence catalog to the extent that it has the effect of treating the peptide of the present invention or the nucleic acid encoding the peptide, for example, antibacterial and / or cancer. It is obvious to ordinary engineers that the present invention is not limited to the above. For example, the biological functional equivalent that can be contained in the peptide of the present invention can be a peptide having a mutation of an amino acid sequence that exhibits biological activity equivalent to that of the peptide of the present invention.

In general, the peptides (including fusion proteins) and polynucleotides referred to herein are those that have been isolated. An “isolated” peptide or polynucleotide is one that has been removed from its original environment. For example, proteins present in the natural state are separated by removing all or part of the substances present together in the state. Such a polypeptide must have a purity of at least 90%, preferably 95%, more preferably 99% or more. Polynucleotides are isolated by cloning in a vector.

The term “vector” as used herein refers to a structure that can be transferred to a host cell, and preferably made to express one or more genes of interest or nucleic acid sequences. For example, vectors were ligated with viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, CCA (cationic condensing agents). Examples include DNA or RNA expression vectors, DNA or RNA expression vectors packaged in liposomes, and specific eukaryotic cells such as producer cells.

As used herein, “expression control sequence” means a nucleic acid sequence that regulates transcription of a nucleic acid. Expression control sequences include promoters such as structural promoters or inducible promoters, enhancers, and the like. The expression control sequence is linked to the nucleic acid sequence to be transcribed. As used herein, “operably linked” refers to a functional linkage between a nucleic acid regulatory sequence (eg, a promoter, signal sequence, or transcription regulatory factor binding site array) and another nucleic acid sequence. means. The regulatory sequence thereby regulates the transcription and / or translation of the other nucleic acid sequence.

As used herein, “pharmaceutically effective amount” or “therapeutically effective amount” means an amount sufficient to achieve the efficacy or activity of the active ingredient peptide or fragment thereof and / or the nucleic acid encoding them. means. For example, the pharmaceutical composition containing the peptide according to the present invention can be used as an active ingredient of an antibacterial agent, antibiotic agent and / or anticancer agent.

<Peptides, nucleic acids, vectors>
The mechanism of action of antimicrobial peptides can be broadly divided into two. One is that most antimicrobial peptides increase the permeability of fungal cell membranes to disrupt membrane potential and stop cell metabolism. Another is that a small number of other antimicrobial peptides penetrate into the strain cells, bind to DNA or RNA, and exhibit a potent mechanism of action that suppresses transcription or translation. The following structural elements are known to be important for the activity of these antimicrobial peptides. First, amphipathic helix structure, second, distribution of residues that stabilize the helix structure, third, distribution of basic residues, fourth, distribution of hydrophobic residues, 5 is an interaction between a charged residue and a dipole having a helix structure, and sixth is a salt bridge between residues having an opposite charge.

In order to synthesize a peptide having an antibacterial activity and / or an anticancer activity as a physiological activity, the present inventors synthesized a peptide variant derived from Ezo abalone (Haliotis discus hannai) and confirmed its activity. Analyzes and expresses the full-length cDNA sequence encoding lipid polysaccharides and glucan-binding proteins (LGBP) as a pattern recognition protein that recognizes pathogen-related molecular patterns (PAMPs) from the gene of Ezo Abalone The amino acid sequence of the full-length protein to be confirmed was confirmed. In the present invention, a peptide variant in which a partial amino acid sequence of a short peptide constituting a motif or domain region recognizing a pathogen is replaced with antibacterial activity among the amino acid sequence of LGBP full-length protein expressed from a full-length cDNA derived from Ezo abalone And / or based on having anti-cancer activity.

Therefore, according to one aspect of the present invention, the present invention relates to a peptide as a mutant having physiological activity. According to one aspect of the present invention, a peptide as a variant can be represented by an amino acid sequence of the following formula (1).

[Formula 2]
(N-terminal) -WLWKAIWKLLL- (C-terminal) (1)

  In the formula (1), X is a basic amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H), or serine (S), threonine (T), asparagus. A polar amino acid selected from the group consisting of gin (N) and glutamine (Q).

In one exemplary embodiment, in the amino acid of formula (1), X constituting the C-terminus may be lysine or threonine, in which case the peptide of formula (1) is SEQ ID NO: 3 or the sequence It consists of the amino acid sequence of No. 5. If necessary, some of the amino acids of the peptide of the present invention can be modified by phosphorylation or the like, and the carboxyl group located at the C-terminus can be amidated. In one exemplary embodiment, the peptides of the invention exhibit potent antibacterial activity against various bacteria or yeasts, eg against various tumor cell lines that induce uterine cancer, cervical cancer and lung cancer. The activity was confirmed to have anticancer activity.

For example, the peptide represented by SEQ ID NO: 3 or SEQ ID NO: 5 synthesized according to an exemplary embodiment of the present invention exhibits antibacterial activity against various Gram-positive and negative strains and yeast (FIG. 3A). And see FIG. 3B). For example, the peptides of the present invention include Bacillus cereus, Staphylococcus aureus RM4220 Streptococcus iniae FP5229 and S. cerevisiae. Peptidomonas aeruginosa KCTC2004, Vibrio angulararum, Vibrio harveyi, Gram-negative bacteria including mutans, yeast Candida albicans KCTC7965, etc. It is not limited to having antibacterial activity. In particular, the activity of the peptide synthesized according to the present invention is not reduced even by heat treatment at high temperature and salt treatment at a high concentration (see FIGS. 4 and 5).

Peptides according to the present invention can be isolated by manufacturing through recombinant means or chemical synthesis. For example, a peptide expressed by the nucleic acid sequence mentioned in the present specification can be easily produced by a known method using any of many known expression vectors. Expression can be achieved in a suitable host cell transformed into an expression vector containing a DNA sequence encoding the peptide. Suitable host cells include prokaryotic cells, yeast and eukaryotic cells. It is preferable to use E. coli, yeast or mammalian cell lines (such as Cos or CHO) as host cells. For protein purification, first, the supernatant containing the recombined protein secreted into the culture medium, obtained with a water-soluble host / vector system, is concentrated using a commercially available filter. Next, the concentrated solution obtained above is purified using an appropriate purification matrix such as an affinity matrix or an ion exchange resin. Finally, pure protein can be obtained by performing one-step or multi-step reverse phase HPLC.

Fragments and mutants composed of 100 amino acids or less, and generally 50 amino acids or less, can be produced by synthesis. For example, such polypeptides can be synthesized by conventional solid-phase techniques, ie, Merrifield solid-phase synthesis method in which amino acids are sequentially added to growing amino acid chains. (Merifield, 1963, J. Am. Chem. Soc. 85: 2146-2149). Equipment for automated synthesis of polypeptides can be purchased from a supplier and can be operated according to the supplier's manual.

For example, peptides described herein that are expressed from microorganisms can be secreted into and recovered from the periplasm of the host cell. In general, protein recovery involves grinding the microorganisms by means such as osmotic shock, sonication or lysis. Once the cells are destroyed, cell debris or pre-cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. As an alternative, the protein can be transferred to a culture medium and separated therefrom. Cells can be removed from the culture, the culture supernatant can be filtered and concentrated, and the protein produced can be further purified. Expressed polypeptides are usually obtained by known methods such as fractional distillation on immunoaffinity or ion exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation exchange resins such as DEAE; Focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using eg Sephadex G-75; addition using hydrophobic affinity resin, ligand affinity and Western blot assay using compatible antigen immobilized on matrix Can be separated and confirmed.

The system, the peptide produced, can be purified to obtain additional detection and a substantially homogeneous formulation. Standard protein purification methods known in the art can be used. For example, fractional distillation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and Gel filtration using Dex G-75.

According to another aspect of the present invention, the present invention relates to a nucleic acid encoding the peptide represented by the formula (1) described above. In one exemplary embodiment, a nucleic acid encoding a peptide according to the present invention comprises a nucleotide composed of the base sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

Nucleic acids that code for the peptide of formula (1) according to the present invention (eg, nucleotides of SEQ ID NO: 4 or SEQ ID NO: 6) may be included in a suitable vector. The vector can also include an expression control sequence linked to the polynucleotide of the present invention. Each vector contains various components depending on its function (either amplification or expression of heterologous polynucleotide, or both) and compatibility with the particular host cell in which the vector is present. In general, vector components include, but are not limited to, origins of replication (especially when the vector is inserted into prokaryotic cells), selectable marker genes, promoters, ribosome binding sites (RBS), signal sequences, heterologous nucleic acid inserts and transcription termination sequences. Is not to be done. For example, the recombination vectors of the present invention include expression control sequences that can affect protein expression, such as initiation codons, termination codons, polyadenylation signals, enhancers, membrane targeting, or signal sequences for secretion, etc. Can do. One type of vector is a “plasmid” that refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vector system of the present invention can be constructed by various methods known in the art, and specific methods for this can be found in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

Various in vitro amplification techniques that amplify sequences subcloned into expression vectors are known. Such techniques include techniques using PCR (polymerase chain reaction), LCR (ligase chain reaction), Qβ-replication enzyme amplification, and other RNA polymerases (Sambrook et al., 1989, Molecular Cloning). A Laboratory Manual (2nd Ed) 1-3; U.S. Patent No. 4,683,202; PCR protocols A Guide to Methods and Applications, Innis et al., Eds. Academic Press Ind. Academic Press. Improved methods of clone ing in vitro amplified nucleic acids are describable in US Patent No. 5,426,039).

The vectors of the present invention can also be fused with other sequences to facilitate the purification of the expressed peptide. Examples of the sequence to be fused include glutathione-S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA), and the most preferable. Is 6x His. Because of the additional sequence for purification, the peptide expressed in the host can be quickly and easily purified through affinity chromatography. If necessary, sequences encoding Fc sections can be fused to facilitate extracellular secretion of these peptides.

According to an exemplary embodiment of the invention, a peptide expressed by a vector comprising a nucleotide sequence encoding a peptide of formula (1) is purified through affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate for this enzyme, can be used. When 6x His is used, Ni-NTA His-conjugated resin column (Novagen, USA) can be used to obtain the desired peptide quickly and easily. Host cells capable of stably and continuously cloning and expressing the above-mentioned vectors are known in the art, and any host cell can be used.

<Composition>
The peptide synthesized according to the present invention exhibits antibacterial activity against various bacterial strains and yeast cell strains (see FIGS. 3A to 3B), and the antibacterial activity does not decrease even when heated at high temperatures (FIG. 4). The activity is not lost even when a high concentration of salt is added (see FIG. 5). In addition, the peptides of the present invention are not toxic to normal cells, but are selectively toxic to cancer cell lines (see FIGS. 6C to 6D) and prevent apoptosis of cancer cell lines. Induced and showed anti-cancer activity (see FIGS. 7A-7E and 8A-8E).

Therefore, the peptide synthesized according to the present invention can be used as an active ingredient in various compositions that require antibacterial and / or anticancer activity. For example, the peptide of the present invention can be applied to an antibacterial and / or anticancer pharmaceutical composition, or can be applied to an active ingredient of a cosmetic composition or food additive that requires antibacterial properties.

In one exemplary embodiment, the present invention provides an antibacterial or antimicrobial agent comprising a pharmaceutically effective amount of a peptide having the amino acid sequence represented by the aforementioned formula (1), and optionally a pharmaceutically acceptable carrier. The present invention relates to a pharmaceutical composition for cancer. For example, the effective volume of the active ingredient is 0.01 to 1000 μg / ml, preferably 0.1 to 500 μg / ml, more preferably 0.1 to 100 μg / ml, and the active ingredient is once a day. It can be administered three times.

According to the present invention, the peptide used as an active ingredient of the pharmaceutical composition can be obtained by any suitable means such as oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, It can be administered by means for intraperitoneal, intrapulmonary, intradermal, intradural and epidural, intranasal and, if desired, local treatment, intralesional administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. The compound which is the active ingredient of the present invention is administered in any convenient dosage form such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. be able to. Such compositions may contain ingredients commonly used in pharmaceuticals such as diluents, carriers, pH adjusters, sweeteners, fillers and additional active agents.

For example, the peptide that is the active ingredient of the pharmaceutical composition according to the present invention can be administered in various parenteral dosage forms, but when formulated, it is usually used as a filler, filler, binding agent. It can be prepared using diluents or excipients such as an agent, a wetting agent, a disintegrant, and a surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

If necessary, the peptide of the present invention can be used in admixture with various pharmaceutically acceptable carriers such as physiological saline or organic solvent, and in order to increase stability and absorbability, glucose, sucrose Alternatively, it can be used with carbohydrates such as dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers.

On the other hand, the present invention relates to an antibacterial cosmetic composition comprising a peptide having the amino acid sequence represented by formula (1) as an active ingredient. For example, the peptides of the present invention can be used as preservatives in cosmetic compositions. The cosmetic composition comprises a dermatologically acceptable medium, ie a medium that is compatible with the skin, mucous membranes, hair and scalp. This is all pharmaceutical dosage forms suitable for topical use, in particular aqueous, aqueous / alcoholic or oily solutions, or aquatic, aqueous / alcoholic or oily gels, or solid or kneaded anhydrous products Or emulsion (O / W) obtained by dispersing oil phase in water phase or vice versa (W / O), or suspension, microemulsion, microcapsule, fine granulocyte, ionic type (liposome) And / or can be provided in the form of a non-ionic sac dispersion. For example, as a solvent for producing a cosmetic composition, ethanol, glycerin, butylene glycol, propylene glycol, glyceres-26, methyl glutes-20, isocetyl myristate, isocetyl octanoate, octyldodecyl myristate, octyldodecanol, One or more selected from the group consisting of stearyl isostearate, cetyl octoate and neopentyl glycol dicaprate can be used.

In the cosmetic of the present invention, if necessary, another component that is usually blended in cosmetics can be blended together with the above components. The ingredients that can be added include fat and oil ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, colorants, organic powders, UV absorbers, antiseptics, bactericides, antioxidants, antioxidants, Examples include plant extracts, pH adjusters, alcohols, foaming agents, fillers, gelling agents or thickeners, fragrances, cooling agents, blood circulation promoters, antiperspirants, and purified water.

The present invention also relates to an antibacterial food additive containing, as an active ingredient, a peptide having the amino acid sequence represented by the above formula (1). When using the peptide of this invention for a food additive, the said peptide may be added as it is, may be used with another food ingredient, and may be used appropriately by a normal method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. Generally, the peptide of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material. However, in the case of long-term ingestion, the amount may be an amount below the above range, and there is no problem in terms of stability. Therefore, the active ingredient may be an amount above the above range.

The type of the food is not particularly limited. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, dairy products, and various soups , Drinking water, tea, drinks, alcoholic beverages and vitamin complexes, etc., including all foods of normal meaning.

EXAMPLES Hereinafter, although this invention is demonstrated in detail through an Example, this invention is not limited to the invention described in the following Example.

<Example 1: Sequence analysis of full length cDNA and full length peptide of abalone LGBP (lipopolysaccharides and glucan binding proteins)>
A cDNA library was constructed from seven tissues obtained from third grade Ezo abalone and the expressed sequence tags were analyzed. Using a clone with high similarity to non-complementary open reading frame (ORF) and black abalone that are expressed sequencing tags of Ezo Abalone, lipid polysaccharide and β-1,3-glucan binding protein derived from Ezo Abalone (HDH-LGBP) was isolated. One EST (expressed sequence tag) clone (DGT-151) obtained from a digestive tract cDNA library is homologous to LGBP of other species, and a 632-bp nucleic acid sequence was obtained from this clone.

To obtain the full length cDNA of the HDH-LGBP gene, use the SMART RACE cDNA amplification kit (BD Biosciences) according to the manufacturer's instructions, and 5'- and 3'-random amplification of cDNA ends (RACE, random amplification of cDNA ends). Gastrointestinal cDNA for each was synthesized. Based on the partial base sequence of DGT-150 clone, we designed gene-specific primers for 5'-RACE and 3'-RACE, adopted the RACE method using gene-specific primers, and changed the amino-terminal coding sequence. Obtained. A 380 bp fragment was amplified by 5'-RACE, and as a result of sequence analysis, a full-length cDNA sequence was synthesized that overlapped with the EST sequence and encoded LG-BP (HDH-LGBP) derived from Ezo abalone. The amplified fragment was subcloned into the pGEM-T Easy vector (Promega), and the sequence was analyzed using an ABI3103 automated DNA sequence analyzer (Applied Biosystems). To complete the full HDH-LGBP cDNA sequence, 5′-terminal, 3′-terminal and DGT-151 partial sequences were combined and aligned using GENETYX version 8.0 (SDC Software Development, Tokyo, Japan).

Subsequently, the sequence was analyzed using a computer program. The amino acid sequence was estimated from the obtained full-length cDNA, and the molecular weight and isoelectric point were calculated using GENETYX version 8.0 (SDC Software Development, Tokyo, Japan). In addition, the BLASTP program was used in NCBI (http://www.ncbi.nim.nih.gov/blast/) to analyze the sequence similarity with other sequences.

The presence of the signal peptide was predicted by SignalP3.0, and the domain search was performed by CD-search and Pfam sequence search in NCBI.

The full length cDNA sequence of HDH-LGBP and the amino acid sequence expressed therefrom are shown in FIG. The entire sequence of HDH-LGBP cDNA is 31-bp 5'-untranslated region (5'-UTR), 162 bp 3'-UTR with poly (A) tail, expected molecular weight 47.8 kDa, It consisted of 1263 bp open reading frame (ORF) coding for a polypeptide composed of 420 amino acids with a target isoelectric point of 5.27. The resulting HDH-LGBP full-length gene contains a putative signal sequence of the first 20 amino acid residues. Therefore, mature HDH-LGBP is composed of 400 amino acids, the measured molecular weight of the protein portion is 45467 Da, and the expected isoelectric point is 4.93.

SMART analysis revealed that the amino acid region from residues 184 to 321 of the immature peptide belongs to the glycoside hydrolase family. Five putative glycation sites (Asn-Xaa-Ser / Thr, NXS / T) associated with N-linked carbohydrate chains in the mature protein sequence are Asn-28, -99, -265 relative to the mature peptide. , -310, -350. Since the N-linked glycation site of HDH-LGBP is located near the β-glucan recognition motif, glycation at this site affects the binding ability to β-glucan. One short putative cell attachment site and integrin binding site, Arg / Lys-Gly-Asp (R / KGD), is present in the mature peptide sequence from Lys-189 to Asp-191. HDH-LGBP has a β-1,3-glucanase site with active residues of mature peptides Trp-209, Glu-214, Ile-215 and Asp-216.

<Example 2: Design, structure prediction and synthesis of peptide>
(1) Design and physiochemical characteristics of antibacterial active peptide Based on the amino acid sequence corresponding to the polysaccharide-binding domain of HDH-LGBP, the natural peptide expected to have antibacterial activity is compared with the natural peptide. Peptide variants were designed in which some amino acids were substituted differently. Peptide variants were designed based on the amino acid sequence located in the polysaccharide binding motif of HDH-LGBP. A natural peptide (HDH-LGBP-N) having the amino acid sequence of SEQ ID NO: 1 (192 to 202 amino acid residues in the mature full-length peptide of FIG. 1) and a part of the natural peptide of SEQ ID NO: 3 It was expected that the peptide variant having the amino acid sequence (HDH-LGBP-A1) and the peptide variant having the amino acid sequence of SEQ ID NO: 4 (HDH-LGBP-A2) had antibacterial activity.

The pI value, net charge value, Boman Index value and secondary structure affecting the molecular weight and peptide activity of natural peptides and peptide variants were evaluated. The theoretical isoelectric point (p.I.) and net charge were evaluated with the ExPAYs server. Boman Index values were calculated using the online Antimicrobial Peptide Database v2.34 (ADP2).

P. Of these peptides. I. Values, net charge values, hydrophobicity, Boman Index values, etc. are shown in Table 1 below. Factors such as hydrophobicity, net charge, protein binding potential, and Boman index can affect peptide activity. Boman index evaluates the ability of a peptide to bind to another protein, such as another receptor, and is the sum of the free energy of the side chains of amino acid residues divided by the total number of amino acid residues. Is defined. A natural peptide with abundant W and P has an acidic PI value of 5.52, a net charge value of 0, and a low Boman index value. In addition, the two selected peptide variants are expected to have a relatively high net charge value and a low Boman index value and have antibacterial activity.

The secondary structure of the peptide was predicted using the GOR method [17ExPASy]. Schiffer Edmundson's helical wheel projection was used to predict the hydrophobic region, hydrophilic region, and α-helix structure in the secondary structure of two artificially deformed peptide variants. The helical wheel diagram used EMBOSS pepwheel (European Bioinformatics Institute Cambridge, UK) to predict the secondary structure. The results are shown in FIG. 2 and the secondary structure is shown in Table 1.

In Table ^{1, *} denotes a C- terminal ^{amidation, **} represents a ^{β-turn, ***} indicates a random ^{coil, ****} denotes the α helix.

(2) Peptide mutant synthetic natural peptide (HDH-LGBP-N) whose antibacterial activity has been confirmed, and two peptide mutants (HDH-LGBP-A1, HDH-LGBP) in which some amino acids of this natural peptide are substituted -A2), and these peptides were purified to Peptron Inc. with a purity of 95% or more. (South Korea, Daejeon) and synthesized commercially. These peptides were synthesized using FmoC solid phase peptide synthesis (SPPS) using ASP48S (Peptron Inc., Korea, Daejeon), and Vydac Everest C18 column (250 mm × 22 mm, 10 μm; Grace, Deerfield, IL, USA) And purified using high performance liquid chromatography (HPLC). Elution was performed using a water / acetonitrile linear gradient (acetonitrile 3-40% (v / v)) containing 0.1% (v / v) trifluoroacetic acid. The molecular weight of the purified peptide was confirmed using a liquid chromatography / mass spectrometer (LC / MS; HP100 series; Agilent, Santa Clara, CA, USA). All the synthesized peptides were dissolved in 0.01% acetic acid to obtain a stock solution of 1000 μg / ml.

Example 3: Highly Sensitive Radiation Diffusion Assay for Antibacterial Performance>
The antimicrobial activity of the synthesized peptide variants described above was measured through a highly sensitive radiation diffusion assay (URDA). Bacillus cereus, Staphylococcus aureus RM4220, Streptococcus inae FP5229, and S. cerevisiae. Synthetic peptide antibacterial properties tested against Gram-positive bacteria including mutans, Gram-negative bacteria including Pseudomonas aeruginosa KCTC2004, Vibrio angulararum, Vibrio harveyi, and Candida albicans KCTC7965 yeast. The strains to be tested were grown in brain heart and exudate medium (BHI, BD Bioscience, USA) at the appropriate temperature (25 ° C for P. aeruginosa and S. iniae, 37 ° C for others). C. a yeast strain albicans KCTC7965 was grown in yeast medium (YM) at 25 ° C. After incubating for 16 to 18 hours, the suspension of the fungus and yeast was added to 10 ⁸ CFU / ml, C.I. Dilute to 0.5 McFarland turbidity standard (McFarland turbidity standard of 0.5, Vitek Colorimeter # 52-1210; Hach, Loveland, CO, USA) corresponding to 10 ⁶ CFU / ml for albicans. Diluted bacteria or C.I. 1/2 ml of an albicans suspension was added to a 10 mM phosphate buffer with 0.03% TSB (Tryptic Soy Broth) or 0.03% SDB (Soybean powder Dextrose Broth) and 1% Type I (low EEO) agarose. The solution was added to 9.5 ml of an underlay gel containing 5 × 10 ⁶ CFU / ml or 5 × 10 ⁴ CFU / ml dissolved in (PB; pH 6.6). Two purified peptides were serially diluted with 5 μl of acidified water (0.01% HAc), and each dilution was added to a 2.5 mm diameter well made of 1 mm thick underlay gel. P. aeruginosa, S. et al. iniae, C.I. After incubation at 25 ° C. for albicans and 37 ° C. for other strains for 3 hours, respectively, 6% BHI or 6% using 10 mM phosphate buffered saline (PBS, pH 6.6) The bacterial or yeast suspension was covered with 1% agarose with 10 ml of double strength overlay gel containing% YM. After incubating the plate for an additional 18-24 hours, the diameter of the clear site was measured. Except for the diameter of the well, the diameter of the transparent part was expressed in units (0.1 mm = 1 U).

URDA was used to measure the minimum effective concentration (MECs) for gram positive bacteria, gram negative bacteria and yeast, and the antibacterial activity against the synthesized HDH-LGBP peptide variants. The minimum effective concentration of the synthetic peptide (MEC, μg / ml) was calculated as the x-intercept of the unit diagram against the log 10 value of peptide concentration. This antibacterial analysis was performed three times, and the average of the results was obtained. The experimental results are shown in Table 2 and FIGS. 3A and 3B, respectively. The HDH-LGBP peptide variant is particularly cereus, S. et al. aureus, S .; mutans and S.M. Gram positive bacteria (MECs 0.008 to 1.92 μg / ml) such as Excellent antibacterial activity against Gram-negative bacteria (MECs 1.92-2.12 μg / ml) such as aeruginosa. Moreover, these peptide variants are C.I. It also shows potential antibacterial activity against albicans yeast (MECs 2.11 to 2.16 μg / ml). Based on these results, it was confirmed that HDH-LGBP peptide derivatives have a wide range of antibacterial activity.

<Example 4: Effect of temperature and salt on antibacterial activity>
To study the effect of temperature and salt on the antibacterial activity of peptide variants (HDH-LGBP-A1, HDH-LGBP-A2) cereus, S. et al. aureus, S .; iniae, P.A. aeruginosa and yeast C.I. albicans was tested for antibacterial activity using a sensitive radiation diffusion assay (URDA). In order to explore thermostability, each of the two peptide variants was incubated at 100 ° C. for 10 minutes. After heat treatment, the peptide was allowed to cool and used for URDA as described in Example 3. The analysis results for thermal stability are shown in FIG. The antibacterial activity of these peptides is not seriously affected by heat treatment. In particular, the two peptide variants are S. cerevisiae. aureus, P.A. aeruginosa and C.I. It shows strong antibacterial activity against microbial strains such as albicans. Thermal stability analysis confirmed that the HDH-LGBP peptide variant is stable to heat.

On the other hand, in order to confirm the stability against salt, the two peptide variants were incubated with different concentrations of salt (0.5%, 1%, 2%) for 30 minutes. Specifically, in order to study the sensitivity of these peptide variants to salts, HDH-LGBP-derived peptide variants were incubated at different concentrations of sodium (NaCl, 0.5%, 1%, 2%). Antibacterial activity was evaluated and compared using URDA. After treatment, the peptides were used for URDA as described above. The analysis results are shown in FIG. The antimicrobial activity of these peptide variants was not significantly affected by the high concentration. It was confirmed that the peptide variant derived from HDH-LGBP has high salt resistance.

<Example 5: Cytotoxic effect of peptide variant>
Based on phase-contrast microscopy and mitochondrial dehydrogenase activity against two human cancer cell lines (HeLa and A549) and normal cell lines (human umbilical vein endothelial cells, Human Umbilical Vein Endothelial Cells, HUVEC) The toxic effects of the synthesized HDH-LGBP peptide variants were studied using MTS analysis to measure living cells. HeLa (human cervical adenocarcinoma) and A549 (human lung carcinoma) cell lines were purchased from the US Microbiology Conservation Center (ATCC; Rockville MD, USA). The HUVEC cell line was provided by Dr. John (National Busan University, Department of Molecular Biology, Busan, Korea). All cells in a 37 ° C. incubator with 5% CO 2, DMEM (Dulbecco's) containing 10% fetal bovine serum (FBS, Gibco) and 100 U / ml antibiotic / antimycotics (Life Technologies) (Modified Eagle's medium, Welgene) medium.

Cultured HeLa and A549 cells (4 × 10 3 cells / well) were cultured overnight at 37 ° C. in 96-well plates. Subsequently, the cells were treated with various concentrations (1, 5, 10, 25, 50 μg / ml) of HDH-LGBP-A1 and HDH-LGBP-A2 peptide variants as antimicrobial peptides (AMPs) for 24 hours. Incubation was performed at 37 ° C. Cell morphology by treatment of peptide variants was analyzed with a phase contrast microscope. The analysis results are shown in FIGS. 6A and 6B, respectively. Untreated control cells showed a normal monolayer shape (left panel in FIGS. 6A and 6B) and were not significantly affected by peptide treatment at a concentration of 1-5 μg / ml. However, when HDH-LGBP-derived peptide variants were treated at a concentration of 10 μg / ml, the number of cells decreased, round-shaped cells increased, and cell contraction began to increase (FIGS. 6A and 6B). In particular, when treated with 50 μg / ml peptide variant, cell separation, swelling and damage were detected in HeLa and A549 cells within 5 minutes (results not shown). Such a result indicates that the peptide variant derived from HDH-LGBP can directly disrupt the cell membrane and lyse cells at a constant concentration, for example, 50 μg / ml.

Subsequently, according to the manufacturer's instructions, MTS analysis was used to measure the cytotoxicity of each of normal cell lines HUVEC (Human universal vein endothelial cells) and cancer cell lines HeLa and A549 cells. HUVEC, HeLa and A549 cells (4 × 10 3 cells / well) were cultured overnight at 37 ° C. in 96 well plates. Subsequently, the cells were treated with various concentrations (1, 5, 10, 25, 50 μg / ml) of HDH-LGBP-A1 and HDH-LGBP-A2 peptide variants as antimicrobial peptides (AMPs) for 24 hours. Incubation was performed at 37 ° C. Tetrazolium compound, 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium 20 μl of a mixture of (MTS) and phenazine methosulfate (PMS, Promega, Mannheim, Germany) as an electron coupling reagent was added, and the cells were incubated at 37 ° C. for 4 hours. A microtiter plate reader was used to detect absorbance at 490 nm. All data were repeated 3 times for 3 independent experiments. The results were expressed as a percentage of inhibition of viable cells and the 0.01% acetic acid treated group (negative control group) value was excluded from the experimental results.

The HDH-LGBP-A1 peptide decreased cancer cell viability in a concentration-dependent manner. In particular, HDH-LGBP-A1 peptide is toxic to HeLa cells, and when exposed to HDH-LGBP-A1 peptide at concentrations of 10 μg / ml, 25 μg / ml and 50 μg / ml, non-viability in HeLa cells is increased. , 35%, 99% and 95%, respectively. Similarly, 25%, 99% and 97% of A549 cells were damaged when exposed to HDH-LGBP-A1 peptides at concentrations of 10 μg / ml, 25 μg / ml and 50 μg / ml (see FIG. 6C). In the case of HDH-LGBP-A2 peptide, cytotoxicity against HeLa cells was 16% and 99% when exposed to HDH-LGBP-A1 peptide at concentrations of 10 μg / ml, 25 μg / ml and 50 μg / ml, respectively. 98%. Similarly, cytotoxicity against A549 cells was 13%, 99%, and 99%, respectively, when exposed to HDH-LGBP-A2 peptides at concentrations of 10 μg / ml, 25 μg / ml, and 50 μg / ml (FIG. 6D). See).

On the other hand, normal cells (HUVEC) exert cell viability even at a high concentration of 50 μg / ml, and the viability when exposed to HDH-LGBP-A1 and HDH-LGBP-A2 is 32.8%, It was 47.9%. From these results, it was confirmed that the peptide variants synthesized according to the present invention can suppress the growth of uterine carcinoma and lung adenocarcinoma by 90% or more. This means that the peptide variants synthesized according to the invention have a potential anticancer effect.

<Example 6: Analysis of anticancer effect of peptide variant on cancer cell membrane>
In this example, the effect of the HDH-LGBP peptide variant on the cancer cell membrane of HeLa cells was studied using the Annexin V-FITC / PI (propidium iodide) staining method. To evaluate the structure maintenance of the cell membrane and the effect of HDH-LGBP on phosphatidylserine (PS) on the cell surface, HeLa cells were seeded in 35 mm dishes and HDH-LGBP-A1 and HDH-LGBP-A2 at various concentrations for 24 hours. (1-50 μg / ml) and incubated with 0.01% acetic acid as a negative control. After treatment with peptide variants at constant concentrations (1, 5, 10, 20 μg / ml), cells were harvested by digestion with trypsin, washed with cold PBS, binding buffer (0.01 M Hepes / NaOH, pH 7. 4) Resuspended in 0.14 M NaCl and 2.5 mM CaCl2. Subsequently, the cells were stained with FITC-annexin V and PI according to the manufacturer's instructions (FITC-Annexin V Apoptosis Detection Kit, BD Biosciences, USA). Stained cells were mixed gently and flow cytometry was measured with a Beckman Coulter FC500 (Beckman Coulter) and the results were analyzed using CellQuest software (BD Biosciences, USA). Phosphatidylserine (PS) moves from the inner layer of the cell membrane to the outer layer in the early stages of apoptosis. Annexin V, a calcium-dependent phospholipid binding protein, binds PS with high affinity, but this binding is a marker for apoptosis. A disrupted membrane of damaged or dead cells allows PI to pass through, while viable cells with an intact membrane exclude PI. The Q1, Q2, Q3, and Q4 gates represent dead cells, late stages of apoptosis, normal cells, and early stages of apoptosis, respectively.

The analysis results using the HDH-LGBP-A1 peptide variant are shown in FIGS. 7A to 7E, and the analysis results using the HDH-LGBP-A2 peptide variant are shown in FIGS. 8A to 8E, respectively. Viable cells (normal cells) captured in the third quadrant, Q3, decreased when treated with the peptide mutant, but cells captured in the first quadrant (Q2) and the fourth quadrant (Q4) (apoptotic cells). Cells) increased in a concentration-dependent manner when treated with the peptide variant. The survival percentage of cancer cells treated with the HDH-LGBP-A1 peptide variant was 90.5% (control) to 86.13% (1 μg / ml), 73.33% (5 μg / ml), 68.01% The survival percentage of cancer cells reduced to (10 μg / ml), 40.06% (20 μg / ml) and treated with the HDH-LGBP-A2 peptide variant was 86.89% (1 μg / ml), 75.21. % (5 μg / ml), 51.55% (10 μg / ml), 29.76% (20 μg / ml). On the other hand, the rate of damage or loss of the cell membrane structure captured by Q2 or Q4 increased in a concentration-dependent manner.

FIGS. 7A to 7E and FIGS. 8A to 8E show that the fraction of cells undergoing apoptosis was greatly increased in HeLa cells treated with a peptide variant at a concentration of 10 μg / ml. From these results, the peptide variant synthesized according to the present invention induces death of HeLa cells by disrupting the membrane structure (PS exposure) and increasing membrane permeability (PI uptake into the cell interior). Can do. Further, PS is exposed by the membrane disruption effect, and cytoplasmic components can be released to the outside of the cell, thereby inducing cell death.

<Example 7: Inhibition analysis of DNA binding and DNA polymerase>
In order to investigate the interaction between the HDH-LGBP peptide variant and the intracellular molecules DNA and DNA polymerase, the electrophoretic mobility shift assay using the HDH-LGBP peptide variant synthesized according to the present invention was used. (EMSA, electrophoretic mobility shift assay) and DNA polymerase inhibition assay (DNA polymerase inhibition assay). In the test according to this example, the inhibition of the migration rate of the DNA band passing through the agarose gel was examined, and the peptide-DNA binding was evaluated. Various concentrations of peptides (0, 0.157,...) In which DNA (50 ng; Roche, Basel, Switzerland) cleaved with λ-Hind III, a commercial molecular weight marker, was dissolved in 0.01% acetic acid. 313, 0.625, 1.25, 2.5 μg), incubated at room temperature for 5 minutes, and then electrophoresed on a 1.0% agarose gel containing 0.5 μg / ml ethidium bromide (EtBr). did. The analysis result is shown in FIG. 9A. Generally, negatively charged DNA moves to the anode by an electric field, but when DNA is densely packed with a cationic peptide, the DNA gradually moves or does not move in the loading well. Compared with DNA that did not form a graft, the mobility was somewhat suppressed by 2.5 μg of HDH-LGBP-A1.

Subsequently, peptide mutants at various concentrations (2.5, 1.25, 0.625, 0.313 μg / ml) were added to each reaction mixture in order to evaluate the DNA polymerase inhibitory activity of the peptide mutants. did. Use the following primer pair: E. coli genetic DNA was used as a template for PCR (16S-F1, 5'-CTCCTACCGGGAGGCAGCAG-3 ', 16S-R3, 5'-CCAGGGTATCTAATCCTG-3'). The expected amplification product was ˜1.5 kb in length. Each PCR consisted of 30 cycles of 90 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute. After PCR, all amplification products were electrophoresed on a 1.5% agarose gel containing EtBr.

The analysis result is shown in FIG. 9B. The two synthesized peptide analogues all show strong DNA polymerase inhibitory activity. In particular, the HDH-LGBP-A2 peptide exhibits stronger DNA polymerase inhibitory activity than the HDH-LGBP-A1 peptide. At all concentrations tested (0.157-5 μg), the activity of DNA polymerase was completely suppressed by HDH-LGBP-A2. HDH-LGBP-A1 showed weak inhibitory activity at a concentration of 0.313 μg compared to HDH-LGBP-A2. The test results of this example indicate that the affinity for DNA polymerase is greater than when the peptide variant derived from HDH-LGBP is DNA alone.

Although the present invention has been described above based on the exemplary embodiments and examples of the present invention, the present invention is not limited to the technical ideas described in the above-described embodiments and examples. Rather, those skilled in the art to which the present invention pertains can easily assume various modifications and changes based on the above-described embodiments and examples. However, it will be further apparent from the claims that all such variations and modifications fall within the scope of the present invention.

As used herein, the term "peptide" includes any separation, or by recombinant techniques (Recombinant technique) by or chemically synthesized proteins, fragments and peptides of the proteins from those that occur naturally. In certain embodiments, compound variants are provided, eg, peptide variants having one or more amino acid substitutions. As used herein, “peptide variants” refers to substitutions, deletions, additions and / or insertions of one or more amino acids in the peptide amino acid sequence (substitutions). insertions) and exhibiting substantially the same biological function as a peptide composed of amino acids of the main body. The peptide variant must have an identity of 70% or more, preferably 90% or more, more preferably 95% or more with the original peptide. Such substitutions can include amino acid substitutions known as “conservative”. Variants can also include nonconservative changes. In one exemplary embodiment, the variant polypeptide sequence differs from the original sequence by substitution, deletion, addition or insertion of 5 or fewer amino acids. Variants can also be altered by deletion or addition of amino acids that have minimal impact on the peptide's immunogenicity, secondary structure and hydropathic nature.

Peptides according to the present invention, by manufacturing through recombinant methods (Recombinant means clustering) or chemical synthesis, it can be separated. For example, a peptide expressed by the nucleic acid sequence mentioned in the present specification can be easily produced by a known method using any of many known expression vectors. Expression can be achieved in a suitable host cell transformed into an expression vector containing a DNA sequence encoding the peptide. Suitable host cells include prokaryotic cells, yeast and eukaryotic cells. It is preferable to use E. coli, yeast or mammalian cell lines (such as Cos or CHO) as host cells. For protein purification, first, the supernatant containing the recombined protein secreted into the culture medium, obtained with a water-soluble host / vector system, is concentrated using a commercially available filter. Next, the concentrated solution obtained above is purified using an appropriate purification matrix such as an affinity matrix or an ion exchange resin. Finally, pure protein can be obtained by performing one-step or multi-step reverse phase HPLC.

Claims (13)

The peptide represented by the amino acid sequence of following formula (1).
[Formula 1]
(N-terminal) -WLWKAIWKLLL- (C-terminal) (1)
(In the formula (1), X is a basic amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H), or serine (S), threonine (T), (A polar amino acid selected from the group consisting of asparadine (N) and glutamine (Q).)   The peptide according to claim 1, wherein the peptide of formula (1) is composed of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.   The peptide according to claim 1, wherein the C-terminus of the peptide represented by the formula (1) is amidated.   The peptide according to claim 1, wherein the peptide has at least one physiological activity selected from antibacterial activity and anticancer activity.   A nucleic acid encoding the peptide of claim 1.   The nucleic acid according to claim 5, wherein the nucleic acid comprises a nucleotide composed of the base sequence of SEQ ID NO: 4 or SEQ ID NO: 6.   A recombination expression vector into which the nucleic acid according to claim 5 or 6 is inserted.   An anticancer pharmaceutical composition comprising the peptide according to any one of claims 1 to 4 as an active ingredient.   The pharmaceutical composition for anticancer according to claim 8, wherein the peptide treats at least one cancer selected from the group consisting of uterine cancer, cervical cancer and lung cancer.   An antibacterial pharmaceutical composition comprising the peptide according to any one of claims 1 to 4 as an active ingredient.   The antimicrobial pharmaceutical composition according to claim 10, wherein the peptide exhibits antibacterial activity against gram positive bacteria, gram negative bacteria and yeast.   An antibacterial cosmetic composition comprising the peptide according to any one of claims 1 to 4 as an active ingredient.   An antibacterial food additive comprising the peptide according to any one of claims 1 to 4 as an active ingredient.