JP4154218B2 - Novel antibacterial polypeptides and their use - Google Patents

Description

【００４４】
上述した各ペプチド（個々のアミノ酸配列は配列表を参照）は、市販のペプチド合成機（ABI 433A peptide synthesizer (Applied Biosystems社製品)）を用いてFast Moc^TM protocolに従い固相合成法（Fmoc法）により合成した。なお、縮合剤としてHATU（Applied Biosystems社製品）を使用し、固相合成法に用いた樹脂およびアミノ酸はNOVA biochem社から購入したものを用いた。また、Fmoc-アミノ酸において、リジンの側鎖官能基の保護基にはtert-butoxycarbonyl (Boc)を、ヒスチジンの側鎖官能基の保護基にはtriphenylmethyl (Trt)を、アルギニンの側鎖官能基の保護基にはpentamethylchroman-6-sulfonyl (Pmc)を使用した。
アミノ酸配列のＣ末端をアミド化する場合には、固相担体として「Fmoc-PAL-Polyethylen (PEG-PS) 樹脂」 を使用した。
【００４５】
上記ペプチド合成機の合成プログラムに準じて脱保護基反応および縮合反応を反復して樹脂に結合するFmoc−アミノ酸からペプチド鎖を伸長していき、目的の鎖長の合成ペプチドを得た。
具体例としては、２０％ピペリジン／ジメチルホルムアミド(DMF)（ペプチド合成用グレード、関東化学（株）製品）によって、アミノ酸のアミノ保護基であるFmocを切断除去し、ＤＭＦで洗浄し、Fmoc−アミノ酸(-OH)各3ｅｑを反応させ、ＤＭＦで洗浄する操作を反復した。そして、ペプチド鎖の伸長反応が全て終了した後、２０％ピペリジン／ＤＭＦによりFmoc基を切断し、ＤＭＦ、メタノールの順で上記反応物を洗浄した。
側鎖官能基の脱保護と同時に樹脂からペプチドを切断した。この方法は、82.5％Trifluoroacetic acid(以下TFA)（TFA：1,2-ethandithiol：m-cresole：Thioanisole：水＝82.5：2.5：5：5：5）（以上、和光純薬）５ｍｌを加え、室温で３時間反応させた。
樹脂を除き、ジエチルエーテル中に沈殿させた後、遠心（３０００ｒｐｍ、４℃、３ｍｉｎ）しエーテル層を取り除いた。この操作を３回繰返した。最後にエーテル層を取り除いた後、ペプチドを風乾させ、純水に溶解させた。純水に溶解しないものは３０％以下のエタノール水溶液に溶解させた。その後、凍結乾燥した。
【００４６】
得られたペプチド沈殿物を真空乾燥し、高速液体クロマトグラフを用いて精製を行った。具体的には、プレカラム（Guard-Pak社製品、製品名Deltapak C18 A300）およびＣ１８逆相カラム（DAISOPAK SP-120-5-ODS-AP、20mmI.D.×250mm（DAISO））を使用し、０．１％トリフルオロ酢酸水溶液と０．１％トリフルオロ酢酸アセトニトリル溶液との混合液を溶離液に用いた。すなわち、溶離液に含まれる上記トリフルオロ酢酸アセトニトリル溶液の分量を経時的に増大させつつ（容積比で１０％から８０％への濃度勾配を設ける）、７ｍｌ／分の流速で上記カラムを用いて４０〜５０分間分離精製を行った。なお、逆相カラムから溶離したペプチドは紫外線検出器を用いて２２０ｎｍで検出し、記録チャート上にピークとして示される。また、溶離した各ペプチドの分子量をKRATOS 質量分析装置 KOMPACT MALDI III（島津製作所製）を用いてMALDI-TOF/MS（Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry：マトリックス支援レーザーイオン化−飛行時間型−質量分析)に基づいて決定した。その結果、目的のペプチドが合成・精製されていることを確認した。
【００４７】
＜実施例2＞
○合成ペプチドの抗菌活性
本発明に係る抗菌性ペプチド（サンプル１〜５）、および比較例について、下記に記載のグラム陰性細菌およびグラム陽性細菌に対する抗菌活性（最小発育阻止濃度：ＭＩＣ）を９６穴(well)マイクロプレートを用いた液体培地希釈法により求めた。
○供試菌
・グラム陽性菌
Bacillus subtilis ATCC6633
Micrococcus luteus IFO12708
Staphylococcus aureus IFO12732
Staphylococcus aureus IID1677(MRSA)
・グラム陰性菌
Escherichia coli IFO12713
Escherichia coli O157:H7 sakai
Klebsiella pneumoniae ATCC4352
Pseudomonas aeruginosa ATCC10145
Salmonella enteritidis IFO3313
【００４８】
ペプチド濃度が５００、２５０、１２５、６２．５、３１．３、１５．６、７．８、３．９、１．９、１．０および０．５μＭとなる液体肉汁培地（DIFCO社製品「NUTRIENT BROTH Dehydrated」）をそれぞれ作製し、９６穴マイクロプレートに１５０μｌずつ分注した。一方、ＬＢ Ｂｒｏｔｈ，Ｌｅｎｎｏｘ（ＤＩＦＣＯ社製品）で１８時間、３７℃にて静置培養した菌液（約２×１０⁶ｃｅｌｌｓ／ｍＬ）を、薬剤溶液（上記ペプチド含有肉汁培地）と等量、９６穴マイクロプレートの各ウェルに接種した。接種後、３７℃の恒温器内で培養し、２４時間後の濁度により菌の生育状況を調べた。この計測時において、菌の増殖が認められない検体の最小の濃度を、本実施例におけるＭＩＣと定めた。
かかる抗菌試験に基づくサンプル１〜５および比較サンプルとして用いたＮｉｓｉｎのＭＩＣを表２に示す。
【００４９】
【表２】

【００５０】
表２に示す結果から明らかなように、本発明に係るペプチド（サンプル１〜５）は、比較例とした世界中で広く利用されている既存の抗菌性ペプチドであるナイシン（Ｎｉｓｉｎ、Sigma Aldrich社より購入）と比べ、グラム陽性菌に対しては遜色のない高い抗菌力を示し、かつグラム陰性菌、薬剤耐性菌に対しても高い抗菌力を示した。
表２に示す結果より、本発明に係る抗菌性ペプチドが、グラム陰性、グラム陽性、薬剤耐性菌に対して優れた抗菌活性と広い抗菌スペクトルを有することが確かめられた。
【００５１】
＜比較例２＞
サンプル６については、Staphylococcus aureus IFO12732およびEscherichia coli IFO12713について抗菌活性を実施例２と同様にして調べたが、ＭＩＣはともに４００μg／ml以上となり、抗菌活性が認められなかった。
【００５２】
塩基性アミノ酸残基が１個であるサンプル６について抗菌活性を調べたが、抗菌活性が認められなかった。しかし、実施例２に記載のサンプル１〜５には、抗菌活性が認められることから、本発明の抗菌性ペプチドには、塩基性アミノ酸残基が２個以上連続している部位が２ヶ所存在する必要があることを示している。
【００５３】
以上、本発明の具体例を詳細に説明したが、これらは例示にすぎず、特許請求の範囲を限定するものではない。特許請求の範囲に記載の技術には、以上に例示した具体例を様々に変形、変更したものが含まれる。また、本明細書に説明した技術要素は、単独であるいは各種の組み合わせによって技術的有用性を発揮するものであり、出願時請求項記載に限定されるものではない。また、本明細書に例示した技術は複数目的を同時に達成するものであり、そのうちの一つの目的を達成すること自体で技術的有用性を持つものである。
【００５４】
【発明の効果】
本発明の抗菌性ペプチドは、グラム陰性、グラム陽性、薬剤耐性菌に対して優れた抗菌活性と広い抗菌スペクトルを有することから、抗菌剤または消毒剤などとして有用である。また、本発明の抗菌性ペプチドの設計方法により、溶解性および抗菌性ペプチド全体の荷電などをコントロールすることができることから、用途に応じた抗菌性ペプチドを得ることが可能となる。
【００５５】
【配列表フリーテキスト】
配列番号1 設計された抗菌性ペプチド。
配列番号2 設計された抗菌性ペプチド。
配列番号3 設計された抗菌性ペプチド。
配列番号4 設計された抗菌性ペプチド。
配列番号5 設計された抗菌性ペプチド。
配列番号６ 設計されたペプチド。
【００５６】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antimicrobial peptide and the design of the antimicrobial peptide.
[0002]
[Prior art]
Antibacterial peptides have a broad antibacterial spectrum and are thought to be resistant to the emergence of drug-resistant bacteria, thus providing antibacterial properties to the prevention and treatment of bacterial infectious diseases in humans and animals, or foodstuffs For this purpose, antimicrobial peptides are expected to be used.
To date, many antibacterial peptides have been isolated from animals and plants. For example, an antibacterial peptide derived from Thai beetle and an antibacterial agent containing the antibacterial peptide as an active ingredient are disclosed (for example, see Patent Document 1).
Further, an antibacterial peptide derived from a scorpion poison and an antibacterial agent containing the antibacterial peptide as an active ingredient are disclosed (for example, see Patent Document 2).
The antibacterial peptides described above contain basic amino acids such as lysine, arginine and / or histidine, and are called cationic antibacterial peptides in which the charge of the whole peptide molecule is positive. The principle of action of these antibacterial peptides is considered to be due to the electrostatic interaction between the positively charged basic amino acid moiety in the antibacterial peptide and the membrane on the surface of the fungus (see, for example, Non-Patent Document 1). .
Antibacterial peptides described in each of the above publications have been discovered and isolated from those originally present as antibacterial peptides in nature (or peptides in which the amino acid sequence of a natural antibacterial peptide has been partially modified) Therefore, it is difficult to develop a product having antibacterial performance exceeding the antibacterial activity and antibacterial spectrum of the antibacterial peptide.
[0003]
○ Prior literature
[Patent Document 1]
JP 2000-063400 A (Claims)
[Patent Document 2]
JP 2001-186887 A (Claims)
[Non-Patent Document 1]
M.M. Zasloff, “Nature”, 2002, 415, p. 389-395
[0004]
[Problems to be solved by the invention]
The present invention is not based on the conventional approach of developing antibacterial peptides that replace amino acid residues of known antibacterial peptides, and has an antibacterial peptide having a different amino acid sequence from peptides that exist and function as antibacterial peptides in nature Is one of the purposes.
[0005]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have incorporated into the molecular design of an antibacterial peptide the fact that the peptide molecule has a sequence part composed of two basic amino acid residues and that both ends of the peptide are hydrophobic. Thus, the present inventors have found that an antibacterial peptide that exhibits high antibacterial activity and has a broad antibacterial spectrum against drug-resistant bacteria such as Gram-negative bacteria, Gram-positive bacteria, and MRSA can be realized, and the present invention has been completed. .
The antimicrobial peptide provided by the design method of the present invention is an artificially synthesized antimicrobial peptide that does not exist in nature. In addition, a non-naturally occurring artifact comprising a nucleotide sequence encoding an antimicrobial peptide provided by the present invention and / or a nucleotide sequence complementary to the sequence (typically substantially composed of the sequence) A synthesized polynucleotide is provided.
[0006]
The present invention provides an antibacterial agent containing the antibacterial peptide of the present invention as a component. Typically, the antimicrobial agent contains a pharmaceutically acceptable carrier in addition to the antimicrobial peptide of the present invention, depending on the application. Further, a non-naturally occurring artificially synthesized polynucleotide comprising a nucleotide sequence encoding the antibacterial peptide of the present invention and / or a nucleotide sequence complementary to the sequence, or substantially constituted by these sequences (Which may be in the form of DNA or RNA segments).
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
In addition, matters other than matters specifically mentioned in the present specification (for example, primary structure and chain length of antibacterial peptides) and matters necessary for the practice of the present invention (for example, peptide synthesis, polynucleotide synthesis, peptide components) General items such as those related to the preparation of drugs are considered as design items by those skilled in the art based on conventional techniques in fields such as organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, pharmacy, and medicine. obtain. The present invention can be carried out based on the contents disclosed in this specification and common technical knowledge in the field. In the following description, amino acids are represented by one-letter code based on the nomenclature related to amino acids shown in the IUPAC-IUB guidelines (however, three-letter code in the sequence listing).
[0008]
Here, “an artificially synthesized antibacterial peptide or polynucleotide that does not exist in nature” means that the peptide chain or nucleotide chain (full length) does not exist by itself in the natural world, but chemical synthesis or biosynthesis ( That is, it refers to an antimicrobial peptide or polynucleotide artificially produced by production based on genetic engineering. Here, the “peptide” is a term indicating an amino acid polymer having a plurality of peptide bonds, and is not limited by the number of amino acid residues contained in the peptide chain. So-called oligopeptides having less than 10 amino acid residues are also encompassed by the peptides herein. The “polynucleotide” is a term indicating a polymer (nucleic acid) in which a plurality of nucleotides are linked by a phosphodiester bond, and is not limited by the number of nucleotides. Various lengths of DNA and RNA fragments are encompassed by the polynucleotides herein.
[0009]
-Design of antibacterial peptides
In 1935, a quaternary ammonium salt having a long-chain alkyl group, which was a cationic surfactant developed by Domagk, was widely used as an antibacterial agent and had a long-chain alkyl group. The bis-type quaternary ammonium salt compound, which has the structural feature of possessing two quaternary ammonium salt moieties in the molecule, has significantly improved antibacterial activity compared to a compound having one quaternary ammonium salt, and has an environment, pH The method for designing the antibacterial peptide of the present invention has been completed on the basis of being hardly affected by the influence of temperature and temperature.
[0010]
That is, it has basic amino acid residue portions at two positions that are not the terminal of the antibacterial peptide, and hydrophobic and / or neutral amino acid residue portions that connect these basic amino acid residue portions (note that the basic amino acid residues) High antibacterial properties by incorporating into the molecular design of antibacterial peptides the characteristics of having hydrophobic amino acid residues at both ends of the peptide. It was possible to obtain antibacterial peptides that exhibited activity and had a broad antibacterial spectrum against gram-negative bacteria, gram-positive bacteria, and drug-resistant bacteria such as MRSA.
[0011]
  That is,The present inventionProvided byAntibacterial peptides areA linear antibacterial peptide having the following features (1) to (5):
(1). The total number of amino acid residues is 10-20;
(2). Having a symmetrical peptide sequence in which the sequence up to the center of the peptide sequence seen from the N-terminal side and the sequence up to the center of the peptide sequence seen from the C-terminal side are identical;
(3). Each of the N-terminal part and the C-terminal part is composed of one or more hydrophobic amino acid residues;
(4). There are sites where two or more basic amino acid residues are continuous adjacent to the N-terminal portion, and sites where two or more basic amino acid residues are continuous adjacent to the C-terminal portion. Is; and
(5). The center of a peptide sequence sandwiched between a portion where two or more basic amino acid residues near the N-terminal portion are continuous and a portion where two or more basic amino acid residues near the C-terminal portion are continuous The part is composed of 2 to 8 amino acid residues, wherein the central part is composed of a hydrophobic amino acid residue and / or a neutral amino acid residue, with the exception of the N-terminal portion and the C-terminal. A basic amino acid residue may be included on the condition that two or more basic amino acid residues adjacent to each other are not adjacent to a continuous site;
  Including both,Antibacterial peptide.
[0012]
[0013]
[0014]
[0015]
  As amino acids corresponding to basic amino acid residues in the antimicrobial peptide of the present invention,GoodPreferably, a site where two or more basic amino acid residues are continuous is constituted by lysine and / or arginine.
[0016]
AntiAmino acids corresponding to hydrophobic amino acid residues in fungal peptides include valine, leucine, isoleucine, phenylalanine, tryptophan, proline, glycine, alanine and methionine, preferably valine, leucine, isoleucine, phenylalanine and alanine. More preferably, the N-terminal part and the C-terminal part are composed of one or more hydrophobic amino acid residues selected from the group consisting of valine, leucine, isoleucine and alanine.
[0017]
AntiAmino acids corresponding to hydrophobic and / or neutral amino acid residues in fungal peptides include valine, leucine, isoleucine, phenylalanine, tryptophan, glycine, alanine, methionine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine Preferably valine, leucine, isoleucine, phenylalanine, tryptophan, glycine, alanine, serine, threonine, more preferably a hydrophobic amino acid residue and / or a neutral amino acid residue constituting the center of the peptide sequence. The group is one or more hydrophobic and / or neutral amino acid residues selected from the group consisting of valine, tryptophan, glycine, alanine, serine and threonine.
[0018]
BookinventionThe method for designing a linear antibacterial peptide provided by the following (1) to (5):
(1). The total number of amino acid residues is 10-20;
(2). Having a symmetrical peptide sequence in which the sequence up to the center of the peptide sequence seen from the N-terminal side and the sequence up to the center of the peptide sequence seen from the C-terminal side are identical;
(3). Each of the N-terminal part and the C-terminal part is composed of one or more hydrophobic amino acid residues;
(4). There are sites where two or more basic amino acid residues are continuous adjacent to the N-terminal portion, and sites where two or more basic amino acid residues are continuous adjacent to the C-terminal portion. Is; and
(5). The center of a peptide sequence sandwiched between a portion where two or more basic amino acid residues near the N-terminal portion are continuous and a portion where two or more basic amino acid residues near the C-terminal portion are continuous The part is composed of 2 to 8 amino acid residues, wherein the central part is composed of a hydrophobic amino acid residue and / or a neutral amino acid residue, with the exception of the N-terminal portion and the C-terminal. A basic amino acid residue may be included on the condition that two or more basic amino acid residues adjacent to each other are not adjacent to a continuous site;
  It is characterized by synthesizing a peptide chain having bothIt is a design method.
[0019]
  In the antibacterial peptide of the present invention, an L-type amino acid and / or a D-type amino acid can be used as an amino acid residue, but an L-type amino acid is preferred.
  The antibacterial peptide of the present invention is linear from the viewpoint that part or all of amino acid residues are unlikely to become an “antigen” unless the antibacterial activity is lost.Shapepreferable. Such a peptide is unlikely to constitute an epitope.
[0020]
  From this viewpoint, the antibacterial peptide is linear and has a relatively low molecular weight., SpecialThose having 10 to 20 amino acid residues are preferred.
[0021]
In addition, the N-terminal amino group and the C-terminal carboxyl group can be chemically modified, for example, formylation or acylation of the N-terminal amino group, and amidation of the C-terminal carboxyl group is antibacterial. It may be effective to increase power. The acylation of the N-terminal amino group may be carried out with a lower fatty acid such as an acetyl group, a propyl group or a butyl group, or with a higher fatty acid, and may have a branch.
[0022]
[0023]
BookThe antimicrobial peptide of the invention preferably has a symmetrical peptide sequence. This symmetrical peptide sequence is a peptide sequence in which the sequence up to the center of the antibacterial peptide sequence seen from the N-terminal side is the same as the sequence up to the center of the antibacterial peptide sequence seen from the C-terminal side. For example,
VLLKRAARKLLV
An array such as That is, from the N-terminal side, the sequence is “VLLKRA”, and from the C-terminal side is the sequence of “ARKLLV”, which is symmetrical from the center of the peptide sequence. In addition, when the number of amino acid residues is odd, it is symmetrical from the central amino acid residue.
  Some amino acids exhibit the same performance even when leucine is replaced with isoleucine, and those with sequences substituted with such amino acids can be regarded as identical..
[0024]
Substitution identity amino acids include:
In valine, isoleucine, leucine
In leucine, isoleucine, valine
In isoleucine, leucine, valine
For tryptophan, tyrosine
For serine, threonine
For threonine, serine
In alanine, glycine, serine
In glycine, alanine
For lysine, arginine
For arginine, lysine
Etc.
[0025]
  Specific examples of the antibacterial peptide of the novel amino acid sequence provided by the present invention are substantially composed of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Peptides are mentioned. All of these five peptides2It is composed of 0 amino acids or less (specifically, 11 to 15 amino acids), and is suitable for maintaining a linear shape. In addition, since it has low immunogenicity, it is suitable as a component of an antibacterial agent (antibacterial component).
[0026]
The antimicrobial peptide of the present invention can be easily produced according to a general chemical synthesis method. For example, any conventionally known solid phase synthesis method or liquid phase synthesis method may be employed. A solid-phase synthesis method using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as an amino-protecting group is preferred.
The antibacterial peptide of the present invention can be obtained by a solid phase synthesis method using a commercially available peptide synthesizer (for example, available from Applied Biosystems, etc.), and a desired amino acid sequence, modified (C-terminal amidation, etc.) moiety. Can be synthesized.
[0027]
The present invention relates to an antibacterial peptide having a different amino acid sequence from a peptide that exists and functions as an antibacterial peptide in nature, and a polynucleotide encoding the peptide, regardless of the conventional approach of developing an antibacterial peptide-containing antibacterial agent. One of the purposes is to provide. Another object is to provide an antibacterial agent (pharmaceutical composition) containing such an antibacterial peptide or a polynucleotide encoding the peptide as a component.
[0028]
Alternatively, the antibacterial peptide of the present invention may be biosynthesized based on a genetic engineering technique. This approach is preferred when producing peptides with relatively long chains. That is, DNA having a nucleotide sequence (including the ATG start codon) encoding the amino acid sequence of the desired antimicrobial peptide is synthesized. And for expression comprising this DNA and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis elements that control the expression level) for expressing the amino acid sequence in the host cell A recombinant vector carrying the gene construct is constructed depending on the host cell. This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, animal (mammalian) cell) by a general technique, and the host cell or a tissue or an individual containing the cell under a predetermined condition Is cultured. Thereby, the target peptide can be expressed and produced in the cell. Then, the target antimicrobial peptide can be obtained by isolating and purifying the peptide from the host cell (in the medium if secreted).
[0029]
A polynucleotide substantially consisting of a nucleotide sequence encoding the antimicrobial peptide of the present invention and / or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) by a conventionally known method. That is, a codon corresponding to each amino acid residue constituting the amino acid sequence of the antibacterial peptide can be selected, and the nucleotide sequence corresponding to the amino acid sequence of the antibacterial peptide can be easily determined. Once the nucleotide sequence is determined, a polynucleotide (single strand) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, using the obtained single-stranded DNA as a template, various enzymatic synthesis means can be employed to obtain the desired double-stranded DNA.
[0030]
The polynucleotide provided by the present invention may be in the form of RNA (such as mRNA) or in the form of DNA. DNA can be provided in double-stranded or single-stranded form. When provided as a single strand, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) having a sequence complementary thereto.
The polynucleotide provided by the present invention can be used as a material for constructing a recombinant gene (expression cassette) for expressing the antimicrobial peptide of the present invention in various host cells.
[0031]
For example, a polynucleotide having a nucleotide sequence encoding the antibacterial peptide according to the present invention and various regulatory elements (promoter, ribosome binding site, terminator, enhancer, expression level for expressing the amino acid sequence in a host cell). Including a variety of cis elements to be controlled) can be used to construct a recombinant vector having a gene construct for expressing a foreign peptide. The construction of the vector and the type of regulatory elements used in its construction can vary depending on the type of host cell of interest. For the construction of the recombinant vector, a polynucleotide restriction technique using various restriction enzymes and a polynucleotide fragment ligation technique well-understood in the field of genetic engineering are employed. These techniques can be easily performed by utilizing various commercially available devices. As a method for constructing a recombinant vector and a method for introducing the constructed recombinant vector into a host cell, a method conventionally used in the art may be employed as it is, and such method itself particularly characterizes the present invention. Since it is not a thing, detailed description is abbreviate | omitted.
[0032]
Since the antibacterial peptide of the present invention has a broad antibacterial spectrum, it is suitably used as a component such as an antibacterial agent, a bactericidal agent, a disinfectant, an antiseptic, and a deodorant. For example, treatment of bacterial infections, wound surface disinfection, eye disease prevention, antibacterial or disinfectant for intracavitary cleaning (eg gargle) and toothpaste, cosmetic preservatives, contact lens preservation, food preservatives It can be used as a freshness-preserving agent, bacteriostatic agent or bactericidal agent such as kitchen utensils, bathroom utensils and toilet articles, and bactericidal or bacteriostatic agents on furniture and sanitary equipment surfaces. In addition, bacteriostatic agents or disinfectants for textile products such as clothing and curtains, bacteriostatic agents or disinfectants for walls and floors, and further, bactericides such as industrial water, building management water and bath water, bacteriostatic agents Alternatively, it can be used as a preservative. Moreover, deodorizing effect can be expected from antibacterial or bactericidal action. Furthermore, it can be used as a bacteriostatic agent or fungicide in the agricultural field, for example, as an agrochemical for crops or agricultural materials. In addition, it can be used as a bacteriostatic agent or bactericidal agent in the field of animal husbandry and beekeeping, for example, as a bacteriostatic agent or bactericidal agent in a beekeeping box as a preservative for livestock feed. Further, as a bacteriostatic agent or a bactericidal agent in the fishery field, it can be used, for example, for treatment of fish infected with bacteria in a fish farm, sterilization of materials such as nets and gloves.
The antibacterial peptide of this invention can be used for the antibacterial agent, the disinfectant, etc. which were described above by containing 1-1000 microgram / ml.
[0033]
There is no limitation in particular regarding the form of the antimicrobial agent containing the antimicrobial peptide of this invention. For example, typical forms of internal preparations and external preparations include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, creams and the like. Moreover, since it uses for injection etc., it can also be set as the freeze-dried material and granulated material for melt | dissolving in a physiological saline etc. just before use and preparing a chemical | medical solution.
[0034]
In a preparation such as an antibacterial agent containing the antibacterial peptide of the present invention, the other carrier, that is, a secondary component (pharmaceutically acceptable) may be appropriately different depending on the use and form of the antibacterial agent, Various fillers, extenders, binders, moisturizers, surfactants, excipients, pigments, fragrances, and the like that are generally used as preparations can be used.
It should be noted that the process itself for preparing various forms of drugs using the antibacterial peptide of the present invention and various carriers as a material may be in accordance with known methods, and the formulation method itself does not characterize the present invention. The detailed explanation is omitted.
[0035]
The antibacterial agent provided by the antibacterial peptide of the present invention can be used in a method or dosage depending on its form and purpose. For example, the solution can be administered by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection. Alternatively, it may be administered by spraying into the nasal cavity or bronchi. A solid form such as a tablet can be administered orally. The dose at this time can be calculated from the antibacterial activity against the target bacteria.
[0036]
In addition, when used for the purpose of disinfecting (sterilizing) the surface of sanitary ware or preserving foods, a liquid containing a relatively large amount (for example, 1 to 100 mg / ml) of the antibacterial peptide of the present invention is directly applied to the surface of the object. It can be performed by spraying or wiping the surface of the object with a cloth or paper wetted with the liquid. These are merely examples, and the same forms and methods of use as antibacterial agents and disinfectants such as agricultural chemicals and quasi-drugs using conventional antibacterial agents can be applied.
[0037]
For example, cancer patients and AIDS patients undergoing radiotherapy may develop immunodeficiency as a complication and have more severe symptoms due to bacterial infections than are the cause of the original disease itself. The antibacterial peptide provided by the present invention exhibits an antibacterial action selectively against bacteria and is not toxic to mammals such as humans. For this reason, the antimicrobial peptide which concerns on this invention is useful as a component of an antimicrobial agent, and the antimicrobial agent provided by this invention can be safely applied with respect to a human body.
[0038]
In the field of regenerative medicine, the antibacterial peptide of the present invention can be used for the purpose of preventing bacterial infection during culturing of skin, bone, various organs and the like. For example, by adding an antibacterial peptide of the present invention alone or an antibacterial agent containing the peptide as one of the components at an appropriate concentration to the culture solution, bacterial infection of tissues or organs in culture can be prevented. .
[0039]
In addition, a polynucleotide encoding the antibacterial peptide of the present invention can be used as a material for gene therapy for cultured cells and cultured tissues. For example, the gene (DNA segment or RNA segment) encoding the antibacterial peptide of the present invention is incorporated into an appropriate vector and introduced into the target cultured tissue (cell), so that the cultured tissue (cells) is always or at a desired time. ), The antimicrobial peptide of the present invention can be expressed. Therefore, the polynucleotide (DNA segment or RNA segment) encoding the antibacterial peptide of the present invention is useful as a drug for preventing bacterial infection of cultured tissues (cells).
[0040]
The polynucleotide encoding the antimicrobial peptide of the present invention can be used as a material used for so-called gene therapy. For example, by incorporating a gene (DNA segment or RNA segment) encoding the antibacterial peptide of the present invention into an appropriate vector and introducing it into the target site, the antibacterial peptide of the present invention is always in vivo (cells). It can be expressed. Therefore, the polynucleotide (DNA segment or RNA segment) encoding the antibacterial peptide of the present invention is useful as a drug for preventing or treating bacterial infection in the above-mentioned patients.
[0041]
【Example】
The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the examples shown in these examples.
[0042]
<Example 1>
○ Peptide synthesis
Peptides (Samples 1 to 5, Sample 6) described in Table 1 below were produced using a peptide synthesizer described below.
[0043]
[Table 1]

[0044]
Each peptide described above (for the individual amino acid sequences, refer to the sequence table) can be obtained using a commercially available peptide synthesizer (ABI 433A peptide synthesizer (Applied Biosystems)).^TMIt was synthesized by the solid phase synthesis method (Fmoc method) according to the protocol. In addition, HATU (Applied Biosystems product) was used as a condensing agent, and the resin and amino acid used in the solid phase synthesis method were those purchased from NOVA biochem. In Fmoc-amino acid, tert-butoxycarbonyl (Boc) is used as the protecting group for the side chain functional group of lysine, triphenylmethyl (Trt) is used as the protecting group for the side chain functional group of histidine, and the side chain functional group of arginine is used. Pentamethylchroman-6-sulfonyl (Pmc) was used as a protecting group.
When amidating the C-terminal of the amino acid sequence, “Fmoc-PAL-Polyethylen (PEG-PS) resin” was used as a solid phase carrier.
[0045]
The peptide chain was extended from the Fmoc-amino acid bound to the resin by repeating the deprotection group reaction and the condensation reaction according to the synthesis program of the above peptide synthesizer to obtain a synthetic peptide having the desired chain length.
As a specific example, 20% piperidine / dimethylformamide (DMF) (grade for peptide synthesis, product of Kanto Chemical Co., Ltd.) was used to cleave off Fmoc, which is an amino protecting group of an amino acid, washed with DMF, and Fmoc-amino acid. (-OH) The reaction of reacting each 3 eq and washing with DMF was repeated. After the peptide chain elongation reaction was completed, the Fmoc group was cleaved with 20% piperidine / DMF, and the reaction product was washed with DMF and methanol in this order.
The peptide was cleaved from the resin simultaneously with deprotection of the side chain functional group. In this method, 82.5% Trifluoroacetic acid (hereinafter TFA) (TFA: 1,2-ethandithiol: m-cresole: Thioanisole: water = 82.5: 2.5: 5: 5: 5) (above, Wako Pure Chemical Industries, Ltd.) 5 ml is added, The reaction was allowed to proceed for 3 hours at room temperature.
After removing the resin and precipitating in diethyl ether, the ether layer was removed by centrifugation (3000 rpm, 4 ° C., 3 min). This operation was repeated three times. Finally, after removing the ether layer, the peptide was air-dried and dissolved in pure water. Those not dissolved in pure water were dissolved in 30% or less ethanol aqueous solution. Thereafter, it was freeze-dried.
[0046]
The obtained peptide precipitate was vacuum-dried and purified using a high performance liquid chromatograph. Specifically, a precolumn (Guard-Pak product, product name Deltapak C18 A300) and a C18 reverse phase column (DAISOPAK SP-120-5-ODS-AP, 20 mm I.D. × 250 mm (DAISO)) A mixture of a 0.1% trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acid acetonitrile solution was used as an eluent. That is, while increasing the amount of the trifluoroacetic acid acetonitrile solution contained in the eluent over time (providing a concentration gradient from 10% to 80% by volume), the column was used at a flow rate of 7 ml / min. Separation and purification were performed for 40 to 50 minutes. The peptide eluted from the reverse phase column is detected at 220 nm using an ultraviolet detector and is shown as a peak on the recording chart. In addition, the molecular weight of each eluted peptide was measured using the KRATOS mass spectrometer KOMPACT MALDI III (manufactured by Shimadzu Corporation). MALDI-TOF / MS (Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry) Mass spectrometry). As a result, it was confirmed that the target peptide was synthesized and purified.
[0047]
<Example 2>
-Antibacterial activity of synthetic peptides
The antibacterial peptide according to the present invention (samples 1 to 5) and the comparative example were subjected to antibacterial activity (minimum growth inhibitory concentration: MIC) against gram-negative bacteria and gram-positive bacteria described below in a 96-well microplate. It was determined by the liquid medium dilution method used.
○ Test bacteria
・ Gram positive bacteria
Bacillus subtilis ATCC6633
Micrococcus luteus IFO12708
Staphylococcus aureus IFO12732
Staphylococcus aureus IID1677 (MRSA)
・ Gram negative bacteria
Escherichia coli IFO12713
Escherichia coli O157: H7 sakai
Klebsiella pneumoniae ATCC4352
Pseudomonas aeruginosa ATCC10145
Salmonella enteritidis IFO3313
[0048]
Liquid gravy medium (product of DIFCO "" with peptide concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.9, 1.0 and 0.5 μM) NUTRIENT BROTH Dehydrated ”) was prepared, and 150 μl was dispensed into a 96-well microplate. On the other hand, a bacterial solution (about 2 × 10 × 10) that was statically cultured at 37 ° C. for 18 hours in LB Broth, Lennox (product of DIFCO).⁶cells / mL) was inoculated into each well of a 96-well microplate in the same amount as the drug solution (the peptide-containing broth medium). After inoculation, the cells were cultured in a 37 ° C. incubator, and the growth of the bacteria was examined by turbidity after 24 hours. At the time of this measurement, the minimum concentration of the specimen in which no bacterial growth was observed was defined as the MIC in this example.
Table 2 shows the MICs of Nisin used as samples 1 to 5 and a comparative sample based on the antibacterial test.
[0049]
[Table 2]

[0050]
As is apparent from the results shown in Table 2, the peptides according to the present invention (samples 1 to 5) are nisin (Nisin, Sigma Aldrich) which is an existing antibacterial peptide widely used around the world as a comparative example. Compared with (purchased more), it showed high antibacterial activity comparable to Gram-positive bacteria and also high antibacterial activity against Gram-negative and drug-resistant bacteria.
From the results shown in Table 2, it was confirmed that the antibacterial peptide according to the present invention has excellent antibacterial activity and a broad antibacterial spectrum against Gram negative, Gram positive and drug resistant bacteria.
[0051]
<Comparative example 2>
For sample 6, antibacterial activity was examined for Staphylococcus aureus IFO12732 and Escherichia coli IFO12713 in the same manner as in Example 2. However, both MICs were 400 μg / ml or more, and no antibacterial activity was observed.
[0052]
Sample 6 having one basic amino acid residue was examined for antibacterial activity, but no antibacterial activity was observed. However, since samples 1 to 5 described in Example 2 have antibacterial activity, the antibacterial peptide of the present invention has two sites where two or more basic amino acid residues are continuous. Shows that you need to.
[0053]
Specific examples of the present invention have been described in detail above, but these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and changes of the specific examples illustrated above. The technical elements described in the present specification exhibit technical usefulness alone or in various combinations, and are not limited to the claims at the time of filing. In addition, the technology exemplified in the present specification achieves a plurality of objects at the same time, and has technical utility by achieving one of the objects.
[0054]
【The invention's effect】
The antibacterial peptide of the present invention is useful as an antibacterial agent or disinfectant because it has excellent antibacterial activity against gram negative, gram positive and drug resistant bacteria and a broad antibacterial spectrum. Further, the antibacterial peptide design method of the present invention can control the solubility and the charge of the whole antibacterial peptide, and therefore, it is possible to obtain an antibacterial peptide according to the application.
[0055]
[Sequence Listing Free Text]
SEQ ID NO: 1 designed antibacterial peptide.
SEQ ID NO: 2 designed antibacterial peptide.
SEQ ID NO: 3 designed antibacterial peptide.
SEQ ID NO: 4 designed antibacterial peptide.
SEQ ID NO: 5 designed antibacterial peptide.
SEQ ID NO: 6 designed peptide.
[0056]
[Sequence Listing]

Claims (2)

An antibacterial peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 in the sequence listing . An antibacterial agent comprising the antibacterial peptide according to claim 1.