JP2016121081A - Antiviral drug and antimicrobial agent - Google Patents
Antiviral drug and antimicrobial agent Download PDFInfo
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- JP2016121081A JP2016121081A JP2014260849A JP2014260849A JP2016121081A JP 2016121081 A JP2016121081 A JP 2016121081A JP 2014260849 A JP2014260849 A JP 2014260849A JP 2014260849 A JP2014260849 A JP 2014260849A JP 2016121081 A JP2016121081 A JP 2016121081A
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- virus
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- amino acid
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- antiviral agent
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Abstract
Description
本発明は、所定のアミノ酸配列からなるペプチドを有効成分として含む、抗ウイルス剤および抗菌剤に関する。 The present invention relates to an antiviral agent and an antibacterial agent comprising a peptide having a predetermined amino acid sequence as an active ingredient.
近年、インフルエンザ等のウイルス性疾患の流行を背景として、ウイルス性疾患の有効な予防・治療法の開発が望まれている。インフルエンザウイルスやヘルペスウイルスなどのエンベロープ型と呼ばれるウイルスは、エンベロープと呼ばれる脂質二重膜を有する。多くのエンベロープ型ウイルスは、エンベロープと、ウイルスと接触した細胞の細胞膜とが融合することにより、細胞内へ侵入すると考えられている。 In recent years, against the epidemic of viral diseases such as influenza, it has been desired to develop effective preventive and therapeutic methods for viral diseases. Viruses called envelope types such as influenza viruses and herpes viruses have a lipid bilayer membrane called an envelope. Many enveloped viruses are thought to enter cells by fusing the envelope and the cell membrane of cells in contact with the virus.
一部のウイルス性疾患に対する別の対処法としては、アシクロビル、タミビル、またはザナミビルのような医薬品(抗ウイルス薬)が用いられている。しかしながら、多くのウイルス性疾患については有効な治療方法が無く、一般的にはワクチン接種による予防的手段がとられている。 As another countermeasure against some viral diseases, pharmaceuticals (antiviral drugs) such as acyclovir, tamivir, or zanamivir are used. However, there are no effective treatments for many viral diseases, and generally preventive measures are taken by vaccination.
このほか、例えば、特許文献1に記載のウイルス性疾患治療用医薬組成物のように、所定の配列を有するペプチドが抗ウイルス活性を呈することも報告され始めており、ウイルス性疾患の新たな予防・治療法として期待される。 In addition, for example, it has begun to be reported that a peptide having a predetermined sequence exhibits antiviral activity, such as a pharmaceutical composition for treating viral diseases described in Patent Document 1, and new prevention and prevention of viral diseases. Expected as a treatment.
一方、ウイルスと異なり、微生物に対しては、医学的には抗生物質による治療が一般的に行われている。さらに、抗菌活性を有するペプチド(抗菌ペプチド)の存在も知られており(例えば、特許文献2)、抗菌ペプチドのなかには、ディフェンシンなど、生体が本来的に防御手段として備えるものも存在する。 On the other hand, unlike viruses, microorganisms are generally treated with antibiotics medically. Furthermore, the existence of peptides having antibacterial activity (antibacterial peptides) is also known (for example, Patent Document 2), and some antibacterial peptides are inherently provided as a defense means, such as defensin.
ワクチン療法については、単回接種では免疫されない場合もあり、複数回の接種が被接種者の経済的な負担となり、侵襲度も高い。さらに、免疫療法は、ワクチンに用いられた特定の抗原に対して特異的に機能する抗体が作られるにとどまる。従って、例えばワクチンに用いられたものとは異なる型のウイルスに対しては十分な予防的効果が得られない、といった問題が存在する。また、アシクロビルのような医薬品については、耐性株の発生や重篤な副作用を引き起こす可能性等の問題が指摘されている。従って、本発明の第一の目的は、ワクチンやアシクロビル等の従来公知な医薬品に代わる、新規な抗ウイルス剤を提供することにある。 As for vaccine therapy, immunization may not be achieved by a single vaccination, and multiple vaccinations are an economical burden on the recipient and are highly invasive. Furthermore, immunotherapy only produces antibodies that function specifically against the specific antigen used in the vaccine. Therefore, for example, there is a problem that a sufficient preventive effect cannot be obtained against a virus of a type different from that used for the vaccine. In addition, for drugs such as acyclovir, problems such as the generation of resistant strains and the possibility of causing serious side effects have been pointed out. Accordingly, a first object of the present invention is to provide a novel antiviral agent that replaces a conventionally known pharmaceutical such as a vaccine or acyclovir.
微生物に関しては、抗生物質の使用による耐性菌の発生が大きな問題となっており、抗生物質に代わる抗菌ペプチド等の抗菌剤の開発が望まれている。従って、本発明の第二の目的は、従来の抗生物質に代わる新規な抗菌剤を提供することにある。 Regarding microorganisms, the generation of resistant bacteria due to the use of antibiotics has become a major problem, and the development of antibacterial agents such as antibacterial peptides that replace antibiotics is desired. Accordingly, a second object of the present invention is to provide a novel antibacterial agent that replaces conventional antibiotics.
本発明者らは、上記の問題を解決すべく、鋭意研究を行った結果、所定の配列を有するペプチドが、抗ウイルス活性、特にエンベロープ型ウイルスに対して高い抗ウイルス活性示すこと見出した。また、本発明者らは、所定の配列を有するペプチドが、抗菌活性、特に肺炎レンサ球菌(Streptococcus pneumoniae)に対して高い抗菌活性を示すことを見出した。 As a result of intensive studies to solve the above problems, the present inventors have found that a peptide having a predetermined sequence exhibits high antiviral activity against an antiviral activity, particularly against an enveloped virus. Further, the present inventors have found that a peptide having a predetermined sequence exhibits high antibacterial activity, particularly against Streptococcus pneumoniae.
本発明によれば、新規な抗ウイルス剤、特にエンベロープ型ウイルスに対して使用される抗ウイルス剤を提供することにある。また、本発明によれば、新規な抗菌剤、特に肺炎レンサ球菌(Streptococcus pneumoniae)に対して使用される抗菌剤を提供することができる。 According to the present invention, it is an object to provide a novel antiviral agent, particularly an antiviral agent used against enveloped viruses. Moreover, according to this invention, the novel antibacterial agent, especially the antibacterial agent used with respect to Streptococcus pneumoniae (Streptococcus pneumoniae) can be provided.
以下、本発明の実施の形態を説明する。なお、本発明は、以下の実施の形態のみには限定されない。 Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.
また、本明細書において、範囲を示す「X〜Y」は「X以上Y以下」を意味し、特記しない限り、操作および物性等の測定は室温(20〜25℃)/相対湿度40〜50%の条件で測定する。 Further, in this specification, “X to Y” indicating a range means “X or more and Y or less”, and unless otherwise specified, measurement of operation and physical properties is room temperature (20 to 25 ° C.) / Relative humidity 40 to 50. Measure under the condition of%.
なお、本発明に係る抗ウイルス剤や抗菌剤に有効成分として含まれる「(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなるペプチド」、および「(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有するペプチド」を、「本発明に係るペプチド」とも称する。また、「(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなるペプチド」を単に「ペプチド(A)」と、「(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有するペプチド」を「ペプチド(B)」とも称する。 In addition, “(A) LNGTSMASPHVAGAAAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGKGLINV (SEQ ID NO: 1) peptide included in the antiviral agent or antibacterial agent according to the present invention” and “(B) in the amino acid sequence (A)” A “peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added and having antiviral activity” is also referred to as “a peptide according to the present invention”. Further, “(A) LNGTSMASPHVGAAAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGKGLINV (SEQ ID NO: 1)” is simply referred to as “peptide (A)” and “(B) one or several amino acids in the amino acid sequence (A)”. A “peptide consisting of a deleted, substituted or added amino acid sequence and having antiviral activity” is also referred to as “peptide (B)”.
<抗ウイルス剤>
本発明の第一の実施形態は、下記(A)および(B)からなる群から選ばれるペプチドを有効成分として含む、抗ウイルス剤に関する。
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有する、ペプチド。
<Antiviral agent>
1st embodiment of this invention is related with the antiviral agent which contains the peptide chosen from the group which consists of following (A) and (B) as an active ingredient.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity.
本発明に係る抗ウイルス剤について推測される作用メカニズムを、以下に示す。なお、下記メカニズムは推測であり、本発明の技術的範囲を制限するものではない。従来公知の抗ウイルス薬の多くは、ウイルス自体を破壊するものではなく、ウイルスの増殖を抑制することにより、抗ウイルス活性を発現するものであった。すなわち、アシクロビルやリバビリンはウイルスの核酸合成を阻害することによって、また、タミビルやザナミビルはウイルスのノイラミニダーゼを阻害することによって、ウイルス増殖を抑制する。インターフェロンもまたウイルス性疾患の治療に用いられる医薬であるが、これは免疫機能を賦活することによって抗ウイルス活性を発現するものであり、やはりウイルスを破壊するものではない。一方、本発明に係るペプチドは、以下に説明するように、ウイルスと相互作用し、カプシドを破壊して核酸を放出させることにより抗ウイルス活性を発現するものと推測される。 The presumed mechanism of action of the antiviral agent according to the present invention is shown below. The following mechanism is speculation and does not limit the technical scope of the present invention. Many of the conventionally known antiviral drugs do not destroy the virus itself, but express antiviral activity by suppressing the growth of the virus. That is, acyclovir and ribavirin inhibit viral growth by inhibiting viral nucleic acid synthesis, and Tamivir and zanamivir inhibit viral neuraminidase. Interferon is also a drug used for the treatment of viral diseases, but it exhibits antiviral activity by stimulating immune function and does not destroy viruses. On the other hand, as described below, the peptide according to the present invention is presumed to interact with a virus and express antiviral activity by destroying the capsid and releasing the nucleic acid.
ウイルスのなかには、エンベロープと呼ばれる脂質二重膜によってカプシドが覆われた構造を有するもの(エンベロープ型ウイルス)が存在する。エンベロープは脂質二重膜であることから、負電荷を有する。本発明に係るペプチドにはアルギニン、リジン、ヒスチジンといった塩基性アミノ酸が含まれる反面、アスパラギン酸やグルタミン酸のような酸性アミノ酸が存在しないため、等電点が10.2程度の正電荷を有する。かような電荷の偏りが一因となって、本発明に係るペプチドはエンベロープと電気的に相互作用することが可能となる。従って、本発明の一実施形態では、エンベロープ型ウイルスに対して使用される、抗ウイルス剤が提供される。 Some viruses (envelope viruses) have a structure in which a capsid is covered with a lipid bilayer membrane called an envelope. Since the envelope is a lipid bilayer membrane, it has a negative charge. The peptide according to the present invention contains basic amino acids such as arginine, lysine and histidine, but has no positive amino acid such as aspartic acid or glutamic acid, and therefore has a positive charge with an isoelectric point of about 10.2. This charge bias contributes to the fact that the peptide according to the present invention can interact electrically with the envelope. Accordingly, in one embodiment of the present invention, an antiviral agent is provided for use against enveloped viruses.
また、本発明に係るペプチドの9〜21番目のアミノ酸(Pro9〜Lys21)(へリックス領域1)、および27〜37番目のアミノ酸(Ala27〜Thr37)(へリックス領域2)はα−へリックス構造をとると考えられる。このため、本発明に係るペプチドは、ウイルスと相互作用した後、上記のへリックス領域がエンベロープやカプシドに穿孔し、ウイルスから核酸が放出されるものと推測される。特に、N末端側に近いへリックス領域1が、カプシドの穿孔について重要な働きを担うものと考えられる。従って、本発明に係る抗ウイルス剤は、ウイルス自体を破壊する機能を有すると考えられ、従来の抗ウイルス薬とは作用機序が相違するものと推測される。 In addition, the 9th to 21st amino acids (Pro 9 to Lys 21 ) (helix region 1) and the 27th to 37th amino acids (Ala 27 to Thr 37 ) (helix region 2) of the peptide according to the present invention are α -It is considered to have a helix structure. For this reason, after the peptide according to the present invention interacts with the virus, it is presumed that the above-mentioned helix region is perforated in the envelope or capsid, and the nucleic acid is released from the virus. In particular, it is considered that the helix region 1 close to the N-terminal side plays an important role in capsid perforation. Therefore, the antiviral agent according to the present invention is considered to have a function of destroying the virus itself, and it is presumed that the action mechanism is different from that of the conventional antiviral agent.
各アミノ酸は、その側鎖の相違に基づいて、類似の性質を有するアミノ酸と置換しうることが、本技術分野においては知られている(保存的置換)。例えば、非極性中性アミノ酸(Gly、Ala、Met、Val、Leu、Ile、Pro)、親水性中性アミノ酸(Asn、Gln、Thr、Ser、Tyr、Cys)、酸性アミノ酸(Asp、Glu)、塩基性アミノ酸(Arg、Lys、His)、および芳香族アミノ酸(Phe、Tyr、Trp)のように類似の性質を有するものにグループ分けでき、これらのグループ内のアミノ酸は相互に置換し得る。 It is known in the art that each amino acid can be substituted with an amino acid having similar properties based on differences in its side chains (conservative substitution). For example, non-polar neutral amino acids (Gly, Ala, Met, Val, Leu, Ile, Pro), hydrophilic neutral amino acids (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), Groups can be grouped into those with similar properties such as basic amino acids (Arg, Lys, His) and aromatic amino acids (Phe, Tyr, Trp), and the amino acids within these groups can be substituted for each other.
生理活性を有するペプチドをそっくり含み、その一端または両端に1個または数個のアミノ酸配列が連結されたペプチドもまた、当該生理活性を維持し得ることが、本技術分野において知られている。従って、上記した抗ウイルス活性を有するペプチドを部分配列として含み、その一端または両端に1個または数個のアミノ酸が連結され、抗ウイルス活性を有するペプチドもまた、抗ウイルス剤に用いることが可能である。 It is known in the art that a peptide including a peptide having a physiological activity and having one or several amino acid sequences linked to one or both ends thereof can also maintain the physiological activity. Therefore, a peptide having the above-described antiviral activity as a partial sequence and having one or several amino acids linked to one or both ends thereof and having antiviral activity can also be used as an antiviral agent. is there.
従って、アミノ酸配列(A)において1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有するペプチド(ペプチド(B))も、本発明に係る抗ウイルス剤の有効成分として用いることができる。上記のペプチド(B)における「置換」とは、「保存的置換」であり得る。 Therefore, a peptide (peptide (B)) comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity is also an antiviral agent according to the present invention. It can be used as an active ingredient. The “substitution” in the above peptide (B) may be “conservative substitution”.
なお、本明細書において、アミノ酸についての「数個」とは、通常2〜5個、好ましくは2〜3個である。 In the present specification, “several” for amino acids is usually 2 to 5, preferably 2 to 3.
ペプチド(B)は、2次構造(α−へリックス構造)保持の観点から、アミノ酸配列(A)における9〜21番目のアミノ酸(Pro9〜Lys21)と相同な領域を有していてもよい。本発明の一実施形態では、ペプチド(B)は、アミノ酸配列(A)において1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、アミノ酸配列(A)における9〜21番目のアミノ酸(Pro9〜Lys21)と相同な領域を有し、かつ抗ウイルス活性を有する。また、アミノ酸配列(A)における9〜21番目のアミノ酸(Pro9〜Lys21)、および27〜37番目のアミノ酸(Ala27〜Thr37)と相同な領域を有していてもよい。本発明の一実施形態では、ペプチド(B)は、アミノ酸配列(A)において1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、アミノ酸配列(A)における9〜21番目のアミノ酸(Pro9〜Lys21)、および/または27〜37番目のアミノ酸(Ala27〜Thr37)と相同な領域を有し、かつ抗ウイルス活性を有する。 The peptide (B) may have a region homologous to the 9th to 21st amino acids (Pro 9 to Lys 21 ) in the amino acid sequence (A) from the viewpoint of maintaining the secondary structure (α-helix structure). Good. In one embodiment of the present invention, the peptide (B) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A), and the 9th to 21st positions in the amino acid sequence (A) It has a region homologous to the amino acid (Pro 9 to Lys 21 ) and has antiviral activity. Also, 9 to 21 amino acids in the amino acid sequence (A) (Pro 9 ~Lys 21 ), and 27 to 37 amino acid (Ala 27 ~Thr 37) and may have regions of homology. In one embodiment of the present invention, the peptide (B) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A), and the 9th to 21st positions in the amino acid sequence (A) Have a region homologous to the amino acid (Pro 9 to Lys 21 ) and / or the 27th to 37th amino acids (Ala 27 to Thr 37 ), and have antiviral activity.
本発明に係るペプチドには、抗ウイルス活性を有する限りにおいて、細胞膜透過性、プロテアーゼ耐性、ペプチド合成・精製工程の簡便性等を考慮し、従来公知の手法により修飾したペプチドも含まれる。ペプチドの修飾としては、例えば、ポリエチレングリコール(PEG、例えば分子量100〜5万程度)の付加、膜透過性ペプチド(例えば、Trans−Activator of Transcription Protein(Tat)ペプチド、オリゴアルギニン、またはペネトラチン)の付加、ゲラニル化およびファルネシル化等のプレニル化(例えば、イソプレン単位数1〜5のプレニル基)、ビオチン化、アルキル化(例えば、炭素数1〜15のアルキル基)、アセチル化およびパルミトイル化等のアシル化(例えば、炭素数1〜15のアシル基)、ベンジルオキシカルボニル化、ならび9−フルオレニルメチルオキシカルボニル化などが挙げられる。 As long as it has antiviral activity, the peptide according to the present invention includes peptides modified by a conventionally known method in consideration of cell membrane permeability, protease resistance, simplicity of peptide synthesis / purification process, and the like. Examples of the peptide modification include addition of polyethylene glycol (PEG, for example, molecular weight of about 100 to 50,000), addition of a membrane-permeable peptide (for example, Trans-Activator of Transcription Protein (Tat) peptide, oligoarginine, or penetratin). , Prenylation such as geranylation and farnesylation (eg prenyl group having 1 to 5 isoprene units), biotinylation, alkylation (eg alkyl group having 1 to 15 carbon atoms), acylation such as acetylation and palmitoylation (For example, an acyl group having 1 to 15 carbon atoms), benzyloxycarbonylation, 9-fluorenylmethyloxycarbonylation and the like.
なお、本明細書において、「抗ウイルス活性」は、例えば、実施例に記載の方法にて評価される。具体的には、試料の終濃度が0.1mg/mlの場合に、実施例の方法にて測定したPFUが、ビヒクルの値を100%としたときに50%以下であればよい。 In addition, in this specification, "antiviral activity" is evaluated by the method as described in an Example, for example. Specifically, when the final concentration of the sample is 0.1 mg / ml, the PFU measured by the method of the embodiment may be 50% or less when the vehicle value is 100%.
本発明の一実施形態では、ペプチド(B)は、ペプチド(A)と少なくとも70%(例えば、80%、90%、95%、98%または100%)の相同性を有し、かつ抗ウイルス活性を有する。なお、本明細書において、ペプチドの相同性はBLASTPプログラムによって評価される。 In one embodiment of the invention, peptide (B) has at least 70% (eg, 80%, 90%, 95%, 98% or 100%) homology with peptide (A) and is antiviral. Has activity. In the present specification, peptide homology is evaluated by the BLASTP program.
本発明の一実施形態においては、(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有するペプチド、が提供される。当該「置換」は、「保存的置換」であり得る。本実施形態の別の側面では、(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列と少なくとも70%(例えば、80%、90%、95%、98%または100%)の相同性を有し、かつ抗ウイルス活性を有するペプチド、が提供される。 In one embodiment of the present invention, (A) an amino acid sequence represented by (A) LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1) comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has antiviral activity. Having a peptide. The “substitution” may be a “conservative substitution”. In another aspect of this embodiment, (A) has at least 70% (eg, 80%, 90%, 95%, 98%, or 100%) homology with the amino acid sequence represented by LNGTSMASPHVAGAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGGKGLINV (SEQ ID NO: 1) And a peptide having antiviral activity.
本発明に係る抗ウイルス剤は各種ウイルスに対して使用されるが、特に、エンベロープ型ウイルスに対して使用されることが、抗ウイルス活性の観点から好ましい。エンベロープ型ウイルスとしては、特に制限されるものではないが、例えば、単純ヘルペスウイルス、水痘・帯状疱疹ウイルス、コイヘルペスウイルス、サケヘルペスウイルス、ウシヘルペスウイルス、インフルエンザウイルス、ヒト免疫不全ウイルス、B型肝炎ウイルス、C型肝炎ウイルス、エボラウイルス、SARSコロナウイルス、ムンプスウイルス、ラッサウイルス、デングウイルス、風疹ウイルス、麻疹ウイルス、黄熱ウイルス、および日本脳炎ウイルス等が例示できる。このうち、本発明に係る抗ウイルス剤は、単純ヘルペスウイルス、水痘・帯状疱疹ウイルス、コイヘルペスウイルス、サケヘルペスウイルス、およびウシヘルペスウイルスのようなヘルペスウイルス;ならびにインフルエンザウイルスに対して有効に使用され、特にヘルペスウイルスに対して有効に使用され得る。 The antiviral agent according to the present invention is used against various viruses, but it is particularly preferable from the viewpoint of antiviral activity that it is used against enveloped viruses. Examples of enveloped viruses include, but are not limited to, herpes simplex virus, varicella-zoster virus, koi herpes virus, salmon herpes virus, bovine herpes virus, influenza virus, human immunodeficiency virus, hepatitis B Examples include viruses, hepatitis C virus, Ebola virus, SARS coronavirus, mumps virus, lassa virus, dengue virus, rubella virus, measles virus, yellow fever virus, and Japanese encephalitis virus. Among these, the antiviral agent according to the present invention is effectively used against herpes viruses such as herpes simplex virus, varicella-zoster virus, koi herpes virus, salmon herpes virus, and bovine herpes virus; and influenza virus. In particular, it can be used effectively against herpes viruses.
(ペプチドの製造方法)
本発明に係るペプチドは、アミノ酸合成装置等を用いた従来公知の手段により製造することができる。また、本発明者らは、本発明に係るペプチドが、バチルス サブチルス(Bacillus subtilis)の発酵物中に質量5〜6kDaのペプチドとして含まれることを見出した。本発明の一実施形態では、バチルス サブチルスの発酵物から、質量5〜6kDaのペプチドを回収することを含む、上記抗ウイルス剤の製造方法が提供される。
(Method for producing peptide)
The peptide according to the present invention can be produced by a conventionally known means using an amino acid synthesizer or the like. The present inventors have also found that the peptide according to the present invention is contained as a peptide having a mass of 5 to 6 kDa in a fermented product of Bacillus subtilis. In one Embodiment of this invention, the manufacturing method of the said antiviral agent including collect | recovering peptides with a mass of 5-6 kDa from the fermented material of Bacillus subtilis is provided.
なお、上記のペプチドの質量は、質量分析、サイズ排除クロマトグラフィー、または電気泳動等公知の手法により測定できるが、本明細書においては、下記条件にてドデシル硫酸ナトリウム(SDS)/Tris−トリシン系電気泳動法にて確認された値である。 In addition, although the mass of said peptide can be measured by well-known methods, such as mass spectrometry, size exclusion chromatography, or electrophoresis, in this specification, sodium dodecyl sulfate (SDS) / Tris-tricine system on condition of the following. It is a value confirmed by electrophoresis.
本発明に係るペプチドは、質量が5〜6kDaであり、生体内のタンパク質と比べて比較的分子量が低い。電気泳動法において低分子量領域の分離を向上させるためには、アクリルアミド濃度の高いゲルを用いる必要がある。一般的な電気泳動法であるSDS/Tris−グリシン系では、アクリルアミド濃度の高いゲルほどひび割れや気泡の混入が起こり易く、ゲルの作製が困難になる。また、アクリルアミド濃度の高いSDS/Tris−グリシン系ゲルでは、泳動時の発熱のコントロールも難しく、低分子量領域の分離が良好ではない場合がある。従って、本発明では、SDS/Tris−グリシン系よりも低いアクリルアミド濃度でゲルの作製が可能であり、低分子量領域の分離に適するSDS/Tris−トリシン系電気泳動法にてペプチドの質量を確認する。 The peptide according to the present invention has a mass of 5 to 6 kDa, and has a relatively low molecular weight as compared to proteins in vivo. In order to improve the separation of the low molecular weight region in the electrophoresis method, it is necessary to use a gel having a high acrylamide concentration. In an SDS / Tris-glycine system that is a general electrophoresis method, a gel with a higher acrylamide concentration is more likely to be cracked or mixed with bubbles, making it difficult to produce the gel. Moreover, in an SDS / Tris-glycine gel having a high acrylamide concentration, it is difficult to control the heat generation during electrophoresis, and the separation of the low molecular weight region may not be good. Therefore, in the present invention, a gel can be prepared with an acrylamide concentration lower than that of the SDS / Tris-glycine system, and the mass of the peptide is confirmed by SDS / Tris-tricine electrophoresis suitable for separation of a low molecular weight region. .
(ペプチド質量の確認のための電気泳動の方法)
下記組成にて、ゲルを作製する。
(Electrophoresis method for confirmation of peptide mass)
A gel is prepared with the following composition.
具体的には、泳動プレートを用いて分離ゲルを重合させる(重合中は、50%(v/v)2−プロパノール水溶液を重層させる。)。分離ゲルの重合後、泳動プレートを水洗し、スペーサーゲルを重合させる(重合中は、50%(v/v)2−プロパノール水溶液を重層させる。)。再び泳動プレートを水洗し、コームを泳動プレートに挿入した状態で濃縮ゲルを重合させる。かような手法により調製したゲルを水洗して電気泳動に用いる。 Specifically, the separation gel is polymerized using an electrophoresis plate (50% (v / v) 2-propanol aqueous solution is layered during polymerization). After the separation gel is polymerized, the electrophoresis plate is washed with water to polymerize the spacer gel (during polymerization, 50% (v / v) 2-propanol aqueous solution is overlaid). The electrophoresis plate is washed again with water, and the concentrated gel is polymerized with the comb inserted in the electrophoresis plate. The gel prepared by such a method is washed with water and used for electrophoresis.
電気泳動は、下記条件にて行う。
陽極側泳動バッファー :0.2MTris−HCl緩衝液(pH8.9)
陰極側泳動バッファー :0.1MTris−0.1Mトリシン−0.1%(w/v)SDS
泳動 :開始から30分間は50V定電圧とし、以降は150V定電圧(ミニゲルで90分泳動)。
Electrophoresis is performed under the following conditions.
Anode-side electrophoresis buffer: 0.2 M Tris-HCl buffer (pH 8.9)
Cathode-side electrophoresis buffer: 0.1 M Tris-0.1 M Tricine-0.1% (w / v) SDS
Electrophoresis: A constant voltage of 50 V for 30 minutes from the start, and a constant voltage of 150 V thereafter (electrophoresis for 90 minutes with a mini gel).
バチルス サブチルスの発酵物得られたペプチドの配列は、従来公知のアミノ酸配列の解析方法により確認することができる。 The sequence of the peptide obtained by fermentation of Bacillus subtilis can be confirmed by a conventionally known amino acid sequence analysis method.
バチルス属が生産するプロテアーゼの一種であるサチライシン類(ズブチリシン類、subtilisin)には、本発明に係るペプチドに相当するアミノ酸配列が含まれる。以下に、バチルス サブチルス(Bacillus subtilis)が生産するサチライシン類(ナットウキナーゼ、およびケラチナーゼ)を例に、clustalWプログラムによりによりペプチド(A)とアライメントを比較した結果を示す。なお、下記アライメント比較結果において、「peptideA」は「ペプチド(A)」を、「nattokinase[B.subtilis]」はバチルス サブチルスのナットウキナーゼ(GenBank: ACJ48969.1)配列を、「keratinase[B.licheniformis]」はバチルス リケニフォルミス(Bacillus licheniformis)のケラチナーゼ(GenBank: AID16241.1)配列を示す。ペプチド(A)は、バチルス サブチルスのナットウキナーゼ(GenBank: ACJ48969.1)と83%の相同性を、バチルス リケニフォルミスのケラチナーゼと98%の相同性を示す。 Subtilisins, which are a kind of protease produced by the genus Bacillus, contain amino acid sequences corresponding to the peptides of the present invention. The results of comparing alignment with peptide (A) using the clustalW program are shown below, taking as an example subtilisins (nattokinase and keratinase) produced by Bacillus subtilis. In the alignment comparison results below, “peptideA” is the “peptide (A)”, “nattokinase [B. subtilis]” is the Bacillus subtilis nattokinase (GenBank: ACJ48969.1) sequence, “keratinase [B. licheniformis]”. "Indicates the Bacillus licheniformis keratinase (GenBank: AID16241.1) sequence. Peptide (A) shows 83% homology with Bacillus subtilis nattokinase (GenBank: ACJ48969.1) and 98% homology with Bacillus licheniformis keratinase.
本発明の技術的範囲を制限するものではないが、バチルス サブチルスの発酵物中に含まれる質量5〜6kDaのペプチドは、バチルス サブチルスが生産するサチライシン類のプロテアーゼに由来するのではないかと推測される。 Although not limiting the technical scope of the present invention, it is presumed that the 5 to 6 kDa peptide contained in the fermentation product of Bacillus subtilis is derived from a protease of the subtilisins produced by Bacillus subtilis. .
上記のバチルス サブチルスの発酵物は、大豆発酵物であることが好ましい。大豆発酵物としては、例えば、大豆粉、大豆かす、脱脂大豆かす、大豆ペプチド、大豆加水分解物、ソイトン、またはソイペプトン等を窒素源として含む培地でバチルス サブチルスを培養した培養物であっても良く、また、納豆、味噌等の発酵食品であっても良い。納豆などのバチルス サブチルスの発酵大豆食品は、古くから食経験があり、安全性の観点で特に優れる。本発明の一実施形態では、納豆から質量5〜6kDaのペプチドを回収することを含む、上記の抗ウイルス剤の製造方法が提供される。 The fermentation product of Bacillus subtilis is preferably a fermented soybean product. The soy fermented product may be, for example, a culture obtained by cultivating Bacillus subtilis in a medium containing soybean powder, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolyzate, soybean, or soybean peptone as a nitrogen source. Moreover, fermented foods, such as natto and miso, may be sufficient. Bacillus subtilis fermented soy foods such as natto have long been eaten and are particularly excellent in terms of safety. In one Embodiment of this invention, the manufacturing method of said antiviral agent including collect | recovering peptides with a mass of 5-6 kDa from natto is provided.
本発明に係るペプチドの製造に用いられるバチルス サブチルスとしては、本発明に係るペプチドを生産するものであれば特に制限されないが、サチライシン類を生産するものが好ましく、例えば、ATCC 15245株、NCIB 3610株、およびATCC 6051株等が挙げられるが、変種である納豆菌(バチルス サブチルス ナットー(Bacillus subtilis natto))であってもよい。このうち、古来より発酵食品の生産に用いられてきた、納豆菌(バチルス サブチルス ナットー(Bacillus subtilis natto))が、安全性の面から特に好ましい。これらの微生物は、ATCCや独立行政法人製品評価技術基盤機構(NITE)等から入手することができる。 The Bacillus subtilis used for the production of the peptide according to the present invention is not particularly limited as long as it produces the peptide according to the present invention, but those that produce subtilisins are preferable, for example, ATCC 15245 strain, NCIB 3610 strain , And ATCC 6051 strain, etc., and may be a natto bacterium (Bacillus subtilis Natto) which is a variant. Of these, Bacillus subtilis Natto, which has been used for the production of fermented foods since ancient times, is particularly preferable from the viewpoint of safety. These microorganisms can be obtained from ATCC, National Institute of Technology and Evaluation (NITE), etc.
バチルス サブチルスを培養して発酵物を得る場合、その培養は従来公知の方法で行えばよく、例えば20〜50℃、好ましくは30〜40℃で培養する。培地のpHは、通常は6〜8である。 When Bacillus subtilis is cultured to obtain a fermented product, the culture may be performed by a conventionally known method, for example, 20 to 50 ° C, preferably 30 to 40 ° C. The pH of the medium is usually 6-8.
バチルス サブチルスの培養には、通常、炭素源、窒素原、および無機塩を含む、合成培地または天然培地を用いる。 For cultivation of Bacillus subtilis, a synthetic medium or natural medium containing a carbon source, a nitrogen source, and an inorganic salt is usually used.
例えば、炭素源としては、コーンスターチ、コーンミール、デンプン、デキストリン、麦芽、ブドウ糖、グリセリン、スロース、糖蜜等が単独でまたは混合物として用いられる。 For example, as the carbon source, corn starch, corn meal, starch, dextrin, malt, glucose, glycerin, sucrose, molasses and the like are used alone or as a mixture.
窒素源としては、大豆粉、大豆かす、脱脂大豆かす、大豆ペプチド、大豆加水分解物、ソイトン、ソイペプトン、硫酸アンモニウム、硝酸ナトリウム、コーンスティープリカー、グルテンミール、肉エキス、脂肉骨粉、酵母エキス、乾燥酵母、綿実粉、ペプトン、ポリペプトン、小麦胚芽、魚粉、ミートミール、脱脂米糠、脱脂肉骨粉、麦芽エキス、コーングルテンミール等の無機または有機の窒素源を単独でまたは混合物として使用できる。ペプチドの生産性の観点から、好ましくは、窒素源は大豆に由来するものが好ましく、具体的には、大豆粉、大豆かす、脱脂大豆かす、大豆ペプチド、大豆加水分解物、ソイトン、およびソイペプトンからなる群から窒素源が選択されることが好ましい。 Nitrogen sources include soybean meal, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolysate, soyton, soy peptone, ammonium sulfate, sodium nitrate, corn steep liquor, gluten meal, meat extract, fat meat bone meal, yeast extract, dried Inorganic or organic nitrogen sources such as yeast, cottonseed meal, peptone, polypeptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat and bone meal, malt extract, corn gluten meal and the like can be used alone or as a mixture. From the viewpoint of peptide productivity, preferably, the nitrogen source is derived from soybean, specifically from soybean flour, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolysate, soyton, and soy peptone. Preferably the nitrogen source is selected from the group consisting of
無機塩としては、炭酸カルシウム、塩化ナトリウム、塩化カリウム、硫酸マグネシウム、臭化ナトリウム、ホウ酸ナトリウム又はリン酸第一カリウム、硫酸亜鉛、硫酸マグネシウム等の各種無機塩が単独でまたは混合物として使用できる。 As the inorganic salt, various inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or monopotassium phosphate, zinc sulfate, magnesium sulfate can be used alone or as a mixture.
また、必要に応じて、鉄、マンガン、亜鉛、コバルト、モリブデン酸等の重金属を微量添加することもできる。さらに、加熱滅菌時および培養中における発泡を押さえるため、大豆油、亜麻仁油などの植物油、オクタデカノール等の高級アルコール類、各種シリコン等の消泡剤を添加してもよい。 Moreover, if necessary, a trace amount of heavy metals such as iron, manganese, zinc, cobalt, and molybdic acid can be added. Furthermore, in order to suppress foaming during heat sterilization and culture, vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, and various antifoaming agents such as silicon may be added.
発酵物が、大豆食品の発酵物である場合、例えば納豆の製造方法を例にとると、洗浄した大豆を必要に応じて水に浸漬し(例えば、10〜20℃で15〜30時間)し、蒸煮する。その後、納豆菌の胞子を大豆に散布し、発酵(35〜50℃、相対湿度85〜95%、15〜30時)を行って発酵物を得る。発酵後、低温(例えば、4〜10℃)で24〜72時間程度さらに熟成させても良い。 When the fermented product is a fermented product of soy food, for example, taking a natto production method as an example, the washed soybean is immersed in water as necessary (for example, at 10 to 20 ° C. for 15 to 30 hours). Cook. Then, the spore of Bacillus natto is sprinkled on soybeans and fermented (35-50 ° C., relative humidity 85-95%, 15-30 hours) to obtain a fermented product. After fermentation, it may be further ripened for about 24 to 72 hours at a low temperature (eg, 4 to 10 ° C.).
質量5〜6kDaのペプチドは、硫安(硫酸アンモニウム)沈殿、カラム精製、限外濾過、透析、塩析等、従来公知の方法を適宜組み合わせることによって行うことができる。 A peptide having a mass of 5 to 6 kDa can be performed by appropriately combining conventionally known methods such as ammonium sulfate (ammonium sulfate) precipitation, column purification, ultrafiltration, dialysis, and salting out.
発酵物からの本発明に係るペプチドの精製方法としては、例えば、上記の納豆等のバチルス サブチルスの発酵物を生理食塩水、または緩衝液中でホモジナイズし、その後、遠心分離等により水溶性画分を回収する。緩衝液としては、リン酸緩衝液、Tris緩衝液のほか、PIPES、TES、HEPES等のグッド緩衝液等も用いることができる。抽出に用いる緩衝液のpHは、好ましくは6.5〜8.5である。 As a method for purifying a peptide according to the present invention from a fermented product, for example, a fermented product of Bacillus subtilis such as natto as described above is homogenized in physiological saline or a buffer solution, and then the water-soluble fraction is separated by centrifugation or the like. Recover. As the buffer solution, besides the phosphate buffer solution and the Tris buffer solution, Good buffer solutions such as PIPES, TES, and HEPES can be used. The pH of the buffer used for extraction is preferably 6.5 to 8.5.
遠心分離により水溶性画分を回収する場合、その条件は特に制限されないが、例えば、10,000〜22,000rpmで5〜60分程度行ない、上清を回収する。もっとも、ここでの操作は、有効成分とその他の成分を大まかに分離するためのものであり、最終的に本発明の効果が得られるのであれば、特に制限されない。 When the water-soluble fraction is collected by centrifugation, the conditions are not particularly limited. For example, the supernatant is collected at 10,000 to 22,000 rpm for about 5 to 60 minutes. However, the operation here is for roughly separating the active ingredient from other ingredients, and is not particularly limited as long as the effects of the present invention can be finally obtained.
続いて、上記の硫安分画、カラムクロマトグラフィー、限外濾過、透析、塩析等、従来公知の方法を適宜組み合わせて、上記の発酵物の水溶性画分から質量5〜6kDaのペプチドを回収する。本発明に係るペプチドは、精製の操作の容易さから、好ましくは、硫安分画によって精製される。 Subsequently, a peptide having a mass of 5 to 6 kDa is recovered from the water-soluble fraction of the fermented product by appropriately combining conventionally known methods such as the above ammonium sulfate fractionation, column chromatography, ultrafiltration, dialysis, and salting out. . The peptide according to the present invention is preferably purified by an ammonium sulfate fraction because of the ease of purification operation.
本発明に一実施形態では、質量5〜6kDaのペプチドの回収が、発酵物から30%飽和硫安に可溶な画分(1)を得、画分(1)から50%飽和硫安に不溶な画分(2)を得ることを含む、上記抗ウイルス剤の製造方法が提供される。本発明においては、発酵物(例えば、上記の水溶性画分)に飽和濃度の30%の硫安を添加して遠心分離(例えば、15,500rpm、4℃で15分間)を行い得られた上清(画分(1))に、飽和濃度の50%の硫安を添加して遠心分離(例えば、15,500rpm、4℃で15分間)を行ない得られた沈殿画分(2)を、30〜50%画分ともいう。なお、本明細書における「画分(2)」には、上記のように飽和濃度の50%の硫安を添加して遠心分離を行ない得られた沈殿を、さらに透析等によって脱塩、および/または乾燥したものも含まれる。 In one embodiment of the present invention, recovery of a peptide with a mass of 5-6 kDa yields a fraction (1) soluble in 30% saturated ammonium sulfate from the fermented product and is insoluble in 50% saturated ammonium sulfate from fraction (1). There is provided a method for producing the above antiviral agent, comprising obtaining fraction (2). In the present invention, the fermented material (for example, the above water-soluble fraction) was added with 30% of ammonium sulfate at a saturated concentration and centrifuged (for example, 15,500 rpm, 15 minutes at 4 ° C.). To precipitate (fraction (1)), 50% of saturated ammonium sulfate was added and centrifuged (for example, 15,500 rpm, 4 ° C. for 15 minutes). Also referred to as ~ 50% fraction. In the “fraction (2)” in the present specification, the precipitate obtained by adding 50% saturated ammonium sulfate and centrifuging as described above is further desalted by dialysis or the like, and / or Or dried ones are also included.
かような手法により得られた30〜50%画分には、本発明に係るペプチドの一種である、質量5〜6kDaのペプチドが含まれる。従って、30〜50%画分を、必要に応じて透析等により脱塩し、バチルス サブチルスの発酵物から回収された本発明に係るペプチドを含む粗精製物として、本発明に係る抗ウイルス剤に用いてもよい。即ち、本発明に係る抗ウイルス剤においては、抗ウイルス効果が得られる限りにおいて、質量5〜6kDaのペプチドのバチルス サブチルスの発酵物に由来する成分が含まれることを妨げない。 The 30 to 50% fraction obtained by such a technique includes a peptide having a mass of 5 to 6 kDa, which is a kind of peptide according to the present invention. Accordingly, 30-50% fraction is desalted by dialysis or the like as necessary, and the crude product containing the peptide according to the present invention recovered from the fermented Bacillus subtilis is used as the antiviral agent according to the present invention. It may be used. That is, the antiviral agent according to the present invention does not prevent the inclusion of a component derived from a fermented product of Bacillus subtilis having a mass of 5 to 6 kDa as long as an antiviral effect is obtained.
また、バチルス サブチルスの発酵物から回収された質量5〜6kDaのペプチドとしては、上記で得られた画分(2)を、再び上記の生理食塩水や緩衝液等に溶解させ、より高度な精製工程を経て回収されたものであってもよい。より高度な精製工程としては、カラムクロマトグラフィー、および限外濾過等が例示できる。カラムクロマトグラフィーにより精製する場合、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、疎水性クロマトグラフィー、吸着クロマトグラフィー(ヒドロキシアパタイト担体)、逆相クロマトグラフィー、および/または親和性クロマトグラフィー等、従来公知の方法を適宜組み合わせて行えばよい。以下、例として、疎水性クロマトグラフィーによる精製条件を例示するが、本発明に係るペプチドが回収可能である限り、下記の方法に制限されるものではない。 In addition, as a peptide having a mass of 5 to 6 kDa recovered from a fermentation product of Bacillus subtilis, the fraction (2) obtained above is dissolved again in the above-mentioned physiological saline, buffer solution, etc. What was collect | recovered through the process may be used. Examples of more sophisticated purification steps include column chromatography and ultrafiltration. When purifying by column chromatography, conventionally known methods such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption chromatography (hydroxyapatite carrier), reverse phase chromatography, and / or affinity chromatography May be combined as appropriate. Hereinafter, as an example, purification conditions by hydrophobic chromatography are exemplified, but the present invention is not limited to the following method as long as the peptide according to the present invention can be recovered.
(疎水性クロマトグラフィーによる精製条件)
画分(2)を、終濃度1%(w/v)となるように10mM Tris−HCl緩衝液(pH7.4)に添加する。さらに、25%飽和濃度となるように硫酸アンモニウムを上記緩衝液に加え、15,000rpmで15分間、4℃で遠心分離を行う。遠心分離後、上清を回収し、孔径0.45μmのフィルターにてろ過し、ろ液を下記の疎水性クロマトグラフィーによる精製の試料として用いる。ブチルセファロースカラム(例えば、Butyl Sepharose High Performance(GEヘルスケア社製))を、平衡化緩衝液(25%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にて平衡化する。平衡化後のカラムに上記の試料をアプライし、洗浄緩衝液(25%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にてカラムを洗浄する。溶出緩衝液(20%、15%、および10%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にて順次溶出される画分のうち、10%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にて溶出された画分(画分(3))を得る。
(Purification conditions by hydrophobic chromatography)
Fraction (2) is added to 10 mM Tris-HCl buffer (pH 7.4) to a final concentration of 1% (w / v). Further, ammonium sulfate is added to the buffer so as to obtain a 25% saturation concentration, followed by centrifugation at 15,000 rpm for 15 minutes at 4 ° C. After centrifugation, the supernatant is collected and filtered through a filter having a pore size of 0.45 μm, and the filtrate is used as a sample for purification by the following hydrophobic chromatography. A butyl sepharose column (eg, Butyl Sepharose High Performance (GE Healthcare)) is equilibrated with an equilibration buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). . The above sample is applied to the column after equilibration, and the column is washed with a washing buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). Of fractions sequentially eluted with elution buffer (10 mM Tris-HCl buffer (pH 7.4) containing 20%, 15%, and 10% saturated ammonium sulfate), containing 10% saturated ammonium sulfate A fraction (fraction (3)) eluted with 10 mM Tris-HCl buffer (pH 7.4)) is obtained.
好ましくは、本発明に係る抗ウイルス剤の製造方法は、上記画分(2)を、疎水性クロマトグラフィーにより分離し、10%飽和硫安溶出画分(3)を得ることをさらに含む。 Preferably, the method for producing an antiviral agent according to the present invention further includes separating the fraction (2) by hydrophobic chromatography to obtain a 10% saturated ammonium sulfate elution fraction (3).
質量5〜6kDaのペプチドを含む当該画分(3)を、抗ウイルス剤に用いることができる。なお、本明細書における「画分(3)」には、上記のような10%飽和硫安溶出画分を、さらに透析等によって脱塩、および/または乾燥したものも含まれる。 The fraction (3) containing a peptide having a mass of 5 to 6 kDa can be used as an antiviral agent. The “fraction (3)” in the present specification includes a 10% saturated ammonium sulfate elution fraction as described above, which is further desalted by dialysis and / or dried.
任意に、上記の画分(3)から、質量5〜6kDaのペプチドである本発明に係るペプチドを更に上記方法により精製し、質量5〜6kDaのペプチドを単離して抗ウイルス剤に用いてもよい。 Optionally, the peptide according to the present invention, which is a peptide having a mass of 5 to 6 kDa, is further purified by the above method from the fraction (3), and the peptide having a mass of 5 to 6 kDa is isolated and used as an antiviral agent. Good.
より好ましくは、本発明に係る抗ウイルス剤の製造方法は、上記画分(3)を、逆相クロマトグラフィーにより分離し、保持時間30分の画分(アセトニトリル濃度30%(v/v)の画分、画分(4))を得ることをさらに含む。なお、本明細書における「画分(4)」には、上記のようなアセトニトリル濃度30%(v/v)の画分を、さらに乾燥したものも含まれる。下記に逆相クロマトグラフィーの方法を記載するが、本発明に係るペプチドが回収可能である限り、下記の方法に制限されるものではない
(逆相クロマトグラフィーによる精製条件)
カラム: オクタデシル(C18)シリカゲルカラム(例えば、製品名:Wakosil(登録商標)− 5C18HG、和光純薬社製)
移動相: A液(HPLC用H2O):B液(HPLC用アセトニトリル)(アセトニトリル濃度0%(v/v)から60%(v/v)のリニアグラジエント)
カラム温度: 30℃
流速: 1ml/min。
More preferably, in the method for producing an antiviral agent according to the present invention, the fraction (3) is separated by reverse phase chromatography, and a fraction having a retention time of 30 minutes (acetonitrile concentration of 30% (v / v)) is used. Further comprising obtaining a fraction, fraction (4)). In addition, the “fraction (4)” in the present specification includes those obtained by further drying the fraction having the acetonitrile concentration of 30% (v / v) as described above. Although the method of reverse phase chromatography is described below, it is not limited to the following method as long as the peptide according to the present invention can be recovered (purification conditions by reverse phase chromatography).
Column: Octadecyl (C18) silica gel column (for example, product name: Wakosil (registered trademark) -5C18HG, manufactured by Wako Pure Chemical Industries, Ltd.)
Mobile phase: Liquid A (H 2 O for HPLC): Liquid B (acetonitrile for HPLC) (linear gradient from acetonitrile concentration 0% (v / v) to 60% (v / v))
Column temperature: 30 ° C
Flow rate: 1 ml / min.
本発明の一実施形態では、本発明に係るペプチドは、単離されたものである。単離されたペプチドとしては、上記の手法により得られた画分(4)を用いても良い。 In one embodiment of the invention, the peptide according to the invention is isolated. As the isolated peptide, the fraction (4) obtained by the above method may be used.
バチルス サブチルスの発酵物からの回収物中に含まれる質量5〜6kDaのペプチドの量は、例えば、回収物の固形分換算で、10〜100質量%であることが、抗ウイルス活性の観点から好ましい。 The amount of the peptide having a mass of 5 to 6 kDa contained in the recovered product from the fermented Bacillus subtilis is preferably 10 to 100% by mass in terms of the solid content of the recovered product, for example, from the viewpoint of antiviral activity. .
精製後は、凍結乾燥等により回収物を乾燥し、本発明に係る抗ウイルス剤に用いても良い。 After purification, the recovered material may be dried by freeze drying or the like and used for the antiviral agent according to the present invention.
<抗菌剤>
本発明の第二の実施形態では、下記(A)および(B)からなる群から選ばれるペプチドを有効成分として含む、抗菌剤が提供される。
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗菌活性を有する、ペプチド。
<Antimicrobial agent>
In 2nd embodiment of this invention, the antibacterial agent which contains the peptide chosen from the group which consists of following (A) and (B) as an active ingredient is provided.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.
本実施形態において用いられるペプチドについては、抗ウイルス剤について上述した記載が適宜修飾されて適用される。 About the peptide used in this embodiment, the description mentioned above about an antiviral agent is modified suitably, and is applied.
本発明に係る抗菌剤は、ストレプトコッカス属の菌に対して好適に用いられ、より具体的には、アルファー・溶血レンサ球菌(alfa−Streptococcus)、ストレプトコッカスsp.(Streptococcus sp.)、ストレプトコッカス アガラクティエ(S.agalactiae)、ストレプトコッカス・アンギノサス(S.anginosus)、ストレプトコッカス・コンステラタス(S.constellatus)、ストレプトコッカス・ゴルドニイ(S.gordonii)、ストレプトコッカス・ミティス(S.mitis)、ストレプトコッカス・インターメディウス(S.intermedius)、ストレプトコッカス・オラリス(S.oralis)、肺炎レンサ球菌(S.pneumoniae)、化膿レンサ球菌(S.pyogenes)、およびストレプトコッカス・サングイニス(S.sanguinis)等が例示できる。 The antibacterial agent according to the present invention is preferably used against bacteria belonging to the genus Streptococcus, and more specifically, alpha-Streptococcus, Streptococcus sp. (Streptococcus sp.), Streptococcus agalactiae (S. agalactiae), Streptococcus anginosus (S. constellatus), Streptococcus gordonii (S. Examples include S. intermedius, S. oralis, S. pneumoniae, S. pyogenes, and S. sanguinis. .
このうち、本発明に係る抗菌剤は、肺炎レンサ球菌(Streptococcus pneumoniae)に対して特に有効に使用される。すなわち、本発明の一実施形態では、本発明に係る抗菌剤は、肺炎レンサ球菌(Streptococcus pneumoniae)に対して使用される。すなわち、本発明の一実施形態では、上記抗菌剤は、抗肺炎レンサ球菌剤である。 Among these, the antibacterial agent according to the present invention is particularly effectively used against Streptococcus pneumoniae. That is, in one embodiment of the present invention, the antibacterial agent according to the present invention is used against Streptococcus pneumoniae. That is, in one Embodiment of this invention, the said antibacterial agent is an anti-pneumonitis streptococcus agent.
<抗ウイルス剤、抗菌剤の応用>
本発明に係る抗ウイルス剤および/または抗菌剤は、例えば各種の食品や飲料に混合して、または医薬品もしくは各種生活用品の成分として利用できる。
<Application of antiviral and antibacterial agents>
The antiviral agent and / or antibacterial agent according to the present invention can be used, for example, as a mixture in various foods and beverages, or as a component of pharmaceuticals or various daily necessities.
本発明に係る抗ウイルス剤および/または抗菌剤を医薬品として用いるときの投与量は、疾患の症状、患者の年齢などにより異なるが、例えば、本発明に係るペプチドとして成人1日あたり0.1〜10000mg/60kg体重、好ましくは10〜10000mg/60kg体重、より好ましくは100〜5000mg/60kg体重である。 The dosage when the antiviral agent and / or antibacterial agent according to the present invention is used as a pharmaceutical varies depending on the symptom of the disease, the age of the patient, and the like. It is 10,000 mg / 60 kg body weight, preferably 10 to 10,000 mg / 60 kg body weight, more preferably 100 to 5000 mg / 60 kg body weight.
医薬品として用いられる場合は、経口投与、または筋肉内、皮内、皮下、静脈内、下部体腔、皮膚などの非経口投与により投与される。また、本発明に係る抗ウイルス剤および/または抗菌剤は、本発明に係るペプチドのみから構成されてもよいし、各投与形態に適した、薬学的に許容される担体および/または希釈剤を用いて製剤化することもできる。製剤化方法および各種担体は、医薬製剤の分野において周知である。薬学的に許容される担体または希釈剤は、例えば、生理緩衝液のような緩衝液や、賦形剤(砂糖、乳糖、コーンスターチ、リン酸カルシウム、ソルビトール、グリシン等)であってもよく、結合剤(シロップ、ゼラチン、アラビアゴム、ソルビトール、ポリビニルクロリド、トラガント等)、滑沢剤(ステアリン酸マグネシウム、ポリエチレングリコール、タルク、シリカ等)等が適宜混合されていてもよい。投与形態としては、錠剤、顆粒剤、散剤、カプセル剤、シロップ剤、点眼剤、トローチ剤、注射剤、坐剤、軟膏などの剤型が採用されうる。これらの製剤は一般的に知られている製法によって製造することができる。 When used as a pharmaceutical, it is administered by oral administration or parenteral administration such as intramuscular, intradermal, subcutaneous, intravenous, lower body cavity and skin. Further, the antiviral agent and / or antibacterial agent according to the present invention may be composed of only the peptide according to the present invention, or a pharmaceutically acceptable carrier and / or diluent suitable for each dosage form. It can also be formulated. Formulation methods and various carriers are well known in the field of pharmaceutical formulation. The pharmaceutically acceptable carrier or diluent may be, for example, a buffer such as a physiological buffer or an excipient (sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine, etc.), and a binder ( Syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth, etc.), lubricant (magnesium stearate, polyethylene glycol, talc, silica, etc.) and the like may be appropriately mixed. As the dosage form, dosage forms such as tablets, granules, powders, capsules, syrups, eye drops, troches, injections, suppositories, ointments and the like can be adopted. These preparations can be produced by generally known production methods.
また、本発明に係る抗ウイルス剤および/または抗菌剤を食用に供する量の目安は、前記の医薬品と同じ程度である。健康食品とするときは、乾固物に換算して健康食品素材の全量に対し、0.0005〜10質量%、好ましくは、0.1〜5.0質量%の割合になるように添加される。0.0005質量%以上とすることにより、目的とする効果が効果的に得られ、呈味の観点から10質量%とすることが好ましい。飲食品は、本発明に係る抗ウイルス剤および/または抗菌剤を単独かまたは食品に一般に使用される原料、例えば糖類、澱粉、脂質などの賦形剤、さらには必要に応じて結合剤、滑沢剤、着色剤、香料などの矯味矯臭剤などを併用することができ、常法により製造される。飲食品としては、例えばキャンディー、ドロップ、錠菓、チューイングガム、カプセル、清涼飲料、乳飲料、果汁飲料、野菜飲料、豆乳飲料、コーヒー飲料、茶飲料、粉末飲料、濃縮飲料、栄養飲料、アルコール飲料などが含まれる。 Moreover, the standard of the quantity which uses the antiviral agent and / or antibacterial agent which concern on this invention for food is the same grade as the said pharmaceutical. When it is used as a health food, it is added in a ratio of 0.0005 to 10% by mass, preferably 0.1 to 5.0% by mass, based on the total amount of the health food material in terms of dry matter. The By setting it as 0.0005 mass% or more, the target effect is acquired effectively and it is preferable to set it as 10 mass% from a viewpoint of taste. The food and drink are prepared by using the antiviral agent and / or antibacterial agent according to the present invention alone or as a raw material generally used in foods, for example, excipients such as sugars, starches and lipids, and if necessary, binders, lubricants. A flavoring agent such as a flavoring agent, a coloring agent, and a fragrance can be used in combination, and it is produced by a conventional method. Examples of foods and drinks include candy, drops, tablet confectionery, chewing gum, capsules, soft drinks, milk drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, nutrition drinks, alcoholic drinks, etc. Is included.
利便性の観点から、本発明の抗ウイルス剤および/または抗菌剤は、噴霧剤の形態であることも好ましい。噴霧剤は、例えば、スプレー、エアロゾル、または乾燥噴霧の形態で使用される。噴霧剤は、圧縮ガスやポンプによる噴霧および/または吐出に適した剤型に加工される。例えば水、pH6〜8程度の緩衝液、または生理食塩水等に本発明に係るペプチドを溶解させたり、揮発性溶媒に懸濁して製剤化したりする。噴霧剤は、口腔用噴霧剤や鼻腔噴霧剤など、生体に直接投与できる他、フェイスマスクや衣類などの繊維製品に塗布したり、加湿器等により室内に霧散させたりして使用できる。 From the viewpoint of convenience, the antiviral agent and / or antibacterial agent of the present invention is preferably in the form of a spray. The propellant is used, for example, in the form of a spray, aerosol or dry spray. The propellant is processed into a dosage form suitable for spraying and / or discharging with a compressed gas or a pump. For example, the peptide according to the present invention is dissolved in water, a buffer solution having a pH of about 6 to 8, or physiological saline, or is formulated in a suspension in a volatile solvent. The spray can be administered directly to a living body, such as an oral spray or a nasal spray, or can be applied to a textile product such as a face mask or clothing, or sprayed indoors with a humidifier or the like.
本発明に係る一実施形態においては、上記の抗ウイルス剤および/または抗菌剤を塗布してなる、繊維製品が提供される。繊維製品は、合成繊維、再生繊維もしくは天然繊維、またはこれらの混紡を加工した衣類、衛生用品、寝装品、フィルム類、およびインテリア用品等である。特に限定されるものではないが、繊維製品としては、例えば、シャツ、外套、ズボン、スカート、ナイトウェア、下着類、靴下、手袋、帽子、白衣、看護衣、介護衣、作業着、フェイスマスク、シーツ、枕カバー、カーペット、布団、座布団、壁紙、各種フィルター類、カーテン、およびカーペット等が例示できる。本発明に係る繊維製品は、例えば、繊維製品または製品加工前の繊維に、本発明に係る抗ウイルス剤および/または抗菌剤をスプレー、浸漬、含浸、またはコーティング等の方法により塗布すればよい。 In one embodiment according to the present invention, a textile product obtained by applying the antiviral agent and / or antibacterial agent is provided. Textile products include synthetic fibers, recycled fibers or natural fibers, or garments, hygiene products, bedding products, films, interior goods, and the like processed from these blends. Although not particularly limited, examples of textile products include shirts, cloaks, trousers, skirts, nightwear, underwear, socks, gloves, hats, white robes, nursing clothes, nursing clothes, work clothes, face masks, Examples include sheets, pillow covers, carpets, futons, cushions, wallpaper, various filters, curtains, carpets, and the like. What is necessary is just to apply | coat the antiviral agent and / or antibacterial agent which concern on this invention to the textiles or fiber before product processing by the method of spraying, immersion, impregnation, or coating etc., for example.
<治療方法等>
本発明の第三の実施形態では、下記(A)および(B)からなる群から選ばれるペプチドの、ウイルス感染症、または細菌感染症の治療または予防のための使用が提供される。
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗菌活性を有する、ペプチド。
<Treatment methods>
In the third embodiment of the present invention, there is provided use of a peptide selected from the group consisting of the following (A) and (B) for treatment or prevention of viral infection or bacterial infection.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.
当該実施形態は、上記の(A)および(B)からなる群から選ばれるペプチドの有効量を患者に投与することを含む、ウイルス感染症の治療または予防方法である。 The embodiment is a method for treating or preventing a viral infection, comprising administering to a patient an effective amount of a peptide selected from the group consisting of (A) and (B).
なお、本明細書において「有効量」とは、ウイルス感染症による健康被害の治療または予防といった所望の効果を発揮するうえで少なくとも必要とされる有効成分の量である。 In the present specification, the “effective amount” is an amount of an active ingredient that is required at least for exerting a desired effect such as treatment or prevention of health damage caused by a viral infection.
また、患者は、例えばヒト、サル、マウス、ラット、ハムスター、ウサギ、モルモット、ウシ、ブタ、イヌ、ウマ、ネコ、ヤギ、ヒツジを含む哺乳類を含む動物を指し、好ましくはヒトである。 The patient refers to animals including mammals including, for example, humans, monkeys, mice, rats, hamsters, rabbits, guinea pigs, cows, pigs, dogs, horses, cats, goats and sheep, and preferably humans.
本実施形態において、上記の(A)および(B)からなる群から選ばれるペプチドは、上記したように、経口投与、または筋肉内、皮内、皮下、静脈内、下部体腔、皮膚などの非経口投与により投与される。また、各投与形態に適した、上記したような薬学的に許容される担体および/または希釈剤を用いて製剤化したペプチドを使用しても良い。 In this embodiment, the peptide selected from the group consisting of the above (A) and (B) is orally administered or non-muscular such as intramuscular, intradermal, subcutaneous, intravenous, lower body cavity, and skin as described above. It is administered by oral administration. In addition, a peptide formulated with a pharmaceutically acceptable carrier and / or diluent as described above suitable for each dosage form may be used.
本発明の第四の実施形態では、ウイルス感染症、または細菌感染症の治療または予防のための医薬品の製造における、下記(A)および(B)からなる群から選ばれるペプチドの使用が提供される。
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗菌活性を有する、ペプチド。
In the fourth embodiment of the present invention, there is provided use of a peptide selected from the group consisting of the following (A) and (B) in the manufacture of a medicament for the treatment or prevention of viral infection or bacterial infection. The
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.
本発明の第三の実施形態や、第四の実施形態においては、上記の抗ウイルス剤や抗菌剤における記載が適宜変更されて適用される。 In the third embodiment and the fourth embodiment of the present invention, the description in the antiviral agent and the antibacterial agent is appropriately changed and applied.
本発明の第三の実施形態や第四の実施形態における、ウイルス感染症は、抗ウイルス剤についての説明において記載される上記のウイルスの感染に起因する疾患である。本発明は特に、エンベロープ型ウイルス感染症、なかでもヘルペスウイルス感染症に有効に使用される。 The virus infection in the third embodiment and the fourth embodiment of the present invention is a disease caused by the infection of the virus described in the description of the antiviral agent. The present invention is particularly useful for enveloped viral infections, particularly herpes virus infections.
本発明の第三の実施形態や、第四の実施形態における細菌感染症は、抗菌剤についての説明において記載される上記のストレプトコッカス属の菌の感染に起因する疾患である。本発明は特に、肺炎レンサ球菌(Streptococcus pneumoniae)感染症に有効に使用される。 The bacterial infection in the third embodiment and the fourth embodiment of the present invention is a disease caused by infection of the above-mentioned Streptococcus spp. Described in the description of the antibacterial agent. The present invention is particularly effectively used for Streptococcus pneumoniae infections.
本発明の効果を、以下の実施例および比較例を用いて説明する。ただし、本発明の技術的範囲が以下の実施例のみに制限されるわけではない。 The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.
<製造例1>
納豆(株式会社ヤマダフーズ製)170gを秤量し、510ml(納豆に対し3倍量)の10mM Tris−HCl(pH7.4)を加え、ホモジナイザー(ポリトロンPT4000、セントラル科学貿易)で均質化した。均質化した納豆は、15分間(15,500rpm、4℃)遠心分離を行い、水溶性画分と不溶性画分とに分離した。上清の水溶性画分を回収し、飽和濃度の30%になるように硫安を添加して、4℃にて30分間攪拌した。攪拌後、溶液を15分間(15,500rpm、4℃)遠心分離した。遠心分離後に回収した上清に対し、飽和濃度の50%になるように硫安を添加し、4℃にて30分間攪拌した。攪拌後、溶液を15分間(15,500rpm、4℃)遠心分離し、沈殿を得た(30〜50%画分)。
<Production Example 1>
170 g of natto (manufactured by Yamada Foods) was weighed, 510 ml (3 times the amount of natto) of 10 mM Tris-HCl (pH 7.4) was added, and homogenized with a homogenizer (Polytron PT4000, Central Science Trade). The homogenized natto was centrifuged for 15 minutes (15,500 rpm, 4 ° C.) and separated into a water-soluble fraction and an insoluble fraction. The water-soluble fraction of the supernatant was collected, ammonium sulfate was added so that the saturation concentration was 30%, and the mixture was stirred at 4 ° C. for 30 minutes. After stirring, the solution was centrifuged for 15 minutes (15,500 rpm, 4 ° C.). To the supernatant collected after centrifugation, ammonium sulfate was added so that the saturation concentration was 50%, and the mixture was stirred at 4 ° C. for 30 minutes. After stirring, the solution was centrifuged for 15 minutes (15,500 rpm, 4 ° C.) to obtain a precipitate (30-50% fraction).
得られた沈殿を、10mlの10mM Tris−HCl緩衝液(pH7.4)で溶解させた。30〜50%画分の溶液を、10mM Tris−HCl緩衝液(pH7.4)に対して24時間、透析膜を用いて透析を行った(画分(2))。 The obtained precipitate was dissolved in 10 ml of 10 mM Tris-HCl buffer (pH 7.4). The solution of the 30-50% fraction was dialyzed against a 10 mM Tris-HCl buffer (pH 7.4) for 24 hours using a dialysis membrane (fraction (2)).
透析後の試料を、終濃度1%(w/v)となるように10mM Tris−HCl緩衝液(pH7.4)に添加した。さらに、25%飽和濃度となるように硫酸アンモニウムを上記緩衝液に加え、15,000rpmで15分間、4℃で遠心分離を行った。遠心分離後、上清を回収し、孔径0.45μmのフィルターにてろ過し、ろ液を下記の疎水性クロマトグラフィーによる精製の試料として用いた。 The sample after dialysis was added to 10 mM Tris-HCl buffer (pH 7.4) so that the final concentration was 1% (w / v). Further, ammonium sulfate was added to the buffer so as to obtain a 25% saturation concentration, and centrifugation was performed at 15,000 rpm for 15 minutes at 4 ° C. After centrifugation, the supernatant was collected and filtered through a filter having a pore size of 0.45 μm, and the filtrate was used as a sample for purification by the following hydrophobic chromatography.
ブチルセファロースカラム(Butyl Sepharose High Performance(GEヘルスケア社製))を、平衡化緩衝液(25%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にて平衡化した。平衡化後のカラムに上記の試料をアプライし、洗浄緩衝液(25%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にてカラムを洗浄した。溶出緩衝液(20%、15%、10%、5%、および2%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4))にて順次溶出した画分のうち、10%飽和濃度の硫酸アンモニウムを含む10mM Tris−HCl緩衝液(pH7.4)にて溶出した画分(画分(3))を得た。 A butyl sepharose column (Butyl Sepharose High Performance (manufactured by GE Healthcare)) was equilibrated with an equilibration buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). The sample was applied to the column after equilibration, and the column was washed with a washing buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). 10% saturation of fractions eluted sequentially with elution buffer (10 mM Tris-HCl buffer (pH 7.4) containing 20%, 15%, 10%, 5%, and 2% saturated ammonium sulfate concentration) A fraction (fraction (3)) eluted with 10 mM Tris-HCl buffer (pH 7.4) containing ammonium sulfate at a concentration was obtained.
疎水性クロマトグラフィーによる精製物を、SDS/Tris−トリシン系電気泳動により展開した結果を図1に示す。図1中、「10%飽和溶出」、「5%飽和溶出」、および「2%飽和溶出」は、それぞれ溶出緩衝液中の硫安飽和濃度(%)を意味する。また、図1における矢印は、5〜6kDaペプチドである。 The result of developing the purified product by hydrophobic chromatography by SDS / Tris-Tricine electrophoresis is shown in FIG. In FIG. 1, “10% saturation elution”, “5% saturation elution”, and “2% saturation elution” mean the ammonium sulfate saturation concentration (%) in the elution buffer, respectively. Moreover, the arrow in FIG. 1 is a 5-6 kDa peptide.
10%飽和濃度の硫酸アンモニウムにて溶出されたペプチド画分(画分(3))を、以下の条件にて逆相クロマトグラフィーに供し、保持時間30分の画分(アセトニトリル濃度30%(v/v)の画分、画分(4))を本発明に係るペプチドとして単離した。 The peptide fraction (fraction (3)) eluted with 10% saturated ammonium sulfate was subjected to reverse phase chromatography under the following conditions, and the fraction with a retention time of 30 minutes (acetonitrile concentration 30% (v / The fraction of v) and the fraction (4)) were isolated as peptides according to the present invention.
(逆相クロマトグラフィーによる精製条件)
カラム: オクタデシル(C18)シリカゲルカラム(製品名:Wakosil(登録商標)− 5C18HG、和光純薬社製)
使用機器: Gulliver series(日本分光社製)
移動相: A液(HPLC用H2O)/B液(HPLC用アセトニトリル) (アセトニトリル濃度0%(v/v)から60%(v/v)のリニアグラジエント)
カラム温度: 30℃
流速: 1ml/min
注入量: 1.0ml
検出器: LKB 2158 Uvicord SD(206nm)。
(Purification conditions by reverse phase chromatography)
Column: Octadecyl (C18) silica gel column (Product name: Wakosil (registered trademark) -5C18HG, manufactured by Wako Pure Chemical Industries, Ltd.)
Equipment used: Gulliver series (manufactured by JASCO)
Mobile phase: Liquid A (H 2 O for HPLC) / Liquid B (acetonitrile for HPLC) (linear gradient from acetonitrile concentration 0% (v / v) to 60% (v / v))
Column temperature: 30 ° C
Flow rate: 1 ml / min
Injection volume: 1.0ml
Detector: LKB 2158 Uvicord SD (206 nm).
上記ペプチドの分子量を、上記条件のドデシル硫酸ナトリウム(SDS)/Tris−トリシン系電気泳動法にて確認した。結果を図2に示す。図2中、矢印は本発明に係るペプチドである。 The molecular weight of the peptide was confirmed by sodium dodecyl sulfate (SDS) / Tris-Tricine electrophoresis under the above conditions. The results are shown in FIG. In FIG. 2, the arrows indicate the peptides according to the present invention.
電気泳動により確認された質量5kDaのペプチドのアミノ酸配列をプロテインシーケンサ(PPSQ−31B/33B、株式会社島津製作所製)にて確認したところ、配列番号1で表されるアミノ酸配列(質量:5480Da)であった。 When the amino acid sequence of the peptide of mass 5 kDa confirmed by electrophoresis was confirmed by a protein sequencer (PPSQ-31B / 33B, manufactured by Shimadzu Corporation), the amino acid sequence represented by SEQ ID NO: 1 (mass: 5480 Da) was obtained. there were.
上記方法にて回収されたペプチドを凍結させた後、凍結乾燥を行い、下記実施例における試料(試料1)とした。なお、試料1には、本発明に係るペプチドが90質量%以上含まれる。 The peptide collected by the above method was frozen and then lyophilized to obtain a sample (sample 1) in the following Examples. Sample 1 contains 90% by mass or more of the peptide according to the present invention.
<実施例1>
上記条件にて得られた試料1を用いて、以下の手法によりヘルペスウイルスに対する抗ウイルス活性を評価した。
<Example 1>
Using the sample 1 obtained under the above conditions, antiviral activity against herpes virus was evaluated by the following method.
(1)以下の方法により、HSV1(Herpes simplex virus type 1)(VR3株、ATCCより購入)のウイルス液を調製した。 (1) A virus solution of HSV1 (Herpes simplex virus type 1) (VR3 strain, purchased from ATCC) was prepared by the following method.
(ウイルス液の調製方法)
細胞:Vero細胞(アフリカミドリサル腎細胞、IFN遺伝子の欠損あり)
培地:10%ウシ胎児血清(FBS)、10μg/ml penicillin−streptomycin含有RPMI1640(以下、「RPMI1640培地」、または単に「培地」とも称する。)(和光純薬社製)
(1)−1: T75フラスコで培養したVero細胞に、HSV1を含むウイルスを接種した。
(1)−2: CO2インキュベーター内で1時間培養し、細胞にウイルスを接触、感染させた。
(1)−3: 培養上清を除き、細胞をPBS(−)(リン酸緩衝生理食塩水)で一度洗浄した。
(1)−4: フラスコに10mlの培地を入れ、CO2インキュベーター内で細胞変性効果(CPE、cytopathic effect)が観察されるまで培養した。(約2〜3日)
(1)−5: 培養細胞全体のうち約90%の細胞にCPEが観察された時点で、細胞を凍結融解させた。その後、上清を回収し、遠心分離した(2000rpm、5分、25℃)。
(1)−6: 遠心分離後、上清を回収した。
(1)−7: 回収した上清をウイルス液とし、500μlずつチューブに分注し、使用時まで−80℃で保存した。
(Method for preparing virus solution)
Cells: Vero cells (African green monkey kidney cells, IFN gene deficient)
Medium: RPMI1640 containing 10% fetal bovine serum (FBS), 10 μg / ml penicillin-streptomycin (hereinafter also referred to as “RPMI1640 medium” or simply “medium”) (manufactured by Wako Pure Chemical Industries, Ltd.)
(1) -1: Vero cells cultured in T75 flasks were inoculated with a virus containing HSV1.
(1) -2: The cells were cultured for 1 hour in a CO 2 incubator, and the cells were contacted and infected with the virus.
(1) -3: The culture supernatant was removed, and the cells were washed once with PBS (−) (phosphate buffered saline).
(1) -4: 10 ml of the medium was placed in the flask and cultured in a CO 2 incubator until a cytopathic effect (CPE) was observed. (About 2-3 days)
(1) -5: When CPE was observed in about 90% of the cultured cells, the cells were freeze-thawed. Thereafter, the supernatant was collected and centrifuged (2000 rpm, 5 minutes, 25 ° C.).
(1) -6: After centrifugation, the supernatant was recovered.
(1) -7: The recovered supernatant was used as a virus solution, dispensed in 500 μl aliquots into tubes, and stored at −80 ° C. until use.
(2)(1)で調製したウイルス液と、試料1(下記(4)における終濃度:0.1〜1mg/ml)を、RPMI1640培地中で混合し、反応液を調製した。 (2) The virus solution prepared in (1) and sample 1 (final concentration in the following (4): 0.1 to 1 mg / ml) were mixed in an RPMI1640 medium to prepare a reaction solution.
(3)反応液を室温(25℃)で30分間静置した。 (3) The reaction solution was allowed to stand at room temperature (25 ° C.) for 30 minutes.
(4)RPMI1640培地を用いて反応液を希釈し、ウイルス希釈液を作製(102〜109(PFU(Plaque Forming Unit)/ml))した。 (4) The reaction solution was diluted with RPMI1640 medium to prepare a virus diluted solution (10 2 to 10 9 (PFU (Plaque Forming Unit) / ml)).
なお、本明細書において、PFUは以下のように定義される。すなわち、60μlのウイルス液と、540μlのRPMI1640培地とを混合し、10倍希釈系列を作製した。24wellプレート上に単層培養されたVero細胞に、上記で調製したウイルス液を接種し、PBSで洗浄した。その後、カルボキシメチルセルロース(CMC)(終濃度1%(w/v))を溶解したRPMI1640培地を添加し、37℃で48時間培養した。その後、細胞をクリスタルバイオレットで染色し、染色されない透明な円(プラーク)の数を目視により計測した。1個のプラークを形成するウイルス量を「1PFU」と定義した。 In the present specification, PFU is defined as follows. That is, 60 μl of virus solution and 540 μl of RPMI 1640 medium were mixed to prepare a 10-fold dilution series. Vero cells cultured in a monolayer on a 24-well plate were inoculated with the virus solution prepared above and washed with PBS. Thereafter, RPMI1640 medium in which carboxymethylcellulose (CMC) (final concentration 1% (w / v)) was dissolved was added and cultured at 37 ° C. for 48 hours. Thereafter, the cells were stained with crystal violet, and the number of transparent circles (plaques) not stained was visually measured. The amount of virus forming one plaque was defined as “1PFU”.
(5)(4)で調製したウイルス希釈液(250μl)を、24wellプレート(グライナー社製)にあらかじめ単層培養(37℃の5%CO2インキュベーターで48時間)させたVero細胞(細胞数:1×105/ウェル)に接種させた。 (5) Vero cells (cell number: cells) prepared by monolayer culture (48 hours in a 5% CO 2 incubator at 37 ° C.) in advance on a 24-well plate (manufactured by Greiner) with the virus diluted solution (250 μl) prepared in (4). 1 × 10 5 / well).
(6)ウイルスに接種させたVero細胞を、37℃の5%CO2インキュベーター内で1時間静置させた。 (6) Vero cells inoculated with the virus were allowed to stand for 1 hour in a 5% CO 2 incubator at 37 ° C.
(7)1時間の静置後、上清を捨て、500μlのPBS(−)にて、細胞を洗浄した。 (7) After standing for 1 hour, the supernatant was discarded, and the cells were washed with 500 μl of PBS (−).
(8)500μl/ウェルのRPMI1640培地(1%(w/v)CMCを含む)中にて、37℃の5%CO2インキュベーター内で細胞を培養した。 (8) Cells were cultured in 500 μl / well of RPMI 1640 medium (containing 1% (w / v) CMC) in a 5% CO 2 incubator at 37 ° C.
(9)培地を2日/回のペースで交換しつつ、プラークが出現する5日目まで培養を継続した。 (9) While changing the medium at a rate of 2 days / time, the culture was continued until day 5 when plaque appeared.
(10)培養開始から5日目に、PFU(plaque forming unit)を算出した。 (10) PFU (place forming unit) was calculated on the fifth day from the start of the culture.
PFUの算出結果を図3に示す。図3において、試料1を含まない反応液中でウイルス溶液を静置した場合は、PFU=1.2×109/mlであった。これに対し、試料1を1mg/mlまたは0.1mg/ml含む反応液中でウイルス溶液を静置した場合は、PFUはそれぞれ8.0×106/mlおよび2.8×108/mlであった。
図3に示す通り、試料1には抗ウイルス活性が認められた。
The calculation result of PFU is shown in FIG. In FIG. 3, when the virus solution was allowed to stand in a reaction solution not containing sample 1, the PFU was 1.2 × 10 9 / ml. In contrast, when the virus solution was allowed to stand in a reaction solution containing Sample 1 at 1 mg / ml or 0.1 mg / ml, the PFU was 8.0 × 10 6 / ml and 2.8 × 10 8 / ml, respectively. Met.
As shown in FIG. 3, antiviral activity was observed in Sample 1.
<実施例2>
実施例1と同じ条件にて得られた試料1を用いて、以下の手法により各種細菌に対する抗菌活性を評価した。
<Example 2>
Using sample 1 obtained under the same conditions as in Example 1, antibacterial activity against various bacteria was evaluated by the following method.
具体的には、下記表2に示す培地により、試料濃度が1280μg/mlから1.25μg/mlまでの2倍希釈系列を調製し、菌体と共に96ウェルプレートに収容した(150μl/ウェル)。37℃のインキュベーター内で一晩培養し、菌体の発育の有無を顕微鏡で確認し、試料1の最小発育阻止濃度(MIC、μg/ml)を求めた。結果を表2に示す。 Specifically, a 2-fold dilution series having a sample concentration from 1280 μg / ml to 1.25 μg / ml was prepared using the medium shown in Table 2 below, and contained in a 96-well plate (150 μl / well) together with the cells. The cells were cultured overnight in a 37 ° C. incubator, and the presence or absence of bacterial cell growth was confirmed with a microscope, and the minimum inhibitory concentration (MIC, μg / ml) of Sample 1 was determined. The results are shown in Table 2.
なお、陽性対照として用いたペニシリン系抗生物質であるアモキシシリンは、和光純薬工業より購入した。 Amoxicillin, a penicillin antibiotic used as a positive control, was purchased from Wako Pure Chemical Industries.
上記より、本発明に係る抗菌剤は、肺炎レンサ球菌(S.pneumoniae)に対して特に強い抗菌活性を有することが示された。 From the above, it was shown that the antibacterial agent according to the present invention has a particularly strong antibacterial activity against S. pneumoniae.
Claims (10)
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗ウイルス活性を有する、ペプチド。 An antiviral agent comprising a peptide selected from the group consisting of the following (A) and (B) as an active ingredient;
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity.
前記画分(1)から50%飽和硫安に不溶な画分(2)を得ることを含む、請求項5または6に記載の製造方法。 Recovery of the 5-6 kDa peptide yielded a fraction (1) soluble in 30% saturated ammonium sulfate from the fermented product,
The production method according to claim 5 or 6, comprising obtaining a fraction (2) insoluble in 50% saturated ammonium sulfate from the fraction (1).
(A)LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV(配列番号1)で表わされるアミノ酸配列からなる、ペプチド、
(B)前記アミノ酸配列(A)において、1または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ抗菌活性を有する、ペプチド。 An antibacterial agent comprising a peptide selected from the group consisting of the following (A) and (B) as an active ingredient;
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.
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