JP2000026499A - New peptide, antimicrobial agent, new peptide gene, new recombinant dna and production of new peptide - Google Patents
New peptide, antimicrobial agent, new peptide gene, new recombinant dna and production of new peptideInfo
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- JP2000026499A JP2000026499A JP10197190A JP19719098A JP2000026499A JP 2000026499 A JP2000026499 A JP 2000026499A JP 10197190 A JP10197190 A JP 10197190A JP 19719098 A JP19719098 A JP 19719098A JP 2000026499 A JP2000026499 A JP 2000026499A
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、抗菌活性を誘導し
たカブトムシ体液中に存在する新規ペプチド、該ペプチ
ドを有効成分として含有することを特徴とする抗菌剤、
該ペプチドをコードする新規ペプチド遺伝子、該遺伝子
を含む新規な組み換え体DNA及び新規ペプチドの製造
法に関する。TECHNICAL FIELD The present invention relates to a novel peptide present in a beetle body fluid having induced antibacterial activity, an antibacterial agent comprising the peptide as an active ingredient,
The present invention relates to a novel peptide gene encoding the peptide, a novel recombinant DNA containing the gene, and a method for producing a novel peptide.
【0002】[0002]
【従来の技術】昆虫の体腔内に細菌が侵入すると、生体
防御反応の一つとして、抗菌性タンパク質あるいはペプ
チドが体液中に誘導される。カブトムシの体液より得ら
れる抗菌性ペプチドとして、デイフェンシン[Biochem.
Biophys.Res.Commun.,Vol.220,526-531,(1996)]が知ら
れているのみである。2. Description of the Related Art When bacteria enter the body cavity of an insect, antibacterial proteins or peptides are induced into body fluids as one of the biological defense reactions. As an antimicrobial peptide obtained from the body fluid of a beetle, defensin [Biochem.
Biophys. Res. Commun., Vol. 220, 526-531, (1996)].
【0003】[0003]
【発明が解決しようとする課題】本発明の目的は、カブ
トムシ由来の新規ペプチド及び該ペプチドを有効成分と
して含有する抗菌剤を提供することにある。更に本発明
の目的は、遺伝子工学的手法を用いて該ペプチドを効率
よく生産するための新規な組み換え体DNAを提供する
ことにある。An object of the present invention is to provide a novel beetle-derived peptide and an antibacterial agent containing the peptide as an active ingredient. It is a further object of the present invention to provide a novel recombinant DNA for efficiently producing the peptide using a genetic engineering technique.
【0004】[0004]
【課題を解決するための手段】本発明者は、抗菌活性を
誘導したカブトムシ体液より、大腸菌、黄色ブドウ球菌
等に対して優れた抗菌活性を示す2種類の新規抗菌性ペ
プチドを単離することに成功し、さらに遺伝子工学の手
法を用いて該ペプチドを効率よく生産するための新規な
組み換え体DNAを作成し本発明を完成した。Means for Solving the Problems The present inventors have isolated two kinds of novel antimicrobial peptides having excellent antibacterial activity against Escherichia coli, Staphylococcus aureus and the like from a beetle body fluid having induced antibacterial activity. Succeeded, and a novel recombinant DNA for efficiently producing the peptide was prepared by using a genetic engineering technique, thereby completing the present invention.
【0005】すなわち本発明は、 (1)配列番号1に記載のアミノ酸配列、もしくは該ア
ミノ酸配列において1または複数のアミノ酸が付加、欠
失もしくは置換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、 (2)第(1)項記載のペプチドを有効成分として含有
する抗菌剤、 (3)第(1)項記載のペプチドを有効成分として含有
する抗大腸菌剤、 (4)配列番号2に記載のアミノ酸配列、もしくは該ア
ミノ酸配列において1または複数のアミノ酸が付加、欠
失もしくは置換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、 (5)第(4)項記載のペプチドを有効成分として含有
する抗菌剤、 (6)第(4)項記載のペプチドを有効成分として含有
する抗黄色ブドウ球菌剤、That is, the present invention relates to (1) a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence, and which has an antibacterial activity. (2) an antibacterial agent containing the peptide according to item (1) as an active ingredient; (3) an anti-Escherichia coli agent containing the peptide according to item (1) as an active ingredient; Or a peptide comprising an amino acid sequence in which one or more amino acids have been added, deleted or substituted in said amino acid sequence, and having an antibacterial activity; (5) The peptide according to (4) as an active ingredient (6) an anti-Staphylococcus aureus agent comprising the peptide according to (4) as an active ingredient;
【0006】(7)第(1)項又は第(4)項記載の新
規ペプチドをコードする遺伝子 (8)第(7)項記載の新規ペプチド遺伝子をベクター
DNAに挿入してなることを特徴とする新規な組み換え
体DNA、 (9)カブトムシ幼虫より第(1)項又は第(4)項記
載のペプチドを取得することを特徴とするペプチドの製
造法を提供する。(7) A gene encoding the novel peptide according to item (1) or (4). (8) A novel peptide gene according to item (7) is inserted into a vector DNA. (9) A method for producing a peptide, which comprises obtaining the peptide according to (1) or (4) from a beetle larva.
【0007】本発明の新規ペプチド(以下、「カブトシ
ン」という)は、例えば抗菌活性を誘導したカブトムシ
体液より、タンパク質を単離する通常の方法を用いて生
産することができる。また、通常のペプチド合成法、あ
るいは遺伝子工学的手法を用いて生産することも可能で
ある。[0007] The novel peptide of the present invention (hereinafter referred to as "beetlesin") can be produced, for example, from a body fluid of a beetle in which antibacterial activity has been induced, using a conventional method for isolating a protein. Further, it can be produced using a conventional peptide synthesis method or a genetic engineering technique.
【0008】遺伝子工学的手法としては、例えば、新規
ペプチド遺伝子が挿入された組み換え体DNAにより形
質転換又は形質導入された宿主細胞を用いて新規ペプチ
ドを生産する方法や、ウサギ網状赤血球、コムギ胚芽等
を用いた無細胞タンパク質合成系を用いて該組み換え体
DNAを翻訳してカブトシンを生産する方法等を用いる
ことができる。[0008] Examples of the genetic engineering techniques include a method for producing a novel peptide using a host cell transformed or transduced with a recombinant DNA into which a novel peptide gene has been inserted, a rabbit reticulocyte, a wheat germ and the like. And a method of producing the buttocin by translating the recombinant DNA using a cell-free protein synthesis system using E. coli.
【0009】[0009]
【発明の実施の形態】以下に、抗菌活性を誘導したカブ
トムシ体液よりカブトシンを単離する方法及び遺伝子工
学的手法によるカブトシンの生産方法について詳細に説
明する。 〔1〕抗菌活性を誘導したカブトムシ体液よりカブトシ
ンを単離する方法 カブトシンを得るための出発原料は、抗菌活性を誘導し
たカブトムシ体液であれば如何なるものでもよい。抗菌
活性の誘導法としては、例えばカブトムシの幼虫の体腔
内に、大腸菌の生理食塩水懸濁液を注入する方法が挙げ
られる。BEST MODE FOR CARRYING OUT THE INVENTION A method for isolating beetosine from a body fluid of a beetle in which an antibacterial activity has been induced and a method for producing beetosine by genetic engineering will be described in detail below. [1] Method of Isolating Beetle from Beetle Body Fluid with Induced Antibacterial Activity The starting material for obtaining beetosine may be any beetle body fluid with induced antibacterial activity. As a method for inducing antibacterial activity, for example, a method of injecting a physiological saline suspension of Escherichia coli into the body cavity of a larva of a beetle can be mentioned.
【0010】抗菌活性の誘導後20〜28時間後にカブ
トムシの幼虫の足を切断して体液を集める。この体液を
加熱した後、遠心分離して上清を得る。Twenty to twenty-eight hours after the induction of the antimicrobial activity, the larval feet of the beetle are cut off and the body fluid is collected. After heating this body fluid, it is centrifuged to obtain a supernatant.
【0011】次いで、得られた上清を逆相カラムクロマ
トグラフィーに供する。吸着画分をトリフルオロ酢酸を
含むアセトニトリルと水との混合液で溶出し抗菌活性画
分を集める。ここで用いる逆相カラムとしては例えば商
品名:セップパックC18カートリッジ(ウオーターズ
社製)等があげられる。Next, the obtained supernatant is subjected to reverse phase column chromatography. The adsorbed fraction is eluted with a mixture of acetonitrile containing trifluoroacetic acid and water, and an antibacterial active fraction is collected. As the reversed phase column used here, for example, a product name: Seppak C18 cartridge (manufactured by Waters) can be mentioned.
【0012】次いで、得られた抗菌活性画分をゲル濾過
のFPLCあるいはHPLCに供する。ここで用いるF
PLC用ゲル濾過カラムとしては例えば商品名:スーパ
ーデックス75 HR 10/30 (ファルマシア社製)、HPL
C用ゲル濾過カラムとしては、例えば商品名:TSKgel S
W2000 (東ソー社製)等があげられる。Next, the obtained antibacterial active fraction is subjected to gel filtration FPLC or HPLC. F used here
Examples of gel filtration columns for PLC include trade names: Superdex 75 HR 10/30 (Pharmacia), HPL
As the gel filtration column for C, for example, trade name: TSKgel S
W2000 (manufactured by Tosoh Corporation) and the like.
【0013】次いで、ゲル濾過によって得られた抗菌活
性画分を、逆相FPLCあるいは逆相HPLCに供す
る。吸着画分をトリフルオロ酢酸を含むアセトニトリル
水溶液の濃度勾配溶出液により溶出することにより二つ
の抗菌活性ピークが得られる。このうちより高いアセト
ニトリル濃度で溶出される抗菌活性を有する画分を集め
る。逆相FPLCカラムとしては例えば商品名:Pep RP
C HR 5/5(ファルマシア社製)、逆相HPLCカラムと
しては例えば商品名:カプセルパック C18 SG300(資生
堂社製)等があげられる。Next, the antibacterial active fraction obtained by gel filtration is subjected to reverse phase FPLC or reverse phase HPLC. Two antimicrobial activity peaks are obtained by eluting the adsorbed fraction with a concentration gradient eluent of acetonitrile aqueous solution containing trifluoroacetic acid. Among these, the fraction having antibacterial activity eluted at a higher acetonitrile concentration is collected. As the reversed-phase FPLC column, for example, trade name: Pep RP
Examples of C HR 5/5 (Pharmacia) and reverse phase HPLC columns include, for example, Capsule Pack C18 SG300 (manufactured by Shiseido) and the like.
【0014】集められた抗菌活性画分を、再度、逆相H
PLCに供する。トリフルオロ酢酸を含むアセトニトリ
ルの濃度勾配により溶出させる。アセトニトリルの濃度
勾配を緩くすることにより、本発明の2種類の抗菌性ペ
プチド、配列番号1(配列番号1をカブトシンAとい
う)と配列番号2(配列番号2をカブトシンBという)
が分離して溶出され、それぞれを単離することができ
る。The collected antibacterial active fraction is again subjected to reverse phase H
Subject to PLC. Elution is carried out with a gradient of acetonitrile containing trifluoroacetic acid. By relaxing the concentration gradient of acetonitrile, two kinds of antimicrobial peptides of the present invention, SEQ ID NO: 1 (SEQ ID NO: 1 is referred to as Kabutosin A) and SEQ ID NO: 2 (SEQ ID NO: 2 is referred to as Kabutosin B)
Are separated and eluted, and each can be isolated.
【0015】なお、本発明の抗菌性ペプチドを精製する
際の抗菌活性の測定は、平板培地での増殖阻止円の形成
を指標とし、検定菌として例えば黄色ブドウ球菌 (Stap
hylococcus aureus subsp. aureus ATCC 6538P) を用い
て行なうことができる。The antibacterial activity of the antibacterial peptide of the present invention is measured by using the formation of a growth inhibition circle on a plate medium as an index.
aureus ATCC 6538P).
【0016】〔2〕遺伝子工学的手法によるカブトシン
の生産方法 以下に、カブトシン遺伝子のクローニング方法及びカブ
トシン遺伝子を含むDNAを挿入した組み換え体DNA
により、宿主細胞を形質転換又は形質導入し、得られた
組み換え微生物をもちいてカブトシンを生産する方法に
ついて説明する。[2] Method for Producing Kabutosin by Genetic Engineering Technique The following describes a method for cloning a katocin gene and a recombinant DNA into which a DNA containing the kabutosin gene has been inserted.
A method for transforming or transducing a host cell to produce beetosine using the obtained recombinant microorganism will be described.
【0017】カブトシン遺伝子を含むDNAはカブトム
シゲノムDNA又はcDNA由来の天然の遺伝子をクロ
ーニングすることにより得られる。該遺伝子のクローニ
ング方法としては、本発明の二種の抗菌性ペプチドのア
ミノ酸配列に基ずき適当なプローブDNAを合成して、
これを用いてカブトムシゲノムDNA又はcDNAのラ
イブラリーからスクリーニングする方法、あるいは該ペ
プチドのアミノ酸配列に基ずき適当なプライマーDNA
を作成して、5’RACE法や3’RACE法等の適当
なPCR法により該遺伝子の断片を含むDNAを特異的
に増幅させて得、これらを連結させて全長の遺伝子を含
むDNAを得る方法等があげられる。The DNA containing the beetosine gene can be obtained by cloning a natural gene derived from a beetle genomic DNA or cDNA. As a method for cloning the gene, a suitable probe DNA is synthesized based on the amino acid sequences of the two antibacterial peptides of the present invention,
A method for screening from a library of beetle genomic DNA or cDNA using the same, or an appropriate primer DNA based on the amino acid sequence of the peptide
And DNA containing the gene fragment is specifically amplified by an appropriate PCR method such as the 5′RACE method or the 3′RACE method, and these are ligated to obtain a DNA containing the full-length gene. Method and the like.
【0018】カブトシン遺伝子がコードするアミノ酸配
列は、抗菌活性を失うことのない限り、1もしくは複数
のアミノ酸が付加、欠失もしくは置換されていてもよ
い。抗菌活性を失うことのない範囲でアミノ酸配列が変
更されたカブトシン遺伝子は、全て本発明に含まれる。
カブトシン遺伝子に変異を導入する方法としては該遺伝
子と変異原となる薬剤、具体的にはヒドロキシルアミ
ン、亜硝酸、亜硫酸、5’−ブロモウラシル等を接触さ
せる方法を挙げることができる。この他、紫外線照射
法、カセット変異法、PCR法を用いた部位特異的変異
導入法等の方法を広く用いることができる。さらには、
化学合成したDNAをアニーリングして所望の部位に変
異を有する変異型カブトシン遺伝子を構築することも可
能である。One or more amino acids may be added, deleted or substituted in the amino acid sequence encoded by the beetosine gene as long as the amino acid sequence does not lose the antibacterial activity. The invention of the present invention includes all butohsin genes whose amino acid sequences have been changed within a range that does not cause loss of antibacterial activity.
Examples of a method for introducing a mutation into a beetosine gene include a method in which the gene is brought into contact with a mutagenic agent, specifically, hydroxylamine, nitrite, sulfurous acid, 5′-bromouracil, or the like. In addition, methods such as ultraviolet irradiation, cassette mutation, and site-specific mutagenesis using PCR can be widely used. Moreover,
It is also possible to anneal chemically synthesized DNA to construct a mutated kabutosin gene having a mutation at a desired site.
【0019】カブトシン遺伝子を含むDNAはその両端
に適当な制限酵素、例えば、EcoRIやSalI等の
認識部位が付与されていることが好ましい。これによ
り、該DNAのベクターDNAへの挿入を効率よく行な
うことができる。It is preferable that the DNA containing the beetosine gene is provided with an appropriate restriction enzyme at both ends thereof, for example, a recognition site such as EcoRI or SalI. Thereby, the DNA can be efficiently inserted into the vector DNA.
【0020】カブトシン遺伝子を含むDNAを化学合成
する場合、該DNAを複数のオリゴヌクレオチドに分け
て化学合成し、これらをアニーリングした後、DNAリ
ガーゼにより結合する。その場合のコドンは、カブトシ
ンのアミノ酸配列に影響を与えないコドンであって、か
つ宿主細胞で頻繁に使用されるコドンに置換することが
好ましい。これにより、天然のカブトシン遺伝子を用い
た場合に比べ、より大量のカブトシンタンパク質を生産
することができる。In the case of chemically synthesizing a DNA containing a beetosine gene, the DNA is divided into a plurality of oligonucleotides, chemically synthesized, annealed, and then ligated by DNA ligase. In such a case, the codon which does not affect the amino acid sequence of cabtocin is preferably replaced with a codon frequently used in a host cell. As a result, a larger amount of a beetosine protein can be produced as compared with a case where a natural beetosine gene is used.
【0021】本発明のカブトシン遺伝子を含むDNAは
容易に増幅することができる。すなわち、該DNAを適
当なベクターDNA、例えばプラスミドDNA、バクテ
リオファージDNA等に挿入して組み換え体DNAを得
る。次いで、該組み換え体DNAを用いて宿主細胞、例
えば大腸菌等を形質転換あるいは形質導入して組み換え
微生物を得る。この組み換え微生物を培養し、常法を用
いて組み換え体DNAを調整した後、適当な制限酵素を
用いてインサートDNAを切り出し、これを精製すれば
よい。尚、組み換え体微生物より組み換え体DNAを調
整する際に、ジデオキシ法[Methods Enzymol.,Vol.101,
20-78(1983)]等により、インサートDNAの塩基配列を
確認することが好ましい。The DNA containing the beetosine gene of the present invention can be easily amplified. That is, the DNA is inserted into an appropriate vector DNA, for example, a plasmid DNA, bacteriophage DNA or the like, to obtain a recombinant DNA. Next, a host cell, for example, Escherichia coli or the like is transformed or transduced with the recombinant DNA to obtain a recombinant microorganism. After culturing the recombinant microorganism and preparing a recombinant DNA using a conventional method, the insert DNA may be cut out using an appropriate restriction enzyme and purified. When preparing recombinant DNA from a recombinant microorganism, the dideoxy method [Methods Enzymol., Vol. 101,
20-78 (1983)] and the like, it is preferable to confirm the base sequence of the insert DNA.
【0022】カブトシンを生産するための宿主細胞とし
ては、真核細胞及び原核細胞のいずれをも用いることが
できる。真核細胞としては動物、植物、昆虫、酵母等の
細胞が、また原核細胞としては大腸菌、枯草菌、放線菌
等が挙げられる。宿主細胞として植物や昆虫の細胞を用
いる場合、培養細胞である必要はなく、植物体や虫体、
具体的にはタバコの植物体やカイコの幼虫等であっても
よい。[0022] Eukaryotic cells and prokaryotic cells can be used as host cells for producing beetosine. Eukaryotic cells include cells of animals, plants, insects, yeasts and the like, and prokaryotic cells include Escherichia coli, Bacillus subtilis, actinomycetes and the like. When plant or insect cells are used as host cells, they do not need to be cultured cells.
Specifically, it may be a tobacco plant or a silkworm larva.
【0023】カブトシンのような抗菌性ペプチドを大腸
菌等の原核細胞に生産させる場合、目的とするペプチド
を、β−ガラクトシダーゼ、β−ラクタマーゼ、マルト
ース結合タンパク質あるいはプロテインA等との融合タ
ンパク質として発現させることが好ましい。融合タンパ
ク質として発現されたカブトシンは抗菌活性を示さない
ので、宿主である原核細胞の増殖を抑制しない。本発明
において宿主として使用できる原核細胞としては、エッ
シェリシア属の菌株、例えばEscherichia coliJM109、E
scherichia coli HB101 (ATCC33694)等を挙げることが
できる。When an antimicrobial peptide such as cabtocin is produced in a prokaryotic cell such as Escherichia coli, the target peptide is expressed as a fusion protein with β-galactosidase, β-lactamase, maltose binding protein or protein A. Is preferred. Since beetosine expressed as a fusion protein does not show antibacterial activity, it does not inhibit the growth of prokaryotic host cells. Prokaryotic cells that can be used as hosts in the present invention include Escherichia strains such as Escherichia coli JM109, E.
scherichia coli HB101 (ATCC33694) and the like.
【0024】融合タンパク質として発現された抗菌性ペ
プチドは、該融合タンパク質が可溶性である場合にはア
フィニティークロマトグラフィーを用いることにより、
また不溶性である場合には遠心分離処理を用いることに
より、容易に精製することができる。The antimicrobial peptide expressed as a fusion protein can be purified by using affinity chromatography when the fusion protein is soluble.
When it is insoluble, it can be easily purified by centrifugation.
【0025】カブトシンペプチドを発現させるためのベ
クターDNAは、宿主細胞で複製可能なものであれば如
何なるものでもよく、例えば、プラスミドDNA、バク
テリオファージDNA等が挙げられる。宿主細胞が大腸
菌である場合のベクターDNAとしては、例えばプラス
ミドpMAL-C2(NEW England Labs社製)、pGEX-5X-1(フ
ァルマシア社製)、pXa1(ベーリンガー社製)等を用い
ることができる。The vector DNA for expressing the beetin peptide may be any vector DNA as long as it can be replicated in a host cell, and examples thereof include plasmid DNA and bacteriophage DNA. As the vector DNA when the host cell is Escherichia coli, for example, plasmid pMAL-C2 (manufactured by NEW England Labs), pGEX-5X-1 (manufactured by Pharmacia), pXa1 (manufactured by Boehringer) and the like can be used.
【0026】次いで、ベクターDNAの適当な制限酵素
部位にカブトシン遺伝子を含むDNAを挿入して組み換
え体DNAを調製する。この組み換え体DNAを用い
て、宿主細胞を形質転換あるいは形質導入することによ
り、組み換え微生物を得ることができる。形質転換はデ
ィー・エム・モーリソン法[ Methods Enzymol.,68巻,3
26-331頁(1979)]、塩化カルシウム法[Gene,6巻,23
頁,(1979)]等により、また形質導入はビー・ホーン法
[Methods Enzymol.,68巻,299-309頁(1979)]等により
行なうことができる。Next, a recombinant DNA is prepared by inserting a DNA containing the beetosine gene into an appropriate restriction enzyme site of the vector DNA. By using this recombinant DNA to transform or transduce a host cell, a recombinant microorganism can be obtained. Transformation was carried out by the DM Morrison method [Methods Enzymol., 68, 3
26-331 (1979)], calcium chloride method [Gene, 6, 23
, (1979)], and transduction can be performed by the Beehone method [Methods Enzymol., 68, 299-309 (1979)].
【0027】次いで、この組み換え微生物を培地に培養
し、培養物からカブトシンを採取する。Next, this recombinant microorganism is cultured in a medium, and cabtocin is collected from the culture.
【0028】宿主細胞として真核細胞を用い、カブトシ
ンを単独の形で発現させる場合には、カブトシンの採取
方法として、通常のタンパク質単離方法を用いることが
できる。すなわち、リゾチーム等の酵素を用いた溶菌処
理、超音波破砕処理、磨砕処理等により菌体を破壊し、
カブトシンを菌体外に排出させる。次いで、カブトムシ
体液よりカブトシンを単離する時と同様の方法、すなわ
ちゲル濾過法、イオン交換樹脂、逆相HPLCを用いる
ことにより高度に精製されたカブトシン標品を得ること
ができる。When eukaryotic cells are used as host cells and cabtocin is expressed in a single form, an ordinary protein isolation method can be used as a method for collecting cabtocin. In other words, bacteriolysis using enzymes such as lysozyme, ultrasonic crushing, disruption of the cells by grinding, etc.,
The buttocin is excreted outside the cells. Next, a highly purified beetosine preparation can be obtained by using the same method as used for isolating beetosine from a beetle body fluid, that is, gel filtration, ion exchange resin, and reverse phase HPLC.
【0029】宿主細胞として真核細胞または原核細胞を
用い、カブトシンを融合タンパク質として発現させた場
合は、菌体を破壊した後、遠心分離処理やアフィニティ
ークロマトグラフィー等の組合せにより、融合タンパク
質を得る。次いで、ファクターXa(ベーリンガー・マ
ンハイム社製)等を用いた酵素処理により融合タンパク
質からカブトシンペプチドを切り出し、これを精製す
る。In the case where eukaryotic cells or prokaryotic cells are used as host cells and cabtocin is expressed as a fusion protein, the fusion protein is obtained by disrupting the cells and then performing a combination of centrifugation, affinity chromatography and the like. Next, a captocin peptide is cut out from the fusion protein by an enzyme treatment using Factor Xa (manufactured by Boehringer Mannheim) or the like, and purified.
【0030】遺伝子工学的手法により得られるカブトシ
ンは、抗菌活性を誘導したカブトムシ体液より単離され
たカブトシンと全く同様の抗菌活性を示すものである。The beetosine obtained by the genetic engineering technique exhibits exactly the same antibacterial activity as that of the beetle isolated from the beetle body fluid in which the antibacterial activity is induced.
【0031】カブトシンは、例えば、米飯、パンなどの
腐敗を起こすバチルス・ズブチリス(Bacillus subtili
s) 、食中毒の原因菌である黄色ブドウ球菌(Staphyloco
ccusaureus) 、大腸菌(Escherichia coli)をはじめ、バ
チルス(Bacillus) 属、スタフィロコッカス(Staphyloc
occus)属、クロストリジウム(Clostridium)属、ストレ
プトコッカス(Streptococcus)属等のグラム陽性細菌、
エシェリシア(Escherichia)属、シュウドモナス(Pseudo
monas)属等のグラム陰性細菌等に広く抗菌活性を発揮し
うる。特に、カブトシンAは大腸菌に、カブトシンBは
黄色ブドウ球菌に抗菌活性を示した。具体的なデーター
を表1に示した。Kabutosin is, for example, Bacillus subtili which causes spoilage of rice, bread and the like.
s), Staphylococcus aureus (Staphyloco
ccusaureus), Escherichia coli, Bacillus, Staphylococcus
occus) genus, Clostridium (Clostridium) genus, Streptococcus (Streptococcus) gram-positive bacteria such as genus,
Escherichia, Pseudomonas
It can exert antibacterial activity widely on Gram-negative bacteria such as Monas). In particular, cabtocin A showed antibacterial activity against Escherichia coli, and cabtocin B showed antibacterial activity against Staphylococcus aureus. Table 1 shows specific data.
【0032】カブトシンは、そのままで、または通常用
いられる固体担体、液体担体、乳化分散剤等により錠
剤、粉剤、水和剤、乳剤、カプセル剤等の形に製剤化し
て抗菌剤として使用することができる。上記担体として
は、水、ゼラチン、澱粉、ステアリン酸マグネシウム、
ラクトース、アラビアゴム、植物油等が挙げられる。カ
ブトシンを抗菌剤として使用する場合には、抗菌活性を
誘導したカブトムシより得られる抽出液や、遺伝子工学
的手法で得られる抗菌活性を有する粗培養液を未精製の
まま使用することもできる。Kabutosin can be used as an antibacterial agent as it is or as a tablet, powder, wettable powder, emulsion, capsule or the like using a commonly used solid carrier, liquid carrier, emulsifying dispersant or the like. it can. As the carrier, water, gelatin, starch, magnesium stearate,
Lactose, gum arabic, vegetable oil and the like. When beetosine is used as an antibacterial agent, an extract obtained from a beetle having induced antibacterial activity or a crude culture having antibacterial activity obtained by a genetic engineering technique can be used without purification.
【0033】上記でいう抗菌剤には、食品用防腐剤、医
療用抗菌剤、建材・塗料用防腐剤、農園芸用抗菌剤、家
畜飼料用防腐剤、養魚飼料用防腐剤等が包含され、広汎
な分野で利用することができる。The antibacterial agents mentioned above include food preservatives, medical antibacterial agents, building materials and paint preservatives, agricultural and horticultural antibacterial agents, livestock feed preservatives, fish feed preservatives, and the like. It can be used in a wide range of fields.
【0034】カブトシンを、食品用防腐剤として食品に
添加する場合は、食品中に混合する方法、食品表面に塗
布する方法等が例示される。その場合は、食品1kg当
たり20mg以上、好ましくは、50〜500mg程度
添加すればよい。When beetosine is added to a food as a food preservative, there are exemplified a method of mixing it into the food, a method of applying it to the surface of the food, and the like. In that case, 20 mg or more, preferably about 50 to 500 mg, may be added per 1 kg of food.
【0035】適用できる食品としては、カマボコ、チク
ワ等の水産練り製品、ハム、ソーセージ等の畜肉加工
品、清涼飲料、果実飲料等の飲料類、ケーキ、プリン、
饅頭等の菓子類が例示される。Examples of applicable foods include fish paste products such as kamaboko and chikuwa, processed meat products such as ham and sausage, beverages such as soft drinks and fruit drinks, cakes, puddings, and the like.
Sweets such as buns are exemplified.
【0036】カブトシンを有効成分として含有する抗菌
剤は、さらに他の殺菌剤、医薬品、防腐剤、食品添加物
等と適宜混合して使用することも可能である。The antibacterial agent containing cabtocin as an active ingredient can be used by being appropriately mixed with other bactericides, pharmaceuticals, preservatives, food additives and the like.
【0037】以下に、抗菌活性を誘導したカブトムシ体
液からカブトシンを単離する場合の実施例、遺伝子工学
的手法によりカブトシンを効率よく生産するための組み
換え体DNAを作成する場合の実施例及び抗菌活性試験
例により本発明を具体的に説明するが、これらにより本
発明の範囲が限定されるものではない。The following is an example in which beetosine is isolated from a beetle body fluid in which an antibacterial activity has been induced, an example in which a recombinant DNA for producing beetosine efficiently by genetic engineering techniques, and an antibacterial activity. The present invention will be specifically described by test examples, but these are not intended to limit the scope of the present invention.
【0038】[0038]
〔実施例1〕カブトシンA及びBの単離及び構造解析 (1)抗菌活性を誘導したカブトムシ体液からのカブト
シンA及びBの単離 カブトムシ(Allomyrina dichotoma)の3令幼虫((有)
つくばチキンより購入)に大腸菌(Escherichia coli JM
109,宝酒造社製)の生理食塩水懸濁液(2.5×10 7個/ml)
を0.02ml/匹の割合で体腔内に注射し、抗菌活性
を誘導した。24時間後、幼虫の足を切断して体液を放
出させ、予めアプロチニン(シグマ社製)を加えてある
遠心管に集めた。なお、アプロチニンの最終濃度は約2
0μg/mlになるようにした。この体液を直ちに遠心
分離して血球成分を沈澱させ、上澄みを回収した。回収
した上澄みを95℃で5分間加熱処理したのち、再度遠
心分離し上清を得た。 Example 1 Isolation and Structural Analysis of Beetle A and B
Isolation of Shin A and B. Third instar larva of beetle (Allomyrina dichotoma)
Escherichia coli JM (purchased from Tsukuba Chicken)
109, manufactured by Takara Shuzo Co., Ltd. in physiological saline (2.5 × 10 7(Pcs / ml)
Was injected into the body cavity at a rate of 0.02 ml / animal, and antibacterial activity
Was induced. Twenty-four hours later, the larvae's feet are cut and the body fluid is released.
Aprotinin (Sigma)
Collected in a centrifuge tube. The final concentration of aprotinin was about 2
It was adjusted to 0 μg / ml. Immediately centrifuge this body fluid
The blood cells were separated and sedimented, and the supernatant was recovered. Collection
After heating the supernatant at 95 ° C for 5 minutes,
Separation was performed to obtain a supernatant.
【0039】得られた遠心上清のうち3mlを0.05
%トリフルオロ酢酸で平衡化したセップパックC18カ
ートリッジ(ウオーターズ社製)に流した。吸着画分を
0.05%トリフルオロ酢酸を含む10、20、30、
40、50、及び60%の各アセトニトリル水溶液、更
に0.05%トリフルオロ酢酸を含むアセトニトリルを
5mlずつ順次流すことにより溶出させた。溶出液はア
セトニトリル濃度に応じて分画した。30、40及び5
0%アセトニトリルで溶出された画分に抗菌活性を認め
た。3 ml of the obtained centrifuged supernatant was added to 0.05
The solution was allowed to flow through a Seppak C18 cartridge (manufactured by Waters) equilibrated with trifluoroacetic acid. 10, 20, 30, containing 0.05% trifluoroacetic acid
Elution was carried out by sequentially flowing 5 ml of each of acetonitrile aqueous solutions of 40, 50 and 60%, and acetonitrile containing 0.05% of trifluoroacetic acid. The eluate was fractionated according to the acetonitrile concentration. 30, 40 and 5
Antimicrobial activity was observed in the fraction eluted with 0% acetonitrile.
【0040】得られた活性画分を1つにまとめて遠心エ
バポレーターで 濃縮したのち、PBS(1L当りNaC
l 8g,Na2PO4 1.44g, KH2PO4 0.
24g,KCl 0.2g,pH7.4)で平衡化した
Superdex 75 HR 10/30(ファルマシア社製)のFPL
Cカラムに流してゲル濾過を行なった。溶出にはPBS
を用いた。 流速は0.5ml/分とし、溶出液は0.
5mlずつ分画した。抗菌活性画分を集め、濃縮した。The obtained active fractions were combined, concentrated by a centrifugal evaporator, and then concentrated in PBS (NaC per liter).
l 8g, Na 2 PO 4 1.44g , KH 2 PO 4 0.
24 g, KCl 0.2 g, pH 7.4)
Superdex 75 HR 10/30 (Pharmacia) FPL
Gel filtration was performed by flowing through a C column. PBS for elution
Was used. The flow rate was 0.5 ml / min, and the eluate was 0.1 ml / min.
Each 5 ml was fractionated. Antibacterial active fractions were collected and concentrated.
【0041】得られた濃縮液を、0.05%トリフルオ
ロ酢酸で平衡化したPepRPC HR 5/5(ファルマシア社
製)の逆相FPLCカラムに供した。 吸着画分を0.
05%トリフルオロ酢酸を含むアセトニトリルの濃度勾
配により溶出させた。すなわち、120分間にわたって
アセトニトリルの濃度を0%から40%に上昇させた。
流速は0.25ml/分とし、溶出液は0.25mlず
つ分画した。抗菌活性は2つのピークに分かれ、このう
ち後に溶出した活性画分を濃縮した。なお先に溶出した
活性画分にはカブトムシの既知の抗菌性タンパク質であ
るデフェンシンが含まれていた。The obtained concentrate was applied to a reverse phase FPLC column of PepRPC HR 5/5 (Pharmacia) equilibrated with 0.05% trifluoroacetic acid. If the adsorbed fraction is 0
Elution was carried out with a gradient of acetonitrile containing 05% trifluoroacetic acid. That is, the concentration of acetonitrile was increased from 0% to 40% over 120 minutes.
The flow rate was 0.25 ml / min, and the eluate was fractionated by 0.25 ml. The antibacterial activity was divided into two peaks, and the active fraction eluted later was concentrated. The active fraction eluted earlier contained defensin, a known antibacterial protein of beetles.
【0042】後から溶出した活性画分の濃縮液を0.0
5%トリフルオロ酢酸で平衡化したmRPC C2/C
18 PC3.2/3(ファルマシア社製)の逆相カラ
ムに供し、更に精製を行なった。吸着画分は0.05%
トリフルオロ酢酸を含むアセトニトリルの濃度勾配によ
り溶出された。すなわち、まず5分間にわたってアセト
ニトリルの濃度を0%から20%に上昇させた後、さら
に60分間にわたって20%から30%に上昇させた。
流速は0.25ml/分とし、225nmの吸光度をモ
ニターしながら溶出させた。60分あたりに溶出した2
つのピークをそれぞれ回収し、先に溶出したものをカブ
トシンB、後に溶出したものをカブトシンAとした。こ
のようにして3mlのカブトムシ体液からカブトシンA
及びBをそれぞれ1μgを得た。なお、タンパク質は2
25nmの吸光度より算出し、11ODを1mgとし
た。The concentrated solution of the active fraction eluted later was added to 0.0
MRPC C2 / C equilibrated with 5% trifluoroacetic acid
It was applied to a reverse phase column of 18PC 3.2 / 3 (manufactured by Pharmacia) and further purified. Adsorption fraction is 0.05%
It was eluted with a gradient of acetonitrile containing trifluoroacetic acid. That is, first, the concentration of acetonitrile was increased from 0% to 20% over 5 minutes, and then from 20% to 30% over 60 minutes.
The elution was performed at a flow rate of 0.25 ml / min while monitoring the absorbance at 225 nm. 2 eluted per 60 minutes
The two peaks were collected, and the one that eluted first was designated as captocin B, and the one that eluted later was designated as captocin A. In this way, 3 ml of beetle body fluid is converted into beetin A
And B were each obtained in an amount of 1 μg. The protein is 2
It was calculated from the absorbance at 25 nm, and 11 OD was defined as 1 mg.
【0043】なお、カブトシンA及びBを精製する際の
抗菌活性の測定は、 平板培地での増殖阻止円の形成を
指標とし [Nature,Vol.292, 246(1981)]、検定菌とし
て黄色ブドウ球菌(Staphylococcus aureus subsp. aur
eus ATCC 6538P)を用いて行なった。The antibacterial activity when purifying beetosine A and B was measured using the formation of a growth inhibition circle on a plate medium as an index [Nature, Vol. 292, 246 (1981)]. Staphylococcus aureus subsp. Aur
eus ATCC 6538P).
【0044】(2)カブトシンA及びBの構造解析 (a)質量分析 質量分析計(M−1200H型LC/MSシステム、日
立製作所社製)を用いてエレクトロスプレーイオン化法
により測定した。測定の結果、カブトシンBは7,73
1Da、カブトシンAは7,740Daの値を得た。(2) Structural Analysis of Kabutosin A and B (a) Mass Spectrometry The mass was measured by an electrospray ionization method using a mass spectrometer (M-1200H LC / MS system, manufactured by Hitachi, Ltd.). As a result of the measurement, 7,73 of beetin B was detected.
1Da and Kabutosin A obtained a value of 7,740 Da.
【0045】(b)N末端アミノ酸配列 カブトシンA及びBのN末端アミノ酸配列を、プロテイ
ンシーケンサー(473A型アプライドバイオシステム
ズ社製)を用いて、エドマン法により分析した。得られ
たカブトシンAのN末端アミノ酸配列を配列番号3に、
カブトシンBのそれを配列番号4にそれぞれ示した。(B) N-terminal amino acid sequence The N-terminal amino acid sequences of the beetins A and B were analyzed by the Edman method using a protein sequencer (type 473A, manufactured by Applied Biosystems). The obtained N-terminal amino acid sequence of beetosine A is shown in SEQ ID NO: 3,
It is shown in SEQ ID NO: 4 for that of buttocin B, respectively.
【0046】(c)プロテアーゼ消化物のアミノ酸配列 カブトシンA及びBをリシルエンドペプチダーゼ(和光
純薬社製)で消化した後、生じたペプチド断片を逆相H
PLC(逆相カラム:カプセルパックC8 SG300
資生堂社製)で単離し、それぞれ得られた断片のアミ
ノ酸配列をプロテインシークエンサーで分析した。カブ
トシンAについては配列番号5及び配列番号6に示した
アミノ酸配列を有する断片を得た。カブトシンBについ
ては前記配列番号4に加えて、配列番号5に示したアミ
ノ酸配列を有する断片を得た。(C) Amino acid sequence of protease digested product After digesting cabtocins A and B with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), the resulting peptide fragment was subjected to reverse phase H
PLC (reverse phase column: Capsule Pack C8 SG300
(Manufactured by Shiseido Co., Ltd.), and the amino acid sequence of each obtained fragment was analyzed using a protein sequencer. With respect to cabtocin A, fragments having the amino acid sequences shown in SEQ ID NOs: 5 and 6 were obtained. With respect to cabtocin B, a fragment having the amino acid sequence shown in SEQ ID NO: 5 in addition to SEQ ID NO: 4 was obtained.
【0047】(3)抗菌活性測定 種々の細菌に対するカブトシンA及びBの抗菌活性は、
[Eur.J.Biochem., 187巻, 381-386 頁 (1990)] 記載の
方法に準じて、最小増殖阻止濃度(M.I.C.)により
求めた。(3) Measurement of antibacterial activity The antibacterial activities of cabtocin A and B against various bacteria are as follows.
According to the method described in [Eur. J. Biochem., 187, 381-386 (1990)], it was determined by the minimum growth inhibitory concentration (MIC).
【0048】(検定菌) 大腸菌(Escherichia coli JM109 宝酒造社製) 黄色ブドウ球菌(Stahpylococcus aureus subsp. aureu
s ATCC 6538P) 枯草菌(Bacillus subtilis ISW 1214 岩城硝子社製)(Test Bacteria) Escherichia coli (Escherichia coli JM109, manufactured by Takara Shuzo) Staphylococcus aureus subsp. Aureu
s ATCC 6538P) Bacillus subtilis ISW 1214 (Iwaki Glass)
【0049】(抗菌活性試験法)検定菌を3%TBS
(triptic soy broth デイフコ社製)の液体培地で37℃
で対数増殖期まで培養した後、遠心分離で菌体を集め、
10mM リン酸ナトリウム(pH7.4)の緩衝液で
洗浄し、同緩衝液に懸濁させた。0.03%TSB,
0.02% Tween20を含む1%アガロース(オートク
レーブ滅菌した後、42℃に保温したもの)10ml
に、菌体の懸濁液を菌体数が 2〜6×106個/10m
lとなるように加えて混ぜ、直ちに直径9cmのペトリ
デイッシュに流し込んで固めた。ゲルパンチャーを用い
て寒天培地上に直径2mmの穴をあけ,この穴に20μ
g/mlの濃度に調整したカブトシンA又はBの水溶液
を5μl入れた。37℃で2時間静置した後、この寒天
培地上に、6%TSBを含む1%アガロース(オートク
レーブ滅菌した後、42℃に保温したもの)10mlを
流し込んで固めた。これを37℃で24時間静置した
後、形成された増殖阻止円の直径を測定した。得られた
結果を表1に示す。なお、阻止円の直径が2.00mm
の場合は阻止円が形成されなかったことを示す。(Testing method for antibacterial activity)
(triptic soy broth, manufactured by Difco) at 37 ° C
After culturing until the exponential growth phase, collect the cells by centrifugation,
The cells were washed with a buffer of 10 mM sodium phosphate (pH 7.4) and suspended in the same buffer. 0.03% TSB,
10 ml of 1% agarose containing 0.02% Tween 20 (autoclaved and kept at 42 ° C)
To a suspension of the bacterial cells cell number 2 to 6 × 10 6 cells / 10m
1 and mixed, and immediately poured into a 9 cm diameter petri dish and hardened. Using a gel puncher, make a 2 mm diameter hole on the agar medium.
5 μl of an aqueous solution of beetosine A or B adjusted to a concentration of g / ml was added. After standing at 37 ° C. for 2 hours, 10 ml of 1% agarose containing 6% TSB (sterilized in an autoclave and kept at 42 ° C.) was poured onto the agar medium and solidified. After allowing this to stand at 37 ° C. for 24 hours, the diameter of the formed growth inhibition circle was measured. Table 1 shows the obtained results. In addition, the diameter of the stop circle is 2.00 mm.
Indicates that no blocking circle was formed.
【0050】[0050]
【表1】 抗菌活性(阻止円の直径(mm)) 検定菌 カブトシンA カブトシンB 黄色ブドウ球菌 2.00 3.67 大腸菌 4.00 2.00 枯草菌 2.00 2.00[Table 1] Antibacterial activity (diameter of inhibition circle (mm)) Test bacteria Cabutosin A Cabutosin B Staphylococcus aureus 2.00 3.67 Escherichia coli 4.00 2.00 Bacillus subtilis 2.00 2.00
【0051】表1の結果から、カブトシンAは大腸菌に
対して抗菌作用を示し、カブトシンBは黄色ブドウ球菌
に対して抗菌作用を示すことが判明した。From the results shown in Table 1, it was found that beetosine A has an antibacterial effect on Escherichia coli and that beetosine B has an antibacterial effect on Staphylococcus aureus.
【0052】〔実施例2〕カブトシンA及びBのcDN
Aのクローニング カブトシンBの全一次構造を明らかにするために、以下
に示すPCR法によりカブトシンBをコ−ドするcDN
Aをクローニングした。カブトシンAについても同様な
方法でクローニングすることができる。Example 2 cDN of Kabutosin A and B
Cloning of A In order to elucidate the entire primary structure of kabutosin B, cDN coding for kabutosin B by the following PCR method
A was cloned. The beetin A can be cloned in the same manner.
【0053】(1)カブトムシ脂肪体mRNA及びcD
NAの調製 カブトムシの3齢幼虫に大腸菌を注射することにより抗
菌活性を誘導した。注射してから18時間後に幼虫を開腹
し、脂肪体を回収した。脂肪体は使用時まで-130℃で保
存した。この脂肪体から QuickPrep mRNA Purification
Kit( ファルマシア社製) を用い て mRNAを精製し
た。cDNAの合成は、この精製して得たmRNAを鋳
型とし、cDNA first-strand synthesis kit(ファルマ
シア社製)を用いて行なった。この際のプライマーとし
てキットに付属のNot I-(dT18) primerを用いた。(1) Beetle fat body mRNA and cD
Preparation of NA Antibacterial activity was induced by injecting Escherichia coli into the third instar larva of the beetle. Larvae were laparotomized 18 hours after the injection, and fat pads were collected. The fat pad was stored at -130 ° C until use. QuickPrep mRNA Purification from this fat body
The mRNA was purified using Kit (Pharmacia). cDNA was synthesized using the mRNA obtained by the purification as a template and a cDNA first-strand synthesis kit (Pharmacia). Not I- (dT18) primer attached to the kit was used as a primer at this time.
【0054】(2)カブトシンBのcDNA断片の取得
とcDNAのクローニング プロテインシークエンサーで明らかになったカブトシン
BのN末端アミノ酸配列をもとに、配列内での上流側、
下流側それぞれに相当するミックスプライマー(プライ
マー1(配列番号:9)及びプライマー2(配列番号:
10))を合成した。前工程で得たcDNAを鋳型とし
てPCRを行なったところ、約 80bp の増幅産物を得
た。増幅反応の際のポリメラ−ゼには Ampli Taq Gold
(パ ーキンエルマー 社製 ) を用い、付属の緩衝液とdN
TP基質を説明書に従って用いた。サーマルサイクラーに
はパーキンエルマー社製の GENE AMP PCR SYSTEM 2400
を用いた。 この産物をプラスミドベクターpCR 2.1(イ
ンビトロゲン 社製)にクローニングした。断片の塩基配
列を Dye Terminator CycleSequencing FS Ready React
ion Kit ( アプライドバイオシステムズ 社製)を用い
てDNAシークエンサー ( Model 373S,アプライドバイ
オ システムズ社製)により調べた。この塩基配列から
推定されるアミノ酸配列は、明らかになっているカブト
シンBの部分アミノ酸配列の 3残基目から30残基目まで
に相当し、目的の遺伝子の断片が増幅されていることが
わかった。 プライマー1:CA(A/G)CC(A/C/G/T)GG(A/C/G/T)GC(A/C/
G/T)CC(A/C/G/T)AA(C/T)TT(C/T) (配列番号:9) プライマー2:(A/G)TT(A/C/G/T)GG(A/C/G/T)CC(C/T)TG
(C/T)TG(C/T)TC(A/C/G/T)AC (配列番号:10)(2) Acquisition of cDNA fragment of Kabutosin B and cloning of cDNA Based on the N-terminal amino acid sequence of Kabutosin B revealed by a protein sequencer,
Mix primers (primer 1 (SEQ ID NO: 9) and primer 2 (SEQ ID NO:
10)) was synthesized. When PCR was performed using the cDNA obtained in the previous step as a template, an amplification product of about 80 bp was obtained. Ampli Taq Gold is used as the polymerase during the amplification reaction.
(Manufactured by PerkinElmer) and the supplied buffer and dN
TP substrate was used according to the instructions. The thermal cycler is GENE AMP PCR SYSTEM 2400 manufactured by PerkinElmer.
Was used. This product was cloned into the plasmid vector pCR 2.1 (Invitrogen). Dye Terminator CycleSequencing FS Ready React
The DNA was analyzed using a DNA sequencer (Model 373S, manufactured by Applied Biosystems) using an ion Kit (manufactured by Applied Biosystems). The amino acid sequence deduced from this nucleotide sequence corresponds to the 3rd to 30th residues of the partial amino acid sequence of the beetle B which has been clarified, indicating that the fragment of the target gene has been amplified. Was. Primer 1: CA (A / G) CC (A / C / G / T) GG (A / C / G / T) GC (A / C /
G / T) CC (A / C / G / T) AA (C / T) TT (C / T) (SEQ ID NO: 9) Primer 2: (A / G) TT (A / C / G / T) GG (A / C / G / T) CC (C / T) TG
(C / T) TG (C / T) TC (A / C / G / T) AC (SEQ ID NO: 10)
【0055】次に、前工程で明らかになった領域を含め
て更に下流の塩基配列を、以下のように 3' RACE法で得
た。まずこの為のPCRの上流側プライマーとして、前
工程で明らかになった塩基配列のなかから次の配列を選
んで合成し、これをプライマー3(配列番号:11)と
した。もう一方の下流側のプライマーとして、cDNA
合成の際に用いた Not I-(dT18)primerの中にある塩基
配列を合成して用いた(プライマー4(配列番号:1
2))。プライマー3とプライマー4とを組み合わせ、
cDNAを鋳型としてPCRを行なったところ、約 300
bpの増幅産物を得た。この産物をpCR 2.1 にクローニン
グして、産物の塩基配列を調べたところ、目的の領域が
増幅されいることがわかった。 プライマー3:GATGCCCGCTTCACAGTTA (配列番号:1
1) プライマー4:AACTGGAAGAAATTCGCGGC (配列番号:1
2)Next, a base sequence further downstream including the region revealed in the previous step was obtained by the 3 ′ RACE method as follows. First, as an upstream primer for PCR for this purpose, the following sequence was selected from the nucleotide sequences identified in the previous step and synthesized, and this was used as primer 3 (SEQ ID NO: 11). As the other downstream primer, cDNA
The base sequence in Not I- (dT18) primer used in the synthesis was synthesized and used (Primer 4 (SEQ ID NO: 1
2)). Combining primer 3 and primer 4,
When PCR was performed using cDNA as a template, about 300
A bp amplification product was obtained. This product was cloned into pCR 2.1, and the nucleotide sequence of the product was examined. As a result, it was found that the target region was amplified. Primer 3: GATGCCCGCTTCACAGTTA (SEQ ID NO: 1
1) Primer 4: AACTGGAAGAAATTCGCGGC (SEQ ID NO: 1
2)
【0056】前工程までに得られた配列より更に上流の
塩基配列を以下のように して5'RACE法により調べた。
この際 5' RACE System for Rapid Amplification of c
DNA End Reagent Assembly, Ver.2 (ギブコ BRL社製)
のキットを用いた。前工程の下流側の解析で明らかにな
った配列をもとに最下流側のプライマー(プライマー5
(配列番号:13))を、プライマー5のすぐ上流の配
列をもとにプライマー6(配列番号:14)を合成し
た。脂肪体より抽出したmRNAを鋳型とし、プライマ
ー5を用いて逆転写酵素により一本鎖cDNAを合成し
た。得られた一本鎖cDNAの3'端にdCのポリマーを付
加した後、これを鋳型とし、キットに付属のアンカープ
ライマーとプライマー6とを組み合わせてPCRを行な
った。産物をクローニングするために、この産物を鋳型
とし、キットに付属のアダプタープライマーとプライマ
ー6とを組み合わせてPCRを行なったところ、約 550
bp の増幅産物を得た。この産物をpCR2.1 にクローニン
グして、塩基配列を調べたところ、目的の領域の塩基配
列を得た。 プライマー5:CCACCTATAAGTTCCACC (配列番号:1
3) プライマー6:AACGCTCCAATTCGGTTTGCTTTTT (配列番
号:14)The nucleotide sequence further upstream than the sequence obtained up to the previous step was examined by the 5 'RACE method as follows.
At this time, 5 'RACE System for Rapid Amplification of c
DNA End Reagent Assembly, Ver.2 (Gibco BRL)
Was used. Based on the sequence revealed in the downstream analysis of the previous step, the most downstream primer (primer 5
(SEQ ID NO: 13)) and primer 6 (SEQ ID NO: 14) were synthesized based on the sequence immediately upstream of primer 5. Using mRNA extracted from the fat pad as a template, single-stranded cDNA was synthesized using a primer 5 and reverse transcriptase. After adding a dC polymer to the 3 ′ end of the obtained single-stranded cDNA, PCR was performed using this as a template and combining an anchor primer and primer 6 attached to the kit. In order to clone the product, PCR was carried out using this product as a template and combining the adapter primer and primer 6 attached to the kit with about 550
A bp amplification product was obtained. This product was cloned into pCR2.1 and the nucleotide sequence was examined. As a result, the nucleotide sequence of the target region was obtained. Primer 5: CCACCTATAAGTTCCACC (SEQ ID NO: 1
3) Primer 6: AACGCTCCAATTCGGTTTGCTTTTT (SEQ ID NO: 14)
【0057】次に、前工程で明らかになったカブトシン
Bの上流側と下流側の部分配列をひと続きの配列、すな
わちカブトシンBのcDNAの全長を含むクローンとし
て得るために以下のPCRを行なった。まず、得られた
カブトシンBの上流側の配列をもとにプライマー7(配
列番号:15)を合成した。次に、プライマー7とプラ
イマー4とを組み合わせ、cDNAを鋳型としてPCR
を行なったところ、約600bpの増幅断片を得た。こ
の産物をpCR2.1にクローニングして塩基配列を調
べたところ、配列番号:7に示した配列を得た。 プライマー7:GAGACGAAATTGTATCGCTC (配列番号:1
5)Next, the following PCR was carried out in order to obtain partial sequences on the upstream and downstream sides of the beetin B identified in the preceding step as a continuous sequence, ie, a clone containing the full-length cDNA of the beetin B. . First, a primer 7 (SEQ ID NO: 15) was synthesized based on the obtained sequence on the upstream side of the beetin B. Next, primer 7 and primer 4 were combined, and PCR was performed using cDNA as a template.
As a result, an amplified fragment of about 600 bp was obtained. This product was cloned into pCR2.1 and the nucleotide sequence was examined. As a result, the sequence shown in SEQ ID NO: 7 was obtained. Primer 7: GAGACGAAATTGTATCGCTC (SEQ ID NO: 1
5)
【0058】カブトシンAについてもカブトシンBと同
様なクローニング操作により塩基配列を調べた。すなわ
ちカブトシンAのcDNAの全長を含むクローンを得る
ために以下のPCRを行なった。まず、カブトシンBと
異なるカブトシンAの上流側の塩基配列部分を利用して
プライマー8(配列番号:16)を合成した。次に、下
流側のプライマーとして先に合成して得られたプライマ
ー4とプライマー8とを組合せ、cDNAを鋳型として
PCRを行なったところ、約600bpの増幅断片を得
た。この産物をpCR2.1にクローニングして塩基配
列を調べたところ、配列番号:8に示した配列を得た。 プライマー8:GAGACGAAATCGTATCGTTC (配列番号:1
6)The nucleotide sequence of beetosine A was examined by the same cloning operation as that of beetosine B. That is, the following PCR was carried out in order to obtain a clone containing the full length of the cDNA of beetosine A. First, a primer 8 (SEQ ID NO: 16) was synthesized using a base sequence portion on the upstream side of beetosine A different from beetosine B. Next, the primer 4 and the primer 8 previously synthesized were combined as the downstream primer, and PCR was performed using cDNA as a template. As a result, an amplified fragment of about 600 bp was obtained. This product was cloned into pCR2.1 and the nucleotide sequence was examined. As a result, the sequence shown in SEQ ID NO: 8 was obtained. Primer 8: GAGACGAAATCGTATCGTTC (SEQ ID NO: 1
6)
【0059】これらの結果から、カブトシンBのアミノ
酸配列は配列番号2と結論され、カブトシンAのアミノ
酸配列は配列番号1と結論された。カブトシンA又はB
は、まず、それぞれ前駆体ペプチドの形で翻訳され、プ
ロセシングにより上流部分のペプチドが除かれ、成熟型
のペプチドであるカブトシンA又はBになるものと考え
られた。From these results, it was concluded that the amino acid sequence of beetosine B was SEQ ID NO: 2, and that the amino acid sequence of beetosine A was SEQ ID NO: 1. Kabutosin A or B
Was first translated in the form of a precursor peptide, and the upstream peptide was removed by processing, resulting in the mature form of the peptide, kabutosin A or B.
【0060】pCR2.1にクローニングされたカブト
シンAの組み換え体DNAを大腸菌に形質転換し、組み
換え微生物 E.coli JM109(pCRKABUA1)を得た。pCR
2.1にクローニングされたカブトシンBの組み換え体
DNAについても同様に、形質転換した組み換え微生物
E.coli JM109(pCRKABUB1)を得た。組み換え微生物 E.c
oli JM109(pCRKABUA1)はFERM P-16859として、組み換え
微生物 E.coli JM109(pCRKABUB1)はFERM P-16860として
工業技術院生命工学工業技術研究所にそれぞれ寄託され
ている。Escherichia coli was transformed with the recombinant DNA of beetosine A cloned into pCR2.1 to obtain a recombinant microorganism E. coli JM109 (pCRKABUA1). pCR
Similarly, for the recombinant DNA of beetosine B cloned in 2.1, the transformed recombinant microorganism
E. coli JM109 (pCRKABUB1) was obtained. Recombinant microorganism Ec
oli JM109 (pCRKABUA1) has been deposited as FERM P-16859, and the recombinant microorganism E. coli JM109 (pCRKABUB1) has been deposited as FERM P-16860 at the Biotechnology Industrial Research Institute, National Institute of Advanced Industrial Science and Technology.
【0061】「配列表フリ−テキスト」 配列番号:9 カブトシンBのN末端アミノ酸配列を
もとに、設計されたDNA。 配列番号:10 カブトシンBのN末端アミノ酸配列を
もとに、設計されたDNA。 配列番号:11 3’RACE法により、カブトシンB
のcDNAの一部を増幅するために設計されたDNA。 配列番号:12 NotI−(dT18)primer
の中の塩基配列に相当するように、設計されたDNA。 配列番号:13 5’RACE法(1st step)
により、カブトシンBのcDNAの一部を増幅するため
に設計されたDNA。 配列番号:14 5’RACE法(2nd step)
により、カブトシンBのcDNAの一部を増幅するため
に設計されたDNA。 配列番号:15 カブトシンBのcDNAの全長を増幅
するために設計されたDNA。 配列番号:16 カブトシンAのcDNAの全長を増幅
するために設計されたDNA。"Sequence List Free Text" SEQ ID NO: 9 DNA designed based on the N-terminal amino acid sequence of cabtocin B. SEQ ID NO: 10 DNA designed on the basis of N-terminal amino acid sequence of beetosine B SEQ ID NO: 11 Kabutosin B was obtained by 3'RACE method.
DNA designed to amplify a part of the cDNA of the above. SEQ ID NO: 12 NotI- (dT18) primer
DNA designed to correspond to the base sequence in. SEQ ID NO: 13 5 ′ RACE method (1st step)
DNA designed to amplify a part of the cDNA of beetosine B SEQ ID NO: 14 5 'RACE method (2nd step)
DNA designed to amplify a part of the cDNA of beetosine B SEQ ID NO: 15 DNA designed to amplify full-length cDNA of buttocin B SEQ ID NO: 16 DNA designed to amplify full-length cDNA of cabtocin A
【0062】[0062]
【発明の効果】本発明により、カブトムシの生産する2
種の新規抗菌性ペプチド(カブトシンA及びB)及び該
ペプチドを効率よく生産するための組み換え体DNAが
提供された。カブトシンAは大腸菌に、カブトシンBは
黄色ブドウ球菌に対して有効な抗菌作用を示すことか
ら、食品用防腐剤、医療用抗菌剤として好適である。According to the present invention, beetle production 2
Kinds of novel antimicrobial peptides (captocins A and B) and recombinant DNA for efficiently producing the peptides have been provided. Since kabutosin A has an effective antibacterial effect on Escherichia coli and cabutosin B has an effective antibacterial effect on Staphylococcus aureus, it is suitable as a food preservative and a medical antibacterial agent.
【0063】[0063]
【配列表】 SEQUENCE LISTING 〈110〉 Director General, National Institute of Sericultural and Entomol ogical Science Noda Institute for Scientific Research 〈120〉 Novel peptides, antibacterial agents, novel peptide genes, novel recombinant DNAs and a process for producing novel peptides 〈130〉 SP211 〈160〉 16 〈210〉 1 〈211〉 72 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 1 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 〈210〉 2 〈211〉 72 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 2 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 〈210〉 3 〈211〉 44 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 3 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr 35 40 〈210〉 4 〈211〉 30 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 4 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn 20 25 30 〈210〉 5 〈211〉 12 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 5 Thr Asp Arg Tyr Asp Val Gly Ala Thr Trp Ser Lys 1 5 10 〈210〉 6 〈211〉 10 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 6 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg 1 5 10 〈210〉 7 〈211〉 591 〈212〉 DNA 〈213〉 Allomyrina dichotoma 〈400〉 7 GaGaCGaaaT TGTaTCGCTC aaCTGaaaaC aTaTTGGTGT aTaTCTCGCC 50 TaaTaGTTaa aTCTTCaaCT aCGaTTTaaa G aTG aTG aaa CTC TaC 96 Met Met Lys Leu Tyr -70 GTT aTC TTT GGT CTT aTT GTa CTT TCT aCT GCT TaC GTG GTT CCa Gaa 144 Val Ile Phe Gly Leu Ile Val Leu Ser Thr Ala Tyr Val Val Pro Glu -65 -60 -55 CGT TaC TTT CaG CCT aTa TaT CCT GaT aCT GCG GCG GTG CaT GCC TTC 192 Arg Tyr Phe Gln Pro Ile Tyr Pro Asp Thr Ala Ala Val His Ala Phe -50 -45 -40 AGA GAC GAG CCT TTC ACG GTG ACA CCT GCC GAA TTG CGC TCA TAT CTA 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Ala Glu Leu Arg Ser Tyr Leu -35 -30 -25 -20 GGT ATC ACC GAT GAA GAT GAA ATA GAA ATG CCG GTA GTT TAT AAT CGT 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Asn Arg -15 -10 -5 GAA AGG AGG TCC TTA CAG CCA GGT GCA CCT AAT TTC CCG ATG CCC GGT 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly -1 1 5 10 TCA CAG TTA CCT ACC AGC ATC ACC TCT AAT GTT GAA CAA CAG GGG CCA 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro 15 20 25 AAT ACT GCA GCC ACT ATC AAT GCC CAA CAT AAA ACT GAT AGG TAC GAC 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 GTT GGA GCC ACC TGG AGC AAG GTC ATT CGC GGA CCA GGA AAA AGC AAA 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 CCG AAT TGG AGC GTT GGT GGA ACT TAT AGG TGG TAACACCTGT ATTTC 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 GTTATTTTTG TAAAATATTT TTTGATATAA ATTAAATTTT GTTCGCAAAA 578 AAAAAAAAAA AAA 591 〈210〉 8 〈211〉 596 〈212〉 DNA 〈213〉 Allomyrina dichotoma 〈400〉 8 GAGACGAAAT CGTATCGTTC AACTGAAAAC ATATTGGTGC ATATCTCGCC 50 TAATAGCTAA ATCTCCAACT ACCACTTAAA G ATG ATG AAA CTC TAC 96 Met Met Lys Leu Tyr -70 ATT ATT TTC GGT CTT ATT GCA CTT TCT ACT GCT TAT GCG GTT CCA GAA 144 Ile Ile Phe Gly Leu Ile Ala Leu Ser Thr Ala Tyr Ala Val Pro Glu -65 -60 -55 CGC TAC TTC CAG CCT CCA TAT CCT GAT ACT GCG GCC GTG CAT GCC TAC 192 Arg Tyr Phe Gln Pro Pro Tyr Pro Asp Thr Ala Ala Val His Ala Tyr -50 -45 -40 AGA GAC GAG CCT TTC ACG GTT ACA CCT ACC GAA TTC CGC TCC TAT CTA 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Thr Glu Phe Arg Ser Tyr Leu -35 -30 -25 -20 GGT ATC ACC GAT GAA GAT GAA ATA GAA ATG CCG GTA GTT TAT ATT CGC 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Ile Arg -15 -10 -5 GAA AGG AGG TCC TTA CAG CCA GGT GCA CCT AAT TTC CCG CTG CCC GGT 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly -1 1 5 10 TCA CAG TTA CCT ACC AGC ATC ACC TCT AAT GTT GAA CGA CAG GGG CCA 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro 15 20 25 AAT ACT GCA GCC ACT ATC AAT GCC CAA CAT AAA ACT GAT AGG TAC GAT 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 GTT GGA GCC ACC TGG AGC AAG GTC ATT CGC GGA CCA GGA AAA AGC AAA 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 CCG AAT TGG AGC GTT GGT GGA ACT TAT AGG TGG TAACACCTGT ATTTC 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 GTTATTTTTG TAAAATATTT TTTGATATAA ATTAAATTTT GTTCGCAAAA 578 AAAAAAAAAA AAAAAAAA 596 〈210〉 9 〈211〉 21 〈212〉 DNA 〈213〉 Artificial Sequence 〈223〉 Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. 〈400〉 9 CARCCNGGNG CNCCNAAYTT Y 21 〈210〉 10 〈211〉 21 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. 〈400〉 10 RTTNGGNCCY TGYTGYTCNA C 21 〈210〉 11 〈211〉 19 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA by 3'RACE. 〈400〉 11 GATGCCCGCT TCACAGTTA 19 〈210〉 12 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA corresponds to a part of the NotI-(dT18)primer. 〈400〉 12 AACTGGAAGA AATTCGCGGC 20 〈210〉 13 〈211〉 18 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA in the 1st step of 5'RACE. 〈400〉 13 CCACCTATAA GTTCCACC 18 〈210〉 14 〈211〉 25 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA in the 2nd step of 5'RACE. 〈400〉 14 AACGCTCCAA TTCGGTTTGC TTTTT 25 〈210〉 15 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of the full length of kabutocin B cDNA. 〈400〉 15 GAGACGAAAT TGTATCGCTC 20 〈210〉 16 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of the full length of kabutocin A cDNA. 〈400〉 16 GAGACGAAAT CGTATCGTTC 20[Sequence List] SEQUENCE LISTING <110> Director General, National Institute of Sericultural and Entomol ogical Science Noda Institute for Scientific Research <120> Novel peptides, antibacterial agents, novel peptide genes, novel recombinant DNAs and a process for producing novel peptides <130 > SP211 <160> 16 <210> 1 <211> 72 <212> PRT <213> Allomyrina dichotoma <400> 1 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 <210> 2 <211> 72 <212> PRT <213> Allomyrina dichotoma <400> 2 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn Thr Ala 20 25 30 Al a Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 <210 > 3 <211> 44 <212> PRT <213> Allomyrina dichotoma <400> 3 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr 35 40 <210> 4 <211> 30 <212> PRT <213> Allomyrina dichotoma <400> 4 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn 20 25 30 <210> 5 <211> 12 <212> PRT <213> Allomyrina dichotoma <400> 5 Thr Asp Arg Tyr Asp Val Gly Ala Thr Trp Ser Lys 1 5 10 <210> 6 <211> 10 <212> PRT <213> Allomyrina dichotoma <400> 6 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg 1 5 10 <210> 7 <211> 591 <212> DNA <213 Allomyrina dichotoma <400> 7 GaGaCGaaaT TGTaTCGCTC aaCTGaaaaC aTaTTGGTGT aTaTCTCGCC 50 TaaTaGTTaa aTCTTCaaCT aCGaTTTaaa G aTG aTG aaa CTC TaC 96 Met Met Lys Leu Tyr -70 GTT aTC TTT GGT TCT GCT TCT GTT GCT CCT GTT CTGT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CTT GTT CGT GCT CTCT Leu Ile Val Leu Ser Thr Ala Tyr Val Val Pro Glu -65 -60 -55 CGT TaC TTT CaG CCT aTa TaT CCT GaT aCT GCG GCG GTG CaT GCC TTC 192 Arg Tyr Pyr Gln Pro Ile Tyr Pro Asp Thr Ala Ala Val His Ala Phe -50 -45 -40 AGA GAC GAG CCT TTC ACG GTG ACA CCT GCC GAA TTG CGC TCA TAT CTA 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Ala Glu Leu Arg Ser Tyr Leu -35 -30 -25 -20 GGT ATC ACC GAT GAA GAT GAA ATA GAA ATG CCG GTA GTT TAT AAT CGT 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Asn Arg -15 -10 -5 GAA AGG AGG TCC TTA CAG CCA GGT GCA CCT AAT TTC CCG ATG CCC GGT 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly -1 1 5 10 TCA CAG TTA CCT ACC AGC ATC ACC TCT AAT GTT GAA CAA CAG GGG CCA 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro 15 20 25 AAT ACT GCA GCC ACT ATC AAT GCC CAA CAT AAA ACT GAT AGG TAC GAC 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 GTT GGA GCC ACC TGG AGC AAG GTC ATT CGC GGA CCA GGA AAA AGC AAA 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 CCG AAT TGG AGC GTT GGT GGA ACT TAT AGG TGG TAACACCTGT ATTTC 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 GTTATTTTTG TAAAATATTT TTTGATATAA ATTAAATTTT GTTCGCAAAA 578 AAAAAAAAAA AAA 591 <210> 8 <211> 596 <212> DNA <213> Allomyrina dichotoma <400> 8 GAGATCATC ATC TAGTATCATCGTC ACT ATG ATG AAA CTC TAC 96 Met Met Lys Leu Tyr -70 ATT ATT TTC GGT CTT ATT GCA CTT TCT ACT GCT TAT GCG GTT CCA GAA 144 Ile Ile Phe Gly Leu Ile Ala Leu Ser Thr Ala Tyr Ala Val Pro Glu -65 -60 -55 CGC TAC TTC CAG CCT CCA TAT CCT GAT ACT GCG GCC GTG CAT GCC TAC 192 Arg Tyr Phe Gln Pro Pro Tyr Pro Asp Thr Ala Ala Va l His Ala Tyr -50 -45 -40 AGA GAC GAG CCT TTC ACG GTT ACA CCT ACC GAA TTC CGC TCC TAT CTA 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Thr Glu Phe Arg Ser Tyr Leu -35 -30 -25- 20 GGT ATC ACC GAT GAA GAT GAA ATA GAA ATG CCG GTA GTT TAT ATT CGC 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Ile Arg -15 -10 -5 GAA AGG AGG TCC TTA CAG CCA GGT GCA CCT AAT TTC CCG CTG CCC GGT 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly -1 1 5 10 TCA CAG TTA CCT ACC AGC ATC ACC TCT AAT GTT GAA CGA CAG GGG CCA 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro 15 20 25 AAT ACT GCA GCC ACT ATC AAT GCC CAA CAT AAA ACT GAT AGG TAC GAT 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 GTT GGA GCC ACC TGG AGC AAG GTC ATT CGC GGA CCA GGA AAA AGC AAA 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 CCG AAT TGG AGC GTT GGT GGA ACT TAT AGG TGG TAACACCTGT ATTTC 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 GTTATTTTTG TAAAATATTT TTTGATATAA ATTAAATTTT GTTCGCAAAA 578 AAAAAAAAAA AAAAAAAA 596 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <223> Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. <400> 9 CARCCTTAANG CNC 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. <400> 10 RTTNGGNCCY TGYTGYTCNA C 21 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of kabutocin B cDNA by 3'RACE. <400> 11 GATGCCCGCT TCACAGTTA 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA corresponds to a part of the NotI- (dT18) primer. <400> 12 AACTGGAAGA AATTCGCGGC 20 <210> 13 <211> 18 <212> DNA < 213> Artificial Sequence <220> <223> Designed DNA for amplification of ap art of kabutocin B cDNA in the 1st step of 5'RACE. <400> 13 CCACCTATAA GTTCCACC 18 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of kabutocin B cDNA in the 2nd step of 5'RACE. <400> 14 AACGCTCCAA TTCGGTTTGC TTTTT 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of the full length of kabutocin B cDNA. <400> 15 GAGACGAAAT TGTATCGCTC 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of the full length of kabutocin A cDNA <400> 16 GAGACGAAAT CGTATCGTTC 20
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【手続補正書】[Procedure amendment]
【提出日】平成10年8月11日(1998.8.1
1)[Submission date] August 11, 1998 (1998.
1)
【手続補正1】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0063[Correction target item name] 0063
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【0063】[0063]
【配列表】 SEQUENCE LISTING 〈110〉 National Institute of Sericultural and Entomological Science Noda Institute for Scientific Research 〈120〉 Novel peptides, antimicrobiol agents, novel peptide genes, novel recombinant DNAs and a process for producing novel peptides 〈130〉 SP221 〈160〉 16 〈210〉 1 〈211〉 72 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 1 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 〈210〉 2 〈211〉 72 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 2 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 〈210〉 3 〈211〉 44 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 3 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr 35 40 〈210〉 4 〈211〉 30 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 4 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn 20 25 30 〈210〉 5 〈211〉 12 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 5 Thr Asp Arg Tyr Asp Val Gly Ala Thr Trp Ser Lys 1 5 10 〈210〉 6 〈211〉 10 〈212〉 PRT 〈213〉 Allomyrina dichotoma 〈400〉 6 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg 1 5 10 〈210〉 7 〈211〉 591 〈212〉 DNA 〈213〉 Allomyrina dichotoma 〈400〉 7 gagacgaaat tgtatcgctc aactgaaaac atattggtgt atatctcgcc 50 taatagttaa atcttcaact acgatttaaa g atg atg aaa ctc tac 96 Met Met Lys Leu Tyr -70 gtt atc ttt ggt ctt att gta ctt tct act gct tac gtg gtt cca gaa 144 Val Ile Phe Gly Leu Ile Val Leu Ser Thr Ala Tyr Val Val Pro Glu -65 -60 -55 cgt tac ttt cag cct ata tat cct gat act gcg gcg gtg cat gcc ttc 192 Arg Tyr Phe Gln Pro Ile Tyr Pro Asp Thr Ala Ala Val His Ala Phe -50 -45 -40 aga gac gag cct ttc acg gtg aca cct gcc gaa ttg cgc tca tat cta 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Ala Glu Leu Arg Ser Tyr Leu -35 -30 -25 -20 ggt atc acc gat gaa gat gaa ata gaa atg ccg gta gtt tat aat cgt 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Asn Arg -15 -10 -5 gaa agg agg tcc tta cag cca ggt gca cct aat ttc ccg atg ccc ggt 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly -1 1 5 10 tca cag tta cct acc agc atc acc tct aat gtt gaa caa cag ggg cca 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro 15 20 25 aat act gca gcc act atc aat gcc caa cat aaa act gat agg tac gac 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 gtt gga gcc acc tgg agc aag gtc att cgc gga cca gga aaa agc aaa 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 ccg aat tgg agc gtt ggt gga act tat agg tgg taacacctgt atttc 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 gttatttttg taaaatattt tttgatataa attaaatttt gttcgcaaaa 578 aaaaaaaaaa aaa 591 〈210〉 8 〈211〉 596 〈212〉 DNA 〈213〉 Allomyrina dichotoma 〈400〉 8 gagacgaaat cgtatcgttc aactgaaaac atattggtgc atatctcgcc 50 taatagctaa atctccaact accacttaaa g atg atg aaa ctc tac 96 Met Met Lys Leu Tyr -70 att att ttc ggt ctt att gca ctt tct act gct tat gcg gtt cca gaa 144 Ile Ile Phe Gly Leu Ile Ala Leu Ser Thr Ala Tyr Ala Val Pro Glu -65 -60 -55 cgc tac ttc cag cct cca tat cct gat act gcg gcc gtg cat gcc tac 192 Arg Tyr Phe Gln Pro Pro Tyr Pro Asp Thr Ala Ala Val His Ala Tyr -50 -45 -40 aga gac gag cct ttc acg gtt aca cct acc gaa ttc cgc tcc tat cta 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Thr Glu Phe Arg Ser Tyr Leu -35 -30 -25 -20 ggt atc acc gat gaa gat gaa ata gaa atg ccg gta gtt tat att cgc 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Ile Arg -15 -10 -5 gaa agg agg tcc tta cag cca ggt gca cct aat ttc ccg ctg ccc ggt 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly -1 1 5 10 tca cag tta cct acc agc atc acc tct aat gtt gaa cga cag ggg cca 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro 15 20 25 aat act gca gcc act atc aat gcc caa cat aaa act gat agg tac gat 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 gtt gga gcc acc tgg agc aag gtc att cgc gga cca gga aaa agc aaa 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 ccg aat tgg agc gtt ggt gga act tat agg tgg taacacctgt atttc 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 gttatttttg taaaatattt tttgatataa attaaatttt gttcgcaaaa 578 aaaaaaaaaa aaaaaaaa 596 〈210〉 9 〈211〉 21 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. 〈400〉 9 carccnggng cnccnaaytt y 21 〈210〉 10 〈211〉 21 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. 〈400〉 10 rttnggnccy tgytgytcna c 21 〈210〉 11 〈211〉 19 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA by 3'RACE. 〈400〉 11 gatgcccgct tcacagtta 19 〈210〉 12 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA corresponds to a part of the NotI-(dT18)primer. 〈400〉 12 aactggaaga aattcgcggc 20 〈210〉 13 〈211〉 18 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA in the 1st step of 5'RACE. 〈400〉 13 ccacctataa gttccacc 18 〈210〉 14 〈211〉 25 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of a part of kabutocin B cDNA in the 2nd step of 5'RACE. 〈400〉 14 aacgctccaa ttcggtttgc ttttt 25 〈210〉 15 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of the full length of kabutocin B cDNA. 〈400〉 15 gagacgaaat tgtatcgctc 20 〈210〉 16 〈211〉 20 〈212〉 DNA 〈213〉 Artificial Sequence 〈220〉 〈223〉 Designed DNA for amplification of the full length of kabutocin A cDNA. 〈400〉 16 gagacgaaat cgtatcgttc 20[Sequence List] SEQUENCE LISTING <110> National Institute of Sericultural and Entomological Science Noda Institute for Scientific Research <120> Novel peptides, antimicrobiol agents, novel peptide genes, novel recombinant DNAs and a process for producing novel peptides <130> SP221 <160 > 16 <210> 1 <211> 72 <212> PRT <213> Allomyrina dichotoma <400> 1 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 <210> 2 <211> 72 <212> PRT <213> Allomyrina dichotoma <400> 2 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala G ln His Lys Thr Asp Arg Tyr Asp Val Gly Ala 35 40 45 Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys Pro Asn Trp 50 55 60 Ser Val Gly Gly Thr Tyr Arg Trp 65 70 <210> 3 <211> 44 <212> PRT <213> Allomyrina dichotoma <400> 3 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro Asn Thr Ala 20 25 30 Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr 35 40 <210> 4 <211> 30 <212> PRT <213> Allomyrina dichotoma <400> 4 Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly Ser Gln Leu 1 5 10 15 Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro Asn 20 25 30 <210> 5 <211> 12 <212> PRT <213> Allomyrina dichotoma <400> 5 Thr Asp Arg Tyr Asp Val Gly Ala Thr Trp Ser Lys 1 5 10 <210> 6 <211> 10 <212> PRT <213> Allomyrina dichotoma <400> 6 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg 1 5 10 <210> 7 < 211> 591 <212> DNA <213> Allomyrina dichot oma <400> 7 gagacgaaat tgtatcgctc aactgaaaac atattggtgt atatctcgcc 50 taatagttaa atcttcaact acgatttaaa g atg atg aaa ctc tac 96 Met Met Lys Leu Tyr -70 gtt atc ttt ggt ctt att gta ctt tctg gct ctt tctg gtt Ile Val Leu Ser Thr Ala Tyr Val Val Pro Glu -65 -60 -55 cgt tac ttt cag cct ata tat cct gat act gcg gcg gtg cat gcc ttc 192 Arg Tyr Pyr Ghe Pro Ile Tyr Pro Asp Thr Ala Ala Val His Ala Phe -50 -45 -40 aga gac gag cct ttc acg gtg aca cct gcc gaa ttg cgc tca tat cta 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Ala Glu Leu Arg Ser Tyr Leu -35 -30 -25 -20 -20 ggt atc acc gat gaa gat gaa ata gaa atg ccg gta gtt tat aat cgt 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Asn Arg -15 -10 -5 gaa agg agg tcc tta cag cca ggt gca cct aat ttc ccg atg ccc ggt 336 Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Met Pro Gly -1 1 5 10 tca cag tta cct acc agc atc acc tct aat gtt gaa caa cag ggg cca 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Gln Gln Gly Pro 15 20 25 aat act gca gcc act atc aat gcc caa cat aaa act gat agg tac gac 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 gtt gga gcc acc tgg agc aag gtc att cgc gga cca gga aaa agc aaa 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 ccg aat tgg agc gtt ggt gga act tat agg tgg taacacctgt atttc 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 gttatttttg taaaatattt tttgatataa attaaatttt gttcgcaaaa 578 aaaaaaaaaa aaa 591 〈210〉 8 〈211〉 596 〈212〉 DNA 〈213〉 Allomyrina dichotoma 〈400〉 8 gagacgaaat cgtatcctatcaggacta gatcgactat atcgacta cctatcct atcgacta gcatact 96 Met Met Lys Leu Tyr -70 att att ttc ggt ctt att gca ctt tct act gct tat gcg gtt cca gaa 144 Ile Ile Phe Gly Leu Ile Ala Leu Ser Thr Ala Tyr Ala Val Pro Glu -65 -60 -55 cgc tac ttc cag cct cca tat cct gat act gcg gcc gtg cat gcc tac 192 Arg Tyr Phe Gln Pro Pro Tyr Pro Asp Thr Ala Ala Val His Ala Tyr -50- 45 -40 aga gac gag cct ttc acg gtt aca cct acc gaa ttc cgc tcc tat cta 240 Arg Asp Glu Pro Phe Thr Val Thr Pro Thr Glu Phe Arg Ser Tyr Leu -35 -30 -25 -20 ggt atc acc gat gaa gat gaa ata gaa atg ccg gta gtt tat att cgc 288 Gly Ile Thr Asp Glu Asp Glu Ile Glu Met Pro Val Val Tyr Ile Arg -15 -10 -5 gaa agg agg tcc tta cag cca ggt gca cct aat ttc ccg ctg ccc ggt Glu Arg Arg Ser Leu Gln Pro Gly Ala Pro Asn Phe Pro Leu Pro Gly -1 1 5 10 tca cag tta cct acc agc atc acc tct aat gtt gaa cga cag ggg cca 384 Ser Gln Leu Pro Thr Ser Ile Thr Ser Asn Val Glu Arg Gln Gly Pro 15 20 25 aat act gca gcc act atc aat gcc caa cat aaa act gat agg tac gat 432 Asn Thr Ala Ala Thr Ile Asn Ala Gln His Lys Thr Asp Arg Tyr Asp 30 35 40 45 gtt gga gcc acc tgg agc aag gtc att cgc gga cca gga aaa agc aaa 480 Val Gly Ala Thr Trp Ser Lys Val Ile Arg Gly Pro Gly Lys Ser Lys 50 55 60 ccg aat tgg agc gtt ggt gga act tat agg tgg taacacctgt atttc 528 Pro Asn Trp Ser Val Gly Gly Thr Tyr Arg Trp 65 70 gttatttttg taaaatat tt tttgatataa attaaatttt gttcgcaaaa 578 aaaaaaaaaa aaaaaaaa 596 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. <400> 9 carccnggng cnccnaaytty 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA degenerated from N-terminal amino acid sequence of kabutocin B. <400> 10 rttnggnccy tgytgytcna c 21 <210 > 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of kabutocin B cDNA by 3'RACE. <400> 11 gatgcccgct tcacagtta 19 <210> 12 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA corresponds to a part of the NotI- (dT18) primer. <400> 12 aactggaaga aattcgcggc 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of ka butocin B cDNA in the 1st step of 5'RACE. <400> 13 ccacctataa gttccacc 18 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of a part of kabutocin B cDNA in the 2nd step of 5'RACE. <400> 14 aacgctccaa ttcggtttgc ttttt 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of the full length of kabutocin B cDNA. <400> 15 gagacgaaat tgtatcgctc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed DNA for amplification of the full length of kabutocin A cDNA. 400> 16 gagacgaaat cgtatcgttc 20
フロントページの続き (72)発明者 原 精一 千葉県野田市野田399 財団法人 野田産 業科学研究所 内 Fターム(参考) 4B024 AA01 AA05 BA67 BA80 CA04 CA06 EA04 GA11 HA01 4C084 AA02 AA06 AA07 BA01 BA20 CA49 CA53 DA42 MA22 MA35 MA37 MA43 MA52 ZB352 4H011 AA02 4H045 AA10 AA20 AA30 BA21 CA51 EA29 FA71 FA74 GA15 GA22 GA25 Continuing from the front page (72) Inventor Seiichi Hara 399 Noda, Noda-shi, Chiba F-term (Reference) in Noda Institute of Industrial Science 4B024 AA01 AA05 BA67 BA80 CA04 CA06 EA04 GA11 HA01 4C084 AA02 AA06 AA07 BA01 BA20 CA49 CA53 DA42 MA22 MA35 MA37 MA43 MA52 ZB352 4H011 AA02 4H045 AA10 AA20 AA30 BA21 CA51 EA29 FA71 FA74 GA15 GA22 GA25
Claims (9)
ド。 (a)配列番号1に記載のアミノ酸配列からなるペプチ
ド。 (b)配列番号1に記載のアミノ酸配列において1もし
くは複数のアミノ酸が付加、欠失もしくは置換されたア
ミノ酸配列からなり、かつ抗菌活性を有するペプチド。1. A novel peptide of the following (a) or (b): (A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1; (B) a peptide comprising an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1, and having an antibacterial activity;
て含有する抗菌剤。2. An antibacterial agent comprising the peptide according to claim 1 as an active ingredient.
て含有する抗大腸菌剤。3. An anti-Escherichia coli agent comprising the peptide according to claim 1 as an active ingredient.
ド。 (a)配列番号2に記載のアミノ酸配列からなるペプチ
ド。 (b)配列番号2に記載のアミノ酸配列において1もし
くは複数のアミノ酸が付加、欠失もしくは置換されたア
ミノ酸配列からなり、かつ抗菌活性を有するペプチド。4. A novel peptide of the following (a) or (b): (A) a peptide consisting of the amino acid sequence of SEQ ID NO: 2; (B) a peptide comprising an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of SEQ ID NO: 2, and having an antibacterial activity;
て含有する抗菌剤。5. An antibacterial agent comprising the peptide according to claim 4 as an active ingredient.
て含有する抗黄色ブドウ球菌剤。6. An anti-Staphylococcus aureus agent comprising the peptide according to claim 4 as an active ingredient.
コードする遺伝子。A gene encoding the novel peptide according to claim 1 or 4.
ーDNAに挿入してなることを特徴とする新規な組み換
え体DNA。8. A novel recombinant DNA obtained by inserting the peptide gene according to claim 7 into a vector DNA.
載のペプチドを取得することを特徴とするペプチドの製
造法。9. A method for producing a peptide, comprising obtaining the peptide according to claim 1 from a beetle larva.
Priority Applications (1)
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JP10197190A JP2000026499A (en) | 1998-07-13 | 1998-07-13 | New peptide, antimicrobial agent, new peptide gene, new recombinant dna and production of new peptide |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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JP10197190A JP2000026499A (en) | 1998-07-13 | 1998-07-13 | New peptide, antimicrobial agent, new peptide gene, new recombinant dna and production of new peptide |
Publications (1)
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JP2000026499A true JP2000026499A (en) | 2000-01-25 |
Family
ID=16370315
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JP10197190A Pending JP2000026499A (en) | 1998-07-13 | 1998-07-13 | New peptide, antimicrobial agent, new peptide gene, new recombinant dna and production of new peptide |
Country Status (1)
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JP (1) | JP2000026499A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100790807B1 (en) | 2006-12-08 | 2008-01-04 | 대한민국 | An anti-bacterial peptide base pairs and an anti-bacterial peptide isolated from dung beetle Copris tripartitus larvae |
CN114507653A (en) * | 2022-02-16 | 2022-05-17 | 河南农业大学 | Phosphodiesterase BSP, biological agent and application thereof |
CN114907466A (en) * | 2022-04-29 | 2022-08-16 | 福建农林大学 | Monochamus alternatus antimicrobial peptide and coding gene and application thereof |
-
1998
- 1998-07-13 JP JP10197190A patent/JP2000026499A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100790807B1 (en) | 2006-12-08 | 2008-01-04 | 대한민국 | An anti-bacterial peptide base pairs and an anti-bacterial peptide isolated from dung beetle Copris tripartitus larvae |
CN114507653A (en) * | 2022-02-16 | 2022-05-17 | 河南农业大学 | Phosphodiesterase BSP, biological agent and application thereof |
CN114507653B (en) * | 2022-02-16 | 2023-06-30 | 河南农业大学 | Phosphodiesterase BSP, biological agent and application thereof |
CN114907466A (en) * | 2022-04-29 | 2022-08-16 | 福建农林大学 | Monochamus alternatus antimicrobial peptide and coding gene and application thereof |
CN114907466B (en) * | 2022-04-29 | 2023-05-12 | 福建农林大学 | Monochamus alternatus antibacterial peptide, and encoding gene and application thereof |
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