JP5107717B2 - 殺虫ポリペプチドおよびその使用方法 - Google Patents
殺虫ポリペプチドおよびその使用方法 Download PDFInfo
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- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical class O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 229940035289 tobi Drugs 0.000 description 1
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
Images
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
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- A—HUMAN NECESSITIES
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- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2217/00—Genetically modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
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Description
本発明は、全内容を参照として本明細書に組み込まれる2004年11月4日に出願された米国仮出願第60/625,297号による優先権を主張する。
米国政府は、米国国立科学財団助成金番号MCB0234638に基づき、本発明において一定の権利を有する。
配列番号1:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Val−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVLLVLATVLGGVEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
を有するブルーマウンテンジョウゴグモ(Hadronyche versuta)からのプレプロペプチドである。
(QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
配列番号3:Gly−Ser−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(GSCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
配列番号4:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAGTTCTAGGAGGAGTTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCCCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAGCGGAATGAAAACGGCCACACTGTTTATTATTGCAGGGCT
である。
配列番号5:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Ile−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg
(MNTATGFIVLLVLATILGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCR)
である。
配列番号6:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg
(QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCR)
である。
配列番号7:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAATTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAGCGGAATGAAAACGGCCACACTGTTTATTATTGCAGG
である。
配列番号8:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg
(MNTATGFIVLLVLATVLGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCR)
である。
配列番号9:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg
(QYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCR)
である。
配列番号10:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAGTTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAACTAAATGAAAACGACAACACTGTTTATTATTGCAGG
である。
配列番号11:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Ile−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVLLVLATILGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
である。
配列番号12:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Arg−Asn−Glu−Asn−Gly−His−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
である。
配列番号13:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAATTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGACGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAGCGGAATGAAAACGGCCACACTGTTTATTATTGCAGGGCT
である。
配列番号14:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Ala−Asn−Pro−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVLLVLATVLGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENANPVYYCRA)
である。
配列番号15:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Ala−Asn−Pro−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQELNENANPVYYCRA)
である。
配列番号16:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAGTTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGCCAATATTACGTTCCAGTTGATCAACCGTGCTCTTTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACCCAAGAGCTAAATGAAAACGCCAACCCTGTTTATTATTGCAGGGCT
である。
配列番号17:Met−Asn−Thr−Thr−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Ile−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTTTGFIVLLVLATILGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号18:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号19:
ATGAATACCACAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAATTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAGCTAAATGAAAACGACAACACTGTTTATTATTGCAGGGCT
である。
配列番号20:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVLLVLATVLGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号21:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号22:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAGTTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAACTAAATGAAAACGACAACACTGTTTATTATTGCAGGGCT
である。
配列番号23:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Phe−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Ile−Glu−Ala−Gly−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVFLVLATVLGGIEAGESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号24:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号25:
ATGAATACCGCAACAGGTTTCATCGTCTTTTTGGTTTTGGCGACAGTTCTCGGAGGTATTGAAGCAGGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAACTAAATGAAAACGACAACACTGTTTATTATTGCAGGGCT
である。
配列番号26:Met−Asn−Thr−Ala−Thr−Gly−Phe−Ile−Val−Leu−Leu−Val−Leu−Ala−Thr−Val−Leu−Gly−Gly−Ile−Glu−Ala−Arg−Glu−Ser−His−Met−Arg−Lys−Asp−Ala−Met−Gly−Arg−Val−Arg−Arg−Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(MNTATGFIVLLVLATVLGGIEARESHMRKDAMGRVRRQYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号27:Gln−Tyr−Cys−Val−Pro−Val−Asp−Gln−Pro−Cys−Ser−Leu−Asn−Thr−Gln−Pro−Cys−Cys−Asp−Asp−Ala−Thr−Cys−Thr−Gln−Glu−Leu−Asn−Glu−Asn−Asp−Asn−Thr−Val−Tyr−Tyr−Cys−Arg−Ala
(QYCVPVDQPCSLNTQPCCDDATCTQELNENDNTVYYCRA)
である。
配列番号28:
ATGAATACCGCAACAGGTTTCATCGTCCTTTTGGTTTTGGCGACAGTTCTCGGAGGTATTGAAGCTAGAGAATCTCATATGAGAAAAGATGCCATGGGAAGAGTTCGTCGACAATATTGCGTTCCAGTTGATCAACCGTGCTCTCTGAATACCCAACCGTGCTGCGATGATGCCACGTGCACACAAGAGCTAAATGAAAACGACAACACTGTTTATTATTGCAGGGCT
である。
トテンチフォームリーフマイナー)、Lymantria dispar(マイマイガ)、Malacosoma neustria(オビカレハ)、Mamestra brassicae(ヨトウガ)、Mamestra configurata(バーサアーミーワーム)、ホーンワームManduca sextaおよびManuduca quinquemaculata、Operophtera brumata(ナミスジフユエダシャク)、Ostrinia nubilalis(アワノメイガ)、Panolis flammea(マツキリガ)、Pectinophora gossypiella(ワタアカミムシガ)、Phyllocnistis citrella(ミカンハモグリガ)、Pieris brassicae(オオモンシロチョウ)、Plutella xylostella(コナガ)、Rachiplusia ni(アオムシ類)、Spilosoma virginica(イェローベアモス)、Spodoptera exigua(シロイチモジヨトウ)、Spodoptera frugiperda(ヨトウガ)、Spodoptera littoralis(ハスモンヨトウ)、Spodoptera litura(ハスモンヨトウ)、Spodoptera praefica(イェローストライプドアーミーワーム)、Sylepta derogata(ワタノメイガ)、Tineola bisselliella(コイガ)、Tineola pellionella(イガ)、Tortrix viridana(ヨーロッパオークリーフローラー)、Trichoplusia ni(イラクサギンウワバ)、Yponomeuta padella(ヨーロッパリンゴスガ)などの鱗翅目;普通のコウロギAcheta domesticus、ツリーローカスト(Anacridium spp.)、トノサマバッタLocusta migratoria、ツーストライプドグラスホッパーMelanoplus bivittatus、ディフェレンシャルグラスホッパーMelanoplus differentialis、レッドレッグドグラスホッパーMelanoplus femurrubrum、マイグラトリーグラスホッパーMelanoplus sanguinipes、ノーザンモルクリケットNeocurtilla hexadectyla、アカトビバッタNomadacris septemfasciata、ショートウィングルドモルクリケットScapteriscus abbreviatus、サザンモルクリケットScapteriscus borellii、トーニィモルクリケットScapteriscus vicinus、およびサバクトビバッタSchistocerca gregariaなどの直翅目;ウシハジラミBovicola bovis、バイティングラウス(Damalinia spp.)、ネコシラミFelicola subrostrata、ウシジラミHaematopinus eurysternus、テールスィッチラウスHaematopinus quadripertussus、ブタジラミHaematopinus suis、フェイスラウスLinognathus ovillus、フットラウスLinognathus pedalis、イヌジラミLinognathus setosus、ウシホソジラミLinognathus vituli、ニワトリオオハジラミMenacanthus stramineus、ニワトリハジラミMenopon gallinae、ヒトジラミPediculus humanus、ケジラミPhthirus pubis、リトルブルーキャットルラウスSolenopotes capillatus、およびイヌハジラミTrichodectes canisなどのシラミ目;チャタテムシ類Liposcelis bostrychophila、Liposcelis decolor、Liposcelis entomophila、およびTrogium pulsatoriumなどのチャタテムシ目;トリノミCeratophyllus gallinae、イヌノミCtenocephalides canis、ネコノミCtenocephalides felis、ヒトノミPulex irritans、およびケオピスネヅミノミXenopsylla cheopisなどのノミ目;ガーデンシンフィランScutigerella immaculataなどのコムカデ目;オナガシミCtenolepisma longicaudata、フォアラインシルバーフィッシュCtenolepisma quadriseriata、普通のシミLepisma saccharina、およびマダラシミThermobia domesticaなどのシミ目;タバコスリップFrankliniella fusca、ヒラズハナアザミウマFrankliniella intonsa、ミカンキイロアザミウマFrankliniella occidentalis、コットンバッドスリップFrankliniella schultzei、クリバネアザミウマHercinothrips femoralis、ソイビーンスリップNeohydatothrips variabilis、ケリーシトルススリップPezothrips kellyanus、アボカドスリップScirtothrips perseae、ミナミキイロアザミウマThrips palmi、およびネギアザミウマThrips tabaciなどのアザミウマ目等および前記の昆虫の1つ以上を含む組合せ。
(U−ACTX−Hv1aおよびホモログの同定)
雌のブルーマウンテンジョウゴグモ(Hadronyche versuta)を、オーストラリアのニューサウスウェールス州のブルーマウテン地域から入手した。雄および雌のシドニージョウゴグモ(Atrax robustus)は、オーストラリアのニューサウスウェールス州のシドニー都市圏地域から収集した。検体は、毒腺の抽出まで気密な収集用広口びん中に入れておいた。これらのジョウゴグモを、−20℃に40から60分間冷却した。毒腺は各検体から独立に切り出した。各一対の毒腺は、独立に抽出緩衝液(Amersham Pharmacia Biotech)に入れた。
配列番号29:CACCCCTAATACGACTCACTATAGG
配列番号30:(A/G)TTNCC(A/G)TT(T/C)TC(A/G)TT(T/C)TC(T/C)TC(A/G)AA
であった。
配列番号31:TGCTGCAATATGAATACCGC
配列番号32:GGGCAGGTTTTTTTTTTTTTTTTT
であった。
(U−ACTX−Hv1aの組換え形の調製)
U−ACTX−Hv1aの予測された成熟ポリペプチド領域の残基3から39をコードする合成遺伝子を、重なるオリゴヌクレオチドのアニーリング、伸長、および増幅により設計した(図2を参照されたい)。最初の段階で、U−ACTX−Hv1aの残基3から39をコードする4つのオリゴヌクレオチドをアニールして、Pfuポリメラーゼで伸長させた。4つのオリゴヌクレオチドのコドンの使用は、大腸菌(Escherichia coli)での最適発現に対して最適化した。4つのオリゴヌクレオチドは、
FW178−1(配列番号33):
TGCGTTCCGGTTGACCAGCCGTGCTCCCTGAACACCCAGCCG、
FW178−2(配列番号34):
CGTTACGCTCCTGGGTGCAGGTAGCGTCGTCGCAGCACGGCTGGGTGTTCAGGGAGC、
FW178−3(配列番号35):
CGCTACCTGCACCCAGGAGCGTAACGAAAACGGTCACACCGTTTACTACTGCCG、および
FW178−R(配列番号36):
GAATTCTCAAGCACGGCAGTAGTAAACGG
と命名した。
(組換えU−ACTX−Hv1aは昆虫にとって致死的である)
rU−ACTX−Hv1aの殺虫活性は、昆虫生理食塩水に溶解したポリペプチドのMusca domestica(イエバエ)への直接注入により、定量的に測定した。性未確認のハエ(体重10から25mg)に、10から105pmol/gの範囲の濃度で1から2μlのポリペプチドを注入した。対照のハエには2μlの昆虫生理食塩水を注入した。10匹のハエに各濃度のポリペプチドを注入した。29ゲージ針を備えたArnoldマイクロアプリケーター(Burkard Scientific Supply、イングランド、Rickmansworth所在)を、胸背注入投与に使用した。注入するために、検体は4℃で一時的に動けなくして、それからただちに室温(24℃)に戻した。
y=(a−b)/[1+(x/LD50)n]
式中、yは注入後24時間における試料集団中の死亡率(%)であり、xはpmol・g−1での毒素用量であり、nは可変勾配係数であり、aは最大応答であり、bは最小応答である。100±4pmol/gのLD50計算値によれば、rU−ACTX−Hv1aは、これまでに発見された最も強力な殺虫ペプチド毒素の1つである。
(rU−ACTX−Hv1aの分子状標的の決定−アメリカゴキブリPeriplaneta americanaのDUMニューロンでの実験)
[プロトコル]
P.americanaのDUMニューロンはカルシウムチャネルを含み、そこからのCaVチャネル電流(ICa)は、全細胞パッチ−クランプ記録技法を使用して記録することができる。DUMニューロン細胞本体は、P.americanaの神経索の末端腹部神経節(TAG)の正中から単離した。ゴキブリは、−20℃に約5分間冷却することにより麻酔した。次に、それらを切開用シャーレ上に背面側を上にしてピンでとめ、背面角質、消化管内容物、および縦走筋を取り出した。神経節の神経索を確認し、TAGを注意深く取り出して、200mM NaCl、3.1mM KCl、5mM CaCl2、4mM MgCl2、10mM N−[2−ヒドロキシエチル]ピペラジン−N’−[2−エタンスルホン酸](HEPES)、50mMスクロースを含み、5容量%の仔ウシ血清および50IU・ml−1ペニシリンおよび50μg・ml−1ストレプトマイシン(Trace Biosiences、オーストラリアNoble Park所在)を添加し、NaOHを使用してpHを7.4に調節した、通常の昆虫生理食塩水(NIS)に入れた。
I=gmax{1−(1/(1+exp[V−V1/2)/s]))}(V−Vrev)
式中、Iは与えられた電位Vにおけるピーク電流の大きさ、gmaxは最大コンダクタンス、V1/2は最大半量活性における電位差、sは勾配係数、およびVrevは逆転電位である。
P.americanaDUMニューロンからのCaVチャネル電流(ICa)は、全細胞パッチクランプ記録技法を使用して記録した。DUMニューロンは、脱分極試験パルスに続き、内向きのテトロドトキシン(TTX)感受性ナトリウムチャネル電流(INa)および多くの電位作動性カリウムチャネル電流(IK)を生ずる。これらの電流は、TTX、テトラエチルアンモニウム(TEA)、およびCsの組合せを使用して遮断され、CaVチャネルを通して流れる本来の電流が残る。
(rU−ACTX−Hv1aの分子状標的の決定−異種発現したゴキブリpSIoチャネルでの実験)
[プロトコル]
ヒト胚腎(HEK293)細胞(American Type Culture Collection、米国ミズーリ州Bethesda所在)を、ダルベッコ変法イーグル培地(DMEM/高変形、JRH Biosciences、米国カンザス州Lenexa所在)に10%仔ウシ血清を補完した培地中で維持した。pSIoチャネル(P.americanaの高コンダクタンスカルシウム活性化カリウムチャネル)の発現は、G418耐性遺伝子も有する発現ベクターpcDNA3.1(InvitrogenBV、米国カリフォルニア州、San Diego所在)中にクローン化したpSIoコード領域を含む構築物によるHEK293細胞のトランスフェクションにより実施した。35mm2のシャーレ中のHEK293単層を、9μlのリポフェクタミン試薬(Gibco、BRL)および5μgのDNAを使用してトランスフェクトした。次いで、安定にトランスフェクトされた細胞を1000μg ml−1のG418(Gibco,米国ニューヨーク州、Grand Island所在)で選択した。これらの細胞を、上記の通常の成長培地中に維持して、以下に記載するパッチクランプ実験に使用する無菌カバーガラス上で培養した。
P.americanaの高コンダクタンスカルシウム活性化カリウムチャネル(pSIo)のαサブユニットを発現しているHEK293細胞におけるIK(Ca)に対するrU−ACTX−Hv1aの効果を試験した。pSIoチャネル発現HEK293細胞に対する1μM rU−ACTX−Hv1aの添加は、約77%のIK(Ca)遮断を惹起した。この結果は、pSIoチャネルを発現しているHEK293細胞に対する1μMのカリブドトキシン添加について以前に報告された約80%遮断(ダースト(Derst)ら(2003年),European journal of Neuroscience,17,1197〜1212頁)と同様である。用量応答曲線(図5)は、rU−ACTX−Hv1aが、579nMのIC50でpSIo電流を遮断することを示す。したがって、rU−ACTX−Hv1aは、昆虫のCaVおよびKCa両チャネルを標的にすると思われる。チャネル活性化の電位差依存性に明確な脱分極シフトはなかったので、rU−ACTX−Hv1aは、チャネル開閉変形剤であるよりもむしろ細孔遮断薬として作用するようである。
Claims (17)
- 昆虫の電位作動型カルシウムチャネルにおけるカルシウム電流の50%以上および昆虫の高伝導カルシウム活性化カリウムチャネルの活性の50%以上を遮断し、配列番号2と少なくとも90%の同一性を有するアミノ酸配列を含むことを特徴とする殺虫ポリペプチド。
- 請求項1に記載の殺虫ポリペプチドであって、アトラクス(Atrax)属またはハドロニュケ(Hadronyche)属のクモ毒成分であることを特徴とする殺虫ポリペプチド。
- 請求項1に記載の殺虫ポリペプチドであって、前記昆虫の電位作動型カルシウムチャネルが、ワモンゴキブリ(P.americana)のDUMニューロン由来のものであることを特徴とする殺虫ポリペプチド。
- 請求項1に記載の殺虫ポリペプチドであって、前記昆虫の高伝導カルシウム活性化カリウムチャネルが、ワモンゴキブリ(P.americana)の高伝導カルシウム活性化カリウムチャネルを含むことを特徴とする殺虫ポリペプチド。
- 請求項1に記載の殺虫ポリペプチドであって、配列番号2、3、6、9、12、15、18、21、24および27のいずれか1つを含むことを特徴とする殺虫ポリペプチド。
- 請求項5に記載の殺虫ポリペプチドであって、プロペプチド配列、シグナルペプチド配列、またはそれらの組合せをさらに含むことを特徴とする殺虫ポリペプチド。
- 殺虫に有効な量の、請求項1に記載の精製したポリペプチドと、農業的に許容し得る担体とを含むことを特徴とする殺虫組成物。
- 配列番号2と90%以上の同一性を有するアミノ酸配列を含み、殺虫活性を有するポリペプチドをコードすることを特徴とする単離されたポリヌクレオチド。
- 請求項8に記載のポリヌクレオチドであって、配列番号2、3、6、9、12、15、18、21、24および27のいずれか1つを含むポリペプチドをコードすることを特徴とするポリヌクレオチド。
- 発現制御配列に作動可能に連結した、配列番号2、3、6、9、12、15、18、21、24および27のいずれか1つをコードするポリヌクレオチドを含むことを特徴とする発現ベクター。
- 発現制御配列に作動可能に連結した、配列番号2、3、6、9、12、15、18、21、24および27のいずれか1つをコードするポリヌクレオチドを含む発現ベクターを含むことを特徴とする宿主細胞。
- 配列番号2と90%以上の同一性を有するアミノ酸配列を含み、殺虫活性を有するポリペプチドをコードするポリヌクレオチドを含むことを特徴とする昆虫ウィルス。
- 昆虫、昆虫の幼虫、または植物を処理する方法であって、前記昆虫、昆虫の幼虫、または植物と、配列番号2と90%以上の同一性を有するアミノ酸配列を含み、殺虫活性を有するU−ACTXポリペプチドの殺虫に有効な量とを接触させることを含むことを特徴とする方法。
- 請求項13に記載の方法であって、前記U−ACTXポリペプチドが、配列番号2、3、6、9、12、15、18、21、24および27のいずれか1つを含むことを特徴とする方法。
- 配列番号2と90%以上の同一性を有するアミノ酸配列を含み、殺虫活性を有するU−ACTXポリペプチドを発現することを特徴とするトランスジェニック植物。
- 配列番号2と90%以上の同一性を有するアミノ酸配列を含み、殺虫活性を有するポリペプチドをコードするポリヌクレオチドを含むことを特徴とするトランスジェニック昆虫。
- 昆虫のチャネルに結合する試験化合物を選択する方法であって、
昆虫の電位作動型カルシウムチャネル、昆虫のカルシウム活性化カリウムチャネル、または前記昆虫のチャネルの1つ以上を含む組合せである昆虫のチャネルを提供すること、および、
配列番号2と90%以上の同一性を有し、かつ殺虫活性を有するU−ACTXペプチドの前記昆虫のチャネルへの結合と、前記試験化合物が競合するかどうかを決定すること、または前記昆虫のチャネルに予め結合している、配列番号2と90%以上の同一性を有し、かつ殺虫活性を有するU−ACTXペプチドの少なくとも一部を、前記試験化合物が遊離させるかどうかを決定すること、
を含むことを特徴とする方法。
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DK354487A (da) * | 1986-07-11 | 1988-01-12 | Noboru Yanaihara | Oncogen-relaterede peptider |
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US7084119B2 (en) * | 1993-06-29 | 2006-08-01 | Chiron Corporation | Truncated keratinocyte growth factor (KGF) having increased biological activity |
US5457178A (en) * | 1993-07-07 | 1995-10-10 | Fmc Corporation | Insecticidally effective spider toxin |
NZ308772A (en) * | 1995-05-17 | 1999-04-29 | Du Pont | Recombinant baculovirus insecticides |
US5756459A (en) * | 1995-06-07 | 1998-05-26 | Fmc Corporation | Insecticidally effective peptides isolatable from phidippus spider venom |
AUPO733397A0 (en) * | 1997-06-13 | 1997-07-10 | University Of Sydney, The | Pesticidal compounds |
US6521454B1 (en) | 1999-06-30 | 2003-02-18 | The United States Of America, As Represented By The Secretary Of Agriculture | Baculoviruses, insecticidal compositions, and methods for control of invertebrates |
US7279547B2 (en) * | 2000-02-10 | 2007-10-09 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
US6583264B2 (en) * | 2000-02-10 | 2003-06-24 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
DE602005010295D1 (de) | 2004-11-04 | 2008-11-20 | Univ Connecticut | Insektizide polypeptide und deren verwendung |
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2005
- 2005-11-04 DE DE602005010295T patent/DE602005010295D1/de active Active
- 2005-11-04 ES ES05818323T patent/ES2318576T3/es active Active
- 2005-11-04 CA CA2595757A patent/CA2595757C/en active Active
- 2005-11-04 AU AU2005304859A patent/AU2005304859B2/en active Active
- 2005-11-04 EP EP08105465A patent/EP2030983A1/en not_active Withdrawn
- 2005-11-04 WO PCT/US2005/040139 patent/WO2006052806A2/en active Application Filing
- 2005-11-04 AT AT05818323T patent/ATE410445T1/de not_active IP Right Cessation
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- 2005-11-04 JP JP2007540098A patent/JP5107717B2/ja active Active
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WO2006052806A3 (en) | 2006-08-17 |
AU2005304859B2 (en) | 2011-06-30 |
EP2030983A1 (en) | 2009-03-04 |
US20080260789A1 (en) | 2008-10-23 |
US7354993B2 (en) | 2008-04-08 |
AU2005304859A1 (en) | 2006-05-18 |
JP2008518624A (ja) | 2008-06-05 |
EP1812464A2 (en) | 2007-08-01 |
DE602005010295D1 (de) | 2008-11-20 |
CA2595757A1 (en) | 2006-05-18 |
US8293868B2 (en) | 2012-10-23 |
US20110237502A1 (en) | 2011-09-29 |
ES2318576T3 (es) | 2009-05-01 |
US7880060B2 (en) | 2011-02-01 |
US20130276155A1 (en) | 2013-10-17 |
ATE410445T1 (de) | 2008-10-15 |
US20060242734A1 (en) | 2006-10-26 |
WO2006052806A2 (en) | 2006-05-18 |
EP1812464B1 (en) | 2008-10-08 |
CA2595757C (en) | 2015-02-03 |
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