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MODIFIED Α-HEMOLYSIN AND NANOPORE SEQUENCER USING SAME UPDATE

外国特許コード F150008482
整理番号 E102P06WO
掲載日 2015年10月26日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2014JP069540
国際公開番号 WO 2015016125
国際出願日 平成26年7月24日(2014.7.24)
国際公開日 平成27年2月5日(2015.2.5)
優先権データ
  • 特願2013-157799 (2013.7.30) JP
発明の名称 (英語) MODIFIED Α-HEMOLYSIN AND NANOPORE SEQUENCER USING SAME UPDATE
発明の概要(英語) Provided is a modified α-hemolysin in which a mutation is introduced to the amino acid sequence of an α-hemolysin monomer and which has arginine at the 147th position or glutamine at the 146th position from the N terminal of the amino acid sequence, and due to this modified α-hemolysin, the ability to form a nanopore in a lipid membrane is enhanced without reducing enzyme activity when compared to the wild form.
従来技術、競合技術の概要(英語) BACKGROUND ART
Α - hemolysin is, secreted from Staphylococcus aureus 7 (Staphylococcus aureus) the amount of the poison protein, the protein is water-soluble, open nanopores in the lipid bilayer membrane, red blood cells to activity. Of a material having a molecular weight of about 3kDa in a bi-directional diffusion (transmission) can be synthesized using a function of promoting biological used in the field. Α - hemolysin is, the amount of the 7 for the first time, the lipid film to form pores (nanopores) can be known.
Α - using such toxins, have been proposed one after another generation sequencer (for example, in Japanese Unexamined Patent Publication 2011-527191 (Patent Document 1)). That is, the amount of 7 α - in the lipid bilayer membrane made of a hemolysin nanopore is formed, through which the chain DNA sequence information technology. That is, a voltage is applied across the nanopore and the ion current flows, and pass through pores of the nano-chain DNA at the moment of change in the current value detected for each base 1, to obtain sequence information. At this time, the kind of the base 4 so as to improve the distinguishing DNA sequences can result can be obtained. Compared with the conventional sequencer, and is easy to adjust the sample, such as on the fragmentation processing is unnecessary, and to some extent in the blood dirty solution can be used to directly analyze DNA. And the nucleotide sequence in real time, the nucleotide sequence obtained by analyzing the user an intended to obtain the result sequencing can be continued, because of their advantages.
However, in current technology is the limited number of nanopore chip 1 (approximately 10000) and, as an array of numbers read unit billion to decrypt the nano pore formation and to improve the efficiency of which require some contrivance such as (for example, protein translocation through an α-hemolysin nanopore J. Nivala et al., "Unfoldase-mediated", Nature Biotechnology,2013,31 (3), (non-patent document 1) pp.247-250 reference.).
From this point of view, the ability of the lipid membrane higher than the nano-pore-forming toxins which is where it is desirable to provide α -.
The past, a hemolysin for α -, enzyme due to the introduction of point mutations have been attempted modification technique is, causes a reduction of the enzymatic activity is in most cases (for example, b. Walker et al., "Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification", The Journal of Biological Chemistry,1995,Vol.270,No.39,pp.23065-23071( non-patent document 2) reference.).
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • YOMO, Tetsuya
  • MATSUURA, Tomoaki
  • KAZUTA, Yasuaki
  • FUJII, Satoshi
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
参考情報 (研究プロジェクト等) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
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