UY27864A1 - Producción de péptidos y proteinas por acumulación de cuerpos proteicos derivados de retículo endoplásmico en plantas - Google Patents
Producción de péptidos y proteinas por acumulación de cuerpos proteicos derivados de retículo endoplásmico en plantasInfo
- Publication number
- UY27864A1 UY27864A1 UY27864A UY27864A UY27864A1 UY 27864 A1 UY27864 A1 UY 27864A1 UY 27864 A UY27864 A UY 27864A UY 27864 A UY27864 A UY 27864A UY 27864 A1 UY27864 A1 UY 27864A1
- Authority
- UY
- Uruguay
- Prior art keywords
- nucleic acid
- protein
- endoplasm
- peptides
- proteins
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title abstract 5
- 102000004169 proteins and genes Human genes 0.000 title abstract 5
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract 3
- 238000009825 accumulation Methods 0.000 title 1
- 102000004196 processed proteins & peptides Human genes 0.000 title 1
- 150000007523 nucleic acids Chemical class 0.000 abstract 5
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 3
- 239000002773 nucleotide Substances 0.000 abstract 3
- 125000003729 nucleotide group Chemical group 0.000 abstract 3
- 108020004707 nucleic acids Proteins 0.000 abstract 2
- 102000039446 nucleic acids Human genes 0.000 abstract 2
- 108010055615 Zein Proteins 0.000 abstract 1
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 abstract 1
- 210000003660 reticulum Anatomy 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
- C07K14/425—Zeins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/585—Calcitonins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8221—Transit peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Se describe una molécula de ácido nucleico como que contiene una primera secuencia de ácido nucleico que comprende una secuencia de nucleótidos que codifica la proteína zeína y, o un fragmento de la misma capaz de dirigir y retener una proteína en retículo en doplásmico (ER) de una célula vegetal; una segunda secuencia de ácido nucleico que contiene una secuencia de nucleótidos que codifica una secuencia de aminoácidos que se puede escindir enzimáticamente por medios enzimáticos o químicos; y una tercera secuencia de ácidos nucleicos que contiene la secuencia de nucleóticos que codifica un péptido o una proteína de interés. También se describen los procedimientos de uso de esta molécula de ácido nucleico para transformar las células huéspedes vegetales y producir el péptido o la proteína de interés.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES200201508A ES2224792B1 (es) | 2002-06-28 | 2002-06-28 | Produccion de peptidos y proteinas por acumulacion de cuerpos proteicos derivados de reticulos endoplasmico en plantas. |
Publications (1)
Publication Number | Publication Date |
---|---|
UY27864A1 true UY27864A1 (es) | 2003-09-30 |
Family
ID=29797406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
UY27864A UY27864A1 (es) | 2002-06-28 | 2003-06-25 | Producción de péptidos y proteinas por acumulación de cuerpos proteicos derivados de retículo endoplásmico en plantas |
Country Status (16)
Country | Link |
---|---|
US (2) | US7575898B2 (es) |
EP (1) | EP1523558B1 (es) |
JP (2) | JP4610334B2 (es) |
KR (2) | KR20100017491A (es) |
CN (1) | CN100460510C (es) |
AR (1) | AR040293A1 (es) |
AT (1) | ATE366317T1 (es) |
AU (1) | AU2002333330B2 (es) |
CA (1) | CA2490348A1 (es) |
DE (1) | DE60221051T2 (es) |
DK (1) | DK1523558T3 (es) |
ES (2) | ES2224792B1 (es) |
MX (1) | MXPA05000290A (es) |
RU (1) | RU2381275C2 (es) |
UY (1) | UY27864A1 (es) |
WO (1) | WO2004003207A1 (es) |
Families Citing this family (30)
Publication number | Priority date | Publication date | Assignee | Title |
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US20050032211A1 (en) * | 1996-09-26 | 2005-02-10 | Metabogal Ltd. | Cell/tissue culturing device, system and method |
IL155588A0 (en) * | 2003-04-27 | 2003-11-23 | Metabogal Ltd | Methods for expression of enzymatically active recombinant lysosomal enzymes in transgenic plant root cells and vectors used thereby |
ES2224792B1 (es) * | 2002-06-28 | 2007-02-16 | Era Plantech, S.L. | Produccion de peptidos y proteinas por acumulacion de cuerpos proteicos derivados de reticulos endoplasmico en plantas. |
US7951557B2 (en) | 2003-04-27 | 2011-05-31 | Protalix Ltd. | Human lysosomal proteins from plant cell culture |
GB0426160D0 (en) * | 2004-11-29 | 2004-12-29 | Era Plantech S L | Production of proteins |
GB0426161D0 (en) * | 2004-11-29 | 2004-12-29 | Era Plantech S L | Protein isolation and purification |
US20090249513A1 (en) * | 2005-03-07 | 2009-10-01 | National University Corporation Nagoya University | Method for Expression and Accumulation of Peptide in Plant |
US8163880B2 (en) * | 2006-02-23 | 2012-04-24 | Era Biotech S.A. | Production of biologically active proteins |
US7732569B2 (en) * | 2006-10-19 | 2010-06-08 | E.I. Du Pont De Nemours And Company | Zein-based peptide tags for the expression and purification of bioactive peptides |
DK2150608T3 (en) | 2007-05-07 | 2018-03-12 | Protalix Ltd | LARGE-SCALE ENTRY BIOR ACTOR |
CA2615372A1 (en) | 2007-07-13 | 2009-01-13 | Marc-Andre D'aoust | Influenza virus-like particles (vlps) comprising hemagglutinin |
CA2707235C (en) | 2007-11-27 | 2013-11-19 | Medicago Inc. | Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin |
DK2294202T3 (en) * | 2008-07-08 | 2015-08-31 | Medicago Inc | SOLUBLE RECOMBINANT INFLUENZA ANTIGENES |
KR20110084906A (ko) | 2008-10-10 | 2011-07-26 | 에라 바이오테크, 에스.에이. | 면역원-특이적 면역보강제로서의 재조합 단백질체들 |
BRPI1015053A2 (pt) | 2009-06-24 | 2019-07-09 | Medicago Inc | partículas similares a vírus da influenza quiméricas compreendendo hemaglutinina |
EP2418284A1 (en) | 2010-08-13 | 2012-02-15 | ERA Biotech, S.A. | Protein body-inducing polypeptide sequences |
EP2468872A1 (en) | 2010-12-23 | 2012-06-27 | Philip Morris Products S.A. | Method for producing mullerian inhibitor substance in plants |
EP2468871A1 (en) | 2010-12-23 | 2012-06-27 | Philip Morris Products S.A. | Method for producing apolipoprotein in plants |
EP2468870A1 (en) | 2010-12-23 | 2012-06-27 | Philip Morris Products S.A. | Method for expressing desxoyribonuclease in plants |
CN103403170B (zh) | 2011-01-17 | 2015-11-25 | 菲利普莫里斯生产公司 | 植物中的蛋白质表达 |
FI20115704L (fi) | 2011-07-01 | 2013-01-02 | Teknologian Tutkimuskeskus Vtt Oy | Vierasproteiinien tuoton parantaminen |
WO2013114374A1 (en) * | 2012-02-01 | 2013-08-08 | Protalix Ltd. | Dnase i polypeptides, polynucleotides encoding same, methods of producing dnase i and uses thereof in therapy |
CA2969891A1 (en) * | 2014-12-05 | 2016-06-09 | Planet Biotechnology Inc. | Dpp4 immunoadhesin compositions and methods |
DE102015107846A1 (de) | 2015-05-19 | 2016-11-24 | Johann Wolfgang Goethe-Universität Frankfurt | Synthetische Organellen in Hefe |
KR101848082B1 (ko) * | 2017-05-11 | 2018-04-12 | 주식회사 바이오앱 | 셀룰로오즈 결합 도메인을 포함하는 재조합 벡터 및 상기 벡터를 사용하여 단백질을 분리정제하는 방법 |
BR112019025373A2 (pt) | 2017-05-30 | 2020-06-30 | Agrivida, Inc. | planta transgênica imunomoduladora e métodos relacionados |
US10947552B1 (en) | 2020-09-30 | 2021-03-16 | Alpine Roads, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US10894812B1 (en) | 2020-09-30 | 2021-01-19 | Alpine Roads, Inc. | Recombinant milk proteins |
AU2021353004A1 (en) | 2020-09-30 | 2023-04-13 | Nobell Foods, Inc. | Recombinant milk proteins and food compositions comprising the same |
CN113683669B (zh) * | 2021-03-25 | 2023-05-16 | 中国农业大学 | 基于与醇溶蛋白的互作在植物中积累目标蛋白的方法 |
Family Cites Families (26)
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US4215040A (en) * | 1978-12-22 | 1980-07-29 | The Procter & Gamble Company | Density separation process |
ATE143694T1 (de) * | 1987-10-20 | 1996-10-15 | Plant Genetic Systems Nv | Verfahren zur herstellung eines biologisch- aktiven peptides durch expression von modifizierten speicherproteingenen in transgenetischen pflanzen |
JP3026985B2 (ja) * | 1987-10-20 | 2000-03-27 | プラント・ジェネティック・システムズ・エヌ・ブイ | 遺伝子導入植物内の修飾された貯蔵種子タンパク質遺伝子の発現による生物学的に活性なペプチドの製造方法 |
US5589615A (en) * | 1988-10-14 | 1996-12-31 | Plant Genetic Systems N.V. | Process for the production of transgenic plants with increased nutritional value via the expression of modified 2S storage albumins |
US5571697A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine Texas Medical Center | Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus |
US5650554A (en) | 1991-02-22 | 1997-07-22 | Sembiosys Genetics Inc. | Oil-body proteins as carriers of high-value peptides in plants |
US5948682A (en) * | 1991-02-22 | 1999-09-07 | Sembiosys Genetics Inc. | Preparation of heterologous proteins on oil bodies |
ATE287947T1 (de) * | 1994-10-24 | 2005-02-15 | Texas A & M Univ Sys | Orale immunisierung durch verwendung von transgenen pflanzen |
US5935822A (en) * | 1995-03-21 | 1999-08-10 | The Regents Of The University Of Colorado | Product and process for membrane and soluble polypeptide segregation |
FR2744134B1 (fr) * | 1996-01-29 | 1998-04-03 | Biocem | Proteines de reserve de plantes enrichies en acides amines, notamment alpha-zeine de mais enrichie en lysine plantes exprimant ces proteines |
US5990384A (en) * | 1996-05-31 | 1999-11-23 | New Mexico State University | Co-expression of proteins |
EP1019517B2 (en) * | 1997-09-30 | 2014-05-21 | The Regents of The University of California | Production of proteins in plant seeds |
AR020090A1 (es) * | 1998-06-15 | 2002-04-10 | John Innes Ct | Celula vegetal monocotiledonea o semilla de ella,planta o parte de una planta, metodo para hacer una celula vegetal monocotiledonea, metodo para hacer unaplanta, uso de una construccion de expresion para producir una celula de planta o semilla monocotiledonea, y uso de una construccion de expresion |
EP1141347A1 (en) * | 1998-12-31 | 2001-10-10 | New Mexico State University Technology Transfer Corporation | Co-expression of proteins |
US20020061309A1 (en) * | 2000-03-08 | 2002-05-23 | Garger Stephen J. | Production of peptides in plants as N-terminal viral coat protein fusions |
US7417178B2 (en) | 2000-05-02 | 2008-08-26 | Ventria Bioscience | Expression of human milk proteins in transgenic plants |
WO2002086077A2 (en) | 2001-04-18 | 2002-10-31 | New Mexico State University Technology Transfer Corporation | Co-expression of zein proteins |
JP3940793B2 (ja) * | 2002-05-15 | 2007-07-04 | 独立行政法人農業生物資源研究所 | 任意のペプチドを植物のタンパク顆粒で蓄積させる方法 |
AU2003239894A1 (en) * | 2002-05-24 | 2003-12-12 | Restoragen Inc. | Methods and constructs for high yield expression of clostripain |
ES2224792B1 (es) * | 2002-06-28 | 2007-02-16 | Era Plantech, S.L. | Produccion de peptidos y proteinas por acumulacion de cuerpos proteicos derivados de reticulos endoplasmico en plantas. |
US20070150976A1 (en) | 2003-12-09 | 2007-06-28 | Ventria Bioscience | High-level expression of fusion polypeptides in plant seeds utilizing seed-storage proteins as fusion carriers |
WO2005086813A2 (en) * | 2004-03-08 | 2005-09-22 | Syngenta Participations Ag | Glutamine-rich maize seed protein and promoter |
WO2005113775A1 (en) | 2004-05-21 | 2005-12-01 | Simon Fraser University | Pproduction of a protein localized to the endoplasmic reticulum protein bodies in a transgenic plant and seed |
GB0426161D0 (en) * | 2004-11-29 | 2004-12-29 | Era Plantech S L | Protein isolation and purification |
GB0426160D0 (en) * | 2004-11-29 | 2004-12-29 | Era Plantech S L | Production of proteins |
WO2008078730A1 (ja) * | 2006-12-26 | 2008-07-03 | Translational Research, Ltd. | 経鼻投与用製剤 |
-
2002
- 2002-06-28 ES ES200201508A patent/ES2224792B1/es not_active Expired - Fee Related
- 2002-08-05 KR KR1020097024928A patent/KR20100017491A/ko not_active Application Discontinuation
- 2002-08-05 EP EP02807566A patent/EP1523558B1/en not_active Expired - Lifetime
- 2002-08-05 ES ES02807566T patent/ES2289185T3/es not_active Expired - Lifetime
- 2002-08-05 KR KR1020047021412A patent/KR100987946B1/ko not_active IP Right Cessation
- 2002-08-05 CN CNB028293754A patent/CN100460510C/zh not_active Expired - Fee Related
- 2002-08-05 RU RU2005101738/13A patent/RU2381275C2/ru not_active IP Right Cessation
- 2002-08-05 AT AT02807566T patent/ATE366317T1/de not_active IP Right Cessation
- 2002-08-05 JP JP2004516544A patent/JP4610334B2/ja not_active Expired - Fee Related
- 2002-08-05 MX MXPA05000290A patent/MXPA05000290A/es active IP Right Grant
- 2002-08-05 AU AU2002333330A patent/AU2002333330B2/en not_active Ceased
- 2002-08-05 WO PCT/EP2002/008716 patent/WO2004003207A1/en active IP Right Grant
- 2002-08-05 DE DE60221051T patent/DE60221051T2/de not_active Expired - Lifetime
- 2002-08-05 CA CA002490348A patent/CA2490348A1/en not_active Abandoned
- 2002-08-05 DK DK02807566T patent/DK1523558T3/da active
- 2002-08-06 US US10/213,462 patent/US7575898B2/en not_active Expired - Fee Related
-
2003
- 2003-06-25 UY UY27864A patent/UY27864A1/es not_active Application Discontinuation
- 2003-06-27 AR ARP030102330A patent/AR040293A1/es active IP Right Grant
-
2009
- 2009-08-17 US US12/542,514 patent/US8802825B2/en not_active Expired - Fee Related
-
2010
- 2010-05-12 JP JP2010110481A patent/JP2010213713A/ja not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
WO2004003207A9 (en) | 2005-02-10 |
WO2004003207A1 (en) | 2004-01-08 |
ES2289185T3 (es) | 2008-02-01 |
EP1523558B1 (en) | 2007-07-04 |
CN100460510C (zh) | 2009-02-11 |
KR100987946B1 (ko) | 2010-10-18 |
DK1523558T3 (da) | 2007-11-05 |
US7575898B2 (en) | 2009-08-18 |
AU2002333330B2 (en) | 2007-07-12 |
CA2490348A1 (en) | 2004-01-08 |
JP2010213713A (ja) | 2010-09-30 |
DE60221051T2 (de) | 2008-03-13 |
AR040293A1 (es) | 2005-03-23 |
ATE366317T1 (de) | 2007-07-15 |
DE60221051D1 (de) | 2007-08-16 |
MXPA05000290A (es) | 2005-03-31 |
AU2002333330A1 (en) | 2004-01-19 |
US20040005660A1 (en) | 2004-01-08 |
ES2224792A1 (es) | 2005-03-01 |
KR20100017491A (ko) | 2010-02-16 |
CN1639342A (zh) | 2005-07-13 |
EP1523558A1 (en) | 2005-04-20 |
JP4610334B2 (ja) | 2011-01-12 |
ES2224792B1 (es) | 2007-02-16 |
JP2005531309A (ja) | 2005-10-20 |
KR20050024419A (ko) | 2005-03-10 |
US20100083403A1 (en) | 2010-04-01 |
RU2005101738A (ru) | 2006-07-20 |
RU2381275C2 (ru) | 2010-02-10 |
US8802825B2 (en) | 2014-08-12 |
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