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公开(公告)号：US08093458B2
公开(公告)日：2012-01-10
申请号：US11631719
申请日：2005-07-07
申请人： Sylvestre Marillonnet , Carola Engler , Stefan Mühlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Stefan Mühlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
CPC分类号： C12N15/8203 , C12N15/8205 , C12N15/8216 , C12N15/8257
摘要： A process of producing a protein of interest by expression of said protein of interest from a sequence of interest in a plant or in plant leaves, comprising: (a) transfecting said plant or said plant leaves by infiltrating said plant or said plant leaves with an Agrobacterium strain in the presence of a complementing factor, said Agrobacterium strain containing in T-DNA a heterologous DNA sequence having a sequence portion encoding a replicon, wherein said sequence encoding a replicon contains sequences necessary for replicon function of said replicon, said sequences being derived from a plant virus, and said sequence of interest to be expressed from said replicon, (b) optionally isolating said protein of interest from said plant or said plant leaves infiltrated in step (a), wherein said Agrobacterium strain is provided with a first genetic modification rendering said Agrobacterium strain defective for transfecting organisms with said T-DNA in the absence of said complementing factor.
摘要翻译： 通过从植物或植物叶中感兴趣的序列表达所述目标蛋白质来产生感兴趣的蛋白质的方法，包括：（a）通过用所述植物或所述植物叶子渗透所述植物或所述植物叶来转染所述植物或所述植物叶 农杆菌菌株在补充因子存在下，所述含有T-DNA的土壤杆菌菌株具有编码复制子的序列部分的异源DNA序列，其中编码复制子的所述序列含有所述复制子的复制子功能所必需的序列，所述序列得自 从所述复制子表达的所述感兴趣的序列，（b）任选地从所述植物或所述在步骤（a）中渗透的所述植物叶分离所述目的蛋白质，其中所述农杆菌菌株被提供有第一遗传 在没有所述补充因子的情况下，使所述农杆菌菌株对所述T-DNA转染生物有缺陷。
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公开(公告)号：US20080057563A1
公开(公告)日：2008-03-06
申请号：US11631719
申请日：2005-07-07
申请人： Sylvestre Marillonnet , Carola Engler , Stefan Muhlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Stefan Muhlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
CPC分类号： C12N15/8203 , C12N15/8205 , C12N15/8216 , C12N15/8257
摘要： A process of producing a protein of interest by expression of said protein of interest from a sequence of interest in a plant or in plant leaves, comprising: (a) transfecting said plant or said plant leaves by infiltrating said plant or said plant leaves with an Agrobacterium strain in the presence of a complementing factor, said Agrobacterium strain containing in T-DNA a heterologous DNA sequence having a sequence portion encoding a replicon, wherein said sequence encoding a replicon contains sequences necessary for replicon function of said replicon, said sequences being derived from a plant virus, and said sequence of interest to be expressed from said replicon, (b) isolating said protein of interest from said plant or said plant leaves infiltrated in step (a), wherein said Agrobacterium strain is provided with a first genetic modification rendering said Agrobacterium strain defective for transfecting organisms with said T-DNA in the absence of said complementing factor.
摘要翻译： 通过从植物或植物叶中感兴趣的序列表达所述目标蛋白质来产生感兴趣的蛋白质的方法，包括：（a）通过用所述植物或所述植物叶子渗透所述植物或所述植物叶来转染所述植物或所述植物叶 农杆菌菌株在补充因子存在下，所述含有T-DNA的土壤杆菌菌株具有编码复制子的序列部分的异源DNA序列，其中编码复制子的所述序列含有所述复制子的复制子功能所必需的序列，所述序列得自 （b）从所述植物或所述在步骤（a）中渗透的所述植物叶分离所述感兴趣的蛋白质，其中所述农杆菌菌株被提供有第一种遗传修饰物 使得所述农杆菌菌株在缺乏所述补体因子的情况下缺陷以用所述T-DNA转染生物体。
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公开(公告)号：US20070300330A1
公开(公告)日：2007-12-27
申请号：US10586998
申请日：2005-01-19
CPC分类号： C12N15/8216
摘要： A process for replicating or for replicating and expressing a sequence of interest in a plant, comprising: (i) an RNA replicon or a precursor thereof, said RNA replicon being derived from a plus-sense single stranded RNA virus and comprising at least one sequence of interest; and (ii) a helper replicon, or a precursor thereof, wherein said helper replicon is (a) incapable of systemic movement in said plant both in the presence and in the absence of said RNA replicon (i) and (b) capable of expressing in a plant one or more proteins necessary for systemic movement of said RNA replicon (i), whereby said RNA replicon (i) is capable of replicating or replicating and expressing said sequence of interest in said plant, but unable to move systemically in said plant in the absence of said one or more proteins expressed by said helper replicon (ii).
摘要翻译： 一种在植物中复制或表达感兴趣的序列的方法，包括：（i）RNA复制子或其前体，所述RNA复制子衍生自正义单链RNA病毒，并且包含至少一个序列 出于兴趣; 辅助复制子或其前体，其中所述辅助复制子是（a）在所述RNA复制子（i）和（b）能够表达的存在和不存在下，在所述植物中不能全身运动 在植物中，所述RNA复制子（i）的系统运动所必需的一种或多种蛋白质，其中所述RNA复制子（i）能够在所述植物中复制或复制和表达所述感兴趣的序列，但不能在所述植物中系统地移动 在不存在由所述辅助复制子（ii）表达的所述一种或多种蛋白质的情况下。
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公开(公告)号：US09267143B2
公开(公告)日：2016-02-23
申请号：US10578962
申请日：2004-11-10
CPC分类号： C12N15/8203 , C12N15/8216
摘要： A process of expressing a sequence of interest in a plant, plant part, or plant cell culture, comprising: (a) providing a plant, plant part, or plant cell culture containing in cell nuclei a heterologous DNA having a sequence encoding an RNA replicon operably linked or linkable to a transcription promoter, wherein said sequence encoding an RNA replicon contains (i) sequences for replicon function of said RNA replicon, said sequences being derived from a sequence of a plant RNA virus, (ii) a sequence of interest, whereby said sequences for replicon function exhibit at selected localities of said sequences of said plant RNA virus function conservative differences from said sequence of said plant RNA virus, said differences causing an increased frequency of replicon formation compared to an RNA replicon not exhibiting said differences; and (b) causing expression of said sequence of interest.
摘要翻译： 在植物，植物部分或植物细胞培养物中表达感兴趣的序列的方法，包括：（a）提供在细胞核中含有编码RNA复制子的序列的异源DNA的植物，植物部分或植物细胞培养物 其可编码RNA复制子的所述序列包含（i）所述RNA复制子的复制子功能的序列，所述序列衍生自植物RNA病毒的序列，（ii）感兴趣的序列， 其中所述复制子功能的序列在所述植物RNA病毒的所述序列的选定位置处表现出与所述植物RNA病毒的所述序列的保守差异，所述差异与不显示所述差异的RNA复制子相比导致复制子形成的频率增加; 和（b）引起所述感兴趣序列的表达。
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公开(公告)号：US08597950B2
公开(公告)日：2013-12-03
申请号：US10586998
申请日：2005-01-19
IPC分类号： C12N15/86 , A61K38/00 , C12N7/00 , C07K14/415 , C07K14/005 , C07H21/04 , C12N5/04
CPC分类号： C12N15/8216
摘要： A process for replicating or for replicating and expressing a sequence of interest in a plant, comprising: (i) an RNA replicon or a precursor thereof, said RNA replicon being derived from a plus-sense single stranded RNA virus and comprising at least one sequence of interest; and (ii) a helper replicon, or a precursor thereof, wherein said helper replicon is (a) incapable of systemic movement in said plant both in the presence and in the absence of said RNA replicon (i) and (b) capable of expressing in a plant one or more proteins necessary for systemic movement of said RNA replicon (i), whereby said RNA replicon (i) is capable of replicating or replicating and expressing said sequence of interest in said plant, but unable to move systemically in said plant in the absence of said one or more proteins expressed by said helper replicon (ii).
摘要翻译： 一种在植物中复制或表达感兴趣的序列的方法，包括：（i）RNA复制子或其前体，所述RNA复制子衍生自正义单链RNA病毒，并且包含至少一个序列 出于兴趣; 辅助复制子或其前体，其中所述辅助复制子是（a）在所述RNA复制子（i）和（b）能够表达的存在和不存在下，在所述植物中不能全身运动 在植物中，所述RNA复制子（i）的系统运动所必需的一种或多种蛋白质，其中所述RNA复制子（i）能够在所述植物中复制或复制和表达所述感兴趣的序列，但不能在所述植物中系统地移动 在不存在由所述辅助复制子（ii）表达的所述一种或多种蛋白质的情况下。
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公开(公告)号：US08415463B2
公开(公告)日：2013-04-09
申请号：US12438743
申请日：2007-09-06
CPC分类号： C12N15/8203 , C12N15/8216 , C12N2770/26011 , C12N2770/26043
摘要： Nucleic acid comprising or encoding an RNA replica comprising, in this order, the following segments (i) to (iii): i) a nucleic acid sequence encoding a potexvirus RNA-dependent RNA polymerase or a function-conservative variant thereof; ii) a nucleic acid sequence comprising: a) a potexvirus triple gene block or a function-conservative variant thereof and b) a sequence encoding a potexviral coat protein or a function-conservative variant thereof; or a sequence encoding a tobago viral movement protein; and iii) a heterologous nucleic acid sequence expressible from said replica in a plant or in plant tissue.
摘要翻译： 包含或编码RNA复制品的核酸，其依次包括以下片段（i）至（iii）：i）编码病毒RNA依赖性RNA聚合酶或其功能保守变体的核酸序列; ii）核酸序列，其包含：a）罐状病毒三重基因嵌段或其功能保守变体，和b）编码潜在外壳蛋白或其功能保守变体的序列; 或编码多巴胺病毒运动蛋白的序列; 和iii）在植物或植物组织中可从所述复制品表达的异源核酸序列。
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公开(公告)号：US20110055976A1
公开(公告)日：2011-03-03
申请号：US12920847
申请日：2009-03-03
CPC分类号： C12N15/8257 , C07K14/415
摘要： A process of producing a protease in a plant or in plant cells, comprising (a) providing a plant comprising a heterologous nucleotide sequence comprising a coding sequence encoding a fusion protein, said fusion protein comprising: an apoplast or plastid signal peptide; a SUMO protein or a derivative of a SUMO protein; and a zymogen of said protease, and (b) expressing said fusion protein.
摘要翻译： 一种在植物或植物细胞中产生蛋白酶的方法，包括（a）提供包含异源核苷酸序列的植物，所述异源核苷酸序列包含编码融合蛋白的编码序列，所述融合蛋白包含：质外体或质体信号肽; SUMO蛋白或SUMO蛋白的衍生物; 和所述蛋白酶的酶原，和（b）表达所述融合蛋白。
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公开(公告)号：US20100071084A1
公开(公告)日：2010-03-18
申请号：US12438743
申请日：2007-09-06
CPC分类号： C12N15/8203 , C12N15/8216 , C12N2770/26011 , C12N2770/26043
摘要： Nucleic acid comprising or encoding an RNA replica comprising, in this order, the following segments (i) to (iii): i) a nucleic acid sequence encoding a potexvirus RNA-dependent RNA polymerase or a function-conservative variant thereof; ii) a nucleic acid sequence comprising: a) a potexvirus triple gene block or a function-conservative variant thereof and b) a sequence encoding a potexviral coat protein or a function-conservative variant thereof; or a sequence encoding a tobago viral movement protein; and iii) a heterologous nucleic acid sequence expressible from said replica in a plant or in plant tissue.
摘要翻译： 包含或编码RNA复制品的核酸，其依次包括以下片段（i）至（iii）：i）编码病毒RNA依赖性RNA聚合酶或其功能保守变体的核酸序列; ii）核酸序列，其包含：a）罐状病毒三重基因嵌段或其功能保守变体，和b）编码潜在外壳蛋白或其功能保守变体的序列; 或编码多巴胺病毒运动蛋白的序列; 和iii）在植物或植物组织中可从所述复制品表达的异源核酸序列。
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公开(公告)号：US20070044170A1
公开(公告)日：2007-02-22
申请号：US10578962
申请日：2004-11-10
CPC分类号： C12N15/8203 , C12N15/8216
摘要： A process of expressing a sequence of interest in a plant, plant part, or plant cell culture, comprising: (a) providing a plant, plant part, or plant cell culture containing in cell nuclei a heterologous DNA having a sequence encoding an RNA replicon operably linked or linkable to a transcription promoter, wherein said sequence encoding an RNA replicon contains (i) sequences for replicon function of said RNA replicon, said sequences being derived from a sequence of a plant RNA virus, (ii) a sequence of interest, whereby said sequences for replicon function exhibit at selected localities of said sequences of said plant RNA virus functionconservative differences from said sequence of said plant RNA virus, said differences causing an increased frequency of replicon formation compared to an RNA replicon not exhibiting said differences; and (b) causing expression of said sequence of interest.
摘要翻译： 在植物，植物部分或植物细胞培养物中表达感兴趣的序列的方法，包括：（a）提供在细胞核中含有编码RNA复制子的序列的异源DNA的植物，植物部分或植物细胞培养物 其可编码RNA复制子的所述序列包含（i）所述RNA复制子的复制子功能的序列，所述序列衍生自植物RNA病毒的序列，（ii）感兴趣的序列， 其中所述用于复制子功能的序列在所述植物RNA病毒功能的所述序列的所选序列的所选位点处表现出与所述植物RNA病毒的所述序列相比的所述差异，所述差异导致复制子形成的频率增加，与不显示所述差异的RNA复制子相比; 和（b）引起所述感兴趣序列的表达。
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公开(公告)号：US08883420B2
公开(公告)日：2014-11-11
申请号：US13121019
申请日：2009-10-08
申请人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
发明人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid is provided. The process comprises amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2, and a nucleic acid sequence segment of known sequence K3. The process further comprises treating the linear double-stranded DNA molecules from the PCR amplification with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA. The process additionally comprises annealing the product of the exonuclease treatment to a linearized double-stranded acceptor nucleic acid which has been designed to complement the single-stranded overhangs of the product of the exonuclease treatment.
摘要翻译： 提供将感兴趣的核酸序列插入受体核酸的过程。 该方法包括通过PCR扩增包含以下顺序的序列片段U，已知核苷酸序列K2的核酸序列片段和已知序列K3的核酸序列片段的DNA。 该方法还包括用外切核酸酶从PCR扩增中处理线性双链DNA分子，以在DNA的第一端获得单链突出端，并且在第二末端包含包含核酸片段K2和K3的单链突出端 的DNA。 该方法还包括将外切核酸酶处理的产物退火至线性化双链受体核酸，其被设计为补充外切核酸酶处理产物的单链突出端。
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