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    Process of clean cloning
    4.
    发明授权
    Process of clean cloning 有权
    清洁克隆的过程

    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

    IPC分类号： C12Q1/68 ,  C12N15/66 ,  C12N15/64

    CPC分类号： C12N15/64 ,  C12N15/66

    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。

    PROCESS OF CLEAN CLONING
    5.
    发明申请
    PROCESS OF CLEAN CLONING 有权
    清洁过程

    公开(公告)号：US20110263024A1

    公开(公告)日：2011-10-27

    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

    IPC分类号： C12N15/87

    CPC分类号： C12N15/64 ,  C12N15/66

    摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: 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 using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3′-end in a nucleotide sequence of nucleic acid sequence segment K3; treating the linear double-stranded DNA molecules contained in the PCR product obtained in the previous step 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; and annealing the product of the previous step to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single-stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA.

    摘要翻译： 包括以下步骤：通过PCR扩增包含以下顺序的DNA序列的已知核苷酸序列K2的核酸序列片段和核酸的方法 已知序列K3的序列片段使用限定扩增DNA第一末端的正向引物和限定扩增DNA第二末端的反向引物，所述反向引物在其核酸序列片段的核苷酸序列的3'末端终止 K3; 用外切核酸酶处理前一步骤中获得的PCR产物中包含的线性双链DNA分子，以在DNA的第一端获得单链突出端，并且在第一端包含核酸片段K2和K3的单链突出端 DNA的第二端; 并将前述步骤的产物退火到线性双链受体核酸，其在第一端具有与DNA的第一末端的单链突出部互补的单链突出端，并且在其第二端具有单链突出端 与DNA的第二末端的单链序列片段K2互补的突出突出端。

    PLANT-BASED RECOMBINANT BUTYRYLCHOLINESTERASE PRODUCTION METHODS
    6.
    发明申请
    PLANT-BASED RECOMBINANT BUTYRYLCHOLINESTERASE PRODUCTION METHODS 有权
    基于植物的重组丁基胆碱酯酶生产方法

    公开(公告)号：US20150184138A1

    公开(公告)日：2015-07-02

    申请号：US14448545

    申请日：2014-07-31

    申请人： Greg Pogue ,  Ernie Hiatt ,  Romy Kandzia ,  Stefan Werner ,  Frank Thieme ,  Tsafrir Mor

    发明人： Greg Pogue ,  Ernie Hiatt ,  Romy Kandzia ,  Stefan Werner ,  Frank Thieme ,  Tsafrir Mor

    IPC分类号： C12N9/18 ,  A61K38/46

    CPC分类号： C12N9/18 ,  A61K38/465 ,  C12Y301/01008

    摘要： A new, reliable, easily scalable and reproducible method for the production of recombinant butyrylcholinesterase (rBuChE) is provided. Through the utilization of a plant transfection procedure, various plant strains have been shown to generate effective and scalable amounts of rBuChE under acceptable manufacturing processes to permit reliable levels of such enzymes for desired nerve agent protection requirements (including tetrameric products). As well, such methods in engineered plant lines have shown suitable production of these enzymes in tetramer form with glycan formation and sialyalation (for terminal groups) to allow for optimal potency against organophosphorus agent exposure as well as proper immunogenic response within the plant sources. The overall production method, including the transfection and production within mammalian cells, as well as the process steps involved for such a reliable sourcing platform from plants is thus encompassed within the invention.

    摘要翻译： 提供了一种用于生产重组丁酰胆碱酯酶（rBuChE）的新的，可靠的，易于扩展和重现的方法。 通过利用植物转染方法，已经显示各种植物菌株在可接受的制造过程中产生有效和可缩放量的rBuChE，以允许可靠水平的这些酶用于所需的神经药物保护要求（包括四聚物产品）。 同样，工程化植物品系中的这些方法已经显示出以四聚体形式合适地生产这些酶，其中聚糖形成和sialyalation（用于末端基团）以允许针对有机磷剂暴露的最佳效力以及在植物来源内的适当的免疫原性反应。 因此，包括哺乳动物细胞内的转染和生产的总体生产方法以及从植物获得这种可靠的采购平台所涉及的工艺步骤。