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    PLANT-BASED RECOMBINANT BUTYRYLCHOLINESTERASE PRODUCTION METHODS
    2.
    发明申请
    PLANT-BASED RECOMBINANT BUTYRYLCHOLINESTERASE PRODUCTION METHODS 审中-公开
    基于植物的重组丁基胆碱酯酶生产方法

    公开(公告)号：WO2015050627A1

    公开(公告)日：2015-04-09

    申请号：PCT/US2014/049387

    申请日：2014-08-01

    Applicant: POGUE, Greg ,  HIATT, Ernie ,  KANDZIA, Romy ,  WERNER, Stefan ,  THIEME, Frank ,  MOR, Tsafrir

    Inventor： POGUE, Greg ,  HIATT, Ernie ,  KANDZIA, Romy ,  WERNER, Stefan ,  THIEME, Frank ,  MOR, Tsafrir

    IPC: C12N9/00

    CPC classification number: C12N9/18 ,  A61K38/465 ,  C12Y301/01008

    Abstract: 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.

    Abstract translation: 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。

    PROCESS OF CLEAN CLONING
    5.
    发明申请
    PROCESS OF CLEAN CLONING 审中-公开
    清洁过程

    公开(公告)号：WO2010040531A3

    公开(公告)日：2010-07-01

    申请号：PCT/EP2009007237

    申请日：2009-10-08

    Applicant: ICON GENETICS GMBH ,  MARILLONNET SYLVESTRE ,  WERNER STEFAN ,  ENGLER CAROLA ,  KANDZIA ROMY ,  THIEME FRANK ,  WEBER ERNST

    Inventor： MARILLONNET SYLVESTRE ,  WERNER STEFAN ,  ENGLER CAROLA ,  KANDZIA ROMY ,  THIEME FRANK ,  WEBER ERNST

    IPC: C12N15/09 ,  C12N15/10

    CPC classification number: C12N15/64 ,  C12N15/66

    Abstract: 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.

    Abstract translation: 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互补的双链突出端。

    PROCESS OF CLEAN CLONING
    6.
    发明申请
    PROCESS OF CLEAN CLONING 审中-公开
    清洁克隆过程

    公开(公告)号：WO2010040531A2

    公开(公告)日：2010-04-15

    申请号：PCT/EP2009/007237

    申请日：2009-10-08

    Applicant: ICON GENETICS GMBH ,  MARILLONNET, Sylvestre ,  WERNER, Stefan ,  ENGLER, Carola ,  KANDZIA, Romy ,  THIEME, Frank ,  WEBER, Ernst

    Inventor： MARILLONNET, Sylvestre ,  WERNER, Stefan ,  ENGLER, Carola ,  KANDZIA, Romy ,  THIEME, Frank ,  WEBER, Ernst

    IPC: C12N15/09 ,  C12N15/10

    CPC classification number: C12N15/64 ,  C12N15/66

    Abstract: 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.

    Abstract translation: 将感兴趣的核酸序列插入受体核酸中的方法，包括以下步骤：通过PCR扩增包含以下顺序的DNA的序列：序列区段U，核酸序列片段 使用限定扩增的DNA的第一末端的正向引物和限定扩增的DNA的第二末端的反向引物的已知核苷酸序列K2和已知序列K3的核酸序列区段，所述反向引物在其3'末端终止于 核酸序列片段K3的核苷酸序列; 用外切核酸酶处理在前一步骤中获得的PCR产物中所含的线性双链DNA分子，以在DNA的第一末端获得单链突出端和单链突出端，所述单链突出端包含位于第一端的核酸片段K2和K3 DNA的第二末端; 和将前一步骤的产物退火成线性化双链受体核酸，所述线性化双链受体核酸在其第一末端具有与DNA第一末端的单链突出端互补的单链突出端，并且在其第二末端具有单一突出端 - 与DNA第二末端的单链序列片段K2互补的多余突出端。

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