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    HOMOLOGOUS RECOMBINATION METHOD, CLONING METHOD, AND KIT
    51.
    发明公开
    HOMOLOGOUS RECOMBINATION METHOD, CLONING METHOD, AND KIT 有权
    志愿者重新安置，KLONVERFAHREN UND KIT

    公开(公告)号：EP2251423A1

    公开(公告)日：2010-11-17

    申请号：EP09717816.4

    申请日：2009-03-06

    申请人： National University Corporation University Of Toyama

    发明人： ISOBE, Masaharu ,  KUROSAWA, Nobuyuki

    IPC分类号： C12N15/09 ,  C12R1/19

    CPC分类号： C12N15/66 ,  C12N15/1082

    摘要： The present invention provides a method which can achieve the homologous recombination of a gene of interest selectively, and a recombinant DNA molecule produced by the method. A homologous recombination method which uses a PCR product and a linearlized vector is disposed. The PCR product comprises a sequence for a target gene and amplification primer sequences P1 and P2 on both terminal ends. The vector contains homologous recombination regions VP1 and VP2 which respectively comprises nucleotide sequences homologous to the amplification primer sequences P1 and P2 of the PCR product, and a homologous recombination region VT1 which comprises a nucleotide sequence homologous to a sequence T1 that is a part of sequence in the PCR product internal to P1 on the terminal side of VP1 and/or a homologous recombination region VT2 which comprises a nucleotide sequence homologous to a sequence T2 that is a part of sequence in the PCR product internal to P2 on the terminal side of VP2 (provided that at least one of T1 and T2 has a nucleotide sequence specific to the target gene). The method comprises subjecting the PCR product to a homologous recombination reaction to insert the PCR product into the vector, thereby producing a recombinant DNA molecule in which the PCR product of interest specifically inserted into the vector. Also disclosed a cloning method for producing the recombinant DNA molecule comprising the vector and the PCR product of interest specifically inserted into the vector by the homologous recombination method, and amplifying the recombinant DNA molecule.

    摘要翻译： 本发明提供了可以选择性地实现感兴趣的基因的同源重组的方法和通过该方法产生的重组DNA分子。 使用PCR产物和线性化载体的同源重组方法。 PCR产物包含两个末端的靶基因序列和扩增引物序列P1和P2。 载体含有同源重组区VP1和VP2，其分别包含与PCR产物的扩增引物序列P1和P2同源的核苷酸序列，以及同源重组区VT1，其包含与作为序列的一部分的序列T1同源的核苷酸序列 在VP1的末端侧的P1内部的PCR产物和/或同源重组区域VT2中，该同源重组区域VT2包含与序列T2同源的序列T2的核苷酸序列，该序列T2是VP2的末端侧的P2内部的PCR产物的一部分序列 （条件是T1和T2中的至少一个具有对靶基因特异性的核苷酸序列）。 该方法包括使PCR产物进行同源重组反应以将PCR产物插入载体中，从而产生重组DNA分子，其中感兴趣的PCR产物特异性插入载体中。 还公开了通过同源重组法将包含载体和感兴趣的PCR产物特异性插入载体的重组DNA分子的克隆方法，并扩增重组DNA分子。

    METHOD FOR CLONING T CELL RECEPTOR
    55.
    发明公开
    METHOD FOR CLONING T CELL RECEPTOR 审中-公开
    VERFAHREN ZUM KLONEN EINES T-ZELL-REZEPTORS

    公开(公告)号：EP2899269A1

    公开(公告)日：2015-07-29

    申请号：EP13823838.1

    申请日：2013-07-24

    申请人： National University Corporation University Of Toyama

    发明人： KISHI, Hiroyuki ,  MURAGUCHI, Atsushi ,  KOBAYASHI, Eiji ,  OZAWA, Tatsuhiko

    IPC分类号： C12N15/09 ,  C12N5/10

    CPC分类号： C12P19/34 ,  C07K14/7051 ,  C12N5/0636 ,  C12N15/85

    摘要： An object is to provide a TCR cloning system that enables not only bias-free analysis of TCR repertoires, but also collection of antigen-specific TCR α/β cDNA pairs and evaluation of functions thereof. There is provided a method for producing a gene of T cell receptor (TCR) specific to an antigen A, which comprises 1) the step of stimulating a group of T cells including a T cell specific to an antigen A or one T cell specific to an antigen A under a condition effective for amplification of a TCR gene; 2) the step of identifying a T cell specific to an antigen A among the group of T cells including a T cell specific to the antigen A, and sorting one T cell specific to the antigen A into a vessel; and 3) the step of subjecting the one activated T cell specific to the antigen A in the vessel to PCR to amplify a gene of TCR specific to the antigen A. According to the present invention, a target TCR gene can be cloned within a shorter time compared with that required by the conventional methods, for example, about ten days. Further, according to the present invention, genes of TCR α chain and β chain can be highly efficiently cloned. Under the conditions of the examples, a pair of a TCR α chain and TCR β chain could be obtained from stimulated T cells sorted as single cells at a ratio of 100%.

    摘要翻译： 目的是提供一种TCR克隆系统，其不仅能够对TCR谱系进行无偏差分析，而且能够收集抗原特异性TCR±/ / 2 cDNA对和其功能的评估。 提供了一种用于产生对抗原A特异性的T细胞受体（TCR）基因的方法，其包括：1）刺激一组T细胞的步骤，所述T细胞包括对抗原A特异性的T细胞或一种特异于T细胞的T细胞 在有效扩增TCR基因的条件下的抗原A; 2）鉴定包含对抗原A特异性的T细胞的T细胞群体中特异于抗原A的T细胞的步骤，将对抗原A特异性的1个T细胞分选成容器; 和3）将所述特异性的一种活化的T细胞对所述血管中的抗原A进行PCR的步骤，以扩增抗原A特异性的TCR基因。根据本发明，靶TCR基因可以在较短的 时间与常规方法所需的时间相比，例如约十天。 此外，根据本发明，可以高效克隆TCR±链和链的基因。 在实施例的条件下，可以从100％的比例分选为单细胞的刺激的T细胞获得一对TCR±链和TCR2链。

    ARCTIGENIN-CONTAINING BARDANAE FRUCTUS EXTRACT AND METHOD FOR PRODUCING SAME
    59.
    发明公开
    ARCTIGENIN-CONTAINING BARDANAE FRUCTUS EXTRACT AND METHOD FOR PRODUCING SAME 审中-公开
    维多利亚州泽西尔赫斯特朗

    公开(公告)号：EP2623107A1

    公开(公告)日：2013-08-07

    申请号：EP11829102.0

    申请日：2011-09-27

    申请人： Kracie Pharma, Ltd. ,  National Cancer Center ,  National University Corporation University Of Toyama

    发明人： OKUBO, Toshiki ,  YOMODA, Satoshi ,  FUSE, Takafumi ,  KAWASHIMA, Takanori ,  ESUMI, Hiroyasu ,  MIYOSHI, Chika ,  KADOTA, Shigetoshi

    IPC分类号： A61K36/28 ,  A61K31/365 ,  A61K31/7048 ,  A61K36/00 ,  A61P35/00 ,  C07D307/33

    CPC分类号： A61K36/28 ,  A61K31/365 ,  A61K31/7048 ,  A61K2236/19 ,  C07D307/33

    摘要： PROBLEM TO BE SOLVED
    The present invention is intended to provide a burdock fruit extract containing arctigenin and arctiin at a definite ratio and a method for producing the same. More particularly, the present invention is intended to provide a method for producing a burdock fruit extract containing arctigenin and arctiin at a weight ratio of approximately 1:1.
    SOLUTION
    A method for producing a burdock fruit extract containing arctigenin and arctiin at a weight ratio of arctigenin/arctiin = 0.7 to 1.3 is provided, including the steps of: cutting a burdock fruit and converting arctiin which is inherent in the burdock fruit into arctigenin by enzymatic conversion by beta-glucosidase which is inherent in the burdock fruit, wherein the enzymatic conversion includes reaction at a temperature from 20°C to 50°C. Also provided is the method further including a step of extracting an extract containing arctigenin and arctiin by adding an organic solvent and heating to reflux.

    摘要翻译： 待解决的问题本发明旨在提供一种含有一定比例的抗坏血酸和铁蛋白的牛蒡果实提取物及其制造方法。 更具体地说，本发明旨在提供一种以大约1:1的重量比生产含有抗坏血酸和铁蛋白的牛蒡果实提取物的方法。 解决方案提供一种含有抗坏血酸素/ arctinin = 0.7〜1.3的重量比的红曲霉素和arctiin的牛蒡果实提取物的方法，包括以下步骤：将牛蒡水果切割成牛蒡果实固有的arctiin， 牛蒡果实固有的β-葡糖苷酶的酶转化，其中酶促转化包括在20℃至50℃的温度下进行反应。 该方法还包括通过加入有机溶剂并加热回流来提取含有抗坏血酸和铁蛋白的提取物的步骤。