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    • 2. 发明申请
    • METHOD FOR ASSAYING REPLICATION OF HBV AND TESTING SUSCEPTIBILITY TO DRUGS
    • 测定HBV复制的方法和测试药物的不良性
    • WO2004029301A3
    • 2005-02-24
    • PCT/EP0312398
    • 2003-09-26
    • INST NAT SANTE RECH MEDDURANTEL DAVIDDURANTEL SANDRATREPO CHRISTIANZOULIM FABIEN
    • DURANTEL DAVIDDURANTEL SANDRATREPO CHRISTIANZOULIM FABIEN
    • C12Q1/70
    • C12Q1/706
    • A method for measuring the replication capacity of HBV, e.g. HBV present in a biological sample, possibly in the presence of a pharmaceutical product, in particular an antiviral agent, the method comprising: (a) possibly extracting nucleic acids contained in the biological sample; (b) PCR amplifying HBV nucleic acids using at least two primer pairs selected so as to obtain at least two different amplified HBV genomic fragments which upon assembly represent a linear continuous DNA sequence transcriptable in pgRNA ; (c) cloning the fragments obtained under (b) into a vector under the control of an heterologous promoter, so producing a vector containing a linear continuous DNA sequence transcriptable in pgRNA under control of said promoter; (d) transfecting or transducing susceptible cells with the vector; (e) culturing the transfected or transduced cells in conditions allowing synthesis of HBV pgRNA from the cloned HBV DNA; (f) possibly treating the cultured cells with the pharmaceutical product, in particular antiviral agent; and (g) determining the replication capacity of the HBV, possibly incidence of the pharmaceutical product, preferably antiviral agent, on viral gene expression and/or viral replication.
    • 测定HBV复制能力的方法,例如 HBV存在于生物样品中,可能存在药物产品,特别是抗病毒剂,该方法包括:(a)可能提取生物样品中所含的核酸; (b)使用选择的至少两个引物对PCR扩增HBV核酸,以获得至少两种不同的扩增的HBV基因组片段,其在组装时表示可转录到pgRNA中的线性连续DNA序列; (c)将在(b)获得的片段克隆到异源启动子控制下的载体中,从而产生含有在所述启动子控制下在pgRNA中转录的线性连续DNA序列的载体; (d)用载体转染或转导敏感细胞; (e)在允许从克隆的HBV DNA合成HBV pgRNA的条件下培养转染或转导的细胞; (f)可能用药物产品,特别是抗病毒剂处理培养的细胞; 和(g)确定HBV的复制能力,药物产品,优选抗病毒剂可能发生在病毒基因表达和/或病毒复制上。
    • 9. 发明申请
    • METHOD FOR ASSAYING REPLICATION OF HBV AND TESTING SUSCEPTIBILITY TO DRUGS
    • 测定HBV复制的方法和测试药物的不良性
    • WO2004029301A2
    • 2004-04-08
    • PCT/EP2003/012398
    • 2003-09-26
    • INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM)DURANTEL, DavidDURANTEL, SandraTREPO, ChristianZOULIM, Fabien
    • DURANTEL, DavidDURANTEL, SandraTREPO, ChristianZOULIM, Fabien
    • C12Q1/70
    • C12Q1/706
    • A method for measuring the replication capacity of HBV, e.g. HBV present in a biological sample, possibly in the presence of a pharmaceutical product, in particular an antiviral agent, the method comprising: (a) possibly extracting nucleic acids contained in the biological sample; (b) PCR amplifying HBV nucleic acids using at least two primer pairs selected so as to obtain at least two different amplified HBV genomic fragments which upon assembly represent a linear continuous DNA sequence transcriptable in pgRNA ; (c) cloning the fragments obtained under (b) into a vector under the control of an heterologous promoter, so producing a vector containing a linear continuous DNA sequence transcriptable in pgRNA under control of said promoter; (d) transfecting or transducing susceptible cells with the vector; (e) culturing the transfected or transduced cells in conditions allowing synthesis of HBV pgRNA from the cloned HBV DNA; (f) possibly treating the cultured cells with the pharmaceutical product, in particular antiviral agent; and (g) determining the replication capacity of the HBV, possibly incidence of the pharmaceutical product, preferably antiviral agent, on viral gene expression and/or viral replication.
    • 测定HBV复制能力的方法,例如 HBV存在于生物样品中,可能存在药物产品,特别是抗病毒剂,该方法包括:(a)可能提取生物样品中所含的核酸; (b)使用选择的至少两个引物对PCR扩增HBV核酸,以获得至少两种不同的扩增的HBV基因组片段,其在组装时表示可转录到pgRNA中的线性连续DNA序列; (c)将在(b)获得的片段克隆到异源启动子控制下的载体中,从而产生含有在所述启动子控制下在pgRNA中转录的线性连续DNA序列的载体; (d)用载体转染或转导敏感细胞; (e)在允许从克隆的HBV DNA合成HBV pgRNA的条件下培养转染或转导的细胞; (f)可能用药物产品,特别是抗病毒剂处理培养的细胞; 和(g)确定HBV的复制能力,药物产品,优选抗病毒剂可能发生在病毒基因表达和/或病毒复制上。