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    Method for reproducing in vitro the Proteolytic activity of the NS3 protease of hepatitis C virus (HCV)
    1.
    发明授权
    Method for reproducing in vitro the Proteolytic activity of the NS3 protease of hepatitis C virus (HCV) 失效
    在体外再现丙型肝炎病毒（HCV）NS3蛋白酶的蛋白水解活性的方法

    公开(公告)号：US5739002A

    公开(公告)日：1998-04-14

    申请号：US700356

    申请日：1996-08-23

    申请人： Raffaele De Francesco ,  Christina Failla ,  Licia Tomei

    发明人： Raffaele De Francesco ,  Christina Failla ,  Licia Tomei

    IPC分类号： A61K38/55 ,  A61P1/16 ,  A61P31/12 ,  C07K14/18 ,  C12N9/50 ,  C12N15/09 ,  C12Q1/37 ,  G01N33/573 ,  G01N33/576 ,  C07H21/04 ,  C12Q1/70

    CPC分类号： C07K14/005 ,  G01N33/573 ,  G01N33/5767 ,  C12N2770/24222

    摘要： This is a method for reproducing in vitro the serine protease activity associated with the HCV NS3 protein, that comprises using both of the sequences contained in NS3 and the sequences contained in NS4A. This method takes advantage of the ability of the HCV NS4A protein, or sequences contained therein, to act as a cofactor of the serine protease activity or more generally of the enzymatic activities associated with NS3. Optimal serine protease activity is obtained when NS4A is present in a molar ratio of at least 1:1 with NS3. NS3 and NS4A can also be incorporated in the reaction mixture as NS3-NS4A precursor, as this precursor will generate, by means of an autoproteolytic event, equimolar amounts of NS3 and NS4A. It is also possible to mutate the cleavage site between NS3 and NS4A in a procursor, so that NS4A remains covalently The sequences that do not influence the proteolytic activity of NS3 can subsequently be removed from protease activity.

    摘要翻译： PCT No.PCT / IT95 / 00018 Sec。 371日期：1996年8月23日 102（e）日期1996年8月23日PCT提交1995年2月14日PCT公布。 公开号WO95 / 22985 日期1995年8月31日这是一种再现体外与HCV NS3蛋白相关的丝氨酸蛋白酶活性的方法，其包括使用NS3中包含的两个序列和NS4A中包含的序列。 该方法利用HCV NS4A蛋白或其中所含的序列的能力作为丝氨酸蛋白酶活性的辅因子或更一般地与NS3相关的酶活性。 当NS4A与NS3以至少1:1的摩尔比存在时，获得最佳的丝氨酸蛋白酶活性。 NS3和NS4A也可以作为NS3-NS4A前体引入反应混合物中，因为该前体将通过自体蛋白水解事件产生等摩尔量的NS3和NS4A。 也可能在前体中突变NS3和NS4A之间的切割位点，使得NS4A保持共价。随后可以从蛋白酶活性中除去不影响NS3的蛋白水解活性的序列。

    Methodology to produce, and purify and assay polypeptides with the proteolytic activity of the HCV NS3 protease
    2.
    发明授权
    Methodology to produce, and purify and assay polypeptides with the proteolytic activity of the HCV NS3 protease 失效
    用于产生和纯化和测定具有HCV NS3蛋白酶的蛋白水解活性的多肽的方法

    公开(公告)号：US06197536B1

    公开(公告)日：2001-03-06

    申请号：US09011961

    申请日：1998-02-23

    申请人： Christian Steinkühler ,  Antonello Pessi ,  Elisabetta Bianchi ,  Marina Taliani ,  Licia Tomei ,  Andrea Urbani ,  Raffaele De Francesco ,  Frank Narjes

    发明人： Christian Steinkühler ,  Antonello Pessi ,  Elisabetta Bianchi ,  Marina Taliani ,  Licia Tomei ,  Andrea Urbani ,  Raffaele De Francesco ,  Frank Narjes

    IPC分类号： C12Q137

    CPC分类号： C07K14/005 ,  C12N9/506 ,  C12N2770/24222

    摘要： The process according to the present invention allows expression and isolation of polypeptides with the proteolytic activity of HCV NS3 protease in a pure, catalytically active form, and in amounts that are sufficient for discovery of NS3 protease inhibitors and for determination of the three-dimensional structure of the NS3 protease. A further subject of the present invention is a procedure that defines the chemical and physical conditions necessary for completion of the proteolytic activity of the above polypeptides. The invention further comprises new compositions of matter (expression vectors) containing nucleotide sequences capable of expressing the above mentioned polypeptides in culture cells. Finally, new compounds of matter are defined, suitable to measure the above proteolytic activity, and useful to develop NS3 protease inhibitors and therefore therapeutic agents for use against HCV. The figure shows the kinetic parameters of HCV NS3 protease using the S3 depsipeptide substrate (SEQ ID NO:45).

    摘要翻译： 根据本发明的方法允许以纯的，催化活性形式表达和分离具有HCV NS3蛋白酶的蛋白水解活性的多肽，并且其量足以发现NS3蛋白酶抑制剂和用于确定三维结构 的NS3蛋白酶。 本发明的另一主题是定义完成上述多肽的蛋白水解活性所需的化学和物理条件的方法。 本发明还包括含有能够在培养细胞中表达上述多肽的核苷酸序列的物质组合物（表达载体）。 最后，定义新的物质化合物，适合于测量上述蛋白水解活性，并且可用于开发NS3蛋白酶抑制剂，因此用于抗HCV的治疗剂。 该图显示了使用S3 depipipeptide底物（SEQ ID NO：45）的HCV NS3蛋白酶的动力学参数。

    Method for producing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities encoded by hepatitis C virus (HCV)
    4.
    发明授权
    Method for producing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities encoded by hepatitis C virus (HCV) 失效
    用于在体外生产RNA依赖性RNA聚合酶和由丙型肝炎病毒（HCV）编码的末端核苷酸转移酶活性的方法

    公开(公告)号：US06383768B1

    公开(公告)日：2002-05-07

    申请号：US08952981

    申请日：1998-03-23

    申请人： Raffaele De Francesco ,  Licia Tomei ,  Sven-Erik Behrens

    发明人： Raffaele De Francesco ,  Licia Tomei ,  Sven-Erik Behrens

    IPC分类号： C12Q148

    CPC分类号： C12N9/1241 ,  C12N9/127 ,  C12N2799/026

    摘要： This is a method for reproducing in vitro the RNA-dependent RNA polymerase activity associated with hepatitis C virus. The method is characterized in that sequences contained in NS5B are used in the reaction mixture. The terminal nucleotidyl transferase activity, a further property of the NS5B protein, can also be reproduced using this method. The method takes advantage of the fact that the NS5B protein, either purified to apparent homogeneity or present in extracts of overproducing organisms, can catalyze the addition of ribonucleotides to the 3′-termini of exogenous or endogenous RNA molecules. The invention also relates to a composition of matter that comprises sequences contained in NS5B, and to the use of these compositions for the set up of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS5B.

    摘要翻译： 这是一种在体外再生与丙型肝炎病毒相关的RNA依赖性RNA聚合酶活性的方法。 该方法的特征在于在反应混合物中使用包含在NS5B中的序列。 末端核苷酸转移酶活性，NS5B蛋白质的另一个性质也可以使用这种方法再现。 该方法利用了纯化至表观均一性或存在于产生过量生物体的提取物中的NS5B蛋白可以催化向外源或内源RNA分子的3'末端加入核糖核苷酸的事实。 本发明还涉及包含NS5B中包含的序列的物质组合物，以及这些组合物用于建立能够为治疗目的选择抑制与NS5B相关的酶活性的化合物的酶学试验的用途。

    MICRORNA EXPRESSION SIGNATURE IN PERIPHERAL BLOOD OF PATIENTS AFFECTED BY HEPATOCARCINOMA OR HEPATIC CIRRHOSIS AND USES THEREOF
    5.
    发明申请
    MICRORNA EXPRESSION SIGNATURE IN PERIPHERAL BLOOD OF PATIENTS AFFECTED BY HEPATOCARCINOMA OR HEPATIC CIRRHOSIS AND USES THEREOF 审中-公开
    肝细胞癌或肝硬化患者外周血中MICRORNA表达标志及其用途

    公开(公告)号：US20120238617A1

    公开(公告)日：2012-09-20

    申请号：US13394530

    申请日：2010-09-06

    申请人： Massimiliano Pagani ,  Raffaele De Francesco ,  Grazisa Rossetti ,  Sergio Abrignani ,  Riccardo Lorenzo Rossi

    发明人： Massimiliano Pagani ,  Raffaele De Francesco ,  Grazisa Rossetti ,  Sergio Abrignani ,  Riccardo Lorenzo Rossi

    IPC分类号： C12Q1/68 ,  A61P35/00 ,  A61P1/16 ,  A61K31/7105

    CPC分类号： C12Q1/6886 ,  C12Q2600/158 ,  C12Q2600/178

    摘要： A method for diagnosing or prognosticating hepatocellular carcinoma, also in the early stages, or for assessing the risk of developing hepatocellular carcinoma, or for monitoring the effectiveness of an anti-tumour therapy against hepatocellular carcinoma by measuring the expression level of at least one miRNA gene product in a peripheral blood sample or in a biological fluid sample. Said method comprises measuring, in an isolated sample of peripheral blood or biological fluid, the expression level of at least one miRNA gene product, and comparing said measured expression level with a reference level. Such method can also be used to diagnose or assess the risk of developing liver cirrhosis in patients affected by chronic hepatitis, or to prognosticate the evolution of cirrhosis in patients affected by cirrhosis, or to monitor the effectiveness of a pharmacological therapy against liver cirrhosis.

    摘要翻译： 用于诊断或预后的肝细胞癌的方法，也可在早期阶段，或用于评估发展中的肝细胞癌的风险，或通过测量至少一种miRNA基因的表达水平监测抗肿瘤治疗对肝细胞癌的有效性 产品在外周血样品或生物流体样品中。 所述方法包括在外周血液或生物流体的分离样品中测量至少一种miRNA基因产物的表达水平，并将所述测量的表达水平与参考水平进行比较。 这种方法也可用于诊断或评估慢性肝炎患者发生肝硬化的风险，或预测肝硬化患者肝硬化的进展情况，或监测药物治疗对肝硬化的有效性。

    MONITORING OF IMMUNE SYSTEM USING PERIPHERAL BLOOD MICRO-RNA EXPRESSION PROFILE ANALYSIS AND USES THEREOF
    6.
    发明申请
    MONITORING OF IMMUNE SYSTEM USING PERIPHERAL BLOOD MICRO-RNA EXPRESSION PROFILE ANALYSIS AND USES THEREOF 审中-公开
    使用外周血微量RNA表达谱分析及其用途监测免疫系统

    公开(公告)号：US20130165497A1

    公开(公告)日：2013-06-27

    申请号：US13703730

    申请日：2011-06-15

    申请人： Sergio Abrignanai ,  Raffaele De Francesco ,  Massimiliano Pagani

    发明人： Sergio Abrignanai ,  Raffaele De Francesco ,  Massimiliano Pagani

    IPC分类号： C12Q1/68 ,  A61K31/7105

    CPC分类号： C12Q1/6883 ,  C12Q2600/158 ,  C12Q2600/178

    摘要： The present invention relates to a method for monitoring the immune system of an individual, which comprises measuring, preferably by quantitative RT-PCR, the expression level of at least one microRNA (miRNA) gene product in a peripheral blood sample or in a biological fluid sample, and comparing said measured expression level with a reference level. In particular, the at least one miRNA gene product, which the method of the invention measures, is expressed by lymphocyte populations of an individual, in particular by naive CD4+T, TH1, TH2 and TH17 lymphocytes. The method of the invention is useful for the diagnosis, prognosis, prevention, control and/or the treatment of a pathological condition caused by or associated with an immune system dysfunction. Moreover, the method of the present invention is useful for monitoring, in an individual, the evolution of conditions mediated by the immune system, such as the response to a vaccination.

    摘要翻译： 本发明涉及一种用于监测个体的免疫系统的方法，其包括优选通过定量RT-PCR测量外周血样品或生物液体中至少一种微小RNA（miRNA）基因产物的表达水平 采样，并将所述测量的表达水平与参考水平进行比较。 特别地，本发明的方法测量的至少一种miRNA基因产物由个体的淋巴细胞群体特别是天然的CD4 + T，TH1，TH2和TH17淋巴细胞表达。 本发明的方法可用于诊断，预后，预防，控制和/或治疗由免疫系统功能障碍引起或与免疫系统功能障碍相关的病理状况。 此外，本发明的方法可用于在个体中监测由免疫系统介导的病症的进化，例如对疫苗接种的反应。