公开(公告)号：US09052321B2

公开(公告)日：2015-06-09

申请号：US13122154

申请日：2009-09-28

申请人： Bertrand Saunier ,  Miriam Triyatni ,  Edward A. Berger

发明人： Bertrand Saunier ,  Miriam Triyatni ,  Edward A. Berger

IPC分类号： C12N5/10 ,  C12N15/85 ,  G01N33/68 ,  C12Q1/68 ,  G01N33/576 ,  C07K14/005

CPC分类号： G01N33/5767 ,  C07K14/005 ,  C12N2770/24144 ,  C12N2770/24222 ,  C12N2770/24243

摘要： Provided herein is a mammalian cell transformed to contain a plasmid encoding a T7 or SP6 promoter operably linked to one or more HCV genes, a subgenomic replicon from a flavivirus and a cytoplasmic T7 and SP6 RNA amplification system. Also provided herein are isolated replication-competent HCV particles produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the replication-competent HCV particles from the cell culture. Provided herein are isolated HCV structural proteins produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the HCV structural proteins from the cell culture. Further provided herein is a system for assaying HCV entry into a cell comprising a first plasmid encoding a T7 or SP6 promoter operably linked to an HCV polynucleotide comprising at least the 5′-UTR to NS2 operably linked to an EMCV IRES in frame with an SP6 or T7 polymerase gene, respectively, a first host cell line expressing a replicon from a flavivirus and comprising a cytoplasmic T7 and SP6 RNA amplification system, a second plasmid encoding a reporter gene operably linked to both T7 and SP6 promoters in tandem, and a second host cell line comprising a cytoplasmic T7 polymerase or SP6 polymerase RNA amplification system.

摘要翻译： 本文提供了转化成含有编码与一种或多种HCV基因可操作地连接的T7或SP6启动子的质粒的哺乳动物细胞，来自黄病毒的亚基因组复制子和细胞质T7和SP6 RNA扩增系统。 本文还提供了通过包括以下步骤的方法产生的分离的可复制的HCV颗粒，所述方法包括提供根据第一实施方案的转化的哺乳动物细胞，培养细胞，以及从细胞培养物中回收可复制的HCV颗粒。 本文提供了通过包括以下步骤的方法产生的分离的HCV结构蛋白，所述方法包括提供根据第一实施方案的转化的哺乳动物细胞，培养细胞，以及从细胞培养物中回收HCV结构蛋白。 本文进一步提供用于测定HCV进入细胞的系统，其包含编码T7或SP6启动子的第一质粒，所述第一质粒可操作地连接至包含至少5'- UTR至NS2的HCV多核苷酸，其可操作地连接到具有SP6的框架的EMCV IRES 或T7聚合酶基因，表达来自黄病毒的复制子的第一宿主细胞系，其包含细胞质T7和SP6RNA扩增系统，编码与T7和SP6启动子串联可操作地连接的报告基因的第二质粒和第二个 包含细胞质T7聚合酶或SP6聚合酶RNA扩增系统的宿主细胞系。

公开(公告)号：US20120100574A1

公开(公告)日：2012-04-26

申请号：US13122154

申请日：2009-09-28

申请人： Bertrand Saunier ,  Miriam Triyatni ,  Edward A. Berger

发明人： Bertrand Saunier ,  Miriam Triyatni ,  Edward A. Berger

IPC分类号： C12P21/02 ,  C12N7/02 ,  C12N7/00 ,  C12N5/10 ,  C07K14/18

CPC分类号： G01N33/5767 ,  C07K14/005 ,  C12N2770/24144 ,  C12N2770/24222 ,  C12N2770/24243

摘要： Provided herein is a mammalian cell transformed to contain a plasmid encoding a T7 or SP6 promoter operably linked to one or more HCV genes, a subgenomic replicon from a flavivirus and a cytoplasmic T7 and SP6 RNA amplification system. Also provided herein are isolated replication-competent HCV particles produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the replication-competent HCV particles from the cell culture. Provided herein are isolated HCV structural proteins produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the HCV structural proteins from the cell culture. Further provided herein is a system for assaying HCV entry into a cell comprising a first plasmid encoding a T7 or SP6 promoter operably linked to an HCV polynucleotide comprising at least the 5′-UTR to NS2 operably linked to an EMCV IRES in frame with an SP6 or T7 polymerase gene, respectively, a first host cell line expressing a replicon from a flavivirus and comprising a cytoplasmic T7 and SP6 RNA amplification system, a second plasmid encoding a reporter gene operably linked to both T7 and SP6 promoters in tandem, and a second host cell line comprising a cytoplasmic T7 polymerase or SP6 polymerase RNA amplification system.

摘要翻译： 本文提供了转化成含有编码与一种或多种HCV基因可操作地连接的T7或SP6启动子的质粒的哺乳动物细胞，来自黄病毒的亚基因组复制子和细胞质T7和SP6 RNA扩增系统。 本文还提供了通过包括以下步骤的方法产生的分离的可复制的HCV颗粒，所述方法包括提供根据第一实施方案的转化的哺乳动物细胞，培养细胞，以及从细胞培养物中回收可复制的HCV颗粒。 本文提供了通过包括以下步骤的方法产生的分离的HCV结构蛋白，所述方法包括提供根据第一实施方案的转化的哺乳动物细胞，培养细胞，以及从细胞培养物中回收HCV结构蛋白。 本文进一步提供用于测定HCV进入细胞的系统，其包含编码T7或SP6启动子的第一质粒，所述第一质粒可操作地连接至包含至少5'- UTR至NS2的HCV多核苷酸，其可操作地连接到具有SP6的框架的EMCV IRES 或T7聚合酶基因，表达来自黄病毒的复制子的第一宿主细胞系，其包含细胞质T7和SP6RNA扩增系统，编码与T7和SP6启动子串联可操作地连接的报告基因的第二质粒和第二个 包含细胞质T7聚合酶或SP6聚合酶RNA扩增系统的宿主细胞系。

公开(公告)号：US20050272029A1

公开(公告)日：2005-12-08

申请号：US10524443

申请日：2003-08-18

申请人： Bertrand Saunier ,  Miriam Triyatni

发明人： Bertrand Saunier ,  Miriam Triyatni

IPC分类号： A61K20060101 ,  A61K39/12 ,  C07K14/18 ,  C07K16/10 ,  C12N7/00 ,  C12N7/02 ,  C12N7/04 ,  C12Q1/68 ,  C12Q1/70

CPC分类号： C07K14/005 ,  A61K39/12 ,  A61K2039/5258 ,  C07K16/109 ,  C07K2317/77 ,  C12N7/00 ,  C12N2710/14143 ,  C12N2770/24222 ,  C12N2770/24223 ,  C12N2770/24234 ,  C12N2770/24251

摘要： Methods for obtaining HCV complexes and HCV-like particles comprising HCV structural genes are provided. In one method, cells containing HCV-like particles are lysed with digitonin in the presence of protease inhibitors. Polyethylene glycol is slowly added to the lysate, to provide a precipitate that comprises complexes of the HCV structural proteins associated with lipid vesicles or micelles and complexes comprising viral structural proteins in the form of insoluble aggregates. In another method, the lysate is centrifuged through a sucrose cushion. Preferably, the pellet is then subjected to equilibrium ultracentrifugation, to provide a preparation of HCV-like particles that are heterogenous in size. The third method comprises subjecting the infected cells to hypertonic/hypotonic shock, and lysing the cells with digitonin in the presence of protease inhibitors. The lysate is pelleted and fractionated to provide a population of HCV-like particles that are substantially homogenous and have an average diameter of about 50 nm. As used herein the term “substantially homogenous” means that the shape of the particles are similar and that the size of the particles vary by 10% or less. Methods of using the HCV complexes and HCV-Iike particles as screening tools, diagnostic tools, and immunogenic compositions are also provided. Methods of treating patients exhibiting symptoms of HCV infection with compounds or substances that interfere with binding or internalization of the present HCV-like particles to asialoglycoprotein receptors are also provided.

摘要翻译： 提供了获得包含HCV结构基因的HCV复合物和HCV样颗粒的方法。 在一种方法中，在蛋白酶抑制剂的存在下，含有类似于HCV的颗粒的细胞用苦参蛋白裂解。 将聚乙二醇缓慢加入到裂解物中，以提供包含与脂质囊泡或胶束相关的HCV结构蛋白的复合物的沉淀物和包含不溶性聚集体形式的病毒结构蛋白质的复合物。 在另一种方法中，将裂解物通过蔗糖垫离心。 优选地，将沉淀物进行平衡超速离心，以提供尺寸异质的HCV样颗粒的制剂。 第三种方法包括使受感染的细胞经受高渗/低渗性休克，并在蛋白酶抑制剂存在下用洋地黄毒素裂解细胞。 将裂解物沉淀并分级分离以提供基本上均匀且具有约50nm平均直径的HCV样颗粒群。 如本文所用，术语“基本均匀”是指颗粒的形状相似，并且颗粒的尺寸变化10％或更小。 还提供了使用HCV复合物和HCV-Iike颗粒作为筛选工具，诊断工具和免疫原性组合物的方法。 还提供了治疗患有HCV感染症状的患者的化合物或干扰本发明的HCV样颗粒与唾液酸糖蛋白受体结合或内化的物质的方法。