公开(公告)号：US5578473A

公开(公告)日：1996-11-26

申请号：US209178

申请日：1994-03-10

申请人： Peter Palese ,  Jeffrey D. Parvin ,  Mark Krystal

发明人： Peter Palese ,  Jeffrey D. Parvin ,  Mark Krystal

IPC分类号： A61K39/00 ,  A61K39/02 ,  A61K39/12 ,  A61K39/13 ,  A61K39/21 ,  A61K39/245 ,  A61K39/29 ,  C07K14/00 ,  C07K14/11 ,  C07K14/16 ,  C07K14/705 ,  C07K19/00 ,  C12N7/00 ,  C12N9/12 ,  C12N9/24 ,  C12N15/09 ,  C12N15/10 ,  C12N15/11 ,  C12N15/86 ,  C12P21/02 ,  C12R1/91 ,  C12N7/01 ,  C12N7/02 ,  C12N7/04

CPC分类号： C07K14/005 ,  C12N15/10 ,  C12N15/11 ,  C12N15/86 ,  C12N9/127 ,  C12N9/2402 ,  C12Y302/01018 ,  A61K2039/5256 ,  A61K39/00 ,  C12N2740/16122 ,  C12N2740/16222 ,  C12N2760/16022 ,  C12N2760/16122 ,  C12N2760/16143 ,  C12N2840/203 ,  C12N2840/85

摘要： Recombinant negative strand virus RNA templates which may be used to express heterologous gene products and/or to construct chimeric viruses are described. Influenza viral polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicative that the promoter lay solely within the 15 nucleotide 3' terminus. Sequences not specific for the influenza vital termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus sense cRNA were copied at low levels. The specificity for recognition of the virus-sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of vital protein were required in order to catalyze both the cap-endonuclease primed and primer-free RNA synthesis from these model templates as well as from genomic length RNAs. This indicated that this reconstituted system had catalytic properties very smilar to those of native viral RNPs. High levels of expression of a heterologous gene was obtained using the constructs and methods described.

摘要翻译： 描述了可用于表达异源基因产物和/或构建嵌合病毒的重组负链病毒RNA模板。 用于制备耗尽病毒RNA的流感病毒聚合酶用于复制由质粒编码序列制备的小RNA模板。 显示仅含有3'末端基因组RNA的模板结构被有效拷贝，表明启动子仅位于15个核苷酸3'末端内。 不特定流感重要终点的序列没有被复制，令人惊讶的是，含有与正义cRNA相同的末端的RNA被拷贝到低水平。 通过位点特异性诱变进一步定义识别病毒感应启动子的特异性。 还发现需要增加水平的重要蛋白质以便从这些模型模板以及来自基因组长度RNA的催化端帽内切核酸酶引物和无引物的RNA合成两者。 这表明这种重构系统具有与天然病毒RNP非常相似的催化性质。 使用描述的构建体和方法获得异源基因的高水平表达。

公开(公告)号：US5166057A

公开(公告)日：1992-11-24

申请号：US527237

申请日：1990-05-22

申请人： Peter Palese ,  Jeffrey D. Parvin ,  Mark Krystal

发明人： Peter Palese ,  Jeffrey D. Parvin ,  Mark Krystal

IPC分类号： A61K39/00 ,  A61K39/02 ,  A61K39/12 ,  A61K39/13 ,  A61K39/21 ,  A61K39/245 ,  A61K39/29 ,  C07K14/00 ,  C07K14/11 ,  C07K14/16 ,  C07K14/705 ,  C07K19/00 ,  C12N7/00 ,  C12N9/12 ,  C12N9/24 ,  C12N15/09 ,  C12N15/10 ,  C12N15/11 ,  C12N15/86 ,  C12P21/02 ,  C12R1/91

CPC分类号： C07K14/005 ,  C12N15/10 ,  C12N15/11 ,  C12N15/86 ,  C12N9/127 ,  C12N9/2402 ,  C12Y302/01018 ,  A61K2039/5256 ,  A61K39/00 ,  C12N2740/16122 ,  C12N2740/16222 ,  C12N2760/16022 ,  C12N2760/16122 ,  C12N2760/16143 ,  C12N2840/203 ,  C12N2840/85

摘要： Recombinant negative strand virus RNA templates which may be used to express heterologous gene products and/or to construct chimeric viruses are described. Influenza viral polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicative that the promoter lay solely within the 15 nucleotide 3' terminus. Sequences not specific for the influenza viral termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus sense cRNA were copied at low levels. The specificity for recognition of the virus-sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap-endonuclease primed and primer-free RNA synthesis from these model templates as well as from genomic lengths RNAs. This indicated that this reconstituted system had catalytic properties very similar to those of native viral RNPs. High levels of expression of a heterologous gene was obtained using the constructs and methods described.

摘要翻译： 描述了可用于表达异源基因产物和/或构建嵌合病毒的重组负链病毒RNA模板。 用于制备耗尽病毒RNA的流感病毒聚合酶用于复制由质粒编码序列制备的小RNA模板。 显示仅含有3'末端基因组RNA的模板结构被有效拷贝，表明启动子仅位于15个核苷酸3'末端内。 不具体针对流感病毒末端的序列没有复制，令人惊讶的是，含有与正义cRNA相同的末端的RNA被复制在低水平。 通过位点特异性诱变进一步定义识别病毒感应启动子的特异性。 还发现需要增加水平的病毒蛋白以便催化来自这些模型模板的帽内切核酸酶引物和无引物的RNA合成以及来自基因组长度的RNA。 这表明这种重构系统具有与天然病毒RNP非常相似的催化性质。 使用描述的构建体和方法获得异源基因的高水平表达。