公开(公告)号：US20070219354A1

公开(公告)日：2007-09-20

申请号：US11714985

申请日：2007-03-07

申请人： Daria Hazuda ,  Elizabeth Chen Dodson ,  Ming-Tain Lai ,  Min Xu ,  Xiao-Ping Shi ,  Adam Simon ,  Guoxin Wu ,  Yueming Li ,  Bruce Register

发明人： Daria Hazuda ,  Elizabeth Chen Dodson ,  Ming-Tain Lai ,  Min Xu ,  Xiao-Ping Shi ,  Adam Simon ,  Guoxin Wu ,  Yueming Li ,  Bruce Register

IPC分类号： C07K16/00 ,  C07K14/00

CPC分类号： C07K7/06 ,  A01K2217/05 ,  A01K2267/0306 ,  A61K38/00 ,  C07K5/1016 ,  C07K5/1019 ,  C07K5/1021 ,  C07K14/4711 ,  C07K16/18 ,  C12P21/06 ,  C12Q1/37 ,  G01N33/5088 ,  G01N33/6896 ,  G01N2500/00 ,  G01N2800/52

摘要： Provided are methods of identifying inhibitors of β-secretase that employ modified β-secretase substrates. The modified β-secretase substrates have β-secretase cleavage sites that are altered from wild type. The amino acid sequences of the altered β-secretase cleavage sites contain different amino acids in at least one of the positions P2-P1-P1′-P2′ of the β-secretase cleavage site. Many of the modified β-secretase substrates are more efficient substrates for β-secretase than are corresponding substrates having wild-type sequences, that is, these modified substrates are more susceptible to enzymatic breakdown by β-secretase. Recombinant polynucleotide molecules encoding the modified β-secretase substrates are provided. Antibodies that recognize cleavage products of the modified β-secretase substrates are provided. Stable cell lines expressing the modified β-secretase substrates are provided. Transgenic animals expressing the modified β-secretase substrates are provided.

公开(公告)号：US20060270841A1

公开(公告)日：2006-11-30

申请号：US10504446

申请日：2003-02-25

申请人： Amy Espeseth ,  Marc Ferrer ,  Osvaldo Flores ,  Daria Hazuda ,  James Inglese ,  Michael Miller ,  Bruce Register ,  Xiao-Ping Shi ,  Adam Simon ,  Paul Zuck

发明人： Amy Espeseth ,  Marc Ferrer ,  Osvaldo Flores ,  Daria Hazuda ,  James Inglese ,  Michael Miller ,  Bruce Register ,  Xiao-Ping Shi ,  Adam Simon ,  Paul Zuck

IPC分类号： C12Q1/68 ,  C07H21/04 ,  C12P21/06 ,  C12N9/64

CPC分类号： C07K14/47 ,  C07K14/4702 ,  C07K14/4711 ,  C07K2319/00 ,  C07K2319/50 ,  G01N2500/10

摘要： The present invention provides DNA constructs, genetically engineered host cells, and methods for identifying inhibitors of amyloid precursor protein (APP) processing. The methods provide for the convenient identification, in a single assay, of inhibitors of β-secretase and γ-secretase as well as other forms of APP processing. The methods rely on fusion proteins of APP and transcription factors in which APP processing releases the transcription factors, allowing the transcription factors to activate transcription of a reporter gene. Inhibitors are identified as substances that block or diminish transcription factor release from the fusion protein, thereby causing a diminution of reporter gene readout.

摘要翻译： 本发明提供了DNA构建体，遗传工程化宿主细胞和用于鉴定淀粉样前体蛋白（APP）处理抑制剂的方法。 该方法提供了在单次测定中方便地鉴定β-分泌酶和γ-分泌酶抑制剂以及其它形式的APP处理。 该方法依赖于APP和转录因子的融合蛋白，其中APP处理释放转录因子，允许转录因子激活报告基因的转录。 抑制剂被鉴定为阻断或减少转录因子从融合蛋白释放的物质，从​​而导致报告基因读出的减少。

公开(公告)号：US07196163B2

公开(公告)日：2007-03-27

申请号：US10427208

申请日：2003-04-30

申请人： Daria Jean Hazuda ,  Elizabeth Chen Dodson ,  Ming-Tain Lai ,  Min Xu ,  Xiao-Ping Shi ,  Adam J. Simon ,  Guoxin Wu ,  Yueming Li ,  Bruce Register

发明人： Daria Jean Hazuda ,  Elizabeth Chen Dodson ,  Ming-Tain Lai ,  Min Xu ,  Xiao-Ping Shi ,  Adam J. Simon ,  Guoxin Wu ,  Yueming Li ,  Bruce Register

IPC分类号： A61K38/07 ,  A61K38/16

CPC分类号： C07K7/06 ,  A01K2217/05 ,  A01K2267/0306 ,  A61K38/00 ,  C07K5/1016 ,  C07K5/1019 ,  C07K5/1021 ,  C07K14/4711 ,  C07K16/18 ,  C12P21/06 ,  C12Q1/37 ,  G01N33/5088 ,  G01N33/6896 ,  G01N2500/00 ,  G01N2800/52

摘要： Provided are methods of identifying inhibitors of β-secretase that employ modified β-secretase substrates. The modified β-secretase substrates have β-secretase cleavage sites that are altered from wild type. The amino acid sequences of the altered β-secretase cleavage sites contain different amino acids in at least one of the positions P2-P1-P1′-P2′ of the β-secretase cleavage site. Many of the modified β-secretase substrates are more efficient substrates for β-secretase than are corresponding substrates having wild-type sequences, that is, these modified substrates are more susceptible to enzymatic breakdown by β-secretase. Recombinant polynucleotide molecules encoding the modified β-secretase substrates are provided. Antibodies that recognize cleavage products of the modified β-secretase substrates are provided. Stable cell lines expressing the modified β-secretase substrates are provided. Transgenic animals expressing the modified β-secretase substrates are provided.

摘要翻译： 提供了鉴定使用修饰的β-分泌酶底物的β-分泌酶抑制剂的方法。 修饰的β-分泌酶底物具有从野生型改变的β-分泌酶切割位点。 改变的β-分泌酶切割位点的氨基酸序列在β-分泌酶切割位点的至少一个位置P2-P1-P1'-P2'中含有不同的氨基酸。 许多修饰的β-分泌酶底物比具有野生型序列的相应底物更有效的β-分泌酶底物，也就是说，这些修饰的底物更易受β-分泌酶的酶分解。 提供了编码经修饰的β-分泌酶底物的重组多核苷酸分子。 提供了识别修饰的β-分泌酶底物的切割产物的抗体。 提供了表达改良的β-分泌酶底物的稳定细胞系。 提供了表达改良的β-分泌酶底物的转基因动物。

公开(公告)号：US5728557A

公开(公告)日：1998-03-17

申请号：US458067

申请日：1995-06-01

申请人： R. Bruce Register ,  Jules A. Shafer

发明人： R. Bruce Register ,  Jules A. Shafer

IPC分类号： C12N9/50 ,  C12N15/10 ,  C12N15/00

CPC分类号： C12N9/503 ,  C12N15/102

摘要： A method for introducing a mutation into a desired site in Herpes Simplex Virus Type 1 uses a set of starting vectors, where each starting vector has a fragment of the substantially complete HSV-1 genome and also DNA which overlaps with a sequential genomic fragment contained in other starting vectors, so that upon co-transfection of a host cell, replication of viral DNA, and recombination, a mutated replicable virus is formed. The starting vector containing a gene which is to be mutated is replaced by a replacement vectors. The replacement vectors contain a copy of the mutated gene and overlapping DNA, and genomic DNA which was present in the starting vector. A host cell is transformed with the replacement vectors and the remaining starting vectors under conditions allowing replication of viral DNA and recombination to form a replicating mutated virus. In preferred embodiments, the protease gene is mutated.

摘要翻译： 将突变引入单克隆单纯疱疹病毒1型中的所需位点的方法使用一组起始载体，其中每个起始载体具有基本上完整的HSV-1基因组的片段，以及与包含在其中的连续基因组片段重叠的DNA 其他起始载体，使得当共转染宿主细胞时，病毒DNA的复制和重组，形成突变的可复制病毒。 含有待突变的基因的起始载体被替换载体所取代。 替换载体含有突变基因和重叠DNA的拷贝，以及起始载体中存在的基因组DNA。 在允许复制病毒DNA并重组以形成复制突变病毒的条件下，用置换载体和剩余起始载体转化宿主细胞。 在优选的实施方案中，蛋白酶基因被突变。