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1. 发明授权

US06428951B1 Protein fragment complementation assays for the detection of biological or drug interactions 失效
标题翻译：用于检测生物或药物相互作用的蛋白质片段互补测定法
公开(公告)号：US06428951B1
公开(公告)日：2002-08-06
申请号：US09499464
申请日：2000-02-07
申请人： Stephen William Watson Michnick , Joelle Nina Pelletier , Ingrid Remy
发明人： Stephen William Watson Michnick , Joelle Nina Pelletier , Ingrid Remy
IPC分类号： C12Q125
CPC分类号： G01N33/6845 , A01K67/0271 , A01K2267/0331 , A01K2267/0393 , C07K14/43595 , C07K2319/00 , C12N15/1055 , G01N33/5008 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/581 , G01N33/68 , G01N33/6803 , Y02A90/26
摘要： We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to-GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (lie to Val, Ala and Gly) resulted in a sequential increase in E. coli doubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and kinetic aspects of molecular interactions including protein-protein, protein-DNA, protein-RNA, protein-carbohydrate and protein-small molecule interactions, for screening cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity. The selection and design criteria applied here is developed for numerous examples of clonal selection, colorometric, fluorometric and other assays based on enzymes whose products can be measured. The development of such assay systems is shown to be simple, and provides for a diverse set of protein fragment complementation applications.
摘要翻译：我们描述了设计和实施蛋白质片段互补测定（PCAs）以检测体内和体外生物分子相互作用的策略。该策略的设计，实现和广泛应用用大量酶进行了说明，具体细节提供了鼠二氢叶酸还原酶（DHFR）的例子。由小鼠DHFR融合到GCN4亮氨酸拉链序列的N-和C-末端片段组成的融合肽在基本培养基中生长的大肠杆菌中共表达，其中内源性DHFR活性被甲氧苄啶抑制。互补融合产物的共表达恢复了菌落形成。存活只发生在两个DHFR片段存在并含有亮氨酸 - 拉链形成序列时，表明重组酶活性需要辅助亮氨酸拉链形成。增加严重程度的DHFR片段 - 接口点突变体（以Val，Ala和Gly表示）导致大肠杆菌倍增时间顺序增加，说明成功的DHFR片段重组而不是片段之间的非特异性相互作用。该测定可用于研究包括蛋白质蛋白质，蛋白质-DNA，蛋白质-RNA，蛋白质 - 碳水化合物和蛋白质 - 小分子相互作用在内的分子相互作用的平衡和动力学方面，用于筛选靶向蛋白质与未知蛋白质结合的cDNA文库或用于生物活性的小有机分子的文库。这里应用的选择和设计标准是针对克隆选择，色度，荧光测定和其他基于可以测量其产物的酶的测定的许多实例开发的。这种测定系统的开发被证明是简单的，并且提供了多种蛋白质片段互补应用的集合。

2. 发明授权

US06828091B2 Method of identifying immunosuppressive agents 失效
标题翻译：识别免疫抑制剂的方法
公开(公告)号：US06828091B2
公开(公告)日：2004-12-07
申请号：US09920332
申请日：2001-08-02
申请人： Shailaja Kasibhatla , Douglas R. Green , Ben Tseng
发明人： Shailaja Kasibhatla , Douglas R. Green , Ben Tseng
IPC分类号： C12Q125
CPC分类号： G01N33/505
摘要： A method for identifying therapeutically effective immunosuppressive agents by screening such agents for those which induce apoptosis in activated T cells is disclosed. T cells were isolated then activated and treating with various test compounds. A caspase substrate is added to detect caspase activation and apoptosis in the cells. Compounds which stimulate caspase activation and apoptosis are also tested against resting T cells to determine those agents which are more effective in activated T cells compared to resting T cells. Compounds with this selectivity are effective in treating immunopathological disorders such as arthritis, graft rejection, graft versus host disease, inflammatory bowel syndrome and the like.
摘要翻译：公开了通过筛选诱导活化的T细胞凋亡的那些试剂来鉴定治疗有效的免疫抑制剂的方法。分离T细胞，然后活化并用各种试验化合物处理。加入半胱天冬酶底物以检测细胞中的半胱天冬酶活化和细胞凋亡。还针对静息T细胞测试刺激半胱天冬酶活化和凋亡的化合物，以确定与静息T细胞相比在活化的T细胞中更有效的那些试剂。具有这种选择性的化合物在治疗免疫病理学疾病如关节炎，移植排斥反应，移植物抗宿主病，炎性肠综合征等方面是有效的。

3. 发明授权

US06699657B2 In vitro system for replication of RNA-dependent RNA polymerase (RDRP) viruses 失效
公开(公告)号：US06699657B2
公开(公告)日：2004-03-02
申请号：US10066130
申请日：2002-01-31
申请人： Robert W. King , Matthew W. Jeffries , Claudio Pasquinelli
发明人： Robert W. King , Matthew W. Jeffries , Claudio Pasquinelli
IPC分类号： C12Q125
CPC分类号： C12Q1/70 , C12N15/85 , C12N2770/24011 , C12N2770/24211 , C12N2840/203 , Y02A50/60
摘要： An in vitro method to conduct genomic replication of the viral genomes of viruses that utilize RNA-dependent RNA polymerase for replication (RDRP viruses), such as HCV. The method employs a construct comprising the 3′ and 5′ untranslated regions (UTRs) of the viral genome which are operably linked on the 5′ and 3′ ends of a reporter sequence, in antisense orientation, such that when viral replication is occurring within the cell which produces RDRP, the reporter protein will be made. The method of the invention provides an efficient means for measuring genomic replication in RDRP viruses, and also for the rapid screening of compounds for their ability to inhibit genomic replication of RDRP viruses, including the Hepatitis C virus (HCV).

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