FUNCTIONAL GENOMICS STUDY OF TREPONEMA PALLIDUM

梅毒螺旋体功能基因组学研究

• 批准号: 6849701
• 负责人: Timothy Palzkill
• 金额: $ 30.1万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6849701
• 关键词: DNA binding protein; Treponema pallidum; bacterial antigens; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; cell adhesion; clinical research; epitope mapping; fibronectins; functional /structural genomics; genetic library; host organism interaction; human subject; mass spectrometry; matrix assisted laser desorption ionization; membrane proteins; protein protein interaction; protein structure function; proteomics; virulence; yeast two hybrid system

DESCRIPTION (provided by applicant): The genome sequences of many microorganisms have now been determined. Several of these organisms are the agents of infectious disease The sequences reveal many genes of unknown function. The genome sequence information should enable new approaches to be developed to determine the function of genes and their possible role in pathogenesis. A functional genomics approach will be used to identify proteins important for the Treponema pallidum host-pathogen interaction. T. pallidum is the causative agent of syphilis. The complete genome sequence of this organism has been completed. Several features of T. pallidum make it an excellent system on which to develop and test functional genomics technologies. First, with a size of 1 million base pairs, the genome is one of the smallest known. Second, there are a total of 1031 open reading frames, which makes it feasible to systematically construct libraries containing each open reading frame in a relatively short period of time. Finally, little is known of the biology or pathogenesis of this organism because a continuous culture system is not available. This severely limits the experimental options for study of the organism. Therefore, new approaches are needed to understand gene function in T. pallidum. During the previous funding period, we have used a topoisomerase-based method to clone PCR products encoding 1008 of the 1031 open reading frames identified in the genome sequence of T. pallidum. In addition, the plasmid vector system used for cloning the open reading frames, the univector system, permits the rapid conversion of the original plasmid clone set to other functional vectors containing various promoters or tag sequences. The conversion to functional vectors is based on a single step Cre-loxP site-specific recombination reaction. Using Cre-loxP recombination, the T. pallidum clone set has been converted to specialized vectors for large scale protein expression, phage display and two-hybrid analysis. These plasmid collections will be used in a functional genomics approach to i) identify proteins involved in adhesion to host cells, ii) systematically identify T. pallidum antigenic proteins, and iii) establish a large-scale protein-protein interaction network among periplasmic and surface localized proteins.

描述（由申请人提供）：许多微生物的基因组序列现已确定。其中一些生物体是传染病的病原体。序列揭示了许多功能未知的基因。基因组序列信息应该能够开发新的方法来确定基因的功能及其在发病机制中可能的作用。功能基因组学方法将用于鉴定对梅毒螺旋体宿主-病原体相互作用重要的蛋白质。梅毒螺旋体是梅毒的病原体。该生物体的完整基因组序列已经完成。梅毒螺旋体的几个特征使其成为开发和测试功能基因组技术的优秀系统。首先，基因组有 100 万个碱基对，是已知最小的基因组之一。其次，共有1031个开放阅读框，这使得在相对较短的时间内系统地构建包含每个开放阅读框的文库成为可能。最后，由于没有连续培养系统，因此对该生物体的生物学或发病机制知之甚少。这严重限制了生物体研究的实验选择。因此，需要新的方法来了解梅毒螺旋体的基因功能。 在之前的资助期间，我们使用基于拓扑异构酶的方法克隆了编码梅毒螺旋体基因组序列中确定的 1031 个开放阅读框中的 1008 个的 PCR 产物。此外，用于克隆开放阅读框的质粒载体系统，univector系统，允许将原始质粒克隆集快速转换为包含各种启动子或标签序列的其他功能载体。向功能载体的转化基于单步 Cre-loxP 位点特异性重组反应。使用 Cre-loxP 重组，梅毒螺旋体克隆组已转化为用于大规模蛋白质表达、噬菌体展示和双杂交分析的专用载体。这些质粒集合将用于功能基因组学方法，以 i) 鉴定参与宿主细胞粘附的蛋白质，ii) 系统地鉴定梅毒螺旋体抗原蛋白，以及 iii) 在周质和表面之间建立大规模的蛋白质-蛋白质相互作用网络局部蛋白质。