WHOLE GENOME PHAGE DISPLAY OF T PALLIDUM GENES

梅毒螺旋体基因的全基因组噬菌体展示

• 批准号: 2894310
• 负责人: Timothy Palzkill
• 金额: $ 21.43万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2894310
• 关键词: DNA binding protein; Treponema pallidum; bacterial genetics; bacterial virus; enzyme linked immunosorbent assay; fibronectins; genetic library; genetic transcription; genome; host organism interaction; nucleic acid sequence; open reading frames; southern blotting; transfection; transfection /expression vector

The function of many genes cannot be deduced from sequence similiarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. Phage display libraries that include all of the open reading frames (ORFs) of Treponema pallidum will be constructed. T. pallidum is the causative agent of syphilis. The complete genome sequence of this organism has recently been completed. Several features of T. pallidum make it an excellent system on which to develop and test genomic phage display technology. First, with a size of 1 million base pairs, the genome is one of the smallest known. Second, there are a total of 1041 open reading frames which makes it feasible to systematically construct libraries containing each ORF in a relatively short period of time. Finally, little is known of the biology or pathogensis 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. Two types of phage display libraries will be constructed. One type will be constructed by systematically inserting each of the 1041 ORFs of T. pallidum into a phage display vector. The second library will be constructed by shotgun cloning of randomly fragmented T. pallidum genomic DNA. The libraries will be used to identify ORFs that are involved in attachment of T. pallidum to fibronectin and host cells. In addition, the libraries will be used to identify T. pallidum DNA binding proteins and locate their binding sites. This information can be used to discover T. pallidum transcriptonal regulatory networks. The work will also serve as a model system for the use of genomic phage display as a tool for functional genomics.

许多基因的功能不能从序列相似性中推断出来，通常需要生化方法。全基因组序列不仅可以被认为是一组基因，而且可以被认为是一组功能结构域的集合。这些结构域可以通过亲和方法进行研究，从而鉴定配体可以提供有关生化功能的信息。为了利用这种方法，必须以一种适合亲和力研究的形式来表达所有的功能域。噬菌体展示技术为实现这一目标提供了一种手段。将构建包含梅毒螺旋体所有开放阅读框(ORF)的噬菌体展示文库。梅毒螺旋体是梅毒的病原体。这种生物的完整基因组序列最近已经完成。梅毒螺旋体的几个特征使其成为开发和测试基因组噬菌体展示技术的极佳系统。首先，基因组的大小为100万个碱基对，是已知最小的基因组之一。其次，开放阅读框架共有1041个，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架的图书馆成为可能。最后，由于没有连续的培养系统，人们对这种生物的生物学或病原学知之甚少。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。将构建两种类型的噬菌体展示文库。一种类型将通过系统地将梅毒螺旋体1041个ORF中的每一个插入到噬菌体展示载体中来构建。第二个文库将通过鸟枪克隆随机碎片化的梅毒螺旋体基因组DNA来构建。这些文库将被用来鉴定参与梅毒螺旋体与纤维连接蛋白和宿主细胞附着的ORF。此外，这些文库还将用于鉴定梅毒螺旋体DNA结合蛋白并定位其结合部位。这些信息可以用来发现梅毒螺旋体转录调控网络。这项工作还将作为使用基因组噬菌体展示作为功能基因组学工具的模型系统。