ISOLATION AND TESTING OF P GINGIVALIS VIRULENCE GENES

此修订应用程序的长期目标是隔离新的 牙龈卟啉单胞菌毒力基因及其特性研究 以及之前分离的基因。预计结果将有所改善。 我们对本病致病机制的一般理解 有机体在牙周病病因中的作用。 该应用程序有三个具体目标。第一本书名为 “构建并利用报告基因分离和鉴定P. 一种无启动子的报告基因编码 四环素耐药性将被随机插入到牙龈假单胞菌中 利用克隆的染色体片段提供的同源性进行染色体分析。这个 得到的文库将得到丰富和筛选，以寻找表达 报告基因在小鼠脓肿模型感染中的作用 但在实验室培养过程中并不表现出来。这样的克隆 Win很可能被标记上重要基因中的报告基因 感染过程中牙龈假单胞菌的存活和毒力 进程。 在题为“克隆和遗传特性”的具体目标2中 报告基因标记的毒力基因“，标记的基因将 通过记号笔营救技术恢复。5个素数的部分排序 基因的末端将使我们能够确定哪些可能是 最重要的是牙龈假单胞菌的致病性。这个 测序数据还将为我们提供必要的信息 合成可用于克隆牙龈假单胞菌全基因的探针 转化为大肠杆菌。选定毒力基因的同源突变株将 使用插入失活方法构建。致病的 这些突变体的潜力将与它们的亲本进行比较 小鼠脓肿模型，在某些情况下，诺生菌大鼠模型。 申请书的具体目标3名为“遗传和生化” 牙龈假单胞菌表面抗原和毒力因子的特征“。 在特定目标2中分离到的毒力基因是从遗传和 具有生化特征的。此外，我们将执行相同的操作 先前克隆的牙龈假单胞菌编码基因的特征 血凝素和另外两种未知的表面抗原。他们的 作为假定的毒力因子的作用将通过安装在 记者基因构建是针对L和检测而发展起来的 它们在小鼠脓肿模型感染过程中的表达。

The long range goal of this revised application is to isolate new virulence genes from Porphyromonas gingivaIis and to characterize them along with previously isolated genes. The results are expected to improve our general understanding of the pathogenic mechanisms employed by this organism in periodontal disease causation. The application has three specific aims. The first is entitled "Construction and Use of a Reporter Gene to Isolate and Characterize P. gingivalis Virulence Genes". A promoterless reporter gene encoding tetracycline resistance will be randomly inserted into the P. gingivalis chromosome using homology provided by cloned chromosomal fragments. The resulting library will be enriched and screened for clones which express the reporter gene function during infection in the murine abscess model but which do not express it during laboratory cultivation. Such clones win likely be labeled with the reporter gene in genes that are important to the survival and virulence of P. gingivalis during the infectious process. In Specific Aim 2, entitled "Cloning and Genetic Characterization of Reporter Gene-Labeled Virulence Genes", the labeled genes will be recovered by marker rescue techniques. Partial sequencing of the 5 prime ends of the genes will enable us to determine those likely to be of greatest importance with regard to P. gingivalis pathogenicity. The sequencing data will also provide us with the necessary information to synthesize probes which can be used to clone the entire P. gingivalis gene into Escherichia coli. Isogeneic mutants of selected virulence genes will be constructed using insertional inactivation methods. The pathogenic potential of these mutants will be compared to their parent using the murine abscess model and, in certain cases, the gnotobiotic rat model. Specific Aim 3 of the application is entitled "Genetic and Biochemical Characterization of P. gingivaIis Surface Antigens and Virulence Factors". The virulence genes isolated in Specific Aim 2 win be genetically and biochemically characterized. In addition, we will perform the same characterizations on previously cloned P. gingivalis genes encoding hemagglutinin and two other unidentified putative surface antigens. Their role as putative virulence factors will be tested by installing them in the reporter gene construct developed in Specific Aim l and examining their expression during infection in the murine abscess model.