ISOLATION AND TESTING OF P GINGIVALIS VIRULENCE GENES

牙龈卟啉单胞菌毒力基因的分离与检测

• 批准号: 2443680
• 负责人: Jeffrey D. Hillman
• 金额: $ 23.58万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443680
• 关键词: Bacteroides gingivalis; Escherichia coli; bacterial antigens; bacterial genetics; chromosomes; gene expression; genetic library; hemagglutinin; immunoelectron microscopy; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; mutant; nucleic acid sequence; oral bacteria; pathologic process; periodontium disorder; protein purification; recombinant proteins; reporter genes; restriction mapping; surface antigens; virulence; western blottings

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.

此修订后的应用程序的长期目标是隔离新的 牙龈卟啉单胞菌的毒力基因及其特征 以及先前分离的基因。 结果预计会有所改善 我们对此致病机制的一般理解 牙周病病因中的有机体。 该应用程序具有三个具体目标。 第一个题为 “构建和使用报告基因来分离和表征 P. gingivalis毒力基因”。编码的无启动子报告基因 四环素抗性将被随机插入牙龈卟啉单胞菌中 使用克隆染色体片段提供的同源性来分析染色体。 这 所得文库将被富集并筛选表达的克隆 小鼠脓肿模型感染期间报告基因的功能 但在实验室培养期间不表达它。 这样的克隆 胜利可能在重要的基因中被标记为报告基因 传染期间牙龈卟啉单胞菌的生存和毒力 过程。 在具体目标 2 中，题为“克隆和遗传特征 报告基因标记的毒力基因”，标记的基因将是 通过标记救援技术恢复。 5个素数的部分排序 基因的末端将使我们能够确定那些可能的基因 最重要的是牙龈卟啉单胞菌的致病性。 这 测序数据还将为我们提供必要的信息 合成可用于克隆整个牙龈卟啉单胞菌基因的探针 进入大肠杆菌。 选定毒力基因的同基因突变体将 使用插入失活方法构建。 致病性 这些突变体的潜力将与它们的亲本进行比较 鼠脓肿模型，在某些情况下，还有限生大鼠模型。 该申请的具体目标 3 的标题是“遗传和生化 牙龈卟啉单胞菌表面抗原和毒力因子的表征”。 Specific Aim 2 中分离出的毒力基因在遗传上和 生化特征。 另外，我们也会进行同样的操作 先前克隆的牙龈卟啉单胞菌基因编码的表征 血凝素和另外两种未鉴定的推定表面抗原。 他们的 作为假定毒力因子的作用将通过将它们安装在 在Specific Aim l中开发的报告基因构建体并检查 它们在小鼠脓肿模型感染期间的表达。