IRON AVAILABILITY IN THE PATHOGENESIS ACTINOBACILLUS ACTINOMYCETEMCOMITANS

伴放线杆菌发病过程中铁的有效性

• 批准号: 3839105
• 负责人: Leslie J Winston
• 金额: --
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3839105
• 关键词: Actinobacillus actinomycetemcomitans; SDS polyacrylamide gel electrophoresis; genetic library; growth media; iron metabolism; membrane proteins; microorganism culture; molecular cloning; molecular pathology; nucleic acid probes; protein structure function

The purpose of the project is to study the role of iron availability on the pathogenesis of Actinobacillus actinomycetemcomitans (A. a.). Little is known about the iron transport systems of anaerobic bacteria, although it is clear that the acquisition of iron from the host is required in order to sustain growth. The identification of an iron regulated membrane protein of 70kD that is antigenic in humans may represent a vaccine candidate for the prevention and treatment of A.a. infections. The specific aims of the study are to identify potential in vivo iron sources utilized by A.a. to help in targeting iron transport systems. In addition, the role of the 70kD iron regulated membrane protein in iron uptake will be studied. To achieve these goals an iron limited growth medium will be developed in order to study iron utilization with a limited number of variables. Separation of the 70kD protein from other membrane proteins will be achieved with sodium dodecyl sulfate gel electrophoresis and the protein will be subsequently transferred to a PVDF membrane. The 70kD band will be excised and sequenced using an automated gas-phase sequencer. The derived N-terminal amino acid sequence will be utilized to generate an oligonucleotide probe specific for the 70kD protein and used for screening a lambama gt11 library of A.a. DNA. As an additional strategy, monospecific antisera will also be utilized to screen the library. Once the wild-type gene has been identified, it will be cloned into an appropriate vector and utilized to generate mutations in the wild type gene in order to characterize the function of the protein in iron uptake.

该项目的目的是研究铁的作用 对静脉细菌的发病机理的可用性 放线菌（A. a。）。对铁知之甚少 厌氧菌的运输系统，尽管很明显 需要从宿主那里获取铁才能 维持增长。 铁调节膜的鉴定 人类抗原的70KD蛋白质可能代表一种疫苗 A.A.的预防和治疗候选人感染。这 该研究的具体目的是确定体内铁的潜力 A.A.使用的资源帮助瞄准铁运输 系统。另外，70kD铁调节的膜的作用 将研究摄取铁中的蛋白质。 为了实现这些目标 铁有限生长培养基将开发以研究铁 使用数量有限的变量。分离 通过 十二烷基硫酸钠凝胶电泳和蛋白质将是 随后转移到PVDF膜。 70kd乐队将 使用自动气相测序仪切除和测序。 这 衍生的N末端氨基酸序列将用于生成 针对70KD蛋白的寡核苷酸探针，用于 放映A.A.的Lambama GT11图书馆脱氧核糖核酸。另外 策略，单特异性抗血清还将用于筛选 图书馆。一旦确定了野生型基因，它将是 克隆到适当的向量中，并用于生成 野生型基因中的突变是为了表征 蛋白质在铁吸收中的功能。