ANALYSIS OF V CHOLERAE GENES INVOLVED IN COLONIZATION

霍乱弧菌定植相关基因分析

• 批准号: 3455253
• 负责人: Kenneth Milan Peterson
• 金额: $ 9.44万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455253
• 关键词: Vibrio cholerae; antibody formation; bacterial genetics; bacterial proteins; chimeric proteins; gene expression; gene induction /repression; genetic manipulation; genetic strain; genetic transcription; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; microorganism growth; molecular cloning; mutant; synthetic protein; virulence; western blottings

The studies outlined in this proposal are aimed at providing a better understanding of Vibrio cholerae virulence determinants which are involved in intestinal colonization. The goal of this project is the molecular analysis of a region of the V. cholerae chromosome which contains a set of four coordinately regulated genes (acf loci, accessory colonization factor) that are involved in the colonization properties of V. cholerae. Insertional mutations in any one of these four genes produces a quantitatively identical colonization defect in suckling mice. The proposed experiments are intended to elucidate the organization, expression, and function of genes specifying the ACF phenotype. The four genes encoding Acf determinants will be isolated and subjected to both DNA sequence analysis and computer aided amino acid similarity search. This region of DNA will be subjected to transposon and deletion mutational analysis in order to: define the limits of each acf gene; locate promoters and controlling elements:; and identify additional genes involved in ACF expression. Acf gene products will be identified, characterized, and localized to specific bacterial compartments with antisera raised against PhoA fusion proteins, purified acf proteins, or synthetic peptides using immunoblot analysis, fluorescence microscopy and colloidal gold labeled antibody immuno-electronmicroscopy. V cholerae strains with defined acf mutations will be constructed in ctxA deletion backgrounds by in vivo marker exchange for use as potential vaccine candidates. An adult rabbit animal model will be assessed for its suitability in characterizing (protective?) host immune responses to Acf determinants. E1 tor strains of V. cholerae (the biotype responsible for the current pandemic) will be characterized with respect to ACF; to date these genes have been characterized only for a classical strain. Knowledge gained from the proposed investigation may provide a basis for the development of efficacious cholera vaccines.

该提案中概述的研究旨在提供更好的 了解颤音霍乱毒力决定因素 参与肠定植。这个项目的目标是 对霍乱绿色葡萄球菌的区域的分子分析该区域 包含一组四个协调调节的基因（ACF基因座，附件 定植因子）参与的定植特性 V.霍乱。在这四个基因中的任何一个中插入突变 在哺乳小鼠中产生定量相同的定植缺陷。 提出的实验旨在阐明组织， 指定ACF表型的基因的表达和功能。 编码ACF决定因素的四个基因将被隔离并进行 DNA序列分析和计算机辅助氨基酸相似性 搜索。该DNA区域将受到转座子和缺失 突变分析以：定义每个ACF基因的限制； 找到启动子和控制元素：并确定其他基因 参与ACF表达。将确定ACF基因产品， 特征并本地定位于特定的细菌室 针对Phoa融合蛋白，纯化的ACF蛋白或 使用免疫印迹分析，荧光显微镜和 胶体金标记的抗体免疫电子显微镜检查。 V霍乱 CTXA缺失将构建具有定义ACF突变的菌株 体内标记的背景交换作为潜在疫苗 候选人。将对成年兔动物模型进行评估 特征（保护性？）宿主对ACF的免疫反应的适用性 决定因素。 V.霍乱的E1 Tor菌株（负责的生物型 当前的大流行）将以ACF为特征；迄今为止 这些基因仅针对经典菌株的特征。 从拟议的调查中获得的知识可能为 有效的霍乱疫苗的开发。