TRYPANOSOMA CRUZI:GENE DISCOVERY USING CDNA MICROARRAYS

克鲁兹锥虫：使用 CDNA 微阵列发现基因

• 批准号: 6207162
• 负责人: TODD A MINNING
• 金额: $ 3.75万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6207162
• 关键词: Trypanosoma cruzi; biotechnology; drug screening /evaluation; genetic library; genetic mapping; high throughput technology; host organism interaction; immune response genes; immunopathology; immunopathology therapy; laboratory mouse; microarray technology; microorganism genetics; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protozoal vaccine; trypanosomiasis; vaccine development; vector vaccine

Chagas' disease remains a significant cause of morbidity and mortality in Central and South America where an estimated 100 million people are at risk for T. cruzi infection. Current control measures for Chagas' disease utilize decades-old technologies. Vaccine development in particular has been slowed by disagreement about the significance of autoimmunity in Chagas' disease pathology and by inefficient methods of gene discovery. The proposed study will utilize recent advances in three areas to accelerate gene discovery efforts for T. cruzi vaccine development. These are the findings that CD8+ T cell-mediated immune responses are critical to the outcome of T. cruzi infections, that genetic vaccination can potentiate protective CTL responses in T. cruzi infected animals, and that DNA microarrays can be used for genome-wide expression analyses and gene discovery. The long-term objective of this project is to identify new T. cruzi vaccine candidates for use in a multi- component genetic vaccine for Chagas' disease. Specifically, the applicants propose to create multiple, high density microarrays of DNAs from 10,000 random, ORF-containing clones from a T. cruzi genomic expression library. Using a two color fluorescence scheme the microarrays will be probed with mixtures of labeled cDNA probes prepared from parasites at different time points during trypomastigote to amastigote differentiation to enable simultaneous, pairwise comparisons of the relative expression levels for the genes represented in the arrays. The signal intensities produced by each fluor tag for each spot on the arrays will be determined using an array scanner. Clones representing genes upregulated early in trypomastigote to amastigote differentiation will be selected for sequencing. Based on the results of the sequence analyses a subset of the upregulated genes will be tested for their ability to elicit protective immune responses in mice. It is anticipated that this project will yield a minimum of 20-30 novel vaccine candidates.

恰加斯病仍然是中美洲和南美洲发病和死亡的一个重要原因，估计那里有 1 亿人面临克氏锥虫感染的风险。 目前针对恰加斯病的控制措施采用了数十年历史的技术。 由于对自身免疫在南美锥虫病病理学中的重要性的分歧以及基因发现方法的低效，疫苗的开发尤其受到阻碍。拟议的研究将利用三个领域的最新进展来加速克氏锥虫疫苗开发的基因发现工作。 这些发现表明，CD8+ T 细胞介导的免疫反应对于克氏锥虫感染的结果至关重要，基因疫苗接种可以增强克氏锥虫感染动物的保护性 CTL 反应，并且 DNA 微阵列可用于全基因组表达分析和基因发现。 该项目的长期目标是确定新的克氏锥虫候选疫苗，用于恰加斯病的多成分基因疫苗。 具体而言，申请人提议从来自克氏锥虫基因组表达文库的10,000个随机的、包含ORF的克隆创建多个高密度DNA微阵列。 使用双色荧光方案，将用在锥鞭毛体向无鞭毛体分化期间的不同时间点从寄生虫制备的标记cDNA探针的混合物来探测微阵列，以便能够同时、成对地比较阵列中代表的基因的相对表达水平。 将使用阵列扫描仪确定阵列上每个点的每个荧光标签产生的信号强度。 将选择代表在锥鞭毛体向无鞭毛体分化早期上调的基因的克隆进行测序。根据序列分析的结果，将测试上调基因的子集在小鼠中引发保护性免疫反应的能力。 预计该项目将产生至少 20-30 种新型候选疫苗。