New molecular techniques for T. cruzi

克氏锥虫的新分子技术

• 批准号: 9132477
• 负责人: Huan Huang
• 金额: $ 0.55万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-13 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9132477
• 关键词: New; molecular; techniques; T.; cruzi

Abstract Trypanosoma cruzi (T. cruzi) causes Chagas Disease (e.g. American Trypanosomiasis) in humans. This infection is endemic to Latin America; however, due to immigration from endemic areas Chagas Disease is found in both Europe and the United States. What is striking about Infection with T cruzi is the development of chronic infection with disease symptoms manifesting decades after the acute infection. Research on T. cruzi has been limited by the difficulties in genetic manipulation. We used a modified pTREX vector with a ligand-controlled destabilization domain (ddFKBP) to regulate a gene/protein of interest. This vector system allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages. Using this technique, we found that two mitogen activated protein kinases (MAPK), TcMAPK1 and TcMAPK3, are essential for T. cruzi. We plan to develop a conditional gene deletion system based on our ddFKBPpTREX vector system. In addition, quickly over- expressing a lethal gene in a regulated fashion should be feasible in the ddFKBP system and this can be done in multiple T. cruzi isolates using the same vector construct without any need to genetically modify the isolates. Such an inducible lethal phenotype T. cruzi would be very useful for pathogenesis studies allowing elimination of the organism at various time points after infection to dissect the mechanisms of disease causation. Such parasites could also represent a new vaccine strain and would provide controlled immune stimulation. We, therefore, propose to: (1) develop a robust conditional knockout vector system using TcMAPK1 and TcMAPK3, essential genes for T. cruzi growth, based on our ddFKBPpTREX vectors. This system should be useful for the manipulation of other essential genes this parasite; and (2) we will also create vector systems for T. cruzi that allow the regulated expression of toxin genes that will kill this parasite when these genes are expressed. These transgenic parasites will be potentially valuable to define pathogeneses or as vaccine strains.

摘要 克氏锥虫引起恰加斯病(如美国锥虫病) 人类。这种感染是拉丁美洲的地方性疾病；然而，由于来自地方性疾病的移民 地区恰加斯病在欧洲和美国都有发现。令人震惊的是什么 克氏支原体感染是以疾病症状为表现的慢性感染的发展 在急性感染几十年后。克氏毛滴虫的研究一直受到以下困难的限制 基因操纵。我们使用了一种改进的pTREX载体，该载体具有配体控制的失稳 结构域(DdFKBP)来调节目标基因/蛋白质。这一载体系统允许快速和 克氏锥虫不同生命周期的可逆蛋白表达及高效功能分析 周期阶段。利用这项技术，我们发现有两种有丝分裂原激活的蛋白激酶 (MAPK)、TcMAPK1和TcMAPK3是克鲁兹毛滴虫的必需基因。我们计划制定一项有条件的 基于我们的ddFKBPpTREX载体系统的基因缺失系统。此外，很快就会超过- 在ddFKBP系统中，以规范的方式表达致死基因应该是可行的，并且 这可以在使用相同载体结构的多个克氏锥虫分离物中完成，而不需要任何 对这些分离株进行基因改造。这样一种可诱导的致死表型克氏锥虫将非常 适用于病因学研究，允许在不同的时间点消除生物体 感染来剖析疾病的致病机制。这种寄生虫也可能代表着 一种新的疫苗株，并将提供受控的免疫刺激。 因此，我们建议：(1)开发一个健壮的条件基因敲除载体系统，使用 TcMAPK1和TcMAPK3是克氏锥虫生长的必需基因，基于我们的ddFKBPpTREX 向量。该系统对其他必需基因的操作具有一定的参考价值 寄生虫；以及(2)我们还将为克氏锥虫创建媒介系统，允许受监管的 表达毒素基因，当这些基因表达时，这些基因将杀死这种寄生虫。这些 转基因寄生虫在确定致病机理或作为疫苗毒株方面具有潜在的价值。