DNA ANALYSIS OF PARASITES

寄生虫的 DNA 分析

• 批准号: 6288834
• 负责人: THEODORE NASH
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/6288834
• 关键词: Giardia; electroporation; genetic mapping; immunofluorescence technique; microorganism genetics; nucleic acid sequence; protein sequence; protozoal antigen; pulsed field gel electrophoresis; reporter genes; surface antigens; transfection; transfection /expression vector

Giardia lamblia is the most common disease-causing parasite in the United States responsible for an estimated 3 million cases a year. Besides causing problems in day care centers, travelers, backpackers, and homosexuals, it not uncommonly contaminates water supplies and has been responsible for massive epidemics. The organism lives in the small intestines and causes diarrhea, abdominal pain, nausea and vomiting. Symptoms are commonly intermittent and long lasting. An environmentally resistant cyst form is passed in the feces, and, because large numbers of cysts are excreted and only a few can initiate infection, infections are common.During the past year, a system for stably placing DNA into Giardia was improved (stable transfection) so that enough protein could be expressed to make it easily detected in the parasite. This allows the expression of any protein in the organism. For instance, a foreign protein, or more of a protein normally present, or a protein defective in some way that would interfere with normal function could be made. We are presently using this system to study a number of questions.Giardia has the ability to change its surface relatively frequently, a process called antigenic variation. Usually only one surface antigen or VSP can be seen on its surface. How only one VSP among many is expressed on the surface of the parasite, how VSPs are made and transported to the surface, and what is their biologic role are of major interest. Using the transfection system we have been able to express a novel VSP that is continually expressed on its surface along with a native VSP. Changes in the protein were then introduced into the VSP to determine the importance of particular regions and whether they could be secreted and placed onto the surface of the parasite. These studies showed that certain changes to conserved regions of the VSP resulted in inability of the parasite to process the VSP and place it on its surface. Substitution of an unrelated sequence in one conserved area unexpectedly showed no effects One question is how Giardia controls antigenic variation. The ability of the parasite to express two VSPs at once indicates that control of VSP expression does not reside in the coding region of the protein. In other studies, a foreign model protein with known and characterized secretion and processing in more developed cells was introduced into Giardia. We are comparing how Giardia handles this protein compared to mammalian cells.In order to survive and infect others, Giardia forms a cyst wall. Two constituents of the cyst wall were previously defined in this laboratory. Both proteins are not normally made or found in the growing form of the parasite but are induced or made when the growing form of the parasite (trophozoite) is cultured under the right conditions. We are presently studying how Giardia controls the production of cyst wall proteins and found that the controlling elements lie immediately upstream to the cyst wall protein gene. The two cyst wall protein genes are induced in exactly the same manner although their upstream regions are not identical. Exactly which small DNA pieces are important is currently being studied. Studies on Microsporidium were begun. This is a parasite that causes diarrhea and other problems in AIDS but recently has been found to cause diarrhea in normal persons as well. The parasite is only found inside the cell it infects. Using very special techniques we have been able to define proteins that are important for the parasite in general, important for infecting the host, and proteins that are specifically seen in the host cell when infected with this parasite. Understanding what proteins are important can lead to a better way to treat or prevent infection. - Giardia lamblia. Microsporidium, transfection, antigenic variation

贾迪亚·兰布利亚（Giardia Lamblia）是美国最常见的引起疾病的寄生虫，估计每年有300万例。除了在日托中心，旅行者，背包客和同性恋者中引起问题外，它并不罕见地污染供水，并且负责大规模的流行病。生物体生活在小肠中，导致腹泻，腹痛，恶心和呕吐。症状通常是间歇性和持久的。粪便中传递了一种具有环境性囊肿的形式，并且由于大量囊肿被排出并且只有少数可以引起感染，因此感染很常见。在过去的一年中，将DNA稳定地放入贾第鞭毛虫中的系统得到了改善（稳定的traint染），因此可以轻松地表达出足够的蛋白质，以使其在羊角岩中轻松地检测到它。这允许在生物体中表达任何蛋白质。例如，可以使外来蛋白质或更多蛋白质通常存在，或以某种方式有缺陷的蛋白质会干扰正常功能。目前，我们正在使用该系统来研究许多问题。吉亚迪亚具有相对频繁地改变其表面的能力，即一种称为抗原变异的过程。通常，在其表面上只能看到一个表面抗原或VSP。在寄生虫的表面上，如何在许多人中只有一个VSP，如何制造和运输到表面，以及它们的生物学作用是主要感兴趣的。使用转染系统，我们已经能够表达出一种新型的VSP，该VSP与天然VSP一起在其表面不断表达。然后将蛋白质的变化引入VSP中，以确定特定区域的重要性以及是否可以分泌并将其放置在寄生虫的表面上。这些研究表明，对VSP的保守区域的某些变化导致寄生虫无法处理VSP并将其放置在其表面上。在一个保守区域中替换一个无关的序列意外地表明，没有一个问题是贾第鞭毛虫如何控制抗原变异。寄生虫立即表达两个VSP的能力表明，对VSP表达的控制不存在于蛋白质的编码区域。在其他研究中，将具有已知和特征性分泌和加工的外国模型蛋白在更发达的细胞中引入了贾第鞭毛虫。我们正在比较贾第鞭毛虫如何处理该蛋白与哺乳动物细胞相比。为了生存和感染其他蛋白质，贾第鞭毛虫形成了囊肿壁。先前在该实验室中定义了囊肿壁的两个成分。两种蛋白质通常都不是以寄生虫的生长形式生产或发现的，而是在适当的条件下培养寄生虫（Trophozoite）的生长形式时诱导或制造。我们目前正在研究贾第鞭毛虫如何控制囊肿壁蛋白的产生，并发现控制元件立即位于囊肿壁蛋白基因上游。尽管它们的上游区域并不相同，但以完全相同的方式诱导了两个囊肿壁蛋白基因。目前正在研究哪些小型DNA碎片很重要。开始有关微孢子虫的研究。这是一种寄生虫，会导致腹泻和艾滋病中的其他问题，但最近发现也会引起普通人的腹泻。该寄生虫仅在IT感染的细胞内发现。使用非常特殊的技术，我们已经能够定义对通常对寄生虫很重要的蛋白质，对于感染宿主的重要性以及在感染该寄生虫时在宿主细胞中特别看到的蛋白质很重要。了解哪种蛋白质很重要可以导致更好的治疗或预防感染的方法。 - 贾迪亚·兰布利亚（Giardia Lamblia）。微孢子虫，转染，抗原变异