A Heterologous Expression System for Apicomplexa Genes

顶端复合体基因的异源表达系统

• 批准号: 7660811
• 负责人: Jose A Fernandez-Robledo
• 金额: $ 17.49万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660811
• 关键词: Antigens; Apicomplexa; Area; Cancer Patient; Categories; Cells; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Cloning; Cryptosporidium; Culture Media; Cyclospora; Development; Development Plans; Dinophyceae; Disease; Disease Outbreaks; Gene Expression; Genes; Genetic Vectors; Genome; Goals; Individual; Infection; Knowledge; Malaria; Mining; Morbidity - disease rate; Oocysts; Oral; Parasites; Parasitic Diseases; Pharmaceutical Preparations; Plasmodium; Plasmodium falciparum; Populations at Risk; Pregnant Women; Process; Production; Proteins; Public Health; Recombinant Proteins; Relative (related person); Resistance; Screening procedure; Signal Transduction; Site; Soil; Structure; System; Time; Toxoplasma; Transfection; Transplantation; Vaccines; Vertebral column; Water; authority; base; drug candidate; enteropeptidase; expression vector; fighting; immunosuppressed; improved; interest; mortality; parasite genome; pathogen; protein expression; protein function; public health relevance; success; tool; transmission process; vaccine development; vector

DESCRIPTION (provided by applicant): The phylum Apicomplexa is constituted exclusively by protozoan parasites, several of which pose a significant threat to public health. The apicomplexan Plasmodium falciparum is the ethiological agent of Malaria and can cause high mortality and morbidity in endemic areas. Toxoplasma, Cryptosporidium, and Cyclospora are also well known to public health authorities since they pose a threat to immunosuppressed individuals (e.g. transplanted and cancer patients), but also because sporadic outbreaks do occur (Cryptosporidium and Cyclospora) and because additional populations are at risk, i.e. pregnant women (Toxoplasma). Cryptosporidium, Cyclospora, and Toxoplasma are also listed as Category B Bioterrorist Threat Pathogens according to the Center of Disease Control, since infection occurs by oral transmission and the resistant oocysts shed by hosts can remain viable for long periods of time in soil and water. With most of the genomes of these parasites available, it becomes evident the need for heterologous expression systems that can generate recombinant proteins, a step required for characterization of protein function, structural studies, production of antigen, screening and profiling drug candidates, and understanding action mechanisms. Available heterologous systems have been used with varying degrees of success; however, there is not a single expression system universally suitable for all applications and all have drawbacks in particular applications. Hence, we propose to develop a heterologous system for production of Apicomplexa proteins using the protozoan P. marinus, a close relative of the Apicomplexa that is easily cultured in a full-defined cell-free medium. Although we have already a vector for transfection, in AIM1 we will incorporate into the vector a selectable marker and mine the P. marinus genome for targeting and secretion signals. In AIM2, we will use selected Apicomplexa genes for proof of concept; these will include well-characterized apicomplexan genes as well as genes that have proven difficult to express in other systems. We expect to overcome some of the past difficulties and to provide a valid alternative when the available heterologous expression systems do not result in expression, optimal protein yield, or functional proteins. The success of this system would result in apicomplexan proteins suitable for protein function studies, crystal structure determination, antigen production, and drug candidates, and will ultimately provide the tools required to more effectively fight and ameliorate the effects of these diseases. PUBLIC HEALTH RELEVANCE: In order to develop new drugs and vaccines for fighting parasitic diseases, a more efficient system for producing recombinant proteins is required. We propose the protozoan Perkinsus marinus as a heterologous expression system for producing Apicomplexa proteins.

描述(申请人提供)：Apicomplexa门完全由原生动物寄生虫组成，其中几种对公共卫生构成重大威胁。恶性尖端复合体疟原虫是疟疾的人种学病原体，在疟疾流行地区可导致高死亡率和高发病率。弓形虫、隐孢子虫和环孢子虫也为公共卫生当局所熟知，因为它们对免疫抑制的个人(例如移植患者和癌症患者)构成威胁，但也因为确实发生零星暴发(隐孢子虫和环孢子虫)，还因为更多的人群处于危险之中，即孕妇(弓形虫)。根据疾病控制中心的数据，隐孢子虫、环孢子虫和弓形虫也被列为B类生物恐怖威胁病原体，因为感染是通过口腔传播的，宿主排出的抗药性卵囊可以在土壤和水中长期存活。随着这些寄生虫的大部分基因组的获得，显然需要能够产生重组蛋白的异源表达系统，这是鉴定蛋白质功能、结构研究、生产抗原、筛选和分析候选药物以及了解作用机制所必需的步骤。现有的异源系统已经得到了不同程度的成功；然而，没有一个单一的表达系统普遍适用于所有应用，并且在特定的应用中都有缺点。因此，我们建议开发一个异源系统，使用原生动物P.marinus来生产Apicomplexa蛋白，P.marinus是Apicomplexa的近亲，很容易在全定义的无细胞培养基中培养。虽然我们已经有了一个可用于转染的载体，但在AIM1中，我们将在载体中加入一个可选择的标记，并挖掘Marinus基因组中的靶向和分泌信号。在AIM2中，我们将使用精选的ApicomplexA基因来验证概念；这些基因将包括特征良好的Apicomplexan基因以及已被证明在其他系统中难以表达的基因。我们希望克服过去的一些困难，并提供一个有效的替代方案，当现有的异源表达系统不能导致表达、最佳蛋白质产量或功能蛋白质时。该系统的成功将产生适合于蛋白质功能研究、晶体结构确定、抗原生产和候选药物的apicplexan蛋白质，并最终将提供更有效地对抗和改善这些疾病的影响所需的工具。公共卫生相关性：为了开发抗击寄生虫病的新药和疫苗，需要一种生产重组蛋白的更有效的系统。我们提出了原生动物Perkinsus marinus作为异源表达系统来生产Apicomplexa蛋白。