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（由申请人提供）：门神经系统仅由原生动物寄生虫组成，其中一些对公共卫生构成了重大威胁。恶性疟原虫是疟疾的属性药物，可能在地方性地区引起高死亡率和发病率。弓形虫，隐孢子虫和环孢子虫也是公共卫生机构的众所周知，因为它们对免疫抑制的个体构成威胁（例如，移植和癌症患者），但也因为零星的爆发确实发生（隐孢子虫和环孢子虫），并且因为其他人口具有风险。根据疾病控制中心，隐孢子虫，环孢子虫和弓形虫也被列为B类生物恐怖主义威胁病原体，因为感染是通过口服传播发生的，并且宿主释放的抗性卵囊可长期保持在土壤和水中的长时间。借助这些寄生虫的大多数基因组，很明显地需要对可以产生重组蛋白的异源表达系统，这是表征蛋白质功能，结构研究，抗原的产生，抗原和筛查药物候选物以及了解动作机制所需的步骤。可用的异源系统已有不同程度的成功使用。但是，没有一个表达式系统普遍适用于所有应用程序，并且在特定应用程序中都有缺点。因此，我们建议使用原生动物P. Marinus开发一种用于生产Apicomplexa蛋白的异源系统，原生动物P. Marinus是Apicomplexa的近亲，很容易在无细胞的培养基中培养。尽管我们已经有了转染的向量，但在AIM1中，我们将在矢量中纳入可选标记，并为靶向和分泌信号开采P. Marinus基因组。在AIM2中，我们将使用选定的APICOMPLEXA基因进行概念证明。这些将包括特征良好的APICOMPLEXAN基因以及在其他系统中很难表达的基因。我们期望克服过去的某些困难，并在可用的异源表达系统不会导致表达，最佳蛋白质产量或功能蛋白时提供有效的选择。该系统的成功将导致适用于蛋白质功能研究，晶体结构测定，抗原产生和候选药物的APICOMPLEXAN蛋白，并最终提供更有效地与这些疾病作用作用和改善这些疾病的作用所需的工具。公共卫生相关性：为了开发与寄生疾病作斗争的新药和疫苗，需要更有效的系统来产生重组蛋白质。我们提出原生动物珀金斯·马里努斯（Perkinsus Marinus）作为用于产生Apicomplexa蛋白的异源表达系统。