A Heterologous Expression System for Apicomplexa Genes

描述（由申请人提供）：顶复合体门完全由原生动物寄生虫组成，其中一些对公众健康构成重大威胁。顶复合体恶性疟原虫是疟疾的病原，在流行地区可引起高死亡率和发病率。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体（例如移植和癌症患者）构成威胁，但也因为确实会发生零星暴发（隐孢子虫和环孢子虫），而且还因为有更多人群（即孕妇）处于危险之中（弓形虫）。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类生物恐怖威胁病原体，因为感染是通过口腔传播的，宿主排出的耐药卵囊可以在土壤和水中长时间存活。由于这些寄生虫的大部分基因组都是可用的，因此很明显需要能够产生重组蛋白的异源表达系统，这是蛋白质功能表征、结构研究、抗原生产、候选药物筛选和分析以及了解作用机制所必需的一步。现有的异源系统已获得不同程度的成功；然而，没有一个单一的表达系统普遍适用于所有的应用程序，并且在特定的应用程序中都有缺点。因此，我们建议利用原生动物P. marinus（顶复合体的近亲，易于在完全确定的无细胞培养基中培养）开发一种生产顶复合体蛋白的异源系统。虽然我们已经有了用于转染的载体，但在AIM1中，我们将在载体中加入一个可选择的标记物，并挖掘P. marinus基因组的靶向和分泌信号。在AIM2中，我们将使用选定的顶复合体基因来证明概念；这些将包括具有良好特征的顶复合体基因以及已被证明难以在其他系统中表达的基因。我们希望克服一些过去的困难，并提供一个有效的替代方案，当可用的异种表达系统不能导致表达，最佳蛋白产量，或功能蛋白。该系统的成功将产生适合于蛋白质功能研究、晶体结构测定、抗原生产和候选药物的顶复合体蛋白，并将最终提供更有效地对抗和改善这些疾病影响所需的工具。公共卫生相关性：为了开发对抗寄生虫病的新药和疫苗，需要一种更有效的生产重组蛋白的系统。我们提出了一种产生顶复合体蛋白的异源表达系统。

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.