A Heterologous Expression System for Apicomplexa Genes

顶端复合体基因的异源表达系统

• 批准号: 7843525
• 负责人: Jose A Fernandez-Robledo
• 金额: $ 18.09万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7843525
• 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（由申请方提供）：顶复门仅由原生动物寄生虫构成，其中几种对公共卫生构成重大威胁。顶端复形类恶性疟原虫是疟疾的病原体，在疟疾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是公共卫生当局熟知的，因为它们对免疫抑制个体（例如移植和癌症患者）构成威胁，但也因为确实发生零星爆发（隐孢子虫和环孢子虫），并且因为其他人群处于风险中，即孕妇（弓形虫）。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，因为感染是通过口腔传播发生的，宿主排出的抗性卵囊可以在土壤和水中长时间存活。随着这些寄生虫的大部分基因组可用，显然需要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、抗原生产、筛选和分析候选药物以及理解作用机制所需的步骤。可用的异源系统已经以不同程度的成功使用;然而，没有一种表达系统普遍适用于所有应用，并且在特定应用中都具有缺点。因此，我们建议开发一种异源系统，用于使用原生动物P. marinus生产Apicomplexa蛋白，这是Apicomplexa的近亲，易于在完全确定的无细胞培养基中培养。虽然我们已经有了用于转染的载体，但在AIM 1中，我们将在载体中掺入选择性标记，并挖掘海原藻基因组的靶向和分泌信号。在AIM 2中，我们将使用选定的顶复门基因进行概念验证;这些基因将包括具有良好特征的顶复门基因以及已被证明难以在其他系统中表达的基因。我们希望克服过去的一些困难，并提供一个有效的替代时，可用的异源表达系统不导致表达，最佳的蛋白质产量，或功能蛋白。该系统的成功将导致apicomplexan蛋白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，并最终提供更有效地对抗和改善这些疾病的影响所需的工具。公共卫生关系：为了开发用于对抗寄生虫病的新药和疫苗，需要用于生产重组蛋白的更有效的系统。我们建议原生动物Perkinsus marinus作为生产Apicomplexa蛋白的异源表达系统。

项目成果

Protozoan parasites of bivalve molluscs: literature follows culture.

• DOI: 10.1371/journal.pone.0100872
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Fernández Robledo JA;Vasta GR;Record NR
• 通讯作者: Record NR

Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay.

使用基于 ATP 的生物发光测定法鉴定 Perkinsus marinus 的 MMV Malaria Box 抑制剂。

• DOI: 10.1371/journal.pone.0111051
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: AlemánResto,Yesmalie;FernándezRobledo,JoséA
• 通讯作者: FernándezRobledo,JoséA

Production of recombinant proteins from protozoan parasites.

• DOI: 10.1016/j.pt.2010.02.004
• 发表时间: 2010-05
• 期刊: TRENDS IN PARASITOLOGY
• 影响因子: 9.6
• 作者: Fernandez-Robledo, Jose A.;Vasta, Gerardo R.
• 通讯作者: Vasta, Gerardo R.

Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity.

• DOI: 10.1371/journal.pone.0087435
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Wijayalath W;Majji S;Kleschenko Y;Pow-Sang L;Brumeanu TD;Villasante EF;Vasta GR;Fernández-Robledo JA;Casares S
• 通讯作者: Casares S