Malaria antigens binding to band 3 in red cell invasion

疟疾抗原与红细胞侵袭中的带 3 结合

• 批准号: 6599178
• 负责人: STEVEN S OH
• 金额: $ 8.25万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599178
• 关键词: Plasmodium falciparum; SDS polyacrylamide gel electrophoresis; antigen antibody reaction; clinical research; enzyme linked immunosorbent assay; erythrocytes; fluorescence microscopy; genetic library; human tissue; intracellular parasitism; laboratory rabbit; malaria; parasite infection mechanism; protein binding; protein protein interaction; receptor binding; vaccine development; western blottings; yeast two hybrid system

DESCRIPTION (provided by the applicant): Plasmodium falciparum merozoite proteins have been considered as a major target for developing a subunit vaccine that could block the malaria parasite invasion of erythrocytes. For most merozoite antigens, however, their molecular function during the invasion process is not well understood. Recently, we have discovered that two non-glycosylated ectoplasmic regions of band 3 function as a crucial erythrocyte receptor and interact with a number of P. falciparum merozoite proteins by a sialic acid-independent mechanism. We were able to identify one of these proteins as merozoite surface protein-1 (MSP1). However, other merozoite proteins binding to the band 3 receptor could not been identified by biochemical methods. Based on our findings, we hypothesize that band 3 is a crucial host receptor interacting with multiple parasite ligands in sialic acid-independent manner during the P. falciparum invasion of erythrocytes. The long-term objective of our study is to identify target antigens for an effective malaria vaccine on the basis of well-defined functions of the antigens. In this study, we propose to test our hypothesis by identifying the merozoite antigens that bind to the band 3 receptor. The specific aims of the study are as follows. (a) Screening of P. falciparum cDNA library: Using the receptor region of human band 3 as the bait, parasite proteins binding to the band 3 receptor will be identified by screening a cDNA library in a yeast two-hybrid system in vivo. (b) Independent confirmation of the interactions: The binding of newly identified parasite ligands to band 3 will be confirmed in solution in vitro using quantitative methods. (c) Production and characterization of antibodies to the identified parasite ligands: Polyclonal antibodies will be generated in rabbits against recombinant proteins and characterized using native parasite proteins. (d) Localization of the parasite ligands to merozoites: Indirect immunofluorescence microscopy will be carried out using the produced antibodies and the parasites at the segmented schizont stage to localize the band 3-binding parasite antigens to the merozoite. We anticipate this study will make a significant contribution towards the identification of potential new targets for a malaria vaccine and provide a better understanding of the sialic acid-independent erythrocyte invasion mechanism in P. falciparum malaria.

描述（由申请方提供）：恶性疟原虫裂殖子蛋白被认为是开发可阻断疟原虫侵入红细胞的亚单位疫苗的主要靶标。然而，对于大多数裂殖子抗原，它们在入侵过程中的分子功能还不清楚。最近，我们发现带3的两个非糖基化的胞外区作为一个重要的红细胞受体发挥作用，并通过唾液酸非依赖性机制与许多恶性疟原虫裂殖子蛋白相互作用。我们能够鉴定这些蛋白质之一为裂殖子表面蛋白-1（MSP 1）。然而，其他裂殖子蛋白结合带3受体不能通过生物化学方法鉴定。基于我们的研究结果，我们假设带3是恶性疟原虫入侵红细胞过程中与多种寄生虫配体以唾液酸非依赖性方式相互作用的关键宿主受体。我们研究的长期目标是在明确抗原功能的基础上确定有效疟疾疫苗的靶抗原。在这项研究中，我们建议通过鉴定与带3受体结合的裂殖子抗原来验证我们的假设。研究的具体目标如下。(a)恶性疟原虫cDNA文库的筛选：以人带3受体区为诱饵，通过酵母双杂交系统在体内筛选cDNA文库，鉴定与带3受体结合的疟原虫蛋白。(b)相互作用的独立确认：将使用定量方法在体外溶液中确认新鉴定的寄生虫配体与条带3的结合。(c)针对已鉴定寄生虫配体的抗体的产生和表征：将在兔中产生针对重组蛋白的多克隆抗体，并使用天然寄生虫蛋白进行表征。(d)将寄生虫配体定位于裂殖子：将使用产生的抗体和处于分节的裂殖体阶段的寄生虫进行间接免疫荧光显微镜检查，以将带3结合寄生虫抗原定位于裂殖子。我们预计这项研究将为确定疟疾疫苗的潜在新靶点做出重大贡献，并更好地了解恶性疟原虫疟疾中唾液酸非依赖性红细胞侵袭机制。