Development of novel proteins as a defence against potential biothreat agents

开发新型蛋白质来防御潜在的生物威胁因子

• 批准号: 7110717
• 负责人: Alexey G Zdanovsky
• 金额: $ 36.51万
• 依托单位: ALPHA UNIVERSE, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-07 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110717
• 关键词: Pseudomonas; antibody; bacterial virus; evolution; library; ligands; neurotoxins; proteins; serotyping

DESCRIPTION (provided by applicant): The ongoing war on terror has created the necessity of a whole new spectrum of systems that are able to detect and inactivate biothreat agents, systems that are inexpensive to produce, robust and stable to allow their use on a massive scale by first responders or even the general population. However, properties of antibodies that are currently used as main components in such systems do not allow their prolonged storage without refrigeration and simple incorporation into electronic devices. Also, antibodies are expensive to produce and when used as antidotes cannot inactivate agents that have been already internalized into the cells. Here, we propose to convert a small and extremely stable domain of bacterial protein into an entity capable of specific binding and inactivation of biothreat agents. We expect that the resulting product will be inexpensive to produce, easy to modify to allow its adaptation to the needs of specific protocols and will be stable enough to survive harsh environmental conditions and even transport through the cell membrane barrier. In Phase I, we will test the feasibility of our approach by developing proteins that will recognize the catalytic domain of botulinum neurotoxin serotype A (BoNT/A-L). For this, we will create several types of phage displays of the chosen bacterial protein and screen them for the ability to recognize a specially created derivative of BoNT/A-L. Also, during Phase I, we will create a system that will allow extremely sensitive monitoring of enzymatic activity of BoNT/A-L. This system will be used to monitor the efficiency of the screening process and to determine affinities of selected constructs. During Phase II, we will prove that the developed protocol allows development of proteins recognizing any biothreat agent of interest. For this, we will apply the protocol to the evolution of proteins capable of recognizing and/or neutralizing botulinum neurotoxins of serotypes B and C.

描述（由申请人提供）：正在进行的反恐战争需要一系列全新的系统，这些系统能够检测和灭活生物威胁制剂，这些系统生产成本低廉，坚固且稳定，可以大规模使用急救人员甚至一般人群的规模。然而，目前在此类系统中用作主要成分的抗体的特性不允许它们在不冷藏的情况下长期储存和简单地并入电子设备中。此外，抗体的生产成本很高，并且当用作解毒剂时，不能使已经内化到细胞中的试剂失活。在这里，我们建议将细菌蛋白的一个小且极其稳定的结构域转化为能够特异性结合和灭活生物威胁剂的实体。我们预计所得产品的生产成本低廉，易于修改以适应特定方案的需求，并且足够稳定，能够在恶劣的环境条件下生存，甚至可以穿过细胞膜屏障运输。在第一阶段，我们将通过开发能够识别 A 型肉毒杆菌神经毒素 (BoNT/A-L) 催化结构域的蛋白质来测试我们方法的可行性。为此，我们将为所选细菌蛋白创建几种类型的噬菌体展示，并筛选它们识别专门创建的 BoNT/A-L 衍生物的能力。此外，在第一阶段，我们将创建一个系统，可以极其灵敏地监测 BoNT/A-L 的酶活性。该系统将用于监测筛选过程的效率并确定所选构建体的亲和力。在第二阶段，我们将证明所开发的协议允许开发识别任何感兴趣的生物威胁因子的蛋白质。为此，我们将将该协议应用于能够识别和/或中和血清型 B 和 C 肉毒杆菌神经毒素的蛋白质的进化。