Development of novel proteins as a defence against potential biothreat agents

描述（由申请人提供）：正在进行的反恐战争创造了一种全新的系统的必要性，这种系统能够检测和灭活生物威胁剂，系统生产成本低廉，强大且稳定，可以让第一响应者甚至普通人群大规模使用。然而，目前在这种系统中用作主要成分的抗体的特性不允许它们在没有冷藏和简单地并入电子设备的情况下长期储存。此外，抗体的生产成本很高，而且当用作解毒剂时，不能使已经内化到细胞中的药物失活。在这里，我们建议将细菌蛋白的一个小而非常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。我们期望最终的产品生产成本低廉，易于修改以适应特定协议的需要，并且足够稳定，可以在恶劣的环境条件下生存，甚至可以通过细胞膜屏障运输。在第一阶段，我们将通过开发识别肉毒杆菌神经毒素血清型A （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，我们将为选定的细菌蛋白创建几种类型的噬菌体展示，并对它们进行筛选，以识别专门创建的BoNT/ a - l衍生物。此外，在第一阶段，我们将创建一个系统，将允许非常敏感地监测BoNT/ a - l的酶活性。该系统将用于监测筛选过程的效率，并确定所选结构的亲和力。在第二阶段，我们将证明开发的方案允许开发识别任何感兴趣的生物威胁剂的蛋白质。为此，我们将把该方案应用于能够识别和/或中和血清型B和C的肉毒杆菌神经毒素的蛋白质的进化。

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.