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已经产生了能够检测和消除生物威胁剂的全新系统谱的必要性，该系统生产成本低，坚固且稳定，以允许第一响应者甚至普通人群大规模使用。然而，目前在这样的系统中用作主要组分的抗体的性质不允许它们在没有冷藏的情况下长期储存并且不允许简单地并入电子装置中。此外，抗体的生产成本很高，并且当用作解毒剂时，不能消除已经内化到细胞中的药剂。在这里，我们建议将细菌蛋白质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。我们期望所得产物将是廉价的生产，易于修改以允许其适应特定协议的需要，并且将是足够稳定的以在恶劣的环境条件下生存，甚至通过细胞膜屏障运输。在第一阶段，我们将测试我们的方法的可行性，通过开发蛋白质，将识别肉毒杆菌神经毒素血清型A（BoNT/A-L）的催化结构域。为此，我们将创建所选细菌蛋白的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，允许对BoNT/A-L的酶活性进行极其灵敏的监测。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剂的蛋白质。为此，我们将应用该方案来进化能够识别和/或中和血清型B和C的肉毒杆菌神经毒素的蛋白质。

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.