Evaluation of Toxicity Score and Precise Snake Venom Analytics for Next Generation Antivenom Development

摘要 这个项目的总体目标是让本科生和研究生在 分子毒物学有可能对我们了解蛇的重要进展做出贡献 毒液病理学和基于抗体的新疗法的发展。根据世卫组织的统计数据， 据估计，到2020年，全球将新增12.5万人死于蛇毒。在……里面 尽管作为一种被忽视的热带病和全球健康问题，蛇咬伤毒液对它很重要， 关于蛇毒的作用机制和内在的毒性/致死性贡献，人们知之甚少。这个 我们研究的科学基础是一种特定的蛇毒毒素组合在 蛇咬伤会导致坏死、凝血障碍、严重出血和最终死亡。因为 对单个毒素进行了评估，缺乏蛇毒病理的协同机制 有权研发解毒剂。我们假设精确的毒性评估和蛇毒 对粗制/分级的毒液和重组毒素的分析将提供一种模型，在该模型中优先考虑 可给予毒性最强的毒液成分(S)。为了检验我们的假设，我们将讨论(3)具体的 目标：特定目标1：分析蛇毒的组成、组合评价和内在特性 毒性。我们将提纯和鉴定鳄鱼蛇种的蛇毒。定性将包括 分析蛇毒以确定每一组分和丰度中的毒素。学生们将调查 不同毒液组分组合的作用机制。我们将描述致命的 每种毒液组分的浓度/剂量对HUVEC细胞、凝血功能的影响，并将验证其毒性 使用活体致死小鼠模型对每一组分进行评分。具体目标2：生产重组人 毒肽。我们将从我们收集的cdna文库中克隆这些毒素，并将产生质粒。 转化感受态大肠杆菌细胞进行蛋白质表达和纯化。开发方法以实现 生产重组毒素对于抗蛇毒血清的开发和研究毒性和靶点是必不可少的 医学上相关的毒肽。这些研究也将是使用毒素来解决结构问题的关键 具有已知目标的复合体中的毒素或结构。具体目标3：鉴定蛇毒 血浆外切体中的生物标志物。我们将使用新的Evtrap技术和 蛋白质组学在基于发现的小鼠血浆外切体蛇毒生物标志物分析中的应用。 全面的蛇毒生物标志物分析将为研究蛇毒的病理生理、代谢、 蛇毒的生物学、细胞学和免疫学反应。

ABSTRACT The overall goal of this project is to engage undergraduate and graduate students in innovative studies in molecular toxinology that have the potential to contribute to important advances in our understanding of snake venom pathology and the development of novel antibody-based therapeutics. According to WHO statistics, it is estimated that in 2020 there will be 125,000 new deaths world-wide reported due to snake envenomation. In spite of its importance for snake bite envenomation as a neglected tropical disease and global health issue, very little is known of the mechanism of action of snake venom and intrinsic toxic/lethal contribution. The scientific foundation for our study is that a specific combination of snake venom toxins plays a critical role in snakebites by causing necrosis, coagulopathy, severe hemorrhaging and eventually death. Inasmuch as individual toxins have been evaluated, synergistic mechanism for snake venom pathology are lacking and warranted for antidote development. We hypothesize that precise toxicity evaluation and snake venom analytics of crude/fractionated venom and recombinant toxins will provide a model in which priority can be given to the most toxic venom component(s). To test our hypothesis, we will address (3) Specific Aims: Specific Aim 1: To profile snake venom composition, combinational assessment and intrinsic toxicity. We will purify and characterize snake venom of Crotalid snake species. Characterization will include profiling snake venom to identify the toxins in each fraction and abundance. Students will investigate the mechanism of action different combinations of venom fractions. We will characterize the lethal concentrations/doses of each venom fraction on HUVEC cells, blood coagulation, and will validate the toxicity score for each fraction using an in vivo lethality mouse model. Specific Aim 2: To produce recombinant venom peptides. We will clone these toxins from our collection of cDNA libraries and will generate plasmids and transform competent E.coli cells for protein expression and purification. Development of methods to produce recombinant toxins is essential for antivenom development and investigating toxicity and targets for medically relevant venom peptide. These studies will also be essential to use toxins in solving structures to venom toxins or structures in complexes with known targets. Specific Aim 3: To identify snake venom biomarkers in plasma exosomes. We will explore the “venom-reactome” using novel Evtrap technology and proteomics in discovery-based analysis of snake venom biomarkers from mouse plasma exosomes. Comprehensive snake venom biomarker analysis will shed new light on the pathophysiology, metabolic, biological, cellular and immunological responses to snake venom.