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

摘要 该项目的总体目标是让本科生和研究生参与创新研究， 分子毒理学有可能有助于我们对蛇的理解取得重要进展， 毒液病理学和基于抗体的新疗法的发展。据世卫组织统计， 据估计，到2020年，全世界将有125，000例新的死亡报告是由于蛇的毒液。在 尽管它对于蛇咬伤毒液蛰入作为一种被忽视的热带疾病和全球健康问题的重要性， 人们对蛇毒的作用机制和内在毒性/致命作用知之甚少。的 我们研究的科学基础是，蛇毒毒素的特定组合在 被蛇咬伤后，会引起坏死、凝血功能障碍、严重水肿，最终死亡。只要 个别毒素已被评估，缺乏蛇毒病理学的协同机制， 研发解毒剂我们假设精确的毒性评估和蛇毒 粗/分级毒液和重组毒素的分析将提供一个模型， 可以给予毒性最强的毒液成分。为了验证我们的假设，我们将讨论（3）具体的 目的：具体目的1：分析蛇毒成分、组合评估和内在 毒性我们将纯化和表征响尾蛇蛇毒。表征将包括 分析蛇毒，以确定毒素的每一个部分和丰度。学生将调查 毒液组分的不同组合的作用机制。我们将描述致命的 在HUVEC细胞上的每种毒液级分的浓度/剂量、血液凝固，并将验证毒性 使用体内致死小鼠模型对每个级分进行评分。具体目标2：生产重组体 毒液肽我们将从我们收集的cDNA文库中克隆这些毒素， 并转化感受态大肠杆菌细胞用于蛋白质表达和纯化。制定方法， 生产重组毒素对于抗蛇毒血清的开发和研究毒性和靶点至关重要。 医学上相关的毒液肽这些研究也将是必不可少的使用毒素在解决结构， 毒液毒素或与已知目标复合的结构。具体目标3：鉴别蛇毒 血浆外泌体中的生物标志物。我们将使用新的Evtrap技术探索“毒液反应组”， 蛋白质组学在小鼠血浆外泌体蛇毒生物标志物的发现分析中的应用。 全面的蛇毒生物标志物分析将揭示新的光的病理生理学，代谢， 对蛇毒的生物、细胞和免疫反应。

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.