SGER: Novel Antimicrobial and Antiviral Properties of the Hok Killer Protein

本研究的目的是研究Hok杀伤肽作为一种全新的抗生素和抗病毒系统的应用。Hok肽作为一种广泛的、非特异性的抗寄生虫药物的潜在功效将通过测试其对相关细菌、真菌和噬菌体的活性来确定。由于Hok肽的细胞外抗生素应用在很大程度上未经测试，因此本研究具有高度的新颖性和探索性。然而，抗生素的想法是合理的，因为迄今为止几乎所有测试的细菌都容易被Hok在细胞内表达时杀死，而且PI最近表明，当Hok蛋白在大肠杆菌细胞中表达时，可以排除噬菌体T4（一种破坏细菌的病毒）。为了研究对人类病原体的广泛有效性，Hok肽将使用重组细菌过表达，使用凝胶过滤和反相高效液相色谱纯化，并通过快速原子轰击质谱进行鉴定。使用纯化的肽，将进行抗菌试验，以确定肽是否能够减缓或防止细菌和真菌的生长。为了阻止病毒在动物细胞中的繁殖，类似于Hok蛋白通过在病毒繁殖之前杀死宿主来排除T4噬菌体的方式，将开发一种用于动物细胞的通用自杀系统来对抗基于病毒的疾病。这将需要杀手蛋白在多种动物细胞中的可控表达和活性。首先，我们将利用杆状病毒转移载体pVL1392在多面蛋白启动子下的昆虫细胞中表达Hok，来评估Hok对昆虫细胞（作为代表性动物细胞）的杀伤能力。＊＊＊

9615938 Wood The goal of this proposed research is to investigate the use of the Hok killer peptide as a completely new antibiotic and antiviral system. The potential efficacy of the Hok peptide as a broad, non-specific, antiparasitic drug will be determined by testing its activity against relevant bacteria, fungi, and phage. This research highly novel and exploratory since the extracellular antibiotic application of Hok peptide is largely untested. However, the antibiotic idea is sound since almost all bacteria tested to date are susceptible to killing by Hok when it is expressed intracellularly, and the PI has recently shown that phage T4 (a virus that destroys bacteria) can be excluded when the Hok protein is expressed in E. coli cells. In order to investigate the broad range effectiveness against human pathogens, the Hok peptide will be overexpressed using recombinant bacteria, purified using gel filtration and reverse-phase, high- performance-liquid chromatography, and identified through fast-atom-bombardment mass spectroscopy. Using the purified peptide, antimicrobial assays will be performed to determine if the peptide is capable of slowing or preventing bacterial and fungal growth. To thwart the propagation of virus in animal cells in a manner analogous to how the Hok protein excludes T4 phage by killing the host before the virus can propagate, a general suicide system for animal cells will be developed to combat virus-based diseases. This will require controllable expression of a killer protein nd activity in a wide variety of animal cells. Initially, the ability of Hok to kill insect cells (as representative animal cells) will be evaluated by using the baculovirus transfer vector pVL1392 for Hok expression in insect cells under the polyhedrin promoter. ***