基于无细胞体外体系和活性蛋白质谱技术的天然产物ε-聚赖氨酸抑制烟草花叶病毒的作用靶点研究

ε-聚赖氨酸（ε-PL）是从项目组开发的特色抗病毒生物制剂嘧肽霉素中分离得到的一种高效抑制烟草花叶病毒（TMV）的活性组分，其关键分子作用机制尚不清楚。本研究拟基于麦胚抽提物（wheat germ extract）以及RdRp assay两种无细胞体外体系分别揭示ε-PL对TMV蛋白质翻译，复制复合体的形成以及核酸RNA合成的关键作用；活性蛋白质谱技术（activity-based protein profiling, ABPP）是能够在复杂的生物样品中标记、检测、分离靶标蛋白质的新技术。本研究拟基于ABPP技术构建活性探针，采用液相色谱串联质谱法明确与ε-PL结合的靶标病毒或宿主蛋白质。利用基因敲除的拟南芥突变体以及病毒诱导基因沉默（VIGS）技术对选取的关键靶基因进行分子生物学功能验证。本项目将为阐明ε-PL抑制植物病毒分子作用机制以及高通量筛选抑制植物病毒的新化合物提供重要理论依。

ε-poly-L-lysine (ε-PL) is a kind of highly effective anti-viral compound against tobacco mosaic virus (TMV), which was isolated from Cytosinpeptidemycin, an antiviral biological agent developed by our research group, but the underling molecular mode of action of ε-PL remains to be clarified. In this study, the effects of ε-PL on TMV protein translation, formation of viral replication complex and viral RNA synthesis will be elucidated using cell-free in vitro systems of wheat germ extract and RdRp assay. Activity based protein profiling (ABPP) is a new technology that can label, detect and separate target proteins in complex biological samples. In this study, the target virus or host proteins that can interact with ε-PL will be pulled down by active probe using ABPP technique and identified by Liquid chromatography-tandem mass spectrometry. Furthermore, the functions of the identified proteins will be verified using gene knockout Arabidopsis and virus induced gene silenced (VIGS) plants. This project will provide important theoretical basis for elucidating the molecular mode of action for ε-PL on plant virus and high-throughput screening of effective and more specific antiviral compounds.