小菜蛾抗菌肽抗流感病毒作用及其机制研究

Antimicrobial peptides (AMPs) are widely expressed in nature with the characteristic of small molecular mass. AMPs have a variety of interesting biological functions including antibacterial and antiviral activities. AMPs are effective at killing both gram-positive and gram-negative bacteria by disrupting bacterial membranes or translocating across both the cellular and nuclear membranes to act on the inside targets which rarely induces resistance. However few reports about molecular mechanism of anti-virus activity were seen in the AMPs research. A growing body of research has focused on how to improve the antimicrobial activity and stability of AMPs, but the lack of a cost-effective means of mass production remains one major obstacle to their further development as therapeutics. In previous study, we established an intein-induced expression system which could produce AMPs in a facile and efficient way. We demonstrated the antibacterial activity and the antibacterial mechanism of pxCECA1, also found that pxCECA1 could suppress influenza virus in vitro. In this project, we intend to clarify the pxCECA1’s anti-influenza virus activity through plaque assay and gene expression analysis. On the other hand, we try to demonstrate the mechanism and find the target site pxCECA1 acts on, hoping provide a theoretical basis for the development of AMPs like antibacterial and antiviral drugs.

抗菌肽是具有抗细菌、抗病毒等多种生物活性的小分子多肽，具有抗菌谱广、作用迅速、不易诱导产生耐药性等特点，是潜在的新一代抗菌药物。而其抗病毒的分子机制则未明确，尤其是天蚕素类抗菌肽抗流感病毒的活性和机制未见报道。我们之前以小菜蛾抗菌肽pxCECA1为研究对象，阐明了pxCECA1抗菌活性及其抗菌作用分子机制，并发现了其具有明显的体外抗流感病毒的活性。本课题计划根据流感病毒感染细胞的关键事件，提出pxCECA1抗流感病毒可能的作用机制；在病毒感染细胞的不同阶段，通过空斑抑制实验、病毒基因载量和表达分析观察病毒抑制情况，研究病毒增殖周期中关键因子的变化，寻找pxCECA1作用的靶位点，最终阐明其抗流感病毒的作用机制。本课题研究意义在于，通过明确pxCECA1抗流感病毒的活性及其作用机制，找到一种兼具抗菌及抗流感病毒活性的生物多肽，并为抗菌肽类及基于以上作用机制的药物研发提供理论基础。

抗菌肽是具有抗细菌、抗病毒等多种生物活性的小分子多肽，具有抗菌谱广、作用迅速、不易诱导产生耐药性等特点，是潜在的新一代抗菌药物。而其抗病毒的分子机制则未明确，尤其是天蚕素类抗菌肽抗流感病毒的活性和机制未见报道。本课题以小菜蛾抗菌肽pxCECA1为研究对象，构建了一种能够高效表达具有生物活性的小分子抗菌肽的表达系统。我们进一步发现了pxCECA1具有明显的体外抗流感病毒的活性，并提出pxCECA1抗流感病毒可能的作用机制；在病毒感染细胞的不同阶段，通过空斑抑制实验、目的蛋白对受感染细胞的保护作用来分析观察病毒抑制情况，研究病毒增殖周期中关键因子的变化，寻找pxCECA1作用的靶位点，并证实pxCECA1并未通过直接作用于病毒包膜以及血凝素和神经氨酸酶的活性来抑制流感病毒。本课题研究意义在于，找到大量稳定制备抗菌肽的方法，通过明确pxCECA1抗流感病毒的活性及其作用机制，找到一种兼具抗菌及抗流感病毒活性的生物多肽，并为抗菌肽类及基于以上作用机制的药物研发提供理论基础。