细胞吞噬相关miRNA在对虾抗白斑综合征病毒中的作用

Studies have shown that miRNAs play a key regulatory role in the virus-host interactions within the host cell, involved in a number of immune cell signaling pathways. We use phagocytic inhibitor to stimulate shrimp, and then screen differentially expressed miRNA by detecting the change of miRNA chip comparing with the miRNA of normal shrimp, thus obtained phagocytosis-related miRNA. Then we test which miRNA can regulate the phagocytosis and verify its antiviral function. The degradome sequencing and RNAi methods will be used for identify the target gene which miRNA may function and further analysis of the corresponding signaling pathways. Cloning of several key genes regulating the phagocytosis of WSSV and combine the screening work of target gene regulated by miRNA with the former study organically, then analysis the signal pathway regulated by miRNA. We explore the application prospect of using RNA to control shrimp viral disease through glucan coated method and screen for small molecules and polysaccharide as antiviral agents using the obtained target gene. This project has important scientific significance for the study of the mechanism of miRNA regulate cellular immunity in shrimp and explore pathways to protect shrimp against virus disease.

研究表明，宿主细胞内的miRNAs在病毒与宿主的相互作用中起关键的调节作用，参与许多免疫细胞信号传导途径。我们用细胞吞噬的抑制剂刺激对虾，利用芯片检测miRNA的变化情况，与正常对虾的miRNA作比较，筛选差异表达的miRNA，从而得到与细胞吞噬相关的miRNA。然后对miRNA进行调控细胞吞噬能力的检测，对确定具有调控功能的miRNA进行抗病毒功能验证。采用了降解组测序、RNAi等方法寻找miRNA可能作用的靶基因，进一步分析相应的信号通路。克隆调控对虾细胞吞噬WSSV的几个关键基因，将前期研究结果和miRNA调控的靶基因筛选工作有机地结合起来，分析miRNA调控的信号通路。利用葡聚糖包被的方法探索RNA调控对虾抗病毒的应用前景，用获得的靶基因进行小分子和多糖抗病毒制剂的筛选。本项目对于研究对虾miRNA调控细胞免疫机制和探寻免疫对虾抗病毒病的途径具有重要的科学意义。

越来越多的研究表明，细胞内的miRNA在病毒与宿主的相互作用中起关键的调节作用。利用microRNA芯片筛选得到与日本对虾血细胞吞噬WSSV相关的microRNA，通过对这些表达量发生显著变化的microRNA进行Northern Blot验证，发现这些microRNA在感染WSSV之后的确有和芯片结果相同的变化趋势。 日本对虾特有的microRNA-S5在病毒或弧菌感染后，其表达量显著增加。microRNA-S5能调控细胞吞噬和细胞凋亡，而WSSV可利用对虾microRNA-S5，通过控制细胞凋亡/细胞吞噬，来逃逸非特异性免疫系统的捕捉，帮助病毒复制。microRNA-100在WSSV或溶藻弧菌感染后，表达量上调，microRNA-100可通过调节凋亡作用、吞噬作用和酚氧化酶活性，来提升对虾抗弧菌免疫，并在一定程度上影响了WSSV感染的进程。进行了与细胞吞噬相关的MCM7基因克隆和功能验证，研究结果显示MCM7对血细胞吞噬具有正向调控作用，并且显著对虾的抗病毒免疫。本项目对于研究对虾microRNA的细胞免疫调控机制和探寻免疫对虾抗病的途径具有十分重要的科学意义。