益智仁多糖抗猪流行性腹泻病毒的作用与机制研究

Porcine epidemic diarrhea virus (PEDV) is one of the most important pathogens which is endangering swine industry in China. Thus, the study of anti-PEDV drug is very important to the clinic. In preliminary work, we studied the effect of the polysaccharides from the alpiniae oxyphyllae fructus on PEDV. The results showed the polysaccharides significantly decreased the adsorption, invasion, and replication, which indicated the polysaccharides inhibited the reproduction of PEDV. Therefore, in this project, we expect to harvest active ingredients from the polysaccharides and to study the inhibition mechanism of the ingredients on PEDV reproduction. The S protein, RNA-dependent RNA polymerase, transcription-regulating sequences of PEDV is significant to its reproduction. Hence, the molecular mechanisms of the inhibition effect on PEDV adsorption, invasion, and replication will be studied from these aspects by laser scanning confocal, co-immunoprecipitation, western blot, northern blot, real-time RT-PCR, and RNA-pull down techniques. The aim of this project is to illuminate the molecular mechanism of the inhibition on PEDV reproduction of alpiniae oxyphyllae fructus polysaccharides, and to provide references of the anti-PEDV drug development and the antiviral study of polysaccharides from traditional Chinese medicine.

猪流行性腹泻病毒（PEDV）是严重危害我国养猪业的病原之一，研究抗PEDV药物具有重要的临床意义。在前期工作中，课题组初步研究了益智仁总多糖对PEDV的作用，结果表明益智仁总多糖显著地抑制了PEDV的吸附、入侵和复制，具有抗PEDV增殖的作用。因此，本项目拟从益智仁总多糖中分离出具有抗PEDV作用的活性部位，并研究其抑制病毒增殖的机制。PEDV的S蛋白、RNA依赖的RNA聚合酶、转录调控序列是PEDV增殖过程中的重要调控因素，因而本项目拟从这些方面及其相关因素入手，采用激光共聚焦、免疫共沉淀、western blot、northern blot、real-time RT-PCR和RNA pull-down等技术研究益智仁多糖抑制PEDV吸附、入侵和复制的分子机制。旨在阐明益智仁多糖抑制PEDV增殖的机制，为抗PEDV药物的研发提供参考，并为中药多糖抗病毒的研究提供借鉴。

猪流行性腹泻病毒（PEDV）是严重危害我国养猪业的病原之一，目前临床上缺乏抗PEDV的特效药，因此研究抗PEDV药物具有重要的临床意义。本项目主要开展了益智仁多糖（AOFP）的抗PEDV作用机制研究。首先，对AOFP进行了分离纯化，共获得三个活性部位（AOFP1、AOFP2和AOFP3）。同时，对AOFP1、AOFP2和AOFP3进行了结构解析，并研究了其免疫调节和抗PEDV作用机制。结果表明，AOFP1和AOFP2没有表现出明显的抗PEDV活性，却表现出了免疫调节的作用：AOFP1能显著促进淋巴细胞的增殖与活化，调控Th1/2免疫反应，并增强特异性免疫反应；AOFP2能促进巨噬细胞的增殖与活化，能同时诱导巨噬细胞的M1和M2极化。AOFP3表现出了明显的抗PEDV作用：AOFP3影响了PEDV S蛋白与宿主细胞pAPN蛋白的结合，从而竞争性地抑制了PEDV的吸附；AOFP3影响了宿主细胞内胆固醇的合成，从而抑制了PEDV的入侵；AOFP3通过影响PEDV RdRp与病毒3'UTR的结合抑制了PEDV RdRp的活性，并且降低了宿主细胞hnRNP A1的合成，从而抑制了PEDV的复制。本项目旨在阐明益智仁多糖抗PEDV的作用机制，并为抗PEDV药物研发提供参考。本项目作为第一资助已发表SCI论文4篇，作为第二或第四资助已发表论文4篇；申报发明专利1项；参加学术会议1次；培养研究生3名。