亲环蛋白A促进牛病毒性腹泻病毒复制的分子机制研究

牛病毒性腹泻是由牛病毒性腹泻病毒（BVDV）引起的一种烈性传染病。BVDV感染宿主后，病毒大量复制是其治病的直接原因，但其致病机制目前尚不清楚。前期研究发现亲环蛋白A（CyPA）特异性抑制剂环孢素A（CsA）和CyPA多抗抑制CyPA的活性后使BVDV复制能力显著降低。为了阐明CyPA调控BVDV复制的分子机制，本项目拟利用RNA干扰、CRISPR/Cas9 基因敲除和蛋白过表达技术调控CyPA的表达以明确CyPA对BVDV复制的影响；通过pull-down、免疫共沉淀、Western-blot等方法阐明与CyPA互作的病毒蛋白，构建与CyPA互作的BVDV蛋白的截短突变体，鉴定与CyPA互作的病毒蛋白的结构域；通过构建蛋白截短或点突变的CyPA突变体，确定与BVDV蛋白作用的CyPA功能区，从而揭示CyPA促进BVDV复制的分子机制，为研发BVDV新型疫苗和抗病毒药物提供理论依据。

Bovine Viral Diarrhea is an infectious disease caused by bovine viral diarrhea virus (BVDV). It is known that virus-host interaction plays a key role in the viral replication and pathogenesis and efficient replication of BVDV is necessary for the viral pathogenesis, therefore, understanding the mechanism of BVDV replication is helpful for the development of prevention and control strategies of BVD. Based on the previous study in our lab, we firstly found that inhibition of cyclophilin A (CyPA) by its specific inhibitor cyclosporine A (CsA) or anti-CyPA polyclonal antibody significantly suppressed the replication of BVDV in vitro, suggesting the positive role of CyPA in the viral replication. In the present project CyPA expression was modulated by RNAi ,CRISPR/Cas9 or protein overexpression to determine the effect of CyPA on the viral replication. To elucidate the regulatory mechanism of BVDV replication by CyPA, techniques such as pull-down, Co-immunoprecipitation and western-blot were used to explore the CyPA-interacting BVDV proteins.Further, the interaction domain of which was determined by analyzing the interaction between CyPA and truncated mutants of CyPA-interacting viral proteins.Further more, truncated mutants or point mutations of CyPA were constructed to analyze the functional domain of CyPA interacting with BVDV proteins. This study will provide a theoretical basis for the development of novel efficient vaccines and antiviral drugs against BVDV.