GCRV内衣壳的自组装及与细胞相互作用机制研究

In order to explore the assembly mechanism of core particles of the grass carp reovirus (GCRV) and their interactions with the host cells during infection, baculovirus expression system will be used to express core proteins VP1, VP3 and VP6 for self-assembly core-like particles of GCRV in vitro. The core protein VP6 fused to biotin acceptor peptide (BAP) will be specifically biotinylated in the presence of biotin ligase (BirA) which is co-expressed with core proteins. Streptavidin-conjugated quantum dots (SA-QDs) will be specifically integrated to the biotinylated core-like particles following one-step reaction in vitro. QDs-labeled core-like particles will be re-coated with baculovirus-expressed outer-capsid proteins VP5 and VP7 in vitro and resulted in QDs-labeled GCRV capsid-like particles. The infection mechanism of cores will be studied by investigating interactions between QDs-labeled GCRV capsid-like particles and host cells. This study will not only contribute to understanding the assembly mechanism of the doubled-shelled capsid and the interaction mechanism between core particles and host cells, but also provide relative scientific evidence to establish reliable diagnostic methods and develop effective prophylactic vaccines for GCRV.

为了探究草鱼呼肠孤病毒内衣壳的组装及其在感染过程中与细胞相互作用的机制，本项目拟采用杆状病毒表达系统共表达GCRV内衣壳蛋白VP1、VP3与VP6，以实现类内衣壳的体外自组装。通过在内衣壳蛋白VP6上融合表达受体肽标签，在共表达的生物素连接酶BirA的作用下对类内衣壳进行特异性生物素化修饰。此外，通过体外一步结合反应实现量子点对类内衣壳的位点特异性标记，然后借助GCRV外衣壳蛋白（VP5/VP7）的体外重构包裹量子点标记的类内衣壳，以重构GCRV类衣壳颗粒。在此基础上，进行量子点标记GCRV类衣壳颗粒与细胞相互作用的具体过程及作用机制研究。本项目不仅对揭示GCRV的双层衣壳组装机制和内衣壳在细胞内的感染机制具有重要意义,而且能够为建立GCRV可靠的诊断方法和研制有效的预防疫苗提供相关的科学依据。

草鱼呼肠孤病毒(GCRV)是呼肠孤病毒科水生呼肠孤病毒属成员，现有资料证表明GCRV为水生呼肠孤病毒属中致病力最强的毒株。为了探讨草鱼呼肠孤病毒内衣壳的组装及其在感染过程中与细胞相互作用的机制，本研究采用杆状病毒表达系统进行了GCRV内衣壳蛋白VP1、VP3与VP6的共表达，并实现了类内衣壳的体外自组装。首先，在内衣壳蛋白VP6上融合表达受体肽标签，在共表达的生物素连接酶BirA的作用下实现了对类内衣壳特异性生物素化修饰。此外，通过体外一步结合反应，实现了量子点对类内衣壳蛋白的位点特异性标记。然后采用GCRV外衣壳蛋白VP5 和VP7的体外重构包裹量子点标记的类内衣壳，实验证实获得重构GCRV类衣壳颗粒。在此基础上，进行了量子点标记GCRV类衣壳颗粒与细胞相互作用的具体过程及作用机制研究。其研究结果为进一步揭示GCRV组装与感染机制提供了重要的实验依据。