病毒性出血败血症病毒基质蛋白M诱导细胞凋亡的关键功能域鉴定及其作用机理研究

Viral Hemorrhagic Septicemia Virus (VHSV) is rhabdovirus which could affect over 80 marine and freshwater fish species. Apoptosis was involved in the infection of animal rhabdovirus, and there is close relationship between virus infection and apoptosis. It has been reported that matrix protein of animal rhabdovirus could induce apoptosis, however, the mechanism of matrix protein of fish rhabdovirus inducing apoptosis is still not clear.So, this project will observe the subcellular localization of matrix protein by constructing recombinant plasmid and indirect immunofluorescence, and the function of matrix protein inducing apoptosis using TUNEL, Annexin V-FITC/PI double staining and Flow Cytometry. In addition, the process and mechanism of matrix protein inducing apoptosis will be determined in the means of using apoptosis detection kit and measuring the expression level of apoptosis-related genes. We will focus on the identification of apoptosis-related functional domain of matrix protein using the method of truncated gene, point mutation and reverse genetics. It will help us to understand the process of virus infection and lay the theoretical foundation for the prevention of VHSV and drug development through studying the molecular mechanism of VHSV matrix protein inducing apoptosis and identifying related functional domain of matrix protein.

病毒性出血败血症病毒（VHSV）是一种能感染80多种海水和淡水鱼类的弹状病毒病原。弹状病毒在感染过程中会引起细胞凋亡现象的发生，且病毒感染与细胞凋亡之间存在着密切的联系。有研究表明动物弹状病毒的基质蛋白M能诱导细胞凋亡，然而有关鱼类弹状病毒特别是VHSV的M蛋白诱导细胞凋亡的机理却并不清楚。为此，本项目拟通过构建重组质粒和间接免疫荧光的方法观察M蛋白在细胞中的定位，并结合TUNEL、Annexin V/PI 双染法和流式细胞术等手段研究M蛋白诱导细胞凋亡的作用；进一步借助细胞凋亡检测试剂盒辅以凋亡相关基因表达水平检测等技术探明M蛋白诱导细胞凋亡的途径和机制；重点利用基因截短、点突变及反向遗传学手段确定M基因上与诱导细胞凋亡相关的关键功能域。通过探明VHSV的M蛋白诱导细胞凋亡的分子机制及关键功能域的确定，将有助于认识病毒感染的发生过程，为鱼类病毒性出血败血症防治和药物研制奠定理论基础。

病毒性出血败血症病毒（Viral haemorrhagic septicemia virus, VHSV）是一种能引起鱼类产生严重疾病的重要病毒病原，也是世界卫生组织要求必须申报的鱼类病原之一。VHSV属于弹状病毒科，它能感染80多种海水和淡水鱼类。迄今为止，人们已分别从北美、欧洲以及亚洲的日本和韩国分离到不同的VHSV菌株。自从1963年分离到这种病毒，VHSV已经对全球的海水、淡水养殖业带来了不可估量的巨大损失。随着全球性贸易的发展和海洋运输业的推进，VHSV将可能被带入新的区域，从而对其它的海水淡水鱼类造成影响。因此，对VHSV开展深入研究，以便于采取有效措施对病毒进行防治迫在眉睫，然而，人们对于VHSV在感染过程与宿主鱼类细胞的互作机制却还不明确。本项目主要研究了VHSV的M蛋白诱导鲤鱼上皮瘤细胞（EPC）发生凋亡的机理。项目完成了M基因的克隆和表达，制备了多克隆抗体，完成了VHSV的体外反向遗传拯救系统，并对M蛋白诱导细胞凋亡的机制进行了探索，揭示VHSV的M蛋白通过线粒体途径诱导EPC细胞凋亡。本项目的研究结果为探明VHS病毒与宿主互作机制及病毒防治打下了基础。