羊口疮病毒Bcl-2样蛋白ORFV125与宿主细胞互作蛋白的筛选及其功能域定位

Orf is a zoonotic disease caused by Orf virus and endemic in recent years, and raise serious security concerns to world's public health. ORFV encodes Bcl-2-like protein ORFV125, sequence analysis showed that ORFV125 is not only has Bcl-2 homologous domain, but also 3D structurally similar to the Bcl-2 protein family, the Bcl-2 protein is known to be a key protein to control cell apoptosis. Therefore ORFV125 may act in a Bcl-2 like manner to control apoptosis, but the necessary studies aimed at dissecting it's interactions with cellular proteins and its function domain site in details have not been carried out. This proposal intends to screen the host proteins that interacted with ORFV125 by using yeast two hybrid, immune histochemistry and conjunction biology. After that the interaction domain will be identified using surface plasma resonance and in vitro interactions experiment. Molecular simulation, co-immunoprecipitation and bioinformatics methods were used to verify the key amino acids in protein interaction, which will resolves ORFV125 protein function and the molecular basis that interacted with the host cellular protein, and lay the foundation for the study of the molecular mechanisms of apoptosis of ORFV encoding apoptotic regulation gene.

羊口疮是由羊口疮病毒(Orf virus,ORFV)引起的一种人兽共患性烈性传染病，近年该病流行广泛，对人类健康及公共卫生造成严重的危害。ORFV编码Bcl-2样蛋白ORFV125，分析显示ORFV125的3D结构及其结构域与Bcl-2家族蛋白高度相似，而Bcl-2家族蛋白是转导细胞凋亡信号的关键分子，作为Bcl-2样蛋白，ORFV125的功能性结构域及其与宿主细胞互作的分子机制尚不清楚。因此本研究拟采用酵母双杂交、免疫组化及生物学等技术从羊睾丸细胞cDNA文库中筛选并鉴定出与其相互作用的宿主细胞蛋白；等离子表面共振(SPR)及体外交互实验明确ORFV125蛋白功能性结构域；分子模拟、免疫共沉淀及生物信息学等方法确定ORFV125蛋白中决定互作活性的关键氨基酸位点，从而解析ORFV125蛋白功能及其与宿主细胞互作的分子基础，为研究ORFV编码的凋亡调节基因调控细胞凋亡的分子机制奠定基础。

羊口疮是由羊口疮病毒(Orf virus,ORFV)引起的一种人兽共患性烈性传染病，近年该病流行广泛，对人类健康及公共卫生造成严重的危害。ORFV编码Bcl-2样蛋白ORFV125，分析显示ORFV125的3D结构及其结构域与Bcl-2家族蛋白高度相似，而Bcl-2家族蛋白是转导细胞凋亡信号的关键分子，作为Bcl-2样蛋白，ORFV125的功能性结构域及其与宿主细胞互作的分子机制尚不清楚。因此本研究采用酵母双杂交、免疫组化及生物学等技术从羊睾丸细胞cDNA文库中筛选并鉴定出与其相互作用的宿主细胞蛋白cytochrome b (cytb) gene，GUCY2C，BIRC5，GTF3C6和SERBP1；免疫共沉淀及生物信息学等方法确定ORFV125蛋白中决定互作活性的关键氨基酸位点，从而解析ORFV125蛋白功能及其与宿主细胞互作的分子基础，为研究ORFV编码的凋亡调节基因调控细胞凋亡的分子机制奠定基础。