基于宿主范围基因ANK的山羊痘病毒跨物种感染的分子机制研究

Goatpox virus virulence is the strongest in animal poxvirus, and the choice of the host is very strict. But It has been reported that a sheeppox or goatpox was cross infected goats or sheeps, even human in practical production. ANK gene family is goatpox virus host range gene. But its molecular mechanism is unclear as host range factor. This study intended to goat pox virus strains in xinjiang as the research object, and each gene of ANK family was cloned respectively, and recombinant virus of missing the genes and their different structure domain were built and screened, and the recombinant virus were infected in different animal cells, and their respective function or function domain were determine and verified by iRNA interference or gene over expression. The influence of host innate immunity factor such as tumor necrosis factor, apoptosis factor were detect by fluorescent quantitative PCR and western blot method and determined the mechanism of action of ANK. Extract the goat pox virus cell cultures total RNA, construct yeast cDNA library and ANK bait plasmid, double hybrid method of screening with yeast ANK the interactions of proteins, and CO - IP, the PULL - DOWM and immune methods of positioning, fluorescence quantitative appraisal and study its biological function, for further research ANK protein molecular mechanism of action, understand the goatpox virus transmission way, provide new ideas and methods for preventive measures.

羊痘病毒是毒力最强的动物痘病毒，对宿主的选择非常严格。但是实际生产中确实有同时感染绵羊和山羊，甚至感染人的报道。ANK基因家族是羊痘病毒的宿主范围基因。但其作用的分子机理还不清楚。本研究拟以山羊痘病毒新疆株为研究对象，分别克隆ANK的各基因，构建并筛选该基因及其不同结构域缺失的重组病毒，分别感染不同动物细胞，确定各自的功能或功能域，并用iRNA干扰或基因过表达法验证。用荧光定量PCR和免疫印迹法检测各病毒感染对干扰素、细胞凋亡因子、肿瘤坏死因子等宿主先天性免疫因子的影响，确定ANK的作用机制。提取感染山羊痘病毒的山羊细胞培养物总RNA,构建酵母cDNA文库和ANK的诱饵质粒，用酵母双杂交的方法筛选ANK的互作蛋白，并用CO-IP、PULL-DOWM和免疫共定位、荧光定量等方法鉴定和研究其生物学功能，为深入研究ANK蛋白分子作用机制，了解羊痘病毒跨物种感染机理，制定高效预防措施奠定理论基础。

为了确定羊痘病毒ANK基因与其宿主范围选择的关系，本课题对6株并羊痘病毒的5个ANK基因进行了克隆测序，并进行了核苷酸和氨基酸序列的对比分析、遗传进化分析和和病毒效价测定；构建了所有30个ANK基因缺失重组表达载体，并成功纯化3株基因缺失重组病毒，对羊睾丸细胞、vero细胞和293T细胞的感染实验证明ANK基因缺失病毒在vero细胞的感染性明显低于原病毒，对羊睾丸细胞和293T细胞的感染性与原病没有著差异（在羊源细胞上生长良好，在293T细胞上不生长）；分别以SS株、M1株和TS株分别感染羊睾丸细胞、vero和293T细胞，以siRNA沉默ANK141.2基因后，qPCR检测转录水平上羊痘病毒三个心蛋白P32、RPO18IMV 膜蛋白和IMV膜蛋白表达情况，结果三个毒株分别在三种细胞中的转录水平为羊睾丸细胞>vero细胞>293T细胞，沉默与不处理的结果对3种病毒株结果不一致，有的显著升高，有的显著降低，有的没有变化；以siRNA沉默ANK141.2基因后，转录组学测序结果也是对病毒各蛋白的影响有升高、降低和无变化三种，说明ANK基因对羊痘病毒宿主的选择有一定影响；以siRNA沉默ANK145基因后，检测了其对宿主细胞免疫因子的影响，结果该基因沉默后会引起肿瘤坏死因子和γ-干扰素的表达显著增加;用His-pull-down方法筛选到ANK140基因与宿主细胞一个蛋白互，且作用位点位于基因C末端，通过课题的研究，初步了解了羊痘病毒ANK基因家族的生物学特性和部分功能，初步确定AHK基因对病毒感染宿主细胞有一定的作用，为深入研究其分子机制奠定基础。. 依托其课题培养硕士研究3名，培养本科生14名，发表学术论文9篇，其中SCI收录1篇，4篇学术论文发表在北大核心期刊。申报专利1项，进入实审阶段。