东方巴贝斯虫AMA-1与宿主互作蛋白的筛选及其黏附机制研究

Babesia species, collectively known as piroplasms, are tick - transmitted apicomplexan parasites that infect wild and domestic animals worldwide. Bovine babesiosis was caused by many different Babesia speices. Babesia orientalis is one of them, which is the causative agent of babesiosis in water buffalo. It is viewed as one of the most important diseases of buffalo on central and southern parts of China, and recently was also reported to the north side of Yangtse river, and causes enormous economic losses. Babesia orientalis was transmitted by Rhipicephapus haemaphysaloides transovarially with the infected stage being adult tick. Because of the complicated life cycle and effective immune escape mechanism of Babesia orientalis, there is no effective prevention and control plan for it. As we know, apicomplexan parasites exhibit an unusual mechanism of host cell penetration. A central player in this process is the protein apical membrane antigen (AMA-1), which interacts with RONs on the extracytosolic side of the moving junction stage (MJ). Although essential for invasion, most of studies are focus on the precise invasion functional roles of AMA-1. The function and mechanism of AMA-1 interacted with proteins of red blood cell remains unknown. In this study, AMA-1 gene of Babesia orientalis was cloned, yeast two hybrid system was used to screen a water buffalo cDNA library, and identified the interacting partner of AMA-1 with red blood cells. Immunoprecipitation, immunofluorescence and pull - down method was used to confirm the interaction of Babesia orientalis AMA-1 with the proteins in red blood cells. The results of this study would not only elucidate the details of AMA-1 interactions with proteins on red blood cellof the MJ, but also provide the evidences for invasion function of apicomplexan.

牛的巴贝斯虫病是由多种巴贝斯虫寄生于牛体内引起的蜱传性血液原虫病，其中东方巴贝斯虫病在我国长江流域多省份均有流行，对畜牧业造成巨大的经济损失。由于巴贝斯虫复杂生活史和有效免疫逃避机制，至今缺乏有效的防控方案。巴贝斯虫如何侵入宿主红细胞并逃避其免疫机制一直是该病的研究热点。研究表明，顶膜抗原1(AMA-1)在入侵过程中与由棒状体分泌的3种RONs蛋白结合成复合物，从而促进移动连接环的形成，是虫体侵入宿主红细胞的关键作用蛋白。但AMA-1如何与宿主蛋白互作并协助虫体进入红细胞仍不清楚。为此本研究拟用东方巴贝斯虫AMA-1蛋白作诱饵，与水牛cDNA文库双杂交，筛选与AMA-1互作的宿主蛋白，并用免疫共沉淀、免疫荧光验证AMA-1与宿主蛋白的互作及互作蛋白细胞定位，初步阐明东方巴贝斯虫AMA-1协助虫体入侵的作用机理，从而为揭示其入侵宿主红细胞的机制奠定基础，并为顶复门寄生虫入侵机制提供理论依据。

牛的巴贝斯虫病是由多种巴贝斯虫寄生于牛体内引起的蜱传性血液原虫病，其中东方巴贝斯虫病在我国长江流域多省份均有流行，对畜牧业造成巨大的经济损失。由于巴贝斯虫复杂生活史和有效免疫逃避机制，至今缺乏有效的防控方案。巴贝斯虫如何侵入宿主红细胞一直是该病的研究热点。研究表明，顶膜抗原1(AMA-1)在入侵过程中与由棒状体分泌的3种RONs 蛋白结合成复合物，从而促进移动连接环的形成，是虫体侵入宿主红细胞的关键作用蛋白。为此本研究以东方巴贝斯虫为研究对象，分别克隆、表达东方巴贝斯虫AMA-1和RON2的蛋白，Far-Western blot实验和黏附ELISA实验等验证了BoRON2纯化肽段可与天然BoAMA-1互作，初步阐明东方巴贝斯虫AMA-1和RON2在虫体入侵过程中存在相互作用，可能是虫体入侵宿主红细胞的关键因子，从而为揭示虫体入侵宿主红细胞的机制奠定基础，为顶复门寄生虫入侵机制提供理论依据。本研究在进行BoAMA-1与BoRON2互作验证的同时，对可能与入侵相关的棒状体蛋白1和顶质体展开了深入的研究。克隆、表达BoRAP1，证明其在虫体中的存在、具有良好的免疫原性和反应原性；获得东方巴贝斯虫顶质体基因组全长，并进行详细注释。以上结果均发表SCI论文。综上所述，该项目已按照项目书计划完成了申请书和任务书中的研究内容，同时为后期研究工作的开展奠定了扎实的基础。