田鼠巴贝斯虫外泌体的分离鉴定和功能研究

Babesia microti is one of the most important tick-borne causative agent that infects human and multiple animals. Babesia microti mainly survive in the red blood cells of corresponding hosts. At present, the prevention and control of Babesiosis still lacks efficient vaccines and drugs, hence it is urgent to understand the parasite-host interaction mechanism and develop new prevention and cure pathways. Exosomes are membrane-like vesicles that carry a variety of bioactive substances, such as proteins, RNA and lipids. Various functions of exosomes have been reported including immunomodulation, cell migration, and cell-to-cell communication. Presently, a variety of parasites exosomes have been identified to play an important role in the parasite-host interaction, however the exosomes of Babesia still remains unknown. In this research, the isolation, morphological identification and protective immunity of Babesia microti exosomes (both in vivo and in vitro) are performed. Subsequently, the proteins and microRNAs of Babesia microti exosomes are deciphered using the proteomics and the transcriptomics techniques. Then some of the identified exosome proteins such as proteins involved in the invasion process and key enzymes are cloned and their functions are analyzed. The sequences of the exosome microRNAs are also confirmed and the diagnostic values are estimated. This study lays the foundation for the further investigation in Babesia parasite-host interaction mechanism.

田鼠巴贝斯虫是最重要的人巴贝斯虫病病原，经蜱传播，寄生于宿主红细胞中。目前，防控巴贝斯虫病仍缺少疫苗和高效药物，了解虫体与宿主的互作机制，研发新型防治技术已刻不容缓。外泌体是一种可携带蛋白质、RNA和脂质等多种生物活性物质的膜性小囊泡，可以介导免疫调节、细胞迁移和细胞间通讯等功能。目前已有多种寄生虫外泌体在寄生虫-宿主互作中发挥重要作用的研究被报道，然而巴贝斯虫外泌体研究仍为空白。本研究将以田鼠巴贝斯虫外泌体为研究对象，开展宿主体内感染和体外培养两个体系条件下，田鼠巴贝斯虫外泌体的分离、形态鉴定及免疫保护力研究，分别应用蛋白组学和转录组学技术解析田鼠巴贝斯虫外泌体的蛋白质和microRNA组分，而后对田鼠巴贝斯虫外泌体中与虫体入侵相关的蛋白或关键酶分子基因克隆并分析其功能，鉴定外泌体microRNA分子的序列并评估其诊断价值。这项研究为深入解析巴贝斯虫与宿主互作机制提供基础。

田鼠巴贝斯虫是最重要的人巴贝斯虫病病原，经蜱传播，寄生于宿主红细胞中。目前，防控巴贝斯虫病仍缺少疫苗和高效药物，了解虫体与宿主的互作机制，研发新型防治技术已刻不容缓。外泌体是一种可携带蛋白质、RNA和脂质等多种生物活性物质的膜性小囊泡，可以介导免疫调节、细胞迁移和细胞间通讯等功能。目前已有多种寄生虫外泌体在寄生虫-宿主互作中发挥重要作用的研究被报道，然而巴贝斯虫外泌体研究仍为空白。本研究以田鼠巴贝斯虫外泌体为研究对象，建立B. microti 短期体外培养体系，通过梯度超速离心，结合标准试剂盒等方法分别收集外泌体，并进行外泌体形态学、免疫特征等鉴定；通过蛋白组学、转录组学以及免疫学等方法，对收集到的B. microti外泌体进行蛋白成分和miRNA鉴定以及生物信息学分析，阐明B. microti 外泌体中重要的蛋白分子和 microRNA 分子的种类。通过以上蛋白组学分析中得到B. microti外泌体中重要蛋白分子PrxQ蛋白，对其进行基因克隆，初步鉴定其表达谱和抗氧化功能，进而初步阐明外泌体重要组分的作用。这项研究本项目填补了目前关于巴贝斯虫外泌体研究的空白，为深入解析巴贝斯虫与宿主互作机制奠定基础，也为其它顶复门原虫的防治提供参考。本项目已以第一标注发表SCI论文1篇，支持培养毕业研究生3名，其中博士1名，硕士2名。