东方巴贝斯虫对宿主特异性致病的差异机制研究

寄生虫宿主差异性机制一直是寄生虫学研究的热点。巴贝斯虫是红细胞内专性寄生虫，大多数巴贝斯虫具有宿主特异性。我们前期研究发现牛巴贝斯虫可以感染黄牛和水牛，而东方巴贝斯虫仅感染水牛，这一差异为我们寻找巴贝斯虫对宿主特异性致病机制提供了条件。为此本研究以仅感染水牛的东方巴贝斯虫为研究对象，一方面建立牛巴贝斯虫基因组文库，将文库通过CRISPR/Cas9转入东方巴贝斯虫中，筛选可在黄牛红细胞中稳定培养的虫株从而获得导致宿主特异性致病的关键因子；另一方面通过建立红细胞cDNA文库，用全虫抗原孵育文库筛选特异的红细胞受体，利用Far-western钓取与红细胞互作的虫体抗原，通过双分子荧光系统、粘附和抑制实验、红细胞花环实验等验证虫体蛋白在虫体感染水牛过程中的作用。从而鉴定东方巴贝斯虫入侵水牛红细胞的关键虫体蛋白和宿主受体，阐明巴贝斯虫对宿主特异性致病的机制，从而为揭示寄生虫宿主特异性的机制提供依据。

The molecular mechanism that determines host specific pathogenesis is always a hot topic in the parasitology research field. Babesia is an apicomplexan parasite that only infected RBCs. There are over 100 species of Babesia have since been identified from livestock, wild and domestic vertebrate animals and occasionally humans. However, most Babesia species can only infect specific host, such as Babesia orientalis only infects water buffalo while Babesia bovis infects both water buffalo and cattle. The host specificity of B. orientalis and B. bovis provide convenience to study the mechanism of why Babesia can only obligated infect specific host RBC. Based on the work from our group and others, we select B. orientalis as study object. In order to identify RBC receptors, a water buffalo RBC cDNA library will be established and screened by using the lysis of B. orientalis. Far-western blot will be performed to identify parasite receptors which interact with the RBC ligand. In order to study the characteristic and functions of the parasite receptors, LC/MS, biomolecular fluorescence complementation (BiFC), adhesion and inhibition assay will be performed. On the other hand, we will establish a B. bovis genome library. The library will be transfer into B. orientalis by using CRISPR/Cas9, and the parasites will be cultured in water buffalo RBC system and cattle RBC system, respectively. In order to identify key factors of the specific pathogenesis of Babesia, the colonies which can be stably cultured in cattle RBC will be selected and subjected to genome sequencing. The biological function of the identified parasite proteins and RBC receptors will be characterized. This study will systematically identified parasite proteins and RBC receptors which play key role during Babesia specific recognize host RBCs. Results from this study has significant implications in the molecular mechanisms of parasites host specificity.