EtRON2蛋白与受体互作介导鸡球虫入侵机制的研究

Because of the mechanism of parasite and its interaction with host cell is little to know, we face a big problem in developing the novel anticoccidiosis drugs and molecular vaccine against avian coccidiosis. The previous study suggested that the RON2 was secreted and anchored to the surface of host cell when the parasite contacted with host cell, this process induced host F-actin polymerization, and helped the parasite invade into the host cell. So we hypothesized that the host F-actin polymerization was induced by the RON2 protein interaction with its receptor, which played an important role in the parasite invasion. Based these current results and hypotheses, in this study, we will study on the Eimeria tenella cell culture model, screening the host cell receptor which interact with EtRON2 and induce host F-actin polymerization using GST-pull down assays, immunoprecipitation, mass spectrometry and bioinformatics technique, the interaction between EtRON2 protein and its host receptor will be studied using sub-cellular localization technique. The mechanism of host cell F-actin polymerization induced by interaction between Et RON2 protein and receptor in the invasion of Eimeria tenella sporozoites, which will be studied using a series of biological technology, such as antibody inhibiting, gene regulation, immunohistochemistry and qRT-PCR, etc. Our results will indicate the host cell invasion mechanism by Eimeria tenella which mediated by interaction between EtRON2 protein and receptor, and it will provide the new way to explore the novel anticoccidiosis drug and vaccine against avian coccidiosis.

目前对鸡球虫与宿主细胞的互作机制缺乏了解，新型抗球虫药物及分子疫苗的研制面临巨大挑战。前期研究发现球虫黏附宿主细胞，可分泌RON2蛋白并锚定至宿主细胞表面，诱导宿主细胞F-actin聚集，推测RON2与宿主细胞表面受体互作启动宿主细胞F-actin聚集是虫体入侵的重要机制。基于此，本项目拟以柔嫩艾美耳球虫分泌蛋白EtRON2为研究对象，基于球虫体外细胞培养模型，利用GST-pull down、免疫共沉淀、质谱及生物信息学技术筛选与EtRON2蛋白互作且诱导宿主细胞F-actin聚集的受体；利用亚细胞共定位技术验证EtRON2蛋白与受体的互作；采用抗体阻断、基因调控、免疫组化及qRT-PCR等技术研究EtRON2及相应受体诱导宿主细胞F-actin聚集对虫体入侵的重要作用。预期结果将阐明经EtRON2与受体互作所介导的鸡球虫入侵机制，为新型抗球虫药物及疫苗的研制奠定理论基础。

目前对鸡球虫与宿主细胞的互作机制缺乏了解，新型抗球虫药物及分子疫苗的研制面临巨大挑战。前期研究发现球虫黏附宿主细胞，可分泌RON2蛋白并锚定至宿主细胞表面，诱导宿主细胞F-actin聚集，推测RON2与宿主细胞表面受体互作启动宿主细胞F-actin聚集是虫体入侵的重要机制。鉴于此，本项目紧密围绕研究目标和研究内容，顺利完成对EtRON2蛋白与受体互作介导鸡球虫入侵机制的相关研究：以柔嫩艾美耳球虫分泌蛋白EtRON2为研究对象，基于球虫体外细胞DF-1的培养模型，利用GST-pulldown、质谱及生物信息学技术筛选与EtRON2蛋白互作且诱导宿主细胞F-actin聚集的受体，利用亚细胞共定位技术验证EtRON2蛋白与受体的互作，采用抗体阻断、免疫组化及qRT-PCR等技术研究EtRON2及相应受体诱导宿主细胞F-actin聚集对虫体入侵的重要作用。结果共筛选获得置信度高于95%且肽段数大于2的潜在互作蛋白共计263个，其中位于宿主细胞膜表面且可诱导宿主细胞F-actin聚集的受体蛋白1个：Annexin A2；子孢子与DF-1细胞接触30分钟后，经过亚细胞定位技术可观察到EtRON2荧光信号与Annexin A2荧光信号发生重叠。间接证实其二者在子孢子对宿主细胞入侵过程中发生互作，成功完成了“筛选获得与EtRON2互作且诱导宿主细胞F-actin聚集的受体”的研究目标；分别利用EtRON2和Annexin A2特异性抗体对子孢子和宿主细胞进行作用，结果相比正常子孢子对正常DF-1细胞的入侵，宿主细胞F-actin的聚集作用被明显减弱，且子孢子对DF-1的入侵率明显降低。间接证实EtRON2与Annexin A2的互作诱导宿主细胞F-actin聚集是介导虫体对宿主细胞入侵的重要作用机制，成功完成了“阐明EtRON2与宿主细胞受体（Annexin A2）互作介导的鸡球虫入侵机制”的研究目标。本研究的完成为新型抗球虫药物及疫苗的研制奠定了理论基础。