柔嫩艾美耳球虫子孢子入侵关键结构 Moving Junction 的分子基础与功能研究

Most Apicomplexa are obligate intracellar parasites and important pathogens of human and domestic animals, such as Toxoplasma, Plasmodium, and Eimeria. Different apicomplexan parasites specifically invade different cells, but the cell invasion machinery is highly conserved. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the establishment of a structure called the moving junction (MJ). The MJ is a highly conserved structure which formed during invasion host cell,it anchors the invading parasite to the host cell and serves as a molecular sieve that protects the parasitophorous vacuole from host lysosomal destruction. So the MJ is very essential for the invasion and survival of the apicomplexa intracellular stages.. Recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ complex, include rhoptry neck proteins (RONs) and micronemal AMA1. More precisely, the parasite exports the AMA1 to its own surface and the RON2 as a receptor inserted into the host cell together with other RON partners, so the RON2 is very important in MJ complex and serve as a bridge. Most proteins of the MJ complex are conserved among Apicomplexa, RONs and AMA1 orthologues can be found in the Eimeria and other apicomplex,Moreover these proteins have no orthologues in host, so given the exact molecular components and interactions between them would clearly open interesting perspectives for a new way of interfering with parasite invasion,with an obvious therapeutic potential against pathogenic Apicomplexa. . Eimeria tenella is one of the most important species causing avian coccidiosis and is frequently used as a model species to study Eimeria spp. The life cycles of Eimeria are complex and consist many differentially deveoplmental stages. Among these stages, sporozoite is the first stage to invade host cells. In our previous report, some differentially expressed genes of sporozoites were analyzed. A full length cDNA of rhoptry neck protein 2 (EtRON2) of E.tenella was obtained based on the EST. A yeast two-hybrid cDNA library of sporozoites of E.tenella has been constructed and a monoclonal antibody against EtRON2 has been produced. . In this study, the MJ complex proteins of E.tenella sporozoites will be analyzed and identified by Co-immunoprecipitation and yeast two-hybrid system.The interactions of these obtained proteins will be tested by immnoprecipitation and western bloting.Immunofluorescence and immunoelectronmicroscopy will be used to locate these proteins during invasion.Invasion inhibition assay will be conducted to analyze the function of these proteins. This study will provided important rationale to understand invasion processes and find new targets for studying new vaccines and drugs against coccidiosis.

顶复器原虫多数是专性细胞内寄生虫,有独特的入侵机制,在宿主细胞膜与虫体表面形成一个高度保守的运动结构（Moving Junction,MJ）,对虫体入侵宿主细胞起关键作用。研究表明弓形虫和疟原虫的MJ是由棒状体颈部蛋白复合体（RON2/4/5/8）与微线顶膜抗原1（AMA1）相互作用形成。基因组分析发现鸡球虫有组成MJ复合体的同源蛋白，而组成MJ的蛋白在宿主体内没有同源蛋白，因此，这些蛋白可成为研究防治顶复器原虫病新药物和新疫苗的重要靶分子。柔嫩艾美耳球虫是致病性最强的鸡球虫，本项目拟在已获得柔嫩艾美耳球虫棒状体颈部蛋白2（EtRON2）基础上，通过CoIP和酵母双杂交捕获EtRON2相互作用蛋白，用免疫荧光、免疫电镜、体外抑制等技术鉴定获得的蛋白是否参与形成MJ及在虫体入侵宿主细胞过程中的功能，为阐明鸡球虫入侵机制、寻找防治鸡球虫病重要"关键"分子奠定基础，具有科学意义和潜在应用价值。

鸡球虫属于顶复器原虫，是专性细胞内寄生虫，有独特的入侵机制，虫体入侵宿主细胞时，在宿主细胞膜与虫体表面形成一个高度保守的运动结构（MJ），对虫体入侵宿主细胞起关键作用。研究发现棒状体颈部蛋白（RONs）和微线顶膜抗原1（AMA1）是组成MJ的关键分子，而与它们相互作用的分子可能也参与形成MJ，在虫体入侵过程中发挥重要作用。因此本研究选择柔嫩艾美耳球虫（E.tenella）的AMA1（EtAMA1）作为诱饵蛋白筛选了E.tenella子孢子酵母双杂交cDNA文库，获得14个可能与EtAMA1相互作用的蛋白。选择其中5个基因利用RACE技术扩增获得了含完整开放阅读框（ORF）的cDNA序列：3个保守蛋白，EtCHP559长1746bp（ORF：1224bp）； EtCHP39长1344 bp（ORF：873 bp）；EtCHP317长1108bp（ORF：501bp）；胱硫醚β合成酶(EtCBS)长2423bp（ORF：1887bp）；丝氨酸/苏氨酸蛋白激酶（EtSTK）长1799bp（ORF：1524 bp）。定量PCR分析发现5个基因在E.tenella 不同发育阶段差异表达，EtCHP559和EtSTK在子孢子阶段高表达，EtCHP39和EtCHP317在孢子化卵囊阶段高表达，而EtCBS在未孢子化卵囊阶段高表达。为进一步研究功能，分别将5个基因的ORF连接到原核表达载体，诱导获得重组蛋白，并制备了相应的抗体。间接免疫荧光结果显示这些蛋白主要位于子孢子的表面或顶端，当虫体入侵细胞时，表达量上升。体外抑制显示5种多抗都能抑制子孢子入侵DF-1细胞，且随着抗体浓度的不断增大，抑制率逐渐升高，最高可达70%。为验证5个蛋白与EtAMA1蛋白的互作关系，分别将5个基因连接到真核表达载体pcDNA3.1-flag、pBiFC-VC155上， EtAMA1连接到pcDNA3.1、pBiFC-VN155上，利用免疫共沉淀和双分子荧光互补技术验证互作关系，结果显示EtCHP559、EtCHP39、EtCBS与EtAMA1没有互作关系，EtCHP317和EtSTK是EtAMA1的互作蛋白。GST pull-down结果也显示 EtCHP317和EtSTK是EtAMA1的互作蛋白。这些结果为阐明鸡球虫入侵机制、寻找防治鸡球虫病重要“关键”分子奠定基础，具有科学意义和潜在应用价值。

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