【作者】 沈晓炯；

【导师】 薛飞群；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 柔嫩艾美耳球虫（E.tenella）是引起鸡球虫病的病原体中毒力最强的一种寄生虫，给全球养禽业造成了巨大的经济损失。目前对于球虫病的预防和治疗措施主要依靠化学药物，但随着球虫对现有药物耐药性的增强，迫切需要研发新的抗球虫药，而药物靶标的鉴定是其中一个重要步骤。本研究通过双向电泳分析了地克珠利作用后E.tenella第二代裂殖子差异表达的蛋白，筛选出数个药物作用靶标。同时对热休克蛋白（Heat Shock Protein90，Hsp90）及激活蛋白激酶C受体（Receptorfor activated protein kinase C，RACK）进行了分子药理实验。主要结果如下：1.通过双向电泳和Real-time PCR分析了地克珠利作用后，E.tenella第二代裂殖子蛋白表达变化。结果显示，13个蛋白受地克珠利影响显著，其中11个蛋白被鉴定为E.tenella或其它顶复门寄生虫已注释蛋白，这些蛋白与多种生物功能相关，包括：代谢、蛋白质合成及宿主细胞入侵等。利用Real-time PCR检测了上述蛋白mRNA水平，结合双向电泳图谱，进一步阐释了地克珠利对E.tenella第二代裂殖子作用机制，同时说明其中几个蛋白可作为E.tenella感染治疗的药物设计靶标。2.地克珠利在mRNA及蛋白水平上降低了E.tenella第二代裂殖子中Hsp90的表达，同时发现相对于对照组，加药组E.tenella第二代裂殖子中Hsp90的亚细胞定位更趋于分散。此外，体外实验证明地克珠利可直接作用于E.tenella第二代裂殖子Hsp90。综上，实验进一步阐释了地克珠利对E.tenella的分子药理机制，也表明Hsp90是抗球虫感染中一个潜在的药物靶标。3.关于E.tenella第二代裂殖子RACK的实验，通过免疫荧光分析发现，RACK主要表达在E.tenella第二代裂殖子的顶部。此外，通过Western blot和Real-time PCR研究了地克珠利对E.tenella第二代裂殖子的作用，尽管Real-Time PCR结果显示E.tenella第二代裂殖子中mRNA水平在地克珠利作用后有所上升，但在Western blot结果中，处理组的RACK蛋白条带完全消失，且地克珠利作用组的裂殖子在免疫荧光实验中亦未检测到RACK荧光，说明RACK可能是新药设计中一个潜在的药物靶点。综上所述，通过实验，得到了地克珠利作用于E.tenella第二代裂殖子的数个蛋白靶标。同时，地克珠利对E.tenella第二代裂殖子Hsp90和RACK的实验，验证了地克珠利对寄生虫分子伴侣及信号转导途径的作用，说明两者均是良好的药物作用靶标。更多还原

【Abstract】 Eimeria tenella, one of the most virulent Eimeria species causing coccidiosis, causes vasteconomic losses in the poultry industry, and is also a major animal welfare problem. At present, themain prophylaxis and treatment strategy remains to be chemotherapy, but with the rising drug-resistanceof Eimeria strains, new drug development is urgently demanded, within which the identification of drugtargets is an essential step. In this study, we analyzed the proteomic pattern of the second-generationmerozoite after diclazuril treatment, finding several affected proteins. Meanwhile, the conformativeexperiments on Hsp90and RACK were also carried out. The results are as the followings:1. According to two dimensional gel electrophoresis and real-time PCR,13proteins were revealedto be significantly affected by diclazuril treatment, further11proteins were identified as annotatedproteins of Eimeira tenella or other Apicomplexa parasites. These proteins are related to variousfunctions including metabolic, protein synthesis and host-cell invasion. Through the method of real-timePCR, we also elucidate the possible regulation pattern of diclazuril, and suggest some promising targetsfor the intervention with E.tenella infection.2. Diclazuril down-regulated the expression of Hsp90both on mRNA and protein level, meanwhile,the subcellular localization of Hsp90in the treated group is relatively more dispersed comparing to thecontrol group. Also, the in vitro experiment proved that diclazuril effecting on merozoites through adirect mode. Generally, our work further elucidated the function mechanism of diclazuril, and verifiedHsp90as a promising drug targets for novel anticoccidial agents design.3. Immunofluorescence assay of EtRACK shows it is localized mainly at the top of parasites. Andthe effect of diclazuril on EtRACK is also analyzed through Real-time PCR and Western blot. Thoughthe result of RT-PCR shows the mRNA level is upregulated with diclazuril adminsitration, the proteinbelt of the treated group in Western blot is entirely invisible, suggesting the protein RACK might be afavourable target for novel drug design.In conclusion, diclazuril treatment altered the proteomic pattern of the second-generationmerozoites in E.tenella, with several proteins affected significantly which may play important roles inthe function mechanism of diclazuril. Also, the experiments on Hsp90and RACK revealed the effects ofdiclazuril on molecular chaperones and the signaling pathways, which appear to be promising drugtargets too.更多还原