针对地克珠利显著下调鸡柔嫩艾美耳球虫（E.tenella）第二代裂殖子Rhomboid （ROM）蛋白酶mRNA表达量，抑制球虫入侵这一前期研究结果。本项目拟扩增EtROMs蛋白酶基因，采用酵母双杂交和免疫共沉淀技术筛选EtROMs蛋白酶作用底物分子；借助COS7细胞共表达体系，采用定点突变和Western blot技术研究EtROMs蛋白酶催化位点及剪切活性；建立地克珠利抗E.tenella子孢子入侵鸡肾细胞模型和E.tenella感染鸡模型，以入侵期球虫（子孢子和裂殖子）为研究对象，采用Real-time PCR和Western blot技术检测EtROMs蛋白酶表达量，利用免疫荧光和免疫电镜技术观察EtROMs蛋白酶和底物粘附分子空间表达情况，探讨地克珠利调控EtROMs蛋白酶，抑制球虫入侵分子机制，为阐明地克珠利抗鸡E. tenella作用机制及筛选研发抗球虫新药靶点提供理论依据。

It is found in our previous study that diclazuril downregulates the Rhomboid (ROM) protease mRNA expression, which in turn, interferes adhesins cleavage and inhibits second-generation merozoite of Eimeria tenella invasion to host. In the present study, based on the ESTs of differentially expressed genes identified by the second-generation high-throughput sequencing, EtROM1, EtROM3，EtROM4 and EtROM5 proteases full-length cDNA were amplified using the rapid amplication of cDNA ends technology. To further study the EtROMs cleavage activity，substrate proteins interacting with EtROMs protease were analyzed by yeast two-hybrid screening and proved by co-immunoprecipitation technique. Animal cells COS7 were transiently transfected with plasmid for the expression of GFP-tagged substrates and HA-tagged EtROM proteases. Media and cell samples were analyzed by western blot. At the same time, mutations on EtROMs active site serine were introduced into the plasmid for the expression of EtROMs to elucidate the function of EtROMs cleavage subtract protein and determine the nature of the responsible amine acid. To test whether the intranmembranouse serine proteases, together with its substance protein, participates in the inhibition of diclazuril on a major invasion pathway, chicken challenged with E. tenella sporulated oocysts and received diclazruil model in vivo and primary chicken kidney cells invaded by E. tenella sporozoite model in vitro models were utilized. The expression of EtROMs mRNA and proteins levels both in sporozoite and merozoites were subjected to Real-time PCR and western blot analysis and the intracellular localization of EtROMs with substrate were discriminated by indirect immunofluorescence and immune electron microscope. This study provides a theoretical basis on the mechanism of diclazuril in antagonizing E. tenella and an attractive target for the development of new anticoccidial drug.