Rhomboid蛋白酶参与地克珠利抗柔嫩艾美耳球虫入侵机制研究

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.

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。

柔嫩艾美耳球虫（Eimeria tenella）Rhomboid蛋白酶参与虫体与宿主细胞间黏附分子的剪切，在球虫入侵宿主细胞过程中发挥着至关重要的作用。针对地克珠利显著下调鸡E. tenella第二代裂殖子Rhomboid蛋白mRNA表达，抑制球虫入侵宿主细胞，显著降低E. tenella第二代裂殖子数量这一前期研究结果。本项目成功克隆获得了EtROM1 蛋白酶基因，该基因ORF编码区为888 bp，编码295个Aa，具有7次跨膜结构，无信号肽结构。借助哺乳动物293T细胞共表达体系，将pcDNA3.1-EtROM1-FLAG和pcDNA3.1-EtMIC1-HA质粒，pcDNA3.1-EtROM1-FLAG和pcDNA3.1-EtMIC4-HA质粒共转染细胞，采用免疫共沉淀技术研究了酶和底物的互作效应，Western blotting检测结果表明EtROM1与EtMIC1和EtMIC4分子之间未见明显的互作效应。采用构建的子孢子入侵鸡胚成纤维细胞模型，研究表明兔源EtROM1特异性抗血清可以抑制E. tenella子孢子入侵鸡胚成纤维细胞。通过建立地克珠利抗E. tenella感染鸡动物模型，收集E. tenella不同期段的虫体，Real-time PCR技术检测表明EtROM1主要在子孢子阶段大量表达，而在未孢子化卵囊、孢子化卵囊和第二代裂殖子阶段表达量较少，推测EtROM1主要在子孢子入侵宿主细胞中发挥作用。进一步采用Real-time PCR技术表明地克珠利处理后E. tenella 第二代裂殖子EtROM1mRNA降低了74.1%，Western blotting检测分析表明EtROM1蛋白表达降低了44.1%。利用免疫荧光技术表明EtROM1蛋白主要分布在第二代裂殖子的表面，地克珠利处理组，裂殖子表面EtROM1蛋白分布减少。研究结果表明地克珠利可以通过调控EtROM1蛋白酶的表达及空间分布，抑制球虫入侵宿主细胞，项目研究成果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。