Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

艾美耳球虫是一种顶复门寄生原生动物，已知可感染全世界的家禽。已在植物、绿藻、纤毛虫和 apicomplexan（例如柔嫩球虫）中发现了一种重要的钙依赖性蛋白激酶 (CDPK)。 CDPK 是参与钙信号通路的效应分子，控制着滑行运动、繁殖和宿主细胞侵袭等重要的生理过程。鉴于宿主体内未发现 CDPK，研究柔嫩柔嫩球虫中 CDPK 的功能可作为开发新治疗药物和疫苗的基础。为了评估 CDPK4 在柔嫩艾美球虫 (EtCDPK4) 中的功能，通过免疫共沉淀 (co-IP) 和 His Pull-down 测定来筛选假定的相互作用因子翻译起始因子 eIF-5A (EteIF-5A)，然后进行质谱分析。 EteIF-5A 和 EtCDPK4 之间的相互作用通过双分子荧光互补 (BiFC)、GST pull-down 和 co-IP 确定。然后分析了EteIF-5A的分子特征。采用实时定量聚合酶链反应和蛋白质印迹法测定EteIF-5A在柔嫩球虫不同发育阶段的转录和蛋白水平。结果表明，EteIF-5A mRNA的转录水平在第二代裂殖子中最高，蛋白表达水平在未孢子卵囊中最高。间接免疫荧光显示，EteIF-5A蛋白遍布于子孢子的细胞质中，但在折射体中未发现。随着DF-1细胞侵袭的进行，EteIF-5A荧光强度在滋养体中增强，在未成熟裂殖体中减弱，在成熟裂殖体中增强。 通过蛋白质印迹分析的分泌测定结果表明，EteIF-5A 是一种分泌蛋白，但不是来自微线体。侵袭抑制实验结果表明，兔抗rEteIF-5A多克隆抗体能有效抑制子孢子对细胞的侵袭，抑制率为48%。