Liu Lianrui, Huang Xinmei, Liu Jianhua, Li Wenyu, Ji Yihong, Tian Di, Tian Lu, Yang Xinchao, Xu Lixin, Yan Ruofeng, Li Xiangrui, Song Xiaokai
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing, Jiangsu 210014, China.
Oncotarget. 2017 May 23;8(21):34935-34945. doi: 10.18632/oncotarget.16824.
Clinical chicken coccidiosis is mostly caused by simultaneous infection of several Eimeria species, and host immunity against Eimeria is species-specific. It is urgent to identify common immunodominant antigen of Eimeria for developing multivalent anticoccidial vaccines. In this study, sporozoite proteins of Eimeria tenella, Eimeria acervulina and Eimeria maxima were analyzed by two-dimensional electrophoresis (2DE). Western bot analysis was performed on the yielded 2DE gel using antisera of E. tenella E. acervulina and E. maxima respectively. Next, the detected immunodominant spots were identified by comparing the data from MALDI-TOF-MS/MS with available databases. Finally, Eimeria common antigens were identified by comparing amino acid sequence between the three Eimeria species. The results showed that analysis by 2DE of sporozoite proteins detected 629, 626 and 632 protein spots from E. tenella, E. acervulina and E. maxima respectively. Western bot analysis revealed 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots from the sporozoite 2DE gels of the three Eimeria species. The immunodominant spots were identified as 33, 27 and 25 immunodominant antigens of E. tenella, E. acervulina and E. maxima respectively. Fifty-four immunodominant proteins were identified as 18 ortholog proteins among the three Eimeria species. Finally, 5 of the 18 ortholog proteins were identified as common immunodominant antigens including elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domain-containing protein (UCE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, our results not only provide Eimeria sporozoite immunodominant antigen map and additional immunodominant antigens, but also common immunodominant antigens for developing multivalent anticoccidial vaccines.
临床鸡球虫病大多由几种艾美耳球虫同时感染引起，且宿主对艾美耳球虫的免疫力具有种特异性。为开发多价抗球虫疫苗，鉴定艾美耳球虫的共同免疫显性抗原迫在眉睫。本研究采用二维电泳（2DE）分析柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫的子孢子蛋白。分别用柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫的抗血清对所得2DE凝胶进行蛋白质免疫印迹分析。接下来，通过将基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）数据与现有数据库进行比较，鉴定出检测到的免疫显性斑点。最后，通过比较三种艾美耳球虫之间的氨基酸序列，鉴定出艾美耳球虫共同抗原。结果显示，2DE分析子孢子蛋白分别从柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫中检测到629、626和632个蛋白斑点。蛋白质免疫印迹分析从三种艾美耳球虫子孢子2DE凝胶中分别揭示出50个（柔嫩艾美耳球虫）、64个（堆型艾美耳球虫）和57个（巨型艾美耳球虫）免疫显性斑点。这些免疫显性斑点分别被鉴定为柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫的33个、27个和25个免疫显性抗原。54个免疫显性蛋白被鉴定为三种艾美耳球虫中的18个直系同源蛋白。最后，18个直系同源蛋白中的5个被鉴定为共同免疫显性抗原，包括延伸因子2（EF-2）、14-3-3蛋白、含泛素结合酶结构域蛋白（UCE）和甘油醛-3-磷酸脱氢酶（GAPDH）。总之，我们的结果不仅提供了艾美耳球虫子孢子免疫显性抗原图谱和额外的免疫显性抗原，还为开发多价抗球虫疫苗提供了共同免疫显性抗原。
