Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

• Published in: Veterinary parasitology 2023-12, Vol.324, p.110060-110060, Article 110060
• Main Authors: Wang, Feiyan, Zhang, Amin, Fan, Xuelian, Feng, Qianqian, Zhang, Zhizhi, Liu, Dandan, Su, Shijie, Hou, Zhaofeng, Xu, Jinjun, Kang, Xilong, Pan, Zhiming, Hu, Hunjie, Tao, Jianping
• Format: Article
• Language: English
• Subjects: antigens; Bacillus cereus; blood serum; Cellular invasion; chickens; coccidiosis; domain; Eimeria necatrix; enzymatic reactions; fluorescent antibody technique; genes; Immunoprotection; membrane proteins; merozoites; molecular weight; monoclonal antibodies; pathogens; phospholipase C; polyclonal antibodies; poultry industry; Prokaryotic expression; quantitative polymerase chain reaction; recombinant proteins; SAG protein; sporozoites; vaccines; veterinary parasitology; Western blotting
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/j.vetpar.2023.110060

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813bp, coding 270 amino acids with a predicated molecular weight of 28.86kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P