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

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 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG_-CAP was about 32 kDa 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 < 0.05). The anti-rEnSAG_-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG_-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG_-CAP. Chickens immunized with rEnSAG_-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG_-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.