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) was amplified and cloned for expression of the recombinant protein (rEnSAG). The full length Ensag 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 was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG could be recognized by anti-rEnSAG monoclonal antibody (anti-rEnSAG McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG was detected in second-generation merozoites (MZ-2) using anti-rEnSAG polyclonal antibody (anti-rEnSAG pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG 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 afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG. Chickens immunized with rEnSAG had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.