A novel acyl-CoA binding protein (EnACBP): Molecular characterization, localization, and immunoprotective efficacy against Eimeria necatrix.

Acyl-CoA binding proteins (ACBPs) are conserved intracellular lipid-binding proteins found across eukaryotes that play essential roles in fatty acid metabolism, membrane biogenesis, and cellular energy homeostasis. In this study, the gene ENH_00086110, which encodes a novel ACBP-family protein designated EnACBP, was cloned and characterized from Eimeria necatrix. The deduced mature protein contains 511 aa with a calculated molecular mass of 56.18 kDa. Bioinformatic analysis indicated that EnACBP contains a conserved acyl-CoA binding (ACB) domain in its C-terminal region (residues 424-506) and lacks any recognizable conserved motifs in its extensive N-terminal region. Recombinant protein (rEnACBP) was expressed predominantly as inclusion bodies in Escherichia coli, with a molecular weight of approximately 60 kDa, and was recognized by a mouse anti-His tag monoclonal antibody. qRT-PCR analysis revealed that the transcription level of EnACBP was significantly higher in gametocytes (GAM) and unsporulated oocysts (UO) than in second- and third-generation merozoites (MZ-2 and MZ-3) (P < 0.05). The native EnACBP was detected in both MZ-2 and GAM extracts, with a prominent band of ~72 kDa in GAM. Immunofluorescence assay localized EnACBP to wall-forming body type 1 (WFB1) and the outer oocyst wall. Following the E. necatrix challenge, the anticoccidial index (ACI) of immunized groups was 184.50 and 195.40 at an immunization dose of 100 µg per chicken per immunization. Compared to the controls, the immunized groups induced higher proportions of splenic CD4+/CD3+ and CD8+/CD3+ T lymphocyte and higher level of IgY antibody. These findings not only lay a foundation for understanding the role of EnACBP protein in gametogenesis and oocyst wall formation, but also present a potential protective antigen for the development of a subunit vaccine against avian coccidiosis.