Comparative analysis of model antigen expression level driven by the microneme protein promoters in Eimeria tenella

Coccidiosis is the most severe parasitic disease affecting the poultry industry, and live vaccines have played a pivotal role in its prevention and control, making them the most successful parasitic disease vaccines in commercial use. The components of live coccidiosis vaccines, which serve as vaccine vectors for presenting antigens from other pathogens, offer several advantages and provide a promising new option for disease control in modern poultry farming. Enhancing the expression of heterologous antigens within the complex components of Eimeria is a key strategy for optimizing coccidial vectors and improving immune efficacy. Since secreted antigens can stimulate strong immune responses in the host, we conducted a comparative analysis of the transcription levels of the microneme protein, the most important secretory organelle in apicomplexan parasites, across different stages of the life cycle. Three candidate genes with high transcription levels throughout the entire life cycle (excluding the unsporulated oocyst stage) were selected: ETH2_1343100, ETH2_0957500, and ETH2_1219600. We amplified the upstream (∼1500 bp) and downstream (∼1000 bp) regions of these genes to serve as promoters and regulatory sequences for the enhanced yellow fluorescent protein (EYFP) reporter gene expression cassette. This cassette was then incorporated into a dual-expression vector, fused with a selection marker consisting of a mutated prolyl-tRNA synthetase (PRS) gene and mCherry. After transfecting the plasmids into sporozoites and obtaining recombinant Eimeria strains with stable EYFP expression, we analyzed the fluorescence intensity of EYFP using laser confocal microscopy combined with fluorescence intensity analysis. Recombinant Eimeria driven by the histone 4 (EtHis4) promoter was used as a control. The results showed that the promoters of the three microneme protein genes regulated fluorescence expression levels approximately three times higher than the EtHis4 promoter, with the ETH2_1343100 promoter proving to be the most effective. These findings provide new regulatory sequence options for the genetic manipulation of Eimeria and the enhancement of foreign gene expression, accelerating its potential application as a vaccine vector.