Identification and Characterization of Eimeria tenella Microneme Protein (EtMIC8).

Microneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite's MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control (P < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3+CD8+ lymphocyte cells.

Monoclonal Antibody Preparation and Epitope Identification for Brucella melitensis Elongation Factor Tu.

Elongation factor thermo-unstable (EF-Tu), an abundant multifunctional protein, is pivotal during protein synthesis and is an important antigen. Few studies have addressed the role of this protein in Brucella species, and the epitopes of this protein have not been reported. Here, we describe a monoclonal antibody (McAb), BD6, for EF-Tu in Brucella melitensis. Using western blotting involving a series of partially overlapping recombinant EF-Tu truncation peptides, a novel linear B-cell epitope, 110QTREHIL116 (EF), was identified. Alanine-scanning mutagenesis revealed that residues Q110, T111, R112, I115, and L116 were core residues involved in recognition. Sequence alignment suggested that the epitope peptide was conserved among bacterial species but differed by one amino acid residue (I115) from the host sequence. The epitope peptide was recognized by sera from B. melitensis-infected mice, and while recombinant epitope peptide induced a strong humoral immune response, the corresponding mouse peptide, QTREHLL, did not. These results suggested that I115 may be the key residue for the host immune system to distinguish between bacterial and self epitope EF sequences. Indirect immunofluorescence and western blotting assays showed that epitope peptide could be used in Saccharomyces cerevisiae, human embryonic kidney cell (HEK-293), and chicken fibroblast cell (DF1) expression systems and immunoprecipitation assay. Together, our results suggested that the McAb BD6 is a useful tool for further investigation of the potential functions of the EF-Tu protein in pathogen-host interactions, and that the epitope tag may be useful for application as a novel affinity tag to identify other bacterial pathogens, especially convenient for the identification of intracellular bacteria.

Identification and Application of Epitopes in EtMIC1 of Eimeria tenella Recognized by the Monoclonal Antibodies 1-A1 and 1-H2.

Eimeria tenella microneme-1 protein (EtMIC1) has been proposed to be a transmembrane protein, but this characteristic has not yet been confirmed experimentally. Furthermore, despite EtMIC1 being an important candidate antigen, its key epitope has not been reported. Here, two linear B-cell epitopes of EtMIC1, 91LITFATRSK99 and 698ESLISAGE705, were identified by Western blotting using specific monoclonal antibodies (MAbs) and were named epitope I (located in the I-domain) and epitope CTR (located in the CTR domain), respectively. Sequence comparative analyses of these epitopes among Eimeria species that infect chickens showed that epitope I differs greatly across species, whereas epitope CTR is relatively conserved. Point mutation assay results indicate that all the amino acid residues of the epitopes recognized by MAb 1-A1 or 1-H2 are key amino acids involved in recognition. Comparative analyses of indirect immunofluorescence assay (IFA) results for MAbs 1-A1 and 1-H2 under both nonpermeabilization and permeabilization conditions indicate that epitope I is located on the outer side of the sporozoite surface membrane whereas epitope CTR is located on the inner side, together providing experimental evidence that EtMIC1 is a transmembrane protein. IFA also labeled the EtMIC1 protein on the parasitophorous vacuole membrane and on the surface of schizonts, which suggests that the EtMIC1 protein may play an important role in parasitophorous vacuole formation and E. tenella development. Immunoprotective efficacy experiments revealed that epitope I has good immunogenicity, as evidenced by its induction of high serum antibody levels, blood lymphocyte proliferation, and CD4+ blood lymphocyte percentage.