The molecular characterization and immune protection of microneme 2 of Eimeria acervulina.

Eimeria acervulina (E. acervulina) is an intestinal protozoon and few of its antigen genes were researched. In this study, the gene of E. acervulina microneme protein 2 (EaMIC2) was cloned and characterized. The degenerate primers for the conserved sequence of microneme protein 2 were designed based on Eimeria tenella microneme 2 (EtMIC2) (AF111839.1), Eimeria maxima microneme 2 (EmMIC2) (FR718971.1) and E. brunetti microneme 2 (EbMIC2) (AB723700.1) to amplify the conserved sequence of microneme protein 2. According to the conserved sequence, specific primers for the rapid amplification of cDNA ends (RACE) were designed to amplify the 3'- and 5'-ends of EaMIC2. The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC2 was 882bp and encoded a protein of 293 amino acids with 29.81kDa. The most part of the mature protein (base: 82-882bp, amino acid: 28-293aa) of EaMIC2 (mpmEaMIC2) was inserted into pCold TF to produce recombinant mpmEaMIC2. Western blotting assay was used to analysis the immunogenicity of mpmEaMIC2 and the result showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of mpmEaMIC2. Immunofluorescence analysis using antibody against recombinant protein mpmEaMIC2 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of mpmEaMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens. All the above results suggested that the EaMIC2 was a novel E. acervulina antigen and could be an effective candidate for the development of new vaccine against this parasite.