Expression and characterization of microneme protein-3 adhesive repeat region 1b (MIC3-MAR1b) of Eimeria tenella

Microneme protein 3 (MIC3) has been identified as an important vaccine target in apicomplexan parasites. The protein is secreted by the micronemes of apical complex of merozoite and sporozoite stages of the parasites and help in host cell invasion. In Eimeria tenella MIC3 contains seven repetitive regions. In this study, microneme adhesive repeat region-1b of MIC3 (MIC3-MAR1b) was PCR amplified from cDNA derived from E. tenella, cloned in pET32a(+) expression vector, sequenced and expressed in BL21(DE3) strain of Escherichia coli. Post sequencing alignment studies revealed 99.8% homology of the MIC3-MAR1b of Indian isolate of E. tenella with the well characterized E. tenella Houghton strain. The expression of recombinant protein was standardized to obtain maximum yield at 4 hours after induction with 1mM IPTG. The expressed protein was purified under native conditions and the expression of the recombinant protein was confirmed by immunoblotting and probing the transferred protein on nitrocellulose membrane with Ni-NTA HRP conjugate as well as E. tenella infected chicken sera. Immunolocalization studies revealed that MIC3-MAR1b protein are expressed in apical region of merozoites of E. tenella and purified recombinant protein maintains its immunogenicity. The rMIC3-MAR1b, expressed in the present study, may be further evaluated for use as a vaccine candidate against E. tenella infection in chickens.