Study On Antigenicity Of Recombinant Microneme Protein 5(MIC5) Of Eimeria Necatrix Expressed In E. Coli

Genomic DNA was extracted from the oocyst of Eimeria necatrix, and the EnMIC-5 gene specific primers were designed using Oligo6.0 software. A 1 470 bp specific fragment was amplified by PCR and ligated into pMD18-T vector. The recombinant plasmid was identified by PCR, restriction endonuclease analysis and sequencing. The similarity of the nucleotide sequence and deduced amino acid sequence of the EnMIC-5 gene was 99.7% and 99.6%, respectively compared with the published sequence in GenBank.According to the characterization and the cloning site of pET-28(a)+ vector, the specific primers were designed. Then the fragment was subcloned into the BamH I and Xho I sites of the prokaryotic expression vector pET-28(a)+ and was transformed into E. coli BL21(DE3). Expression of the recombinant plasmid was induced by IPTG in E. coli, and the expressed products were analyzed by SDS-PAGE and Western-blotting. The results of SDS-PAGE showed that the fusion protein with molecular weight of about 57.5 kDa was over-expressed. Western-blotting demonstrated that the expressed recombinant protein was reacted with anti E. necatrix positive sera from chickens. The Kunming mice were immunized with the purified recombinant protein and their sera were examined by ELISA. The results suggested that the recombinant protein have good immunogenicity, and make mice produce a high level of specific antibody.This study can be used for understanding of structure and function of microneme proteins and development of genetically modified vaccine.