Cloning and expression of Eimeria necatrix microneme5 gene in Escherichia coli

Background: Coccidiosis caused by Eimeria necatrix has the most economic impact on poultry production. Micronemal proteins in Eimeria necatrix are thought to be critical ligands determining host cell specificity at the time of invasion. OBJECTIVES: Isolation and purification of Eimeria necatrix oocysts from Khuzestan province of Iran was performed. A cDNA encoding microneme 5 (EnMIC5) protein was cloned and expressed as recombinant protein before the evaluation of its immunogenicity by Western blotting. METHODS: A primer pair was designed based on the published nucleotide sequence of Eimeria necatrix LZ strain micronem5 gene. A Partial cDNA sequence fragment of 758 bp coding for microneme 5 protein (EnMIC5) was amplified by semi- Nested RT-PCR. PCR products were cloned and expressed in a Maltose Binding protein (MBP) containing expression vector (pMAL-c2x) in Escherichia coli. The cDNA which is encoded for 252 amino acids shows high degree of conservation. It contains the adhesive plasma pre-kallikrein and seven hydrophilic motifs. RESULTS: The results of SDS-PAGE revealed that the recombinant protein with a molecular weight of 70 kDa was over-expressed after induction with IPTG. Western blotting results revealed that the expressed recombinant protein was reacted with sera of the chicks infected with Eimeria necatrix. It was suggested that this protein should have a good immunogenicity and can be used for further studies. CONCLUSIONS: In conclusion, the high degree of sequence homology indicates that this protein is immunogenic and might be an interesting vaccine target, and deserves further investigation