Cloning and expression of Eimeria necatrix microneme5 gene in Escherichia coli

Abstract

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