Shahid Chamran University of Ahvaz, Iran
Title: Cloning and Expression of Eimeria necatrix Microneme5 Gene in E. coli
Mansour Mayahi has completed his DVM, 1983, Shahid Chamran University of Ahwaz, MVSc, 1992, Indian Veterinary Research Institute Izatnagar, U.P., India. and PhD, 1998. Tehran University, Tehran, Iran.. He is the dean of Faculty of Veterinary Medicine of Ahvaz. He has published more than 45 papers in reputed journals and published 9 Scientific books and has been serving as an editorial board member of reputed Iranian journal. Dr. Mayahi research mostly are avian diseases.
Micronemes are the smallest secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. Molecular identification and immunogenic potency of these proteins can be used for developing of recombinant vaccine. In this study, after isolation of Eimeria necatrix from Khuzestan province, a pair of primers was designed based on the published nucleotide sequence of micronem5 gene of Eimeria necatrix LZ strain. partial sequence of a cDNA encoding 758 bp fragment of microneme 5 protein (EnMIC5) was amplified by semi- Nested RT-PCR, cloned and expressed in a Maltose Binding protein (MBP) containing expression vector (pMAL-c2x) in E. coli TG1 strain. The amplified fragment contains an open reading frame of 252 amino acids with high degree of conservation with adhesive plasma pre-kallikrein and seven hydrophilic motifs. The results of SDS-PAGE revealed that the fusion protein with molecular weight about 70 kDa was over-expressed after induction of IPTG. Western-blot results demonstrated that the expressed recombinant protein was reacted with sera of infected chicks with Eimeria necatrix, suggesting that this protein should have good immunogenicity and can be used for further studies.