Partial sequence determination of a cDNA encoding microneme 5 protein of Eimeria necatrix isolated in Khuzestan province, Iran

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. Even though coccidiosis is a complex disease that can be caused by any combination of mainly seven species, most of the molecular researches concerning characterization of host and parasite genes and proteins have been limited to Eimeria tenella. The present study describes isolation and purification of Eimeria necatrix oocysts that can be used for an inexpensive and simple total RNA extraction method to partial sequence determination of a cDNA encoding microneme 5 protein. Using the extracted total RNA as template and oligo(dT) as primer, cDNA was synthesized. In order to amplify cDNA encoding the micronem 5 protein (EnMIC- 5), RT-PCR was applied with the specific primers based on the known EST sequence. Following amplification, the unique and thick 758 bp fragment was seen. Domain analysis of EnMIC-5 revealed that the sequence contains the conserved domain of PAN/APPLE superfamily between amino acid resides 130 to 201. This domain has strong similarity to the adhesive plasma pre-kallikrein. Despite sequence similarity of EnMIC-5 with those sequences in database, differences may represent some allelic polymorphism.