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

Document Type : Research Articles

Authors

Shahid Chamran University of Ahvaz

Abstract

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.

Keywords


Ayub Ali, M., Patra, G., Rajkhowa, T.K.,Sailo, L., Singh, W.R., Das, G., Chanu,Kh.V. (2009) A study on biochemical composition of the inner wall of the oocyst of Eimeria necatrix .International Journal of Poultry Science 8 , 1214-1217.
Badran, I. and Lukeshova, D. (2006) Control of Coccidiosis and Different Coccidia of Chicken in Selected Technologies Used in Tropics and Subtropics.Agricultura Tropica et Subtropica 39,39-44.
Brecht, S., Carruthers V. B., Ferguson, D.J.P.(2001) The Toxoplasma micronemal protein MIC4 is an adhesin composed
of six conserved apple domains.Journal of Biological Chemistry 276,4119-4127.
Brown, P.J., Billington, K.J., Bumstead, J.M.,Clark J.D., Tomley, F.M. (2000) A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and
plasma pre-kallikrein. Journal of Molecullar Biochemical Parasitology 107, 91-102.
Cai, X.P., Liu, H., Jia W., Guo, A., Zhang Sh.,Yan, H., Yue, Ch. (2008) Recombinant expression of microneme protein 5
gene of Eimeria necatrix LZ strain in E. coli and antigenicity of recombinant protein. Acta Veterinaria et Zootechnica Sinica 39, 1575 -1580.
Carruthers, V.B. and Tomley, F.M. (2008) Receptor-ligand interaction and invasion: Microneme proteins in Apicomplexans. Subcellular Biochemistry 47, 33–45.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-
chloroform extraction.Analitical Biochemistry 162, 156-159.
De Venevelles, P. J., Chich, F., Faigle, W.,Lombard, B., Loew, D., Pery, P.,Labbe, M. (2006) Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach. International Journal of Parasitology 36, 1399–1407.
Farrell, R.E.J. (1993) RNA methodologies: a laboratory guide for isolation and characterization, Academic Press, San Diego, pp 47-76.
Johnston, D.A., Bratty. J. , Fernando, M.A.(1998) A fast and inexpensive method for isolation of high-quality RNA from
eimerian oocysts. Parasitology Research 84, 549-551.
Klein, H., Mehlhorn H., Ruger, W. (1996) Characterization of genomic clones encoding two microneme antigens of
Sarcocystis muris (Apicomplexa). Parasitology Research 82, 230–237.
Lee, E. H. (1979) Single and low-level oocyst infections of drug-resistant field strains of Eimeria tenella in medicated birds.
Canadian Veterinary Journal 20, 102–104.
Li, L., Brunk, B.P., Kissinger, J.C., Pape, D.,Tang, K., Cole, R. H., Martin, J.,Wylie, T., Dante, M., Fogarty, S. J.(2003) Gene discovery in the Apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genomic Research 13, 443–454.
Marchler-Bauer, A., Anderson, J.B.,DeWeese-Scott, C., Fedorova, ND., Geer, LY., He,S., Hurwitz, DI., Jackson, JD., Jacobs, AR.,Lanczycki, CJ., Liebert, CA., Liu, C.,Madej, T., Marchler, G.H., Mazumder,R., Nikolskaya, AN., Panchenko, AR.,
Rao, BS., Shoemaker, BA., Simonyan,V., Song, JS., Thiessen, RA.,
Vasudevan, S., Wang, Y., Yamashita,RA., Yin, JJ., Bryant, SH. (2003) CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Research 31, 383-387.
McDougald, L.R. (2003) Coccidiosis: Diseases of poultry, 11th ed.,
Blackwell Publishing Company, Iowa State Press, Iowa. pp 979-980.
Miska, K.B., Fetterer R.H., Barfield, R.C.(2004) Analysis of transcripts expressed by Eimeria tenella oocysts using subtractive hybridization methods. Journal of Parasitology 90,
1245–1252.
Qin ZongHua, Xie MingQuan, Cai JianPing,Elhag, A. B, Peng XingYu, Wei WengKang, Wu HuiXian, (2005) Expressing microneme-2 gene of Eimeria necatrix in Escherichia coli.
Chinese Journal of Veterinary Science 25, 22-25.
Refeaga, S.,Girard-Misguich, F., Bourdieu,C., Pery P., Labbe, M. (2003) Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies.Veterinary Parasitology 113, 19–33.
Shirley, M.W. (1995) Eimeria species and strains of chickens: Biotechnology Guidelines on Techniques in Coccidiosis Research. European Commission DGXII, Luxembourg. pp 1- 24.
Shirley, M.W., Smith A.L., Tomley, F.M.,(2005) The biology of avian Eimeria with an emphasis on their control by vaccination. Advanced Parasitology 60, 285–330.
Su, Y.C., Fei A.C., Tsai, F.M. (2003) Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence. Veterinary Parasitology 117,221–227.
Tomley, F.M. (1997) Techniques for Isolation and Characterization of Apical Organelles from Eimeria tenella Sporozoites. Methods: A Companion to Methods in Enzymology 13, 171–176.
Zhao, X.M., Duszynski D.W., Loker, E.S.(2001) A single method of DNA extraction for Eimeria species. Journal of Microbiologic Methods 44, 131- 37.