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Cloning and Expression of Immunogenic Regions of EMA-1 Gene of Theileria equi From Infected Horses | ||
| Archives of Razi Institute | ||
| مقاله 4، دوره 73، شماره 4، اسفند 2018، صفحه 295-303 اصل مقاله (477.57 K) | ||
| نوع مقاله: Original Articles | ||
| شناسه دیجیتال (DOI): 10.22092/ari.2017.110581.1132 | ||
| نویسندگان | ||
| M. Ebrahimi1؛ Hossein Hamidinejat2؛ M. R. Tabandeh* 3؛ M.H. Razi Jalali1؛ A. Rasouli4 | ||
| 1Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran | ||
| 2Department of Parasitology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran | ||
| 3Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran | ||
| 4Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran | ||
| چکیده | ||
| Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in Iran. The immunogenic region of EMA-1 gene was amplified using the blood of infected horses. EMA-1 gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent E. coli BL21 (DE3) cells. Cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme assays, and DNA sequence analysis. The recombinant protein was expressed using isopropyl β-D-1-thiogalactopyranoside as an inducer, purified using nickle-nitrilotriacetic acid column, and then confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot analysis utilizing Anti-His Tag antibody. Furthermore, the immunoreactivity of recombinant protein against the serum of the infected horses was evaluated using dot blot analysis. The PCR product analysis showed a 750-bp band belonging to immunogenic regions of EMA-1 gene. Sequence analysis revealed that cloned EMA-1 and protein had 94% and 97% homology to EMA-1 sequences submitted to GenBank from different countries, respectively. Restriction enzyme and sequence analyses confirmed the subcloning and correction of the orientation of inserted gene. The SDS-PAGE analysis confirmed the expression of EMA-1 protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against T. equi could react with the purified recombinant protein. Purified EMA-1 protein can be used as a reliable tool for the future development of diagnostic tests or vaccines. | ||
| کلیدواژهها | ||
| Theileria equi؛ EMA-1؛ Cloning | ||
| مراجع | ||
|
Baldani, C.D., Hilario, E., Nakaghi, A.C., Bertolini, M.C., Machado, R.Z., 2011. Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from Sao Paulo State, Brazil. Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria 20, 54-60.
Chhabra, S., Ranjan, R., Uppal, S.K., Singla, L.D., 2012. Transplacental transmission of Babesia equi (Theileria equi) from carrier mares to foals. J Parasit Dis 36, 31-33.
Friedhoff, K.T., Tenter, A.M., Muller, I., 1990. Haemoparasites of equines: impact on international trade of horses. Rev Sci Tech 9, 1187-1194.
Heuchert, C.M.S., de Giulli Jr, V., de Athaide, D.F., Böse, R., Friedhoff, K.T., 1999. Seroepidemiologic studies on Babesia equi and Babesia caballi infections in Brazil. Vet Parasitol 85, 1-11
Huang, X., Xuan, X., Yokoyama, N., Xu, L., Suzuki, H., Sugimoto, C., et al., 2003. High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis. J Clin Microbiol 41, 1147-1151.
Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129 Suppl, S3-14.
Kamikawa, C.K., Vicentini, A.P., 2015. Dot-Blot Methodology for Rapid Diagnosis of Paracoccidioidomycosis Caused by Paracoccidioides brasiliensis. J Infect Dis Ther 3.
Knowles, D.P., Kappmeyer, L.S., Perryman, L.E., 1997. Genetic and biochemical analysis of erythrocyte-stage surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria species. Mol Biochem Parasitol 90, 69-79.
Kumar, S., Yokoyama, N., Kim, J.Y., Huang, X., Inoue, N., Xuan, X., et al., 2004. Expression of Babesia equi EMA-1 and EMA-2 during merozoite developmental stages in erythrocyte and their interaction with erythrocytic membrane skeleton. Mol Biochem Parasitol 133, 221-227.
M., V., Flores, C., Garcia, J., Chipres, D., Rivera, T., Estrella, R., et al., 2014. Nested PCR detection of Theileria equi infection and frequency in horses imported into Mexico. J Anim Vet Adv 13, 859-863.
Mahmoud, M.S., El-Ezz, N.T., Abdel-Shafy, S., Nassar, S.A., El Namaky, A.H., Khalil, W.K., et al., 2016. Assessment of Theileria equi and Babesia caballi infections in equine populations in Egypt by molecular, serological and hematological approaches. Parasit Vectors 9, 260.
Nicolaiewsky, T.B., Richter, M.F., Lunge, V.R., Cunha, C.W., Delagostin, O., Ikuta, N., et al., 2001. Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Vet Parasitol 101, 9-21.
Nizoli, L.Q., Conceicao, F.R., Silva, S.S., Dummer, L.A., Santos, A.G., Jr., Leite, F.P., 2009. Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris. Rev Bras Parasitol Vet 18, 1-4.
Papadopoulos, B., Brossard, M., Perié, N.M., 1996. Piroplasms of domestic animals in the Macedonia region of Greece 2. Piroplasms of cattle. Vet Parasitol 63, 57-66.
Salim, B.O., Hassan, S.M., Bakheit, M.A., Alhassan, A., Igarashi, I., Karanis, P., et al., 2008. Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR. Parasitol Res 103, 1145-1150.
Schwint, O.N., Knowles, D.P., Ueti, M.W., Kappmeyer, L.S., Scoles, G.A., 2008. Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host. J Med Entomol 45, 1152-1155.
Short, M.A., Clark, C.K., Harvey, J.W., Wenzlow, N., Hawkins, I.K., Allred, D.R., et al., 2012. Outbreak of equine piroplasmosis in Florida. J Am Vet Med Assoc 240, 588-595.
Steinman, A., Zimmerman, T., Klement, E., Lensky, I.M., Berlin, D., Gottlieb, Y., et al., 2012. Demographic and environmental risk factors for infection by Theileria equi in 590 horses in Israel. Vet Parasitol 187, 558-562.
Xuan, X., Larsen, A., Ikadai, H., Tanaka, T., Igarashi, I., Nagasawa, H., et al., 2001a. Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. J Clin Microbiol 39, 705-709.
Xuan, X., Nagai, A., Battsetseg, B., Fukumoto, S., Makala, L.H., Inoue, N., et al., 2001b. Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens. J Vet Med Sci 63, 1159-1160. | ||
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