Bacterial expression of Beet curly top Iran virus coat protein in Escherichia coli

Document Type : Research paper-Persian

Authors

1 Ph.D. of Plant Pathology, Shiraz Univeristy

2 Professor, Shiraz University

3 Assistant Professor, Shahid Chamran University of Ahvaz

Abstract

Background and Objectives
Beet curly top Iran virus (BCTIV) and Beet curly top virus (BCTV) are the causal agents of beet curly top disease in Iran. It has been reported that both viruses are distributed in different geographical regions and host plants in Iran. Development of a simple, fast and low cost serological method such as ELISA for detection of these viruses, is a necessary demand. To do serological tests such as ELISA, an antibody is needed. Production of polyclonal antibodies against geminiviruses is a hard candidate because of low concentration and localization of their particles in the phloem tissue of their hosts. Therefore, expression and production of geminiviral recombinant proteins in bacterial system is an alternative method to obtain polyclonal antibodies.
Material and Methods
Specific forward and reverse primers containing BamHI/HindIII digestion sites at the 5′ end of each primer, respectively, were designed to amplify the full-length fragment of BCTIV V1 open reading frame encoding the viral coat protein (CP). The amplified 751 bp fragment was cloned into the pTZ57R/T cloning vector. V1 gene was released from pTZ57R/T by enzymatic digestion and cloned into the expression vector pQE30. The recombinant pQE30-BCTIV V1 was transformed into Escherichia coli strain M15. The expression of protein was induced by adding 1 mM of isopropylthio-d-galactoside (IPTG) to the transformed bacterial cells and grown at 37 ºC in liquid LB medium. Expression of recombinant protein was analyzed by SDS-PAGE electrophoresis and Western blot hybridization.
Results
Analysis of the expressed proteins by SDS-PAGE electrophoresis, revealed a protein band with a position corresponding to molecular weight of approximately 30 kDa that was consistent with the predicted molecular weight of BCTIV CP.Time course of induction by IPTG influenced on level of the new recombinant protein expression, thus four hours induction had the highest level of expression. Western blot analysis confirmed the identity of expressed protein via using anti-His antibody to detect expressed recombinant His-BCTIV CP.
 
 
Discussion
One of the most popular bacterial protein expression system is IPTG inducible system wherein IPTG regulate level of new recombinant protein expression. Our results by SDS-PAGE electrophoresis and Western blot analysis revealed a high level of expression of the fusion protein in IPTG-induced bacterial cells. However, a negligible rate of expression of recombinant protein was also observed in non-induced bacterial cells. This suggests low appearance and activity of responsible promotor even without inducing protein expression with IPTG. Expression of conserved plant viral proteins in bacterial systems following purification of the resulted recombinant proteins would facilitate production of specific polyclonal antibodies.

Keywords


Anabestani, A., Izadpanah, K., Tabein, S., Hamzeh-Zarghani, H., and Behjatnia, S. A. A. 2016. Beet curly top viruses in Iran: Diversity and incidence in plants and geographical regions. Iranian Journal of Plant Pathology, 51: 493-504.
Anabestani, A., Behjatnia, S. A. A., Izadpanah, K., Tabein, S., and Accotto, G. P. 2017. Seed transmission of beet curly top virus and beet curly top Iran virus in a local cultivar of petunia in Iran. Viruses, 9: 299.
Behjatnia, S. A. A., Dry, I. B., Rezaian, M. A. 1998. Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus. Nucleic Acids Research, 26: 925-931.
Bennett, C. W. 1971. The curly top disease of sugarbeet and other plants. American Phytopathological Society, 7: 26-28.
Bolok Yazdi, H. R., Heydarnejad, J., and Massumi, H. 2008. Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide. Virus Genes, 36: 539-545.
Brown, J. K., Fauquet, C. M., Briddon, R. W., Zerbini, M., Moriones, E., and Navas-Castillo, J. 2012. Geminiviridae. In: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press. pp. 351-373.
Cheong, D. E., Choi, J. H., Song, J. J., Kim, G. J. 2013. Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins. Bioprocess and Biosystems Engineering, 36: 667-676. 
Gharouni-Kardani, S., Heydarnejad, J., and Zakiaghl, M. 2013. Diversity of Beet curly top Iran virus isolated from different hosts in Iran. Virus Genes, 46: 571-575.
Hanley-Bowdoin, L., Elmer, J. S., and Rogers, S. G. 1990. Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants. Proceedings of the National Academy of Sciences of the United States of America, 87: 1446-1450.
Heydarnejad, J., Hosseini Abhari, E., Bolok Yazdi, H. R., and Massumi, H. 2007. Curly top of cultivated plants and weeds and report of a unique curtovirus from Iran. Journal of Phytopathology, 155: 321–325.
Hirano, H., and Watanabe, T. 1990. Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis, 11: 573-580.
Lima, A. T., Sorbinho, R. R., Gonzales-Aguilera, J., Roch, C. S., Silva, S. J., Xavier, C. A., Silva, F. N., Duffy, S., and Zerbini, F. M.2012.Synonymous site variation due to recombination explains higher variability in begomovirus populations infecting non-cultivated hosts. Journal of General Virology 94: 418-431.
Jahanbin, D., Izadpanah, K., and Behjatnia, S. A. A. 2016. Comparison of natural and experimental host range of Beet severe curly top, Beet curly top Iran and tomato yellow leaf curl viruses. Iranian Journal of Plant Pathology, 51: 505-521.
Saida, F., Uzan, M., Odaert, B., and Bontems, F. 2006. Expression of Highly Toxic Genes in E. coli: Special Strategies and Genetic Tools. Current Protein & Peptide Science, 7: 47-56.
Soleimani, R., Matic, S., Taheri, H., Behjatnia, S. A. A., Vechiatti, M., Izadpanah, K., and Accotto, G. P. 2012. The unconventional geminivirus Beet curly top Iran virus: Satisfying Koch's postulates and determining vector and host range. Annals of Applied Biology, 162: 174-181.
Thommes, P.A., and Buck, K. W. 1994. Synthesis of the tomato golden mosaic virus AL1, AL2, AL3 and AL4 proteins in vitro. Journal of General Virology, 75: 1827-1834.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., and Rozen, S. G. 2012. Primer3: new capabilities and interfaces. Nucleic Acids Research, 40: e115.
Varsani, A., Navas-Castillo, J., Moriones, E., Hernandez-Zepeda, C., Idris, A., Brown, J. K., Murilo Zerbini, F., and Martin, D. P. 2014. Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Archives of Virology, 159: 2193-2203.
Zerbini, F.M., Briddon, R. W., Idris, A., Martin, D. P., Moriones, E., Navas-Castillo, J., Rivera-Bustamante, R., Roumagnac, P., Varsani, A., and ICTV Report Consortium. 2017. ICTV Virus Taxonomy Profile: GeminiviridaeJournal of General Virology, 98: 131-133.
Zhang, M., Chen, R., Zhou, X., and Wu, J. 2018. Monoclonal antibody-based serological detection methods for wheat dwarf virus. Virologica Sinica, 33: 173-180.