Expression of the recombinant coat protein of Potato virus X in Escherichia coli

Document Type : Research Paper

Authors

1 Department of Plant Pathology, Shahid Bahonar University of Kerman

2 Department of Plant Pathology, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

3 Professor, Department of Plant Pathology, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.

4 Associate professor, Department of Plant Biotechnology, College of Agriculture, Shahid Bahonar University of Kerman, Kerman,

Abstract

Objective
Potato virus X (PVX) is a member of the genus Potexvirus in the family Alfaflexiviridae. This virus is one of the most common and widespread viruses infecting potato worldwide. Due to the wide distribution and economic damage of the virus in Iran, identification and detection of the virus is necessary using non-expensive methods. Production of polyclonal antibody through molecular approaches can be a useful method for detection of virus in the nature.
 
Materials and methods
 In this research, an infected potato sample was collected from Zarand (Kerman province) and its PVX infection was confirmed by ELISA test. This sample was inoculated on test plant and the coat protein (CP) gene of this isolate was amplified in RT-PCR test with specific primers comprising BglII and NcoI restriction enzymes. Amplified product was cloned into expression prokaryotic vector (pQE60 plasmid). followed by transformation of the E. coli strain M15 competent cells.
 
Results
Subsequent to induction of CP expression, total protein was extracted and run onto 12.5% SDS-PAGE. An approximate 24 kDa band was observed on the gel corresponded to the PVX coat protein. Furthermore, expression of the PVX CP was confirmed by dot blot technique.
 
Conclusions
In this study, a recombinant antigen suitable for the detection of potato X virus was prepared which was well identified by antiserum produced by injection of complete virus particles into rabbits in dot blot analysis. The production of multiclonal antibodies by recombinant viral antigen will be an important step in accelerating and facilitating the serological identification process of plants infected with this virus.

Keywords

References

References
Abdel-Salam AM, Abdel-Kader HS, El-Saghir SM (2005) Biological, serological, and molecular detection of banana streak badnavirus in vegetatively propagated banana plants in Egypt. Egypt J Virol 2, 255-268.
Abdel-Salam AM, Abdel-Kader HS, El-Saghir SM, Hussein MH (2004) Purification, serology, and molecular detection of Egyptian isolates of banana bunchy top babuvirus and faba bean necrotic yellows nanovirus. Arab J Biotechnol 7, 141-155.
Abdel-Salam AM, EI-Attar AK, Gambley CF (2014) Production of polyclonal antisera to a recombinant coat protein of Potato viruas Y expressed in Escherrichia coli and its application for immunodiagnosis. Int J Virol 10, 1-16.
Abouzid AM, Freitas-Astua J, Purcifull DE et al. (2002) Serological studies using polyclonal antisera prepared against the viral coat protein of four begomoviruses expressed in Escherichia coli. Plant Dis 86, 1109-1114.
Adamas MJ, Accotto GP, Agranovsky AA, Bar-Joseph M, Brunt AA (2005) Family Flexiviridae, In: Fauquet CM, Mayo MA, Maniloff  J, Desselberger  U, Ball La (eds), Virus taxonomy: the eighth report of the ICTV. Academic press, New York, p. 1089-1124.
Amersham Pharmacia Biotech (2000) The Recombinant Protein Handbook – Protein amplification and simple purification. Amersham Pharmacia Biotech AB, Sweden.e.
Baneyx F (1999) Recombinant proteins expression in Escherichia coli. Curr Opin Biotechnol 10, 411-421.
Beemster ABR, Rozendaal A (1972) Potato viruses: Properties and symptoms, In: J.A. de Bokx (ed.), Viruses of potatoes and seed-potato production. PUDOC, Wageningen, p. 115-143.
Beuve M, Sempé L, Lemaire O (2007) A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine. J Virol Methods 141, 117-124.
Bostan H, Haliloglu K (2004) Distribution of PLRV, PVS, PVX and PVY (PVYN, PVYO, and PVYC) in the seed potato tubers in Turkey. PJBS 7, 1140-1143.
Buchen-Osmond C (1987) Potato X Potexvirus. Description and lists from the VIDE database. From http://bio-mirror.im.ac.cn/mirrors/pvo/vide/descr651.htm.
Cerovska N, Moravec T, Plchova H et al. (2010) Production of polyclonal antibodies to Potato virus X using recombinant coat protein.  J Phytopathol 158,66-68.
Cerovska N, Moravec T, Rosecka P et al. (2003) Production of polyclonal antibodies to a recombinant coat protein of Potato map-top Virus. J Phytopathol 151, 195-200.
Clark MF, Adams SAN (1997) Characteristics of microplates method of enzyme-linked-immunosorbent assay for detection of plant viruses. J Gen Virol 34, 475-483.
Cockerham G (1955) Strains of potato virus X, In: Streutgers E, Beemster ABR, van der Want JPH (eds.), Proceeding of 2nd conference on potato virus diseases. Lisse-Wageningen p. 89–92.
Cox AB, Jones RAC (2010) Genetic variability in the coat protein gene of Potato virus X and the current relationship between phylogenetic placement and resistance groupings. Arch 155, 1349–1356
Fajardo TVM, Barros DR, Nickel O, et al. (2007) Expression of Grapevine leafroll-associated virus-3 coat protein gene in Escherichia coli and production of polyclonal antibodies. Fitopatol. Bras 32, 496-500.
Feigelstock DA, Tozzini AC, Hopp HE (1995)Coat protein sequence of a resistance-breaking strain of Potato virus X isolated in Argentina. Virus Genes 10, 289-292.
Fischer B, Sumer I, Goodenough P (1993) Isolation, renaturation and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol Bioeng 41, 3-13.
Folwarczna J, Plchová H, Moravec T et al. (2008) Production of polyclonal antibodies to a recombinant coat protein of Potato virus Y. Folia Microbiologica 53, 438-442.
Janknecht R, de Martynoff G, Lou J et al. (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. PNAS 88, 8972-8976.
Kalendal R (2005) Fast PCR primer design and report sequence searching program with additional tools for the manipulation and analysis of DNA and protein. From http://www.Biocenter.helsinki.fi/bi/programs/fastPCR.html
Khan S, Jan AT, Mandal B et al. (2012) Immunodiagnostics of cucumber mosaic virus using antisera developed against recombinant coat protein. Archives of Phytopathology and Plant Protection 45, 561-59.
Koening R, Lesemann DE (1989) Potato virus X, potexvirus group. AAB 345, 1–5.
Koohapitagtam M, NualsriC (2013) Production of polyclonal antibodies specific to the recombinant coat protein of Blackeye cowpea mosaic virus and its use in disease detection. Kasetsart J (NaturalScience) 47, 603-613.
Ling KS, Zhu HY, Jiang ZY, Gonsalves D (2000) Effective application of DAS-ELISA for detection of grapevine leaf roll associated closterovirus-3 using a polyclonal antiserum developed from recombinant coat protein. Eur 106, 301-309.
Massumi H, Poormohammadi S, Pishyar S et al. (2014) Molecular characterization and field survey of Iranian potato virus X isolates. Virusdisease 25, 338-344.
Qiagen (2003) The QIAexpressionistTM - A handbook for high-level expression and purification of 6xHis-tagged proteins. Qiagen, Hilden, Germany.
Roland, J (2008) Bacterial protein expression – test induction. From: http://josephroland.com/lab/protocols/induction.html Last accessed 30 August 2019.
Sadeghan A, Shams-bakhsh M, Yakhchali B (2013) Expression of Citrus tristeza virus coat protein gene in Escherichia coli. Prot 2, 387-393.
Schwarz E, Scherrer G, Hobom G, Kossel H (1978) Nucleotide sequence of cro, cll and part of the 0 gene in phage lambda DNA. Nature 272, 410-414.
Shamsadden-Saeed F, Massumi H, Moradi S, et al. (2014) Incidence and characterization of Potato virus V infections in Iran. Virusdisease 25,78-84
Shams-bakhsh M, Khakvar R, Poor-Rahim R (2001) Purification, preparation of antiserum and serological diagnosis of potato virus X and Y isolated from Khuzestan province farms. J Agric Sci 24, 47-53.
Soliman AM, Barsoum BN, Mohamed GG et al. (2006) Expression of coat protein gene of the Egyptian isolate of Potato virus X in Escherichia coli and production of polyclonal antibodies against it. "Arab J Biotechnol 9, 115-128.
Van Regenmortel MHV (1982) Serology and immunochemistry of Plant Viruses. Academic Press, INC. New York.e.
Verchot-Lubicz J,Ye CM, Bamunusinghe D (2007) Molecular biology of potexviruses: recent advances. J Virol 88,1643-1655.
Yu XQ, Wang HY, Lan YF (2008) Complete genome sequence of a Chinese isolate of Potato virus X and analysis of genetic diversity. Phytopathol 156, 346–351.
Agricultural Biotechnology Journal
Volume 11, Issue 4
Volume 11, No 4
December 2019
Pages 175-192

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