The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in <i>Escherichia coli</i> DH5<i>α</i> strain
- 1 Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Tehran-Iran, Tehran, Iran
- 2 Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Tehran-Iran, Tehran, Iran
- 3 Medical Faculty and Cellular and Molecular Research Center, Mazandaran University of Medical Sciences, Sari, Iran
- 4 School of Animal and Veterinary Sciences, The University of Adelaide, Adelaide, Australia
Abstract
Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.
- Bouvier, N.M. and Palese, P. (2008) The biology of influenza viruses. Vaccine, 26, 49-53. doi:10.1016/j.vaccine.2008.07.039
- Webster, R.G., Bean, W.J., Gorman, O.T., Chamber, T.M. and Kawaoka, Y. (1992) Evolution and ecology of influenza A viruses. Microbiological Reviews, 56, 152-179.
- Wright, P.E., Neumann, G. and Kawaoka, Y. (2007) Orthomixoviruses. In: Fields Virology, 5th Edition, Lippincott, Willams, Philadelphia, 1692-1731.
- Nichols, J.E. and Leduc, J.W. (2009) Influenza. In: Alan, D.T.B. and Lawrence, R.S., Eds., Vaccines for Biodefence and Emerging and Neglected Diseases, Academic Press, London, 497-525. doi:10.1016/B978-0-12-369408-9.00027-5
- FAO, AIDEnews (2011) Bird flu rears its head again: Increased preparedness and surveillance urged against variant strain. 7 September 2011, 1-8.
- Alexander, D.J. (2007) An overview of the epidemiology of avian influenza. Vaccine, 25, 5637-5644. doi:10.1016/j.vaccine.2006.10.051
- Alexander, P.E., De, P. and Rave, S. (2009) Is H9N2 avian influenza virus a pandemic potential? Canadian Journal of Infectious Disease & Medical Microbiology, 20, 35-36.
- Swayne, D.E. (2003) Vaccines for list A poultry diseases: Emphasis on avian influenza. Developmental Biology, 114, 201-212.
- Capua, I. and Alex-ander, D.J. (2008) Avian influenza vaccines and vaccination in birds. Vaccine, 26S, 70-73. doi:10.1016/j.vaccine.2008.07.044
- Halvorson, D.A. (2002) The control of H5 or H7 mildly pathogenic avian influenza—A role for inactivated vaccine. Avian Pathology, 31, 5-12. doi:10.1080/03079450120106570
- Capua, I., Cattoli, G. and Marangon, S. (2004) DIVA—A vaccination strategy enabling the detection of field exposure to avian influenza. Developmental Biology, 119, 229- 233.
- Suarez, D.L. (2005) Overview of avian influenza DIVA test strategies. Biologicals, 33, 221-226. doi:10.1016/j.biologicals.2005.08.003
- Suarez, D.L., Lee, C.W. and Swayne, D.E. (2006) Avian influenza vaccination in north America: Strategies and difficulties. Developmental Biology, 124, 117-124.
- Beard, C.W. (1989) Serological procedures. In: Friedman, H., Ed., A Laboratory Manual for the Isolation and Identification of Avian Pathotypes, Kendall Hunt Publishing, Dubuque, 192-200.
- Avellaneda, G., Mundt, E., Lee, C.W., Jadhao, S. and Suarez, D.L. (2010) Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus. Avian Diseases, 54, 278-286. doi:10.1637/8644-020409-Reg.1