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 Escherichia coli DH5α strain

Ramin Soleimani, Mehdi Vasfi Marandi, Mohammad Bagher Hashemi-Soteh, Farhid Hemmatzadeh

Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Tehran-Iran, Tehran, Iran.
Medical Faculty and Cellular and Molecular Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
School of Animal and Veterinary Sciences, The University of Adelaide, Adelaide, Australia.

DOI: 10.4236/abb.2012.33040                

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.

Keywords

Avian Influenza Virus; H9N2 Subtype; Recombinant Protein; Non-Structural Protein 1; DIVA

Conflicts of Interest

The authors declare no conflicts of interest.

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