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 DH5 alpha strain

Abstract

Avian influenza is a viral contagious disease that af- fects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradi- cation of AI, but it causes some limitations including trade embargoes and interfering with serologic sur- veillance in differentiation between infected and vac- cinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limi- tations. 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 puri- fication of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plas- mids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screen- ed 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 molecu- lar weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reac- tion using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera pre- pared 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 in- fected and vaccinated chickens.