Cloning and expression of nonstructural gene 1(NS1) of local Egyptian strain H5N1
2012
Soliman, Z. M. A.
The highly pathogenic avian influenza (HPAI-H5N1) is a viral contagious disease that affects poultry industry and human health, The Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance, the vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serological surveillance in differentiation between infected and vaccinated animals (DIVA strategy). In This study the open reading frame of NS1 gene of the Egyptian virus A/chicken/Egypt/01112-NLQP/2011 (H5N1) was used as a template to produce DNA clone of the NS1 gene via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 gene. PCR Products were cloned into pJET1.2� plasmid and subsequently sub cloned into pQE2� expression vector to construct a recombinant plasmid. The Recombinant plasmid designated as pQE2�-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into DH5� E. coli strain by heat shock and expressed protein was isolated either a native or denatured form and further purified by immobilization on a nickel ion affinity column. Then the fusion protein was identified by SDS-PAGE, western Immunoblotting revealed the antigenic band of 26kDa NS1 of HPAI using polyclonal antibody. from this work we established a rapid, easy, and reproducible protocol for cloning and expression of (NS1) gene of H5N1egyptian virus, and indicated that the NS1 protein suitable in a DIVA for differentiation of AI infected and vaccinated chickens and further applications.
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