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Cloning and expression of equine influenza virus nonstructural gene in prokaryotic system


Citation

Abdulhadi, Abdulrahman Dahham and Fateh Mohamed, Bashir Ahmad and Omar, Abdul Rahman and Bejo, Mohd Hair (2012) Cloning and expression of equine influenza virus nonstructural gene in prokaryotic system. In: International Conference on One Health and 24th VAM Congress 2012, 21-23 Sept. 2012, Marriott Putrajaya. (pp. 296-298).

Abstract / Synopsis

Equine influenza virus (EIV) is a highly contagious and widely distributed viral respiratory disease of equiadae caused by type A influenza virus from the family Orthomyxovirus. Rapid diagnoses of affected horses are the front line of defence against outbreaks and an important step for epidemiological investigation. The objective of the present study was to clone, express, and purify the non-structural (NS1) protein of EIV subtype (H3N8). To obtain and develop an effective diagnostic method for equine influenza virus, the NS1 gene of H3N8 subtype was amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Products were cloned into pCR2.0 using the TOPOTM TA cloning vector and subsequently sub-cloned into a prokaryotic expression vector, pRSET B. Recombinant plasmid designated as pRSET B-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion and nucleotide sequence analysis. The results showed that the recombinant NS1 was expressed in the E. coli strain BL21 (DE3)plysS after induction with IPTG. The 6x His-tagged recombinant fusion proteins were purified using the ProBondTM purification system and the expressed protein was identified by SDS-PAGE and western-blotting. A recombinant protein of approximately 13 kDa was produced.


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Additional Metadata

Item Type: Conference or Workshop Item (Poster)
Divisions: Faculty of Veterinary Medicine
Institute of Bioscience
Keywords: Equine influenza virus; Non-structural protein; Recombinant plasmid; SDS-PAGE; Western-blotting
Depositing User: Nabilah Mustapa
Date Deposited: 02 Feb 2017 12:44
Last Modified: 02 Feb 2017 12:44
URI: http://psasir.upm.edu.my/id/eprint/50206
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