Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications

Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

To inhibit the silencing of foreign gene expression in tobacco, the constructed expression vectors were coinfiltrated with Agrobacterium strain LB4404, harboring the p19 silencing-suppressor gene (42) from tomato bushy stunt virus (TBSV) (GenBank accession number: M21958), which was previously cloned into pCAMBIA 1304 vector (CAMBIA Co., Australia) in our lab (P19-pCAMBIA 1304 vector). The agroinfiltration protocol was according to Kapila et al. study (43) . In brief, the Agrobacterium cultures were first adjusted to OD 600 of 1.0 with MS medium supplemented with 2% (w/v) sucrose, 10 mM MES pH = 5.5, 10 mM MgCl 2 , and induced for an additional 3 hours with 400 µM acetosyringone. The Agrobacterium suspensions were subsequently mixed in equal ratios (half for p19 plasmid and half for each of the expression constructs, either PVX-core or pBI-core) and the N. tabacum (cultivar Jafarabadi; kindly provided by Dr. Tohidfar, Agricultural Biotechnology Research Institute of Iran) leaves were immersed in the bacterial suspension while a vacuum of 0.5 mbar was applied for two minutes. The infiltrated leaves were placed adaxial side down on wet Whatman paper in glass containers and subsequently incubated at 25°C with a 16-hour photoperiod for five days.