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

A). Dot blotting; at control column: row 1: negative control (25 μg TSP of untransformed tobacco leaves); row 2: 5 μg positive control (eHCVcp). At PVX column: negative control (tobacco leaves transformed by PVX vector alone; ie, without Tr-HCVcp). PVX-core and pBI-core columns: the extracts from the PVX-core and pBI-core P19 co-agroinfiltrated leaves, respectively. Rows 2 and 1 in these two last columns correspond to 5 µg and 25 µg of TSP, respectively. B) Coomassie-stained 12% SDS-PAGE gel, loaded with; lane 1: 5 μg concentrated plant-purified HCVcp (from the PVX-core expression system). Lane 2: 5 μg of purified eHCVcp. Lane 3: crude protein extract from agroinfiltrated leaves with PVX-core (20 µg). Lane 4: untransformed leaves (20 µg). C) Western blotting; lane 1: positive control (700 ng purified eHCVcp). Lane 2: purified pHCVcp (700 ng from the PVX-core expression system). Lanes 3 and 4: the extracts from P19 co-agroinfiltrated PVXcore and pBI-core leaves, respectively (50 µg of plant TSP was applied in each lane). Lane 5: negative control (50 µg of plant TSP of untransformed tobacco leaves). Lane M: prestained protein ladder (Fermentas). HCVcp denote to HCV core protein, eHCVcp and pHCVcp dnote to E.coli-derived and plant-derived HCVcp, respectively. In western blot and SDS-PAGE figures, the location of HCVcp under the 20 kDa molecular weight range is shown by arrows. The reason for multiple bands in the case of eHCVcp is explained in the corresponding result section of the text. The results of SDS-PAGE showed that Ni-NTA pull-downs of plant extract contained endogenous nonspecific plant proteins besides pHCVcp. According to ELISA data, the concentration of pHCVcp was 1/20 of the column-purified protein. Therefore, although 1 µg (100 µL of 10 μg/mL coated) was coated, only a small amount reacted (less than 50 ng) (Figure 5 B) . However, none of these endogenous nonspecific plant proteins reacted with anti-HCVcp in western blotting of the purified protein fraction.