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

The expression levels of pHCVcp by each of the vectors (pBI-core as a classical plant binary vector and PVX-core as a viral-based vector) in the presence or absence of P-19 co-agroinfiltration were compared by ELISA. Sandwich ELISA is the assay of choice and is usual in quantification of antigen proteins (when an exact and precise standard curve is prepared and a specific antibody against the antigen is used) and a number of diagnostic ELISA kits work on this basis (45, 46) . In this context and to perform an exact and precise quantitative ELISA, we used a specific monoclonal antibody against the core antigen (at the bottom of ELISA plates) and polyclonal anticore antibodies (at the top of the pHCVcp) in a sandwich ELISA-based protocol and also prepared a precise standard curve by applying serial dilution (0.1-1 µg/mL; data not shown) of purified eHCVcp to calculate the expression levels of pHCVcp. They were expressed in form of the percentage of TSP, which was determined using a Bradford protein assay. The leaves transformed by PVX (vector without HCVcp) and co-agroinfiltrated with P19 constructs were used as negative control (Figure 5 A) . The results indicated that the expression of pBI-core and PVX-core was around 0.004% of TSP in the absence of P19 co-agroinfiltration, whereas in its presence, the expression level of pBI-core and PVX-core increased to 0.019% (210 ng/g) and 0.022% (240 ng/g) of TSP fresh leaf weight, respectively, which was almost five times improvement for pHCVcp expression (Figure 5 A) .