Antiviral Ability of Kalanchoe gracilis Leaf Extract against Enterovirus 71 and Coxsackievirus A16
Figure 8
In vitro recombinant 2A protease enzymatic assay. SDS-PAGE (a) and Western blot analysis (b) of the purified 2A protease. Recombinant 2A protease protein was expressed after induction of IPTG treatment and purified through fusion His-tag bound to Ni-NTA agarose column. Purified recombinant 2A protease proteins were resolved on 10% SDS-PAGE and transferred onto nitrocellulose paper. The blot was probed with anti-His-tag and developed with an alkaline phosphatase-conjugated secondary antibody and NBT/BCIP substrates. Lane 1 represents protein molecular weight markers. Lanes 2–9 show the fractions of the purified 2A protease proteins. For characterization of recombinant 2A protease activity in vitro (c), purified 2A protease at concentrations of 0, 0.75, 1, 2.5, 5 and 10 μg/mL were incubated with HRP (0.5 μg/mL concentration) for 2 h at 37°C. Mixtures were developed with ABTS/H2O2 and measured at OD405. For inhibitory enzymatic assays (d), K. gracilis leaf extract at concentration of 1, 10, 50, 100, or 150 μg/mL was added into the mixture of purified 2A protease and HRP then incubated for 2 h at 37°C in 96-well plates in vitro. Mixtures were developed with ABTS/H2O2 and measured at OD405. Percentage of inhibition of 2A protease activity was calculated as ()/() . *P value < 0.05; **P value < 0.01; ***P value < 0.001 by Scheffe’s test.
