Fig. 1
From: Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus
(A) Schematic diagram of the parental plasmids (PP) used in the study. DNA sequences encoding sHBsAg_412–425, NS3/4A and NS5B were cloned into pMC.CMV-MCS-SV40polyA, containing attB and attP recombination sites. KpnI restriction sites are marked with black lines. (B) Analysis of MC DNA. Lanes contain pMC.CMV-MCS-SV40polyA plasmid with NS3/4A, NS5B and sHBsAg_412–425 sequences. Plasmids were purified from bacteria cultures pre-induction ([−] L-Arabinose) and after induction ([+] L-Arabinose) and were digested using KpnI enzyme. M—Gene O’Ruler DNA ladder mix. (C) Transfection of HEK293T cells with MC. HEK 293 cells were transfected with 2 µg of NS3/4A, NS5B and sHBsAg_412–425 MCs. For the detection of NS3/4A, NS5B and sHBsAg_412–425 recombinant proteins, anti-NS3, anti-NS5 antibodies and AP33 monoclonal antibody binding 412–423 region of HCV E2 glycoprotein were used. (D) Western blot analysis of the proteins expressed in HEK293T cells after transfection with PP (parental plasmid) and MC (minicircle). Cell lysates were separated using SDS-PAGE and detected with the anti-HBsAg, anti-NS3 and anti-NS5 antibodies. The lysate from HEK293T cells was used as the negative control (NC). On the right, the results of transfection with empty MC (eMC) are shown. On the left, protein ladder, the molecular weight in kDa is given. The original blots are presented in Supplementary Fig. S1.
