Figure 1

Design and expression of HBcAg-EDIII-2 antigen in E. coli . (A) A map of the HBcAg-EDIII-2 expression vector. The synthetic HBcAg-EDIII-2 gene is inserted under the control of the phage T7 promoter (pT7) in pET29a. The organization of different segments of this fusion gene is indicated in colour as follows. The HBcAg- and EDIII-2-encoding regions are shown in red and blue, respectively. The 6x His tag-encoding sequences at the 5’end is shown in black. The two linker-encoding sequences, the first following the 6x His tag and the second after the EDIII-2 encoding sequences, are shown in grey. Other abbreviations are as follows. Lac I: Lac repressor gene; Kan^R: Kanamycin marker; Ori: Replication origin sequences. (B) SDS-PAGE analysis of recombinant HBcAg-EDIII-2 expression. This panel displays the Coomassie-stained polypeptide profiles of lysates prepared from un-induced (U) and induced (I) E. coli cells harboring the plasmid shown in A. Pre-stained protein molecular weight markers were run in lane ‘M’. Their sizes (in kDa) are shown at the left of the panel. The arrow on the right indicates the position of the recombinant HBcAg-EDIII-2 protein. (C) Immunoblot analyses of recombinant protein expression. Aliquots of un-induced and induced cell lysates (described in panel ‘B’) were electrophoresced, electroblotted onto nitrocellulose membranes and probed with anti-EDIII mAb 24A12 (lane 2), penta His mAb (lane 3), or anti-HBcAg mAb ab8638 (lane 4). An aliquot of the un-induced cell lysate was probed with mAb 24A12 (lane 1). Pre-stained protein size markers were run in lanes marked ‘M’. Their sizes (in kDa) are indicated to the left of the first blot. The arrow to the right indicates the position of the recombinant HBcAg-EDIII-2 protein.