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. 2014 Jan 7;4:487. doi: 10.3389/fimmu.2013.00487

Figure 1.

Figure 1

Purification and immunological characterization of the recombinant HIV-1 p24 protein. (A) SDS-PAGE analysis of whole-cell extracts of the recombinant bacterial strain and purified p24 protein. Samples: lane 1, whole-cell extract of the E. coli BL21(DE3) strain transformed with the plasmid encoding the recombinant p24 cultured without addition of the inducer; lane 2, whole-cell extract of the E. coli BL21(DE3) strain transformed with the plasmid encoding the recombinant p24 cultured after addition of the inducer; lane 3, purified p24 after affinity chromatography in a nickel-containing resin. MW, PageRuler™ pre-stained Protein Ladder (Fermentas). One to two lines were loaded with 10 μL of whole-cell extract of E. coli at D.O.600nm 4 before (line 1) or after (line 2) IPTG addition or 3 μg of purified p24 protein (line 3); (B) Immunoblot developed with p24-specific rabbit polyclonal serum. Protein samples were the same as described above. (C) SDS-PAGE analysis of purified p24 protein not completely separated on 15% polyacrylamide gels. (D) Antigenicity of the recombinant p24. The protein was employed as solid phase-bound antigen in ELISA plates developed with sera obtained from an HIV-1 infected subject (open squares) or from a rabbit immunized with recombinant p24 (closed squares). (E) Antibodies raised in mice immunized with the recombinant p24 recognize the native viral protein. Cells of the A3.01 lineage were transfected with the pHXB2 vector encoding the complete HIV-1 genome. After 7 days, the cells were fixed and incubated with serum from mice immunized with p24 combined with Freund’s adjuvant (lower panel series) or PBS (upper panel series), at a final dilution of 1:500. Cells were labeled with FITC-conjugated anti-mouse IgG antibody and DAPI. The experiments were performed twice showing the same results.