Journal: Clinical and Vaccine Immunology
Article Title: Generation of Mucosal Anti-Human Immunodeficiency Virus Type 1 T-Cell Responses by Recombinant Mycobacterium smegmatis▿
Figure Lengend Snippet: Expression of HIV-1 CON6 gp120 and gp140CF envelope proteins in M. smegmatis . (A) Western blot analysis of CON6 Env protein expression in various M. smegmatis designs, including molecular weight (Mr.) standards (lane 1), untransformed M. smegmatis (lane 2), and recombinant M. smegmatis transformed with either empty plasmid (lane 3), the surface expression plasmid pJH152-CON6 gp120 (lane 4), the intracellular expression plasmid pJH153-CON6 gp120 (lane 5), the secreted expression plasmid pJH154-CON6 gp120 (lane 6), or the surface expression plasmid pJH152-CON6 gp140 (lane 7) or pJH222-CON6 gp140 (lane 8). Both the intact (89 kDa for gp140, 79 kDa and 74 kDa for gp120) and partially cleaved (49 kDa and 34 kDa) CON6 Env protein products, as indicated by arrows, were found with gp120 MAb T8 (anti-C1 gp120 region), gp41-specific MAb 7B2, and V3 loop-specific MAb 7B9. (B) Schematic representation of the full-length (top) and cleaved (bottom) gp140, indicating the binding locations of MAbs T8, 7B2, and 7B9. Full-length gp140 could be detected by T8, 7B2, or 7B9, while the cleaved 34-kDa Env protein band could be detected only by MAb T8. A 50-kDa protein band could be detected by both MAbs 7B2 and 7B9. (C) Western blot analysis of supernatants from untransformed M. smegmatis (lane 1) and recombinant M. smegmatis transformed with the empty plasmid (lane 2) or pJH154-CON6gp140CF (lane 3) for detecting secreted CON6 Env protein expression. A truncated gp140 protein (lane 3) could be detected by 7B9, as indicated by the arrow. A high-molecular-weight protein band, as indicated by an arrowhead, was nonspecific, as it was also detected in recombinant M. smegmatis transformed with pJH154-CON6gp140CF (lane 3) and in the negative control lanes 1 and 2.
Article Snippet: Figure shows that the expression of the intact CON6 gp120 was detected in recombinant M. smegmatis transformed with either the surface expression plasmid pJH152 (Fig. , lane 4), the intracellular expression plasmid pJH153 (lane 5), or the secreted expression plasmid pJH154 (lane 6).
Techniques: Expressing, Western Blot, Molecular Weight, Recombinant, Transformation Assay, Plasmid Preparation, Binding Assay, Negative Control