Comparative analysis of subcellular distribution of protein antigens in Mycobacterium bovis bacillus Calmette-Guérin.
The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.
Florio
W
W
Dipartimento di Biomedicina Sperimentale, Infettiva e Pubblica, Università degli Studi di Pisa, Italy. florio@biomed.unipi.it
Freer
G
G
Daila Casa
B
B
Batoni
G
G
Maisetta
G
G
Senesi
S
S
Campa
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis* / Q000302:isolation & purification / Q000648:ultrastructure; D001329:Autolysis; D001426:Bacterial Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D018834:Chaperonin 60 / Q000276:immunology / Q000302:isolation & purification; D017077:Culture Media, Conditioned / Q000032:analysis / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D007211:Indoles / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009163:Mycobacterium bovis / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D013482:Superoxide Dismutase / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002457:Cell Extracts; D018834:Chaperonin 60; D017077:Culture Media, Conditioned; D007211:Indoles; C093656:AG 85; D013482:Superoxide Dismutase