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. 2004 May;72(5):2791–2802. doi: 10.1128/IAI.72.5.2791-2802.2004

FIG. 1.

FIG. 1.

Map of the cilium adhesin of M. hyopneumoniae and immunoblot analysis using MAb F1B6 and anti-peptide, P97 N-terminal, and R2 antisera. (A) The map shows antibody epitope locations, repeat regions, and selected cleavage sites identified by peptide mass fingerprint analysis, fragment masses, and N-terminal sequences. The major cleavage event at amino acid 195 (large arrow) and another event at amino acid 891 (small arrow) are shown. The locations of the R1 and R2 repeat regions are represented by gray-shaded boxes. The locations of the epitope for MAb F1B6, ΔNP97 peptide, and NP97, P97 N-terminal (P97 N-term), and P28 antisera are shown as bars above or below the map. The coiled-coil regions are represented by the black boxes. The cleavage products of strains J and 232 are shown as gray-shaded bars below the map. Their molecular masses and N-terminal sequences are shown to the right of the map. ND, not determined. *, the peptide TSSQKDPSTLR was identified by peptide mass fingerprinting P70 from strain 232, suggesting that this molecule has the same N-terminal sequence as P97. (B) Immunoblot patterns of M. hyopneumoniae strain J reacted with F1B6 (1:3,000), ΔNP97 (1:20), and NP97 (1:20). An SDS-12% polyacrylamide gel was used to resolve J strain cell lysates. Molecular mass markers (MBI Fermentas) are shown on the left. Equivalent amounts of M. hyopneumoniae cell lysate were loaded in lanes reacted with ΔNP97 (lane 2) and NP97 (lane 3) antibodies. The amount of protein loaded in lane 1 (reacted with MAb F1B6) was approximately one-third of that loaded in lanes 2 and 3. (C) Immunoblot patterns (12% polyacrylamide gel) of synchronized M. hyopneumoniae strain J cultures harvested at different times postinoculation. The blot was reacted with MAb F1B6 (1:1,000). Equivalent amounts of protein were loaded in each lane. Lane 1, 8 h of growth (late lag phase); lane 2, 16 h of growth (early exponential phase); lane 3, 20 h of growth (mid-log phase); lane 4, 24 h of growth (mid-log phase); lane 5, 28 h of growth (mid-log phase); lane 6, 40 h of growth (late log-early stationary phase); lane 7, 48 h of growth (stationary phase); lane 8, 52 h of growth (stationary phase); lane 9, 56 h of growth (stationary phase). (D) Immunoblot patterns (12% polyacrylamide gel) of synchronized M. hyopneumoniae strain J (left panel) and 232 (right panel) cultures harvested at different times postinoculation. The blot was reacted with P97 N-terminal serum (1:100). Equivalent amounts of protein were loaded in each lane. Lane 1, 20 h of growth (mid-log phase); lane 2, 28 h of growth (mid log phase); lane 3, 40 h of growth (late log-early stationary phase); lane 4, 56 h of growth (stationary phase). (E) Immunoblot patterns (10% polyacrylamide gel) of synchronized M. hyopneumoniae strain J (left panel) and 232 (right panel) cultures harvested at different times postinoculation. The blot was reacted with R2 serum (1:100). Equivalent amounts of protein were loaded in each lane. (Left panel) Lane 1, 8 h of growth (late log phase); lane 2, 16 h of growth (early exponential phase); lane 3, 20 h of growth (mid-log phase); lane 4, 24 h of growth (mid-log phase); lane 5, 28 h of growth (mid-log phase); lane 6, 40 h of growth (late log-early stationary phase); lane 7, 48 h of growth (stationary phase). (Right panel) Lane 1, 8 h of growth (late log phase); lane 2, 16 h of growth (early exponential phase); lane 3, 24 h of growth (mid-log phase); lane 4, 28 h of growth (mid-log phase); lane 5, 32 h (mid-log phase); lane 6, 40 h of growth (late log-early stationary phase); lane 7, 48 h of growth (stationary phase). (F) Comparison (using NP97 and ΔNP97 antisera) of immunoblot patterns of strains 232 and J. Molecular weight markers (Bio-Rad Precision Proteins Standards) (broad range) are shown on the right of the panel. An 8 to 16% Bio-Rad Criterion gradient gel was used to separate the proteins.