Purification and partial characterization of the major outer membrane protein of Haemophilus somnus.
We purified the major outer membrane protein (MOMP), which is the most abundant OMP (with an apparent molecular mass of 40 kDa), from Haemophilus somnus strain 8025. The method involves solubilization of the MOMP with Zwittergent 3-14 and further purification accomplished by ion-exchange and molecular-sieve chromatographies. The amino-terminal sequence of the MOMP showed considerable similarity to those of porin proteins from other gram-negative bacteria. The MOMP of H. somnus is immunogenic to rabbits and calves. Hyperimmune sera from rabbits and calves reacted with both the MOMP and lipopolysaccharides in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The rabbit antiserum to the MOMP was cross-reactive with whole-cell preparations from strains 8025, D1238, NT2301, and 540 at a band with a molecular mass of 40 kDa in immunoblot analysis, although the reactivity of the rabbit antiserum with strain 540 was lower than those with the other strains tested. Two murine monoclonal antibodies (MAbs) to the MOMP were developed. ELISA with the OMP fractions as the antigens showed that one MAb was cross-reactive with the four strains but that the other MAb was reactive with the three strains other than strain 540. These results indicate that the MOMP of H. somnus possesses at least two antigenic determinants and that the MOMP of strain 540 is antigenically different from those of the other strains. The antigenic heterogeneity of the H. somnus MOMP has implications regarding the development of a serotyping system with MAbs that is based on the MOMP epitopes.
Tagawa
Y
Y
Division of Systematic Diagnosis Research, National Institute of Animal Health, Ibaraki, Japan.
Ishikawa
H
H
Yuasa
N
N
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006190:Haemophilus; D015151:Immunoblotting; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; C078911:major outer membrane protein, Haemophilus