Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment.
A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K. S. Wise and M. F. Kim, J. Bacteriol. 169:5546-5555, 1987). In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes. Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments. Graded carboxypeptidase-Y digestion of intact M. hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane. However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli. A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates. Serum antibodies from swine taken after, but not before, experimentally induced M. hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product. This confirmed that the p65 lipoprotein is a major immunogen of M. hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain.
Kim
M F
MF
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia 65212.
Heidari
M B
MB
Stull
S J
SJ
McIntosh
M A
MA
Wise
K S
KS
D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D015151:Immunoblotting; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D009174:Mycoplasma / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013552:Swine
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D008074:Lipoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins