Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library

Abstract

A genomic library of Mycoplasma hyopneumoniae was constructed by cloning random DNA fragments approximately 300 base pairs long in a fusion expression plasmid, pEx29, containing the N terminus of the phage MS2 polymerase under the control of the PL promoter of phage lambda. Clones that produced fusion proteins carrying surface-specific antigenic determinants were identified by using antiserum raised in a pig by intranasal inoculation of viable mycoplasmas. Rabbit antisera produced against gel-purified fusion proteins synthesized in Escherichia coli were analyzed by Western blotting to identify antigenically related mycoplasma components. Distinct mycoplasma proteins termed P90, P68, P50, P30, and P26 were identified. Evidence for the surface location of P90, P68, and P50 was provided by their sensitivity to trypsin and their comigration with lactoperoxidase-catalyzed iodinated proteins of intact mycoplasmas. Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, also showed that its antigenic determinant is associated with the mycoplasma surface.