Date of Award
Master of Science
Mycoplasma hyopneumoniae causes a chronic respiratory disease in swine. It is an important component of the porcine respiratory disease complex and has been shown to enhance disease due to other pathogens. Few definitive studies of the surface architecture of M. hyopneumoniae have been reported although mycoplasmas in general abound with antigenic surface lipoproteins and other structures that contribute to disease. Hindering studies in M. hyopneumoniae are the inability to grow the organism on agar surfaces and to genetically manipulate the organism. Some monoclonal antibodies are available, but they have not been fully characterized to identify the gene product recognized. To further study the surface proteins of M. hyopneumoniae it was necessary to raise polyclonal antibodies against specific gene products. This approach was difficult because mycoplasma gene sequences contain TGA codons, usually a stop codon, that code for tryptophan. Also, mycoplasma gene sequences express poorly in Escherichia coli, the normal expression host. With the completion of the M. hyopneumoniae genome sequence, the entire repertoire of lipoprotein genes was now available. A DNA vaccine approach was chosen to induce monospecific, polyclonal antisera in rabbits. Putative open reading frames from M. hyopneumoniae were cloned successively into pCR2.1 and then into the expression vector pSTBO78 after being corrected for TGA codon usage. The expression vector constructs were then used to vaccinate rabbits in an effort to raise polyclonal antibodies against the encoded gene. Rabbit serum was tested by immunoblot and immunofluorescence to verify the presence of antibodies against the protein encoded by the DNA vaccine. Results showed positive results in twenty-eight of the thirty-four of the ORFs used for vaccination. Immunoblots showed protein products of the expected size in eighteen ORFs and only smaller cleaved products in four. Three ORFs showed regular banding patterns of smaller protein products, alluding to a mechanism of size variation in the final protein. Immunofluorescence gave positive results that reflected surface proteins available to the host during infection for antibody recognition.
David Claude Oneal
Oneal, David Claude, "Analysis of the lipoproteins of Mycoplasma hyopneumoniae" (2004). Retrospective Theses and Dissertations. 17514.