Purification and Inactivation of Recombinant Phospholipase D: Approaches to Developing a Corynebacterium Pseudotuberculosis Vaccine
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Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes one of the most frequent equine infectious diseases in the Western United States. Horses exposed to the equine C. pseudotuberculosis biovar develop a disease known as ?pigeon fever? or ?dryland distemper,? indicative of the arid California setting where it occurs. Pigeon fever manifests in three main forms: large external abscesses in the pectoral and ventral regions of the abdomen, internal abscesses associated with a high mortality rate, and ulcerative lymphangitis. Dr. Pollock?s research focuses on developing a vaccine that will combat the major exotoxin produced by this bacterium: phospholipase D (PLD). This study focuses on purifying and inactivating PLD, needed for various assays and vaccine development. PLD was purified from E. coli bacteria containing a recombinant histidine-tagged PLD (His-PLD) gene. To maximize yield and purity, the protocol was revised to include longer incubation times for bacteria growth, as well as altered concentrations of imidazole washes. Purified PLD was then inactivated, reducing toxic function and allowing better antibody-production by the immune system, since greater doses of PLD can be used to immunize animals. There are two methods of inactivation: chemically with formalin and genetically via site-specific mutagenesis. Though the former is currently used, the latter would be a more effective, potentially safer technique and is a major focus of our research. The His-PLD plasmid was isolated and digested with EcoRI and PvuI to confirm its identity. Once the PLD gene sequence has been confirmed, the plasmid will be sent out for site-specific mutagenesis.