Browsing by Author "Ly, Kristina Elisabeth"

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    Expression and Purification of the C-Terminal Domain of Porcine Epidemic Diarrhea Virus (PEDV) S1 Protein
    Ly, Kristina Elisabeth (Virginia Tech, 2024-10-29)
    Porcine Epidemic Diarrhea Virus (PEDV) was first detected in Europe in the 1970s, but did not emerge in the United States until 2013. When it arrived, it ran rampant due to the lack of previous exposure, causing the death of 7-8 million neonatal piglets and $900 million to $1.8 billion in losses to the U.S. pork industry in 2013 and 2014. This virus causes diarrhea and vomiting which leads to dehydration and in extreme cases, death. Neonatal piglets rely heavily on passive lactogenic immunity from their mother's milk, thus making them especially vulnerable to this disease. Within 2-3 days of infection during the initial outbreak, there was a 90-95% mortality rate among these weaning piglets. Additionally, this virus is highly contagious, with high rates of fecal shedding during infection. To control the outbreak, the USDA had approved two emergency-relief vaccines, but both have proved to be ineffective at preventing disease or reducing fecal shedding. These vaccines are still available today. As such, it is necessary to develop a vaccine that will be effective at preventing illness and viral shedding. PEDV is a single-stranded RNA virus made of four major subunits: a structural spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The one most studied and of particular interest is the S protein as it facilitates the virus' attachment and entry into the host cell. The S protein is made of two domains, the S1 domain which allows for protein interactions between the virus and the host cell, and the S2 domain which allows for membrane fusion. Because of the S1's role in protein interaction, it is often the target of potential vaccines. Within the S1 domain, it's C-terminal domain encodes for the receptor binding domain (RBD), which is why the S1 CTD is the target of this study. In this study we focused on the expression, purification, and immunogenicity testing of the CTD protein using T7 Express E. coli as the expression host. We used PCR, gel electrophoresis, Sanger Sequencing, western blots, and mass spectrometry to ensure that the protein was being expressed properly. The future goal is to use this protein as the antigen in a future nanoparticle-based PEDV vaccine.