Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: January 13, 2005
Publication Date: June 1, 2005
Citation: Nemchinov, L.G., Paape, M.J., Sohn, E.J., Bannerman, D.D., Zarlenga, D.S., Hammond, R. 2005. Plant virus-based expression of functional cd14 lps receptor in plants. American Society for Virology Meeting. p.85. Technical Abstract: Transient expression of therapeutic proteins utilizing plant virus-based vectors is a rapid and efficient method for molecular farming in plants. The goal of our study was to express a soluble CD14 (sCD14) protein for prevention of mastitis in lactating dairy cows. The secretory form of CD14 antigen, a high affinity receptor for the complex of lipopolysaccharides (LPS) and LPS-Binding protein (LBP), is capable of binding to the LPS endotoxin of E. coli and other coliform bacteria and neutralizing them. This can prevent the cow's immune system from acute overreacting shock as well as from infection itself, thereby protecting against mastitis-causing bacteria. The CD14 recombinant gene, engineered to carry affinity tags for ease of purification, was incorporated into a plant virus-based vector. Plants were infected with in vitro synthesized viral RNA transcripts and tested for protein expression after showing signs of systemic infection. RT-PCR assays with CD14-specific primers confirmed the presence of CD14 mRNA in the experimentally-infected plants. Western Blots probed with CD14-specific antibodies demonstrated that crude plant extracts as well as affinity-purified samples contain immunoreactive recombinant protein of predicted molecular mass. Functional behavior of sCD14 was tested in vitro by microscopic analysis of apoptosis and by production interleukin-8 (IL-8) production in bovine endothelial cells. In the presence of plant-derived sCD14 (PrbosCD14), LPS induced apoptosis and IL-8 production, demonstrating biological activity of the protein. Infusion of LPS and PrbosCD14 into mammary glands caused increased levels of leukocytes. Quarters of mammary glands challenged with E. coli and injected with PrbosCD14 exhibited no clinical symptoms of infection.