Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of a pandemic that has been devastating the U.S. and global swine industry for more than twenty years. PRRSV vaccine development is challenging due to virus heterogeneity. Evidence indicates that the major envelope protein, GP5, is the primary target for a subunit vaccine. In native virions GP5 primarily exists as a disulfide linked complex with the membrane protein, M, which also possesses immunogenic properties. Recent studies report that the GP5/M complex is a more significant vaccine candidate. Currently, no bulk purification methods have been reported for PRRSV proteins. The objective of this research was to develop a purification process for GP5 or GP5/M from native virions.
PRRS virions were isolated and concentrated through sucrose cushion ultracentrifugation and target envelope proteins were solubilized with Triton X-100 detergent for further processing. GP5/M was not consistently identified in samples and was therefore abandoned. GP5 was identified by Western blot throughout processing with a αORF5 antibody. Cation exchange chromatography (CEX) was utilized for partial fractionation of GP5, although the viral nucleocapsid protein, N, was a major impurity in CEX elution fractions. As a second chromatographic step, hydrophobic interaction chromatography (HIC) further purified GP5 by means of a two-stage elution scheme. Pure GP5 was eluted from the HIC resin in the second HIC elution stage by Triton X-100 displacement; however the protein is present as a homodimeric/tetrameric aggregate. This process will be useful in PRRSV vaccine development and the purified GP5 product could be used as much needed positive controls in animal studies. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/33194 |
| Date | 13 June 2007 |
| Creators | Matanin, Brad Matthew |
| Contributors | Biological Systems Engineering, Zhang, Chenming Mike, Meng, Xiang-Jin, Mallikarjunan, Parameswarakumar |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | Matanin_Thesis_Final.pdf |
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